WO2016028028A1 - Vaccine composition against mycobacterial infection comprising new temperature sensitive mycobacteria strain - Google Patents

Abstract

Disclosed are an M. paragordonae (strain 49061) strain, which is a temperature sensitive mycobacteria, and a vaccine composition comprising the same. Upon vaccination, the vaccine according to the present invention proliferates in the skin and hypodermis, thereby stimulating immunity, and is inhibited in proliferation due to the characteristic of being able to grow only at temperatures lower than body temperature in the center of the body, and thus the possibility of infection is low. The vaccine of the present invention can be used safely and usefully as a vaccine for prevention of diseases caused by mycobacteria, and for immunotherapy.

Description

Vaccine composition for mycobacterial infections comprising novel temperature sensitive mycobacterial strains

The present application is in the technical field related to vaccines against mycobacterial infections using temperature sensitive mycobacterial strains.

Mycobacterium is a group of tuberculosis bacteria, bovine tuberculosis bacteria and leprosy, and A. tuberculosis ( M. avium). complex) and M. abscessus - chelonae It is composed of approximately 150 strains, including the complex. Among these, tuberculosis bacillus, bovine tuberculosis bacillus, and leprosy are classified as absolute pathogenic bacteria and remaining mycobacteria as non-tuberculous mycobacteria (NTM), causing tuberculosis leprosy in humans and causing various diseases in birds and livestock. Doing.

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

Tuberculosis can usually be controlled through prolonged antibiotic therapy, but it is difficult to monitor resistance to antibiotics and to ensure that the patient is following treatment procedures, and carriers can infect others without symptoms. It is not enough to prevent the spread of bacteria.

Therefore, prevention with effective vaccines is a more effective way to prevent tuberculosis from spreading.

In addition, among the non-tuberculosis mycobacterium tuberculosis species except M. avium complex, M. abscessus-M. Chelonae is the most common isolate in Korea . , Shin S, Shim MS et al., Spread of nontuberculous mycobacteria from 1993 to 2006 in Koreans.J Clin Lab Anal. 2008; 22 (6): 415-20). Recent reports suggest that among these, M. massiliense ( M. abscessus subsp. bolletii ) species account for about half (46.5%) of the M. abscessus- M. chelonae complex (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 high rate of treatment failure and mortality due to innate resistance to antibiotics. Therefore, the development of a new vaccine against M. abscessus species is required.

The most widely used preventive vaccine against conventional tuberculosis is BCG ( Mycobacterium bovis Bacille Calmette-Guerin). BCG is effective against tuberculosis in some infants, but its effect is limited for the highly infectious tuberculosis found in adults. BCG has also been reported 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 specific bacteria. In particular, the development of vaccines for temperature-sensitive strains is of greater interest because of the presence of temperature gradients in vitro (such as skin) from humans and animals (such as skin). Although it can grow at relatively low temperatures such as skin and has the ability to stimulate local immunity, the development of temperature-sensitive strains that cannot grow in the body such as organs (Barry et al., Temperature-sensitive bacterial pathogens) generated by the substitution of essential genes from cold-loving bacteria: potential use as live vaccines.J Mol Med. 2011; 89: 437-444). The best known of these are 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 are being 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., Avian Dis. 1998; 42: 667-670). However, temperature-sensitive strains through artificial genetic modifications have low stability and are difficult to maintain.

WO 1998-056931 relates to immunogenic or vaccine compositions using attenuated recombinant mycobacteria and discloses vaccine compositions using M. tuberculosis recombinant strains in which genes associated with purine and pyrimidine biosynthesis are inactivated.

U.S. Patent Application Publication No. 2013-0101623 relates to novel mycobacterial strains and tuberculosis vaccine compositions and discloses vaccine compositions comprising attenuated M. tuberculosis .

There is a need for development of new vaccine compositions based on new strains different from the prior art.

The present application is to provide a vaccine against Mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium tuberculosis.

In one embodiment the present application provides Mycobacterium paragordonae ( strain 49061) strain of Accession No. KCTC 12628BP.

In another aspect the present disclosure also provides a vaccine composition against mycobacterial infection comprising a temperature sensitive strain according to the present application and a pharmaceutically acceptable excipient.

Mycobacterial infections that are effective in the vaccine composition according to the present invention are known in the art and those skilled in the art will be able to easily determine or diagnose diseases caused by infection. Such mycobacterial infections include, but are not limited to, for example, lung diseases, lymphadenitis, dermatological infections, osteoinfections or disseminated diseases. Mycobacteria causing these diseases for which the vaccine according to the present invention is effective include nontuberculous mycobacteria (NTM), rapid growth NTM, Mycobacterium tuberculosis ) or Mycobacterium includes bovis), is the turtle tuberculosis NTM County (Mycobacterium chelonei complex), Mycobacterium Oh Away complex (M. avium complex) (MAC) , Mycobacterium Oh Away (M. avium), Mycobacterium M. intracellulrare , M. kansasii , M. abscessus , or M. massiliense or M. massiliense It is not limiting.

Strains included in the vaccine according to the present application may be live, attenuated live or inactivated strains. In particular, the strain according to the present application can be safely used as live bacteria as a temperature sensitive strain.

The vaccine composition according to the present invention may further comprise an adjuvant if necessary, and those skilled in the art will be able to select an appropriate kind and amount in consideration of the effect. In one embodiment the adjuvant comprises but is not limited to one or more of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, crosslinked polyacrylic acid polymers, dimethyldioctadecyl-ammonium bromide (DDA), immunomodulators, lactoferrins, or IFN-gamma inducers It is not limiting. In another embodiment the crosslinked polyacrylic acid polymer as an adjuvant comprises a Carbopol® homopolymer or copolymer and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN- gamma inducers include poly I: C. In another embodiment, aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate as an adjuvant may be included in an amount of 0.05 to 0.1% by weight, and the crosslinked polyacrylic acid polymer may be included in an amount of 0.25% by weight.

In another aspect the present disclosure also provides a method for preventing or treating mycobacterial infection, comprising administering to a subject in need thereof an effective amount of a vaccine composition according to the present invention.

In one embodiment according to the present invention mycobacterial infection is NTM (nontuberculous mycobacteria), Mycobacterium tuberculosis ) or Mycobacterium bovis . In another embodiment, the NTM is Mycobacterium chelonei complex, Mycobacterium avium complex ( M. avium). complex) (MAC), Mycobacterium Oh Away (M. avium), Mycobacterium intra-cellular records (M. intracellulrare), Mycobacterium Khan strabismus (M. kansasii), Mycobacterium app's revenues ( M. abscessus ), or M. massiliense .

Mycobacterial infections for which the vaccines according to the present invention are effective include lung diseases, lymphadenitis, dermatological infections, osteoinfections or disseminated diseases, and those skilled in the art can precisely grasp the definitions and criteria of these diseases according to the present application. Subjects or patients in need of vaccine treatment will be able to be determined.

The bacteria included in the vaccine used in the method according to the present invention are live bacteria, attenuated live bacteria or inactivated bacteria, and particularly as temperature-sensitive strains, they can be used safely and more effectively as live bacteria. Those skilled in the art will be able to administer appropriate amounts in consideration of the specific type of infectious organism or disease, the degree of infection or the extent of the disease, the underlying disease of the subject, and the like.

The vaccine used in the method according to the present invention may further comprise an adjuvant, and those skilled in the art can determine the adjuvant to be used with the vaccine in consideration of the specific type of infectious organism or disease, the degree of infection or the extent of the disease, the underlying disease of the subject, and the like. For example, the adjuvant may be one or more of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymer, dimethyldioctadecyl-ammonium bromide (DDA), an immunomodulator, lactoferrin, or IFN-gamma inducer. It includes. In one embodiment the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer poly I: C. In another embodiment the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer poly I: C.

The temperature-sensitive mycobacterial strain M. paragordonae (strain 49061) and the vaccine composition comprising the same can be effectively used for the prevention of mycobacterial infections including the Mycobacterium tuberculosis group and Mycobacterium tuberculosis and Mycobacterium tuberculosis. The body temperature tends to increase in temperature from the skin to the organs of the body. The vaccine according to the present invention stimulates immunity by proliferating in the skin and subcutaneous at the time of inoculation, but due to the characteristics that can grow only at a temperature lower than the body temperature in the center of the body The proliferation is inhibited and the possibility of infection is low, and thus it can be safely and usefully used in prophylactic vaccines and immunotherapy against all mycobacterial infections including Mycobacterium tuberculosis. In particular, it can be used safely and effectively in prophylactic vaccines and immunotherapy for mycobacterial infections as live bacteria.

1 is a result showing that the strain according to the present application is novel, Figure 1a is the result of the phylogenetic analysis of 16sRNA, rpoB and hsp65 , Figure 1b to 1d is the sequence of 16sRNA, rpoB and hsp65 of the strain according to the present application, respectively.

Figures 2a and 2b characterizing M. paragordonae , the growth rate in 7H9 medium at 30 ℃ and 37 ℃ M. asiaticum , M. gordonae , and M. marinum It is measured and compared with.

Figure 3 shows the results of performing in vitro infection test against J774 murine macrophage line of M. gordonae , M. marinum and M. paragordonae in accordance with an embodiment of the present application. After the infection was carried out at 30 ℃ and 37 ℃ 1, 3, 5 days later cells were lysed and plated in appropriate dilution in 7H10 solid medium and incubated at 30 ℃. This is a graph of counting colonies grown after that. The graph on the left is a graph including the results of M. paragordonae , M. gordonae and M. marinum , and the graph on the right is a graph comparing the difference between M. paragordonae and M. gordonae .

Figure 4 is infected with M. gordonae , M. marinum and M. paragordonae in accordance with an embodiment of the present invention after the liver and spleen were extracted, homogenized and plated in a suitable dilution in 7H10 solid medium and cultured at 30 ℃ to be.

5 is a vaccine test results for M. abscessus associated strains using M. paragordonae . Figure 5a schematically shows the vaccination procedure, Figure 5b is M. paragordonae mice (BALB / c, 7 weeks old, n = 4) according to Figure 5a after the subcutaneous inoculation twice in the base of the tail M. Attack with the abscessus associated strain ( M. abscessus subsp. bolletii 50594) (tail vein injection), by extracting the organs on each day, M. abscessus subsp. This is the result of measuring the CFU of bolletii 50594. 5C shows M. abscessus subsp. The results of IFN-gamma ELISPOT were performed on 3 and 14 days after bolletii 50594 bacteria attack.

Figure 6 is a vaccine test results for M. tuberculosis H37Ra M. Using paragordonae. Figure 6a schematically shows the vaccination procedure, Figure 6b is BCG and M. paragordonae in accordance with the experimental group in the base of the tail of the mouse (BALB / c, 7 weeks old, n = 6) tail according to Figure 6a, M CFU of the M. tuberculosis H37Ra in the organ was measured by attacking with tuberculosis H37Ra (tail vein injection), and extracting the organs on each day. Figure 6c is the result of performing IFN-gamma ELISPOT on splenocytes obtained 4 weeks, 8 weeks after the attack of M. tuberculosis H37Ra bacteria. Figure 6d shows ELISA for IFN-, TNF- and IL-2 in cell culture after culture by reacting splenocytes obtained at 4 and 8 weeks after M. tuberculosis H37Ra bacteria attack with antigen (whole protein of M. tuberculosis H37Ra). Is the result of FIG. 6E shows the result of confirming EL2 IgG2a antibody reacting with antigens in serum obtained at 4 and 8 weeks after M. tuberculosis H37Ra bacteria attack ( M. tuberculosis H37Ra protein and Ag85B protein).

The present application is based on the discovery of novel mycobacterial strains, which are slow-growing oncolytic bacteria, and the discovery that these strains are useful as vaccines against mycobacterial infections.

The strain according to the present application was isolated from lung infection patients, and 16sRNA analysis, acid fastness analysis, growth characteristics and phylogenetic analysis revealed that the strain was close to (99.0% sequence homology) Mycobacterium gordonae belonging to the mycobacteria group. .

The strains isolated herein are morphologically similar to Mycobacterium gordonae , but on the contrary, the strains herein were found to be temperature sensitive strains with an optimal growth temperature of 25-30 ° C. and no growth at 37 ° C. In addition, lipid analysis, phylogenetic analysis based on hsp65 and rpoB, and DNA-DNA association analysis resulted in a new strain, named Mycobacterium paragordonae , and on July 17, 2014, the deposit number KCTC 12628BP was submitted to the Korea Institute of Bioscience and Biotechnology. Deposited.

The novel temperature sensitive mycobacterial M. paragordonae (strain 49061) according to the present disclosure can be effectively used for the prevention of infections against mycobacteria, and in one embodiment the present application comprises the new temperature sensitive mycobacteria M. paragordonae (strain 49061). It relates to a composition for immunogenicity, vaccines or immunotherapy.

Mycobacteria are known as causative agents of lung diseases such as tuberculosis, lymphadenitis, skin and soft tissue infections, osteoinfections or disseminated diseases. For example, mycobacteria belonging to the Mycobacterium chelonae - M. abscessus complex, cause immunoinvasive skin and soft tissue infections, tuberculosis, hematitis or abscesses in subjects with immune or weakened immunity (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 application can be used as a vaccine against diseases for such mycobacterial infections. In one embodiment, it shows a protective effect against the infection of mycobacteria including NTM (nontuberculous mycobacteria), Mycobacterium tuberculosis or Mycobacterium bovis . In another embodiment NTM is for example the Mycobacterium chelonei complex, M. avium complex (MAC), M. avium , M. intracellulrare , M. kansasii , M. abscessus , or M. massiliense . In addition, NTMs to which the vaccine of the present invention is effective include rapid development NTM.

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

The vaccine composition according to the present invention may induce an immune response against mycobacteria, for example, antibody production and / or a T-cell response, thereby preventing mycobacterial infections including the Mycobacterium tuberculosis group and Mycobacterium tuberculosis, Mycobacterium tuberculosis or Symptoms resulting from infection can be alleviated, alleviated and / or treated.

Mycobacteria M. paragordonae used in the compositions according to the present application Strain 49061 is as described above, and live live bacteria, chemically or thermally killed or inactivated bacteria may be used. For live bacteria, attenuated processes may be used. Attenuation can be performed according to known methods, which can reduce or eliminate pathogenicity of bacteria. In one embodiment, the vaccine according to the present application can be used without undergoing an attenuation process because it is safe as live bacteria as temperature sensitive strains, and has a distinct advantage compared to vaccines using other kinds of strains.

In one embodiment the present application is used for live bacteria. M. paragordonae used in the compositions according to the invention may be particularly advantageous to be used in the state of live bacteria as a temperature sensitive strain. Probiotic vaccines can induce innate and adaptive immune responses as compared to inactivated vaccines, which is advantageous for protective immunity. Probiotic vaccines can also form memory immune cells through sustained antigen stimulation, which is advantageous for immunity. M. paragordonae according to the present invention has a lower CFU (colony forming unit) compared to M. gordonae , M. marinum , M. bovis BCG used as comparative strains in organs after infection as described in the Examples according to the present application. This is because the vaccine according to the present application is safe and thus can be used as a live vaccine, which has a distinct advantage compared to vaccines using other strains.

Vaccine compositions according to the present invention may be formulated for systemic or topical administration and include pharmaceutically possible excipients, carriers and / or media such as phosphate buffered saline solutions, distilled water, water / oil emulsions, wetting agents, emulsifiers, pH But may include, but are not limited to.

The vaccine composition according to the invention may also comprise an adjuvant, in particular any substance or compound capable of promoting or increasing a T-cell mediated response. In one embodiment, the adjuvant may be used when the composition includes chemically or thermally killed bacteria. Adjuvants are known in the art and include, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymers, dimethyldioctadecyl-ammonium bromide (DDA), immunomodulators, lactoferrins, IFN-gamma inducers, and the like. This can be used. In one embodiment, the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer, the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma Inducers may include, but are not limited to, poly I: C. In one embodiment, the synthesized sugar polymer, the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate is included in about 0.05 to 0.1% by weight, the cross-linked polyacrylic acid polymer may be included in 0.25% by weight but not limited thereto. It is not.

The compositions according to the present application may be administered topically or systemically, may be administered once or multiplely, 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 invention is administered once by intramuscular injection. The dosage of the vaccine will depend on the route of administration, age, weight, underlying disease, sex and / or health of the subject or patient, and those skilled in the art will be able to determine appropriate amounts in view of the description herein and common sense in the art. will be.

Vaccine compositions according to the invention can be formulated in solid form, prepared with a liquid solution or suspending agent suitable for the route of administration or dissolved or suspended in solution prior to injection.

In another aspect the invention also relates to a method for preventing or treating mycobacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a vaccine composition according to the invention.

By therapeutically effective amount herein is meant the dosage during the dosing period necessary to exhibit the effect according to the invention, eg, the prophylactic or therapeutic effect of mycobacterial infection. The therapeutically effective amount may vary depending on the route of administration, frequency of administration, disease state, sex, underlying disease, weight, and / or degree of effectiveness of the vaccine composition according to the subject in the subject. A therapeutically effective amount is determined by an amount in which the therapeutic effect exceeds the side effects or toxicity. Those skilled in the art will be able to experimentally determine therapeutically effective amounts for each subject in consideration of knowledge in the art.

A subject herein is a mammal, particularly a human, in need of treatment or prevention of mycobacterial infection.

By treatment is meant any action that improves or advantageously alters the condition of a disease by administration of a composition according to the present application. Those skilled in the art to which the present application belongs, will be able to determine the exact criteria of the disease, and determine the degree of improvement, improvement and treatment with reference to the data presented by the Korean Medical Association.

In one embodiment according to the present invention mycobacterial infection is NTM (nontuberculous mycobacteria), Mycobacterium tuberculosis ) or Mycobacterium bovis . In another embodiment, the NTM is Mycobacterium chelonei complex, Mycobacterium avium complex ( M. avium). complex ) (MAC), Mycobacterium Avium ( M. avium ), Mycobacterium Intracellulrare , Mycobacterium Kansasii , Mycobacterium Absesus ( M. abscessus ), or M. massiliense .

Mycobacterial infections for which the vaccines according to the present invention are effective include lung diseases, lymphadenitis, dermatological infections, osteoinfections or disseminated diseases, and those skilled in the art can precisely grasp the definitions and criteria of these diseases according to the present application. Subjects or patients in need of vaccine treatment will be able to be determined.

The bacteria included in the vaccine used in the method according to the present invention are live bacteria, attenuated live bacteria or inactivated bacteria, and particularly as temperature-sensitive strains, they can be used safely and more effectively as live bacteria. Those skilled in the art will be able to administer appropriate amounts in consideration of the specific type of infectious organism or disease, the degree of infection or the extent of the disease, the underlying disease of the subject, and the like.

The vaccine used in the method according to the present invention may further comprise an adjuvant, and those skilled in the art can determine the adjuvant to be used with the vaccine in consideration of the specific type of infectious organism or disease, the degree of infection or the extent of the disease, the underlying disease of the subject, and the like. For example, the adjuvant may be one or more of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymer, dimethyldioctadecyl-ammonium bromide (DDA), an immunomodulator, lactoferrin, or IFN-gamma inducer. It includes. In one embodiment the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer poly I: C. In another embodiment the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer poly I: C.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Example  1 strain isolation and identification

As symptoms are described in the previous from the sputum samples of lung infections resulting in (Mun et al., (2008 ). Mycobacterium senuense sp. Nov., A slowlygrowing, non-chromogenic species closely related to the Mycobacterium terrae complex. Int Strains were isolated as in J Syst Evol Microbiol 58, 641-646.

The isolated strains were subjected to biochemical analysis as previously described for the associated strains 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.In Manual of Clinical C Microbiology, 9th edn, vol. 1, p. 559, 573-578.Edited by PRMurray, EJ Baron, JH Jorgensen, ML Landry & MA Pfaller.Washington: American EX Society for Microbiology.

In summary, colony form, pigmentation in the dark, photoinduction and growth at various temperatures, niacin accumulation, nitrate reductase, Tween 80 hydrolysis, urease and pyrazineamidase, thiophene-2-carboxylic acid hydrazide (TCH) Resistance, PNB (p-nitrobenzoate), 5% sodium chloride, etabutol and picric acid assays were performed by incubation for 6 weeks in Middlebrook 7H10 agar plates 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, as follows: 1, Strain M. paragordonae (strain 49061); 2, M. asiaticum ATCC 25276 ^T ; 3, M. gordonae ATCC 14470 ^T. Code descriptions are as follows: Strong growth; ++, good growth, +, positive growth; 2, negative / no growth; ±, variable. All strains exhibited cotton colonies and did not grow at 45 ° C.

The morphologically curved, rod-shaped anti-acidic strain showed no spores or filaments under the microscope. The optimum growth temperature is 25-30 ° C. and did not grow at 37 ° C. or 45 ° C. Middlebrook 7H10 agar plates showed orange colony colonies. It did not grow in medium containing picric acid, and urease activity was positive. The most relevant species in the DNA-DNA relevance analysis was less than 70%. Lipid analysis using MALDI-TOF MS showed different from other species, including 16S rRNA (1393 bp), rpoB (306 bp) and It was shown to have a unique sequence in the hsp65 (603 bp) gene (see Figures 1B, 1C and 1D). In addition, strains isolated through phylogeny were found to be slow growing mycobacteria. 16sRNA is a genus, hsp65, RPOb is a species that can distinguish between species, 16S rRNA, hsp65 gene sequence was close to M. gordonae , but the sequence sequence homology is 99%, 95.9% specific to M. gordonae RpoB The gene sequence was close to M. asiaticum , but the sequence homology was 95.4%, indicating that it also has a specific sequence. (FIG. 1A). These results indicate that the strain according to the present application is novel.

The bacterium was named Mycobacterium paragordonae and on July 17, 2014 it was deposited with KCTC 12628BP at the Korea Research Institute of Bioscience and Biotechnology.

Example 2 Mycobacterium paragordonae  Identification of temperature sensitive characteristics and safety analysis

The following experiment was performed to prove the safety of inoculation with live vaccines.

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

Next, infection analysis was performed. Infection analysis was performed to examine whether the M. paragordonae strains did not grow at 37 ° C in liquid and solid medium cultures, and therefore, whether the actual macrophages were affected by temperature. To this end, the bacteria were infected (~ 1 × 10 ⁷ , 10 MOI) with macrophages (J774 cell line, ATCC TIB-67 ^TM ) at 30 ° C and 37 ° C, and the cells were fused (0.5% Triton ^TM X-100). Remove the cells by pipetting by adding the included PBS), and plated in 7H10 solid medium and then cultured at 30 ℃ was measured the number of colonies generated.

The results are described in FIG. As shown in this figure, unlike the comparative strain, M. paragordonae infected at 37 ° C showed little growth.

In addition, in vivo experiments were performed by intravenous injection of M. paragordonae and comparative strain (10 ⁶ CFU) into BALB / c mice (7 weeks old, n = 3), and liver and spleen were extracted and homogenized on day 1, 4 and 7. After diluting with phosphate buffer solution, plated in 7H10 solid medium and incubated at 30 ° C. to measure CFU.

The results are described in FIG. 4A, and as shown therein, when the growth of M. paragordonae was measured in the liver or spleen, it was found that little growth was observed in comparison with other strains, indicating that the strains of the present invention were safe for use in vivo. Indicates.

In addition, in vivo, BALB / c mice (7 weeks old, n = 3) were injected intravenously with M. paragordonae and comparative strains ( M. gordonae , M. marinum or M. bovis BCG) (10 ⁶ CFU in 100 μl PBS). After infection, liver and spleens were extracted and homogenized on day 1, 4 and 7 (compared to M. gordonae and M. marinum ) or day 1, 7, and 14 (compared to M. bovis BCG) and diluted with phosphate buffer solution. After plating on 7H10 solid medium and incubated at 30 ℃ CFU was measured.

The results are described in FIG. 4B, and as shown therein, when the growth of M. paragordonae was measured in the liver, lung or spleen, it was found that little growth compared to other strains.

Example 3 Mycobacterium paragordonae  Vaccine Analysis

Vaccine analysis was then performed as follows. First, ALB / c mice (7 weeks old, n = 4) were intravenously injected 10 ⁶ CFU of M. paragordonae (strain 49061) into the tail twice (0 and 8 weeks) as in the procedure of FIG. 5A. PBS and BCG were used as a control. Then, at 12 weeks, M. abscessus- associated strain ( M. abscessus subsp. Bolletii 50594)-10 ⁶ CFU was injected subcutaneously into the base of the tail. After 3 and 14 days, M. abscessus subsp. CFU of bolletii 50594 was measured as in Example 2. M. abscessus subsp. Bolletii 50594 bacteria and spleens were extracted after 14 days, and M. abscessus subsp. ELISPOT was performed to measure the number of cells secreting IFN-γ when the whole protein of bolletii 50594 strain was treated. Specifically, IFN-γ antibody (eBiosciense, 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 following day each well was washed and incubated at 37 ° C. with complete medium (DMEM + antibiotics + 10% FBS). Spleens of each mouse were extracted, splenocytes were obtained with a 70 μm cell strainer, and the number of splenocytes in each group was adjusted to about 5 × 10 ⁵ cells, and then added to each well of the PVDF-coated 96 well plate. Followed by M. abscessus subsp. 10 μg / ml of bolletii 50594 total protein was treated to the antigen-treated group, and the rest were treated with complete medium. Positive control group was treated with PMA (Phorbol myristate acetate) (5 ng / ml) and iononmycin (500 ng / ml). Thereafter, the cells were incubated at 37 ° C. for one day, and then washed with the detection antibody (eBiosciense, Cat., 13-7311-85, 3 μg / ml) the following day. Subsequently, each well was washed after incubation at 4 ° C., and then treated with streptavidin-HRP for 2 hours at room temperature, followed by color development using the AEC substrate color development kit (Dako) according to the manufacturer's method. . Second, subcutaneously subcutaneously with 10 ⁶ CFU of M. paragordonae (strain 49061) at the base of the tail twice (0 and 8 weeks) in BALB / c mouse (7 weeks old, n = 6) as described in FIG. 6A. Vaccine analysis was performed by injection. The experimental groups were as follows: Experimental group 1: two subcutaneous injections of PBS, experimental group 2: two subcutaneous injections of BCG, experimental group 3: one subcutaneous injection of M. paragordonae after one subcutaneous injection of BCG, experimental group 4: two subcutaneous injections of M. paragordonae injection. Then, at 12 weeks, tail vein was injected with non-pathogenic Mycobacterium tuberculosis, M. tuberculosis H37Ra ~ 10 ⁶ CFU. Four weeks later, M. tuberculosis for each organ (liver, spleen, lung) CFU of H37Ra was measured as in Example 2. M. tuberculosis After 4 and 8 weeks of attack with H37Ra, spleens were extracted, and cell culture after ELISPOT and antigen treatment to measure the number of cells secreting IFN-γ when the whole protein of M. tuberculosis H37Ra strain was treated in splenocytes. ELISA was performed on TNF-α, IFN-γ and IL-2 released into the solution. For ELISA, kits are provided by R & D systems (TNF-α, Cat. No., DY410) or BioLegend (IFN-γ, Cat. No., 430805; IL-2, Cat. No., 431002). It was carried out according to the method. Serum was isolated from blood samples collected 4 and 8 weeks later, and ELISA was performed for IgG2a to analyze the difference in expression of immunoglobulin G2a type in response to M. tuberculosis H37Ra whole protein and Ag85B antigen in serum.

The results are described in FIGS. 5 and 6. In Fig. 5b, M. abscessus subsp in mouse organs in which the vaccine M. paragordonae. bolletii CFU of 50594 showed lower CFU in mouse organs vaccinated with PBS or other M. gordonae and M. bovis BCG. In addition, more spots were observed in M. paragordonae- inoculated mice during ELISPOT for IFN-γ. Through the CFU results after the vaccine test, M. paragordonae was isolated from M. abscessus subsp. bolletii 50594 protection was given.

In addition, the CFU of the M. abscessus associated strains is further reduced compared to the existing M. bovis BCG, indicating higher protection against M. abscessus associated strains. In addition, M. abscessus subsp. ELISPOT confirmed that the number of protective immune cells against bolletii 50594 was greater than BCG when M. paragordonae was used as a vaccine. M. abscessus subsp. It can be given effective protection against the group of turtle Mycobacterium tuberculosis containing bolletii 50594.

In FIG. 6B, the CFU of M. tuberculosis H37Ra was much lower in the organs vaccinated with M. paragordonae than in the PBS treatment group, and vaccinated with M. paragordonae twice compared with the group vaccinated with BCG twice. The CFU of the group showed statistically low CFU. More spots were observed in the mouse splenocytes vaccinated with M. paragordonae twice compared to BCG and mouse splenocytes vaccinated with both strains when the ELISPOT was performed against IFN- (FIG. 6C). In addition, IFN- involved in defensive immunity in splenocytes vaccinated with M. paragordonae strains when the spleen cells extracted from each experimental group were stimulated with the entire protein of M. tuberculosis H37Ra, and the cytokines expressed in the cell culture were confirmed by ELISA. It was confirmed that the expression of γ, TNF-α, and IL-2 was significantly increased compared to the PBS experimental group as well as other experimental groups (FIG. 6D). Finally, the expression level of IgG2a antibody against M. tuberculosis H37Ra in serum of each experimental group was confirmed by ELISA. IgG2a antibodies are mainly formed by Th1-type immune responses and are known as antibody isotypes that are associated with vaccine efficacy. As a result of ELISA of IgG2a antibody, it was confirmed that there were significantly more IgG2a antibodies against M. tuberculosis H37Ra protein and Ag85B antigen in serum samples of experimental group vaccinated with M. paragordonae compared to the experimental group vaccinated with PBS or BCG. (FIG. 6E). These results indicate that M. paragordonae has been isolated from M. abscessus subsp. protection against bolletii 50594 and M. tuberculsos H37Ra.

Furthermore, since the vaccine according to the present invention is naturally temperature-sensitive, safety is also ensured, and it indicates that it can be effectively used as a vaccine against mycobacterial infections including turtle group, tuberculosis and tuberculosis in both humans and animals. .

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

[Accession number]

Name of Trustee: KCTC Korea Biological Resource Center

Accession number: KCTC 12628BP

Trust Date: 20140717

Claims (18)

Vaccine composition for mycobacterial infection comprising Mycobacterium paragordonae strain strain 49061 of Accession No. KCTC 12628BP and a pharmaceutically acceptable excipient. The method of claim 1, wherein the mycobacterial infection is NTM (nontuberculous mycobacteria), Mycobacterium Mycobacterium tuberculosis ) or Mycobacterium bovis . The method of claim 2, wherein the NTM is Mycobacterium tuberculosis group chelonei complex), Mycobacterium avium complex ( M. avium) complex) (MAC), Mycobacterium Oh Away (M. avium), Mycobacterium intra-cellular records (M. intracellulrare), Mycobacterium Khan strabismus (M. kansasii), Mycobacterium app's revenues ( M. abscessus ), or M. massiliense . The method of claim 1, The mycobacterial infection is a lung disease, lymphadenitis, skin tissue, osteoinfection or disseminated disease, vaccine composition. The method of claim 1, The strain is live or dead, vaccine composition. The method of claim 1, The composition of claim 1, further comprising an adjuvant. The method of claim 6, The adjuvant is at least one of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymer, dimethyldioctadecyl-ammonium bromide (DDA), an immunomodulator, lactoferrin, or IFN-gamma inducer. The method of claim 7, wherein The crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer, the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer is poly I: A vaccine composition comprising C. The method of claim 7, wherein The aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate is included in 0.05 to 0.1% by weight, the cross-linked polyacrylic acid polymer is included in 0.25% by weight, vaccine composition. A method for preventing or treating mycobacterial infection, comprising administering to a subject in need thereof an effective amount of a vaccine composition according to any one of claims 1 to 9. The method of claim 10, wherein the mycobacterial infection is NTM (nontuberculous mycobacteria), Mycobacterium Mycobacterium tuberculosis ) or Mycobacterium bovis ( Mycobacterium bovis ), a method for preventing or treating mycobacterial infection. The method of claim 11, wherein the NTM is Mycobacterium Mycobacterium group chelonei complex), Mycobacterium avium complex ( M. avium) complex) (MAC), Mycobacterium Oh Away (M. avium), Mycobacterium intra-cellular records (M. intracellulrare), Mycobacterium Khan strabismus (M. kansasii), Mycobacterium app's revenues M. abscessus , or M. massiliense , a method for preventing or treating mycobacterial infection. The method for preventing or treating mycobacterial infection according to claim 10, wherein the mycobacterial infection is lung disease, lymphadenitis, dermal tissue infection, osteoinfection or disseminated disease. The method for preventing or treating mycobacterial infection according to claim 10, wherein the strain is live, attenuated live or inactivated. The method of claim 10, wherein the composition further comprises an adjuvant. The method of claim 15, wherein the adjuvant is one or more of aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymer, dimethyldioctadecyl-ammonium bromide (DDA), an immunomodulator, lactoferrin, or IFN-gamma inducer, How to prevent or treat mycobacterial infections. The method of claim 16, wherein the crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer, and the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN- The gamma inducer comprises poly I: C. A method for preventing or treating mycobacterial infection. The method of claim 17, The crosslinked polyacrylic acid polymer comprises a Carbopol® homopolymer or copolymer, the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer is poly I: A method for preventing or treating mycobacterial infection, comprising C.

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