RU2318872C2 - Recombinant influenza virus strains expressing mycobacterial protective esat-6 antigen and uses thereof in tuberculosis treatment and prophylaxis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to living attenuated recombinant influenza virus strains expressing earlier secretory antigen Mycobacteriun tuberculosis ESAT-6. Invention is useful in tuberculosis treatment and prophylaxis.

EFFECT: recombinant influenza virus vector with increased protective effect.

7 cl, 14 dwg, 11 tbl, 8 ex

Description

The invention relates to medicine and can be used to obtain recombinant vaccine strains for the prevention and treatment of tuberculosis.

The present invention is aimed at solving one of the most important tasks of modern medicine - the creation of a new, more effective than BCG vaccine against tuberculosis. The need for such a vaccine is dictated by several reasons. First, the only BCG vaccine currently used is effective in preventing tuberculosis in children, but according to many sources, it is not able to prevent the reactivation of the disease in adults, which is the cause of most cases of pulmonary tuberculosis (Trends Immunol, 2001; 22: 160- 8). Secondly, the introduction of a live BCG vaccine can pose a serious threat to vaccines with impaired immunity, for example, in HIV-infected patients with AIDS. In these cases, the attenuated strain of mycobacteria is capable of causing a true infectious process with severe complications (Clin. Infect. Dis., 1997; 24: 1139-46).

Currently, several new approaches to improve the existing BCG vaccine are known:

1) recombinant attenuated mycobacteria, which are genetically engineered deletion mutants of virulent strains of mycobacterium tuberculosis (Science, 1998; 282: 759-762). In contrast to empirically based BCG, these strains carry smaller deletions of the genome regions responsible for virulence factors, but they express important protective antigens of M. tuberculosis and M. bovis lost by BCG. At the same time, such vaccines are potentially unsafe for people with immunosuppression;

2) recombinant BCG vaccines expressing additional protective proteins lost during prolonged cultivation of this strain (Nature medicine, 2003; V.9, N5: 533-539). This type of vaccine has the inherent disadvantages of BCG vaccine in terms of its insecurity;

3) subunit protein preparations based on protective proteins, administered parenterally. It should be noted that the existing experience in the development of subunit and peptide vaccines indicates the need for the introduction of powerful adjuvants in the vaccines, since protein or peptide antigens alone are not sufficiently immunogenic and often create only short-term immunity even after repeated immunizations (Infect. Immun., 2000 ; 68: 791-795). Another disadvantage is the inability of this type of vaccine to stimulate the production of cytotoxic cellular immunity;

4) DNA vaccines. Designed for intracellular expression of protein antigens and therefore able to stimulate the T-cell immunity (Infect. Immun., 1999; 67: 4780-4786). However, DNA vaccines are low immunogenic, require repeated administration and have not yet been licensed for use in humans.

An alternative to these approaches, which enhances the immunogenicity of protective mycobacterial antigens, is the use of a vector system based on a recombinant attenuated influenza virus expressing the ESAT-6 early secretory mycobacterial antigen as an intranasal vaccine. The activation of lymphocytes and the beginning of the production of interferon, which play an important role in the activation of macrophages, are associated with this antigen, thereby exerting a pathogenetic effect on the formation of immunity during infection with tuberculosis mycobacteria (Infect. Immun., 62: 2536-2544).

Unlike the BCG vaccine, intended primarily for parenteral administration, the influenza vector vaccine can be administered intranasally in the form of drops or aerosol. As a result, the development of mucosal immunity (mucosal immunity) is achieved at the entrance gate of tuberculosis infection. The existence of many antigenic subtypes of influenza virus allows us to count on the possibility of several immunizations to achieve the maximum preventive or therapeutic effect.

The purpose of the invention is the creation of recombinant vaccine strains that provide the development of protective immunity against tuberculosis when used as a prophylactic vaccine or serving the purpose of treating tuberculosis, that is, using them as a therapeutic vaccine.

The problem was solved using reverse genetics methods, namely, the creation of live recombinant strains of influenza A virus (influenza vectors) containing an insert of a foreign genetic sequence in the NS1 gene of the influenza virus and expressing protective antigens of Mycobacterium tuberculosis.

The essence of this invention is to obtain a chimeric product based on strains of influenza A virus of various antigenic subtypes and extraneous genetic sequence, immunodominant mycobacterial protein ESAT-6, which is applicable in medical practice.

The achievement of the main objective of the invention is illustrated by the following examples.

OBTAINING AND CHARACTERISTIC OF RECOMBINANT STRAINS OF THE INFLUENZA VIRUS.

Recombinant strains of influenza viruses expressing the ESAT-6 mycobacterial antigen were obtained using reverse genetics methods (J. Virol., 1996; 70: 4188-4192; Adv. Vims Res., 1999; 53: 265-300; J. Virol. , 1999; 73: 9679-82).

Construction of plasmids. Influenza A / PR8 / 34 genomic RNA was extracted using Ultraspec reagent (Biotecx Laboratories). A cDNA copy of the NS gene was obtained by reverse transcription using a primer (5'-ACTACTTCTAGAGAAGACAAAGCAAAAGCAGGGTGACA-3 ') and M-MuLV reverse transcriptase (Fermentas). The obtained cDNA was used to amplify a fragment of the NS1 gene in the polymerase chain reaction (PCR) using the following primers:

direct: (5'-ACTACTTCTAGAGAAGACAAAGCAAAAGCAGGGTGACA-3 ') and

reverse: (5'-ACTACTCTGCAGATTAACCCTCACTAAAAGTAGAAACAAG-3 ').

The amplified fragment was cloned into the pQBI 25/50-fC1 plasmid vector (Qubiogene) at the blunt ends between the Poll promoter and the hepatitis delta ribozyme sequence (HDV), which plays the role of a transcription terminator (J. Virol., 1999; 73: 9679-9682; Adv Virus Res., 1999; 53: 265-300; J. Virol, 1996; 70 (6): 4188-4192). The resulting plasmid was called pPolI-NS-HDV.

The nucleotide sequence of ESAT-6 was synthesized by hybridization of an equimolar amount of six synthetic overlapping oligonucleotides (10 pmol / ml each) (CODON Genetic Systems) by heating to 100 ° C for 4 minutes, followed by slow cooling of the mixture to room temperature.

The resulting DNA fragment, 288 nucleotides in length, was amplified using end primers in PCR (forward primer: 5'ATGACAGAGCAGCAGTG 3 'and reverse primer: 3'CTATGCGAACATCCCAG 5'), and cloned into the pPolI-NS-400V plasmid between nucleotides 401 in the open reading frame of the NS1 gene of the influenza virus without deleting portions of the gene. This design is called pPol-NS-ESAT-6-HDV.

In order to post-translationally separate the ESAT-6 protein from the N-terminal region of the NS1 protein, a synthesized sequence (51 nucleotides) was inserted between them that encodes a portion of the protein protease recognition site 2A (NFDLLKLAGDVESNLG / P) of picornovirus, which has the property of self-cutting before the last proline ( J. ViroL, 1994; 68: 4486-4492). This design is called pPol-NS125-2A-ESAT-6-HDV.

The structure of the recombinant NS1 gene is shown in the scheme (Figure 1). The nucleotide sequence of the NS1 gene encoding 125 amino acids of the influenza virus NS1 protein is followed by the sequence encoding a portion of picornovirus 2A (NFDLLKLAGDVESNLG / P) along with the ESAT-6 sequence (95 amino acids). The arrow marks the self-cutting site in front of the last proline, which provides the post-translational yield of ESAT-6 protein.

The expression plasmids for the PB1, PB2, RA and NP proteins of the influenza virus were obtained by cloning the corresponding PR8 genes into the plasmid pTriEx-1 (Novagene) under the active chicken promoter and named pTriEx-PB1, pTriEx-PB2, pTriEx-PA and pTriEx-NP, respectively .

Obtaining recombinant strains of influenza viruses. The system for producing influenza viruses (H1N1, H2N2) containing the recombinant NS1 gene is shown in the scheme (Figure 2). Vero cells (American Type Culture Collection (ATCC) were transfected with pPol-NS125-2A-ESAT-6-HDV plasmid (1) expressing the chimeric influenza virus NS gene RNA (2), together with the plasmid set pTriEx-PBl, pTriEx-PB2 , pTriEx-PA, and pTriEx-NP expressing proteins of the influenza virus polymerase complex (1 μg of each plasmid). Transfection was performed by electroporation (Nucleofection technique, Amaxa) according to the instructions for use. 24 hours later, transfected Vero cells were infected with helper influenza viruses A / PR / 8-delNS (H1N1) or A / Sing / delNS87 (H2N2) (3), which contained partially or completely the NS1 gene was therefore sensitive to interferon (Virology; 2004, 324 (1): 67-73). 8 hours after infection, human leukocyte interferon-alpha was added as a selective preparation (NIBSC 1 ^st International Standard 1999; 3 units ./ml of culture medium) and the cells were incubated for 48 hours at 37 ° C. The resulting virus crop was twice passaged on Vero cells in the presence of interferon-alpha at a concentration of 3 units / ml. The final virus population (4) contained the recombinant NS1 gene with an ESAT-6 insert, as evidenced by sequencing of the RT-PCR product. After a twofold purification, the viruses were isolated.

The recombinant influenza virus belonging to the H1N1 subtype is called FLU / H1-ESAT-6, the recombinant influenza virus belonging to the H2N2 subtype is called FLU / H2-ESAT-6.

Recombinant influenza virus subtype H3N2 was obtained by genetic reassortment of the FLU / H1-ESAT-6 virus with A / Aichi / 1/68 (H3N2) virus on Vero cells in the presence of the rabbit antiserum to A / PR / 8/34 (H1N1) virus. The resulting viral clone contained 6 fragments of the genome (PB2, PB1, PA, NP, M, recNS) from the vector A / PR8-2A-ESAT-6, and the glycoprotein genes (HA, NA) from strain A / Aichi / 1/68 ( H3N2). Reassortant genotyping was performed by RT-PCR followed by comparative restriction analysis of cDNA copies of each segment. The resulting virus was named FLU / H3-ESAT-6.

Example 1. Recombinant virus A / PR8-2A-ESAT-6 (H1N1) was obtained using methods of reverse genetics in cell culture Vero and is characterized by the presence of a recombinant NS1 gene containing an insert of the nucleotide sequence of ESAT-6 M. tuberculosis between positions 400-401 open reading frame of the NS gene, as evidenced by sequencing of the RT-PCR product.

Due to the presence of the insert, the A / PR8-2A-ESAT-6 virus differs from the wild-type influenza virus by the presence of an increased size of the NS 1 genomic fragment, which was determined by agarose gel electrophoresis after amplification in PCR with specific primers:

5'-ACTACTTCTAGAGAAGACAAAGCAAAAGCAGGGTGACA-3 'and 5'-ACTACTCTGCAGATTAACCCTCTCTATAAAAGTAGAAACAAG-3' (Figure 3). The drawing shows that the size of the amplified recombinant NS 1 fragment exceeds the size of the amplified NS1 fragment of the wild-type influenza virus gene by the calculated value.

Another genetic marker that determines the recombinant nature of this virus is the expression of ESAT-6 protein, as determined by immunofluorescence analysis of infected cells using monoclonal antibodies (Figure 4A). The luminescence of ESAT-6 protein accumulated in the nucleus of Vero virus-infected recombinant cells is clearly visible in the drawing.

Expression of the ESAT-6 insert upon infection of Vero cells was also confirmed by Western blot analysis of lysates of virus-infected cells using monoclonal antibodies against the ESAT-6 protein (Figure 4B). In the drawing, colored zones corresponding to products with a molecular weight of 26 kDa and 24 kDa are visible. The presence of several zones is determined by post-translational cleavage of the carboxyl part of ESAT-6, previously shown for this protein.

The resulting virus can multiply in Vero and MDCK cell cultures to titers of 7.5 log PFU / ml and in 10 day old chicken embryos to titer 9.2 log EID 50 / ml at an optimum temperature of 37 ° C. The virus is not temperature sensitive and reproduced to similar titers at a temperature of 39 ° C.

In case of intranasal infection of mice at a dose of 5.5 log PFU / mouse, the resulting virus has the ability to multiply in the lungs of animals up to titers of 1.5 PFU / ml of 10% lung suspension without causing death of animals i.e. is attenuated compared to the original parent strain. The absence of pathology in the lungs of immunized animals and the absence of weight loss in mice confirm attenuation. The virus has genetic stability for at least 5 consecutive passages in a Vero cell culture.

The characteristics of the recombinant strain of the influenza virus A / PR8-2A-ESAT-6 (H1N1) obtained by the authors are given in the attached certificate of the deposit of the strain (No. 147) into the collection of the Museum of Viruses of the Research Institute of Influenza RAMS.

Example 2. Recombinant virus A / Singapore / -2A-ESAT-6 (H2N2) was obtained using methods of reverse genetics in cell culture Vero and is characterized by the presence of a recombinant NS1 gene containing an insert of the nucleotide sequence of ESAT-6 M. tuberculosis between positions 400-401 open reading frame of the NS gene, as confirmed by sequencing of the RT-PCR product.

When Vero cells are infected with this viral strain, the expression of ESAT-6 protein is observed, which is confirmed by immunofluorescence analysis and immunoblotting of lysates of infected cells using monoclonal antibodies to ESAT-6 protein (Figure 4).

The resulting virus is able to propagate in Vero and MDCK cell cultures up to titers of 7.5 log PFU / ml and in 10 day old chicken embryos up to a titer of 8 log EID 50 / ml at an optimum temperature of 37 ° C. The virus is not temperature sensitive and reproduced to similar titers at a temperature of 39 ° C.

With intranasal infection of mice at a dose of 5.5 log PFU / mouse, the resulting virus has the ability to multiply in the lungs of animals to titers of 3.5 PFU / ml of 10% lung suspension without causing death of the animals i.e. is attenuated compared to the original parent strain.

The virus has genetic stability for at least 5 consecutive passages in a Vero cell culture.

The characteristics of the recombinant strain of the influenza virus A / Singapore / -2A-ESAT-6 (H2N2) obtained by the authors are given in the attached certificate of the deposit of the strain (No. 149) into the collection of the Museum of Viruses of the Research Institute of Influenza RAMS.

Example 3. Recombinant influenza virus A / Aichi-2A-ESAT-6 (H3N2) was obtained by genetic reassortment of virus A / PR8-2A-ESAT-6 with virus A / Aichi / 1/68 (H3N2) on Vero cells in the presence of rabbit antiserum to virus A / PR / 8/34 (H1N1). The resulting viral clone contained 6 fragments of the genome (PB2, PB1, PA, NP, M, recNS) from the vector A / PR8-2A-ESAT-6, and the glycoprotein genes (HA, NA) from strain A / Aichi / 1/68 ( H3N2).

Due to the presence of an insert of the ESAT-6 sequence, the resulting virus differed from wild-type influenza virus by the presence of an increased size of the NS1 genomic fragment, which was determined by agarose gel electrophoresis after its amplification in the NDP with specific primers:

5'-ACTACTTCTAGAGAAGACAAAGCAAAAGCAGGGTGACA-3 'and

5'-ACTACTCTGCAGATTAACCCTCACTAAAAGTAGAAACAAG-3 '(Figure 3). The drawing shows that the amplified recombinant fragment of NS1 has an insert of the ESAT-6 sequence, exceeding the size of the NS1 gene of the wild-type virus by the size of the insert.

This virus is temperature-sensitive and adapted to growth in Vero cell culture.

When Vero cells are infected with the viral strain A / Aichi-2A-ESAT-6 (H3N2), expression of the ESAT-6 protein is observed, which is confirmed by immunofluorescence analysis and immunoblotting of lysates of infected cells using monoclonal antibodies to the ESAT-6 protein (Figure 4).

The resulting virus is able to multiply in Vero cell culture and MDCK to titers of 7.5 log PFU / ml at an optimum temperature of 34 ° C, reproduced to titers of 3 log PFU / ml at 39 ° C.

In case of intranasal infection of mice at a dose of 5.5 log PFU / mouse, the resulting virus has the ability to multiply in the lungs of animals up to titers of 1.5 PFU / ml of 10% lung suspension without causing death of animals i.e. is attenuated compared to the original parent strain. The absence of pathology in the lungs of immunized animals and the absence of weight loss in mice confirm attenuation.

The virus has genetic stability for at least 5 consecutive passages in a Vero cell culture.

The characteristics of the recombinant strain of the influenza virus A / Aichi-2A-ESAT-6 (H3N2) obtained by the authors are given in the attached certificate on the deposit of the strain (No. 148) into the collection of the Museum of Viruses of the Institute of Influenza RAMS.

METHOD FOR PREVENTION OF LUNG TUBERCULOSIS.

Currently, there is a significant deterioration in the epidemiological situation of tuberculosis in Russia and around the world due to unfavorable environmental conditions, negative social factors, the growth of immunodeficiency diseases of various origins and the lack of reliable specific prevention of the disease.

The classic BCG vaccine, which is a live attenuated strain of M. bovis, is effective in preventing tuberculosis in children, but is not able to prevent the reactivation of the disease in adults, which is the cause of most cases of pulmonary tuberculosis (Trends Immunol. 2001; 22: 160-8).

It is known that the protective effect in the development of tuberculosis infection is provided by cytokines produced by type 1 T-helpers (TX-1) - gamma interferon (IFN-γ) interleukin-2 (IL-2), which are mediators of cell-mediated immunity. With an active progressive infection, stimulation of T-2 helper subpopulation of lymphocytes (TX-2) is detected, with a high level of IL-4 production, regarded recently as an indicator of a decrease in resistance to tuberculosis and a decrease in the activity of CD4 ⁺ TX-1.

Among the variety of assumptions that explain the inability of the BCG vaccine to protect the adult population from tuberculosis, the absence of important (protective) antigens in the BCG genome (including ESAT-6), the inability to stimulate the optimal combination of T-lymphocyte subpopulations to create protection, and undesirable stimulation of IL production can be highlighted -four.

In addition, when the BCG vaccine is introduced, the human immune system encounters an extremely complex set of antigens competing for presenting cells, and immunodominant antigens do not always induce maximum protection (J. Immmunol, 1996; 157: 3039-3045) or their expression is transient (Parasite Immunol., 1993; 15: 187-193).

A justified and promising direction in the development of new vaccines against tuberculosis is the formation of a Th1 immune response characteristic of a favorable course and reverse development of the infection, ensuring its high activity and targeted switching to Th1 in the case of Th2 cytokine dominance.

Example 4. The effect of preventive immunization with recombinant influenza vectors on the development and course of acute tuberculosis infection in mice.

The study was conducted on 240 outbred white male mice with an initial body weight of 18-20 g obtained from the Rappolovo nursery, Vsevolozhsk district. Leningrad region. For immunization, virus-containing suspensions of recombinant influenza vectors FLU / ESAT-6, belonging to various antigenic subtypes of influenza A virus (H1N1, H3N2 and H2N2), were used.

Animals were divided into 5 groups. In the first group of mice, they were twice immunized with a mixture of three recombinant influenza vectors of different antigenic subtypes (FLU / ESAT-6 (Tri + Tri)). In the second group, the first immunization was carried out with a recombinant influenza vector of the H1N1 subtype, the second with the influenza vector of the H3N2 variety (FLU / ESAT-6 (H1 + H3)). In the third experimental group, the first immunization was carried out with a recombinant influenza vector of the H1N1 subtype, the second with the influenza vector of the H2N2 variety (FLU / ESAT-6 (H1 + H2)). Immunization was carried out intranasally, without anesthesia, at a dose of 10 ⁶ PFU / mouse, with an interval of 3 weeks. The controls were intact animals and unvaccinated MBT infected mice (infection control).

Generalized tuberculosis was induced by injecting 0.2 ml of a suspension of a three-week culture of M. bovis bovinus 8 into a lateral tail vein of mice at a dose of 0.1 mg / mouse 6 weeks after the first immunization. We used a virulent strain of M. bovis bovinus 8, obtained in the form of a lyophilized culture from the State Research Institute of Standardization and Control of Medical Biological Preparations named after Tarasevich (laboratory of drugs for the prevention and diagnosis of tuberculosis and mycobacteriosis). When white strain of outbred mice is infected with this strain, foci of specific inflammation in the lungs form on days 14-20, and animals die on days 30-45 from the moment of inoculation of the infection, which is close to the corresponding values in mice infected with M. tuberculosis H37Rv (Pershin G. N., 1971).

The effect of the protective effect of the vaccine was evaluated by the following indicators of the severity of the experimental tuberculosis process: dynamics of the mass of mice (weighing 1 time per week), macroscopic evaluation of the lungs and spleen (indices of lung damage, lung and spleen mass ratios), histological examination of lung sections. Comparison with control indicators (intact animals and infection control) was carried out in dynamics 30 and 40 days after infection of the office.

The index of lung lesions was calculated in accordance with the number and severity of foci of specific inflammation detected by visual inspection. Moreover, single submiliary foci were estimated at 0.5 points; numerous submiliary foci (no more than 20) - 1 point; numerous submiliary (more than 20) - 1.5 points; single miliary - in 1.75 points; numerous merging submiliary and single miliary ones - 2 points; miliary (no more than 10) - 2.25 points; numerous miliary, merging - 2.75 points, the appearance of small caseous necrotic tricks - 3 points; extensive caseosis - 4 points; continuous lung damage - 5 points. In cases of serous impregnation of lung tissue, 0.25 to 1 points were added to the calculated lesion index, depending on the area of the lesion.

The mass coefficients of organs were calculated based on the ratio of the mass of the organ and the body mass of the animal (KM = organ mass (g) × 100 / animal mass (g)) and expressed in arbitrary units (conventional units).

For histological examination of the lungs, the organ was fixed in 10% formalin. After fixation and posting, the material was poured into paraffin and step histological sections were made from paraffin blocks. Hematoxylin and eosin stains were used.

The results were subjected to statistical processing, the significance of the differences was evaluated by unpaired t-student test.

An analysis of the results showed that in mice twice immunized with recombinant influenza vectors, the development of experimental tuberculosis infection was slowed down, and the course was more favorable than in infected unvaccinated animals.

On the 30th day after infection, FLU / ESAT-6 vaccinated mice showed a tendency to decrease in the mass coefficients of organs (lungs and spleen) and a statistically significant (p <0.001, p <0.02 and p <0.05) decrease in the indices of damage lungs compared with infected unvaccinated animals, as shown in table 1.

Table 1.
The severity of experimental tuberculosis on the 30th day after infection of the office Experience Conditions The coefficient of mass of the lungs (conventional units) Index of lung lesions (conventional units) Infection control (n = 11) 1.46 ± 0.13 2.25 ± 0.07 FLU / ESAT-6 (H1 + H3) (n = 5) 1.35 ± 0.2 1.6 ± 0.1 p <0.001 FLU / ESAT-6 (Tri + Tri) (n = 5) 1.98 ± 0.48 1.8 ± 0.16 p <0.05 FLU / ESAT-6 (H1 + H2) (n = 5) 1.26 ± 0.11 1.65 ± 0.21 p <0.02 P - significance of differences with unvaccinated animals (infection control) according to the unpaired t-student criterion.

On the 40th day after infection, the protective effect was noted when using three variants of the studied vaccine. The lung mass coefficients, as in the previous observation period, had only a downward trend, and the lung lesion index in the two experimental groups was significantly lower than in the control group (p <0.05). By this time, mice immunized with the trivalent influenza vaccine and the FLU / ESAT-6 vaccine (H1 + H2) also showed a decrease in the spleen mass coefficient (p <0.02), which is shown in Table 2.

Table 2.
The severity of experimental tuberculosis on the 40th day after infection of the office Experience Conditions % increase in body weight by day of slaughter The coefficient of mass of the lungs (conventional units) Index of lung lesions (conventional units) The spleen mass coefficient (conventional unit) Infection control (n = 10) 6.78 2.3 ± 0.27 2.98 ± 0.31 3.17 ± 0.29 FLU / ESAT-6 (H1 + H3) (n = 7) 12.9 1.77 ± 0.2 2.21 ± 0.11 p <0.05 2.6 ± 0.26 FLU / ESAT-6 (Tri + Tri) (n = 7) 35,4 1.69 ± 0.11 2.21 ± 0.11 p <0.05 2.25 ± 0.14 p <0.02 FLU / ESAT-6 (H1 + H2) (n = 7) 18.8 1.82 ± 0.22 2.39 ± 0.11 2.11 ± 0.23 p <0.02 P - significance of differences with unvaccinated animals (infection control) according to the unpaired t-student criterion.

In this case, in the lungs of mice of the control group, multiple, well-defined tuberculous foci, a large number of large confluent necrotic foci were detected, which is reflected in macrographs of the lungs of mice of the compared groups (Fig. 5A). Tuberculous changes in the lungs of vaccinated animals were less pronounced and were represented by single medium-sized tuberculous foci of gray color (Fig. 5 B). It should be noted that by the value of the index of lung lesions by the second control period (40th day after infection) in all groups of animals there was an increase in the prevalence of specific lung lesions, but in vaccinated mice it was less pronounced and delayed in time (Tables 1, 2 )

The study of the dynamics of the mass of mice revealed that by the end of the observation period, the body weight of the vaccinated FLU / ESAT-6 (Tri + Tri) animals corresponded to the data of the mice of the intact group (27.9 g versus 28.4 g) (Figure 6). The percentage of body weight gain in vaccinated mice by day of slaughter (12.9-35.4%) was 1.9 (FLU / ESAT-6 (H1 + H3), 2.8 (FLU / ESAT-6 ( H1 + H2) and 5.2 (FLU / ESAT-6 (Tri + Tri) times, respectively, higher than in the control group (7.8%), which indicates a lower severity of intoxication symptoms in both groups of vaccinated mice.

Histological examination of lung sections on the 40th day after infection showed that all infected animals in the lung tissue had specific infiltrative changes of varying severity with accumulation in the alveoli of serous, and more often fibrinous exudate, large vacuolated macrophages and lymphoid elements (alveolar-macrophage granulomatosis ) In infected unvaccinated mice, they were represented by fields and large confluent foci of infiltration (Table 3). In this case, fresh infiltrative changes prevailed, extending both to the air parenchyma and to the peribronchial and perivascular spaces. Alveoli, interalveolar septa, and lumens of dilated capillaries were infiltrated by clusters of macrophages, epithelioid cells, and lymphoid elements.

Table 3.
The prevalence of specific lung tissue lesions in mice with experimental tuberculosis Groups (number of mice) Decrease (more than 50%) in the airiness of the lung tissue Large foci of infiltration Small areas of infiltration Contamination Control (10) 10 (100%) 10 0 FLU / ESAT-6 (H1 + H3) (7) 1 (14%) 1 (14%) 6 (86%) FLU / ESAT-6 (Tri + Tri) (7) 3 (41%) 3 (41%) 4 (59%) FLU / ESAT-6 (H1 + H2) (7) 1 (14%) 1 (14%) 6 (86%)

In all animals of the control group, accumulations of neutrophilic granulocytes were detected in the lung parenchyma, which in 6 out of 10 cases partially disintegrated and formed nuclear detritus, and in 2 cases, the focus of destruction in the center of the granuloma, which is reflected in the presented micrographs of a histological section of the lung of the mouse control group (Figa).

Table 4.
Peculiarities of the cellular composition of alveolar-macrophage granulomatosis sites in mice with experimental tuberculosis Groups (number of mice) Clusters of epithelioid cells Single neutrophilic granulocytes Accumulations of neutrophilic granulocytes Nuclear detritus Areas of necrosis Contamination Control (10) 10 (100%) 10 (100%) 10 (100%) 6 (60%) 2 (20%) FLU / ESAT-6 (H1 + H3) (7) 7 (100%) 6 (86%) 1 (14%) 0 0 FLU / ESAT-6 (Tri + Tri) (7) 7 (100%) 4 (59%) 0 0 0 FLU / ESAT-6 (H1 + H2) (7) 7 (100%) 4 (59%) 0 0 0

The cell composition of the granuloma was represented by areas of caseous necrosis, nuclear detritus, clusters of decaying neutrophilic granulocytes, macrophages, epithelioid cells, single lymphocytes (Fig. 7B).

In animals with severe necrotic changes, nuclear detritus and desquamated epithelial cells were found in the lumen of the bronchi. Lymphohistiocytic infiltration, indicating the intensity of local immunity of the lung tissue, was very weak, significant perivascular infiltrates were registered only in 4 out of 10 (40%) cases, large peribronchial infiltrates were not observed (Table 5).

Table 5.
The severity of signs of tension of local immunity of lung tissue in mice with experimental tuberculosis Groups (number of mice) Large perivascular infiltrates Large peribronchial infiltrates Contamination Control (10) 4 (40%) 0 FLU / ESAT-6 (H1 + H3) (7) 7 (100%) 4 (59%) FLU / ESAT-6 (Tri + Tri) (7) 7 (100%) 4 (59%) FLU / ESAT-6 (H1 + H2) (7) 7 (100%) 7 (100%)

Compared with the control group of animals, the vaccinated mice of the experimental groups showed an increase in the airiness of the lung tissue, a decrease in the prevalence of specific inflammation, which in most cases (59% -86%) was represented by small individual foci of infiltration (Table 3). In the foci of infiltration, practically no accumulations of neutrophilic granulocytes were detected, nuclear detritus and areas of necrosis were not detected, the occurrence of single non-decaying neutrophils was significantly reduced (Table 4). Only in one case (14%) in mice vaccinated with FLU / ESAT-6 (H1 + H3), small accumulations of neutrophilic granulocytes were noted.

As an illustration (Fig. 8), in a microphotograph of a lung tissue preparation (two magnifications) of one of the vaccinated FLU / ESAT-6 (H1 + H3) mice, a small lesion of alveolar-macrophage granulomatosis with preserved alveoli having somewhat thickened septa and infiltrated lymphocytes is presented (Figa). The cellular composition of the infiltrate is represented by lymphoid elements, macrophages, single epithelioid cells and single non-decaying neutrophils (Figv).

In all experimental animals, more often than in the control group, large perivascular and peribronchial lymphohistiocytic infiltrates were detected, which indicates the activation of local immunity of lung tissue (Table 5).

Thus, double intranasal vaccination of white outbred mice with influenza vectors expressing the protective mycobacterial antigen ESAT-6, carried out 3 weeks before infection of the mice with the virulent strain of M. bovis bovinus 8, delayed the development of experimental tuberculosis infection and its more favorable course compared to a control group of infected unvaccinated animals.

Significant protective effect was noted both with double intranasal administration of a mixture of three recombinant influenza vectors (FLU / ESAT-6 (Tri + Tri), and with sequential intranasal immunization of animals with recombinant influenza vectors related to H1N1 and H3N2, as well as H1N1 and anti-H2N2 the flu virus.

Under the influence of vaccination, a marked decrease in the severity of the course of the infection was observed: favorable body weight dynamics, lower lung mass ratios (p <0.05) and spleen (p <0.02), a decrease in lung injury indices (p <0.001).

An assessment of the morphodynamics of acute tuberculosis infection of white outbred mice revealed a regression of inflammatory changes in the lungs of vaccinated animals, which manifested itself in a decrease in the prevalence of specific inflammation and a decrease in its alterative component, as well as activation of local immunity of lung tissue.

Example 5. Immunogenicity of influenza vectors and the effectiveness of prophylactic immunization with recombinant influenza vectors for sluggish tuberculosis infection in C57 black / 6 mice.

A comparative assessment of the effectiveness of vaccination with recombinant influenza strains FLU / ESAT-6 and standard BCG vaccine for the development and course of sluggish tuberculosis infection was carried out on 165 male C57 black / 6 mice with an initial body weight of 18-20 g, obtained from the Rapolovo nursery, Vsevolozhsk district, Leningrad region.

In order to induce a systemic immune response and immunity of the mucous membranes of mice, one of the experimental groups was successively immunized with influenza vectors belonging to two antigenic subtypes of the influenza virus: (H1N1) and (H3N2). The controls were infected unvaccinated mice (infection control); mice immunized with wild influenza virus A / PR / 8/34 (vaccine control), BCG vaccinated mice and intact animals.

Twice immunization with FLU / ESAT-6 (H1 + H3) and wild flu virus was carried out intranasally, without anesthesia, at a dose of 10 ⁶ PFU / mouse with an interval of 3 weeks. The BCG vaccine was administered once subcutaneously at a dose of 10 ⁵ PFU / mouse on the day of the first immunization.

Generalized tuberculosis was caused by introducing into the lateral tail vein of mice a suspension of a three-week culture of M. bovis bovinus 8 (10 ⁶ CFU / mouse) 6 weeks after the first immunization.

The effect of the protective effect of the studied vaccine was evaluated on the 22nd and 35th days after MBT infection by the following indicators of the severity of the experimental tuberculosis process: MBT inoculation from the lungs and spleen, macroscopic evaluation of the lungs and spleen (lung lesion indices, lung and spleen mass ratios) lethality. The data obtained were compared with similar indicators in the control groups of animals.

During bacteriological examination of the lungs and spleen, metered sowing of organ tissue homogenate was carried out on dense Levenshtein-Jensen egg medium using 10-fold serial dilutions. The growth massiveness of Mycobacterium tuberculosis (MBT) was expressed in decimal logarithms (Log ₁₀ ) of the number of colony forming units (CFU).

An immunological study was carried out 3 weeks after the second immunization (before infection of animals with M. bovis) and in the dynamics of the infection process (on the 15th and 35th days after infection with M. bovis).

Proliferation of spleen lymphocytes and tracheobronchial lymph nodes of mice was evaluated on 96-well plates in a blast transformation reaction upon induction of ConA cells (1 μg / ml) and recombinant ESAT-6 protein (Fusion Antibodies Ltd, Northern Ireland; 5 μg / ml) on a liquid scintillation counter the amount of ³ H-thymidine incorporated into the DNA of proliferating cells. The results for each sample were expressed as absolute values of cell proliferative activity (number of pulses per 1 minute).

The production of IL-4 and IFN-γ in the supernatants of spleen cell cultures and tracheobronchial lymph nodes was determined using the Quantikine ™ enzyme-linked immunosorbent assay systems (R&D Systems, Minneapolis) in accordance with the instructions for use. The level of IFN-γ and IL-4 (PCG / ml) in the studied samples was calculated from the calibration curve of the dependence of the optical density on the concentration of cytokine.

Determination of the level of IL-2 (spontaneous and induced) was determined in primary splenocyte cultures after 24 hours of cell cultivation at 37 ° C in an atmosphere containing 5% CO ₂ according to the level of proliferation of IL-2-dependent cell line CTLL-2 (J.Immunol. 120 (6): 2027-2032; 1978).

Statistical data processing was carried out using the Student's parametric test.

Immunogenicity of recombinant influenza vectors.

In order to induce a systemic immune response, C57black / 6 mice were immunized twice with recombinant strains of influenza A virus expressing an early secretory mycobacterial antigen (ESAT-6). To enhance the immune response, re-immunization was carried out with the influenza vector of another subtype.

The T-cell immune response was determined by measuring the proliferative activity of splenocytes and their ability to produce IFN-γ in response to stimulation with recombinant ESAT-6 protein (rESAT-6). 3 weeks after the second immunization in a suspension of spleen cells of immunized FLU / ESAT-6 (H1 + H3) mice, a significant production of IFN-γ was observed in the amount of 13 ng / ml supernatant, which is shown in Fig.9. However, cytokine production in response to ESAT-6 was not observed in supernatants of spleen cell cultures of mice immunized with wild-type viruses (vaccine control) or BCG vaccine. Thus, double immunization with recombinant influenza vectors belonging to different antigenic subtypes (H1N1, H3N2) and expressing mycobacterial protein ESAT-6 allowed us to obtain a pronounced specific T-cell response to the insert (ESAT-6 antigen) at the systemic level with intranasal administration of the vaccine .

Efficacy of preventive immunization with recombinant influenza vectors for sluggish tuberculosis infection in C57 black / 6 mice.

The effect of immunization with recombinant influenza vectors on the development and course of experimental tuberculosis infection was studied in C57BL / 6 mice, which are the low-response line of mice to Mycobacterium tuberculosis infection. This determined the development of a sluggish tuberculosis infection in this experiment.

The results of the study showed that immunization with FLU / ESAT-6 (H1 + H3) significantly affected the mortality and contamination of mycobacteria in the organs of mice, which are integral indicators of the severity of the infection.

Assessment of MBT inoculation from lungs and spleen on the 22nd day after infection revealed a significant decrease in MBT growth in the group of mice immunized with FLU / ESAT-6, compared with infection control (1.37 log ₁₀ CFU in the lungs; 0.6 log ₁₀ CFU in the spleen), which is shown in table 6. There was no decrease in MBT growth from parenchymal organs using wild influenza virus (vaccine control).

It is noteworthy that the protective effect of FLU / ESAT-6 according to seeding data (protection index) exceeded the effect of the standard BCG vaccine (in the lungs 1.37 log ₁₀ versus 1.17 log ₁₀ ; in the spleen 0.6 log ₁₀ against 0.52 log ₁₀ ), which was confirmed by morphometric data organ assessment at a given follow-up period.

Table 6.
The growth of M. bovis bovinus from parenchymal organs (lungs, spleen) of mice on the 22nd day after infection Group (number of animals) Log ₁₀ CFU in the lungs Protection Index (Log ₁₀ ) Log ₁₀ CFU in the spleen Protection Index (Log ₁₀ ) Infection control (n = 5) 5.146 ± 0.06 5.459 ± 0.08 Vaccine control (PR / 8 wild influenza virus) (n = 5) 5.279 ± 0.19 -0.133 5.491 ± 0.01 -0.077 FLU / ESAT-6 (H1 + H3) (n = 5) 3.778 ± 0.04 * 1.369 4.859 ± 0.12 * 0.60 BCG (n = 5) 3.873 ± 0.12 * 1.173 4.939 ± 0.12 * 0.520 * - significance of differences with unvaccinated animals (infection control) according to the unpaired Student t-test (*, P <0.05).

Significant decrease in lung mass coefficient (0.98 ± 0.06 conventional units vs. 0.83 ± 0.05 conventional units, p <0.05) and a decrease in the index of lung damage (1.75 ± 0.11 conventional units against 1.66 ± 0.09 conventional units) .) compared with the control of infection were only registered in the group of mice immunized with influenza vectors.

A decrease in the severity of the tuberculosis process in the group of animals immunized with FLU / ESAT-6 was also observed on the 35th day after infection. By this time, compared with infected unvaccinated animals, vaccinated mice showed a tendency to a decrease in the mass coefficients of organs (lungs and spleen) and a statistically significant decrease in the index of lung damage (2.4 ± 0.11 conventional units vs. 2.1 ± 0.05 conventional units, p < 0.05).

Analysis of animal survival data showed that in the FLU / ESAT-6 group, the onset of death from tuberculosis was noted on the 59th day after infection, whereas in infected unvaccinated animals and BCG vaccinated with the vaccine it was recorded on 69th and 64th days, respectively (Figure 10). This allows us to talk about a delay in the death of mice of the studied group. At the same time, by 137 days after infection, when the lethality control group reached 100% in the infection control group, 45% remained in the group of immunized FLU / ESAT-6, and 25% of the surviving animals in the group of BCG vaccinated. By the end of the observation period (> 250 days after infection), 10% of the surviving mice remained in the vaccine group inoculated with the test vaccine. By this time, mortality in the group of mice that received BCG vaccine reached 100%. The increase in life expectancy of immunized FLU / ESAT-6 mice compared with the infection control group was statistically significant for the Log Rank test (p <0.0008).

Thus, a significant protective effect of immunization with recombinant influenza vectors for sluggish tuberculosis in C57BL / 6 mice was shown. It is noteworthy that, according to MBT inoculation from parenchymal organs and animal survival, the protective effect of FLU / ESAT-6 exceeded that of the standard BCG vaccine.

Kinetics of the immune response during the development of tuberculosis infection.

An immunological examination of mice revealed marked changes in immunity indicators during the development of the tuberculosis process.

Already on the 15th day after infection, when visual examination of the lungs of infected MBT mice did not yet reveal pronounced foci of tuberculosis, significant spontaneous suppression was recorded in their splenocyte cultures (1327 ± 132 cpm versus 6034 ± 733 cpm, p <0.001), and induced (Con A) proliferative activity of lymphoid cells compared with intact animals (13406 ± 1835 cpm versus 52399 ± 3087 cpm, p <0.01).

Moreover, in vaccinated mice, the level of spontaneous proliferative activity of spleen lymphoid cells was significantly higher than in animals from the infection control group (4106 ± 778 cpm versus 1327 ± 132 cpm, respectively, p <0.02).

In the proliferative response of cells to a specific antigen of ESAT-6, there were no significant differences between vaccinated animals and control of infection, which indicates the formation of a pool of lymphocytes that recognize this antigen and respond in vitro to ESAT-6 stimulation with protein by enhancing the mitotic activity of cells at an early stage of tuberculosis infection (Figa).

By the 35th day after infection, the degree of inhibition of proliferation (both spontaneous and ConA-induced) in infected unvaccinated mice increased even more and the significance of differences with the intact group reached <0.001 according to the unpaired student t-criterion.

In the proliferative response of cells to a specific antigen ESAT-6, significant stimulation (p <0.002) of mitotic activity of splenocytes of vaccinated animals was noted compared with the infection control (13185 ± 1720 cpm versus 2223 ± 1080 cpm) (Fig. 11B).

Thus, in the dynamics of the infectious process, the presence of an antigen-specific proliferative response of splenocytes was characteristic of relatively early infection (15 days from infection); at a later date (35 days from infection) due to the development of immunosuppression due to an increase in the tuberculosis process, the ability of splenocytes of infected unvaccinated animals to respond to ESAT-6 decreased.

When assessing the production of IFN-γ by splenocytes on the 15th day of infection in all infected animals, a significant decrease in the spontaneous level of cytokine production and an increase in Con A and ESAT-6 induced (p <0.001) were noted. The data on increasing the level of induced IFN-γ production at the first stage of the examination are consistent with the literature, where a high level of IFN-γ production by T-lymphocytes in the early stages of tuberculosis development is noted. Moreover, the differences between the groups were unreliable.

On day 35 after infection, significant stimulation of Con A-induced IFN-γ production was observed in supernatants of spleen cell cultures in a group of mice vaccinated with FLU / ESAT-6 when compared with infected unvaccinated animals in which cytokine production was significantly inhibited when compared with the intact group (p <0.002).

On day 35 after infection by induction of recESAT-6 splenocytes (5μg / ml), the level of IFN-γ secretion in all vaccinated animals was significantly higher than in infected unvaccinated mice (Fig. 12A), with a maximum cytokine production (20.1 ng / ml supernatant) was determined in the FLU / ESAT-6 group of immunized mice with a cytokine production level of 4.1 ng / ml in the infection control group.

The greatest differences in IFN-γ production in response to rESAT at this time were noted in the supernatants of lymphocyte cultures of tracheobronchial lymph nodes. Significant production of IFN-γ in an amount of 7.5 ng / ml supernatant was observed in immunized FLU / ESAT-6 (H1 + H3) mice, which is shown in Figv. Moreover, IFN-γ production in supernatants of mouse lymphatic cell cultures immunized with wild-type viruses (vaccine control) or BCG vaccine in response to ESAT-6 was significantly lower (p <0.001), and no cytokine production was observed in the control group.

The decrease in the expression of the inhibition of the Th-1 response of lymphocytes accompanying the development of tuberculosis infection as a result of vaccination with FLU / ESAT-6 (H1 + H3) is also confirmed by the results obtained by evaluating the production of IL-2 in supernatants of splenocyte cultures. Moreover, on the 15th day after infection, when the inhibition of cytokine production in response to Con A in infected unvaccinated mice relative to intact mice was still unreliable, the increase in this indicator in mice vaccinated with FLU / ESAT-6 (H1 + H3) was insignificant. On day 35, when inhibition of IL-2 secretion in the control group became significant (p <0.01), IL-2 production in response to Con A in the group of mice vaccinated with FLU / ESAT-6 was at the level of intact animals. Moreover, the calculation of IL-2 production in% of the level of the intact group showed that while the infection control mice with the development of infection on the 22nd and 35th days after infection, a decrease in the level of cytokine secretion was observed (72.5% and 57.8%), this decrease was not registered in vaccinees (77.5% and 82.1%, respectively).

Immunization with FLU / ESAT-6 also prevented the inhibition of IL-2 production in response to the specific recESAT 6 antigen observed in the natural course of infection by 35 days after infection. In vaccinated mice, in response to recESAT-6, IL-2 production was significantly higher than the level of infection control (p <0.001).

On the production of IL-4 splenocytes, the effect of immunization with FLU / ESAT-6 was detected 15 days after infection against the background of increased cytokine production in all infected animals. It manifested itself in a significant decrease in the level of IL-4 secretion in response to Con A in immunized mice compared with the control group of animals (88.9 ± 1.9 pg / ml versus 141.8 ± 10.9 pg / ml supernatant).

The noted shifts in the production of IFN-γ and IL-4 are confirmed by changes in the IFN-γ / IL-4 index on the 15th day of tuberculosis infection. In the culture of splenocytes during Con A induction in infected unvaccinated animals, the index was significantly reduced (p <0.02), while during vaccination with FLU / ESAT-6 (H1 + H3) it was at the level of the intact group. The highest IFN-γ / IL-4 index in this group of mice was also noted in response to recESAT-6.

Thus, an immunological study during the development of the tuberculosis process showed that immunization with influenza vectors expressing the ESAT-6 antigen elicited an antigen-specific cell response that modified the Th-1 immune response to infection in MBT mice.

Under the influence of vaccination, the following were noted: in the early stages of infection development - a significant decrease in the increased level of IL-4 secretion and restoration of the IFN-γ / IL-4 index on Con A in splenocyte culture; in later, against the background of marked inhibition of Th-1 lymphocytes in unvaccinated infected mice, stimulation of spontaneous and induced (Con A and recESAT-6) IFN-γ production by splenocytes, increase in their proliferation level (spontaneous and in response to rESAT 6), activation recESAT-6-induced splenocyte production of IL-2. The most significant stimulation of IFN-γ secretion in response to recESAT-6 was observed in the tracheobronchial lymph nodes of immunized FLU / ESAT-6 animals.

Conclusion Immunization with influenza vectors expressing ESAT-6 protein, carried out 3 weeks before infection with C57BL / 6 mice M. bovis, led to the development of a pronounced antigen specific cell response and provided a significant protective effect in experimental sluggish tuberculosis infection. It is noteworthy that, according to MBT inoculation from parenchymal organs and animal survival, the protective effect of FLU / ESAT-6 exceeded that of the standard BCG vaccine.

The prophylactic effect is apparently implemented due to the directed correction of the pronounced inhibition of Tx-1 established by us during this course of the tuberculosis process with a significant decrease in the production of cytokines produced by type 1 T-helpers (TX-1) - gamma interferon (IFN-γ ), interleukin-2 (IL-2), which are mediators of cell-mediated immunity and determine the favorable course and reverse development of infection.

Example 6. Preventive effect of recombinant influenza vectors in tuberculosis infection in guinea pigs.

A comparative study of the protective effect of recombinant influenza vectors (FLU / ESAT-6) and the standard BCG vaccine for tuberculosis was carried out in an experiment on guinea pigs, which, due to their high susceptibility to infection, are considered the most suitable animals for experiments on the treatment and prevention of tuberculosis. In this experiment, the effectiveness of preventive immunization in the intranasal and subcutaneous routes of administration of the influenza vaccine was evaluated.

The study was conducted on 16 male guinea pigs with an initial body weight of 250-300 g, obtained from the nursery "Rappolovo", Vsevolozhsk district, Leningrad region. Animals were divided into 4 groups: immunized with a flu vaccine administered intranasally; immunized with a subcutaneous flu vaccine; BCG vaccinated unvaccinated MBT infected animals (infection control). Animals were kept in separate cages (4 guinea pigs per cage) and had free access to water and food.

Twice immunization with FLU / ESAT-6 was carried out without anesthesia, at a dose of 10 ⁶ PFU / guinea pig with an interval of 3 weeks. For immunization, a virus-containing suspension of a mixture of recombinant influenza vectors belonging to various antigenic subtypes of influenza A virus (H1N1, H3N2 and H2N2) was used. The BCG vaccine was administered once subcutaneously in the left inguinal region at a dose of 10 ⁴ CFU / guinea pig on the day of the first immunization.

Generalized tuberculosis was caused by subcutaneous injection of 10 CFU of M. Tuberculosis H37Rv into the right inguinal region of animals 6 weeks after the first immunization.

The impact of immunization on the development and course of tuberculosis infection was assessed 9 weeks after infection with M. tuberculosis H37Rv, when one of the guinea pigs from the infection control group died from generalized tuberculosis infection.

The effect of the protective effect of the vaccine was evaluated by the following indicators of the severity of experimental tuberculosis: the degree of damage to the internal organs and lymph nodes (indexes of organ damage); changes in body weights; seeding MBT from parenchymal organs (lungs, spleen).

During bacteriological examination of the lungs and spleen, metered sowing of organ tissue homogenate was carried out on dense Levenshtein-Jensen egg medium using 10-fold serial dilutions. MBT growth mass was expressed in decimal logarithms (Log ₁₀ ) of the number of colony forming units (CFU).

The value of the total lesion index was calculated according to the results of a visual assessment of the damage to organs of guinea pigs by the tuberculosis process (Pershin GN, “Methods of experimental chemotherapy (practical guide)” - M., 1971, p. 182). It is the sum of the indices of lesions of the lungs, spleen, liver and inguinal lymph nodes (right and left). In each organ, single submiliary foci were estimated at 0.5 points; single miliary - 1 point; numerous merging submiliary or up to 20 miliary - 2 points; numerous merging miliary or up to 20 caseous necrotic tricks - 3 points; more than 20 caseous necrotic tricks or large areas of necrosis - 4 points. The maximum value of the lesion index for each organ was 4 points, and the total index was 20 points. The results were processed using the nonparametric Wilcoxon-Mann-Whitney test.

The coefficients of mass of the lungs and spleen were calculated based on the ratio of the total weight of the animal and the mass of the organ and expressed in arbitrary units (conventional units). The significance of differences was assessed by the unpaired student t-test.

For histological examination of the lungs, the organ was fixed in 10% formalin. After fixation and posting, the material was poured into paraffin and step histological sections were made from paraffin blocks. Hematoxylin and eosin stains were used.

As the results of the study showed, 9 weeks after infection with M. Tuberculosis H37Rv, one guinea pig from the infection control group died from generalized tuberculosis infection. By this time, all animals in the experimental groups were alive. A visual assessment of the prevalence of tuberculosis in guinea pig organs (lungs, liver, spleen and inguinal lymph nodes) by this time showed a decrease in the total index of animal damage in all vaccinated groups (Table 7).

Table 7.
The severity of experimental tuberculosis in guinea pigs 9 weeks after infection with M. tuberculosis H37 Rv Groups (number of animals) Lung lesion index Spleen lesion index Liver damage index Right inguinal l lesion index Left inguinal lesion index Total lesion index Contamination control (n = 3) 4 (4-4) 4 (4-4) 1.5 (1-2) 4 (4-4) 4 (4-4) 17.5 (17-18) BCG vaccinated (n = 4) 1.2 * (0.5-2) 1.17 * (0.5-2) 0.83 (0-2) 4 (4-4) 1.33 (0-4) 8.5 * (5-10.5) FLU / ESAT-6 I.N. (n = 4) 2.0 * (2-2) 1.0 * (1-1) 1.5 (1-2) 4 (4-4) 0 * 8.5 * (8-9) FLU / ESAT-6 p.k. (n = 4) 1.3 * (0.5-3) 0.66 * (0-1) 0.5 * (0-1) 4 (4-4) 0 * 6.53 * (4.5-9) The results were processed using the nonparametric Wilcoxon-Mann-Whitney test (* p <0.05).

It is noteworthy that the lowest total index of organ damage was observed in the group of animals immunized subcutaneously with FLU / ESAT-6 (6.53 points at 8.5 points for BCG vaccinated and intranasally immunized with influenza vaccine), which was associated with a lesser degree of damage to the spleen and liver, the specific lesion of which was represented by single submiliary foci, and the absence of foci of specific inflammation in the collateral inguinal lymph nodes in animals of this group.

In parallel, a biometric assessment of organs revealed a significant decrease in lung mass ratios in all vaccinated animals compared with infection control, while the spleen mass coefficient was significantly lower only in the group of guinea pigs immunized subcutaneously with the FLU / ESAT-6 vaccine. At the same time, in the groups vaccinated with BCG and intranasally immunized with FLU / ESAT-6, the spleen mass coefficients had only a tendency to decrease (Table 8).

Table 8.
Body mass ratios (lungs, spleen) of guinea pigs 9 weeks after infection with M. tuberculosis H37Rv Groups Lung mass ratio Spleen mass coefficient Infection control 1.35 ± 0.03 1.6 ± 0.02 BCG vaccinated 0.73 ± 0.12 ** 0.64 ± 0.27 FLU / ESAT-6 (I.N.) 0.97 ± 0.05 * 0.66 ± 0.27 FLU / ESAT-6 (s.c.) 0.83 ± 0.11 ** 0.58 ± 0.26 * * - significance of differences with unvaccinated animals (infection control) according to the unpaired Student t-test (*, P <0.05; **, P <0.02).

Assessment of MBT inoculation from lungs and spleen 9 weeks after infection revealed a significant decrease in MBT growth in vaccinated animal groups compared with infection control (Table 9). Moreover, the protection index was higher in the groups immunized with BCG vaccine and the influenza vaccine with its subcutaneous administration compared with the group of animals immunized intranasally. These results were confirmed by the analysis of histological sections of the lungs of guinea pigs (presented below).

Table 9.
Inoculation of MBT from parenchymal organs (lungs, spleen) of guinea pigs 9 weeks after infection with M. tuberculosis H37Rv Groups Log ₁₀ number of viable bacteria in the lungs Protection Index (Log ₁₀ ) Log ₁₀ number of viable bacteria in the spleen Protection Index (Log ₁₀ ) Infection control 6.18 (6.13-6.23) 6.32 (6.23-6.4) BCG vaccinated 4.98 * (4.72-5.28) 1.2 4.93 * (4.34-5.37) 1.39 FLU / ESAT-6 (I.N.) 5.23 * (5.11-5.35) 0.95 5.35 * (4.63-6.08) 0.97 FLU / ESAT-6 (s.c.) 5.24 * (4.79-5.8) 0.94 5.03 * (4.43-5.64) 1.29 The results were processed using the nonparametric Wilcoxon-Mann-Whitney test (* p <0.05).

In the lung tissue of infected unvaccinated guinea pigs, specific changes were presented in the form of fused tubercles with foci of caseous necrosis in the center and zones of macrophage-alveolar granulomatosis perifocally (Figa). Infiltrative changes very significantly reduced the airiness of the lung tissue and spread to both the lung parenchyma and the peribronchial and perivascular spaces. In the lumen of the bronchi found clusters of desquamated epithelial cells, necrotic masses and individual lymphoid elements. Lymphohistiocytic infiltration is not marked.

Intranasal administration of FLU / ESAT-6 had little effect on the prevalence of specific pneumonia. Only in two of four cases, perivascular and peribronchially located small foci of macrophage-alveolar infiltration were observed, which did not have accumulations of nuclear detritus and areas of necrotic lung tissue. In two other cases, the merging tubercles and infiltrative changes were of the same nature as in the control of infection. However, large perivascular lymphohistiocytic infiltrates were found in preparations of this group, which is one of the signs of activation of local immunity of the lung tissue.

In guinea pigs vaccinated with BCG and FLU / ESAT-6 (with subcutaneous administration), as in experiments with mice, a decrease in the prevalence of lung tissue lesion and an increase in its airiness was noted. The foci of specific inflammation were represented by foci of macrophage-alveolar granulomatosis, they were small, diffuse, without a pronounced local structure (Fig.13B). The alternative component of inflammation in them was much less common than in unvaccinated animals. In two of the four guinea pigs in both groups, clusters of alveolar macrophages, histiocytes, and epithelioid cells were found in the lesions of the alveoli and interalveolar septa in the lesion foci; in one case, individual neutrophilic granulocytes were found in the focus of infiltration, and only one of the four guinea pigs had a histological section lung tissue was found accumulation of nuclear detritus. Large perivascular and peribronchial lymphohistiocytic infiltrates were noted in all vaccinated animals, which indicates the activation of local immunity of lung tissue.

Thus, double immunization of guinea pigs with a mixture of three antigenically different recombinant influenza vectors, carried out 6 weeks before infection with M. Tuberculosis H37Rv, led to a more favorable course of experimental tuberculosis infection compared with the control group of infected unvaccinated animals. Under the influence of vaccination, a decrease in the severity of the course of the infection was observed, which was reflected in a significant decrease in the indexes of organ and lymph node damage in vaccinated animals compared with the infection control (p <0.05), the absence of mortality in the experimental groups, and lower weight ratios. Evaluation of the morphodynamics of tuberculosis infection revealed a regression of inflammatory changes in the lungs of vaccinated guinea pigs, which manifested itself in a decrease in the prevalence of specific inflammation and a decrease in its alterative component, as well as activation of local immunity of lung tissue.

An unexpected fact was the advantage of the subcutaneous route of administration of the influenza vaccine over the intranasal, which may be due to the reduced susceptibility of guinea pigs to infection with the influenza infection (Med. Microbiol. Immunol. (Berl.) 1979; 165: 241-54).

It is noteworthy that, according to a number of indicators, the protective effect of the studied vaccine when administered subcutaneously exceeded the protective effect of the standard BCG vaccine, which was reflected in lower indexes of organ and lymph node damage and better weight ratios in the group of subcutaneously immunized FLU / ESAT-6 animals.

METHOD FOR TREATING LUNG TUBERCULOSIS.

Currently, new approaches to the treatment of tuberculosis using therapeutic vaccines are being sought. It is expected that the use of a therapeutic vaccine will make it possible to reduce the duration of the use of tuberculostatic drugs, which will delay the appearance of mutant forms of mycobacterium tuberculosis that are resistant to complex antibiotic therapy and achieve a break in many-month treatment courses. The goal of therapeutic vaccination for tuberculosis is, first of all, to increase the reliability and effectiveness of the applied etiotropic drug therapy.

A comparative study of the results of the use of recombinant influenza vectors and BCG vaccine in the treatment of tuberculosis infection was carried out.

Evaluation of the therapeutic effect of FLU / ESAT-6 on the course of an experimental tuberculosis infection.

The study of the therapeutic effect of FLU / ESAT-6 monotherapy on the course of the tuberculosis process was carried out on 50 male mice of the C57black / 6 line, weighing 16-18 g, obtained from the Rappolovo nursery, Vsevolozhsk district. Leningrad region.

We used a model of sluggish generalized tuberculosis caused by inoculation into the tail lateral vein of M.Erdman culture at a dose of 10 ⁴ CFU / mouse. The sequential immunization of FLU / ESAT-6 with influenza vectors related to the antigenic subtypes of the influenza virus (H1N1) and (H3N2) was carried out twice (subcutaneously or intranasally), without anesthesia, with a three-week break from the 15th day after infection, against the background of detection on autopsy of infected mice with multiple submiliary foci of specific inflammation. The therapeutic effect of FLU / ESAT-6 was compared with the action of the BCG vaccine (10 ⁶ CFU / mouse), which was administered once subcutaneously on the day of the first immunization of FLU / ESAT-6.

The therapeutic effect of FLU / ESAT-6 during etiotropic therapy was evaluated on 100 male C57BL / 6 mice when simulating classical experimental tuberculosis caused by the introduction of 10 ⁶ CFU / mouse culture of M.Erdman into the tail lateral vein. Immunization of FLU / ESAT-6 with vaccine control was carried out twice (subcutaneously or intranasally), with a three-week break from the 11th day after infection, against the background of the detection of multiple submiliary foci at the test autopsy of infected mice and bacteriological control of MBT contamination. BCG vaccination (10 ⁶ CFU / mouse) was administered once subcutaneously on the day of the first administration of FLU / ESAT-6. Etiotropic treatment (isoniazid 10 mg / kg daily, sc) started from the first immunization with FLU / ESAT-6.

The effect of the therapeutic effect was evaluated by the following indicators of the severity of the experimental tuberculosis process: mortality, dynamics of the mass of mice, macroscopic evaluation of the lungs and spleen (index of lung damage, mass coefficients of the lungs and spleen), histological examination of lung sections, seeding of MBT from the lungs and spleen.

For histological examination of the lungs, the organ was fixed in 10% formalin. After fixation and posting, the material was poured into paraffin and step histological sections were made from paraffin blocks. Hematoxylin and eosin stains were used.

During bacteriological examination of the spleen, metered sowing of organ tissue homogenate on a dense Levenshtein-Jensen egg medium was carried out by serial dilution. MBT growth mass was expressed in decimal Log ₁₀ of the number of CFU.

To assess phagocytic activity, a cell suspension of peritoneal macrophages (PMF) was obtained by washing the abdominal cavity with 199 medium (Biolot) with 10% cattle serum and 5 units / ml heparin. Cells (1 · 10 ⁶ cells / ml) were incubated at 37 ° C in disposable Petri dishes (1 hour in an atmosphere containing 5% CO ₂ ). After removal of non-adherent cells, a suspension of yeast of the genus Saccharomyces cerevisiae (1 · 10 ⁷ yeast per dish) was added to the PMF monolayer, pre-opsonized with mouse serum, incubated for 1 and 2.5 hours, washed twice, fixed with 70 ° ethanol, stained with hematoxylin and eosin . The calculation was carried out at 80x magnification. According to the data obtained, the following indicators were calculated: phagocytic activity Mf (FA) - percentage of Mf involved in phagocytosis; phagocytic number (PS) - the average number of yeast absorbed by one phagocytic cell; completeness of phagocytosis (PZF) - the number of yeast digested by macrophages in 1.5 hours of cultivation; phagocytosis completion index (ISF) is the ratio of the phagocytic number for 1 hour of cultivation to the phagocytic number for 2.5 hours of cultivation.

Example 7. The effect of therapeutic vaccination FLU / ESAT-6 on the course of experimental tuberculosis infection.

The therapeutic effect of FLU / ESAT-6 monotherapy was evaluated using a low infectious dose of M.Erdman, which caused the development of a sluggish tuberculosis infection. The appearance of foci of specific inflammation in this experiment was recorded on the 15th day after infection, the beginning of death in the infection control group - on the 19th day after inoculation of the infection.

As the results of the study showed, the introduction of FLU / ESAT-6 in monotherapy mode had a positive effect on the mortality rate of mice. By the end of the observation period (71 days after infection) in the group of untreated animals (column 10) and in group 5 (BCG), 3 out of 8 mice died from a generalized tuberculosis process. With the introduction of FLU / ESAT-6 in the subcutaneous mode (8 mice), death was not observed, with intranasal administration, one mouse out of 8 died.

Evaluation of the dynamics of the mass of mice (Fig) revealed a tendency to decrease the severity of the course of infection when using FLU / ESAT-6. Thus, the percentage of body weight gain by the end of the observation period in immunized FLU / ESAT-6 mice was 24.5% with subcutaneous administration of the vaccine, 18.3% with intranasal and 35% with BCG vaccine. In the group of infected untreated animals, there was practically no increase in body weight by this time (2%), which indicates the severity of intoxication symptoms in this group of animals.

Histological examination of lung sections performed 10 weeks after MBT infection (8 weeks from the start of FLU / ESAT-6 therapy) in three surviving untreated mice in the pulmonary parenchyma revealed a large number of fresh infiltrative changes perivascular and peribronchial. The foci of infiltration were mostly medium-sized, merging in places, with blurred borders without a clear spatial orientation of the cells. The alveoli and interalveolar septa in them are filled with serous and fibrinous exudate and loosely infiltrated by lymphocytes, large vacuolated macrophages, epithelioid cells and neutrophilic granulocytes. At the same time, granulomas were also found in the lung tissue - dense foci of infiltration with clear boundaries, where lymphocytes were concentrically located around very large clusters of epithelioid cells. A large number of clusters of neutrophilic leukocytes was noted in the infiltrates, in one of them the cells partially disintegrated and formed nuclear detritus. Perivascular and peribronchial infiltrates were very mild.

Treatment with BCG vaccine mice increased the incidence of lung tissue lesions. In all animals, foci of infiltration were very common, had a confluent character and included, along with cells of the lymphocyte-macrophage series, neutrophilic leukocytes and their accumulations, which in 3 out of 5 cases decay to nuclear detritus. Granulomas, like untreated mice, consisted of a large number of epithelioid cells and contained neutrophilic white blood cells and their clusters.

Monotherapy FLU / ESAT-6 reduced the lesion of the lung tissue both with subcutaneous (column 3A) and with intranasal administration (column 3B). The lungs of mice in these groups were more airy, in 1 of 4 cases in gr. 3A and in 2 of 4 cases in column 3B, there were only small foci of infiltration located around the bronchi and blood vessels. Granulomas consisted mainly of epithelioid cells. Under the influence of FLU / ESAT-6 monotherapy in the foci of infiltration, a decrease in the severity of the alternative component of inflammation was observed. In mice c. 3A (subcutaneous administration of the vaccine) neutrophilic granulocytes in the foci of infiltration were detected in 2 out of 4 cases, their accumulations - in only one case. In gr. 3B (intranasal administration of the vaccine), the signs of alteration were more significant, but their frequency was lower than in the control of infection: single neutrophilic leukocytes were detected in 3 out of 4 mice, neutrophil accumulations in 2 animals, the formation of nuclear detritus and foci of caseosis in one the mouse. Lymphohistiocytic infiltration was noted, however, large perivascular and peribronchial infiltrates were not recorded.

Thus, the introduction of FLU / ESAT-6 in monotherapy regimen both subcutaneously and intranasally revealed the therapeutic effect of immunization of animals on the course of the tuberculosis process according to a decrease in mortality and led to a decrease in the severity of the alternative component of inflammation during histological examination of the lungs. The use of BCG vaccine in a therapeutic regimen compared with infection control led to an increase in the prevalence of specific lung tissue lesions.

Example 8. The effectiveness of therapeutic vaccination FLU / ESAT-6, conducted against the background of etiotropic therapy, during experimental tuberculosis.

The effect of including FLU / ESAT-6 in the complex therapy of experimental tuberculosis was evaluated on the model of the classical course of tuberculosis infection in mice. On test autopsies of untreated mice that were carried out every two to three days (from the 7th day after inoculation of the infect), macroscopically noticeable single submiliary foci of specific inflammation were detected from the 9th day, and multiple submiliary foci from the 11th day after infection. The death of untreated mice was recorded from the 17th day after infection.

The results of immunization with FLU / ESAT-6 under these conditions with subcutaneous (column 4A) and intranasal administration (column 4B) were compared with the data of the treatment control group (column 1), vaccine control groups (subcutaneous - column 7A, intranasal - column .7 B), infection control (column 10) and groups of mice immunized with BCG vaccine (column 6). All mice of the experimental groups, with the exception of infection control (column 10), received isoniazid in an average therapeutic dose (10 mg / kg, daily, subcutaneously).

Analysis of the severity of the infection revealed a clear effect of FLU / ESAT-6 on the value of the index of lung damage and the seeding rate of the office.

Table 10.
The severity of experimental tuberculosis after 12 weeks from the start of complex therapy using FLU / ESAT-6 No. gr. Experience Conditions Coef. lung masses (conventional units) Index of lung lesions (conventional units) Coef. spleen mass (conventional unit) 10 Infection control 2.39 ± 0.1 2.25 ± 0 0.87 ± 0.11 one PTP 1.25 ± 0.1 2.09 ± 0.03 0.39 ± 0.04 4A FLU / ESAT-6 (s / c) + PTP 1.35 ± 0.13 1.89 ± 0.06
P _1-4a <0.01 0.41 ± 0.03 4 V FLU / ESAT-6 (i / n) + PTP 1.27 ± 0.12 1.94 ± 0.03 p _1-4v <0.002 0.43 ± 0.04 6 BCG + PTP 1.6 ± 0.18 1.97 ± 0.33 0.5 ± 0.06 7A Vaccine control (s / c) + PTP 1.46 ± 0.22 1.95 ± 0.11 0.44 ± 0.02 7 V Vaccine control (i / n) + anti-TB drugs 1.21 ± 0.06 1.81 ± 0.07
P _1-7V <0.01 0.46 ± 0.03 PTP is an anti-TB drug.

On the growth of M. Erdman in the spleen of mice, immunization with FLU / ESAT-6 had a significant effect after 6 weeks from the start of treatment. Moreover, the protection index in gr was the highest. 4A for subcutaneous use of the vaccine (2.32 Log ₁₀ ). A slight increase was also observed in the corresponding vaccine control group (0.47 Log ₁₀ ), which is shown in Table 11.

Table 11.
The growth of M. Erdman in the lungs of mice treated with FLU / ESAT-6 as part of complex therapy No. gr. Experience Conditions 6 weeks from the start of therapy 12 weeks from the start of therapy Log ₁₀ CFU / spleen Protection index Log ₁₀ CFU / lung Protection index 10 Infection control - 5.505 one PTP 2.32 2.851 2.654 _(1-10) 4A FLU / ESAT-6 (s.k.) + PTP 0 2.32 0 2.851 _(4A-1) 4 V FLU / ESAT-6 (in.n.) + PTP 2.74 0.42 0 2.851 ₍ 4v _-1) 6 BCG + PTP 2.45 -0.13 4.299 -1.448 (6-1) 7A Vaccine control (PC) + PTP 1.85 0.47 2.32 0.531 (7A-1) PTP is an anti-TB drug.

In the second control period (12 weeks from the start of treatment), M. Erdman growth in the lungs was not observed in both main experimental groups (protection index was 2.851 Log ₁₀ ). In the control of the vaccine with subcutaneous administration (gr. 7A), a protective effect was also noted, although the protection index was slightly lower and amounted to 0.531 Log ₁₀ .

Histological examination of lung sections performed 10 weeks after infection (8 weeks from the start of FLU / ESAT-6 therapy) showed that etiotropic therapy had a pronounced effect on the prevalence of specific inflammation, which had lost its confluent character and was present in all mice treated with anti-tuberculosis drugs medium-sized foci of infiltration.

When using only specific therapy (treatment control, column 1) in the infiltrates, consisting of lymphocytes, macrophages, epithelial cells and their clusters, distinct signs of the alterative nature of the inflammatory process were recorded: in 3 out of 6 cases, accumulations of neutrophilic granulocytes were recorded, in two mice accumulations of nuclear detritus, in one - foci of caseous necrosis. Numerous granulomas in the lungs of all mice of this group contained large accumulations of epithelioid cells, that is, they had a predominantly epithelioid-cell character.

The administration of BCG vaccine to mice during etiotropic therapy (column 6) practically did not reduce specific inflammation in the lung tissue. As in the treatment control group, neutrophilic granulocytes (in 4 of 6 cases), their accumulations (in 3 of 6), and nuclear detritus sites (in 1 case) were noted in the foci of infiltration. Granulomas were also primarily epithelioid-cell in nature.

In mice treated with FLU / ESAT-6 (subcutaneously, gr. 4A; intranasally, gr. 4B), neutrophilic granulocytes, nuclear detritus, and necrotic foci were not found in the composition of the infiltrates. Granulomas in these groups of mice also had their own characteristics: they consisted mainly of lymphocytes, in 4 of 6 animals concentrically located around single (two three) epithelioid cells, and only in two cases around their small clusters. Therefore, we can talk about the predominantly lymphoid nature of granulomas.

In the groups receiving vaccine control against the background of anti-tuberculosis therapy (gr. 7A and gr. 7B), the direction of the changes was similar to the main experimental group, but the severity of the shifts was less: the alteration indicators were neutrophilic granulocytes, their accumulations and areas of nuclear detritus were recorded in one of the mice; the incidence rate of predominantly lymphoid granulomas is lower (column 7A in 2 out of 5 cases, column 7B in 2 out of 4 cases).

The effect of immunization FLU / ESAT-6 on the phagocytic activity of peritoneal macrophages.

An analysis of the phagocytosis of PMF showed that infected untreated mice (column 10), against the background of widespread tuberculous lesion of the lung tissue (53rd day after infection), develop significant inhibition of both the absorption and digestion capacity of MF according to all studied parameters. The phagocytic activity decreased on average to 32.3% against 54.3% in intact mice (p <0.05), the phagocytic number to 4.14 yeast cells versus 6.39 yeast cells (p <0.05) , PZP - up to 30.0 yeast cells versus 217.67 yeast cells (p <0.05), phagocytosis completion index - up to 1.03 arb. against 1.65 conventional units (p <0.05).

The treatment of mice with anti-TB drugs (treatment control, column 1) after 6 weeks from the start of treatment led to a significant increase in 3 of the 4 studied parameters: phagocytic number, PZP and IZF, without restoring only the PZP value to the level of the intact group (157 , 0 yeast cells versus 217.67 yeast cells).

The use of FLU / ESAT-6 as a part of complex therapy with intranasal administration of the drug (column 4B) and BCG vaccine (column 6) led to a complete normalization of phagocytosis activity. Subcutaneous administration of FLU / ESAT-6 (gr. 4A) at this time had a less pronounced effect, slightly increasing only the completion index of phagocytosis compared to infection control (up to 1.94 conventional units against 1.59 conventional units).

Some inhibitory effect on the absorptive function of phagocytes was observed in group 7B (intranasal administration of the vaccine control), where compared with gr. 1, significantly reduced PZP (to 83.4 yeast cells versus 157.0 yeast cells, p <0.05) and IZP (to 1.12 conventional units versus 1.59 conventional units, p <0.05 )

After 12 weeks from the start of treatment, the activity of phagocytosis of peritoneal MF in the treatment control group fully recovered to the level of intact animals. Under these conditions, the vaccines used in all variants of their use stimulated both the absorption and digestion ability of Mf. So, in mice treated with FLU / ESAT-6, there was a significant increase above the data of the intact group of phagocytic activity, phagocytic number and completion rate of phagocytosis. Compared to gr. control of treatment (column 1), the introduction of FLU / ESAT-6 intranasally (column 4B) significantly increased the phagocytic number, and with subcutaneous administration of the vaccine, there was a significant increase in both the phagocytic number and the rate of completion of phagocytosis.

Thus, the use of anti-tuberculosis drugs FLU / ESAT-6 led to a decrease in the prevalence of tuberculous inflammation in the lungs by the lesion index, to sanitation of the lung tissue by M. Erdman, as well as to the complete disappearance of the alternative component of inflammation and a change in the nature of the cellular composition of granulomas with epithelioid on predominantly lymphoid.

The use of FLU / ESAT-6 as part of complex therapy led to the stimulation of phagocytosis activity of cell suspension of yeast by peritoneal macrophages. During the suppression of phagocytosis reactions, FLU / ESAT-6 accelerated their recovery; during the normalization of this function, it increased it to a level significantly higher than in intact mice.

The activating effect of FLU / ESAT-6 on the functional state of Mf is probably the result of its stimulating effect on the differentiation and functional activity of Th-1, which, producing IFN-γ, realize one of the most important ways of Mf activation during the development of tuberculosis infection.

Claims (7)

1. Live attenuated recombinant strain of influenza A virus Influenza virus A (H1N1), containing an insert of the nucleotide sequence of ESAT-6 M.tuberculosis between positions 400-401 in the open reading frame of the NS1 gene, deposited in the collection of the Museum of viruses of the Research Institute of influenza RAMS under No. 147, intended for the prevention and treatment of tuberculosis. 2. Live attenuated recombinant strain of influenza A virus Influenza virus A (H2N2), containing an insert of the nucleotide sequence of ESAT-6 M.tuberculosis between positions 400-401 in the open reading frame of the NS1 gene, deposited in the collection of the Museum of viruses of the Research Institute of influenza RAMS under No. 149, intended for the prevention and treatment of tuberculosis. 3. Live attenuated recombinant strain of influenza A virus Influenza virus A (H3N2), containing an insert of the nucleotide sequence of ESAT-6 M. tuberculosis between positions 400-401 in the open reading frame of the NS1 gene, deposited in the collection of the Museum of viruses of the Institute of influenza RAMS under No. 148, intended for the prevention and treatment of tuberculosis. 4. A method for the prevention of tuberculosis infection by vaccination, characterized in that the vaccination is carried out by the introduction of intranasally and / or subcutaneous live attenuated recombinant strains of influenza A virus subtypes H1 / N1, and / or H2N2, and / or H3N2, expressing the early secretory mycobacterial antigen ESAT- 6. 5. A method for the treatment of tuberculosis infection by introducing intranasally and / or subcutaneous live attenuated recombinant strains of influenza A virus, subtypes H1 / N1, and / or H2N2, and / or H3N2 expressing ESAT-6 early secretory mycobacterial antigen. 6. A method of treating a tuberculosis infection according to claim 5, characterized in that the treatment is carried out against the background of tuberculostatic therapy. 7. A method of treating a tuberculosis infection according to claim 6, characterized in that isoniazid is used as the medicament.

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