WO2016013873A1 - Composition de vaccin pour la prévention de la tuberculose contenant une protéine rv3112, mtbk 20620 ou mtbk 24820 - Google Patents

Composition de vaccin pour la prévention de la tuberculose contenant une protéine rv3112, mtbk 20620 ou mtbk 24820 Download PDF

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WO2016013873A1
WO2016013873A1 PCT/KR2015/007631 KR2015007631W WO2016013873A1 WO 2016013873 A1 WO2016013873 A1 WO 2016013873A1 KR 2015007631 W KR2015007631 W KR 2015007631W WO 2016013873 A1 WO2016013873 A1 WO 2016013873A1
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tuberculosis
mtbk
protein
vaccine composition
mycobacterium tuberculosis
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PCT/KR2015/007631
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Korean (ko)
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신성재
한승정
차승빈
김우식
김종석
김홍민
권기웅
조상래
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연세대학교 산학협력단
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Priority claimed from KR1020140093510A external-priority patent/KR101564323B1/ko
Priority claimed from KR1020140093499A external-priority patent/KR101568736B1/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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  • the present invention was made by the task number NRF-2013R1A2A1A01009932 under the support of the Ministry of Science, ICT and Future Planning, and the research management specialized organization of the project is "Korea Research Foundation", the research project name is "basic research project, mid-sized researcher support project, leap research_ Challenge “, research project titled” Development of immunochemotherapeutic therapy through the regulation of immune diseases specific to the disease stage of tuberculosis ", host organization” Yonsei University Industry-Academic Cooperation Group “, research period” 2015-06-01 ⁇ 2016-05- 31 ".
  • the present invention relates to a vaccine composition for preventing tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • Tuberculosis is one of the three major infectious diseases defined by the WHO, discovered by microbiologist Robert Koch in 1882. It is a fatal disease with high incidence and mortality caused by Mycobacerium tuberculosis. Around 60 million people worldwide are infected with active TB, an estimated 50 million to 100 million new TB infections occur each year, with at least 9 million new TB cases annually, and more than 1.5 million annually. It is said to die of tuberculosis. The incidence rate of tuberculosis is 146 per 100,000 population, and tuberculosis mortality rate is 49 per 100,000 population, making it the largest cause of death among single infectious diseases.
  • Mycobacterium tuberculosis with the predominant genotypes of tuberculosis bacteria is widespread all over the world, mainly in East Asia.
  • genotypes belonging to the Beijing family are estimated to be 92% in China, 72% in Korea, and 40% in Vietnam (Glynn et al., 2002).
  • the genotypes of Beijing and Mycobacterium tuberculosis are associated with drug resistance of Mycobacterium tuberculosis, including monodrug resistance and multidrug resistance (Buu et al., 2009).
  • the genotype with Beijing is also closely associated with the relapse of latent Mycobacterium tuberculosis (Burman et al., 2009).
  • the K strain a Korean highly pathogenic tuberculosis bacterium, exhibits a specific pathogenesis that can overcome early innate immunity during mouse infection, which represents a 10-fold growth rate of Mycobacterium tuberculosis H37Rv strain. .
  • the immune pathology of the host's immune response is considered to be very important as a etiology. Therefore, it is important to raise the acquired immunity through preventive vaccines rather than innate immunity. It was judged.
  • tuberculosis is mainly prevented using live bacteria of Bacillus Calmette-Guerin (BCG), a non-toxic strain of Mycobacterium bovis.
  • BCG Bacillus Calmette-Guerin
  • the BCG vaccine is not known to be effective against the Korean type of highly pathogenic Mycobacterium tuberculosis, and controversy over safety and efficacy has led some countries, such as the United States, to ban it.
  • BCG is also not known to be suitable for use in children with HIV. Therefore, there is a need for the development of a new tuberculosis prevention vaccine that is safer and more effective than BCG.
  • the present inventors continued to develop a new tuberculosis prevention vaccine, and as a result, the Korean-type tuberculosis K strain, which is not present in the existing tuberculosis standard strains, is present only in Korean tuberculosis K strains, and the Korean tuberculosis standard strains.
  • the present invention was completed by finding and identifying a gene specifically overexpressed in the K strain, a highly pathogenic tuberculosis bacterium, and confirming its availability as a vaccine.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the present invention provides a vaccine composition for preventing tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the Rv3112, MTBK 20620 or MTBK 24820 protein according to the present invention When used as an antigen, it exhibits high IFN- ⁇ production ability in the spleen and lung of an animal model that caused infection by Mycobacterium tuberculosis, and results in rapid acquired immune defense formation and T cell Maintaining memory can effectively prevent tuberculosis.
  • the Rv3112, MTBK 20620 or MTBK 24820 protein according to the present invention can be used in the treatment of tuberculosis and can be usefully used to prevent the recurrence or re-infection of high pathogenic tuberculosis with high recurrence rate and to increase the treatment efficiency.
  • FIG. 1 is a diagram showing the result of comparing the same region of the Mycobacterium tuberculosis H37Rv of the tuberculosis standard strain Mycobacterium tuberculosis K strain MTBK 20620 gene and surrounding genes and tuberculosis standard strain.
  • Figure 2 is a diagram showing the result of comparing the same region of the Mycobacterium tuberculosis H37Rv tuberculosis standard strain Mycobacterium tuberculosis K strain MTBK 24820 gene and surrounding genes and tuberculosis standard strain.
  • Figure 3 is a diagram confirming the recombinant Rv3112 protein isolated through cloning via SDS-PAGE.
  • FIG. 4 is a diagram illustrating the recombinant MTBK 20620 protein isolated through cloning through SDS-PAGE.
  • FIG. 5 is a diagram illustrating the recombinant MTBK 24820 protein isolated through cloning through SDS-PAGE.
  • Figure 6 shows the ability of IFN- ⁇ production after treatment with recombinant Rv3112 protein in splenocytes of Mycobacterium tuberculosis-infected mice.
  • Figure 7 shows the ability of IFN- ⁇ production after treatment with recombinant Rv3112 protein in lung cells of Mycobacterium tuberculosis-infected mice.
  • FIG. 8 is a diagram showing a memory T cell analysis method using a flow cytometer after Mycobacterium tuberculosis infection in mice.
  • Figure 9 shows that when treated with recombinant Rv3112 protein to spleen cells of Mycobacterium tuberculosis-infected mice, memory CD4 T cells increase compared to the control (WT).
  • Figure 10 shows that when treated with recombinant Rv3112 protein in lung cells of Mycobacterium tuberculosis infected mice, memory CD4 T cells increase compared to the control (WT).
  • FIG. 11 is a graph showing the results of H & E staining of lung tissue and the area of inflammatory response after 3 weeks of infection with a K strain of Korean highly pathogenic Mycobacterium tuberculosis in mice immunized with recombinant Rv3112 protein (A) : Adjuvant control (GLA-SE only) (negative control), B: BCG only (positive control), C: Rv3112 + GLA-SE (experimental)).
  • FIG. 12 is a diagram showing the results of comparing the bacterial counts of Mycobacterium tuberculosis in lung tissue 4 weeks after the K strain, a Korean highly pathogenic Mycobacterium tuberculosis, was immunized with recombinant Rv3112 protein.
  • FIG. 13 is a diagram showing the results of comparing the bacterial counts of Mycobacterium tuberculosis in spleen tissues after 4 weeks of respiratory infection with K strains of Korean highly pathogenic Mycobacterium tuberculosis in mice immunized with recombinant Rv3112 protein.
  • FIG. 14 is a diagram illustrating an animal experiment design.
  • FIG. 15 is a diagram showing the ability of IFN- ⁇ production after treatment with recombinant MTBK 20620 or MTBK 24820 protein to spleen cells of mice 4 weeks after infection with Mycobacterium tuberculosis.
  • FIG. 16 is a diagram showing the ability of IFN- ⁇ production after treatment with recombinant MTBK 20620 or MTBK 24820 protein to mouse lung cells 4 weeks after infection with Mycobacterium tuberculosis.
  • Figure 17 shows the ability of IFN- ⁇ production after treatment with recombinant MTBK 20620 or MTBK 24820 protein to spleen cells of mice 8 weeks after infection with Mycobacterium tuberculosis.
  • FIG. 18 is a diagram showing the ability of IFN- ⁇ production after treatment with recombinant MTBK 20620 or MTBK 24820 protein to mouse lung cells 8 weeks after infection with Mycobacterium tuberculosis.
  • FIG. 19 is a diagram showing a memory T cell analysis method using a flow cytometer after Mycobacterium tuberculosis infection in mice.
  • FIG. 20 is a diagram showing that memory CD4 T cells were increased in comparison with the control (WT) when the spleen cells of the mice were treated with recombinant MTBK 20620 or MTBK 24820 protein after 4 weeks.
  • FIG. 21 is a diagram showing that memory CD4 T cells increase when compared to control (WT) when treated with recombinant MTBK 20620 or MTBK 24820 protein to lung cells of the mice 4 weeks after infection with Mycobacterium tuberculosis.
  • FIG. 22 is a diagram showing that memory CD4 T cells were increased in comparison with the control group (WT) when the spleen cells of the mice were treated with recombinant MTBK 20620 or MTBK 24820 protein after 8 weeks.
  • FIG. 23 is a diagram showing that memory CD4 T cells were increased in comparison with the control group (WT) when 8 weeks after TB infection, the mice treated with recombinant MTBK 20620 or MTBK 24820 protein.
  • the present invention provides a vaccine composition for preventing tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the present invention also provides a method for preventing tuberculosis, comprising administering Rv3112, MTBK 20620 or MTBK 24820 protein to a subject.
  • Rv3112 (moaD1) is composed of 252 bp gene and 84 amino acid sequences, the size of the protein is 9.2 KDa, PI value is 4.57.
  • Rv3112 is molybdenum cofactor biosynthesis protein D, the main function of which is the function of molybdopterin synthase.
  • MTBK 20620 (Genbank NO. AIB48607.1) is composed of a 279bp gene and 92 amino acid sequences, the protein size is 8.6KDa and the PI value is 8.36.
  • the function of MTBK 20620 is not yet known as a hypothetical protein and does not have specific domains or motifs, but it does not exist in the tuberculosis standard strain, but it is not present in M. tuberculosis 210, M. tuberculosis X122, M. tuberculosis HN878, M. tuberculosis CTRI- 4, M. tuberculosis CCDC5180, M. tuberculosis CCDC5079, M.
  • tuberculosis R1207, M. tuberculosis W-148, etc. were found to be present in the same tuberculosis.
  • the MTBK 20620 gene derived from Mycobacterium tuberculosis K strain and surrounding genes and the same region of Mycobacterium tuberculosis H37Rv, a tuberculosis standard strain, are shown in FIG. 1.
  • MTBK 24820 (PPE39) (Genbank NO. AIB49026.1) is composed of 1869bp gene and 622 amino acid sequences, the size of the protein is 59KDa and PI value is 4.05.
  • PPE is named PPE because it is composed of Pro-Pro-Glu structure at the N-terminal part of the protein. It has MPTRs (major polymorphic tandem repeats) and includes Asn-X-Gly-X-Gly- Asn-X-Gly has a repeating structure.
  • the MTBK 24820 gene and its surrounding genes derived from the Mycobacterium tuberculosis K strain were compared with the same region of Mycobacterium tuberculosis H37Rv, a tuberculosis standard strain.
  • Rv3112 protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by the nucleotide sequence represented by SEQ ID NO: 2, and a functional equivalent of the protein.
  • MTBK 20620 protein includes a protein having an amino acid sequence represented by SEQ ID NO: 3 or a protein encoded by the nucleotide sequence represented by SEQ ID NO: 4, and functional equivalents of the protein.
  • MTBK 24820 protein includes a protein having an amino acid sequence represented by SEQ ID NO: 5 or a protein encoded by the nucleotide sequence represented by SEQ ID NO: 6, and functional equivalents of the protein.
  • the “functional equivalent” means at least 70%, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 1, 3 or 5 as a result of the addition, substitution or deletion of the amino acid. More preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NOs: 1, 3, and 5.
  • the proteins of the present invention include not only proteins having their natural amino acid sequence, but also variants thereof, are also included in the scope of the present invention.
  • the variant refers to a protein in which the amino acid sequence of Rv3112, MTBK 20620 or MTBK 24820 protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • Amino acid exchange in proteins and fragments that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979).
  • Rv3112, MTBK 20620 or MTBK 24820 proteins or variants thereof may be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or produced by genetic recombination methods based on DNA sequences. Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989.
  • the Rv3112 protein may be encoded by the nucleotide sequence of SEQ ID NO: 2
  • the MTBK 20620 protein may be encoded by the nucleotide sequence of SEQ ID NO: 4
  • the MTBK 24820 protein may be encoded by the nucleotide sequence of SEQ ID NO: 6, and has the same function as the nucleotide. Variants that can be included are included within the scope of the present invention.
  • the Rv3112 protein of the present invention is derived from Mycobacterium tuberculosis
  • the MTBK 20620 or MTBK 24820 protein of the present invention is derived from the Korean Mycobacterium tuberculosis K strain, and the protein can be produced by a recombinant method based on a DNA sequence.
  • a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.
  • Tuberculosis of the present invention is eye tuberculosis, skin tuberculosis, adrenal tuberculosis, kidney tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, cholestatic tuberculosis, bone tuberculosis, throat tuberculosis, lymph node tuberculosis, lung Syndrome, breast tuberculosis or spinal tuberculosis.
  • Tuberculosis of the present invention includes those caused by K strain, which is a Korean type of highly pathogenic tuberculosis bacteria.
  • the vaccine composition according to the present invention is not induced an immune response against a pathogen by inoculating an attenuated strain of a pathogenic organism, but by preventing the subsequent pathogen infection by administering a protein antigen having a vaccine effect. Therefore, it is characterized by a safer immunity method than the known BCG immunity method.
  • Rv3112, MTBK 20620 or MTBK 24820 protein according to the present invention can be used as a booster vaccine after BCG vaccine administration.
  • the vaccine composition of the present invention may be administered alone but is preferably administered with an adjuvant.
  • Adjuvant is a substance that promotes an immune response to an antigen in a specific manner during the initial activation of immune cells, and means an agent, a molecule, etc. that enhances immunity by increasing the activity of cells of the immune system, although it is not an immunogen to the host (Warren et. al., 1986, Annu. Rev. Immunol ,. 4: 369).
  • Adjuvants that can be administered with the vaccine composition of the present invention to enhance an immune response include any of a variety of adjuvants, and typical adjuvant includes Freund adjuvant, aluminum compound, mura Wheat dipeptide, lipopolysaccharide (LPS), monophosphoryl lipid A, or coil A and the like are known.
  • the adjuvant may be administered simultaneously with the vaccine composition or sequentially at intervals.
  • the vaccine composition of the present invention may further include a solvent, an excipient, and the like.
  • the solvent includes physiological saline, distilled water and the like
  • the excipient includes, but is not limited to, aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and the like, and is generally used for preparing vaccines in the field of the present invention. It may further include.
  • Vaccine compositions of the invention can be prepared by methods commonly used in the art to which this invention pertains.
  • the vaccine composition of the present invention may be prepared in oral or parenteral preparations, preferably in parenteral preparations, injectable solutions, and in dermis, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural ( administration by the eidural route.
  • the vaccine composition of the present invention can be administered to an individual in an immunologically effective amount.
  • the "immunologically effective amount” means an amount sufficient to exhibit a tuberculosis prevention effect and an amount not to cause a side effect or a serious or excessive immune response, and the exact dosage depends on the specific immunogen to be administered, and the vaccination
  • the subject's age, weight, health, sex, sensitivity to the individual's drug, route of administration, method of administration and the like can be easily determined by those skilled in the art and can be administered once or several times.
  • the Rv3112, MTBK 20620 or MTBK 24820 protein according to the present invention When used as an antigen, it exhibits high IFN- ⁇ production ability in the spleen and lung of an animal model that caused infection by Mycobacterium tuberculosis, and results in rapid acquired immune defense formation and T cell Maintaining memory can effectively prevent tuberculosis.
  • the Rv3112, MTBK 20620 or MTBK 24820 protein according to the present invention can be used in the treatment of tuberculosis and can be usefully used to prevent the recurrence or re-infection of high pathogenic tuberculosis with high recurrence rate and to increase the treatment efficiency.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of tuberculosis comprising Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the present invention also provides a method for treating tuberculosis comprising administering to a subject Rv3112, MTBK 20620 or MTBK 24820 protein.
  • the pharmaceutical composition of the present invention may include one or more known active ingredients having a prophylactic and therapeutic effect of tuberculosis together with Rv3112, MTBK 20620 or MTBK 24820 protein.
  • compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • compositions according to the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, respectively.
  • Can be. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.
  • simple diluents such as water and liquid paraffin
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • witepsol macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of tuberculosis.
  • Genomic DNA was extracted from Mycobacterium tuberculosis H37Rv, a Mycobacterium tuberculosis standard strain, and the Rv3112 gene was cloned therefrom.
  • the Rv3112 gene region was amplified by PCR using a forward primer (5'-CATATGATTAAAGTGAATGTTC-3 ', SEQ ID NO: 7) and a reverse primer (5'-AAGCTTGCCTCCGGCTACCT-3', SEQ ID NO: 8).
  • the PCR product was digested with NdeI and HindIII restriction enzymes and inserted into the expression vector pET-22b (+) vector (Novagen, Madison, Wis.).
  • coli BL21 was transformed using the pET-22b (+) vector into which the Rv3112 gene was inserted, followed by incubation at 30 ° C. until absorbance (OD) at 600 nm was 0.4 to 0.5, and then 1 mM. Isopropyl-D-thiogalactopyranoside (IPTG) was added and incubated again at 30 ° C. for 8 hours. After the transformed E. coli was disrupted using an ultrasonic cell crusher, the crude extract was separated by centrifugation at 15000 rpm for 30 minutes at 4 ° C., the supernatant was recovered, and the manufacturer's method using ALON Metal Affinity Resin (Clontech). Purification according to. Finally, the purified recombinant protein was confirmed by SDS-PAGE. The results are shown in FIG.
  • Genomic DNA was extracted from the Mycobacterium tuberculosis K strain, and the MTBK 20620 or MTBK 24820 gene was cloned therefrom.
  • MTBK_20620 a forward primer (5'-CATATGGTGACCGACCCCGACAT-3 ', SEQ ID NO: 9) and a reverse primer (5'-AAGCTTCCACCAT CTGCGCTTTC-3', SEQ ID NO: 10) were used.
  • a forward primer (5'-CATATGGTGAATTTTTCGGT-) was used.
  • IPTG isopropyl-D-thiogalactopyranoside
  • Example 1 In order to verify the efficacy of the Rv3112 protein isolated in Example 1 can be used as a tuberculosis prevention vaccine, animal experiments were performed.
  • mice Female C57BL / 6 mice (Japan SLC, Inc. Shijuoka, Japan) without specific pathogens 5 to 6 weeks of age were used, and the mice were BL-3 biohazard animal room in Yonsei University Medical Research Center. Breeding in the limited space of. Mice were optionally given sterile commercial mouse food and water.
  • Mycobacterium tuberculosis H37Rv a Mycobacterium tuberculosis standard strain
  • the cultured strains were collected and then disrupted with gentle vortexing with 6 mm glass beads. After cell aggregates had settled, the supernatants were harvested and stored separately at -70 ° C. After thawing, live bacteria were cultured and counted in staged dilution plates in Middlebrook 7H11 agar (Difco, Detroit, MI, USA).
  • the bacterial suspension was sonicated lightly in a sonic bath and diluted with phosphate buffered saline (PBS) at pH 7.2 to obtain the desired number of bacteria, Glas-Col inhalation
  • PBS phosphate buffered saline
  • the device (Terre Haute, IN) was used to infect 200-250 Mycobacterium tuberculosis H37Rv per mouse to enter.
  • splenocytes and lung cells of the mouse were isolated to confirm IFN- ⁇ production and increase of memory T cells by antigen stimulation.
  • T lymphocytes specific for Mycobacterium tuberculosis antigens Upon infection with Mycobacterium tuberculosis, T lymphocytes specific for Mycobacterium tuberculosis antigens replicate and differentiate into effector T cells. Effector T cells die most of the immune response and some survive, forming long-lasting memory T cells. These induced memory T cells are known to play a role in the formation of rapid acquired immune immunity and maintaining T cell memory. Therefore, the following experiment was performed to confirm the effect of Rv3112 protein on memory T cells.
  • mice 4 weeks after Mycobacterium tuberculosis H37Rv infection were isolated, and then, native T cells (CD3 + CD4 + CD62L + CD44-CD127 +) were detected through Rv3112 protein stimulation.
  • central memory T cell CD3 + CD4 + CD62L + CD44 + CD127 +
  • effector memory T cell CD3 + CD4 + CD62L-CD44 + CD127 +
  • effector T cell CD3 + CD4 + CD62L-CD44 + CD127-
  • Induction was analyzed via flow cytometry (FACSverse, BD) (FIG. 8).
  • mice were maintained under the same conditions as in Experimental Example 1-1. Each mouse was immunized (immunization) three times at three-week intervals with an experimental vaccine containing 5 ⁇ g of recombinant Rv3112 protein per injection, along with adjuvant.
  • the adjuvant used 5 ⁇ g of GLA-SE (glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion) per mouse.
  • GLA-SE was dissolved in sterile PBS according to the manufacturer's instructions, diluted with the antigen, and used after the final volume was prepared to be 100 ⁇ l per injection.
  • BCG Pasteur 1173P2 (2 ⁇ 10 5 CFU / mouse) strains were administered subcutaneously and used as a positive control to assess the protective ability of tuberculosis.
  • Negative controls were injected subcutaneously with 100 ⁇ l of sterile PBS or 100 ⁇ l of adjuvant containing no antigen.
  • Korean tuberculosis K strains highly pathogenic to the immunized mice were infected by inhalation of 200-250 tuberculosis bacteria per mouse using a Glas-Col inhalation device (Terre Haute, IN) (Aerosol Infection).
  • each mouse was infected with Korean Mycobacterium tuberculosis K strain and three weeks later, lung tissues were extracted and stored in 10% neutral-buffered formalin, and then fixed in paraffin. The fixed tissue was sectioned to a thickness of 4-5 ⁇ m, stained with hematoxylin and eosin (H & E), and observed under a microscope. The results are shown in FIG.
  • each mouse was infected with the Korean type Mycobacterium tuberculosis K strain, and 4 weeks later, lung and spleen tissues were extracted from each group of mice, and the number of bacteria was confirmed. More specifically, after preparing a homogeneous suspension of lung and spleen tissue, the suspension was serially diluted and plated on Middlebrook 7H11 agar. This was incubated at 37 ° C. for 3-4 weeks and counted colonies to determine the number of live bacteria in the tissue. The results were analyzed by comparison with the control group, and the average log 10 CFU ⁇ standard deviation per whole lung or spleen tissue was shown, which is shown in Figures 12 and 13.
  • the group immunized with the Rv3112 protein as an antigen significantly reduced the number of bacteria in lung and spleen tissue similar to the positive control BCG immunized group (BCG). It was confirmed (p ⁇ 0.001). Through this, it was confirmed that the subunit vaccine containing the Rv3112 protein of the present invention exhibited an excellent preventive effect on pulmonary tuberculosis caused by K strain, which is a Korean high pathogenic tuberculosis bacterium.
  • Example 2 In order to verify the efficacy of the MTBK 20620 or MTBK 24820 protein isolated in Example 2 can be used as a tuberculosis prevention vaccine, animal experiments were performed.
  • mice Female C57BL / 6 mice (Japan SLC, Inc. Shijuoka, Japan) without specific pathogens 5 to 6 weeks of age were used, and the mice were BL-3 biohazard animal room in Yonsei University Medical Research Center. Breeding in the limited space of. Mice were optionally given sterile commercial mouse food and water.
  • Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis K strain Mycobacterium tuberculosis H37Rv, which are the standard strains of Mycobacterium tuberculosis, were incubated for 15 days in 7H9-OADC broth. Cultured strains were collected and then disrupted with gentle vortexing with 6 mm glass beads. After cell aggregates had settled, the supernatants were harvested and stored separately at -70 ° C. After thawing, live bacteria were cultured and counted in staged dilution plates in Middlebrook 7H11 agar (Difco, Detroit, MI, USA).
  • mice 4 and 8 weeks after infection with Mycobacterium tuberculosis H37Rv or Korean Mycobacterium tuberculosis K strain were isolated, and then MTBK 20620 was in vitro (X-vivo).
  • MTBK 20620 was in vitro (X-vivo).
  • the ability to generate antigen-specific IFN- ⁇ through MTBK 24820 protein stimulation was confirmed by ELISA.
  • the control group is known to be an antigen induced during early tuberculosis infection, and ESAT-6 antigen, which is contained in both Mycobacterium tuberculosis H37Rv and Korean Mycobacterium tuberculosis K strain, was used as a control.
  • the results are shown in FIGS. 15 to 18.
  • MTBK 20620 protein did not show strain-specific IFN- ⁇ production in splenocytes after 8 weeks of infection, but it was confirmed that the specificity was observed in lung cells of Korean tuberculosis K strain infected mice. It was.
  • MTBK 24820 protein was confirmed to increase the production capacity of the specific mycobacterium tuberculosis K strain specific IFN- ⁇ in both the spleen and lung cells of the mouse 8 weeks after infection with the Korean tuberculosis K strain.
  • T lymphocytes specific for Mycobacterium tuberculosis antigens Upon infection with Mycobacterium tuberculosis, T lymphocytes specific for Mycobacterium tuberculosis antigens replicate and differentiate into effector T cells. Effector T cells die most of the immune response and some survive, forming long-lasting memory T cells. These induced memory T cells are known to play a role in the formation of rapid acquired immune immunity and maintaining T cell memory. Therefore, the following experiment was performed to confirm the effects of MTBK 20620 or MTBK 24820 protein on memory T cells.
  • T cell CD3 + CD4 + CD62L + CD44-CD127 +
  • central memory T cell CD3 + CD4 + CD62L + CD44 + CD127 +
  • effector memory T cell CD3 + CD4 + CD62L-CD44 + CD127 +
  • effector T cell Induction of CD3 + CD4 + CD62L-CD44 + CD127 ⁇ was analyzed via flow cytometry (FACSverse, BD) (FIG. 19).
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
  • the amount of the above ingredient is prepared per ampoule (2 ml).

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Abstract

La présente invention concerne une composition de vaccin pour la prévention de la tuberculose contenant une protéine Rv3112, MTBK 20620 ou MTBK 24820. Lors de l'utilisation d'une protéine Rv3112, MTBK 20620 ou MTBK 24820 selon la présente invention comme antigène, une capacité de production élevée d'IFN-γ est observée dans la rate et les poumons d'un animal modèle dans lequel une infection par Mycobacterium tuberculosis a été induite, et la tuberculose peut être efficacement prévenue par le maintien de la formation d'une immunité protectrice et de la mémorisation des cellules T rapidement acquises. En outre, une protéine Rv3112, MTBK 20620 ou MTBK 24820 selon la présente invention peut être utilisée dans le traitement de la tuberculose, et peut donc être utilement utilisée pour prévenir la rechute ou la réinfection de la tuberculose hautement pathogène ayant un taux de récurrence élevé et pour augmenter l'efficacité de son traitement.
PCT/KR2015/007631 2014-07-23 2015-07-22 Composition de vaccin pour la prévention de la tuberculose contenant une protéine rv3112, mtbk 20620 ou mtbk 24820 WO2016013873A1 (fr)

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KR10-2014-0093499 2014-07-23
KR10-2014-0093510 2014-07-23
KR1020140093510A KR101564323B1 (ko) 2014-07-23 2014-07-23 Ｒｖ３１１２ 단백질을 포함하는 결핵 예방용 백신 조성물
KR1020140093499A KR101568736B1 (ko) 2014-07-23 2014-07-23 Ｍｔｂｋ ２４８２０ 단백질을 포함하는 결핵 예방용 백신 조성물

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CN111148996A (zh) * 2017-07-28 2020-05-12 百时美施贵宝公司 用于检查点抑制剂的预测性外周血生物标志物

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WO2012031752A2 (fr) * 2010-09-07 2012-03-15 Institut Pasteur Korea Mutants de la tuberculose

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WO2012031752A2 (fr) * 2010-09-07 2012-03-15 Institut Pasteur Korea Mutants de la tuberculose

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111148996A (zh) * 2017-07-28 2020-05-12 百时美施贵宝公司 用于检查点抑制剂的预测性外周血生物标志物
US11899017B2 (en) 2017-07-28 2024-02-13 Bristol-Myers Squibb Company Predictive peripheral blood biomarker for checkpoint inhibitors

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