Pharmaceutical composition and method of stimulating immune response to mycobacterium avium subspecies paratuberculosis

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of invention refers to medicine and concerns a pharmaceutical composition, and a method of stimulating an immune response to Mycobacterium avium, subspecies paratuberculosis (MAP) in a mammal. The composition contains a recombinant polypeptide containing from N-terminal to C-terminal: a C-terminal fragment of MAP_3527 protein, an amino acid sequence of MAP_1519 protein, and then an N-terminal protein of MAP 3527 protein. The method involves administering the pharmaceutical composition in animals in the amounts sufficient to stimulate the immune response on MAP.

EFFECT: group of inventions provides either eliminating, or delaying the MAP proliferation over a period of infection.

18 cl, 3 ex, 31 dwg, 2 tbl

 

The technical field to which the invention relates.

The present invention relates to the stimulation of immune responses, in particular, to pharmaceutical compositions and methods for stimulating preventive and/or therapeutic immune response against Mycobacterium avium subspecies paratuberculosis.

The level of technology

Mycobacterium avium subspecies paratuberculosis (next MAP) is the etiological factor of the disease John, sickness which first explored and described (in 1907) of the German etymologist Heinrich Yon), which causes a chronic granulomatous enteritis of cattle. In animals with clinical manifestations of the disease develop chronic diarrhea, progressive weight loss, which eventually leads to death, while subclinical infected animals produce less milk. The illness of John is of great economic importance for the global dairy industry, causing major losses due to reduced production and early culling of animals, it is estimated that 20% of dairy herds affected by the disease, which brings the loss of the dairy industry $ 220 million per year (Wells, et al. 2000. J. Am. Vet. Med. Assoc.216: 1450-1457). Cattle are most susceptible to infection by this organism in the first 6 months of life, but the disease usually does not manifest early the E. age 3 or 5 years. Infection occurs by ingestion of infected manure, colostrum or milk from infected cows (Sweeney, 1996. Vet. Clin. N. Am. Food Anim. Pract. 12:305-312). Also occurs in utero infection, especially in pregnant cows with progressive disease (Sweeney, et al. 1992. Am. J. Vet. Res. 53: 477-480). Moreover, the importance MAP is greatly increased because of their potential role as an etiological factor in Crohn's disease in humans (Chamberlin, et al. Aliment oil displayed pure Pharmacol Ther 2001; 15(3): 337-46; Naser SA, et al. Mol Cell Probes 2002; 16(1): 41-8).

As currently approved by the vaccine against the disease John for application in this field is an oil suspension of the killed strain MAP, which has significant limitations. First of all, the effectiveness of this vaccine is controversial, with variable results in different studies of vaccination. Another problem is a mutual influence of the whole cell bacterial vaccines and diagnostic tests, because vaccinated animals showed a false-positive reaction to the TB and tuberculin. Thus, there is a need for improved vaccine, but it should be effective and at the same time should not interfere with the diagnosis of tuberculosis and diseases of John. To achieve this goal have been tried several approaches, which include recombinant vaccines, the NC vaccines and vaccine subunit 13 (Shin SJ, et al. Infect Immun 2005; 73(8): 5074-85). However, there is a need to develop improved MAP vaccines.

Disclosure of inventions

The present invention provides pharmaceutical compositions and methods of stimulating an immune response against MAP in mammals. The compositions include a new recombinant polypeptide with a mass of 79 kDa, designated in the application as “Map74F”. Map74F was obtained by binding the C-terminal fragment of the protein MAP_3527 with a mass of about a 17.6 kDa fragment of the protein MAP_1519, for which C is the end follows the N-terminal site of the protein MAP_3527 weight of 14.6 kDa.

In addition Map74F, compositions of the invention can also include proteins MAP, such as antigens 85A, 85B, 85C, a protein with a mass of 35 kDa, superoxiddismutase (hereinafter SOD), proteins MptC, MptD and ESAT-6 like protein, or combinations thereof.

The method comprises introducing the composition mammal in a quantity sufficient to stimulate an immune response against MAP. It is expected that the method will be beneficial to any animal susceptible to infection MAP, but especially favorable for ruminants.

Pharmaceutical compositions can include standard pharmaceutical excipients and may be entered in any traditional way. The composition may be administered at any time the animal susceptible to infection with MAP infection, or animal infected MAP. However, predpochtitel is but to enter the composition of the invention to infection MAP for example, the introduction of pregnant animals that can deliver preventive immunological components to their newborns through colostrum, or introduction during the period from one to five weeks after birth.

The present invention provides pharmaceutical compositions and methods for stimulating an immune response against MAP in animals. Compositions include recombinant polypeptide, referred to in the application as Map74F. Open reading frame (ORF)encoding a Map74F represents 2397-membered nucleotides and encodes a polypeptide containing 799 amino acids. The sequence of the polypeptide Map74F shown in SEQ ID NO: 1. Molecular weight Map74F calculated on the basis of its amino acid composition is 79 kDa, although the actual molecular mass determined by the method of SDS-PAGE analysis is 74 kDa. The sequence shown in SEQ ID NO: 1, is given without any optional tags for purification, such as his-tag tag.

Map74F was created by stitching, in the direction from N-Terminus to the C-end C-terminal fragment of the protein MAP_3527 with a mass of about a 17.6 kDa protein fragment MAP_1519 by weight of 46.8 kDa, and then N-terminal fragment of the protein MAP_3527 with a mass of about 14.6 kDa. Schematic representation of Map74F given in figure 1. Complete amino acid sequence MAP_3527 and MAP_1519 are presented respectively as SEQ ID NO: 2 and SEQ ID NO: 3. C-con the eve protein fragment MAP_3527 mass to 17.6 kDa, present in Map74F represented by amino acids 183-361 sequence SEQ ID NO: 2. Fragment MAP_1519 by weight of 46.8 kDa, present in Map74F represented by amino acids 1-460 of the sequence SEQ ID NO: 3. N-terminal protein fragment MAP_3527 with a mass of 14.6 kDa, present in Map74F represented by amino acids 33-180 sequence SEQ ID NO: 2. It is expected that longer fragments MAP_1519 and N - and C-ends MAP_3527 can be included in the recombinant protein, which could be used in the method of the invention. In addition Map74F, the pharmaceutical compositions of the invention may include other substances that can stimulate an immune response against Marbacher. For example, the composition can include one or more other proteins of the MAP, such as antigens 85A, 85B, 85C, protein mass of 35 kDa, superoxide dismutase (SOD), protein MptC, MptD and ESAT-6 like protein, and combinations thereof. Such proteins are described in the application U.S. No. 11/816,365, and description of these proteins and DNA sequences encoding them, and methods of using the proteins and their coding DNA in pharmaceutical compositions for stimulating an immune response against MAP included in the application by reference.

In one embodiment, the pharmaceutical composition comprises Map74F and one or more of the proteins of the MAP antigens 85A, 85B or SOD. A DNA sequence encoding a gene MAP of antigen 85A, and amino acid sequence, sootvetstvuyshee gene MAP of antigen 85A, given in GenBank access number AF280067 (recording from October 10, 2003). A DNA sequence encoding a gene MAP of antigen 85B and amino acid sequence corresponding to the gene MAP of antigen 85B, is given in GenBank access number AF219121 (recording from November 21, 2002). A DNA sequence encoding a gene MAP of antigen 85C, and the amino acid sequence corresponding to the gene MAP of antigen 85C, is given in GenBank access number AF280068 (recording from November 21, 2002). A DNA sequence encoding a superoxide dismutase gene MAP, and amino acid sequence corresponding to the gene superoxide dismutase MAP, is given in GenBank access number AF 180816 (record dated 30 November 2001).

The method of the invention includes the introduction of a pharmaceutical composition comprising Map74F, mammal in an amount sufficient to stimulate an immune response against MAP. Stimulated immune response may include the promotion of any component of the immune system, including, but not limited to, the production of antibodies reactive to antigens of MAP, stimulation of lymphocyte proliferation, production of Th-1-associated cytokines such as gamma interferon, and a combination of the above.

Map74F can be administered to animals in the form of a vector. For example, the sequence of nucleic acids encoding Map74F, can be cloned into the genome of bacteria (such as Salmonella) or virus (e.g., in the Rus bovine herpes 1 (BHV-I)), and the resulting recombinant bacterium or virus may be injected animals. Thus, the present invention also includes bacterial or viral vectors expressing Map74F. Also within the scope of the present invention includes methodology, DNA vaccines, in which the nucleic acid molecule that encodes a Map74F, entered directly to the animals either in pure form or in combination with auxiliary substance or substance, to facilitate transfection.

The method according to the invention can be beneficial to any animal susceptible to MAP infection. Pharmaceutical compositions and methods are particularly suitable for the prevention or treatment of MAP infection in ruminants, including cattle, sheep, goats, deer and elk, antelope and Buffalo, but not limited to. In one embodiment, the method can be used for prevention or treatment of disease John.

The pharmaceutical compositions can be administered by any infected or uninfected MAP animals. It is believed that the introduction of the compositions of the infected animal in accordance with the method of the invention, stimulates therapeutic immune response. However, the invention also includes the introduction of the pharmaceutical compositions of the invention to infection MAP to promote preventive response. For example, com is osili can be given to pregnant animals, who can pass preventive immunological components to their uninfected infants through colostrum or milk during lactation. Alternatively, the composition can be entered during the period from one to five weeks after birth to ensure preventive effect, which can prevent the infection of MAP or reduce the severity of disease if infection occurs.

Thus, in one embodiment, the method of the invention is the prevention of infection MAP, while in another embodiment, the method is the treatment of infection MAP.

The pharmaceutical compositions can be formed together with standard pharmaceutical excipients and can be entered using any of a variety of conventional routes of administration. Some examples of acceptable pharmaceutical excipients for use with the protein described in Remington''s Pharmaceutical Sciences (18th edition, A.R. Gennaro et al. Eds., Mack Publishing Co., Easton, Pa., 1990).

The pharmaceutical compositions used in the method of the invention can also include an excipient. Can be used with any standard auxiliary substance.

In one embodiment, the excipient may be monophosphoryl lipid A (IFF), which can cover the change in combination with synthetic Dimineata trehalose. In another embodiment, the excipient moretraditional dimethyldioctadecylammonium (the DDA). The pharmaceutical compositions of the invention can be administered in any acceptable way. Suitable routes of administration include oral, mucosal and parenteral (for example, intravascular, intramuscular and subcutaneous injections). The composition can be entered at any time to any animal susceptible to infection with MAP infection or animal infected MAP.

The person skilled in the art it is obvious that the number Map74F and any other antigenic substance in the pharmaceutical composition introduced specific animal will depend on a number of factors such as route of administration, and the size, physical condition and status MAP of the animal, and may be selected by the specialists in this field to achieve the desired result. The composition can be applied by a single injection or series of injections to stimulate the immune response. In General, you may enter a total dose in the amount of 10-200 μg of protein.

Brief description of drawings

Figure 1 is a schematic representation of the design Map74F. Map74F was obtained consistent stitching in the direction from N to the end of the open reading frames coding for C-terminal protein fragment MAP_3527 made with an open frame please take the tion, the coding amino acids 1-460 of protein MAP_1519, and terminated an open reading frame that encodes a N-terminal fragment MAP_3527. ORFS encoding Map74F represents 2397-membered nucleotide which encodes 799-amino acid polypeptide with a predicted molecular weight of about 79 kDa.

Figure 2 is a photograph of a colored Kumasi blue 10% SDS-PAGE electrophoresis of lysates from strain E. coli E. coli (BL21/pLysE), expressing transformed with the vector coding for Map74F. Cells were grown and induced by 1 mm IPTG (isopropylthioxanthone). The lines represent the lysates before (line UI) or 3 hours after (line I) induction IPTG. Purified recombinants Map74F shown on line R along with a token of molecular weights (line M).

Figure 3-6 shows the graphical image data obtained in mice killed 3 weeks after secondary vaccination. Cells from the spleen of mice treated with Marr + IFF or only IFF were stimulated for 2 days 10 mg/ml Map74F, CONA (concanavalin a) and the environment.

Figure 3 presents data that show the level of IFN-γ in supernatant cultures measured using enzyme-linked immunosorbent assay (ELISA).

Figure 4 presents data that show a specific method ELIspot (enzyme-linked immunosorbent spot assay) relative levels of IFN-γ-expressing cells in suspense is maloletok spleen of immunized and control mice with or without stimulation by antigen.

Figure 5 presents data FACS (fluorescent sorting cells) analysis of a population of spleen lymphocytes obtained from vaccinated (Map74F + IFF) and control animals (IFF, con-IFF) after stimulation with recombinant and control antigens.

Figure 6, presents the data of mRNA expression of cytokines in response to Map74F normalized with respect to the “gene of the household” GAPDH. Presents data from three independent experiments.

Figure 7 and 8 presents the data obtained from the serum of vaccinated and control animals collected at different times (revaccinate/ primary vaccination (PV), secondary vaccination (In), before infection (SU), and 4, 8, 12, and 16 weeks after infection) and tested for response to the antibody.

7 shows the data for the serum tested for the presence of specific antibodies to Map74F.

On Fig shows the relationship lgGl/lgG2, obtained by ELISA. Data represent three independent experiments.

Fig.9-11 reflect the data in the expression of protective immunity in the spleen (Figure 9), liver (Figure 10) and mesenterina lymph node (hereinafter MDR) (11)caused by vaccination Map74F + IFF. Map74F significantly reduces the action MAP in the spleen (8-16 weeks after infection), liver (12-16 weeks after Sergey) and MDR (8-16 weeks after infection). Presents data from three independent the x experiments.

On Fig-15 presents pictures histopathological evaluation of tissues from vaccinated and unvaccinated mice.

On Fig shows the liver unvaccinated control mice. Numerous large granuloma is randomly distributed throughout the liver. Hematoxylin and eosin. The thickness of 100 μm. Insert: Higher magnification of granuloma showing numerous acid-fast bacilli. Staining of Till two.

On Fig shows the liver of mice vaccinated Map74F. Visible only a single small lymphoid complexes, surrounded by several macrophages.

On Fig shown spleen from unvaccinated control mice, with the presence of occasional granulomas in the white pulp.

On Fig shown spleen mice vaccinated Map74F. White and red pulp free from granulomas. Hematoxiline and eosinophile staining. The thickness of 100 nm. Insert: Higher magnification of the granuloma, demonstrating the absence of acid-fast bacilli. Staining of Till two.

On Fig-18 presents data showing the lymphoproliferative response of peripheral blood mononuclear cells (hereinafter MCPC) immunized (group I and II) and control (group III) groups of animals on recombinant antigens (85A, 85B, Map74F and SOD), Kona, and tuberculin. The results are expressed as stimulation index (SI), and Planck the errors indicate standard deviation from the mean.

Fig-21 display data showing antigen-specific IFN-Kotlik Mkpco immunized (group I and II) and control (group III) groups of animals. The results are shown as values of optical density, and error bars show the standard deviation from the mean.

Fig-27 display data showing the expression of lymphocyte subpopulations in MCPC taken from immunized (group I and II) and control (group III) groups of animals at a specified time after stimulation with recombinant and control antigens and analyzed using flow cytofluorometry. The results are expressed as the percentage of cells that gives positive staining, relatively reinducing samples (processed by the environment). Error bars show the standard deviation from the mean.

Fig and 29 display data showing the expression of Munchen cytokine in response to recombinant antigens for immunized groups (I and II) and control group (III). The results are expressed as the average fold increase compared to restimulating MCPC, which serves as a control. Error bars indicate the standard deviation from the mean.

Fig reflects the immune response to individual recombinant antigens in vaccinated animal groups I and II and the control is the group of animals III. Error bars indicate the standard deviation from the mean.

Fig shows a summary table of results MAP cultures (colony forming units, then KOE), measured in various tissues collected during necropsy.

The implementation of the invention

The following examples describe various embodiments of the invention. These examples are illustrative and should not be construed as limiting.

Example 1

This Example provides a description of the cloning, expression and purification Map74F.

Education landesbanki OCR, coding Map74F

Map74F was formed by successive staple in tandem LFS C-terminal fragment MAP_3527 with a mass of 16.6 kDa with OCR, coding MAP_1519 protein having a molecular weight of 46.8 kDa, and then on the C-end N-end portion MAP_3527 with a mass of about a 14.6 kDa. Education design MAP_3527 with devoid of a stop codon.

5' and 3' oligonucleotides to C-terminal MAP_3527 (MAP_3527 C) were designed as follows: 5' (THE CATATG CAT CAT CAT CAT CAT CAT CTC AAC CAG AGC GTC TCG GC 3" (SEQ ID NO: 4)and (5," THAT GAATTC GGC CGG CGG CCC CTC CGC 3' (SEQ ID NO: 5)). After 5' of the oligonucleotide containing the Ndel restriction site preceding the ATG initiating codon, followed nucleotide sequence encoding six his-tag residues (italics). the 3' Oligonucleotide contained the EcoRI site restrictively oligonucleotides were used to amplificatoare MAP_3527 with, carboxyl 540-nucleotide segment (a 14.6 kDa, 180 amino acid residues) MAP_3527, and the resulting PCR amplificatory product ligated into PCR Toro vector. Plasmid DNA with the correct insert was treated with Ndel and EcoRI, and ligated into pET17b expressing vector cleaved with the same enzymes. The products of ligation transferred into cells of Escherichia coli DH5ct and one transformant with the correct insert (MAP_3527c) was performed using restriction mapping and DNA sequencing.

PCR amplification of full coding sequences MAP_1519 and ligation in MAP_3527 c-pET plasmas go.

5' and 3' oligonucleotides MAP_1519, contained the following sequence: 5'(5'-CTA ATC GAATTC ATG TTC TAT GGG GCC TTT C-3' SEQ ID NO: 6)) and 3'(5'-TA ATC GATATC CAG GAC CTT GGA CTT GTC-3' SEQ ID NO: 7)). the 5' Oligonucleotide contained the EcoRI restriction site. 3' Oli the rutting cleated contained the EcoRV restriction site, followed by sequence containing six C-terminal amino acid residues, and did not contain a stop codon. Amplificatoare coding sequence MAP_1519 (1380 base pairs; length 460-amino acids with a predicted size of about of 46.8 kDa), and the resulting PCR amplificatory product ligated into PCR 2.1 maintenance vector cleaved with the same enzymes. A clone with the correct insert was treated with EcoRI and EcoRV and ligated into the pre-treated with enzymes EcoRI/EoRV plasmid MAP_3527 c-pET. The ligation products are then transferred into cells of E. coli DH5α and transformants with the correct insert (MAP_3527c-MAP_I 519) identified using restriction mapping and confirmed by DNA sequencing. Cloning of N-terminal fragments MAP_3527 in MAP_3527C-MAP_1519 design.

5' and 3' oligonucleotides N-terminal fragment MAP_3527 were designed as follows: 5'(5'-AT GATATC GGG CTG GCG CCG GCG TCC-3' SEQ ID NO: 8)and 3'(5'-AT CTCGAG TCA CGC GAC CTT GCC GGC-3' SEQ ID NO: 9)). the 5' oligonucleotide contained the EcoRV restriction site. the 3' oligonucleotide contained the Xhol restriction site. They were designed in order to amplify the N-terminal containing 447-base pairs (149-amino acid residues) fragment MAP_3527. The obtained PCR amplificatory product was treated with EcoRV and Xhol, and Legerova in MAP_3527 with-MAP_1519 fused pET plasmid, treated with EcoRV and Xhol. Ligiously mixture was used to tranformirovanie of DH5α E.coli cells and positive clones were identified by restriction mapping and confirmed by Dnsservicebrowse. The final design encodes a polypeptide with a mass of 75 kDa (Map74F), including the only LFS, organized in a linear order MAP_3527C-MAP_1519-MAP_3527N. Expression and purification Map74F recombinant polypeptide

Construction of plasmid DNA was transformed into cells of E. coli BL21 (DE3 pLysE). The transformed E.coli cells were placed on LB agar (environment Loreburn), supplemented with ampicillin (100 µg/ml). Each colony was planted in 10 ml of LB culture medium containing ampicillin (100 μg/ml)and were grown at 37°C overnight with shaking. The culture was diluted 1:100 in LB culture medium containing ampicillin (100 μg/ml) and Chloromycetin (34 μg/ml)and were grown at 37°C with shaking. After 3 h induction with 1 mm IPTG (Invitrogen Co., Carlsbad, CA), cells were separated by centrifugation (5000 g) and once washed with phosphate-saline buffer (FSB). Cells suspended in buffer A (50 mm HCl, 1 mm EDTA, 50 mm NaCl, pH 8.0) and literally in cell disintegrator high pressure or cell disintegrator. After sedimentation by centrifugation Taurus inclusions, precipitates were washed three times with wash buffer for Taurus enable (buffer A + 1% Triton X-100) and twice - CHAPS buffer (Sigma-Aldrich, St.Louis, MO) (1% CHAPS in 10 mm HCl, pH 8.0) to remove lipopolysaccharide. Taurus inclusion was dissolved in buffer (100 mm phosphate, 10 mm Tris-HCl, 8 M urea, 2 mm PMSF (Sigma) and 20 μg/ml leupeptin (Sigma), pH 8.0) and purified on Ni-NTA agarose column (Invitrogen). Erwerbende fractions was investigated using SDS-PAGE electrophoresis, and fractions containing the recombinant polypeptide, United and deliberately against 10 mm Tris-HCl (pH 7.8) overnight at 40C° twice. The polypeptide was passed through Detoxi-GelTM Endotoxin Removing Gel (Pierce, Rockford, IL) and purified polypeptid is investigated on the level of endotoxin using the LAL test (Limulus amoebocyte assay). The purified polypeptide was negligible level (10 PG/ml) of endotoxin.

Figure 1 is given a schematic Map74F showing the structure and sites of enzymatic enzyme used in the construction of the polypeptide. ORS Map74F is 2397 nucleotides in length, encoding a polypeptide consisting of 799 amino acids (Figure 2), with the predicted molecular weight of about 79 kDa. The design of the LFS resulted in the introduction of two hinge sequence (EcoRI and EcoKV) of the six nucleotides that encode two amino acids in each site connections, connecting three of the original LFS. In addition, LFS (Map74F) was designed so that it contains six his-tag residues on the N-end for purification using Ni-NTA matrix. After expression in E. coli, the recombinant polypeptide was purified from calf inclusion and analyzed using SDS-PAGE electrophoresis (Figure 2) with output in the range of about 1.5-2.0 mg of purified polypeptide per liter of induced culture.

Example 2

This example illustrates the use of Map74F to stimulate an immune response against MAP in the model in mice. It was found that mice are a suitable model for the study of MAP infection. Bacterial load and pathology of specific organs are good indicators of infectious status of mice after infection MAP. In mice, the liver, lazenka and mesenteric lymph nodes are the best bodies to assess bacterial load after infection MAP. In this example, it was estimated load MAP on these bodies to determine the effectiveness of the protective properties Map74F after infection. From the results presented below, it is obvious that Map74F immunized animals were able to either eliminate or slow down the proliferation of MAP during the 16-week period of infection. In the spleen, liver and mesenteric lymph nodes, the number of colony forming units (CFU) were decreased at 8 weeks after infection, which assumed the destruction of the MAP. In addition to the bacterial load in liver and spleen of vaccinated animals was observed fewer granulomas and fewer acid-fast microorganisms, indicating a protective effect of polyprotein. Significantly reduced load MAP on the bodies and the improvement of the pathology of the liver and spleen indicate that immunization Map74F protected mice from infection MAP by rapid reduction or elimination of the load MAP. Map74F also resulted in the establishment of a high level of γ-interferon. Map74F evoked good response T-helper cells type 1 (Th1 response).

Materials and methods of Example 2.

Animals

The experiments were performed using female mice C57/BL6 (Harlan Sprague, Indianapolis, Indiana) at the age of 6-8 weeks. The animals were kept in the laboratory level II biological safety and had unlimited access to food and water. All the experiment is Antalya work was carried out in accordance with the rules, norms and principles of the legislation on protection of animals control of the Ministry of health for the humane treatment and use of laboratory animals used for research, study and training, the Director of the National Institutes of Health (NIH) study and use of laboratory animals and the new York state Department of health.

Bacterial strains

MAP isolated from infected cows, designated as MAP 661 15-98, was used to infect mice and to isolate genomic DNA. MAP 661 15-98 crop failure and N, with the addition of 10% oleic acid - albumin - dextrose - catalase (Becton, Dickinson and Co, Sparks, MD) and mycobactin J (Allied Monitor Inc., Fayette, MO). After kulturarbeit within 6-8 weeks, the organisms were harvested by centrifugation at 10 000 g and washed twice with phosphate-saline buffer (FSB; pH of 7.2). The microorganisms were diluted FSB to the desired concentration and used to infect mice.

Immunization of mice

Mice were divided into two groups of 36 mice in each group. Animals of group I were immunized twice, with an interval of three weeks, 50 μg/animal, recombinant polypeptide together with SIMULIA IFF + TDM (Ribi-adjuvant, Corixa, Hamilton, MT) in a total volume of 100 μl per dose by subcutaneous injection in the back. Animals of group II were left as newac innovantage control, they were introduced only emulsion IFF + TDM. Three weeks after the second immunization 12 animals from each group designed to determine the immunogenicity were scored, and the spleen cells were extracted in the usual way. The spleen cells were grown in RPMI medium 1640 (Gibco, Grand Island, NY)containing 10%FBS (Gibco) at 37°C with 5% CO2. Experiments on the immunizations were repeated twice with the same dose and in the same mode. Infection of mice MAP

Three weeks after the second immunization 24 animals from groups I and II were infected via the intraperitoneal injection of 109 colony forming units (CFU) of Mycobacterium avium subspecies paratuberculosis. Six animals in each group were euthanized at 4, 8, 12 and 16 weeks after infection, spleen, liver and mesenteric lymph nodes were collected and divided into two parts. One set of tissues was homogenized in FSB (100 mg/ml) and 100 μl of homogenates of individual tissues were sown on egg-yolk slanted agar (Herald''s egg yolk (HEY), Becton, Dickinson and Co, Sparks, MD)containing mycobactin J., for the assessment of bacterial load. Culture on the sloped agar were studied on the growth of bacteria by counting colonies after 8-12 weeks after vysielanie. Another set of tissues were used for histopathological studies.

Histopathological study

Sections of spleen, liver and MDR what was exireuil by immersion in 10% neutral buffered formalin, they were placed in paraffin, did sections of 4 µm and stained with hematoxylin and eosin, were stained by Zn Nelsen using conventional histological methods, and then studied using optical microscopy.

Enzyme-linked immunosorbent assay humoral immune response Antigen specific lgG response was measured using a conventional method enzyme immunoassay ELISA. On ELISA plates (Nunc-measurement module, Nunc, Roskilde, Denmark) was applied at 200 ng/well of recombinant polypeptide, and incubated at 4°C over night. After a single washing of the FSB-T (0.05% of Tween 20 in the FSB), was added 300 μl of blocking buffer (1% bovine serum albumin (BSA) in FSB-T) and incubated at 25°C for 1 hour. Plates were washed three times FSB-T and added to each well 100 ál of diluted serum samples and incubated at 37°C for 1 hour. For measurement of total immunoglobulin G (lgG) response after washing each well was added 50 ng conjugated with alkaline phosphatase goat antimisting lgG (KPL, Gaithersburg, MD) and incubated at 25°C for 30 minutes. After washing was added 50 μl of BluePhos substrate (KPL) and incubated for 10 minutes. After adding 50 μl of stop solution (KPL) the plates were read on an ELX 808 Ultra tablet reader (Bio-Tek Instruments, Inc. Winooski, VT) at 630 nm. To measure the response isotypes, after washing each well was added 25 ng conjugated with bio is another goat antimisting lgG1 or lgG2a (Southern Biotech, Birmingham, AL), and incubated at 25°C for 30 minutes. After washing was added to 0.2 mg/ml of streptavidin labeled with peroxidase (KPL) and incubated at 25°C for 30 minutes. After washing each well was added 50 μl of 3,3',5,5'-tetramethylbenzidine, and incubated for 15 minutes. Tablets read on ELX 808 Ultra microplate reader (Bio-Tek Instruments) using the method of endpoints at 450 nm after addition of 50 μl of 1N H2SO4as the stop solution.

Analysis of interferon-gamma

The spleen cells obtained by standard methods were planted in the replay with a concentration of 5×105cells/well and were cultured with or without recombinant polypeptide as antigen within 48 hours Supernatant cultures were collected and analyzed for γ-interferon using the set for a solid phase enzyme immunoassay sandwich method (Biosource, Camarillo, CA) according to the manufacturer's instructions. Briefly, 50 μl of culture supernatant was added to wells coated with monoclonal antibodies specific for murine γ-interferon. After 2 h the joint incubation at room temperature with biotinylating polyclonal antibody, the wells were washed and added streptavidin-peroxidase. After 30 minutes incubation and washing the wells was added a solution of tetramethylbenzidine and the results of read is whether at 450 nm in ELx 808 Ultra microplate reader (Bio-Tek Instruments).

Enzyme-linked immunosorbent spot assay (ELIspot)

Set for enzyme-linked immunosorbent spot assay (KPL) was used to determine the relative number of cells expressing γ-interferon, a suspension of single cells of the spleen in accordance with the manufacturer's instructions. Briefly, 10 μg/ml of antibody, immobilized interferon (BD Biosciences, San Jose, CA) were deposited on Multiscreen 96-well filter tablet (Millipore, Bedford, MA) overnight at 4°C. After one wash with wash solution, the plates were blocked with 1×BSA solution for 1 hour at 25°C and again washed. The spleen cells were placed in repetition at a concentration of 5×105cells/well and were grown with and without antigen for 48 hours at 37°C. Plates were washed once with wash solution and incubated for 1 h at 25°C with 5 µg/ml secondary rat artemisinin antibodies to gamma-interferon conjugated with Biotin (BD Biosciences). Plates were washed and incubated for 30 min at 25°C with 0.2 μg/ml HRP-streptavidin. The filters showed TureBlue substrate for 15 minutes, dried in the dark and counted spots.

Analysis of the excited fluorescence sorted cells (FACS analysis) on the cell surface markers

Cells of the spleen was applied in a repeat on 96-well culture microplates at a concentration of 1×106cells/well and were konturirovany 24 hours. The analysis in sbordoni fluorescence sorted cells was performed after stimulation of spleen cells with the polypeptide Map74F and concavalin A suitable estimulante control cells. After triple washing FACS buffer (1% BSA and 0.05% of sodium azide in the FSB), cells suspended in 50 μl of FACS buffer and mixed with 0.5 μg of CD3, CD4 and CD8 antibodies conjugated with FITZ (fluorescein isothiocyanate) or PV (phycoerythrin, eBioscience, San Diego, CA), and incubated on ice 30 minuted twice washed with FACS buffer and suspended in 100 μl of 3% formaldehyde in the FSB, and then transferred to FACS tubes containing 500 µl of the FSB. Data were obtained for 10,000 events using flow-zitomer FACS caliber (Becton-Dickinson, San Jose, CA) and were processed using the Cellquest program. The results were expressed as the increase in the average percentage of cells with positive staining compared to cells reinducing sample stained with the same antibody. Real-time PCR to analyze the expression of cytokine mRNA.

Total RNA was isolated from the tissues of the spleen immunogenic mice using minnebar RNeasy mini kit (Qiagen, Valencia, CA). Messenger RNA was back-transcribed using Superscript™ II (Invitrogen) and was used as a matrix cDNA. Sequences of primers and samples are presented in Table 1. In Table 1 used the following abbreviations: FW, direct primer; RV, reverse primer; TP, TaqMan probe, double-labeled 5'FAM and 3TAMRA;andThe length of the amplicon pairs of bases.bIncoming number of cDNA and according to the corresponding gene in Genbank. cAntisense sample.

The samples were in the state of a fluorescent dye, 6-carboxyfluorescein on the 5' end and a quencher of luminescence, N', N ',N',N'-6-carboxytetramethyl on the 3' end. The reaction was carried out in repetition in a volume of 25 µl containing 2 µl of 10 PM forward and reverse primers, 2 μl of 2 PM samples, and 12.5 μl of TaqMan PCR master mix and 9.5 ál of diluted cDNA, using the following conditions: 10 min at 94°C and then 40 full-temperature cycles (15 seconds at 95°C and 1 min at 60°C), automatic fluorimeter (7700 Sequence detector, Applied Biosystems, Foster city, CA). Quantification was performed using the comparative threshold method cycles and was expressed as relative transcription or the ratio of n-fold compared to the calibration cDNA.

Statistical analysis

The data were statistically processed using Excel. The difference between groups and individual antigens were analyzed by one-factor variance analysis, followed by the criterion of multiple comparisons Tukey-Kramer or the student criterion. Differences were considered significant when they were obtained probability values less than 0.05.

Using materials and methods described in this Example, the following results were obtained.

Immune about the response in mice immunitary the polypeptide Map74F

Three weeks after secondary vaccination four mice from each group were killed, and assessed education anti-Map74F antibodies and T-cell immune response. Mice immunized Map74F, had significantly (P<0,01) stronger lgG1, but not lgG2a response against Map74F. On the contrary, in the control group was not detected Map74F-specific antibodies.

IFN-γ response was evaluated using methods of IFN-γ by ELISA of the culture supernatant and IFN-γ ELISPOT. Antigen-specific IFN-response was significantly higher (P<0,05) in mice immunized Map74F compared to control animals receiving only IFF (Figure 3). Results IFN-γ ELISPOT was also comparable with the results of IFN-γ by ELISA (Figure 4), which further confirmed the increased IFN-γ response in vaccinated animals. The vaccinated group had a mean number of colonies formed by a single cell is equal to 20, in contrast to 7 in the control group. The distribution of antigen-specific CD3+, CD4+and CD8+T cells in the spleen cells was assessed by FACS. The number of CD3+ and CD4+T cells was significantly higher (P<0,01) in mice immunized Map74F (Figure 5) compared to control mice. On the contrary, between vacciniforme and control animals was not observed any significant differences in populations of CD8 T cells. The levels of expression of cytokine genes was assessed by real-time PCR. Found no significant difference (P>0.05) in the levels of expression of cytokine genes IL-2, IL-12, TNF-α and IFN-γ between vaccinated and control animals (6).

Map74F protects C57/BL6 mice against infection MAP

On the basis of immune responses in vaccinated mice was planned to evaluate the protective effect of the polypeptide Map74F against MAP infection in mice. Serum taken at different time points was analyzed using ELISA for total lgG response (Fig.7) and the ratio of the lgG1/lgG2a (Fig). Mice immunized 74F, showed a significant (P<0.01) increase in the level 74F specific antibodies at 3 and 7 weeks (4 weeks after infection) after the second vaccination compared with the control animals. Despite the fact that animals from the control antibody levels were increased after infection, the response was higher in vaccinated animals at 8, 12 and 16 weeks after infection. Significant differences were found in the ratio of lgG1/lgG2a for vaccinated and control animals 3 weeks after secondary vaccination. In vaccinated animals, the ratio of lgG1/lgG2a increased after secondary vaccination until 4 weeks after infection. After that vaccinated animals showed a decrease in the ratio of lgG1/gG2a until the end of the observation period of 16 weeks after infection.

To assess the protective effect 74F, made crops on MAP cells of the spleen, liver and MDR at different time points after infection MAP. In the spleen loading MAP was significantly (P<0,01) lower in vaccinated animals compared to control animals at 8, 12 and 16 weeks after infection (Figure 9). In the liver vaccinated animals had lower MAP load than control animals, and the load was significantly (P<0,01) lower at week 12 after infection (Figure 10). In vaccinated animals, MAP_ load MLN was significantly lower at 8 weeks after infection (11). Histopathologically the study of the liver (Fig and 13) and spleen (Fig and 15) showed that intraperitoneal infection with MAP led to a greater number of granulomas in control rats at 8, 12 and 16 weeks after infection. The unvaccinated control animals in the liver contained numerous, large, randomly located granuloma (Fig), while the liver vaccinated animals contained only a small number of small lymphoid formations (Fig). Similarly, spleen unvaccinated control animals had more granulomas (Fig)than the spleen of vaccinated animals (Fig). When the liver and spleen from control animals were stained by Zn Nelson, were seen numerous kiloto the sustainable bacilli. On the contrary, the number of granulomas and acid-fast bacilli was smaller in animals immunized Map74F.

Thus, in this example, we used spleen cells from vaccinated and control animals to determine the type T-cell response induced by recombinant polypeptide. When mycobacterial infections Th1 cells are responsible for the protection in the early phase of infection. It is believed that the most effective strategy of vaccination against intracellular pathogens is a vaccination that causes as CD4+and CD8+T-cell response in developing Th1-associated cytokines. In General, γ-interferon is considered as the main component in the activation of macrophages and their production of Th-1 CD4+T cells is required for the contained MAP infection. The results indicate that vaccination with recombinant polypeptide Map74F causes significant IFN-γ response. Similarly, it was found that immunization Map74F causes severe CD3+and CD4+T cell response in vaccinated animals, in contrast to CD8+T cells, indicating that increased levels of IFN-γ may occur due to the increase in CD4+T cell populations. The results confirm that the expression of IFN-γ is mainly going on the La in activated CD4 +T cells. Increased CD4+T cell response and protection levels disclosed in the present invention after infection MAP, indicate a protective effect Map74F.

Presented in this example, the immunogenicity study also indicates that Map74F with IFF caused a good immune response in vaccinated animals. The ratio of lgG1/lgG2aincreased to 4 weeks after infection MAP, indicating a Th2-specific response. However, the ratio of the lgG1/lgG2agradually decreased from 8 weeks, indicating a possible shift in the Th1 response. From the results presented in this example, it is obvious that Map74F causes kctm and Th2 responses, the first response is more pronounced, which demonstrates a significant IFN-γ response.

Example 3

This example illustrates the use of Map74F and other selected proteins MAP to stimulate an immune response against MAP in ruminants.

The following materials and methods were used to obtain data presented in this example.

Animals. This study used a total of 25 kids ages 5 to 10 days from the herd is not infected with MAP. Samples fekale taken in kids before experiments immunization, were negative on the MAP, both by culture and by PCR for IS900 gene. All experimental work was performed in accordance with PR the fork, norms and principles of the legislation on protection of animals control of the Ministry of health for the humane treatment and use of laboratory animals used for research, study and training, the Director of the National Institutes of Health (NIH) study and use of laboratory animals and the new York state Department of health.

The bacteria. Clinical strain MAP 66115-98 was used to infect kids after immunization. This strain was IS900 positive and mycobactin-dependent. MAP 66115-98 was culturious in N environment with the addition of 10% oleic acid - albumin - dextrose - catalase (Becton Dickinson, Sparks, MD) and mycobactin J (Allied Monitor Inc., Fayette, MO). After kulturarbeit within 8 weeks of the microorganisms were separated by centrifugation at 4000×g for 10 min and washed twice in phosphate-saline buffer (FSB; pH of 7.2). The microorganisms were diluted FSB to the desired concentration and used for infection of calves.

Antigens and excipients. Three recombinant antigen MAP - 85A, 85B, SOD and recombinant polypeptide Map74F, were cloned, expressed and purified using standard procedures and as described in the application U.S. No. 11/816,365, description of the cloning, expression and purification of which is included in the application by reference. Expressed polypeptides about what imali using Ni-NTA agarose columns (Qiagen, Valencia, CA). Impurities of endotoxin was removed using Affinity Cancer Detoxi Gel (Pierce, Rockford, IL), and antigens had a negligible level (10 PG/ml) of endotoxin in the LAL-test. DDA (Sigma, USA) were mixed in sterile distilled water to a concentration of 2.5 mg/ml, was heated up to 80°C with constant stirring for 20 min and cooled to room temperature. DCA was thoroughly mixed with recombinant antigens to a final concentration of 250 μg/dose.

Immunization of animals. Goats were divided into three groups, eight animals in groups I and II and 9 animals in group III (as was more the kid). All the kids stayed with their mothers until the age of three months. The group of animals I were immunized 100 μg each of the four antigens (85A, 85B, SOD and Map74F) in DDA in the form of a subcutaneous injection. Animal group II were immunized 100 μg of each antigen without DDA. Group III was left as control animals and it was introduced only DDA. Three weeks after the primary immunization kids were vaccinated with a second time the same set of antigens and adjuvants.

Infected animals MAP. Three weeks after the second vaccination all 24 kid were infected orally with 5×108MAP_ cells/animal in 10 ml of milk replacer during 7 consecutive days. For each animal was the held microscopy of faeces for 2, 4, 6, 8 and 10 days after each exposure, and then once every month.

Isolation and culturing mononuclear cells of peripheral blood. MCPC were allocated in experimental goats using standard techniques. Briefly, 10-15 ml of peripheral blood was collected from the jugular vein into EDTA evacuated tubes (Becton Dickinson and Co, Franklin Lakes, NJ). Lymphocytes were separated by differential centrifugation using Histopaque of 1.077 (Sigma-Aldrich, St. Louis, MO). Mononuclear cells were washed three times with phosphate-saline buffer (FSB, pH of 7.2). The washed cell sediment suspended in the FSB and analyzed for viability after staining with 0.4% Trifanova blue. Lymphocytes are again suspended in the medium RPMI-1640 containing 10% fetal calf serum (Gibco, Grand Island, NY), 2 mm L-glutamine, 100 mm HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamicin (Gibco)to a final concentration of 2×106viable cells/ml Cells were seeded (200 µl/well) in 96-well round - or flat-bottomed microplates, depending on the type of experiment.

The study of lymphocyte proliferation. The study of lymphocyte proliferation was performed using standard techniques. Briefly, MCPC in 96-well flat-bottom microplates were incubated at 37°C in a humidified atmosphere of 5% CO2and stimulated each h the four purified recombinant antigens (10 μg/ml), concavalin A (CONA; 10 μg/ml, Sigma-Aldrich, St. Louis, MO) and purified protein derivative (tuberculin; 10 μg/ml, DBL, National Veterinary Services Laboratory, Ames, IA) within 72 hours. DNA synthesis in stimulated and unstimulated control cells was measured by introducing bromodeoxyuridine (BrdU) using BrdU colorimetric kit enzyme-linked immunosorbent assay (Cell proliferation ELISA BrdU colorimetric kit (Roche Diagnostics, Indianapolis, IN) according to manufacturer's instructions. Briefly, cells were labeled for 2 h, 10 μl of BrdU solution for applying labels. Added anti-BrdU antibody labeled with peroxidase and incubated for 90 minutes Then added the substrate solution, enzyme, and incubated at room temperature for 15 minutes the Enzymatic reaction was stopped by 1 M H2SO4and read the optical density (OD) at 450 nm, using ELX808 Ultra microplate reader (Bio-Tek Instruments Inc, Winooski, VT). The tests were carried out in the triplet, and the results were expressed as the mean stimulation index (SI), calculated as the ratio between the average OD of cells, culturebound with antigens and the average OD of cells, culturebound without antigens.

IFN-γ research. The levels of γ-interferon was determined in air-conditioned supernatant MCPC using ELISA method double bind using monoclonal antibodies (BOVIGAM; Biocor Animal Health, Omaha, NE), as specified in tools is the manufacturer. The tablets were read at 450 nm using ELX808 Ultra microplate reader (Bio-Tek Instruments, Inc). The results are interpreted by comparing the optical density (OD) of the negative and positive control. The results were either positive (if OD was greater than that for the positive control) or as negative (OD was less than for the positive control), relative to the boundary values, as suggested by the manufacturer.

Running cytometrics analysis lymphocytic markers.

Was held monochrome flow cytometrics analysis of the surface differencirovannyh antigens of lymphocytes using specific kids monoclonal antibodies (CD2-MUC2A; CD4-17D-lgG1; CD4-GCIAI-lgG2a; CD8-CACT80C-lgG1; CD8-7C2B-lgG2a; CD25-CACT116A-lgG1; CD45RO-ILA116A-lgG3; γδSTCR alpha chain specific lgG2b-GB21 AI; ACTI-CACT7A-lgM; ACT16-GB110A-lgM) (VMRD Inc., Pullman, WA) in accordance with standard protocols. Briefly, cells were washed three times in buffer cell sorting device with activation of fluorescence (FACS), and incubated with the primary antibody, pre-titrated for optimal reactivity for 30 min at 4°C. Following this, the samples three times washed, and incubated with artemisinin immunoglobulin labeled with the fluorescein-isothiocyanato (Vector Laboratories, Burlingame, CA)for 30 minutes at 4°C. the Cells three times washed with FACS buffer and suspe who was denovali in 100 μl of 3% neutral buffer formalin in the FSB. Finally, the cells were transferred to FACS tube and brought up to 500 μl with the FSB before measurement using FACS calibrated flow cytomer (Becton Dickinson, San Jose, CA). Data were collected for 5,000-10,000 events and were analyzed using the Cellquest program.

Quantitative real-time PCR to analyze the expression of cytokine genes. The allocation of total RNA, cDNA synthesis and quantitative real-time PCR was carried out according to standard methods. Briefly, RNA was isolated from lysed MCPC using RNeasy mini Kit (Qiagen, Valencia, CA). Selected samples of RNA was treated with 10 u/ál by Dnazol I, not containing RNase (Qiagen) at 37°C for 10 min, and then thermally iactiveaware at 95°C for 5 min, then cooled on ice. Reverse transcription of RNA samples was performed in a reaction volume of 20 µl (1,6 μl of total RNA, 200 E. Superscript II reverse transcriptase from Invitrogen, 50 mm Tris-HCl, 75 mm KCl, 3 mm MgCl2, 0.01 M of dithiothreitol and 0.6 mm dNTP) at 42°C for 50 min, and then inaktivirovanie at 70°C for 15 min Samples and primers for quantitative real-time PCR were designed according to the program Primer Express (Applied Biosystems, Foster city, CA) using bovine beta-actin and sequences of cytokine gene obtained from GenBank. Detailed description of the samples and primers used in this study is AI, presented in Table 2.

The samples were too efficient fluorescent label, 6-carboxyfluorescein (FAM) on the 5' end and a quencher of fluorescence, N',N',N',N',N'-6-carboxytetramethyl (TAMRA) at the 3' end. PCR was performed in a reaction volume of 25 μl with 10 μl of diluted cDNA, and the concentration of primers 400 nm, 80 nm of TaqMan sample (Integrated DNA Technologies, Inc., Coralville, IO) and universal master mix PCR (Applied Biosystems), containing 10 mm Tris-HCl (pH 8,3), 50 mm KCl, 5 mm MgCl2, 2.5 mm concentration of deoxynucleotidase, of 0.625 E AmpliTaq Gold DNA polymerase and 0.25 E AmpErase uracil-N-glycosylase for the reaction. The samples in the replay was in 96-well-microplate and amplificadores in automatic fluorimeter (7700 Sequence Detector, Applied Biosystems). The PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C, then 40 cycles at 95°C for 15 sec and 60°C for 1 min quantification was done using the comparative threshold method cycles and was expressed as relative transcription or the ratio of n-fold compared to the calibration cDNA.

The immune response to recombinant antibodies. Immune responses for four recombinant antigen, Kona and tuberculin was evaluated using ELISA according to standard Protocol. Indirect ELISA was performed in 96-well flat-bottomed tablets coated with the diversified 100 µl of each of the antigens, kept at 4°C during the night and three times washed FSB containing 0.05% Tween 20 (FSB-T). Unbound sites were blocked with 5% skimmed milk in the FSB-T at 37°C for one hour and washed twice FSB-So To each well was added 100 μl diluted 1:25,000 conjugated to horseradish peroxidase antimisting immunoglobulin G (Sigma) and incubated at 37°C for one hour. Tablets three times washed FSB-T and to each well was added 200 μl of 2,2'-azinobis-thiazoline-6-sulfonic acid (Sigma). The plates were incubated at 37°C for 30 min in the dark, and then added the stop solution (1 M HCl), and three times was read at 405 nm with 2 min intervals using a microplate reader (BioTEK Instruments, Inc, Winooski, VT). In each plate included appropriate positive and negative serum and antigen, and control antibodies.

Crops on MAP faeces and organs. After infection, attempts were made to select the MAP organisms from faeces using egg-yolk medium (Herald''s egg yolk (HEY), Becton, Dickinson and Co. Sparks, MD) in accordance with standard protocols. Samples fekale were taken from all animals at 2, 4, 6, 8 and 10 days after infection and then every month for planting on the MAP. Similarly, 23 of the sample tissues were taken from each of the 24 animals at necropsy and examined on the MAP with the help of crops. Crops conducted in the Bacteriology Department at the Cornell Animal Health Diagnostic center,blind.

Cross-pathology and histopathological study. All animals were euthanized at 38 weeks after primary vaccination, and autopsy was performed. Each animal was taken 23 sample tissues, including spleen, tonsils, mesenteric lymph nodes (3), mandibular lymph nodes, ileocecal lymph nodes, liver lymph node, duodenum (ascending and descending), small intestine (3 phases over an approximately equal intervals from the proximal to distal end), ileum (2 plots at the proximal end 2 of the plot in the middle of the ileum, 2 plots at the distal end), the entrance to the blind colon, blind intestine and spiral colon, which were taken during the autopsy. The collected tissues were fixed by immersion in 10% neutral buffered formalin, were sealed in paraffin, did the slice thickness of 4 μm and stained with hematoxylin and eosin using standard histological techniques. The sections were examined by a certified veterinary pathologist who had no idea about the vaccinated group.

The statistical analysis. The data were statistically processed using Excel. The difference between groups and individual antigens were analyzed by single-factor dispersion of analyze the subsequent criterion for multiple comparisons Tukey-Kramer or the student criterion. Differences were considered significant when they were obtained probability values less than 0.05. Using materials and methods described in this Example, the following results were obtained.

Lymphoproliferative response to antigens: Although antigen-specific lymphoproliferative responses were detected at 6 weeks after primary vaccination (APV), responses were significantly higher (P<0.05) in both vaccinated groups (I and II) compared with unvaccinated control group (III) at 10 weeks after primary vaccination APV (Fig-18). However, we detected no significant differences in the responses between the different studied antigens.

Antigen specific IFN-γ responses Were detected significant differences (P<0.01) in IFN-γ responses between vaccinated and control animals at 6 and 10 weeks after primary vaccination (Fig-21). In vaccinated animals, the best response was recorded for Map74F. IFN-γ responses were significantly higher (P<0,05) for antigens 85A and Map74F in animal group I compared to group animals II 10 week APV. However, it was not found significant differences between other investigational recombinant genes. Although the levels of IFN-γ-specific recombinant antigens was reduced from 18 weeks to 38 weeks after primary vaccination, they remained significantly higher (P<0,05) vaktsinirovat the different animal compared to control.

The distribution of sub-populations of cells in response to recombinant antigens: a Subpopulation of antigenstimulated lymphocytes were studied for differences in their shares for a method of flow cytometry. There was a significant (P<0.01) increase in CD4+lgGI, CD4+lgG2a, CD8+lgGI, CD8+lgG2a cell subtypes in immunized groups compared with the control group (Fig-25). The increase was begun at 6 weeks after primary vaccination and continued throughout the experiment up to 38 weeks after primary vaccination. Populations of T cells, CD4+and CD8+specific to the recombinant antigens were more from immunized animals, but the proportion of CD4+cells was higher than the proportion of CD8+notches. However, there was an increase, albeit insignificant, populations of CD8+T cells 26 and 38 weeks after primary vaccination. While all recombinant antigens investigated in our study increased γδTCR+cell population in both vaccinated groups compared to the control animals, the increase was significantly higher (P<0,05) for 85A and Map74F antigens. Again, as in the case of CD4+and CD8+T cell populations, there has been a steady increase in γδTCR+gunning population until the end of the experiment (Fig). The immunized animals share the antigen is pacificnew CD25 +T cells was higher for the four investigated recombinant antigens. Although there was a slight difference in CD25+T cell responses between different target antigens, it was not significant.

The expression of specific montitoring gene: it Was found a significant increase in expression of IFN-γ specific to the recombinant antigen in immunized animals (P<0.01) compared with control animals starting 6 weeks after primary vaccination (Fig). Although the level of expression reached a maximum at 10 weeks after primary vaccination, up to 38 weeks, the levels remained significantly higher in immunized animals compared to control animals. On the contrary, found no significant differences in the expression of IL-10 between immunized and control animals in any point of our study, except for a General increase in the level of expression of IL-10 on 26-38 weeks after primary vaccination in all three groups (Fig).

Immune response specific to the recombinant antigen: All four recombinant antigen was studied in the present invention, called the immune response in both vaccinated groups. Responses were significantly higher (P<0.01) in vaccinated groups compared with the control group who ince in the sixth week APV until the end of the experiment (Fig). There were no significant differences in immune responses between different used recombinant antigens.

Histopathology tissue taken at the time of necropsy:

Histopathological examination of tissues taken from each animal at the time of necropsy, indicates that 75% of the unvaccinated control animals (Group III) had granulomas in at least one tissue, while among the animals of groups I and II only 25 and 50% of animals, respectively, had a granuloma. Granulomas were found in the mesenteric lymph node and the small intestine 1 and ileocecal lymph node of a different animal from Group I. In contrast, granulomas in groups II and III were localized primarily in the ileum, the ileocecal junction or in the caecum, while ileocecal lymph node was raised from 5 animals from these two groups. Never found one granulomas in the duodenum, and only 2 animals, one from Group I and one from Group III had granulomas in the small intestine. Other damaged tissue included the connection of a blind gut and hepatic lymph nodes in two unvaccinated control animals of Group III.

Load MAP in tissues after infection: loading MAP 23 tissues taken from each animal during Necroscope, assess who was using bacterial culture (Fig). Among the vaccinated animals, only one animal in group I showed a positive result at sowing with very low KOE. In group II, while 5 out of 8 animals were positive for MAP, bacterial load was very low in four of the five animals (less than 5), which was isolated MAP. In the unvaccinated group III, all nine animals were positive for MAP, while most of the animals had at least 5 of tissues positive for MAP with more than 300 KOE (too many to count).

As can be seen from the above, in this example, the estimated protective effect of recombinant antigens 85A, 85, SOD and Map74F in the model for the kids. Since MAP is an intracellular organism, it seems that YOU response, which is mediated by sensitized T-cells and, in particular, γ-interferon, produced by sensitized T-cells, plays an essential role in the protection. Among various methods, the measurement of the response in proliferation of lymphocytes specific study antigen is a widely applicable method for determining the cellular immune response. See the definition of proliferation of lymphocytes specific to the recombinant antigen, three weeks after re-vaccination, which was significantly higher in the vaccinated group after infection compared to the control, that indicates the manifestation of antigen-specific cellular immunity.

K-Interferon is one of the main cytokine genes activated in response to MAP infection in cattle. In addition, a major histocompatibility complex class I restricted CD8+T cells, which produce cytokines such as IFN-γ, is required for resistance to other mycobacterial infections, such as M. tuberculosis. In this example, mediated Map74F IFN-γ response was detected after secondary vaccination and infection. Not wishing to be bound to any theory, it is assumed that the increased IFN-γ could play an important role in protective immunity kids after infection alive MAP_ using mediated IFN-γ signaling of macrophage.

The results presented in this example, also show higher CD4+and CD8+T cell response of immunized animals. The results also confirm the contribution of CD4+T cells in peripheral levels of γ-interferon and proliferative response after immunization with recombinant antigens. CD25 is expressed activated T-cells. Our results clearly indicate the growth of activated T-kitoku vaccinecompensation. Although the population of γδTCR+cells remains relatively small compared to CD4 +and CD8+T cells, it is significantly higher (P<0,05) from immunized animals compared to control animals. CD4+T cell effector mechanism is associated with the secretion of γ-interferon, which activates the bactericidal activity in macrophages, lymphotoxin, perforin and granulysin. CD8+and γδ+T cells also produce granulysin. This is consistent with the results disclosed here studies on the infection and confirms the protective effect of these recombinant antigens. An increased level of mRNA expression of γ-interferon clearly indicates that there is a certain antigen-specific Th1 response in immunized animals, which reflects the low level of expression of Th2-specific IL-10.

Along with lymphoproliferative response, γ-interferon response, CD4+T and CD8+T cell response shows evidence of a significant Th1 response in immunized groups. Were analyzed the results of experiments on the infection in order to evaluate the protective effect of recombinant antigens in kids. It is believed that in the absence of typical clinical symptoms, histopathology and bacterial load in tissues collected during necropsy, are the best standards for the evaluation of protective effects of vaccination MAP. Were collected 2 types of tissues from each of the 25 animals and analyzed the tissue for histopathological changes and load the MAP with the help of crops. There was a significant decrease in the number of animals and tissue damage in the group treated with recombinant antigens and DDA, indicating a protective effect.

The allocation MAP in the sowing is the most sensitive method for the determination of MAP in tissues compared with acid-fast staining and microscopic examination, especially during the early phase of infection. However, the distribution of granulomatous lesions in General reflects the results of the crop MAP. The results of the seeding MAP presented in this example, clearly show a protective effect used four recombinant antigens. Protection was almost full when the antigens were given together with the DDA. MAP was recovered from only one of the eight animals of this group (I), and this animal had a significantly lower potential MAP in only one positive tissue, namely, in the distal ileum. The use of antigens without auxiliary substances showed a protective effect, although the effectiveness of protective effect was somewhat smaller when the antigens were used without excipients. It can be observed from the results of the crops, animals of group II, which received antigens without excipients. Although 5/8 animals were positive for MAP, the bacterial load was in these animals significantly neither the e compared to the control group of animals III pointing to the protective nature of antigens, even without excipients. Significantly higher MAP was found in all animals unvaccinated control group, which again indicates a protective effect of antigens. Immunization kids recombinant antigens has led to a sustained immune response during a long period. The results presented in this Example, therefore, indicate that recombinant antigens stimulate as mediated cellular and humoral immune system. After secondary vaccination, a significant increase in immune response was found for all recombinant antigens in both vaccinated groups compared to the unvaccinated control group. The answer tends to be higher in the group of animals I, who received the antigens with an auxiliary additive, albeit only slightly. The early appearance of reactivity of cell-mediated immunity (CMI) after sero-conversion is a permanent characteristic of mycobacterial infections in ruminants. However, our multicomponent subunit used in this Example, give significant protection in terms of reduction of bacterial load on the target organs compared to monomaniacally kids.

The present image is the buy is not limited to the specific embodiments, as described in the application. In fact, from the above description, the person skilled in the art will be apparent various modifications of the invention other than those described in the application. It is implied that such modifications also fall within the scope of the attached claims.

All references provided in this application is included in the application by reference in its entirety and for all purposes to the same extent as if each individual publication, patent or patent application mentioned specifically and individually for inclusion by reference in its entirety and for all purposes.

Citation of any publication is to reveal their essence before the filing date and should not be seen as a recognition that the present invention gives the right Dating backdating such publication in accordance with the previous invention.

1. Pharmaceutical composition for stimulating an immune response to Mycobacterium avium subspecies paratuberculosis (MAP) in a mammal, comprising a recombinant polypeptide containing, from the N-Terminus to the C-end:
i) C-terminal fragment of the protein BAR, with the indicated C-terminal fragment MAP_3527 contains amino acids 183-361 sequence SEQ ID NO: 2;
ii) the sequence of the protein BAR, and the sequence of the protein BAR contains aminos the slots 1-460 sequence SEQ ID NO: 3; and
iii) N-terminal protein fragment MAR, with the indicated N-terminal protein fragment MAR contains amino acids 33-180 sequence SEQ ID NO: 2.

2. The composition according to claim 1, characterized in that the sequence of the protein BAR contains the amino acid sequence of SEQ ID NO: 3.

3. The composition according to claim 1, characterized in that the recombinant protein contains the amino acid sequence of SEQ ID NO: 1.

4. The composition according to claim 1, characterized in that the composition further comprises an auxiliary substance.

5. The composition according to claim 4, characterized in that the auxiliary substance selected from the group consisting of monophosphorylated And (IFF), dimethyldioctadecylammonium (DDA) and their combinations.

6. The composition according to claim 1, additionally containing protein of Mycobacterium avium subspecies paratuberculosis (MAP)is selected from the group consisting of a MAP protein 85A, MAP protein 85C, superoxide dismutase (SOD) MAP protein and combinations thereof.

7. Method of stimulating an immune response to Mycobacterium avium subspecies paratuberculosis (MAP) in a mammal, comprising administration to the mammal of a composition according to claim 1, stimulating the immune response to Mycobacterium avium subspecies paratuberculosis (MAP) in a mammal.

8. The method according to claim 7, characterized in that the composition further comprises an auxiliary substance.

9. The method according to claim 8, characterized in that the auxiliary substance selected isgroup, consisting of monophosphorylated And (MFA) and dimethyldioctadecylammonium (DDA).

10. The method according to claim 7, characterized in that the mammal is a ruminant, and the specified composition is administered specified ruminant animal.

11. The method according to claim 10, wherein the ruminant is a bovine cattle, sheep, goat, deer or elk.

12. The method according to claim 11, wherein the ruminant is a cattle.

13. The method according to claim 10, wherein the ruminant is not infected with MAP.

14. The method according to claim 10, wherein the ruminant is a positive MAP.

15. The method according to item 12, characterized in that the cattle suffers from John.

16. The method according to claim 7, characterized in that the mammal is pregnant.

17. The method according to claim 7, characterized in that the composition further comprises a protein of Mycobacterium avium subspecies paratuberculosis (MAP)is selected from the group consisting of a MAP protein 85A, MAP protein 85C, superoxide dismutase (SOD) MAP protein and combinations thereof.

18. The method according to claim 7, characterized in that the recombinant protein in a given composition contains the sequence of SEQ ID NO: 1.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine, and concerns the use of a monobactam antibiotic of formula

,

wherein an oxyimino group, i.e. >C=N-O-, is Z-oriented, or a pharmaceutically acceptable salt thereof for preparing a therapeutic agent for treating a bacterial infection, in a combination with a carbapenem antibiotic specified in a group consisting of meropenem, imipenem, ertapenem and doripenem.

EFFECT: invention provides the high clinical effectiveness.

24 cl, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of formula (I) or pharmaceutically acceptable salts thereof wherein A, R1, R2, R3 and m are specified in the patent claim. The present invention also refers to the number of specific compounds, and to a pharmaceutical composition containing the above compounds effective for inhibition of kinases, such as glycogen synthase kinase 3 (GSK-3), Rho kinase (ROCK), Janus kinase (JAK), AKT, PAK4, PLK, CK2, KDR, MK2, JNK1, aurora, pim 1 and nek 2.

EFFECT: preparing the specific compounds and pharmaceutical composition containing the above compounds effective for kinase inhibition.

18 cl, 393 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and aims at the preventive therapy of children who had close domestic contact with the patients suffering the infectious stage of syphilis. The method involves administering a single oral dose of a flavoured suspension of azithromycin (Sumamed) 10 mg per kg of children's body weight for 5 days.

EFFECT: use of the declared method prevents the development of the infection in children and reduces the length of the preventive therapy by two times.

4 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science and concerns treating pneumonia in piglets. That is ensured by using a compositional preparation containing active agents in the form of tylosin tartrate and the antibiotic tetracycline in ratio 1:1. The preparation is administered in the amount providing effective doses of tylosin tartrate 5 mg/kg and tetracycline 5 mg/kg.

EFFECT: use of the above compositional preparation provides the effective treatment of pneumonia ensured by a synergetic effect of the ingredients.

5 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacology, and may be used in veterinary science for preventive and intensive treatment of gastrointestinal and purulent surgical diseases. A method for preparing an antiseptic preparation with metabolic and hepatoprotective activity comprises dissolving polyvinyl alcohol, potassium iodide, crystalline iodine, succinic acid in distilled water in certain proportions (wt %).

EFFECT: invention allows enhancing stimulation of the metabolic processes and hepatoprotective function of the liver.

3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science, and aims at treating respiratory diseases in young farm animals. The complex preparation tylocolin is administered to the animals once a day for 3-5 days intramuscularly. A dose is 0.05 ml/kg of body weight in calves, and 0.075 ml/kg of body weight in piglets.

EFFECT: use of the method enables higher clinical effectiveness in the respiratory diseases of young farm animals.

5 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary medicine, namely to medicines for treating mastitis in animals; a medicine for treating mastitis in animals contains the antibiotic tylosin, a gelling agent, water, with the gelling agent presented by carbopol and triethanolamine; the medicine further comprises Siberian fir essential oil and glycerol; an additional aseptic ingredient is plumepoppy juice prepared by pressing a vegetative part of a plant brought in a blossom time, in a step-loaded mechanical press with the first pressing force of 25-30 kN/cm2, and the exposure of 5 minutes, the second pressing force of 55-60 kN/cm2, and the exposure of 2.5 minutes; and the third pressing force of 80 kN/cm2, and the exposure of 1 minute; the ingredients are related as, %; tylosin 0.05-1.0; Cok plumepoppy juice 0.05-1.0; glycerol 5.0-10.0; carbopol 0.15-0.20; triethanolamine 0.02-0.03; Siberian fir essential oil 0.1-0.3; water up to 100.

EFFECT: agent for treating mastitis in animals has a combined therapeutic and repellent effect and a prolonged shelf life.

4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine, and concerns an agent for treating or disinfecting of alkyl, aryl-(3,5-di-tert-butyl-4-hydroxybenzyl)phosphonium bromides and nitrates of general formula , having simultaneous bactericidal, fungicidal and antioxidant activity at low concentrations, high thermal stability and low toxicity, which can find application in medicine and veterinary science.

EFFECT: what is presented is a new therapeutic agent.

7 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of veterinary microbiology and deals with method of obtaining specific immunomodulator. Claimed method includes the following stages: cultivation of vaccine strain of BCG, destruction of culture with ultrasound, separation of antigenic complex, mixing supernatant liquid with formalin and conjugation of reaction mixture on polyelectrolytes: polyvinylpyrrolidone (PVP) four parts and polyethyleneglycol (PEG) one part by weight, at ratio 1 mh/ml of protein - 480 mg PVP and 120 mg of PEG by weight on magnetic mixer at room temperature until homogenous liquid is obtained.

EFFECT: invention makes it possible to restore impaired immune reactivity, remove secondary immune deficits, obtain harmless for animals substances, which possess expressed protective properties.

6 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the use of a live mycobacterium of the complex M. tuberculosis wherein a zmpl gene function is inactivated for making a drug, pharmaceutical compositions prepared of such mycobacteria, as well as a method for prevention and/or treatment of diseases or conditions involved in antigen and/or immunogen expression in the mycobacterium. The mycobacterium used according to the invention may contains a genetic material coding an antigen and/or immunogen exogenic or xenogenic for the mycobacterium.

EFFECT: invention provides the live mycobacterium based drug with improved immunogenicity and protection effectiveness.

12 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: passage vector contains fused DNA under control of a fused promoter. Fused DNA consists of a DNA fragment coding protein antigen M. tuberculosis, and a gene DNA fragment coding an amino acid sequence under accession number GenBank No.L22858. The double promoter consists of a polyhedrin promoter and a CMV promoter. What is also described is a vaccine for tuberculosis containing a recombinant nuclear polyhedrosis virus Autographa californica (AcNPV) as an active agent. The recombinant AcNPV contains a sequence coming into the vector described above.

EFFECT: more effective use in medicine.

6 cl, 15 dwg, 1 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: recombinant method is used to produce a microorganism of Mycobacterium tuberculosis complex which involves phoP gene inactivation or deletion and fadD26 gene inactivation or deletion. The produced microorganism is used for preventing tuberculosis in humans and animals.

EFFECT: invention allows producing the vaccine microorganism showing the properties of high attenuation and immune protection against tuberculous infection.

7 cl, 27 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary microbiology. A method for growing atypical mycobacteria cultures on a liquid nutrient medium, culture inactivation by autoclaving at 120°C for 30 minutes, protein settling from a culture filtrate of each strain separately by trichloroacetic acid to the final concentration of 4%, re-settling by ammonium sulphate at 50% saturation, protein dialysis through a cellophane coating as against deionised water to remove salts; analysis of the protein solution for its activity in each atypical mycobacteria culture, mixing of the protein solutions in equal portions by the "ED" concentration, sterilising filtration of the solution, packaging and lyophilisation. As atypical mycobacteria cultures, M. intracellulare, M. scrofulaceum, M. fortuitum and M. smegmatis are used, while the atypical mycobacteria cultures are grown on the liquid nutrient medium for 2-3 weeks.

EFFECT: method provides high-quality differential diagnostic response on PPD tuberculin for mammals and cutting economic loss of induced PPD tuberculin responded animal killing.

3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of veterinary. Essence of method of determination of sensitising properties of atypical microbacteria lies in carrying out allergic research of live stock. Separation of atypical microbacteria from biomaterial and determination of their sensitising properties on guinea pigs are realised by hypodermic infection with separated culture. Atypical cultures are introduced fractionally, trice, by equal parts after 24 hours in dose 20 mg/head with the following hypodermic introduction on 30-th day of only tuberculin-PPD. In case if after 48 hours allergic reaction, hyperemia, increase of over 5 mm of tissues volume and ulcers in place of introduction, as well as hyperplasia of regional lymph nodes are presents, sensitising properties of atypical microbacteria are determined.

EFFECT: application of claimed method makes it possible to increase efficiency of determination of sensitising properties of atypical microbactriua.

4 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: method for producing an antigene preparation from Micobacterium tuberculosis involves mycobacteria cultivation on a Lowenstein-Jensen medium, three-fold washing of bacterial cells from the residual nutrient medium. The washed bacterial mass is treated by aqueous acetone solutions of the concentrations 25 % and 50 %, and each acetone treatment the cells are precipitated by centrifugation. Further, the extracts containing an antigen are produced by three-fold sequential treatment of the bacterial mass deposited by centrifugation, by aqueous dimethylsulphoxide solutions with the increasing concentrations 5 % to 20 % with an insoluble fraction every time separated by centrifugation. The produced three portions of dimethylsulphoxide extracts are mixed and exposed to in-depth dialysis against distilled water. The prepared solution is used as an antigen-containing material for preparing a nitrocellulose membrane immunosorbent.

EFFECT: antigen preparation according to the invention exhibits extended-spectrum serum-positive fractions in a western blotting reaction.

4 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: according to the invention, the method provides combined cultivation of nonadherent fraction of mononuclear cells (MNCs) recovered from peripheral blood of the patients with pulmonary tuberculosis, with dendritic cells (DCs) processed with the antigen Mycobacterium tuberculosis and prepared of monocytes of adherent MNC fraction with using mature DCs being prepared by adding a maturing inducer representing lipopolysaccharide E coli to antigen-activated immature DCs. The combined cultivation of specified cells is carried out with recombinant human interleukine-18 (IL-18) added.

EFFECT: intensified proliferative potential of specific cytotoxic cells of mononuclear origins, stimulation of IFN -γ production level, formation of cytotoxic cells in response to the specific antigen M tuberculosis.

3 tbl

FIELD: veterinary science.

SUBSTANCE: method includes intracutaneous injection of PPD-tuberculin in dose of 0.2 ml to animals into right part of neck with simultaneous injection of atypical mycobacteria complex (AMC) into left part of neck, accounting and evaluation of reaction to tuberculin and AMC in 72 hours by measurement of skin fold of injection spot, division of animals into groups that "do not react to tuberculin and AMC", "react to AMC (-)", "react to tuberculin (+)", and "react to tuberculin and AMC (=)" and definition of statistical validity of differences in reactions to tuberculin and AMC, on the basis of which conclusion is made on infection of animals with mycobacteria of tuberculosis or atypical mycobacteria. Additionally each animal of diagnosed group independently from its diagnostic index "do not react to tuberculin and AMC", "react to AMC (-)", "react to tuberculin (+)", and "react to tuberculin and AMC (=)" is exposed to venous blood draw on an empty stomach with immediate injection of solution of hydrocortisone-acetate in dose of 5 ml intramuscularly with further draw of venous blood in 3 and 6 hours for the possibility to detect indices of activity of blood serum ferment - lactate dehydrogenase (LDG) in dynamics. Afterwards indices of LDG activity in dynamics are detected, average indices of LDG fermentative activity are compared in dynamics, those that are defined in group of animals who "do not react to tuberculin and AMC", to indices of fermentative activity of each animal in all other groups. If LDG activity increases in 3 hours by less than 8%, and in 6 hours by less than 17%, compared to average indices of fermentative activity of LDG, animals that "do not react to tuberculin and AMC", "react to AMC (-)", "react to tuberculin (+)", and "react to tuberculin and AMC (=)" are included in group of truly tuberculosis diseased animals of diagnosed group.

EFFECT: method makes it possible to improve accuracy of tuberculosis diagnostics.

2 tbl, 1 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: BCG strain is cultivated with the prepared culture sonicated. Further antigenic complex representing cytoplasmic and cell membranes is recovered from said sonicated culture. At 15000 rpm, made supernatant fluid (namely the antigenic complex) is mixed with formalin. The reaction mixture is incubated at temperature 37°C in a thermostat, analysed for protein content, and conjugated with polyvinylpyrrolidone (PVP) in mass ratio of protein 1 mg/ml - PVP 600 mg (1:600) in a magnetic stirrer at a room temperature until homogeneous liquid is made.

EFFECT: specific immunomodulatory is capable to restore lost immune responsiveness, to eliminate secondary immunodeficiency, to strengthen protective properties of BCG vaccine.

6 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention discloses a bispecific antibody or its functional fragment, specifically binding with IL-4 and IL-13, which contain variable domains of light and heavy chains with established amino acid sequence. The invention also includes use of antibodies or its functional fragments within a pharmaceutical composition for treatment of diseases or disturbances mediated by IL-4 and/or IL-13, including allergic diseases, asthma, cancer. The invention discloses a molecule of nucleic acid, which codes a bispecific antibody or its fragment, an expression vector and a master cell for production of a bispecific antibody and its functional fragment.

EFFECT: invention makes it possible to produce and use new inhibitors of cytokines, preserving stability in process of production and use in vivo.

17 cl, 2 dwg, 8 tbl, 8 ex

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