Antituberculous vaccine

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

 

The invention relates to the selected microorganism of the genus Mycobacterium, characterized in that it inactivated gene Rv0757 that gives phenotype PhoP-and inactivated second gene that prevents the production of DIM (DIM phenotype-). In addition, the present invention relates to the use of this microorganism to obtain vaccines for immunization or prophylaxis of tuberculosis.

The LEVEL of TECHNOLOGY

Currently, the use of vaccines for the prevention of tuberculosis, which are used by people for almost a century, is questionable. BCG derived from .bovis, is currently the single most widely used used TB vaccine in the world. The development and widespread use of BCG vaccine since the early 20-ies of XX century is a significant achievement, as expected, is able to eliminate tuberculosis in the world. However, these initial hopes were not justified, and the results of a large number of effective tests it became clear that the BCG vaccine, in its present form, is of limited use to control the spread of diseases, in particular respiratory forms of the adult population in developing countries, where the disease is endemic [4]. Thanks to his profound knowledge in the field of virulence of M. tuberculosis and models of the immune response, to the that give rise to protective immunity, you can develop more effective than BCG vaccine. The observation that in a BCG vaccination can lead to elevated levels of protection, suggesting that the viability and long-term effectiveness are fundamental properties required for effective TB vaccine. In the present invention as an experienced dosed once a live vaccine, the authors used a strain of M. tuberculosis with inaktivirovannye genome Rv0757 (phoP) and the second independent phoP, a mutation that prevents the synthesis DIM, and showed that in addition, this vaccine is more attenuated than BCG in immunocompromising SCID mice, it gives protection levels comparable to the levels of BCG in mice, and Guinea pigs even more protection than BCG.

Gene phoP along with phoR forms part of a two-component system, which shows a high degree of homology with other two-component systems, which control the transcription of key genes virulence of intracellular pathogens. It also controls the expression of many other genes that are not directly involved in the formation of virulence [19]. Elimination of virulence genes by itself, apparently, is not the only way attenuatio M. tuberculosis. It was shown that auxotrophic for Pantothenate mutant of M. tuberculosis, not who can synthesize Pantothenic acid de novo, peristerona SCID mice, without causing disease [17]. Separate auxotrophy on Latino are also significantly attenuated and is not capable of replication in vivo in SCID mice [28]. So now suppose that vaccine strains based on M. tuberculosis can be successfully attenyerevan while keeping genes that are suppressed in M. bovis BCG.

Earlier research more effective than BCG vaccine was based on the belief that the loss of virulence of BCG is thus a factor that led to the lack of a more powerful protective efficacy [32]. Therefore, it was concluded that the new attenuated mutants of M. tuberculosis with reduced virulence could be more effective as a vaccine. However, a recent study showed that natural infection with M. tuberculosis and BCG vaccination did not differ in their ability to induce protective immunity against tuberculosis [34]. This raises the question about the possibility of improving BCG by rational attenuatio M. tuberculosis. In this connection, the most unexpected and important observation is the fact that a mutant strain of M. tuberculosis according to the present invention combines 2 independent mutations, 1 is related to the PhoP protein synthesis and 2 is related to the synthesis of DIM, it is more attenuated than BCG in SCID mice, even when used in the research Institute in the dose 10 times higher than the dose of BCG, and causes a more powerful protection than BCG model of Guinea pigs.

BRIEF description of the INVENTION

The first aspect of the invention relates to the selected microorganism belonging to the genus Mycobacterium, characterized in that it contains the inactivation of the gene Rv0757 (phoP) and a second gene that prevents the production of DIM (prioritydecrement). In this paper this selected microorganism is designated as the microorganism of the present invention.

The second aspect of the present invention refers to the selected microorganism belonging to the genus Mycobacterium, characterized in that it contains an inactivated gene Rv0757 (phoP) and the second independent from phoP mutation that prevents product DIM. In a preferred aspect of the present invention, the specified second mutation is in a gene Rv2930 (fadD26) and represents a deletion of the gene fadD26, which is crucial in the synthesis of DIM.

The third aspect of the present invention refers to the use of the selected microorganism of the present invention to obtain vaccine for prevention of tuberculosis in animals and, more preferably, for the prevention of tuberculosis in humans, as well as other variants of use, which currently have TB vaccines for the treatment of diseases in humans, the same is as bladder cancer.

In the context of the present invention the strain of M. tuberculosis SO2 can be used to indicate the selected microorganism strain of M. tuberculosis, which iactiveaware by gene Rv0757 obtained from a clinical strain of M. tuberculosis MT by insertions resistant to kanamycin marker in the Bcll site of the gene Rv0757 M. tuberculosis, using homologous recombination in accordance with the method described Pelicic et al. (1997) (Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94: 10955-10960), and which further comprises the inactivation of the second gene that prevents the production of DIM (prioritydecrement). Thus, the strain according to the invention contains two independent mutations in the live attenuated vaccine derived from M. tuberculosis, and independent phoP mutation does not affect the properties of the vaccine produced by inactivation of the specified gene. In example 9 describes the creation of the selected microorganism of the genus Mycobacterium independent double mutation that produces the same phenotype as described for strain M. tuberculosis SO2.

In the context of the present invention is a vaccine refers to drugs, the introduction of which stimulates the body's defenses against disease.

In the context of the present invention BCG will be used to refer to the existing vaccine use and for TB since 1921. BCG vaccine is a live attenuated vaccine derived from a strain of M. bovis, that has lost its virulence after subculturing in the laboratory and which, as is known, has more than a hundred deletirovannykh genes (5).

In the context of the present invention 37Rv used to indicate a pathogenic strain of M. tuberculosis, which was sequenced, Cole et al., and identified these genes as Rv (Link Cole et al. 1998 Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537-544).

In the context of the present invention also MT will be used to denote a clinical isolate of M. tuberculosis (reference 15 Camacho et al.).

In addition, in the context of the present invention, the strain DIM - will be used to denote a strain of M. tuberculosis complex, which is not able to synthesize prioritydecrement, which are important lipids associated with the pathogenicity of M. tuberculosis. Figure 11 shows the use of strain A, which consists of a strain MT gene Rv2930 (fadD26), inaktivirovannye using transposon 1096 described in reference 15 (Camacho et al. 1999 Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Environ Mol 34: 257-267).

In the context of the present invention, the strain SO2+ pSO5 will be used to denote a strain of M. tuberculosis SO2, in which a mutation in Rv0757 complement genome Rv0757 by transforming replicates the second plasmid with mycobacterial gene phoP, and he is not able to additional synthesis DIM, its phenotype is phoP+DIM-.

In the context of the present invention of M. tuberculosis phoP - will be used to denote a strain of M. tuberculosis, which is inactivated by gene Rv0757 by deletions between sites EcoRV-BspEI, its phenotype is phoP-DIM+.

In the context of the present invention Rv2930 (fad26) will be used to denote a gene, which is located at the beginning of the operon responsible for the synthesis of prioritydecrement (reference 15 Camacho et al. 1999), and deletion of this gene in M. tuberculosis leads to sustainable phenotype DIM-.

DESCRIPTION of FIGURES

Figure 1 - Western blot. Using polyclonal antibodies directed against the PhoP and ESAT-6 were Western blot extracellular protein of strain MT, SO2 strain of the present invention and BCG Pasteur. Strain MT has the phenotype genes esat6+ and hoP+, SO2 strain has the phenotype PhoP - and genes esat6+, and the strain of BCG vaccine is PhoP+ and genes esat6-.

Figure 2 - the attenuation of the SO2 strain of the present invention in SCID mice. a) Graph the degree of survival of SCID mice (n=10)infected with aerosols containing the following strains: SO2, SO2, supplemented pSO5 (SO2 + pSO5) and MT in the amount of 20 KOE. The average life was more than 245 days (SO2), 62,1±5,88 (SO2 + pSO5) and 36.7±0,67 (MT). Mice infected by aerosol with the SO2 strain, survived within 245 days of the experiment, whereas mice infected MT and strain SO2, supplemented by the output hoP, died up to 62 days. b) - Graphs the degree of survival of SCID mice (n=7), infected by the intravenous injection of SO2 strain of 5,4x106SOME and BCG Pasteur in the number 2x105SOME. This shows that the level of attenuation strain SO2 exceeds the level of attenuation BCG tuberculosis vaccine, currently used for vaccination of people.

3 - cell immune responses in mice vaccinated with the SO2 strain of the present invention and BCG. Balb/c mice were vaccinated by subcutaneous injection of BCG (Phipps) 8x103SOME or SO2 strain of the present invention in quantities of 2,5x103SOME. The results are presented as the percentage of the total number of populations of CD4+/CD8+ in the spleen at intervals after vaccination and as the percentage of cells that Express IFN-γ from the General population of CD4+/CD8+ after stimulation full antigen of M. tuberculosis. * denotes statistically significant differences between groups at these time points (p<0,005). The results in cellular immunity have shown that animals vaccinated with strain SO2, compared with mice vaccinated with BCG, in the days 14, 30, 45 and 60 CD4+ lymphocyte much more, and on days 45 and 60 is a significant product-specific IFN-γ, directed against antigens of M. tuberculosis. The number of CD8+ lymphocytes in animals vaccinated with the SO2 strain, b is the larger on days 45 and 60, and product-specific IFN-γ, directed against antigens of M. tuberculosis, significant at day 14 compared with mice vaccinated with BCG.

Figure 4 - protective efficiency of the SO2 strain of the present invention in comparison with BCG vaccinated Balb/c mice. Values of CFU obtained in the analysis of lung (a) and spleens (b) Balb/c mice vaccinated with the SO2 strain of the present invention and BCG infected intravenously with M. tuberculosis H37Rv. The decrease in the values of CFU in the lung and spleen of the mice vaccinated with SO2, similarly obtained from mice vaccinated with BCG, which indicates considerable protection compared with non-vaccinated mice.

5 is a protective efficacy in Guinea pigs vaccinated with the SO2 strain of the present invention and BCG in relation to low doses of M. tuberculosis H37Rv. The average values of log10CFU/ml in the lungs (a) and spleen (b) vaccinated Guinea pigs and control pigs, which introduced saline infected with low doses of M. tuberculosis H37Rv. Data represent average values of SOME of the animals (n=6)scored in 6 weeks. Error bars indicate standard deviation. The decrease in the values of CFU in the lungs and spleen of Guinea pigs infected with M. tuberculosis in low doses and vaccinated SO2, similarly obtained in Guinea pigs vaccinated with BCG, and I what is significant compared with non-vaccinated Guinea pigs.

6 is a protective efficacy in Guinea pigs vaccinated with the SO2 strain of the present invention and BCG against infection with high doses of M. tuberculosis H37Rv. a) due To the fact that the experiments associated with protection in mice and Guinea pigs infected with low doses, showed the presence of explicit protection in mice vaccinated with SO2 and BCG, but without differences between BCG and SO2, the model was used Guinea pigs to infection with high doses. Graph the degree of survival of Guinea pigs after aerosol infection with M. tuberculosis H37Rv. b) the severity of lung disease and the spread of infection, estimated using the total consolidation of the lung tissue. The value of each individual animal scored at the end point, a certain man, mark an x in the. The dashed line indicates the average percentage for this group (# in SO2 corresponds to two animals). (C) Low resolution (h) image characteristic histological sections of the lobes of the lung obtained from Guinea pigs to each of the treated groups. Trait represents 1 mm. d) the Average CFU count in the spleen and lungs of vaccinated and non-vaccinated Guinea pigs. This experiment shows that the model of Guinea pigs with high doses of M. tuberculosis infection Guinea pigs vaccinated with SO2, having survived what do considerably longer than with BCG vaccination, and they also formed less pulmonary lesions and had fewer CFU in the spleen and lungs compared to the currently used BCG vaccine.

7. The attenuation of intravenous infection with the SO2 strain of the present invention in mice Balb/C not restored by addition phoP. A study on mice Balb/C mice intravenously infected with strain SO2(phoP-DIM-) in quantity of 105SOME compared with infection with strain MT wild type and strain, supplemented by phoP (SO2 + pSO5). The decrease in the number of colonies (CFU) were observed in the spleen (7a - spleen) and in the lung (7b - easy), the evaluation was made after 3 and 6 weeks. Levels of CFU of the wild-type strain was not restored in the complemented strain. These experiments in immunocompetent mice indicate that unexpected attenuation could be caused by a second mutation, which is not restored by supplementing phoP.

Fig. The SO2 strain of the present invention does not produce DIM, and synthesis DIM is independent of the phoP mutation. Analysis of lipids of different M. tuberculosis strains by thin-layer chromatography. a) In strain MT can be detected products are DIM, while DIM is not reproduced strain SO2 and augmented gene phoP strain (SO2 pSO5). This shows that the lack of product DIM in strain SO2 is not dependent on phoP. On Fig.8b shown shtam MT and strain MT, inactivated only in respect of the phoP gene (MT ∆phoP::hyg), and both are capable of synthesizing DIM that confirms that the product DIM is not dependent on phoP mutation.

Fig.9 - construction of plasmids for gene inactivation fadD26.

Figure 10 - construction of plasmids for inactivation of phoP gene.

11 - study of attenuation in mice: a graph of the degree of survival of Balb/C mice, intratrahealno infected with the purpose of studying attenuate different strains of M. tuberculosis. 37Rv and MT correspond to the M. tuberculosis strains without mutations, and all mice infected with these strains, died before 10 weeks. With the strain of M. tuberculosis DIM- (A) 50% of mice survived after 20 weeks. All animals infected with the SO2 strain (mutant phoP - and DIM-), survived for 20 weeks of the experiment.

Fig is a graph of degree of survival and weight of Guinea pigs to study the toxicity of SO2 strain (50-fold dose of the vaccine). To show that the SO2 strain is non-toxic, six Guinea pigs were infected with 50-fold dose of the vaccine. After 6 months of experiment, the degree of survival was 100%. The increase in body weight was observed in all animals during the 6 months that shows the toxicity of SO2 strain (Y=mass in grams of each week after infection. X=time in weeks).

Fig - the degree of survival of vaccinated Guinea pigs after infection with M. tuberculosis. Study of protection in Guinea pigs, the degree of survivability h is cut 300 days: graph the degree of survival have not been vaccinated Guinea pigs (physiological solution), vaccinated with the currently available vaccine BCG strain of M. tuberculosis phoP - or-SO2 strain (mutant phoP - and DIM-). To study the degree of survival after subcutaneous vaccination of animals were infected by the virulent strain of M. tuberculosis (H37Rv) in high dose. After 60 days, 6 Guinea pigs, which were not vaccinated, died, while the group vaccinated with SO2, phoP - and BCG, have survived. After 300 days after infection, 3 Guinea pigs vaccinated with BCG and phoP-died, while in the group vaccinated with SO2, died only one pig, which indicates that the level of protection of the phoP mutant is similar to the level of protection currently available TB vaccine, at the same time, the vaccine strain SO2, double mutant phoP - and DIM-on the model of Guinea pigs protects better.

Fig - study of protection in Guinea pigs, the degree of survival after 400 days: continuation of the experiment is presented on Fig. 6 not vaccinated Guinea pigs died after 60 days. After 400 days after infection survived 3 Guinea pigs from the group vaccinated with strain SO2 (Figa), whereas survived only 1 Guinea pig vaccinated with BCG (Figa and Fig.14b) and phoP- (Fig.14b), which again indicates that the protection of the phoP mutant is similar to the BCG protection, while vaccination with strain SO2, double mutant phoP - and DIM-through 400 days of the experiment protects better.

DETAILED description of THE INVENTION

In one aspect the present invention relates to a selected microorganism belonging to the genus Mycobacterium, characterized in that it contains the inactivation of the gene Rv0757 that gives phenotype PhoP-and inactivation of the second gene that prevents the production of DIM (DIM phenotype-). In addition, the present invention relates to the use of this microorganism to obtain vaccine for prevention of tuberculosis and directly to the vaccine itself.

In the description of the present invention shows that the selected strains phoP - DIM - genus Mycobacterium have characteristics that make them particularly effective for use as vaccines due to the level of attenuation, which they achieve, and the level of protection that they create.

To demonstrate attenuation immunodepression SCID mice were infected with strain SO2 (phoP - DIM-) of the aerosol. These mice survive (Figa) much longer than mice infected with the wild-type strain. Moreover, this attenuation is retained when the addition phoP in strain SO2 + pSO5 (phoP+ DIM-) (Figa).

Moreover, when conducting research related to attenuation, immunocompetent Balb/C mice by intravenous (Fig.7)shows the apparent attenuation of the SO2 strain compared with the strain MT wild type, but, surprisingly, this attenuation is not complemented by phoP, because the strain SO2 + pSO5 (phoP+ DIM) is as virulent for immunocompetent mouse as the wild-type strain. Research survival in mice Balb/C, when comparing the SO2 strain (DIM-, phoP) strain only DIM-demonstrate, surprisingly, a higher degree of survivability for SO2 (11).

A comparative study of survival rates of SO2 and BCG in SCID mice infected intravenously, show that the level of attenuation strain SO2 is higher than the BCG vaccine, which is currently used in humans for TB (Fig.2b). Toxicity studies on Guinea pigs with a 50-fold dose of the vaccine used for quality control of batches of BCG vaccine, show that within 6 months of the study Guinea pigs gained weight and they neither macroscopically nor microscopically was not observed histological lesions consistent with tuberculosis, thus, confirming the attenuation and the non-toxic strain of SO2 (Fig). This unexpected attenuation and loss of toxicity due to the phenotype of PhoP - DIM-and, in addition, these mutations retain sensitivity to drugs that would allow for a standard treatment.

In the present description it is shown that in experiments with vaccination carried out on Balb/C mice, the protection levels created by the strain of M. tuberculosis SO2 according to the present invention and BCG were similar as in the lungs and spleen up to even the PEX weeks after infection. When comparing the relative proportions of CD4+ and CD8+ cells obtained from the spleens of vaccinated mice, in mice vaccinated with the SO2 strain of the present invention, was found in a higher percentage of both CD4+and CD8+ cells compared with mice vaccinated with BCG. Moreover, the stimulation of these cells by antigen, obtained from the culture filtrate, in mice vaccinated with the SO2 strain of the present invention, at 45 and 60 days after vaccination were determined significantly higher percentage of CD4+/IFN-γ+. Although this percentage is not significant at each time point, a similar trend was determined for CD8+/IFN-γ+ mice vaccinated with the SO2 strain of the present invention. These data indicate that vaccination with the SO2 strain of the present invention leads to a more efficient activation of T cells compared with BCG, which was determined on the synthesis of IFN-γ. Provided that protective immunity to M. tuberculosis in General depends on the cellular immune response of type T1characterized by secretion of IFN-γ-specific T-cells in response to antigen, we can conclude that the relatively high levels of T-cell activation induced by SO2 strain of the present invention, provide him the ability to create a strong protective response.

In addition, using different systems and is dressed for the experiment, and a large number of conditions, the authors were able to show comparative possibility of using mouse models to study differences in the protection created BCG compared to SO2. In the mouse model it was shown that protection creates two vaccines, SO2 (phoP - DIM-) and BCG.

For comparison of vaccines in a more meaningful and at each stage of more labor-intensive experiment with Guinea pigs was undertaken certain strategy. This systematic approach for comparing vaccines may represent a valuable starting point to determine the best candidate vaccines, which must be carried out further experiments. As a rule, I think that Guinea pigs are more sensitive to infection with tuberculosis and may therefore be more meaningful model of the disease [30]. The advantage of Guinea pigs compared with mice is that the pathology of the disease is similar pathology detected with tuberculosis in humans, and therefore it is an appropriate model for testing the efficacy of vaccines. In a recent study of vaccines administered by aerosol containing double auxotrophic mutant of M. tuberculosis, Pantothenate and Latino, five weeks after aerosol application of M. tuberculosis in the lungs and spleen of vaccinated Guinea pigs were detected levels of protection equivalent to levels causing M. bovis BCG, limited the soap the prevalence of infection in the spleen, induced by both vaccines [34]. In another study, which used recombinant BCG, Express tropic ESAT-6, higher than that of M. bovis BCG, protection levels were detected only in the spleen [6], indicating that enhanced protection is associated with its ability to prevent the spread of infection from the lung.

For the implementation of infection Guinea pigs in the low dose was inoculable M. tuberculosis H37Rv, and the protection levels created by the vaccination with the SO2 strain of the present invention and BCG, was the same as in the lungs and spleen within 4 weeks after infection. Both vaccines were created extremely effective protection, reducing the values of CFU in the lungs and spleen by approximately 2 log compared with the control groups, which were injected with saline. However, between the two vaccinated groups were not statistically significant differences. The authors suggested that in such a short period of time after infection would be more difficult to prove the effectiveness of a new vaccine compared with BCG. This is due to the fact that at the moment CFU (colony forming units) of the bodies of animals vaccinated with BCG, are so small that the test does not have differential ability to demonstrate significant additional reduction in CFU. In the other, and the implication survival in Guinea pigs was shown to that despite the fact that BCG provides the creation of a statistically significant protection compared with non-vaccinated controls (vaccinated or ineffective vaccines), this protection is only partial even against infection with low doses of M. tuberculosis. In studies using low doses, held within 60 to 80 weeks after infection, some controls with BCG did not create protection for a single Guinea pig [35], while others have created protection low percentage (between 20 and 30%) animals [36] [37]. Applique high doses, on the other hand, can lead to more serious illness than are typically used to assess the protective efficacy of vaccines against TV.

For the present invention, the authors used aerosol infection with a relatively high dose of M. tuberculosis H37Rv, and the study period was extended until 180 days. The authors have done is to create a more stringent degree of contamination, which can demonstrate the possible protective efficiency of the SO2 strain of the present invention, and at the same time, to assist in establishing the degree of difference with BCG. In terms of the survival of animals in the group vaccinated with BCG were significantly more secure in comparison with non-vaccinated controls, and they showed the highest degree for which the ITA, similar found in other studies, despite the relatively high dose of infection, used in our study. Moreover, the authors also found a statistically significant increase in the protective efficiency of the SO2 strain (phoP - DIM-) according to the present invention in comparison with BCG defined using several indicators, including increased longevity and the degree of consolidation of the lungs. This less severe form of the disease could be directly responsible for a higher degree of survival of animals vaccinated with the SO2 strain of the present invention.

The results described in this invention show that, in accordance with many evaluation criteria the SO2 strain, and hence the microorganism belonging to the genus Mycobacterium (in particular related to the M. Tuberculosis complex), with a phenotype phoP - DIM - is a more effective vaccine than BCG. The vaccine is more attenuated than BCG in SCID mice, it creates mice protective immunity, which, at least, not worse immunity generated BCG, and it provides a stronger cellular immune response. In addition, in experiments on defense research conducted on Guinea pigs infected with high doses of H37Rv strain with phenotype DIM - phoP - leads to 100% survival of Guinea pigs in conditions in which BC is reaches only 33% survival rate. I believe that this protection is associated with lower disease severity and bacterial load.

To determine, due to whether the level of protection of the SO2 strain (phoP - DIM-) the phoP mutation or could be caused by an additional mutation in the DIM, Guinea pigs conducted another experiment vaccination with infection in high doses. Groups of 6 animals were vaccinated with BCG, SO2 (hoP - DIM-) and M. tuberculosis phoP - DIM+, and 6 animals, used as control, were not vaccinated. The experiment lasted 400 days.

In another experiment not vaccinated Guinea pigs died within 70 days. After 300 days after infection died 3 Guinea pigs vaccinated with BCG and phoP - DIM+, compared with only one in the group vaccinated with the SO2 strain, which indicates that the protection created by the phoP mutant - DIM+, similar to the protection of the currently used BCG vaccine, as the vaccine strain SO2, double mutant phoP - and DIM-on the model of the Guinea pig provides the best protection (Fig). After 400 days in the group vaccinated with SO2, survived 3 Guinea pigs (Figa), whereas survived only 1 Guinea pig vaccinated with BCG (Figa and Fig.14b) and phoP - DIM+ (Fig.14b), indicating that after 400 days of the experiment protection mutant phoP - DIM+ is similar to the protection of BCG, whereas vaccination with strain SO2, dual mutant the phoP - and DIM-, protects better, an unexpected effect of a higher protection than BCG is not only one mutation in phoP-but double mutation in phoP - DIM - SO2 strain.

Thus, the first aspect of the present invention refers to the selected microorganism belonging to the genus Mycobacterium, characterized in that it contains the inactivation or deletion:

(a) gene phoP - or of one or more genes that regulate gene phoP-, or regulated gene phoP-and

b) a second gene that prevents the production of DIM.

In a preferred embodiment of the invention selected microorganism according to the invention differs in that the phoP gene is inactivated by inactivation or deletion of the gene Rv0757.

In a more preferred embodiment of the invention selected microorganism according to the invention is characterized by the fact that the products are DIM inactivate by deletion or inactivation of a gene Rv2930 (fadD26).

In an even more preferred embodiment of the invention selected microorganism according to the invention differs in that it contains a deletion or inactivation of genes Rv2930 and Rv0757.

In another embodiment of the invention selected microorganism according to the invention is characterized by the fact that species of the genus Mycobacterium belongs to the Mycobacterium tuberculosis complex.

The second aspect of the invention relates to a method for obtaining selected is microorganism according to the invention, which includes:

a) inactivation or deletion of the gene phoP or one or more genes that regulate gene phoP, preferably, inactivation or deletion of the gene Rv0757, and

(b) inactivation or deletion of the second gene that prevents the production of DIM, preferably a deletion or inactivation of a gene Rv2930 (fadD26).

The third aspect of the invention relates to a vaccine (in this document the vaccine according to the invention) for immunization of an individual against symptoms caused by tuberculosis, with this vaccine contains at least one selected microorganism according to the invention.

In a preferred embodiment of the invention, the vaccine also contains pharmacologically acceptable excipients.

A fourth aspect of the invention relates to a method for obtaining a medicinal product, preferably, the vaccine, which includes the introduction of a selected microorganism according to the invention in a suitable environment for use on humans or animals in therapeutically effective dose and, optionally, addition of excipients that are pharmaceutically acceptable for the receipt of vaccines.

Specified drug suitable for the treatment of bladder cancer, for the treatment or prevention of tuberculosis or as a vector or adjuvant. Preferably for immunization of individuals is big, the but against the onset of symptoms, caused by tuberculosis.

The fifth aspect of the invention relates to the application of the selected microorganism according to the invention to obtain the vaccine according to the invention for the prophylaxis and/or treatment of tuberculosis in humans or animals.

Throughout the description and claims the word "contains" and its variants do not mean to exclude other technical characteristics, additives, components or steps. For the specialist in this field for other purposes, advantages and features of the invention will in part follow from the description and partly in the practice of the invention. As a non-limiting illustrative example of the present invention offers the following examples and figures.

EXAMPLES

Example 1. Materials and methods

1.1. The selection of protein and Western blot turns. To protein PhoP were obtained polyclonal antibodies, which received four doses of PhoP (0.5 mg) at 0, 4, 8, 12 and 16 weeks, respectively. Antibodies against PhoP was determined using the ELISA test (ZEU-Immunotec Zaragoza, Spain). Monoclonal antibodies against ESAT-6 were kindly provided by S. Cole [24]. Cell-free protein extracts of mycobacteria were obtained from early cultures in log phase, which was grown in liquid medium Middlebrook 7H9 broth suspensions-ADC, then in the usual way [25]. Protein extracts of M. tuberculosis was filtered through a filter Millex-GP pore size 0,22 is km (Millipore, Bedford, MA). Collected the culture filtrate of M. tuberculosis H37Rv, cultivated for 5-6 weeks, and filtered proteins culture precipitiously 45% (weight/volume) solution of ammonium sulfate. In accordance with standard methods conducted analysis of the Western blot. As secondary antibodies used goat antibodies against rabbit antibodies labeled with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA).

1.2. Infection of SCID mice with M. tuberculosis. Work with SCID mice was performed under the supervision of the Committee for the protection of animals at the University Hospital “Germans Trias i Pujol” in accordance with EU laws on the protection of laboratory animals. Free from specific pathogens (spf) mouse SCID CB-17/Icr Ico were obtained from Charles River (Bagneux Cedex, France). For aerosol infection of mice was placed in a camera for exposure of the infection, transmitted by airborne droplets (Glas-col Inc., Terre Haute, IN, USA). The nebulizer was filled with 7 ml suspension of M. tuberculosis to enter into the lungs of about 20 viable bacilli. For each experimental group were used for ten mice. For intravenous infection via the lateral tail vein group 7 mice were infected with 200 μl of PBS containing a dose equivalent to 2×105, 2×104and 2×103viable BCG and 5.4×106that is 5.4×105, and 5.4×104viable strain of M. tuberculosis phoP. Using test Mantel-Haenszel between processing is nymi mice was determined by the significance of the differences in life. Counts of viable cells was performed by serial dilutions of the homogenate, placed on agar Middlebrook 7H11+OADC and evaluated after 3 weeks of growth. For histological analysis, tissues were fixed in buffered formularium solution and embedded in paraffin. Did the slice thickness of 5 μm and stained by Zn.

1.3. Determination of the activation of cellular immunity in Balb/c mice after subcutaneous vaccination with the SO2 strain of the present invention and BCG. Groups of four Balb/c mice were scored at 7, 14, 21, 28, 45 and 60 days after subcutaneous BCG vaccination (Phipps) in the amount of 8×103SOME or SO2 strain of the present invention in the amount of 2.5×103. The spleen was removed and placed in 2 ml of RPMI medium and 10% fetal calf serum (GIBCO, Invitrogen Corporation)containing 0.5 mg/ml collagenase type II (Worthington, NJ, USA) and 2 U/ml Gnkazy (GIBCO), and incubated for 1 hour at 37°C in an atmosphere of 5% CO2. Then ran them through a cell filter with a pore size of 70 μm (Falcon, Becton Dickinson 70 µm Nylon 35-2350), pressed the plunger of the syringe and washed with medium. Cells were centrifuged, supernatant was removed, and the red cells were removed lytic buffer [26]. After centrifugation and washing medium RPMI cells resuspendable in FACS buffer (1-fold PBS, pH value of 7.2, 1% BSA) and counted their number. The surface of the cells were labeled, incubare 106cells so µl of monoclonal antibodies against CD4-FITC or anti CD8-FITC, diluted in the ratio 1:20 in PBS containing 1% BSA and 0.1% sodium azide for 20 min at 4°C and analyzed using cytometer FACScan.

Strain M. tuberculosis H37Rv was cultured in the medium Middlebrook 7H9 broth suspensions (Difco Laboratories), supplemented with OADC (Difco Laboratories). After culturing for 1 month of bacterial mass was separated and collected culture filtrate. Antigens specified filtrate precipitiously 45% solution (weight/volume) of ammonium sulfate, washed and re-dissolved in PBS. For stimulation of cells with spleen cells (1×106resuspendable in 100 μl of RPMI medium per well and incubated with the antigens of the culture filtrate of M. tuberculosis, taken at 10 mg, suspended in 100 μl PBS for 72 hours at 37°C in an atmosphere of 5% CO2. Cells and culture medium was centrifuged, supernatant was removed and after counting and checking the viability of 2.5×105cells per tube were marked on the surface of CD4+ or CD8+ cells, as described above. After washing the cells resuspendable and incubated for 20 min at 4°C in 0.1% saponin dissolved in PBS. Extracellular IFN-γ was determined by incubating cells for 20 min at 4°C in the dark with 100 μl of monoclonal antibodies against IFN-γ, taken in a dilution of 1/20, labeled with phycoerythrin (PE). The cells were fixed with 100 μl of 4% solution of paraformaldehyde in PBS. 20 minutes later analyzed arr is sci, using cytometer FACScan. Controls isotypes were Ab-FITC (dilution 1:20) +Ab-PE (dilution 1:20).

1.4. The protective efficiency of the SO2 strain of the present invention in Balb/c mice. All animals were kept under controlled conditions in laboratory animals high level of biological protection P3 at the Institute Pasteur in Paris in accordance with the EU regulations on the protection of laboratory animals. Groups of Balb/c mice (7 per group) were vaccinated subcutaneously in the base of the tail with the SO2 strain of the present invention or BCG (Pasteur), taken in the quantity of 107SOME. Eight weeks after vaccination, all mice intravenously injected with M. tuberculosis H37Rv in the amount of 2.5×105SOME. Four weeks after injection, the mice were scored. Counts of viable cells was performed by serial dilutions of the homogenate, cultured on liquid medium Middlebrook 7H11 + OADC agar, and after 3 weeks was estimated growth of M. tuberculosis H37Rv, excluding the SO2 strain of the present invention on the basis of resistance to kanamycin phenotype of the latter strain.

1.5. The protective efficiency of the SO2 strain of the present invention in Guinea pigs. Experimental work with Guinea pigs was carried out in accordance with UK laws on animal experimentation and approved by the local ethical Committee of the organization for the protection of health, Porton Down, UK. Females Mor is such pigs Dunkin-Hartley received from licensed commercial suppliers (UK Home Office) (David Hall, Burton-on-Trent, UK, or Harlan UK Ltd, Bicester, UK) and diluted in complete isolation. The results presented in Fig.6 show that the SO2 strain creates more protection than BCG. The results presented in Fig and 14, show that this unexpected protection mutant SO2 due to its double DIM phenotype-/PhoP-.

1.6. The use of low dose. Groups of 6 Guinea pigs were vaccinated subcutaneously in the scruff of the neck following drugs in the amount of 250 μl: 5×104CFU BCG Pasteur; 5×104SOME of the SO2 strain of the present invention; or a saline solution. The animals were left alone for 12 weeks before infection by aerosol using the apparatus Henderson, as described previously [27]. Using a Collison nebulizer, got aerosols of fine particles of M. tuberculosis H37Rv with an average diameter of 2 μm (the range of diameter: 0.5 to 7 μm) and brought directly to the nose of the animal. The aerosol was obtained from a water suspension containing 2×106CFU/ml, to achieve a fixed inhalation dose, which is expected about 10-50 CFU/lung.

Four weeks after the introduction of the estimated level of protection. Animals were scored by peritoneal overdose pentobarbital sodium. From the spleen and lungs were aseptically removed tissue (left and middle cranial lobe, right middle portion, and the right caudal lobe) and placed in a sterile container is. The material was stored at -20°C and then was preparing to count the number of bacteria. The tissue is homogenized in 10 ml (for light) or 5 ml (spleen) sterile deionized water, using a grinding system with a rotating blade (Ystral). Counts of viable cells was performed by serial dilutions of the homogenate, cultured in the medium Middlebrook 7H11 + OADC agar, and after 3 weeks was estimated growth of M. tuberculosis. Data for analysis were transferred to the log10and using t-student criterion the number of viable M. tuberculosis for each vaccinated group compared with the control group, which received saline.

1.7. Test protection in Guinea pigs after infection with a high dose of M. tuberculosis. For 10 weeks prior to spray application of M. tuberculosis groups of 6 Guinea pigs were vaccinated subcutaneously with the SO2 strain of the present invention or BCG (Danish 1331) in an amount of 5×104SOME. Spray application was carried out as described in the previous paragraph, using a suspension of 5×107CFU/ml to deliver in light of about 500 CFU. After application the animals were kept in conditions of protection level 3 (ACDP), weight change was regularly controlled and scored a humane way through 180 days after application or at the end point, a certain man (loss of 20% of maximum body weight). Collecting and processing the woven samples after the autopsy was carried out, as described above, except that the consolidation of the lung tissue was measured using image analysis of sections of lung tissue, fixed in formalin, stained with hematoxylin and eosin (H+E). Survival of animals was compared using survival rates by Kaplan-Meier, and to determine statistically significant differences used the analysis of the distribution of Log Rank. Data values and SOME consolidation of the lesions were analyzed using ANOVA, using paired comparisons Fisher for comparison of mean values of groups.

Example 2. Characterization of the M. tuberculosis phoP

The proof of the involvement of the gene phoP in the General regulation of genetic cycles of mycobacteria was obtained by monitoring changes in the size of the bacilli and the consequent properties of growing cells containing an inactivated gene phoP. Considering the basic properties of secreted antigens as determinants of protection against tuberculosis, the authors hope to identify, apply whether the pleiotropic effects of mutations in phoP gene for the synthesis of the main immunodominant antigens: ESAT-6. Using antigens directed against the PhoP protein and ESAT-6, carried out the Western blot of the SO2 strain, BCG and MT. The results clearly showed that the protein PhoP constantly expressively in M. tuberculosis strains MT and BCG, whereas it was completely absent in strain SO2 infusion is his invention. In contrast, the levels of expression of ESAT-6 in the supernatant of cultures of strain SO2 were similar to the levels defined for the parent strain MT and, as expected, in BCG protein ESAT-6 has not been defined.

Example 3. The survival of mice infected with strains of the present invention and BCG

Survival immunocompromised SCID mice was evaluated after aerosol infection (about 20 CFU) of strain MT, SO2 and SO2, supplemented phoP gene (SO2 + pSO5) [23]. All mice infected with the strain SO2, survived for more than 245 days. On the contrary, all SCID mouse infected MT or supplemented with M. tuberculosis SO2-SO5, died after 62 days after infection, indicating restoration of virulence complemented strain (Figa).

In SCID mice after intravenous also compared the attenuation of strain SO2 with BCG. Groups of SCID mice were infected via the lateral tail vein of multiple doses (2×105, 2×104and 2×103CFU) of BCG Pasteur or strain SO2 (5,4×106that is 5.4×105and 5.4×104CFU). Histological staining of infected alveolar macrophages subgroups of mice scored three weeks after infection, showed fewer alcohol-acid-fast bacilli in the lungs of mice infected with a strain of M. tuberculosis SO2, compared with BCG. All mice infected with higher doses of BCG (2×105SOME), died after 92 days of the settlement of the e infection (average life time: 89±3.5 days) (Fig.2b). On the contrary, all mice infected with a maximum dose of SO2 strain (5,4×106SOME), remained alive after 120 days (Fig.2b). At the time of death of the bacterial load of the lungs of mice infected with BCG at 2×105SOMETHING that was at least 100 times higher in comparison with loads of mice infected with strain SO2 in the amount of 5.4×106SOME.

Example 4. Quantitative CD4+ and CD8+ responses in vaccinated Balb/c mice

For comparison, the activation of cellular immunity induced by vaccination with the SO2 strain of the present invention and BCG, 7, 14, 30, 45 and 60 days after vaccination from the spleen of groups consisting of at least four Balb/c mice, subcutaneously vaccinated with SO2 strain of the present invention and BCG Phipps, collected cell suspension and cytofluorometric was determined by the ratio of CD4+ and CD8+ (Figure 3). Vaccination strain of SO2 compared to BCG vaccination 14 days after vaccination induced a significantly higher number of CD4+and 45 days and significantly greater number of cells CD8+. These splenocytes stimulated complete antigens obtained from the culture filtrate of M. tuberculosis. After 3 days the population of lymphocytes was analyzed using flow cytometry and combined specific antibodies to determine the CD4+/CD8+ cells and intracellular synthesis of IFN-γ. Vaccination strain of SO2 compared the structure with BCG 45 days after vaccination induced a significantly greater proportion of cells, producing CD4+/IFN-γ+ (Figure 3). After a certain period has constantly been higher proportion of cells that were produced CD8+/IFN-γ+, in the group that received SO2 (significant difference on day 14).

Example 5. Protective immunity produced by the SO2 strain of the present invention in mice Balb/c

Having evidence that the SO2 strain of the present invention was attenuated in SCID mice, the authors were interested to determine created if the observed decrease in the virulence of some kind of protective properties of the mutant strain. Were vaccinated subcutaneously in Balb/c mice with the SO2 strain of the present invention or BCG (Pasteur). Eight weeks after vaccination, all mice intravenously introduced M. tuberculosis H37Rv in the amount of 2.5×105SOME. 4 weeks after injection, the mice were scored. Protection levels were determined by estimating the number of viable M. tuberculosis H37Rv obtained from lung and spleen of both groups of mice (Figure 4). Compared with controls treated with saline, both vaccines have led to the creation of similar and still significant levels of protection (p<0,05). Inhibition of growth of M. tuberculosis H37Rv was noted in the lungs, and spleen, the decrease amounted to about 1.5 log10and 1.3 log10CFU, respectively.

Example 6. Protective immunity produced by the SO2 strain of the present invention in Guinea pigs

The results obtained in experiments with vaccinated mice, indicate that the attenuation of the SO2 strain of the present invention has created the vaccine properties analogous to properties of BCG Pasteur. However, it is usually assumed that Guinea pigs are a more appropriate model for tuberculosis person, with many analogies from the point of view of progression and pathology of the disease. Therefore, this animal model is a more suitable system for evaluating the effectiveness of the vaccine. To study the protective efficacy of the SO2 strain of the present invention, the authors conducted experiments that included aerosol application to vaccinate animals at low doses (10-50 CFU) and high doses (500 CFU). Groups of six Guinea pigs were vaccinated subcutaneously with the SO2 strain of the present invention or BCG. Ten weeks after vaccination, all Guinea pigs were injected inhalation dose of M. tuberculosis H37Rv.

Animals that received the lower dose were scored after 4 weeks and counting bacterial load in lungs and spleen. Protective efficacy was determined by comparing the number of viable M. tuberculosis H37Rv obtained from organs of Guinea pigs in each treated group. In this experiment, the reduction values CFU in the lungs and spleen were significantly different between the non-HAC is inrevenue control animals and vaccinated with BCG or M. strain tuberculosis SO2 (p=0.005). However, between the vaccinated groups were not found significant differences (Figure 5).

Guinea pigs that received a high dose, scored 180 days after application or when he registered the reduction of body weight by 20%. Levels of protection were determined by comparing the lifetimes of Guinea pigs each treated group. Vaccinated/infected Guinea pigs also investigated the progression of lesion development and compared with what was observed in unvaccinated/non-infected animals. During the phase of the experiment, following the inhalation, not all vaccinated Guinea pigs and four Guinea pigs vaccinated with BCG, were scored at the end point, a certain man, to the end point the end of the experiment (180 days) due to development of severe progressive disease (Figo). On the contrary, all Guinea pigs vaccinated with the SO2 strain of the present invention, remained alive throughout the study. Guinea pigs vaccinated with the SO2 strain of the present invention, survived much longer than vaccinated with BCG (p=0,018), which, in turn, remained alive much longer than the control Guinea pigs, which were treated with saline (p=0,0049). Moreover, Guinea pigs vaccinated with strain SO2,had an increase in weight and they have not observed any visible or clinical symptom of the disease.

The severity of lung disease, the estimated total consolidation of lung tissue, also varied between the different treated groups. The maximum level of disease progression was observed, as expected, have not been vaccinated Guinea pigs, and in this group of animals was determined the average percentage of consolidation 76% (Fig.6b, 6c). In Guinea pigs vaccinated with BCG, it was also clearly expressed fusion granulomas with an average of consolidation 70%, measured in the lungs. On the contrary, in Guinea pigs vaccinated with the SO2 strain of the present invention, watched less consolidation (50%), this consolidation is much less (p<0.05)than in non-vaccinated animals and vaccinated with BCG (Fig. 6C). This relieved the severity of the disease is also reflected in the number of bacteria homogenates of lung and spleen. In the vaccinated groups, the difference in the levels of growth inhibition of M. tuberculosis H37Rv found in both bodies. Values of CFU obtained from Guinea pigs vaccinated with strain SO2 have been reduced by more than 1×log10compared with SOME values from Guinea pigs vaccinated with BCG, and this decrease was statistically significant (p<0.05) in spleen (Fig.6d). These data indicate that the SO2 strain of the present invention was more effective than BCG in related and more high degree of survival of infected Guinea pigs, reduce the severity of lung disease and prevent the spread of infection in the spleen.

Example 7. The attenuation of the SO2 strain of the present invention is a double mutation PhoP-DIM-

Studies with infected Balb/c mice by the intravenous injection of SO2 strain (phoP-DIM-) in comparison with the strain MT wild type and strain, supplemented by phoP (SO2 + SO5)showed that the attenuation of the infection SO2 in Balb/c mice by intravenous injection is not reproduced by the addition phoP. Reduction of colonies (CFU) in the spleen (7a spleen)and in the lung (7b easy), determined after 3 and 6 weeks, never played in the augmented strain, although he and Neverwinter for immunocompetent mice, these experiments indicate that unexpected attenuation could be caused by the second additional mutation (Fig.7).

Studies of lipids of different M. tuberculosis strains by thin-layer chromatography showed that the strain does not produce SO2 DIM, and it does not depend on the phoP mutation (Fig).

To show that the SO2 strain is non-toxic, six Guinea pigs were infected with 50-fold dose of the vaccine. After the 6-month experiment, the degree of survival was 100%. After 6 months in all animals was discovered weight gain, indicating that the toxicity of SO2 strain (Y= mass in grams of each week after infection. X= time in weeks the order) (Fig).

Also investigated the sensitivity to anti-TB drugs. For the following anti-TB drugs: ethambutol, isoniazid, rifampicin and streptomycin, was determined by minimum inhibitory concentration (MIC) against the following strains: H37Rv, MT (wild type) as a control and strain SO2. Data values (micrograms/ml) indicate that after inactivation of phoP gene candidate vaccine strain SO2 retains its sensitivity to many common medicines used to treat tuberculosis.

EthambutolIsoniazidRifampicinStreptomycin
H37Rv20,5<0,004<0,5
MT20,5<0,004<0,5
SO220,5<0,004<0,5

Research attenuatio on intratrahealno infected Balb/c mice showed that strain of M. tuberculosis DIM- (1A29) after 20 weeks survived 50% of mice. All animals infected with the SO2 strain (mutant phoP - and DIM-), miraculously survived for 20 weeks of the experiment (11).

Example 8. Protection of the SO2 strain of the present invention is a double mutation PhoP-DIM-

Patronage was investigated in Guinea pigs vaccinated and infected by aerosol with M. tuberculosis H37Rv. The survival of Guinea pigs after 300 days. To study the survival rate after subcutaneous vaccination of animals infect virulent strain of M. tuberculosis (H37Rv) in high dose. After 60 days, 6 Guinea pigs, which were not vaccinated, died, while the group vaccinated with strains of SO2, phoP - and BCG, have survived. After 300 days after infection died 3 Guinea pigs vaccinated with BCG and phoP-compared with only one pig from group vaccinated with strain SO2, which indicates that the protection of the phoP mutant is similar to the protection of the currently used BCG vaccine, at the same time, the vaccine strain SO2, double mutant phoP - and DIM-on the model of the Guinea pig protects more efficiently (Fig).

These studies protection in Guinea pigs lasted 400 days, while 6 are not vaccinated Guinea pigs died after 60 days. After 400 days after infection from the group in kinyruanda strain SO2, survived 3 Guinea pigs (Figa), whereas survived only 1 Guinea pig vaccinated with BCG (Figa and Fig.14b) and phoP- (Fig.14b), again indicating that protection of the phoP mutant is similar to the BCG protection, while vaccination with strain SO2, double mutant phoP - and DIM-through 400 days of the experiment protects more efficiently.

Example 9. Creating a candidate TB vaccine, based on the mutation is a deletion of the gene fadD26

M. tuberculosis strains used to create the mutant with deletion of the gene fadD26 (∆fadD26)are the SO2 strain, which contains the phoP gene, an inactivated by insertion resistant to kanamycin cassette, and a clinical strain MT.

1. Creating plasmids

1.1. Cloning of the gene fadD26, which is involved in the synthesis of DIM. Gene fadD26 amplified by PCR using genomic DNA from M. tuberculosis H37Rv and using primers fadD26Fw (SEQ ID NO:1) and fadD26Rv (SEQ ID NO:2). To create plasmids AZ1 the PCR product was integrated into the vector GEM-T Easy (Promega).

1.2. The deletion of the gene fadD26 and the insertion resistant hygromycin cassette. To create plasmids AZ3 in plasmid AZ1 between sites BamHI-EcoRV gene fadD26 built in fragment BamHI-EcoRV plasmids pWM27 (Malaga et al. 2003), which contains the cartridge res-Ωhyg-res (res sites recognized resoluti γδ will make it possible to remove resistant marker at the second subculture).

1.3. The creation of the suicide vector for inactivation of the gene petergabriel recombination. Plasmid AZ3 were digested with restriction enzyme XhoI, releasing the insertion of fadD26::Ωhyg, which was built in the vector pJQ200X, linearized by the same enzyme. The final plasmid was named AZ5.

2. The development of strains of M. tuberculosis DIM-

2.1. Plasmid AZ5 built in strains of M. tuberculosis SO2 and MT.

2.2. Selection of single recombinants. Cultivation in hygromycin (20 µg/ml) bacteria, which include plasmid and tested for their resistance to gentamicin (10 μg/ml).

2.3. Selection of double recombinants. Cultivation of single recombinants in a 2% sucrose solution (Pelicic et al. 1997) and hygromycin and tested for their sensitivity to gentamicin.

3. Remove from the mutation ∆fadD26 token resistance to the antibiotic.

3.1. To remove the cassette res-Ωhyg-res and production of mutations without token resistance to the antibiotic embed plasmid pWM19, which contains resolvase γδ, and are breeding for resistance to gentamicin, after which the plasmid is removed, incubare at 39°C in a 2% sucrose solution (Malaga et al. 2003).

Example 2.2. The strain of M. tuberculosis was used to create the double mutant by deletion ∆phoP ∆fadD26, is MT ∆fadD26.

4. Creating plasmids

4.1. Cloning of the gene phoP. Gene phoP amplified by PCR using genomic DNA of M. tuberculosis H37Rv and primers phoPF (SEQ ID NO:3) and phoPR (SEQ ID NO:4). To create plasmids AZ11 the PCR product vstra the Wali in the vector pGEM-T Easy (Promega).

4.2. The deletion of phoP gene and the insertion resistant to kanamycin cassette. To create plasmids AZ13 fragment BamHI-EcoRV plasmids pCG122 (Malaga et al. 2003), which contains the cartridge res-Ωkm-res, was built between sites Bc/I-EcoRV phoP gene in the plasmid AZ11.

4.3. The creation of the suicide vector for inactivation of the gene using homologous recombination. Plasmid AZ13 were digested with restriction enzyme XhoI, releasing the insertion of phoP::Ωkm, which was built in the vector pJQ200X, linearized by the same enzyme. The final plasmid was named AZ15.

5. The creation of a strain of M. tuberculosis with a double mutation ∆phoP ∆fadD26.

5.1. Plasmid AZ15 should be integrated into a strain of M. tuberculosis MT ∆fadD26.

5.2. Selection of single recombinants. Cultivation in kanamycin (20 μg/ml) bacteria, which include plasmid and tested for their resistance to gentamicin (10 μg/ml).

5.3. Selection of double recombinants. Cultivation of single recombinants in a 2% sucrose solution (Pelicic et al. 1997) and kanamycin and tested for their sensitivity to gentamicin.

6. Remove from the mutation ∆phoP token resistance to the antibiotic.

6.1. To remove the cassette res-Ωkm-res and production of mutations without token resistance to the antibiotic you need to embed plasmid pWM19, which contains resolvase γδ, and make a selection on resistance to hygromycin (20 μg/ml), after which the plasmid has to be removed by incubation pri°C in 2% sucrose solution (Malaga et al. 2003).

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39. McShane, H., Pathan, A.A., Sander, C.R. et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004, 10(11), 1240-1244.

40. Kamath, A.T., Fruth, U., Brennan, M.J. et al. New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development. Vaccine 2005, 23(29), 3753-3761.

1. The recombinant microorganism of Mycobacterium tuberculosis complex, characterized in that it contains the inactivation or deletion of the gene phoP and inactivation or deletion of the gene fadD26 for prevention of tuberculosis in humans or animals.

2. Method of constructing a recombinant microorganism according to claim 1, which includes:
a) inactivation or deletion of the gene phoP; and
(b) inactivation or deletion of the gene fadD26.

3. Vaccine for immunization against tuberculosis, which contains an effective amount of the recombinant microorganism according to claim 1 and a pharmacologically acceptable excipients.

4. A method of obtaining a vaccine according to claim 3 for immunization against tuberculosis, which includes at least:
a) introducing the recombinant microorganism according to claim 1 in a suitable environment for introducing a person or animal in t is repitions effective dose;
b) adding the excipients that are pharmaceutically acceptable for the receipt of vaccines.

5. The application of the recombinant microorganism according to claim 1 for the prevention of tuberculosis in humans or animals.

6. The application of the recombinant microorganism according to claim 1 as a vaccine for prevention of tuberculosis in humans or animals.

7. The application of the recombinant microorganism according to claim 1 to obtain a vaccine according to claim 3 for the prevention of tuberculosis in humans or animals.



 

Same patents:

FIELD: agriculture.

SUBSTANCE: invention represents reference strains Francisella tularensis of tularensis subtype, including two genetic groups AI and AII, of holartica subtype, including biovars japonica, erythromycin-sensitive and erythromycin-resistant, Erys and EryR, and of mediasiatica subtype. Strains are deposited in the State Collection of Pathogenic Microbes "Microbe". All strains are natural, apart from the strain of holartica subtype, biovar japonica, which is extracted from a human being. Strains are produced by selection of clones that stably preserve phenotypic properties and containing genes, nucleotide sequence of which is specified for the type and subtype. The set of reference DNA preparations is created on the basis of these strains and may be used for genetic and immunological research.

EFFECT: using the invention will make it possible to increase efficiency of diagnostic research, to standardise and to increase quality of diagnostic preparations at production stages.

4 cl, 4 dwg, 1 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: invention method shall be implemented by inoculation of clinical material from incubation to the surface of a plate-like chromogenic medium Uriselect which is a non-selective agar medium rich in peptone and tryptophane, produced by Bio-Rad (France) by sectorial inoculation method. The culture plate shall be incubated in anaerobic and microaerophilic (5%-10% CO2) conditions, topt =+35+37ºC, for 48-72 hours. If required, repeated inoculation of the grown colonies of Leptotrichia isolates shall be performed, wherein the passage shall be performed by hatching and/or by a sterile cotton pad. The passages shall be incubated in the same conditions. The method for Leptotrichia cultivation using the Uriselect medium enhances the efficiency of Leptotrichia isolates from the clinical material and enables presumable identification of Leptotrichia by substrate color changes, cut strain loss in the event of repeated passages. The cultivation efficiency of the difficult-to-cultivate Leptotrichia - oral microbiocenosis residents in a human oral cavity is over 30%, the method being easy to implement and does not require costly equipment.

EFFECT: increased efficiency of Leptotrichia cultivation using the Uriselect medium, significant cost reduction.

3 cl, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is offered in application of at least one strain of probiotic bacteria specified in Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillus plantarum HEAL 99, DSM 15316, Lactobacillus paracasei 8700:2, DSM 13434 and Lactobacillus paracasei 02A, DSM13432 for preparing a composition for treating and/or preventing a viral infection caused by 'cold' virus, and a related method for treating and/or preventing. A number of days for which 'cold' symptoms are experienced has been also reduced in a group of patients having been taking a probiotic.

EFFECT: what is shown is intensified immune protection against antiviral infection that promotes decreasing 'cold' episodes in comparison with the controls.

16 cl, 13 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is offered in application of at least one strain of probiotic bacteria specified in Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillus plantarum HEAL 99, DSM 15316, Lactobacillus paracasei 8700:2, DSM 13434 and Lactobacillus paracasei 02A, DSM13432 for preparing a composition for treating and/or preventing a viral infection caused by 'cold' virus, and a related method for treating and/or preventing. A number of days for which 'cold' symptoms are experienced has been also reduced in a group of patients having been taking a probiotic.

EFFECT: what is shown is intensified immune protection against antiviral infection that promotes decreasing 'cold' episodes in comparison with the controls.

16 cl, 13 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological fertilisers for plants, specifically to a microbiological composition based on Rhizobium (symbiotic nitrogen fixer) legume bacteria. The composition contains Rhizobium lupini bacteria and associative nitrogen-fixing bacteria Flavobakterium, with components in weight ratio of 1.0-1.5:1.5-2.0. Due to synergetic interaction of the components, the composition has high physiological activity and has not phytotoxic effect on crop plants. The invention enables to efficiently increase plant biomass in mixed legume-grass seeds by approximately 7.1-7.8% compared to inoculation of seeds with legume bacteria, and by 8.9-9.9% compared to inoculation of seeds with associative bacteria. The degree of inhibition of crop plants or damage falls 1.4-8-fold compared to control crop plants.

EFFECT: microbiological composition increases accumulation of green material by 87-91 centner per hectare or 20-23% compared to control plants - yield in mixed lupine seeds and barley without inoculation of seeds with microbiological preparations.

2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological fertilisers for plants, specifically to a microbiological composition based on Rhizobium (symbiotic nitrogen fixer) legume bacteria. The composition contains Rhizobium lupini bacteria and associative nitrogen-fixing bacteria Flavobakterium, with components in weight ratio of 1.0-1.5:1.5-2.0. Due to synergetic interaction of the components, the composition has high physiological activity and has not phytotoxic effect on crop plants. The invention enables to efficiently increase plant biomass in mixed legume-grass seeds by approximately 7.1-7.8% compared to inoculation of seeds with legume bacteria, and by 8.9-9.9% compared to inoculation of seeds with associative bacteria. The degree of inhibition of crop plants or damage falls 1.4-8-fold compared to control crop plants.

EFFECT: microbiological composition increases accumulation of green material by 87-91 centner per hectare or 20-23% compared to control plants - yield in mixed lupine seeds and barley without inoculation of seeds with microbiological preparations.

2 tbl

FIELD: medicine.

SUBSTANCE: method by invention includes cloning if analysed sequence in gene gyrB DNA M. tuberculosis (MBT) by method of polymerase chain reaction (PCR), with further denaturation of PCR-product in presence of denaturing solution for obtaining single-stranded DNA fragments. After that mutations are detected in them by separation of said fragments in polyacrylamide gel. In order to carry out PCR a pair of primers was selected: "external" F1 -5' AGA GTT GGT GCG GCG TAA GA 3', R1 - 5' AAC ACA TGC CCG TTC TCG AT 3' and "internal" F2 - 5' TAA GAG CGC CAC CGA CAT CG 3', R2 -5' GCA TGA ACC GGA ACA ACA AC 3', and amplification modes (1-st stage -94° - 4 min; 2-nd stage - 94° - 30 sec, 59° - 40 sec, 72° - 40 sec (25 cycles); 3-rd stage: 72° - 4 min; 10° - storage), making it possible to clone DNA M. tuberculosis from not only grown cultures, but directly from biological secretions (sputum, BAL). Denaturation of obtained amplicons is carried out in denaturing solution, destabilising double helix at heating. Electrophoretic separation of obtained single-strand fragments of DNA is carried out in 8% polyacrylamide gel with 5% glycerol with voltage 400 V during 5 hours at 8°C. Presence of mutations in examined gene is determined by comparing degree of divergence of denatured DNA strands of analysed and sensitive strains of M. tuberculosis to fluorochinolones.

EFFECT: method makes it possible to determine sensitivity of MBT to fluorochinolones in shorter terms, it is available and safe.

1 dwg

FIELD: medicine.

SUBSTANCE: nutrient medium contains chicken eggs, 3-% extract of wood ash, 2-% water solution of malachite green and peat oxidate.

EFFECT: invention makes it possible to reduce term of paratuberculosis mycobacteria detection.

4 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: sample is prepared: an additional weight of nanocarbon forms is dispersed in 1 ml of organic solvents with a degree of polarity smaller than that of water - dimethyl sulphoxide or ethanol. Then it is mixed and exposed to ultrasound for 30 minutes. The prepared nanocarbon suspension is transferred in an aqueous medium to the final concentration of the used solvent 2.5 %. The produced and control samples are added with a viable sensor recombinant luminescent Escherichia coli K12 strain with cloned luxCDABE genes of luminescent Photobacterium leiognathi system. It is followed by incubation for 60 - 180 minutes, measuring luminous intensity and evaluating optical properties of the analysed suspension simultaneously. A toxicity index (T) is calculated with evaluating an actual luminous intensity of the strain (Iact) in comparison with the control of the same concentration of the solvent, considering light absorbing properties of the analysed suspension (D) and an experimental luminescence level of the bacterial luminescent biosensor (Iexp).

EFFECT: invention allows providing higher accuracy and sensitivity of nanocarbon biotoxicity analysis ensured by the introduction of a correction value - the actual luminous intensity of the strain Iact, considering a common factor of emitted light distribution in the analysed suspension.

1 ex

FIELD: medicine.

SUBSTANCE: method involves cultivation of an obligate methanol-assimilating bacterium Methylophilus methylotrophus or Methylobacillus glycogens in a fluid medium with the bacterium secreting an end protein from a bacterial cell where said bacterium has a DNA structure containing a promoter sequence functioning in the methanol-assimilating bacterium, a nucleotide sequence coding a polypeptide containing a signal sequence which functions in the methanol-assimilating bacterium, and a sequence of the end protein functionally connected with the promoter sequence.

EFFECT: method allows producing the protein effectively by means of extracellular secretion, difficult-to-produce by means of secretory production with application of Escherichia coli bacteria.

5 cl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention refers to the area of biotechnology and dairy products industry. Streptococcus thermophilus strains shall be obtained from the surface of the plant called stone pink. The strain is deposited with the Russian National Collection of Industrial Microorganisms (RNCIM) at registration number RNCIM-B 10089. The strain shall be cultivated in culture medium of firm and liquid texture milk.

EFFECT: enhanced fermented milk products preparation- Mechnkov curdled milk and sour cream with 10% fat content - using lactic bacteria with high antagonistic activity.

2 ex

FIELD: immunology.

SUBSTANCE: invention refers to the area of microbiology and immunology and provides attenuated Mycoplasma gallisepticum bacteria, vaccine composition as well as methods of vaccinating an animal, which strain was deposited with the American Type Culture Collection (FNCC) and has been assigned Accession No. PTA-8485.

EFFECT: reduced expression of protein, identified as MGA_0621, which was shown to exhibit significantly reduced expression of MGA_0621, and was shown to be safe and effective when administered as a vaccine against M. gallisepticum infection.

4 cl, 1 dwg, 5 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention method shall be implemented by inoculation of clinical material from incubation to the surface of a plate-like chromogenic medium Uriselect which is a non-selective agar medium rich in peptone and tryptophane, produced by Bio-Rad (France) by sectorial inoculation method. The culture plate shall be incubated in anaerobic and microaerophilic (5%-10% CO2) conditions, topt =+35+37ºC, for 48-72 hours. If required, repeated inoculation of the grown colonies of Leptotrichia isolates shall be performed, wherein the passage shall be performed by hatching and/or by a sterile cotton pad. The passages shall be incubated in the same conditions. The method for Leptotrichia cultivation using the Uriselect medium enhances the efficiency of Leptotrichia isolates from the clinical material and enables presumable identification of Leptotrichia by substrate color changes, cut strain loss in the event of repeated passages. The cultivation efficiency of the difficult-to-cultivate Leptotrichia - oral microbiocenosis residents in a human oral cavity is over 30%, the method being easy to implement and does not require costly equipment.

EFFECT: increased efficiency of Leptotrichia cultivation using the Uriselect medium, significant cost reduction.

3 cl, 1 tbl, 2 ex

FIELD: biochemistry.

SUBSTANCE: method includes separate culturing of strains of lactic bacteria and bifidus bacteria on bouillon containing digest of casein produced by hydrolysis using cattle pancreas, carbohydrate component containing lactose and raffinose in ratio 1:1. Raffinose is both a carbohydrate component and probiotic component. The medium contains peptone, agar-agar, ascorbic acid and distilled water. The mixture of strains is added to sorbing component prepacked in vial in proportion of ½ - ¼ from total volume of created complex system. Process of immobilization on sorbent - Litovit M dietary supplement is performed at temperature +6°C±2°C for 18-24 hours.

EFFECT: production of immobilized synbiotic product with high content of live microbial cells, several strains of lactic bacteria and bifidus bacteria with increased protective and treatment activity.

3 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: nutrient medium base for lactobacilli strain selection either by histamine reduction in a culture medium, or by histamine gain reduction in the culture medium represents a sterile deoxidated citric casein lactoserum containing copper (II) cations in amount 30-1000 mcmol/l, histamine in amount 200-800 mcmol/l. Said nutrient medium base has pH 6.5-7.4. In addition, the base can contain target additives for preparing liquid, or semi-liquid, or solid nutrient medium in amount 0.2-300 g/l. When culturing a passed Lactobacillus acidophilus KAA strain version, the histamine level in the nutrient medium makes 512.2±201.9 mcmol/l, Lactobacillus acidophilus NKJC strain - 64.7±23.9 mcmol/l, Lactobacillus acidophilus JCH strain - 135.6±52.6 mcmol/l.

EFFECT: invention enables lactobacilli strain selection by histamine reduction or by histamine gain reduction in the differential diagnostic high-histamine nutrient medium during passing these strains.

2 cl, 4 ex

FIELD: medicine.

SUBSTANCE: nutrient medium base for lactobacilli strain selection either by histamine reduction in a culture medium, or by histamine gain reduction in the culture medium represents a sterile deoxidated casein lactoserum containing copper (II) cations in amount 30-1000 mcmol/l, histamine in amount 200-800 mcmol/l. Said nutrient medium base has pH 6.5-7.4. Serum is deoxidated chloride casein lactoserum, deoxidated lactic casein lactoserum or their mixture. In addition, the base can contain target additives for preparing liquid, or semi-liquid, or solid nutrient medium in amount 0.2-300 g/l. When culturing a passed Lactobacillus acidophilus KAA strain version, the histamine level in the nutrient medium makes 485.9±195.1 mcmol/l, Lactobacillus acidophilus NKJC strain - 69.2±28.5 mcmol/l, Lactobacillus acidophilus JCH strain - 129.6±48.4 mcmol/l.

EFFECT: invention enables lactobacilli strain selection by histamine reduction or by histamine gain reduction in the differential diagnostic high-histamine nutrient medium during passing these strains.

2 cl

FIELD: food industry.

SUBSTANCE: invention is related to food industry, in particular, to production of mince. In the process of the semi-products production one introduces Lactococcus lactis technological bacterial suspension into the composition of raw material which consists of chopped components. Prior to introduction of the bacterial suspension one performs its preparation no more than 24 hours in advance. The chopped components are represented by chopped components of raw meat and/or by-products and/or poultry and/or wild fowl and/or fish and/or seafood and/or reptiles.

EFFECT: invention ensures development of conditions for cultivation and adaptation of cell population during production of an active starter based on Lactococcus lactis bacteria and promoting improvement of taste-and-flavour properties of meat mince.

16 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to strain of microorganism, ensuring recovery of soil and animal gastrointestinal tract (GIT) microbiocenosis, possessing antibacterial and fungicidal activity, and to based on them preparation form and can be used in biotechnology, veterinary medicine and plant protection. Strains Bacillus licheniformis IC-831-1-2, Bacillus licheniformis IC-832-1-2, Bacillus licheniformis IC-833-1-2, Bacillus licheniformis IC-834-1-2 are obtained by method of directed selection and are deposited in All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration numbers All-Russian collection of industrial microorganisms respectively: B-10561, B-10562, B-10563 and B-10564. Preparation is characterised by containing filling agent with bacteria biomass in spore form Bacillus licheniformis All-Russian collection of industrial microorganisms B-10561, or All-Russian collection of industrial microorganisms B-10562, or All-Russian collection of industrial microorganisms B-10563, or All-Russian collection of industrial microorganisms B-10564, or their mixture with titre of each bacteria strain not less than 1·103 CFU/g or 1·103 CFU/ml. As filling agent it contains water or powder-like sorbent.

EFFECT: invention makes it possible to increase efficiency of restoring soil, animal GIT microbiocenosis and extend spectrum of antibacterial and fungicidal activity.

9 cl, 9 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological fertilisers for plants, specifically to a microbiological composition based on Rhizobium (symbiotic nitrogen fixer) legume bacteria. The composition contains Rhizobium lupini bacteria and associative nitrogen-fixing bacteria Flavobakterium, with components in weight ratio of 1.0-1.5:1.5-2.0. Due to synergetic interaction of the components, the composition has high physiological activity and has not phytotoxic effect on crop plants. The invention enables to efficiently increase plant biomass in mixed legume-grass seeds by approximately 7.1-7.8% compared to inoculation of seeds with legume bacteria, and by 8.9-9.9% compared to inoculation of seeds with associative bacteria. The degree of inhibition of crop plants or damage falls 1.4-8-fold compared to control crop plants.

EFFECT: microbiological composition increases accumulation of green material by 87-91 centner per hectare or 20-23% compared to control plants - yield in mixed lupine seeds and barley without inoculation of seeds with microbiological preparations.

2 tbl

FIELD: medicine.

SUBSTANCE: nutrient medium contains chicken eggs, 3-% extract of wood ash, 2-% water solution of malachite green and peat oxidate.

EFFECT: invention makes it possible to reduce term of paratuberculosis mycobacteria detection.

4 tbl, 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

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