EA031495B1 - ANTITUBERCULOSIS VACCINE BASED ON RECOMBINANT Ag85B, TB10.4, FliC PROTEINS - Google Patents

Abstract

The invention relates to molecular biology, biotechnology, medicine. An antituberculosis vaccine is provided, containing M. tuberculosis recombinant Ag85B and TB10.4 proteins and also a recombinant adjuvant, Salmonella enterica serovar Typhimurium FliC flagellin, and also a pharmaceutically acceptable excipient, for solving an urgent modern problem consisting in development of a safe-to-produce and safe-to-use antituberculosis vaccine having efficiency, which is at least comparable with that of BCG. The above properties of provided vaccine are confirmed by examples.

Description

The invention relates to molecular biology, biotechnology, medicine. An anti-tuberculosis vaccine containing recombinant proteins Ag85B and TB 10.4 M. tuberculosis, as well as recombinant adjuvant — flagellin FliC Salmonella enterica serovar Typhimurium, as well as a pharmaceutically acceptable excipient, are proposed for solving the acute problem of modernity, which is to create a safe, disruptive effect, which creates a safe, discharging, a safe, discharging, a safe, discharging, a safe, discharging, a safe, discharging, to create a safe, prophylactic effect in the development of a safe, prophylactic euphoria enterophilus Typhimurium. having an efficacy of at least comparable to that of BCG. These properties of the proposed vaccine are confirmed by examples.

031495 B1

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

Tuberculosis is an infectious disease that causes the greatest number of fatal cases. According to the World Health Organization (WHO), about 8 million people get sick with tuberculosis every year, and about 3 million people get sick. Treatment of tuberculosis is complicated by the fact that highly resistant strains of mycobacteria causing tuberculosis with multidrug-resistant (MDR) and extensive drug-resistant (XDR) are widespread. In this regard, a promising approach in the fight against this disease is prevention. Since 2006, at the initiative of WHO, the Global Plan to Stop TB program has been announced, according to which the development of a new generation of vaccines against tuberculosis is one of the leaders. According to the latest WHO report on tuberculosis in 2014, a new vaccine that will help prevent the transmission of tuberculosis among adolescents and adults in developing countries will be the most cost-effective tool in mitigating the epidemic [1].

Thus, the problem of tuberculosis is extremely acute and requires a speedy solution in the form of safe for the production and use of the vaccine, which would have an effectiveness at least comparable to that of BCG.

There are several directions for the development of new tuberculosis vaccines, including live vaccines with improved properties, inactivated vaccines, protein-based vaccines. The authors of the present invention consider the most promising use of vaccines based on proteins, and obtained using recombinant DNA technology. The advantages of such vaccines are that the preparation containing the purified immunogenic protein is stable and safe, its chemical properties are known, there are no additional molecules, including nucleic acids, that could cause undesirable effects in the host organism. However, their effectiveness varies and is determined by a host of factors, one of the most important of which is the selection of components.

To create the present invention, immunodominant secreted antigens of M. tuberculosis were selected — Ag85B protein of protein complex 85 and protein TB10.4 (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 a cross-reactive immune response may be obtained [2]. It is important that these proteins do not have homology with known proteins of other organisms, which ensures the specificity of their action. It has also been shown that the protein TB10.4 has an adjuvant effect when administered in combination with mycobacterial proteins [3].

The following few solutions are known on the basis of the M. tuberculosis proteins listed above.

A known bacterium in one of the embodiments of the invention is a mycobacterium containing plasmid DNA encoding Ag85b and TB10.4 in one of the embodiments of the invention [4]. However, the introduction of a live bacterium, especially mycobacteria, even if attenuated, carries risks. Using a protein-based vaccine is a safer alternative.

The candidate vaccine based on the adenoviral vector, which encodes OPC of at least two proteins from Ag85A, Ag85B and TB10.4, is known, in addition an adjuvant can be used [5]. However, the use of adenovirus also carries risks. Viral vectors have several disadvantages, including the fact that they are expensive and can cause an inflammatory reaction, which eliminates the re-introduction of the vector. The use of recombinant highly purified proteins will allow to induce a specific response without side effects.

The disadvantages of these analogues are the fact that viral vectors and modified pathogens have risks of inheritance, which may prevent their use in humans [6].

A vaccine against a chronic disease, for example, a bacterial infection, containing a mixture of overlapping peptides covering the entire amino acid sequence of a protein, including Ag85B or TB10.4, which may also contain an adjuvant — cationic liposomes — may also be injected, the full-length protein covered by the peptides in the first introduction [7]. It is indicated that in one embodiment of the invention peptides are obtained by protein hydrolysis using more than two proteolytic agents, however, in this case, the process of obtaining a peptide mixture for the vaccine without impurities is complicated. Moreover, the storage conditions of such a peptide mixture can mediate problems with the implementation of this invention. Also, when using chemically synthesized molecules, including peptides, it should be understood that part of them can be represented by enantiomers, which can lead to unpredictable results in the body when used. The concept of enantiomer plays an important role in the pharmaceutical industry, since different enantiomers of medicinal substances, as a rule, have different biological activity. The use of living systems to obtain peptide molecules makes it possible to obtain molecules of natural structure, a homogeneous mixture. However, the use of a vaccine with a significantly smaller number of components is a more optimal option for both production and use in the latter case, since the probability of developing complications due to possible non-specific reactions is less.

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The closest analogue is the vaccine HyVAC IV / Aeras, which contains the proteins Ag85B and TB10.4, as well as the complex adjuvant IC31, containing the synthetic antimicrobial peptide KLKL5KLK, which is aimed at enhancing the interaction of antigen-presenting cells with the antigen, and the TLR9 ligand ODN1a, which has immunostimulating action. This vaccine is in phase IIa clinical trials [1]. However, the production of an active agent of a vaccine, represented by several components, obtained using completely different mechanisms, is technically difficult. The effectiveness of such a vaccine can also be reduced, and safety is not achieved due to the presence of enantiomers in it.

The authors of the present invention proposed an unexpected original solution, which is devoid of these shortcomings.

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 most specific pathogen-specific immune response, in this case capable of stimulating the T-cell immune response, along with B-cell, and also will contribute to the formation of local immunity and which will be safe.

The authors of the present invention found that when Ag85B and TB10.4 proteins are used together with FliC Salmonella enterica serovar Typhimurium (hereafter - FliC) flagellin, obtained as well as mycobacterial antigens using recombinant DNA technology, parenteral and mucosal immunization occurs as T cell-mediated immune response, along with Vcellular, and induction of local humoral immunity, without the formation of antibodies directly to flagellin, which is also a significant factor when used and adjuvant. The formation of local immunity is important to prevent infection with mycobacteria. Immunization of the mucosal surface also greatly facilitates the delivery of the immunogen.

Flagellin is a protein of bacterial flagella present in many bacteria, including pathogens. Flagellin is a TLR5 ligand (Toll-like receptor 5). 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 [8; 9]. Flagellins induce the formation of serum and secretory antibodies, the Th1 and Th2 response both to flagellins themselves and to the co-administered antigens.

Flagellin, depending on the origin of a particular microorganism, is different and consists of 259-1250 amino acid residues, which certainly affects its properties.

Differences in the structure of flagellin are also observed among a single bacterial species. So, most of the Salmonella enterica serovars are biphasic, i.e. they alternate the expression of different flagellated antigens (phases). This is due to the possibility of being in the active state only 1 of 2 flagellin genes (fliC and fljB), localized in different chromosome loci [10], FliC - phase 1 flagellin (STF), fljB - phase 2 flagellin (STF2). These flagellins have the same N-terminus, homologous, differing by several ao, C-terminus, as well as different domain D3, respectively, have a different structure and may have different effects.

Thus, depending on which microorganism flagellin is used, as well as the type of flagellin used (for example, for Salmonella), the effect will vary. You should also consider the possible interaction of the components in the mixture, in connection with which the selection of the flagellin molecule as an adjuvant, as well as its feasibility in each case is a complex process.

The technical result from the use of the invention is expressed in increasing safety in the production and use of a vaccine through the use of exclusively recombinant proteins, including a recombinant adjuvant, as vaccine components, which levels the negative effects from the use of protein molecules obtained in a different way.

The technical result from the use of the invention is expressed in increasing the effectiveness of the vaccine due to the use of exclusively recombinant proteins, including recombinant adjuvant, as components of the vaccine, which eliminates the reduction in the effectiveness of each component of the vaccine due to its loss due to the formation of a heterogeneous mixture, also including enantiomers, as well as through the use of an adjuvant, which allows to significantly enhance the specific response to mycobacterial antigens, including the formation of local immunity, which is usually quite difficult to achieve with vaccination.

The technical result from the use of the invention is expressed in simplifying the production of a vaccine due to the use of a unified technology for producing components, respectively, without the need to create different production sites.

Finally, the technical result from the use of the invention is expressed in the expansion of the spectrum of tuberculosis vaccines. When contraindications to the use of analogues or unwillingness to use analogues due to their shortcomings described above, this vaccine will allow for protection against tuberculosis. Due to the fact that the problem of tuberculosis is very serious, and the introduction of the drug to the market is not possible for many, this invention will increase the chances of fighting this infection.

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Summary of Invention

The solution of the acute task of modernity, which is to create a vaccine that is safe for the production and use of a tuberculosis vaccine that is at least comparable to that of BCG, is in the proposed tuberculosis vaccine containing, in addition to, of course, a pharmaceutically acceptable excipient, recombinant proteins - the Ag85B and TB10 mycobacterium antigens. 4, as well as the recombinant adjuvant - flagellin FliC Salmonella enterica serovar Typhimurium. The vaccine can be administered parenterally or on mucous membranes. The proposed vaccine is effective for the prevention of tuberculosis. These properties of the proposed vaccine are confirmed by examples.

Recombinant proteins were obtained using recombinant DNA technology. The average person skilled in the art knows that at the moment there are many systems for producing such proteins, and all of them, in one way or another, allow us to obtain vaccine components of the present invention, and therefore the concept of recombinant protein is used for each component, and not specified specific producer.

Also, laboratory studies were conducted, reflecting specific examples of the implementation of this invention. The results of laboratory studies are illustrated in FIG. 1-6 and example 2.

Brief Description of the Drawings

FIG. 1-3 illustrates the severity of tuberculosis infection in mice that are prophylactically immunized with BCG and vaccine candidates, 4.5 weeks after infection with M. tuberculosis Erdman, FIG. 4-6 - seeding from the lungs of such mice, groups of animals are designated as follows: K- - intact, K + - infection control, 1 - BCG, 2 - Ag85B, Tb10.4 proteins, FliC, intramuscular injection, 3 - proteins Ag85B, Tb10.4, FliC, and / n introduction.

FIG. 1 - lung mass ratio in arbitrary units;

FIG. 2 - lung lesion index in arbitrary units;

FIG. 3 - spleen mass ratio in arbitrary units;

FIG. 4 is the decimal logarithm of the number of viable bacteria in the lungs;

FIG. 5 - protection index in comparison with unvaccinated animals, decimal logarithm. On the abscissa axis are the groups - 1 - BCG, 2 - proteins Ag85B, Tb10.4, FliC, i / m administration, 3 - proteins Ag85B, Tb10.4, FliC, and / n introduction;

FIG. 6 - protection index in comparison with BCG, decimal logarithm. On the abscissa axis are designated the groups - 2 - Ag85B, Tb10.4, FliC proteins, i / m administration, 3 - Ag85B, Tb10.4 proteins, FliC, and / n introduction.

Example 1. Getting anti-tuberculosis vaccine.

1.1. Production of Ag85B, Tb10.4 Mycobacterium tuberculosis and FliC Salmonella enterica serovar Typhimurium proteins using a prokaryotic system.

The amino acid sequences of Ag85B and Tb10.4 Mycobacterium tuberculosis proteins, taken from the specialized Tuberculist database [11], as well as the amino acid sequence of the Salmonella enterica serovar Typhimurium FliC protein, the number in the GenBank BAA02846.1 database, were translated into nucleotide sequences, simultaneously performing codon optimization for Escherichia coli (E. coli), as well as adding sites for cloning into a vector for expression in this microorganism, pET28a (+). Chemically synthesized genes.

Each gene obtained was cloned into plasmid pET28a for subsequent expression. For this, restriction of the synthesized gene was performed, as well as plasmids, followed by ligation of the obtained DNA fragments. The restriction reaction was carried out in 70 μl of a solution containing 100 μg of DNA, 6 units. Fast Digest NdeI and Fast Digest XhoI enzymes (ThermoScientific, USA). The reaction was carried out in the corresponding FastDigest buffer for 15 min at 37 ° C. Inactivated by heating to 65 ° C for 5 min. The gene ligation reaction and the plasmid pET28a were performed in a volume of 70 μl. The reaction mixture contained purified restricted PCR product — 4.25 μL (1300 ng), restriction vector pET28a (+) —3.5 μL (425 ng), 10X ligase buffer (Thermo Fisher Scientific Inc.) —7 μL, 50% PEG - 7 μl, T4 ligase (Thermo Fisher Scientific Inc.) - 10 μl (7 units of activity), the volume of the mixture was adjusted to 70 μl with non-nucleated water. The reaction mixture was incubated at 22 ° C for 4 h.

The resulting ligase mixture transformed the prepared E. coli cells of the strain BL21 Star (DE3) (Invitrogen, USA) with the genotype F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3).

To prepare competent cells for subsequent transformation by electroporation, a separate colony grown on LB agar (Gibko BRL, USA) was taken and placed in 5 ml of LB medium (Gibko BRL, USA). Cells were grown overnight at 37 ° C with constant stirring (250 rpm). 2 ml of the obtained night culture were placed in 200 ml of LB medium. Cells were grown at 37 ° C with constant stirring (250 rpm) to an OD _{600 of} 0.6, and then precipitated by centrifugation for 10 minutes at 4000 g at 4 ° C. Cells were washed 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. To the cell pellet was added 3 vol. (from the volume of the cell sediment) 15% glycerin solution, the precipitate was resuspended. Cells ready for transformation were stored at -70 ° C.

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Transformation of competent cells was carried out by electroporation. 1 μl of a 10-fold diluted ligase mixture was added to 100 μl of competent cells, mixed, and portions were performed on an Eporator electroporator (Eppendorf, Germany). The transformation was carried out in sterile electroporation cuvettes (Eppendorf, Germany) with a volume of 100 μl, a slit of 1 mm, with an electric pulse of 1.7 kV with a duration of 5 ms.

After transformation, the cells were placed in 1 ml of SOC medium (2% bacterotrypton; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl ₂ , 10 mM MgSO ₄ ; 20 mM glucose) and incubated for 40 min at 37 ° WITH.

The grown clones were screened for the presence of targeted gene insertion. For each gene, one clone of E. coli cells was selected.

Induction of gene expression in each of the obtained strains was carried out using IPTG, having previously determined that the optimal induction protocol is the same for all genes used. For this, a single colony of the producer strain was inoculated into standard liquid LB medium (1% tryptone, 1% yeast extract and 1% sodium chloride) containing kanamycin at a concentration of 25 μg / ml, and incubated at 37 ° С in a thermostatic rotary type shaker overnight at 250 rpm The culture was diluted with fresh LB medium to OD ₆₀₀ 0.1 OE and incubated in a thermostatic rotary type shaker at 250 rpm at 37 ° C until the cell culture OD _{600 was} 0.6-0.8 OE. After this, the expression of the target gene was induced: the IPTG (Invitrogen, USA) was added to the culture to a final concentration of 0.5 mM and incubated at 16 ° C in a thermostatic rotary type shaker at 200 rpm overnight, after which the cells were concentrated centrifugation.

The obtained proteins were purified using the method of immobilized metal affinity chromatography using the Ni-NTU Sepharose sorbent.

The precipitated cells of the producers were lysed using 3 cycles of sonication for 30 seconds with a break of 2 minutes on ice. Then there was the destruction of Taurus inclusion by incubation with lysis buffer containing 500 mm sodium phosphate buffer, pH 8.0, 6 M guanidine hydrochloride, 500 mm sodium chloride, for 1 h. To cells collected by centrifugation from 50 ml of culture, was added 8 ml lyse buffer.

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

1.2. Production of Ag85B, Tb10.4 Mycobacterium tuberculosis and FliC Salmonella enterica serovar Typhimurium proteins using a eukaryotic system.

The amino acid sequences of Ag85B and Tb10.4 Mycobacterium tuberculosis proteins, taken from the specialized Tuberculist database [11], as well as the amino acid sequence of the Salmonella enterica serovar Typhimurium FliC protein, the number in the GenBank BAA02846.1 database, were translated into nucleotide sequences, simultaneously performing codon optimization for Saccharomyces cerevisiae (S. cerevisiae), as well as adding cloning sites into the vector for expression in this microorganism, pYES2 / NT (Invitrogen, USA). Chemically synthesized genes.

Each gene obtained was cloned into the pYES2 / NT vector according to a procedure similar to that used with the prokaryotic system. Further, transformation of S. cerevisiae cells was performed. The yeast was cultivated in YPD medium until the middle of the logarithmic growth phase was reached, washed with 10 mM TE buffer. Cells contained in 100 ml of medium were resuspended in 5 ml of 10 mM Tris HCl buffer (pH 7.5) containing 40.5% PEG-4000, 0.1 M lithium acetate. 1 μg of plasmid DNA was added to 0.5 ml of the cell suspension, mixed, and incubated overnight at room temperature. Next, 50 μl of the cell suspension were sown on selective medium to select transformants. Clones were screened.

The obtained producer strains were grown in YPD medium on a rotary shaker at a speed of 250 rpm at a temperature of 28 ° C for 21-24 h. 80 ml of seed were injected into a 5-liter Biostat fermenter containing 2000 ml of YPD medium. Fermentation was carried out at a temperature of 28 ° C, aeration of 1 l / min and a stirring speed of 1000 rpm. 24 hours after seeding, the fermenter fed the culture medium with 50% glucose solution at a rate of 2 ml / h and set the culture pH to pH 6.8 ± 0.1, using 10% sulfuric acid and 10% NaOH solutions for adjustment. The average total fermentation time was 70 hours.

Spent the isolation and purification of each protein from the water-insoluble fraction of cells of the strain

- 4 031495 producers. When increasing the biomass of S. cerevisiae in a 5-liter fermenter on YPD medium without feed in the presence of 2% glucose in the starting medium with a single fermentation, an average of 800-900 g of wet cell biomass was obtained. 1.5 kg of the washed wet biomass was resuspended in the buffer for destruction, the cells were destroyed with the help of glass beads in a flow-mill for 1.5 h, the resulting suspension was centrifuged and the precipitate was collected. Extraction of the target protein from the precipitate was carried out using a solution of 10% lithium chloride in 90% formic acid for 1518 h, followed by centrifugation. The precipitate was discarded, and the supernatant was subjected to ultrafiltration, followed by diafiltration through an M15 membrane to convert to 10 mM sodium acetate, pH 4.0 and remove the proteins of the producer strain cells.

The proteins were deglycosylated using endo-3-N-acetylglucosaminidase H in 50 mM sodium citrate buffer (pH 5.5) at 37 ° for 13 h in the presence of 0.01% LTO.

Final protein purification was performed using ion exchange chromatography on a HiPrep 16/10 SP FF cation-exchange column (GE Healthcare) in the FPLS system. After passing the filtrate through the column and then washing the column with 10 mM Na-phosphate buffer, pH 7.0 and then 10 mM sodium acetate buffer, pH 4.0, the protein was eluted from the column with 10% NaCl solution in the same buffer and identified electrophoretic in 10, 12% PAAG-DDS-Na, the fractions with the target protein were combined, the protein concentration in them was determined by the Bradford method.

To obtain immunogenic protein agents of the vaccine of the present invention, other prokaryotic and eukaryotic systems can be used with further specific purification and processing.

The use of living systems to obtain proteins - components of the vaccine allows you to get proteins of natural structure, a homogeneous mixture in large quantities in a short time, while neither their production nor the use does not bear the risks associated with safety. Production is not difficult and cost-effective, which has a positive effect on the cost of the vaccine.

The resulting highly purified proteins were mixed in equal proportions and used to immunize laboratory animals.

Example 2. Demonstration of the preventive effect of the vaccine.

The experiment was performed on white mice of non-inbred lines (the nursery of laboratory animals Rappolovo). Animals weighing 19-22 g were kept under standard conditions at an ambient temperature of 27 ± 2 ° C with a constant humidity of 55%, with a 12-hour daylight, dry standardized food and water were obtained without restriction.

Groups of animals (number of mice per group): intact (5) - K-, infection control (15) - K +, BCG vaccination (25) - 1, Ag85B + Tb10.4 + FliC vaccination in / m (20) - 2 , vaccination Ag85B + Tb10.4 + FliC and / n (15) - 3.

BCG was immunized once and vaccine based on recombinant proteins Ag85B, Tb10.4, FliC, twice immunized at intervals of 2 weeks. A dose of 50 μg of protein per mouse was used (in equal parts of Ag85B, Tb10.4 and FliC), injected in a volume of 100 μl intramuscularly or 10 μl intranasally, 5 μl in each nostril. To bring the required volume used saline.

Infection was carried out 10 days after the last vaccination. To simulate tuberculosis, the standard M. tuberculosis Erdman test strain was used. Mycobacterial suspension for infection of mice prepared ex tempore from a three-week strain, cultivated on the environment Levenshteyn-Jensen. Infecting dose - 10 ⁶ colony forming units (CFU) / mouse in 0.2 ml of saline, the route of administration in the lateral tail vein.

Mice removed from experience six weeks after infection by decapitation in accordance with the guidelines of the Ministry of health of the USSR (1985).

Indicators of severity of tuberculosis infection were determined.

1. The coefficients of lung mass (CML) and spleen (CMS) were calculated by the formula in arbitrary units: organ weight (g) × 100 body weight (g).

2. The lung lesion index (IPL) was determined by a combination of exudative and productive changes in arbitrary units — points [12].

Exudative changes:

lungs are airy - 0, single airless foci - 0.25, light airless by 1/2 - 0.5, light airless by 2/3 - 0.75, light airless throughout - 1.0.

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Productive foci:

single submiliary foci - 0.5, numerous (not more than 20) - 1.0, numerous submillary (more than 20) - 1.5, single miliary - 1.75, numerous confluent 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 caseous - 4.0, a continuous lesion of the lungs - 5.0.

3. Bacteriological indicators.

A dosed seeding of a lung tissue homogenate on Levenshtein-Jensen dense egg medium was carried out by serial dilution (3 and 4 dilution). The massive growth of mycobacteria was determined and the organ protection index was calculated. The massive growth of mycobacteria was expressed in decimal log (lg) of the number of CFUs per lung mass. The index of protection of the organ was calculated according to the difference between lg CFU of immunized mice and lg CFU of the infection control group. When evaluating the results, a protection index of> 0.5 lg is considered a positive effect on the delay in the growth of mycobacteria.

The results are shown in FIG. 1-6. Studies have shown the effectiveness of vaccination with Ag85B, Tb10.4, FliC proteins is greater than BCG, both with intramuscular and intranasal administration. It was also found that the differences between the groups are significant.

Thus, in terms of lung mass ratio (CML) (Fig. 1), the vaccine on the basis of proteins Ag85B, Tb10.4, FliC, administered intramuscularly, group 2, took the leading position; this indicator was the lowest among the compared groups, BCG took the second place, group 1, in the third, a vaccine based on Ag85B, Tb10.4, FliC proteins, administered intranasally, group 3. However, it was the vaccine based on Ag85B, Tb10.4 proteins, FliC, administered intranasally, group 3, showed the best result when measuring the lesion index lung (IPL) (Fig. 2) and the ratio of the mass of the spleen (CMR) (Fig. 3). In the case of IPL, the second group by efficiency was group 2, also represented by a mixture of proteins Ag85B, Tb10.4, FliC, but administered intramuscularly, last place was taken by BCG. In the case of the KMS indicator, the second in efficiency was group 1, BCG, then a mixture of proteins Ag85B, Tb10.4, FliC, administered intramuscularly. In terms of the decimal logarithm of the number of viable bacteria in the lungs, the mixture of Ag85B, Tb10.4, FliC proteins with both methods of administration proved to be more effective than BCG (Fig. 4), the best indicator achieved with intranasal administration. It is shown that a positive effect on the delay in the growth of mycobacteria, the index of protection compared with unvaccinated animals, FIG. 5, has only a mixture of proteins Ag85B, Tb10.4, FliC, and with intranasal administration (1.08) group 3, - almost twice as large as compared with that with intramuscular administration (0.58) group 2, and practically three times larger than BCG, group 1. This BCG protection index turned out to be significantly lower> 0.5 lg, - 0.37 lg, which indicates the absence of a positive effect on the growth retardation of mycobacteria by this vaccine. The protection index compared with BCG (Fig. 6) of the mixture of proteins Ag85B, Tb10.4, FliC, administered intramuscularly, group 2, was 0.21, and that with intranasal administration, group 3, -0.71.

Thus, the high efficacy of the anti-tuberculosis vaccine based on recombinant proteins - the antigens Ag85B and TB10.4 M. tuberculosis, as well as the recombinant adjuvant - flagellin FliC Salmonella enterica serovar Typhimurium, which turned out to be larger than that of BCG, was demonstrated in the preventive model on laboratory animals , and with intramuscular and intranasal administration, with the highest rates observed in the latter case. Other parenteral or other mucosal routes 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: animals of the respective groups (groups 2 and 3) survived and were forcibly removed from the experiment, and no side effects were observed during the test.

List of used literature.

1. http://www.who.int/immunization/research/forums_and_initiatives/07_Evans_GVIRF_TB_Vacc ine_Progress.pdf.

2. WO 96/15241, priority date 11/14/94 US.

3. ЕА 012037 В1, the priority date is 16.11.2004.

4. WO 2008150969 A9, priority date 31.05.2007.

5. WO 2006053871 A2, the priority date is November 16, 2004.

6. R.R. Redfield et al., New Engl. J. Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149, 1569 (1989).

7. WO 2008/000261 A2, priority date 06/28/2006.

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8. Mc Dermott P.F. High-affinity interaction between flagellin and the cell surface polypeptide results in human monocyte activation. Infect. Immun. - 2000. - V. 68. - p. 5525-5529.

9. Means TX et al. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol. - 2003. - V. 170. - p. 5165-5175.

10. Baker S., Hardy J., Sanderson K.E. et al. A novel linear plasmid of Salmonella Typhi. PLoS Pathog, 2007, 3 (5): e59. doi: 10.1371 / journal.ppat.0030059.

11. Lew J.M., Kapopoulou A., Jones L.M., Cole S.T. TubercuList - 10 years after. Tuberculosis (edinb). Jan 91 (1): 1-7 (2011).

12. Aleksandrova A.E., Ariel B.M. Assessment of the severity of the tuberculous process in the lungs of mice // Probl. tub., 1993. - № 3. - p. 52-53.

Claims (2)

CLAIM 1. A tuberculosis vaccine containing recombinant proteins Ag85B and TB10.4 M. tuberculosis, recombinant adjuvant flagellin FliC Salmonella enterica serovar Typhimurium, as well as a pharmaceutically acceptable excipient. 2. The vaccine according to claim 1, characterized in that it is mucosal or parenteral. IPL, cu to- o