KR101696514B1 - Temperature Sensitive Mycobacterial strain and its use as vaccine against mycobacterial infection disease - Google Patents

Abstract

This disclosure discloses a strain of M. paragordonae (strain 49061) which is a temperature-sensitive mycobacteria and vaccine compositions comprising it. The vaccine according to the present invention suppresses proliferation due to the characteristic that the vaccine stimulates the immune by proliferation in the skin and subcutaneously but can grow only at a temperature lower than the body temperature in the body, and the possibility of infection is low and the vaccine for preventing a disease caused by mycobacteria And can be used safely and usefully in immunotherapy.

Description

[0001] The present invention relates to a novel thermosensitive mycobacterial strain and a vaccine composition for mycobacterial infection including the same,

The present invention relates to vaccines against mycobacterial infections using temperature sensitive mycobacterial strains.

Mycobacterial (Mycobacterium) is composed of approximately 150 species of note, including Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis coaxial bird tuberculosis group (M. avium complex) and tu Mycobacterium tuberculosis (M. abscessus-chelonae complex). Among them, tuberculosis, tubercle bacillus, and mycobacteria are classified as absolute pathogens, while the remaining mycobacteria are classified as nontuberculous mycobacteria (NTM). Causing tuberculosis leprosy in humans, and various other diseases such as algae and livestock.

 Tuberculosis (TB) is still the leading cause of death worldwide, and the situation is getting worse due to the emergence of multi-drug resistance (MDR) Mycobacterium tuberculosis.

Tuberculosis can be controlled through long-term antibiotic treatment, but it is difficult to monitor the occurrence of resistance to antibiotics and the patient's compliance with the treatment procedure. In addition, carriers may infect others without symptoms, It is not enough to prevent the spread of bacteria.

Thus, effective vaccine prevention is a more effective way to prevent the spread of tuberculosis.

Among the non-tuberculous antibiotics except M. tuberculosis ( M. avium complex), the most frequently isolated isolate is tuberculosis M. abscessus-M. chelonae (Ryoo SW , Shin S, Shim MS et al., Spread of nontuberculous mycobacteria from 1993 to 2006 in Korean. J Clin Lab Anal. 2008; 22 (6): 415-20). Recently, it has been reported that M. massiliense ( M. abscessus subsp. Bolletii ) accounts for about half of the M. abscessus-M. chelonae complex (46.5%) in domestic isolated turtle tuberculosis (Kim HY , Kook Y, Yun YJ et al., Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-Mycobacterium abscessus group isolates. J Clin Microbiol 2008; 46 (10): 3384-90). Infections of M. abscessus are known to have a significantly higher rate of failure and mortality due to congenital resistance to antibiotics. Therefore, it is necessary to develop a new vaccine for M. abscessus .

BCG ( Mycobacterium bovis Bacille Calmette-Guerin) is the most widely used anti-tuberculosis vaccine. BCG is effective for tuberculosis in some infants, but its effect is limited for strong contagious tuberculosis found in adults. In addition, BCG has been reported to have side effects of lymphadenitis, local bone tissue infection, and systemic infection.

Recently, temperature-sensitive strains and attenuated live bacteria have been developed as vaccines against certain bacteria. In particular, the development of vaccines for temperature-sensitive strains is receiving more attention because there is a temperature gradient from the in vitro (skin, etc.) to the body (organs, etc.) of animals as well as humans. Although the ability to grow at relatively low temperatures, such as skin, has the ability to stimulate local immunity, a thermosensitive strain has been developed that can not grow in the body, such as organs (Barry et al., Temperature-sensitive bacterial pathogens J Mol Med. 2011; 89: 437-444). The most well-known of these is the Sabin polio vaccine and the FluMist iinfluenza vaccine (Maassab HF and Bryant ML, The development of live attenuated cold-adapted influenza virus vaccine for humans, Rev Med Virol 1999; 9: 237-244). In addition, temperature-sensitive strains have been developed and studied for veterinary purposes (Chalmers et al., Vet Rec. 1997; 141: 63-67, Jackwood and Saif, Avian Dis 1985; 29: 1130-1139, Morrow et al. al., Avian Dis. 1998; 42: 667-670). However, temperature sensitive strains through artificial genetic modification are low in stability and difficult to maintain.

WO 1998-056931 discloses immunogenic or vaccine compositions using attenuated recombinant mycobacteria, and discloses vaccine compositions using M. tuberculosis recombinant strains in which genes related to purine and pyrimidine life events are inactivated.

US Patent Publication No. 2013-0101623 discloses a novel mycobacterial strain and tuberculosis vaccine composition, which discloses a vaccine composition comprising attenuated M. tuberculosis .

Development of new vaccine compositions based on new strains different from the prior art is required.

The present invention provides vaccines against mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium tuberculosis including a group of turtle tuberculosis bacteria based on new strains.

In one embodiment herein there is provided a pick mycobacterial para accession number KCTC 12628BP Tone (Mycobacterium paragordonae, strain 49061) strain.

In another aspect, the disclosure also provides a vaccine composition for mycobacterial infection comprising a temperature sensitive strain according to the invention and a pharmaceutically acceptable excipient.

The vaccine composition according to the present invention may be used as a vaccine composition for mycobacterial infection such as lung disease, lymphadenitis, soft tissue, osteoporosis or disseminated disease. Mycobacteria causing such diseases include NTM (nontuberculous mycobacteria), fast growing NTM, Mycobacterium tuberculosis or Mycobacterium bovis , and the NTM is Mycobacterium chelonei complex, ≪ RTI ID = 0.0 > M. avium complex (MAC), M. avium , M. intracellulrare , M. kansasii , M. kansasii , M. abscessus , or M. massiliense , but not limited thereto.

The temperature sensitive mycobacteria M. paragordonae (strain 49061) according to the present invention and the vaccine composition containing the same can be effectively used for prevention of mycobacterial infections including tubercle bacilli, tubercle bacillus, and right tubercle bacillus. The body temperature has a grading tendency of increasing the temperature from the skin to the body organs. The vaccine according to the present invention stimulates the immune by proliferating in the skin and subcutaneously in the inoculation. However, in the center of the body, The proliferation is suppressed and the possibility of infection is low. Therefore, it can be safely and usefully used for prevention vaccine against all mycobacterial infections including Mycobacterium tuberculosis and immunotherapy.

Fig. 1 shows the results of a systematic analysis of 16 sRNA, rpoB and hsp65 , and Fig. 1b to Fig. 1d are sequences of 16sRNA, rpoB and hsp65 of the strain according to the present invention, respectively, as a result showing that the strain according to the present invention is novel.
FIGS. 2A and 2B are graphs illustrating the growth characteristics of M. paragordonae in the 7H9 liquid medium at 30 ° C. and 37 ° C., respectively, in comparison with M. asiaticum , M. gordonae , and M. marinum .
Figure 3 shows the results of an in vitro infection test on the J774 murine macrophage cell line of M. gordonae , M. marinum, and M. paragordonae according to one embodiment of the invention. The cells were lysed at 30 ° C and 37 ° C, and after 1 day, 3 days and 5 days, the cells were lysed in 7H10 solid medium and cultured at 30 ° C. And then counting the number of grown colonies. The left graph is a graph comparing the results of M. paragordonae , M. gordonae and M. marinum , and the right graph is a graph comparing the differences between M. paragordonae and M. gordonae .
FIG. 4 shows the results of infection of mice with M. gordonae , M. marinum and M. paragordonae according to one embodiment of the present invention , followed by liver and spleen harvesting, homogenization, and culturing at 7O < 0 > to be.
Figure 5 shows the results of a vaccine test using M. paragordonae . Figure 5a will schematic representation of the vaccination process, Figure 5b were inoculated two times with subcutaneous injection of M. paragordonae the mouse (BALB / c, 7-week-old, n = 4) tail base part according to Figure 5a M. abscessus associated strains (M. abscessus subsp. bolletii 50594) to attack (tail vein injection), and organs in each organ by date in M. abscessus subsp. bolletii 50594 bacteria were measured. Figures 5c / 5d show M. abscessus subsp. The results of IFN-gamma ELISPOT on days 3 and 14 after bolletii 50594 challenge.

The present invention is based on the discovery of a novel mycobacterial strain which is a slowly growing cancer-causing microorganism.

The strain according to the present invention was isolated from a patient suffering from pulmonary infection. As a result of 16 sRNA analysis, acid fastness analysis, growth characteristics and phylogenetic analysis, it was found to be a strain close to Mycobacterium gordonae (99.0% sequence homology) belonging to the mycobacterial group .

The strains isolated from this study were morphologically similar to Mycobacterium gordonae , but the strains of this strain were temperature - sensitive strains that did not grow at 37 ℃ and optimum growth temperature was 25-30 ℃. Also, as a result of analysis of gene analysis based on lipid analysis, hsp65 and rpoB, and DNA-DNA association analysis, it was found to be a new strain, named Mycobacterium paragordonae and deposited on July 17, 2014 with the deposit number KCTC 12628BP Was deposited.

The novel temperature sensitive mycobacteria M. paragordonae (strain 49061) according to the present invention can be effectively used to prevent infection against mycobacteria, and thus in one embodiment, the present invention encompasses novel temperature sensitive mycobacteria M. paragordonae (strain 49061) A vaccine or a composition for immunotherapy.

Mycobacteria are known to be causative agents of pulmonary diseases such as tuberculosis, lymphadenitis, skin and soft tissue infections, bone infections or disseminated diseases. For example, mycobacteria belonging to the Mycobacterium chelonae M. abscessus complex cause immunosuppressive skin and soft tissue infections, tuberculosis, blood inflammation or abscesses in immunologically or immunocompromised subjects (Griffith DE et al. , Am Rev Respir Dis 1993; 147: 1271-8; Wallace RJ Jr., et al., Rev Infect Dis 1983; 5: 657-79).

The vaccine according to the present invention can be used as a vaccine against such a disease for mycobacterial infection. In one embodiment it exhibits a protective effect against infections of mycobacteria including NTM (nontuberculous mycobacteria), Mycobacterium tuberculosis , or Mycobacterium bovis . In other embodiments, the NTMs include, for example, Mycobacterium chelonei complex, M. avium complex (MAC), M. avium , M. intracellulrare , M. kansasii , M. abscessus, or M. massiliense . Also, the NTMs in which the vaccine of this invention is effective include the fast-growing NTM.

In one embodiment according to the present application, the vaccine composition of the present invention is a vaccine against a disease caused by mycobacteria, including Mycobacterium chelonae M. abscessus complex, Mycobacterium tuberculosis or Mycobacterium bovis , Can be used.

The vaccine composition according to the present invention can induce an immune response against mycobacteria, for example, antibody production and / or T-cell response, thereby preventing mycobacterial infection including tubercle bacilli, tubercle bacillus, Alleviate, relieve and / or treat symptoms due to infection.

Mycobacteria M. paragordonae (strain 49061) used in the composition according to the present invention is as described above, and live bacteria or chemically or thermally killed dead bacteria can be used. In the case of live cells, those that have undergone attenuation can be used. The attenuation can be performed according to known methods, thereby reducing or eliminating the virulence of the bacteria.

The vaccine composition according to the present invention may be formulated for systemic or topical administration and may be formulated with pharmaceutically acceptable excipients, carriers and / or vehicles such as phosphate buffered saline solution, distilled water, water / oil emulsion, wetting agent, emulsifying agent, pH And the like, but are not limited thereto.

Vaccine compositions according to the present invention may also comprise any substance or compound that is capable of promoting or augmenting a multisite, particularly T-cell mediated response. In one embodiment, the zvant may be used in the case where a chemical or thermally killed microorganism is included in the composition. Azubent is well known in the art and includes, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, crosslinked polyacrylic acid polymers, dimethyldioctadecyl-ammonium bromide, immunomodulators, lactoferrin, or IFN- Can be used. In one embodiment, the crosslinked polyacrylic acid polymer comprises a Carbopol 占 homopolymer or copolymer, wherein the lymphocaine comprises IFN-gamma, IL-1, IL-2 or IL-12, Inducers can include, but are not limited to, poly I: C. In one embodiment, the synthesized sugar polymer comprises about 0.05 to 0.1 wt% of the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate, and the crosslinked polyacrylic acid polymer may include, but is limited to, 0.25 wt% It is not.

The compositions according to the present invention may be administered topically or systemically and may be single or multiple doses and may be administered by various routes such as subcutaneous, intradermal, intramuscular or intravenous, or by oral, nasal or inhalation routes have. In one embodiment according to the present application, it is administered once by intramuscular injection.

The vaccine composition according to the present invention may be formulated as a liquid solution or suspension suitable for the route of administration or as a solid form dissolved or suspended in a solution prior to injection.

The dosage of the vaccine will depend on the route of administration as well as the age, weight and / or health condition of the patient. For example, a suitable dose may be, for example, 1 to 10 cfu.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Example 1 Strain Isolation and Identification

As described previously ( M. et al., (2008). Mycobacterium senuense sp. Nov., A slowly growing, non-chromogenic species closely related to the Mycobacterium terrae complex. Int J Syst Evol Microbiol 58, 641-646).

The isolated strains were subjected to biochemical assays as described previously for Mycobacterium asiaticum ATCC 25276T and M. gordonae ATCC 14470T (Kent & Kubica, (1985).) Public Health Mycobacteriology: a Guide for the Level III Laboratory Atlanta: Centers for Disease Control and Prevention, 1985; Pfyffer, Mycobacterium : General characteristics, laboratory detection, and staining procedures. PRMurray, EJ Baron, JH Jorgensen, ML Landry & MA Pfaller, Washington: American EX Society for Microbiology.).

Nitrate accumulation, nitrate reductase, Tween 80 hydrolysis, urease and pyrazinamidase, thiophene-2-carboxylic acid hydrazide (TCH) Resistant, PNB (p-nitrobenzoate), 5% sodium chloride, ethambutol and picric acid analyzes were performed for 6 weeks on a Middlebrook 7H10 agar plate supplemented with OADC (Oleic Albumin Dextrose Catalase).

The results are shown in Table 1.

[Table 1]

In Table 1, 1, 2, and 3 represent the strains used: 1, the strain M. paragordonae (strain 49061); 2, M. asiaticum ATCC 25276 ^T ; 3, M. gordonae ATCC 14470 ^T. The reference characters are as follows: Strong growth; ++, good growth, +, positive growth; 2, negative / no growth; ±, variable. All strains showed an active colonization and did not grow at 45 ℃.

Microbial spores and filaments were not observed in the strains showing rod - shaped acid - fastness with many morphologically bent shapes. The optimum growth temperature was 25-30 캜, and did not grow at 37 캜 or 45 캜. On the Middlebrook 7H10 agar plate, orange-colored spike colonies were observed. It did not grow in the medium containing picric acid, and the urease activity was positive. The most relevant species in the DNA-DNA association analysis was less than 70%. In lipid analysis using MALDI-TOF MS, it was found to be different from other species and had a unique sequence in 16S rRNA (1393 bp), rpoB (306 bp) and hsp65 (603 bp) And 1d). In addition, strains isolated through phylogenetic analysis were found to be slow-growing mycobacteria. 16S rRNA and hsp65 were closely related to M. gordonae , but their nucleotide sequence homology was 99% and 95.9%, respectively . Specificity was similar to that of M. gordonae , And the rpoB gene sequence was close to that of M. asiaticum. However, it was confirmed that the rpoB gene sequence was 95.4% homologous with this specific sequence. (Fig. 1A). These results indicate that the strain according to the present invention is novel.

This bacterium was named Mycobacterium paragordonae and deposited on July 17, 2014 with the deposit number KCTC 12628BP at the Center for Microbial Resources, Korea Research Institute of Bioscience and Biotechnology.

Example 2 Mycobacterium paragordonae  Identification of temperature sensitivity of strains and safety analysis

The following experiments were performed to demonstrate the safety of inoculation with live vaccine.

First, in order to confirm that the temperature-sensitive Mycobacterium strain ( Mycobacterium paragordonae , strain 49061) did not grow at 37 ° C, the initial inoculum amount with the other comparative strains ( M. gordonae , M. asiaticum and M. marinum ) (OD ₆₀₀ ) at 30 ° C and 37 ° C in a liquid medium (Middlebrook 7H9), and the growth was measured by time (1, 3, 5 and 7 days). The results are shown in FIG. As shown, the temperature sensitive mycobacteria M. paragordonae (strain 49061) did not grow at a temperature corresponding to normal body temperature (37 ° C) but it was found to grow well at a lower temperature (30 ° C).

Next, infection analysis was performed. Infection analysis showed that M. paragordonae strains did not grow at 37 ℃ in liquid medium and solid medium culture. Therefore, it is necessary to confirm whether or not the M. paragordonae strain shows a temperature-dependent difference even when infected with large macrophages. To this end, the cells were infected (at a concentration of 1 × 10 ⁷ , 10 MOI) in a macrophage (J774 cell line, ATCC TIB-67 ^™ ) at 30 ° C. and 37 ° C. PBS was added and the cells were removed by pipetting). The cells were cultured at 7O < 0 > C in a 7H10 solid medium and counted.

The results are shown in FIG. As shown in the figure, M. paragordonae infected at 37 ℃ did not grow much, unlike the comparative strains.

In addition, M. paragordonae and comparative strain (10 ⁶ CFU) were intravenously injected into BALB / c mice (7 weeks old, n = 3) and infected with liver and spleen at 1, 4 and 7 days Phosphate buffer solution, plated in 7H10 solid medium, and cultured at 30 ° C to measure CFU.

The results are shown in FIG. 4A, and as shown therein, the growth of M. paragordonae in the liver or spleen was found to be almost inactive compared to the other strains, indicating that the strains were safe for use in vivo .

In addition, M. paragordonae and a comparative strain ( M. gordonae , M. marinum or M. bovis BCG) (10 ⁶ CFU) were intravenously injected into BALB / c mice (7 weeks old, n = 3) The liver and spleen were harvested, homogenized on days 1, 4 and 7 (when compared to M. gordonae , M. marinum ) or on days 1, 7 and 14 (compared to M. bovis BCG), diluted with phosphate buffer, Plates were plated on solid medium and cultured at 30 캜 to measure CFU.

The results are shown in FIG. 4b, and as shown therein, the growth of M. paragordonae in the liver, lung or spleen was found to be scarcely grown compared to other strains.

Example 3 Mycobacterium paragordonae  Vaccine analysis

Subsequently, the vaccine analysis was performed as follows. First, 10 ⁶ CFU of M. paragordonae (strain 49061) was intravenously administered to the tail twice (0 and 8 weeks) in an ALB / c mouse (7 weeks old, n = 4) as in the procedure of FIG. PBS and BCG were used as controls. After 12 weeks, M. abscessus ( M. abscessus subsp. Bolletii 50594) ~ 10 ⁶ CFU was intravenously injected into the tail vein. After 3 days and 14 days, each organ (liver, spleen, lung) M. abscessus subsp. The CFU of bolletii 50594 was measured as in Example 2, and after 14 days the spleen was used, the M. abscessus subsp. The ELISPOT was performed to measure the number of IFN-γ-secreting cells treated with the whole protein of the bolletii 50594 strain. Specifically, IFN-γ antibody (eBioscience, Cat., 16-7313-85, 3 μg / ml) was added to a 96-well plate with PVDF membrane and incubated at 4 ° C for one day. The next day, each well was washed and then filled with complete medium (DMEM + antibiotic + 10% FBS) and incubated at 37 ° C. Spleen cells from each mouse were harvested and spleen cells were obtained with a 70 μm cell strainer. Spleen cells of each group were adjusted to about 5 × 10 ⁵ cells and added to each well of a 96-well PVDF-coated plate. Following M. abscessus subsp. bolletii 50594 Total protein 10 μg / ml was treated with antigen and the remainder treated with complete medium. Phosphoryl myristate acetate (5 ng / ml) and ionomycin (500 ng / ml) were treated as a positive control. After incubation at 37 ° C for one day, the cells were treated with the detection antibody (eBioscience, Cat., 13-7311-85, 3 μg / ml) for each well. After incubation at 4 ° C for one day, each well was washed, treated with streptavidin-HRP, incubated at room temperature for 2 hours, washed, and spotted using an AEC substrate color development kit (Dako) .

The results are shown in Figures 5b, 5c and 5d. In Fig. 5b, M. abscessus subsp in mouse organs in which the vaccine M. paragordonae. The CFU of bolletii 50594 showed lower CFU in mouse organs vaccinated with PBS or other M. gordonae and M. bovis BCG. In addition, more spot was observed in mice inoculated with M. paragordonae when ELISPOT was performed on IFN-γ. After the vaccine test, CFU results showed that M. paragordonae was resistant to M. abscessus subsp. bolletii 50594, which indicates the ability to defend against.

It is also shown that the ability of M. abscessus strains to protect against M. abscessus strains is higher by decreasing the CFU of M. abscessus strains compared with the existing M. bovis BCG strains. In addition, M. abscessus subsp. When the number of defense immune cells against bolletii 50594 was higher than that of BCG when M. paragordonae was used as a vaccine, ELISPOT confirmed that M. abscessus subsp. bolletii 50594, which is known to be effective against the tubercle bacillus.

Furthermore, since the vaccine according to the present invention is naturally sensitive to temperature, safety is secured and it can be effectively used as a vaccine against mycobacterial infections including tubercle bacilli, tuberculosis, .

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> Seoul National University R & DB Foundation <120> Temperature Sensitive Mycobacterial strain and its use as vaccine          against mycobacterial infection disease <130> DP201406003P <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1462 <212> DNA <213> M. paragordonae 16S rRNA sequence <400> 1 ggcggcgtgc ttaacacatg caagtcggac ggtaaggccc ttcggggtac acgagtggcg 60 aacgggtgag taacacgtgg gtaatctgcc ctgcacatcg ggataagcct gggaaactgg 120 gtctaatacc gaataggacc acagaacaca tgtcctgtgg tggaaagctt ttgcggtgtg 180 ggatgggccc gcggcctatc agcttgttgg tggggtgatg gcccaccaag gcgacgacgg 240 gtagccggcc tgagagggtg tccggccaca ctgggactga gatacggccc agactcctac 300 gggaggcagc agtggggaat attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt 360 gggggatgac ggccttcggg ttgtaaacct ctttcaccat cgacgaaggt tcgggttttc 420 tcggattgac ggtaggtgga gaagaagcac cggccaacta cgtgccagca gccgcggtaa 480 tacgtagggt gcgagcgttg tccggaatta ctgggcgtaa agagctcgta ggtggtttgt 540 cgcgttgttc gtgaaatctc acggcttaac tgtgagcgtg cgggcgatac gggcagactt 600 gagtactgca ggggagactg gaattcctgg tgtagcggtg gaatgcgcag atatcaggag 660 gaacaccggt ggcgaaggcg ggtctctggg cagtaactga cgctgaggag cgaaagcgtg 720 gggagcgaac aggattagat accctggtag tccacgccgt aaacggtggg tactaggtgt 780 gggtttcctt ccttgggatc cgtgccgtag ctaacgcatt aagtaccccg cctggggagt 840 acggccgcaa ggctaaaact caaagaaatt gacgggggcc cgcacaagcg gcggagcatg 900 tggattaatt cgatgcaacg cgaagaacct tacctgggtt tgacatgcac aggacgccgg 960 cagagatgtc ggttcccttg tggcctgtgt gcaggtggtg catggctgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgtctcat gttgccagcg 1080 ggtaatgccg gggactcgtg agagactgcc ggggtcaact cggaggaagg tggggatgac 1140 gtcaagtcat catgcccctt atgtccaggg cttcacacat gctacaatgg ccggtacaaa 1200 gggctgcgat gccgcgaggt taagcgaatc cttttaaagc cggtctcagt tcggatcggg 1260 gtctgcaact cgaccccgtg aagtcggagt cgctagtaat cgcagatcag caacgctgcg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcacg tcatgaaagt cggtaacacc 1380 cgaagccagt ggcctaaccc tttgggaggg agctgtcgaa ggtgggatcg gcgattggga 1440 cgaggtcgta acaaggtagc cg 1462 <210> 2 <211> 306 <212> DNA <213> M. paragordonae rpoB gene sequence <400> 2 tgcgtaccgt gggcgagctg atccagaacc agatccgggt cggcatgtcc cgcatggagc 60 gcgtcgtccg cgagcggatg accactcagg acgtcgaggc gatcacgccg cagaccctga 120 tcaacatccg gccggtcgtc gccgcgatca aggagttctt cggcaccagc cagctctcgc 180 agttcatgga ccagaacaac ccgctttcgg gcctcaccca caagcgtcgt ctgtcggcgc 240 tggggcccgg cggtctgtcc cgtgagcggg ccggcctgga ggtccgtgac gtccacccgt 300 cgcact 306 <210> 3 <211> 603 <212> DNA <213> M. paragordonae hsp65 gene sequence <400> 3 gaggacccgt acgagaagat cggcgccgag ctggtcaagg aagtcgccaa gaagaccgac 60 gacgtcgccg gtgacggcac cacgacggcc accgtgctgg cgcaggcgct ggtcaaggaa 120 ggcctgcgca acgtcgcagc cggtgccaac ccgctgggcc tgaagcgcgg catcgagaag 180 gccgtggaga aggtcaccca gaccctgctc tcctcggcca aggacgtcga gaccaaggag 240 cagatcgcgg ccaccgcggg catctccgcg ggcgaccagt cgatcggtga cctgatcgcc 300 gaggcgatgg acaaggtcgg caacgagggc gtcatcaccg tcgaggagtc caacaccttc 360 ggcctgcagc tcgagctcac cgagggcatg cggttcgaca agggctacat ctcgggctac 420 ttcgtcaccg acgccgagcg tcaggaagcg gtcctggaag acccctacat cctgctggtc 480 tccagcaagg tgtcgaccgt caaggacctg ttgccgttgc tggagaaggt cattcagggc 540 ggcaagccgc tgctgatcat cgccgaggac gtcgagggtg aggcgctgtc caccctggtg 600 gtg 603

Claims (10)

Mycobacterium paragordonae strain 49061 of Accession No. KCTC 12628BP.
M. the accession number KCTC 12628BP Te Solarium para pick by Tone (Mycobacterium paragordonae, strain 49061) strain and a pharmaceutically acceptable excipient containing, Mycobacterium app revenue's (M. abscessus) or M. tuberculosis (Mycobacterium tuberculosis) Vaccine composition for mycobacterial infection.
delete delete 3. The method of claim 2,
Wherein the mycobacterial infection is a pulmonary disease.
3. The method of claim 2,
Wherein said strain is alive or dead.
3. The method of claim 2,
Wherein the composition further comprises a multitude.
8. The method of claim 7,
Wherein the azbut is at least one of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, a crosslinked polyacrylic acid polymer, dimethyldioctadecyl-ammonium bromide (DDA), an immunomodulator, a lactoferrin, or an IFN-gamma inducing agent.
9. The method of claim 8,
Wherein said crosslinked polyacrylic acid polymer comprises a Carbopol 占 homopolymer or copolymer and wherein said immunomodulating agent comprises IFN-gamma, IL-1, IL-2 or IL-12, said IFN- 0.0 &gt; C. &Lt; / RTI &gt;
9. The method of claim 8,
Wherein the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate is included in an amount of 0.05 to 0.1 wt%, and the crosslinked polyacrylic acid polymer is contained in an amount of 0.25 wt%.

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