EA037849B1 - Fusion protein, dna, genetic construct, producer, fusion protein-based vaccine for tuberculosis prevention and treatment (versions) - Google Patents

Abstract

The invention relates to molecular biology, biotechnology, medicine and cane be used for preventing and treating tuberculosis. Fusion proteins are proposed including immunogenic fragments of Ag85B proteins, Tb10.4 M.tuberculosis, FliC S.Typhimurium, connected by flexible bridges, encoding polynucleotides, genetic constructs for expression of polynucleotides, producers and vaccine options for tuberculosis prevention and treatment based on the described fusion protein, vaccine is developed for humans or animals. The proposed vaccine has an efficiency exceeding that of BCG. The invention claimed for producing said vaccine provides its security and simplicity in production and application.

Description

The group of inventions relates to molecular biology, biotechnology, medicine and can be used for the prevention and treatment of tuberculosis.

Tuberculosis is the infectious disease with the highest number of deaths. According to the World Health Organization (WHO), about 8 million people fall ill with tuberculosis every year, and about 3 million die. Treatment of tuberculosis is complicated by the widespread prevalence of highly resistant strains of mycobacteria that cause multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. In this regard, prevention is a promising approach in the fight against this disease. At the initiative of WHO, since 2006, The Global Plan to Stop TV has been announced, according to which the development of a new generation of vaccines against tuberculosis takes one of the leading places. According to the latest WHO report on tuberculosis, 2014, a new vaccine to help prevent TB transmission among adolescents and adults in developing countries will be the most cost-effective tool in mitigating the epidemic [http://www.who.int/immunization/research/ forums_and_initiatives / 07_Evans_GVIRF_TB_Vaccine_Progress.pdf].

To date, the only registered and used vaccine is BCG (BCG), derived from an attenuated strain of Mycobacterium bovis and first introduced to humans in 1921. Since 2006, several countries have stopped using BCG for mass vaccination following the outbreak of BCG infection in Finland. The United States and the Netherlands have never used BCG in large quantities. The dubious efficacy of BCG vaccination and side effects create an urgent need to develop an effective and safe vaccination against Mycobacterium tuberculosis.

Currently, they are trying to solve this problem in different ways: alternative BCG vaccines are being developed and studied, such as live vaccines with improved properties, inactivated vaccines, subunit vaccines, vaccines obtained using recombinant DNA technology, combined variants. However, a vaccine that is safe and easy to manufacture and use, which would have an efficacy at least comparable to that of BCG, does not exist at the moment. In this regard, the object of the present invention is to provide a vaccine against tuberculosis with the above properties, which will also have a low cost.

Of all the directions in the development of new anti-tuberculosis vaccines, the authors of the present group of inventions consider the most promising use of vaccines obtained using recombinant DNA technology. However, their effectiveness varies and is determined by a variety of factors, one of the most important of which is the selection of components and their shape (structure).

To create the present invention, the immunodominant secreted antigens of M. tuberculosis were selected - the Ag85B protein of the 85 protein complex and the TB 10.4 protein (culture-filtrate protein-7). These proteins are highly conserved among various strains of mycobacteria, and are also capable of eliciting an immune response, which suggests that it is possible to obtain a cross-reactive immune response [WO 96/15241, priority date 14.11.94 US]. It is important that these proteins have no homology with known proteins of other organisms, which ensures the specificity of their action. It has also been shown that the TB 10.4 protein has an adjuvant effect when administered in combination with mycobacterial proteins [EA 012037 B1, priority date 20041116].

The following solutions are known based on the above M. tuberculosis proteins.

Known bacterium, in one embodiment of the invention - mycobacterium containing plasmid DNA, encoding in one embodiment of the invention Ag85b and TB 10.4 [WO 2008150969 A9, priority date 31.05.2007]. However, the introduction of live mycobacteria, albeit attenuated, carries risks. Using a protein-based vaccine is a safer alternative.

Known vaccine HyVAC IV / Aeras, which contains proteins Ag85B and TB 10.4, as well as a complex adjuvant IC31, containing a synthetic antimicrobial peptide KLKL5KLK, whose action is aimed at enhancing the interaction of antigen-presenting cells with antigen, and the ligand TLR9 ODN1a, which has an immunostimulating action. This vaccine is in phase IIa clinical trials [http://www.who.int/immunization/research/fomms_and_initiatives/07_Evans_GVIRF_TB_ Vaccine_Progress.pdf]. However, the production of an active vaccine agent consisting of four components, full-length proteins and peptides obtained using completely different mechanisms, is technically difficult. Moreover, when using chemically synthesized molecules, including protein ones, it should be understood that some of them can be represented by enantiomers, which can lead to unpredictable results in the body when applied. The concept of enantiomerism plays an important role in pharmaceuticals, since different enantiomers of medicinal substances, as a rule, have different biological activities. The use of living systems to obtain peptide molecules makes it possible to obtain molecules of a natural structure, a homogeneous mixture.

Known nucleic acid encoding the proteins Ag85A, Ag85B and TB 10.4 of Mycobacterium tuberculosis under the control of a promoter, as well as an adenoviral vector carrying such a nucleic acid, as a result, a fusion protein is synthesized in the administration organism, and an anti-tuberculosis vaccine based on such an adenoviral vector is known. additionally contain an adjuvant

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[US 8609402 B2, priority date 20041116].

Known adenoviral vector for the treatment of active tuberculosis, carrying a nucleic acid encoding proteins Ag85A, Ag85B and TB 10.4, in one embodiment of the invention in the body of administration, a fusion protein is synthesized from it, an adjuvant can also be added [WO

2012/038367 A1, priority date 20100920].

However, the use of adenovirus to deliver the immunogen also carries risks. Viral vectors have several disadvantages, including the fact that they are expensive and can cause an inflammatory response, which precludes re-introduction of the vector. Also, viral vectors have the ability to replicate, which reduces the degree of control over the expression of the target protein, which, in turn, is not always desirable and applicable.

Viral vectors and modified pathogens also carry risks of inheritance, which may impede their use in humans [R.R. Redfield et al., New Engl. J. Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149,1569 (1989)].

The use of a recombinant fusion protein will make it possible to reduce the number of vaccine components, and accordingly, to simplify and reduce the cost of its production and, accordingly, its cost, while communicating safety to the vaccine.

There is a known method of obtaining a hybrid construct TB10.4-Hsp16.3, according to which the genes encoding these proteins are cloned at different restriction sites in the plasmid, the resulting plasmid transforms E. coli cells [WO 201288739 A1, priority date 29.12.2010].

A hybrid construct is known that contains at least one domain of a Mycobacterium protein capable of eliciting an immune response in a mammal, for example Ag85B, and a phagolysosome exit domain, for example from Listeria [WO 9910496 A1, priority date 22.08.1997].

Known hybrid construct TB10.4-Ag85B-antigen caused by starvation of mycobacteria (produced at least 6.5 times more after exposing mycobacterium to starvation) [WO 2006136162 A2, priority date 23.06.2005].

However, it is possible to further increase the immunogenicity of the vaccine using an adjuvant.

Known hybrid polypeptide, one of the variants of the invention is a protein based on a protein of the ESAT-6 family and protein MPT59 (Ag85B), or one T-cell epitope of MRT59, as well as a composition additionally containing an adjuvant from DDA, Quil A, poly I: C, incomplete Freund's adjuvant, IFN-γ, IL-2, IL-12, MPL and MDP [US7867502 B1, priority date 13.07.1999].

A vaccine containing a fusion protein containing amino acid sequences encoding fusion proteins Ag85B-TB10.4 is known, an adjuvant can additionally be used [WO 2005/061534 A2, priority date 23.12.2003].

Known pharmaceutical composition containing a fusion protein containing amino acid sequences Rv0288 (TB 10.4) and at least one agent from a fragment, including Ag85B, can additionally be used as an adjuvant [US 6991797 B2, priority date 02.07.1993].

Known immunogenic composition containing mycobacterial antigens in the form of separate polypeptides, or in the form of one or more fusion proteins, or together of separate polypeptides and fusion proteins, the fusion protein contains antigens M. tuberculosis Ag85B, TB 10.4 and ESAT6, can also additionally be used an adjuvant [WO 2014/009438 A2, priority date 10.07.2012].

Known immunogenic composition containing a fusion protein, which additionally contains Ag85B, TB10.4, and also containing an adjuvant [US 8101193 B2, US 7968105 B2, US 8293250 B2, US 8703151 B2, priority date 23.06.2005].

Known hybrid protein, in one of the variants hypothetically containing the protein Rpf (Mycobacterium Resuscitation Promoting Factor) as an adjuvant and proteins Ag85B, TB10.4 [WO 2014/009433 A1, priority date 10.07.2012]. However, this construct is not structurally characterized, the sequence is shown only for the fusion protein containing the proteins RpfB-Dhyb, Ag85B, TB 10.4 and ESAT6, RpfB-Dhyb-Ag85B-TB10.4-ESAT6 from 849 a.o. The latter protein is large enough, which makes it difficult to accept a conformation in which all the required protein components can function simultaneously, which can lead to difficulties in the implementation of the declared functions.

The use of an adjuvant makes it possible to enhance the immunogenicity of vaccines, however, it is important to choose an adjuvant that will induce the immune response most specific to the pathogen, in this case, capable of stimulating the T-cell immune response, along with the B-cell one, and which will also contribute to the formation of local immunity. The inventors of the present invention considered flagellin to be the most optimal option for these purposes.

Flagellin is a protein of bacterial flagella present in many bacteria, including pathogenic ones. Flagellin is a TLR5 (Toll-like receptor 5) ligand. The interaction of flagellin with TLR-5 stimulates the maturation of macrophages and dendritic cells - antigen-presenting cells, which leads to the development of an immune response [Mc Dermott P.F. High-affinity interaction between Gram-negative flagellin and a cell surface polypeptide results in human monocyte activation. Infect. Immun. - 2000. - V. 68. - p. 5525-5529; Means T.K. et al. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine produc- 2 037849 tion in human dendritic cells. J. Immunol. - 2003. - V.170. - p.:5165-5175]. Flagellins induce the formation of serum and secretory antibodies, Th1 and Th2 responses both to the flagellins themselves and to co-administered antigens.

Known vaccine composition containing one or more sources of DosR proteins and, in one of the variants, an adjuvant, in one of the variants of bacterial flagellins [WO 2006/104389 A1, priority date 31.03.2005].

A known vaccination composition containing a polypeptide comprising antigenic portions in one of the embodiments of Ag85B and TB 10.4 proteins, also containing a nucleic acid encoding a CD40 ligand, or a peptide that has the ability to bind to the CD40 ligand and activate the CD40 receptor expressed on the antibody producing B-lymphocytes, which in one embodiment of the invention also contains flagellin [WO 2010/034974 A2, priority date 09.24.2008]. This invention is considered by the authors of the present invention as a prototype. The disadvantage of the prototype of the invention is the multicomponent, three different components in one vaccine. Moreover, the fusion protein is not structurally characterized, only its components are indicated. It is also proposed to use full-size flagellin, and its belonging to a particular organism is not indicated, and the type of flagellin is not indicated, for example, for flagellin Salmonella FliC or FljB.

Flagellin, depending on the origin from a particular microorganism, differs and consists of 259-1250 amino acid residues, which, of course, affects its properties.

Differences in the structure of flagellin are also observed among the same bacterial species. Thus, most of the serovars of Salmonella enterica are biphasic, i.e. they have alternating expression of different flagellar antigens (phases). This is due to the possibility of finding in an active state only 1 of 2 flagellin genes (fliC and fljB), localized at different loci of the chromosome [Baker S., Hardy J., Sanderson K.E. et al. A novel linear plasmid mediates flagellar variation in Salmonella Typhi. PLoS Pathog, 2007, 3 (5): e59. doi: 10.1371 / journal.ppat.0030059], FliC - phase 1 flagellin (STF), fljB - phase 2 flagellin (STF2). These flagellins have the same N-terminus, homologous, differing by several amino acid residues, the C-terminus, as well as a different D3 domain, respectively, have a different structure and can have different effects.

Thus, the indication of the microorganism whose flagellin is used and the type of flagellin used (eg for Salmonella) is essential.

Moreover, the known flagellin adjuvants exhibit significant side effects and, in particular, intrinsic antigenic activity and systemic pro-inflammatory properties when administered in vivo.

The binding of flagellin with TLR-5 occurs only in monomeric form [Abaturov A.E. The role of TOLL-like receptors in the recognition of pathogen-associated molecular structures of infectious pathogenic agents and the development of inflammation. Ch.Z. Recognition of TLR / A ligands. E. Abaturov, A.P. Volosovets, E.I. Yulish // Child Health. - Donetsk, 2012, No. 7.-S. 157-164], however, when obtaining full-size flagellins, they can polymerize, and their preservation in solution in monomeric form, while maintaining the correct conformation, requires special conditions. At the same time, the joint use of such a solution and other agents, for example, antigen (s), can negatively affect the conformation of the latter and, accordingly, properties. It turns out that the combined use of full-length flagellin and antigen (s) may not lead to the desired result, due to inactivation of one of the agents when the conditions for retention of activity do not coincide.

In view of the foregoing, the implementation of the invention described in WO 2010/034974 A2 is questioned.

The authors of the present invention have proposed an unexpected original solution that is free of these disadvantages.

The technical result from the use of the invention is to simplify, reduce the cost and shorten the production time of the anti-tuberculosis immunogen - an active agent for the vaccine and, accordingly, the vaccine against tuberculosis, due to the production of only one component, a fusion protein, instead of the production of many individual components of the vaccine mixture (analogs), while maintaining the required activity of the proteins selected for inclusion in the fusion protein due to the length and the created structure of the protein, in which the immunogenic epitopes are connected by flexible bridges.

The technical result from the use of the invention is to increase the efficiency and predictability of the result of using an anti-tuberculosis immunogen - an active agent for a vaccine and, accordingly, a vaccine against tuberculosis, due to the use of one component, which combines the functions of a specific mycobacterial antigen, and an adjuvant that mediates optimal for suppression the development of tuberculosis infection, immunity, the correct conformation of which, mediating the full functioning of all its components, is easy to ensure also due to the absence of other molecules that require different conditions for maintaining activity.

Finally, the technical result from the use of the invention is expressed in the expansion of the range of anti-tuberculosis immunogens - active agents of vaccines and, accordingly, vaccines against tuberculosis. With contraindications to the use of analogs, or unwillingness to use analogs due to their

- 3 037849 of the above-described disadvantages, this vaccine will provide protection against tuberculosis. Due to the fact that the problem of tuberculosis is very acute, and not many people succeed in bringing the drug to the market, this invention will increase the chances of fighting this infection.

The administration of the immunogen or vaccine is preferably intramuscular. It is also possible to use for immunization of the mucosal surface, since immunity induced in one region of the mucosa can induce the development of immunity in the distal region of the mucosa [McGhee J.R. et al. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 1992,10: 75-88]. The formation of local immunity is important to prevent infection with mycobacteria. The content of flagellin fragments in the fusion protein also contributes to this process, so it is known that in the respiratory tract, flagellin induces IgA synthesis and causes a Th2-associated response [Soloff A.C., Barratt-Boyes S.M. Enemy at the gates: dendritic cells and immunity to mucosal pathogens. Cell Res. 2010 Aug; 20 (8): 872-85. doi: 10.1038 / cr.2010.94. Epub 2010 Jul 6]. Immunization of the mucosal surface also greatly facilitates delivery of the immunogen.

The essence of the invention

A group of inventions is proposed: a hybrid protein comprising immunogenic proteins Ag85B, Tb10.4 of Mycobacterium tuberculosis, fragments of flagellin FliC Salmonella enterica serovar Typhimurium, connected by flexible bridges, characterized by the amino acid sequence SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO : 3 or SEQ ID NO: 4; a polynucleotide encoding such a fusion protein, codon optimized for expression in the cells of the fusion protein producer; a genetic construct for expression in the cells of the producer of such a polynucleotide, including, in addition to the polynucleotide, elements allowing to realize the specified purpose; a recombinant cell for the production of the described fusion protein, represented by a prokaryotic or eukaryotic organism; a vaccine for the prevention and treatment of tuberculosis containing the described fusion protein as an active agent in an effective amount, as well as a physiologically acceptable carrier or buffer solution suitable for humans and animals. The proposed vaccine has an efficacy exceeding that of BCG. The inventions claimed for the production of this vaccine ensure its safety and ease of manufacture and use.

Detailed description of the invention

A group of inventions is proposed: a hybrid protein, a polynucleotide, a genetic construct, a recombinant cell, a vaccine based on a hybrid protein for the prevention and treatment of tuberculosis (variants).

Fusion proteins are proposed, each comprising the immunogenic proteins Ag85B, Tb10.4 of Mycobacterium tuberculosis and fragments of the Salmonella enterica serovar Typhimurium FliC protein, connected by flexible bridges; proteins are characterized by the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

The proposed polynucleotides encoding the described hybrid proteins, codon optimized for expression in the cells of the producer of the hybrid protein. With the knowledge of the amino acid sequence of the protein, one skilled in the art will be able to obtain the nucleotide sequence. Codon optimization is carried out independently, using information on the frequency of occurrence of codons in the producer, for example, in a database [for example, Nakamura Y., Gojobori T., Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res. 2000 Jan 1; 28 (1): 292], or using specialized programs, for example, presented on the site http://www.encorbio.com/protocols/Codon.htm.

The proposed genetic constructs for expression in the cells of the producer of the described polynucleotides; each construct includes, in addition to the polynucleotide, elements allowing the indicated purpose to be realized. A genetic construct means a recombinant vector, which can be presented, depending on compatibility with the producer, with a viral, plasmid, phosmid, cosmid or other possible vector. For example, when choosing E. coli cells as a producer, the gene can be in the composition of a bacteriophage, for example, based on phage X, or a plasmid, for example, based on pBR, or pUC, and the like. When used as a producer of a Bacillus subtilis organism, the gene can be, for example, in a pUB-based plasmid. When used as a yeast producer, the genetic construct can be represented, for example, by a plasmid based on YEp, or YRp, or YCp, or YIp.

A genetic construct that ensures protein synthesis in producer cells must contain elements for maintaining and amplifying, mainly in large quantities, and efficiently functioning as intended, as well as for selecting a transformant.

Elements for maintenance and amplification refers to the origin of replication. A suitable origin of replication for, for example, a plasmid vector is, for example, pM1 (der.), ColE1 (der.) And F1, pUC and F1, SV40, but is not limited to them. An element for integrating the construct into the genome of the producer may also be contained. For example, 3 'AOX1 or 18S rRNA for yeast.

Elements for efficient functioning, for the expression of the encoded protein, are meant transcription initiation signals, promoter, translation initiation signals, start codon and stop codon (s), transcription termination sequences, regulatory sequences.

When used as a producer of the organism Escherichia coli, the promoter can be, for example, the promoter of the lactose operon, tryptophan operon; yeast, for example the promoter of the acid phosphatase gene, the alcohol dehydrogenase gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the galactose metabolism gene; fungi, for example the promoter of the cellobiohydrolase gene, or α-amylase, or glucoamylase, or glyceraldehyde-3-phosphate dehydrogenase, or the abp1 gene; plants, for example, the CaMV 19S RNA or CaMV 35S RNA promoter, or the nopaline synthetase gene promoter. When used as a producer of mammalian cells, for example, the promoter can be a natural promoter (for example, CaM kinase II, CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of kainate receptor and subunit B of the glutamate receptor and others), or synthetic. An example of a termination sequence for mammalian cells is that of bovine growth hormone (BGH), for plant cells - the nopaline synthetase gene (NOS T).

Translation initiation signals - Shine-Dalgarno sequence [Kapp L.D., Lorsch J.R. The molecular mechanics of eukaryotic translation // Annual Review of Biochemistry, 73/2004, 657-704] in prokaryotes and the Kozak sequence in eukaryotes [Kozak M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292]. When using mammalian cells, the Kozak sequence just before the ATG start codon allows for increased expression [Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J. 1997; 16 (9): 2482-2492].

Elements for the selection of a transformant are usually understood to be a gene that causes antibiotic resistance or a gene that completes auxotrophy. When a bacterium is used as a producer, the selectable marker can be represented, for example, by a gene for resistance to ampicillin, or kanamycin, or tetracycline; yeast, for example the LEU2 or TRP1 or URA3 genome; fungi, for example the genome of resistance to bialaphos, or hygromycin, or aureobasidin, or bleomycin; plants, for example the genome of resistance to kanamycin or bialaphos; mammalian cells, for example the neomycin resistance genome.

Regulatory sequences are nucleotide sequences that can affect gene expression at the level of transcription and / or translation, as well as the mechanisms that ensure the existence and maintenance of the functioning of a genetic construct. Possible regulatory sequences in relation to the promoter are an enhancer, to increase the level of expression through improving the interaction of RNA polymerase and DNA, an insulator, to modulate the functions of an enhancer, silencers, or their fragments, to reduce the level of transcription, for example, for tissue-specific expression, 5'- untranslated region before the promoter, including the intron. The genetic construct of the present invention, in one of the variants, additionally contains from one of the above regulatory sequences, depending on the variant of the vector, based on the choice of the promoter and the desired parameters of the target gene expression.

To obtain secreted fusion proteins, characterized by SEQ ID NO: 1-4, a signal peptide suitable for the producer used is placed at the N-terminus of the sequence. Examples of such secretory sequences are described in the literature [for example, for E. coli - in RF patent No. 2198179, priority date 09/15/1999, for yeast - in RF patent No. 2143495, priority date 07/08/1994, US patent No. 4546082, priority date 06/17 .1982, European applications for invention No. 116201, 123294, 123544, 163529, 123289, application for invention of Denmark 3614/83, priority date 08.08.1983, for plants - in articles Kapila J., Rycke RD, Van Montagu M., Agenon G. (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 122: 101-108, Stiefel V., RuizAvila L., Raz R., Valles MP, Ghez J., Pages M., Martinez-Izquierdo JA, Ludevid MD, Langdale JA, Nelson T., and Puigdomenech P. (1990 ). Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation. Plant Cell 2, 785-793].

Other elements required for the expression system to function may also be contained.

Producers of the described hybrid proteins are proposed - a recombinant cell for the production of a hybrid protein, represented by a prokaryotic organism, or a cell of a eukaryotic organism. A prokaryotic organism is represented, for example, by Escherichia coli, Bacillus subtilis, a eukaryotic organism - by fungi, plants, mammals.

Examples are given for clarity of the implementation of inventions (genetic construct, recombinant cell), but the implementation is not limited to them. At the moment, in the scientific literature (for example, textbooks on molecular biology, biotechnology, articles, for example, available at http://www.labome.ru/method/Recombinant-Protein-Expression-Vector-Host-Systems.htmL specialized sites on the Internet with databases, for example, vectors) describes a variety of expression systems, as well as methods for their creation and work with them, in connection with which the specialist understands that the presented description of the group of inventions allows them to be implemented.

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The proposed vaccine variants for the prevention and treatment of tuberculosis, each variant contains one of the described fusion proteins as an active agent, in an effective amount, as well as a physiologically acceptable carrier or buffer solution. Pharmaceutically acceptable carriers or buffers are known in the art and include those described in various texts, such as, for example, Remington's Pharmaceutical Sciences. Both a person and an animal, including domestic or farm animals, can act as a consumer of the vaccine.

The authors of the present invention conducted laboratory studies confirming the possibility of implementing the described inventions. The obtained research results are illustrated by the following examples.

Example 1. Modeling of fusion proteins (SEQ ID NO: 1 - SEQ ID NO: 4) for an anti-tuberculosis vaccine.

To model proteins, the following steps were performed.

1) Search for immunogenic epitopes of Ag85B, Tb10.4, FliC proteins using the CPREDS program (http://ailab.cs.iastate.edu/bcpreds/predict.html).

2) Building a whole protein model to determine the orientation of the domains.

3) Building models for each domain (using sample 3D structures and ab initio).

4) Docking models using the whole protein model.

To obtain the most realistic results in an automatic mode, we used the I-Tasser algorithm for protein modeling.

The modeled fusion proteins consist of 546 amino acid residues, their amino acid sequences are presented - SEQ ID NO: 1 - SEQ ID NO: 4. Analysis of the amino acid sequences of these proteins using the ProtParam program (http://au.expasy.org/tools/ protparam.html) showed that the fusion proteins have a molecular weight of 57.2 kDa, pI 4.79.

Example 2. Obtaining highly purified hybrid proteins using a prokaryotic organism.

We converted the amino acid sequences of the calculated fusion proteins into nucleotide sequences (1731 bp), simultaneously performing codon optimization for expression in E. coli cells in the manual mode and adding the NdeI / XhoI restriction sites flanking the gene. The calculated genes were synthesized chemically.

The resulting genes were cloned into the bacterial expression vector pET28a (+) at the NdeI and XhoI restriction sites according to the instructions for the vector.

To create a producer strain, we used E. coli cells of the BL21 Star (DE3) strain (Invitrogen, USA), with the F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) genotype, containing a lysogen in the λDe3 genome and the rne131 mutation. The mutated me (rne131) gene encodes a truncated form of RNase E, which reduces intracellular destruction of mRNA, leading to an increase in its enzymatic stability. Ion- and ompT-mutations in protease genes make it possible to obtain non-proteolyzed recombinant proteins in large quantities.

Prepared cells of E. coli strain BL21 with genotype F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) as follows. The cells were incubated at + 37 ° C overnight in 5 ml of L-broth containing 1% tryptone, 1% yeast extract, and 1% sodium chloride. The culture was diluted with fresh L broth 50-100 times and grown on a shaker at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 min. We transferred 100 ml of the culture into a sterile centrifuge tube and pelleted the cells at + 4 ° С at 5000 g for 10 min. The supernatant was discarded, the cells were resuspended in deionized water in the original volume, followed by centrifugation. The washing procedure was repeated three times. After washing, the cell pellet was resuspended in a small volume of deionized water and centrifuged for 30 s at 5000 rpm in a microcentrifuge.

The transformation of competent cells was carried out by electroporation. For this, 1 μL of a tenfold diluted ligase mixture was added to 12 μL of competent cells, mixed and electroporation was performed on an Eporator electroporator (Eppendorf, Germany) in sterile electroporation cuvettes (Eppendorf, Germany), 100 μL, 1 mm slit, with an electric pulse of 1 , 7 kV with a duration of 5 ms.

After transformation, cells were incubated in SOC medium (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) for 40 min. at + 37 ° C. 10-100 μl of cell suspension was plated on selective LB medium (Gibko BRL, USA) containing kanamycin (100 μg / ml) to select clones containing plasmids (producer strains).

The grown E. coli colonies were checked for the presence of plasmids with the insertion of the target gene. A clone of cells containing the desired plasmid DNA was considered a fusion protein producing strain. Thus, four strains producing fusion proteins characterized by SEQ ID NO: 1 SEQ ID NO: 4 were obtained.

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For the cultivation of the obtained producer strains, a standard agar LB medium containing kanamycin at a concentration of 100 μg / ml and glucose at a concentration of 1% was used to block nonspecific expression.

Expression was induced when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm. 0.2% lactose was used as an inducer (Studier, 2005).

For autoinduction of expression by the method of Studier (Studier, 2005), we used PYP-5052 medium consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K ₂ HPO ₄ , 25 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose, and 0.2% lactose.

A single colony of the producer strain was inoculated into PYP-5052 medium containing kanamycin at a concentration of 100 μg / ml. Fermentation was carried out at + 37 ° C in a thermostated rotary-type shaker at 250 rpm for 20 h until there was no significant change in OD ₆₀₀ for 1 h. An aliquot of cells was taken for analysis of the expression of the gene encoding the fusion protein by electrophoresis in PAGE, and the remaining the biomass was precipitated by centrifugation at 9000 g.

The precipitated cells were lysed using 3 sonication cycles of 30 s with a break of 2 min on ice. Then, the destruction of inclusion bodies was carried out by incubation with lysis buffer containing 500 mM sodium phosphate buffer, pH 8.0, 6M guanidine hydrochloride, 500 mM sodium chloride, for an hour. To the cells harvested by centrifugation from 50 ml of culture was added 8 ml of lysis buffer.

The column containing Ni-NTU Sepharose was pre-equilibrated with loading buffer (500 mM sodium phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 10 mM imidazole). The destroyed inclusion bodies were applied to the column. The column was then washed with two volumes of loading buffer (500 mM sodium phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 10 mM imidazole). The column was then washed with three volumes of wash buffer (500 mM sodium phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 30 mM imidazole). The protein was eluted with 5 ml of elution buffer (500 mM sodium phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 200 mM imidazole). Fractions of 1 ml were collected, analyzed by electrophoresis in 12% PAGE-DDs-Na, fractions with the target protein were combined, the protein concentration in them was determined by the Bradford method.

Received preparations of proteins (SEQ ID NO: 1-4) with a purity of about 97-98%, according to SDS-PAGE, the concentration of the fusion protein in each preparation was 1-2 mg / ml.

Example 3. Obtaining highly purified hybrid proteins using a eukaryotic organism.

3.1. Obtaining highly purified hybrid proteins using fungal cells.

We converted the amino acid sequences of the calculated fusion proteins into nucleotide sequences (1731 bp), simultaneously performing codon optimization for expression in Pichia pastoris yeast cells in the manual mode and adding the gene flanking regions to obtain the secreted protein, according to the instructions for the cloning vector. The calculated genes were synthesized chemically.

The resulting genes were cloned into the eukaryotic expression vector pHIL-S1, according to the instructions for the vector.

Yeast cells were prepared for transformation. Cultivation and freezing of cells of Pichia pastoris strain SMD1163, defective in several yeast proteases, was carried out, which ensures the stability of the secreted protein. The cells were seeded under sterile conditions on agar in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 1 mM dithiothreitol), cultured at 30 ° C, then subcultured into suspension and cultured for 16 h. resuspended in YPD medium supplemented with 15% glycerol and frozen at -86 ° C. To obtain competent cells, cell colonies were preliminarily grown on an agar plate in YPD medium at 30 ° C for two days. Then the contents of one colony were grown in 10 ml of YPD medium at 30 ° C for 16 h. The suspension was diluted in YPD to OD ₆₀₀ 0.2 and a final volume of 10 ml, and the culture was grown to OD ₆₀₀ 0-8 for 4 h. The cell suspension was centrifuged 5 min at 500 g, the supernatant was poured out, resuspended in 10 ml of solution I from the EasyComp Transformation Kit, centrifuged again, and the pellet was resuspended in solution I. temperature of -90 ° C before use.

The EasyComp Transformation Kit included in the Pichia Easy Select Kit (Invitrogen) was used for transformation; the reaction was carried out according to the instructions for the kit. The resulting cell suspension was plated into a sterile dish on an agar gel prepared in YPD medium supplemented with 1 M sorbitol and the antibiotic ampicillin at a final concentration of 100 μg / ml. After 3 days, several dozen colonies were obtained per dish. Cells from the grown colonies were transferred onto a plate with MMD (minimal medium dextrose) agar and cultured for 2 days at 30 ° C.

The cells of the colonies grown on the selective medium were transferred into flasks and cultured in 5 ml of MGY medium on a shaker (250 rpm) for 1 day until OD ₆₀₀ 5. After that, the cells were pelleted by centrifugation at 3000 g for 10 min. Control of the target gene expression was performed by SDS-PAGE.

After the cells were precipitated, the culture medium was filtered (pore diameter 45 μm), then

Tris-HCl pH 6.0 to a final concentration of 20 mM. The culture medium containing the fusion protein was concentrated 5-10 fold using Millipore concentrators for proteins with molecular weight over kDa.

After concentration, the fusion protein preparation was heated in a water bath to boiling (t = 100 ° C) and boiled for 2 min, after which it was centrifuged at 4 ° C, 15000xg for 15 min.

Ion exchange chromatography was carried out on CM-Sepharose. The column with CM-Sepharose was equilibrated with a buffer containing 20 mM Tris-HCl pH 6.0. The fusion protein preparation was applied at a rate of 60 ml / h. The column was washed with 20 mM Tris-HCl pH 6.0; 20 mM Tris-HCl pH 6.0, 200 mM NaCl. Elution was performed with 20 mM Tris-HCl pH 6.0, 1 M NaCl, and fractions of 1 ml were collected.

The preparation of the obtained fusion protein was diluted 2 times, phosphate pH 8.0 was added to a concentration of 50 mM, and applied to the column. After washing the column with loading buffer, ballast proteins were removed by washing with 20 mM imidazole solution in the same buffer. The protein was eluted with a solution containing 200 mM imidazole.

As a result, preparations of hybrid proteins with a purity of over 95% were obtained.

Four producer strains of four target hybrid proteins were selected, for which the presence of bands on the electropherograms corresponding to the molecular weight of the target proteins was revealed. The resulting strains are characterized by a high level of expression of targeted proteins.

3.2. Obtaining highly purified fusion proteins (SEQ ID NO: 1-4) using mammalian cells.

We converted the amino acid sequences of the calculated fusion proteins into nucleotide sequences (1731 bp), simultaneously performing codon optimization for expression in CHO cells in the manual mode and adding cloning sites into the pEGFP-N1 vector. The calculated genes were synthesized chemically.

The resulting genes were cloned into the pEGFP-N1 eukaryotic expression vector according to the instructions for the vector. The expressed fusion proteins were fused to an eGFP protein optimized for brighter fluorescence and high expression in mammalian cells.

The CHO-Ki cell line was cultured in DMEM medium containing 10% inactivated fetal bovine serum, 2 mM L-glutamine, 35 mg / L L-proline, and the commercial antibiotic Mycokill-AB (PAA Laboratories) at a working concentration. Cells were subcultured once every 5 days at a cell concentration of 10 ⁵ cells per 1 cm ² , with a dilution of 1:20. The cells were cultured at 37 ° C, in an atmosphere of 5% CO2 and high humidity.

This cell culture was transfected with the resulting plasmid encoding a fusion protein for each fusion protein.

Cells in the amount of 70-80% of the monolayer (8x10 ⁴ cells per 1 cm ² ) were washed with a culture medium that did not contain serum. A transfection mixture was prepared: 3-4 μg of linearized plasmid DNA was mixed with 375 μl of serum-free culture medium. The commercial transfection reagent Lipofectamine 2000 was mixed in a separate tube with the same amount of the same medium, the amount of which was determined by the ratio of 3 μl of the reagent to 1 μg of plasmid DNA. Thereafter, the resulting solutions were combined and incubated at room temperature for 3040 min.

The resulting transfection mixture was layered on washed cells. After 4-6 hours after the start of transfection, the transfection mixture was replaced with DMEM culture medium with serum.

The level of expression of the target gene was assessed by the intensity of fluorescence on the second day after transfection (36-48 h) using flow cytometry. Untransfected CHO-Ki cells were used as a negative control.

For cytometric analysis, cells were washed with phosphate-buffered saline, trypsinized, and removed from Petri dishes. Then it was washed twice to remove trypsin and carefully resuspended in phosphate buffered saline. The resulting suspension (106 cells in 1 ml of buffer) was transferred into 5 ml round-bottom tubes.

The voltage of the channels of the flow cytometer was selected in such a way that the autofluorescence of nontransfected cells was not taken into account during measurements. For this, the voltage in channels FL1 (in which eGFP fluorescence is detected) and FL2 (which was used as a control empty channel) were changed so that during measurements the vast majority of cells that did not contain the eGFP gene were in a region not exceeding 10 per logarithmic scale delayed for FL1 and FL2 channels.

After calibration, all samples were measured at constant voltages along the FL1 and FL2 channels. Thus, if eGFP fluorescence was detected in cells, the appearance of cells in a region exceeding 10 was recorded on a logarithmic scale along the FL1 channel, and all quantitative values of the gene expression level were measured in this region.

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After transfection, the resulting pools were cultured according to the protocol described above.

In order to get rid of cells that do not contain the transgene, the resulting pools of transfected cells were selected using the commercial antibiotic neomycin administered at a concentration of 800 μg / ml. Selected stable cell lines, each obtained from one transfected cell and, thus, characterized by a homogeneous genotype, which allows you to select the most effective clone.

Individual cell clones were obtained from total cell populations by limiting dilutions after 50 and 75 days of cultivation.

The cultured pools were removed from the plates and diluted in 10 ml of culture medium. After that, the cell concentration was counted using Garyaev's chamber, and additional dilutions were made so that the cell concentration was 2-3 cells per milliliter of medium. The resulting suspension was transferred into 24-well plates at 1 ml per well.

After 2 weeks of cultivation in DMEM medium containing the antibiotic neomycin at a concentration of 800 μg / μl, cytofluorometric analysis of the surviving clones was performed. Further maintenance of the obtained lines and analysis of the level of EGFP expression was performed according to the procedure described for the pools of transfected cells.

In this case, the median distribution of cells by fluorescence intensities served as the main quantitative indicator of the expression activity of the hybrid gene.

A sufficiently high level of EGFP expression was observed in 2 clones with the introduced construct containing the gene corresponding to SEQ ID NO: 1, 1 clone with the introduced construct containing the gene corresponding to SEQ ID NO: 2, 2 clones with the introduced construct containing the gene corresponding to SEQ ID NO: 3 and 2 clones with the introduced construct containing the gene corresponding to SEQ ID NO: 4.

After two and a half months of cultivation, the expression activity of clones containing the construct with the gene of interest averaged about 70% of the initial one. The produced protein was purified using Q-Sepharose anion exchange chromatography at pH 7.0. The yield of the target protein for the obtained producers was 13-16 mg / l.

It is possible to obtain the fusion proteins according to the invention in a secreted form.

The TPA signal sequence (tissue-type plasminogen activator isoform 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP000921.1) was placed at the N-terminus of the amino acid sequence of each fusion protein. We converted the amino acid sequences of proteins into nucleotide ones, codonically optimizing the latter for expression in mammalian cells (CHO) using the program on the website http://www.encorbio.com/protocols/Codon.htm and adding cloning sites into the pcDNA3.1 vector (+ ). The calculated genes were synthesized chemically. The synthesized gene was cloned into the pcDNA3.1 (+) vector according to the instructions for the vector.

Transfection of mammalian cells with the created plasmids was carried out by the method of calcium phosphate precipitation.

To transform mammalian cells (CHO) with plasmid DNA, the cells were seeded in 12-well plates (Costar, USA) with a seeding density of 5x104 cells / cm ² . The next day, the culture medium was changed to synchronize cell division. Three hours later, calcium phosphate-precipitated plasmid DNA was added to the cells. To prepare a precipitate, 250 μl of a solution containing 50 μg of DNA in 250 mM CaCl ₂ was slowly mixed with 250 μl of a solution (1.64% NaCl, 1.13% HEPES pH 7.12, and 0.04% Na ₂ HPO ₄ ). After 24 h of incubation at 37 ° C in an atmosphere of 5% CO _{2, the} medium was replaced with a similar medium containing 100 μg / ml of neomycin for selection of clones containing plasmids with the insertion of the target gene and, therefore, expressing hybrid proteins, the selection was carried out for 20 days, in the wells containing live cells, the medium was changed (in this case, the previous culture medium was not poured out, but was used to determine the amount of secreted proteins by ELISA), and after another day the cells were removed from the substrate and analyzed for the expression of the transformed genes. Transfection efficiency was analyzed using an EPICS XL Beckman Coulter flow cytometer (Beckman Coulter, USA).

The level of fusion proteins in the culture medium of the obtained stable transfect line CHO was assessed using standard solid phase ELISA.

As a result of cloning, stable CHO transfectomes were obtained, which were accumulated for cryopreservation and production of an experimental batch of fusion proteins. The productivity of transfected CHO expressing fusion proteins was 400-520 μg / 10 ⁷ cells / day.

Cultivation of producer cells was carried out using a BIOSTAT® Bplus bioreactor and an autoclaved IMDM medium supplemented with 45 g of DFBS (0.5%) and 25.8 g (100 mM) of zinc heptahydrate (ZnSO ₄ x7H ₂ O) per 9 L of medium. The operating mode was set: temperature 37 ° C, pH 6.9-7.2, oxygen concentration 50% of air saturation. After reaching the specified mode, the bioreactor was inoculated, for which the inoculum was introduced into it under aseptic conditions. The cultivation time was 3 days.

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At the end of the cultivation, the culture liquid was filtered through a sterile capsule.

Sartopure (Sartorius, Germany), with a pore diameter of 1.2 microns, at a rate of 1 l / min. Then the clarified liquid was concentrated on a Viva Flow 200 system (Sartorius, Germany) using a filter.

Concentration was carried out until a total volume of 200 ml was reached.

Chromatographic purification was carried out in two stages using sterile solutions. At the first stage, a BioLogic DuoFlow Pathfinder (Bio-Rad) system with an automatic BioFract fraction collector and a YMC TriArt semi-preparative chromatographic column, 250x4.6 mm, C18 sorbent were used. Before starting work, the column was equilibrated using 200 ml of buffer (1 kg of water for injection and 1 g of trifluoroacetic acid) in manual mode through a chromatograph pump at a rate of 2 ml / min.

The prepared material in a volume of 200 ml was introduced into the chromatograph through the chromatograph pump at a rate of 0.5 ml / min. Elution was performed with a buffer (2 kg of acetonitrile, 2 g of trifluoroacetic acid) at a rate of 0.5 ml per minute. Collected the fraction at the maximum absorption at 260 nm. The fraction volume was approximately 500 ml.

The second stage of purification was carried out using a BioSil SEC 125-5 gel chromatography column, 30x7.8 mm. The column was pre-equilibrated with 0.02M PBS buffer. The resulting material was introduced into the chromatograph through the chromatograph pump at a rate of 0.5 ml / min. Elution was performed with a buffer (0.6M NaCl solution) with a concentration gradient from 0.1 to 0.6M. A fraction was collected with an absorption at A280 nm of at least 3.4 optical units. The fraction was collected in vials. The volume of the resulting solution for each protein preparation was approximately 1 L with a fusion protein concentration of 1.8-2.5 mg per 1 ml.

The fusion proteins according to the invention can also be obtained using other mammalian cells, for example HEK293, COS.

3.3. Obtaining highly purified fusion proteins (SEQ ID NO: 1-4) using plants.

We converted the amino acid sequences of the calculated fusion proteins into nucleotide sequences (1731 bp), simultaneously performing codon optimization for expression in Nicotiana benthamiana cells in the manual mode and adding the gene flanking regions according to the instructions to the cloning vector. The calculated genes were synthesized chemically and cloned into the pTRV1 eukaryotic expression vector. It is also possible to use a viral vector (for example, described in the article Komarov T.V., Skulachev M.V., Zverev A.S., Schwartz AM, Dorokhov Yu.L., Atabekov I.G. (2006) New virus vector for effective production of target proteins in plants Biochemistry, 71 (8), 1043-1049).

The resulting vector was introduced into the Agrobaterium tumefaciens GV3101 strain, which was used for infiltration of N. benthamiana leaves. The resulting Agrobaterium tumefaciens strain carrying hybrid genes was cultured for 12 h at 30 ° C in a shaker. Cells (1.5 ml) was pelleted by centrifugation (4000 g, 5 min), the pellet was resuspended in buffer (1.5 ml of 10 mM MgCl _2, 10 mM MES (pH 5.5)), OD ₆₀₀ was adjusted to 0.2. A suspension of Agrobacteria was applied with a syringe without a needle to the leaves of growing N. benthamiana plants. The maximum level of protein synthesis was observed on days 7-11 after infiltration.

The expression of fusion proteins in the leaf cells of producing plants was analyzed using SDS Na electrophoresis in PAGE. A leaf fragment was triturated in buffer (10 mM KCl, 50 mM Tris pH 8.0, 5 mM MgCl ₂ , 10 mM P-mercaptotanol, 0.4 M sucrose, 10% glycerol) on day 10 after infection. The obtained extract was subjected to centrifugation (14000g, 10 min), the pellet and supernatant were analyzed using SDS-PAGE. The electrophoretogram revealed proteins corresponding in molecular weight to the hybrid proteins according to the invention in the membrane fraction of cells. In the control, plants that did not undergo transformation, the corresponding proteins were not identified. The protein yield was about 10-12% of the fraction of insoluble proteins.

Based on the results obtained, the claimed hybrid proteins can be obtained using both prokaryotic and eukaryotic cell systems, a highly purified preparation of each protein can be obtained using various types of protein purification. The isolation and purification conditions given were selected experimentally and may vary within the values known to the average person skilled in the art.

Example 3. Demonstration of the preventive effect of a vaccine based on a fusion protein characterized by SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4.

The experiment was carried out on non-inbred white mice (Rappolovo laboratory animal nursery). Animals, weighing 19-22 g, were kept under standard conditions, at an ambient temperature of + 27 ± 2 ° C with a constant air humidity of 55%, with a 12-hour light day, received dry standardized food and water without restriction.

The standard test strain M. tuberculosis Erdman was used to model tuberculosis. A mycobacterial suspension for infecting mice was prepared ex tempore from a three-week-old strain cultured on Lowenstein-Jensen medium. The infecting dose is 10 ⁶ CFU / mouse in 0.2 ml of saline, the route of administration is in the lateral tail vein.

Animal groups (number of mice per group).

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Intact (6) - K-;

contamination control (4) - K +;

BCG vaccination (14) - 1;

vaccination with a fusion protein characterized by SEQ ID NO: 1 (11), i / m - 2;

vaccination with a fusion protein characterized by SEQ ID NO: 1, and / n (12) - 3.

A single immunization with BCG and a double immunization with a vaccine based on the fusion protein characterized by SEQ ID NO: 1 were carried out with an interval of two weeks. A dose of 50 μg of protein per mouse was used, injected in a volume of 100 μl intramuscularly or 10 μl - intranasally, 5 μl in each nostril. Saline was used to adjust the volume to the required volume. Infection was carried out two weeks after the last vaccination.

Mice were withdrawn from experience six weeks after infection by decapitation in accordance with the Methodological Recommendations of the USSR Ministry of Health (1985).

The indicators of the severity of the course of tuberculosis infection were determined.

The index of lung damage (IPL) was established by the totality of exudative and productive changes in arbitrary units - points [Aleksandrova A.E., Ariel B.M. Assessment of the severity of tuberculous process in the lungs of mice // Probl. tub., 1993. - No. 3. - S. 52-53].

Exudative changes.

The lungs are airy - 0;

single airless foci - 0.25;

lungs are airless by 1 / 2-0.5;

lungs are airless by 2 / 3-0.75;

the lungs are airless throughout - 1.0.

Productive foci.

single submiliary foci - 0.5;

numerous (no more than 20) - 1.0;

numerous submiliary (more than 20) - 1.5;

single miliary - 1.75;

numerous merging submiliary and single miliary - 2.0;

numerous miliary (no more than 10) - 2.25;

numerous miliary, merging - 2.75;

the appearance of small caseous necrotic foci - 3.0;

extensive caseosis - 4.0;

continuous damage to the lungs - 5.0.

Bacteriological parameters were determined. A dosed inoculation of lung tissue homogenate was carried out on a dense Levenshtein-Jensen egg medium by the method of serial dilutions (3 and 4 dilutions). The massiveness of the growth of mycobacteria was determined and the organ protection index was calculated. The massiveness of mycobacterial growth was expressed in decimal log (lg) of the number of CFUs per lung weight. The organ protection index was calculated based on the difference between lg CFU of immunized mice and lg CFU of mice of the infection control group. When evaluating the results, a protection index> 0.5 lg is considered a positive effect on the growth retardation of mycobacteria.

The results are shown in FIG. 1-4. So, according to the index of lung damage (IPL) (Fig. 1), the leading position was taken by the vaccine based on the fusion protein, administered intramuscularly, group 2, this indicator was the lowest among the compared groups, the second place was taken by the vaccine based on the fusion protein, administered intranasally, group 3, on the third - BCG, group 1. However, it was the vaccine based on the fusion protein, administered intranasally, group 3, showed the best result in terms of the decimal logarithm of the number of viable bacteria in the lungs (Fig. 2) and protection index, as compared with unvaccinated animals (Fig. 3) and compared with BCG (Fig. 4). The protection index of the vaccine based on the fusion protein both with intramuscular and intranasal administration was higher than> 0.5 lg, which indicates the presence of a positive effect on the growth retardation of mycobacteria by this vaccine. The protection index compared with BCG (Fig. 4) of the vaccine based on the fusion protein when administered intranasally, group 3, was 0.35.

Thus, a high efficacy of an anti-tuberculosis vaccine based on a fusion protein based on M. tuberculosis Ag85B and TB 10.4 antigens, as well as a recombinant adjuvant flagellin FHC Salmonella enterica serovar Typhimurium, which turned out to be higher than that of BCG in a prophylactic model in laboratory animals, was demonstrated. , and with intramuscular and intranasal administration. Other parenteral or other mucosal methods of administration of this vaccine are also possible. The differences between groups of animals are significant.

The safety of the proposed vaccine was also demonstrated: the animals of the corresponding groups (groups 2 and 3) survived and were forced out of the experiment; during the tests, no side effects were observed.

Similar studies using a protein vaccine characterized by SEQ

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ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 showed similar results.

Example 4. Demonstration of the therapeutic effect of a vaccine based on a fusion protein characterized by SEQ ID nO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4.

The experiment was carried out on non-inbred white mice (Rappolovo laboratory animal nursery). Animals, weighing 18-22 g, were kept under standard conditions, at an ambient temperature of + 27 ± 2 ° C with a constant air humidity of 55%, with a 12-hour light day, received dry standardized food and water without restriction.

The standard test strain M. tuberculosis Erdman was used to model tuberculosis. A mycobacterial suspension for infecting mice was prepared ex tempore from a three-week-old strain cultured on Lowenstein-Jensen medium. The infecting dose is 10 ⁶ CFU / mouse in 0.2 ml of saline, the route of administration is in the lateral tail vein.

The immunogen was administered 6 days after infection. A dose of 50 μg of fusion protein per mouse was used, injected in a volume of 100 μl intramuscularly or 10 μl - intranasally, 5 μl in each nostril. Saline was used to adjust the volume to the required volume.

Mice were withdrawn from experience six weeks after infection by decapitation in accordance with the Methodological Recommendations of the USSR Ministry of Health (1985).

Groups of animals (n is the number of mice in a group).

Intact (8) - K-;

contamination control (5) - K +;

isoniazid 10 mg / kg (15) - 1.

Therapeutic vaccination with a fusion protein characterized by SEQ ID NO: 2, 5 times a week, i / m (15) - 2.

Etiotropic treatment was started from the 6th day after infection of the animals. The duration of treatment was 36 days.

In animals that received, along with etiotropic therapy, a vaccine based on a fusion protein, a significant decrease in the lung mass coefficient and IPL was noted, as well as a decrease in the seeding rate of mycobacteria from the spleen. Similar studies using a vaccine based on the protein characterized by SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 showed similar results.

Thus, the use of a fusion protein vaccine together with etiotropic therapy for experimental tuberculosis significantly increases its effectiveness.

Claims (8)

CLAIM 1. A fusion protein comprising immunogenic fragments of proteins Ag85B, Tb10.4 Mycobacterium tuberculosis, flagellin FliC Salmonella enterica serovar Typhimurium, the components of the construct are connected by flexible bridges, characterized by the amino acid sequence SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO : 3 or SEQ ID NO: 4. 2. A polynucleotide encoding a fusion protein according to claim 1, codon optimized for expression in the cells of the fusion protein producer. 3. A genetic construct for expression in the cells of a producer of a polynucleotide according to claim 2, comprising said polynucleotide and elements allowing for said purpose. 4. A recombinant cell for the production of a fusion protein according to claim 1. 5. A recombinant cell according to claim 4, characterized in that it is a prokaryotic organism. 6. A recombinant cell according to claim 4, characterized in that it is a cell of a eukaryotic organism. 7. Vaccine for the prevention and treatment of tuberculosis, containing a fusion protein according to claim 1 as an active agent in an effective amount, as well as a physiologically acceptable carrier or buffer solution. 8. A vaccine according to claim 7, suitable for humans and animals.