WO2017123025A1 - Régulateur pour troubles de la fonction immunitaire des voies respiratoires, utilisant des vésicules extracellulaires d'origine bactérienne - Google Patents

Régulateur pour troubles de la fonction immunitaire des voies respiratoires, utilisant des vésicules extracellulaires d'origine bactérienne Download PDF

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WO2017123025A1
WO2017123025A1 PCT/KR2017/000428 KR2017000428W WO2017123025A1 WO 2017123025 A1 WO2017123025 A1 WO 2017123025A1 KR 2017000428 W KR2017000428 W KR 2017000428W WO 2017123025 A1 WO2017123025 A1 WO 2017123025A1
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extracellular vesicles
bacteria
derived
derived extracellular
bacterial
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Korean (ko)
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김윤근
박해심
박한기
최영우
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주식회사 엠디헬스케어
아주대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an agent for regulating airway immune dysfunction by bacterial-derived extracellular vesicles, and in particular, comprising a bacterial-derived extracellular vesicle loaded with bacterial-derived antigen protein as an active ingredient, a composition for preventing or treating respiratory diseases, and It relates to a manufacturing method thereof.
  • An allergy can be defined as a phenomenon in which an acquired immune response occurs in the body when exposed to an antigen present in the surrounding environment, and the pattern of the immune response changes when repeated exposure to the antigen by the memory of the antigen. Allergens caused by exposure to harmful antigens have a beneficial effect on our bodies and induce immune responses through vaccines. On the other hand, when the antigen enters our body, if the immune response to the antigen is excessively induced and the hypersensitivity reaction occurs, the inflammatory reaction occurs in our body to cause irritable inflammatory disease.
  • Chronic rhinitis and sinusitis are conditions in which chronic inflammation occurs in the nasal and sinuses due to repeated exposure to the causative antigen in the air or in the nasal cavity, resulting in symptoms such as sneezing, runny nose and stuffy nose.
  • Asthma is a disease characterized by chronic inflammation of the respiratory tract caused by hypersensitivity of the airway to the antigen, which causes symptoms such as wheezing and dyspnea due to structural changes and functional changes caused by inflammation.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • indoor air includes Bacillus sp., Staphylococcus aureus , Staphylococcus epidermidis , and Pseudomonas Pseudomonas. stutzeri), Streptomyces three tests (Streptomycetes), Corey and four are many types of bacteria, such as (Corynebacteriaceae) inhabit the bacteria Seah [J Appl Microbiol. 1999 Apr; 86 (4): 622-34, BMC Microbiol. 2007 Apr 5; 7:27.
  • Bacterial-derived extracellular vesicles are spherical phospholipid bilayers with diameters of 20-100 nm, and for gram-negative bacteria-derived extracellular vesicles, biochemical and proteomic studies of outer membrane proteins and lipopolysaccharides. LPS), outer membrane lipids, DNA, RNA, and cytoplasmic proteins. It has recently been reported that Gram-positive bacteria also secrete extracellular vesicles, and that proteins contained in extracellular vesicles cause disease.
  • active immunotherapy which induces immune cell activity present in the body by directly administering an antigen-causing substance to the body, and manual control of an immune response by administering substances produced in vitro, such as monoclonal antibodies It can be divided into passive immunotherapy. Active immunotherapy is considered to be more efficient in terms of cost and frequency of administration than passive immunotherapy as a method for preventing disease. Active immunotherapy is intended to induce protective immunity by employing a strategy to induce an acquired immune response with the ability to induce specific long-term protective memory that is characteristic of acquired immunity. Key to regulating immune dysfunction is the key to antigen-specific regulation of immunological adverse events against antigens such as humoral (or antibody mediated) and cellular (T-cell mediated) immunity.
  • LPS Lipopolysaccharide
  • TLR4 receptors present in large amounts in vascular endothelial cells.
  • Protoplasts are living cell substances of plants or bacteria, and the extracellular and cell walls of these cells are removed mechanically and enzymatically, and the endotoxin content in the bacterial outer membrane is removed, thus toxic to the extracellular vesicles itself. You can solve the problem.
  • drugs such as polymyxin B can be treated to remove the activity of endotoxins present in extracellular vesicles.
  • the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a bacterium expressing an antigenic protein in a bacterial-derived extracellular vesicle, and an extracellular vesicle derived therefrom, and a method for preparing the same.
  • the present invention is to provide a method for controlling the airway immune function adverse reaction by bacterial derived extracellular vesicles using the composition.
  • the present invention provides a pharmaceutical composition for preventing or treating respiratory diseases containing bacteria-derived extracellular vesicles as an active ingredient.
  • the bacterium is a bacterium that overexpresses an antigenic protein gene in a bacterial-derived extracellular vesicle, and the respiratory disease is a respiratory disease caused by bacterial-derived vesicles.
  • the bacteria that produce the extracellular vesicles are characterized in that the Escherichia coli (E. coli) or in Salmonella bacteria (Salmonella spp.).
  • the bacteria-derived respiratory disease caused by extracellular vesicles is characterized in that selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), lung cancer, rhinitis, and sinusitis.
  • COPD chronic obstructive pulmonary disease
  • the antigen-derived extracellular vesicle-derived antigen protein is Gram-negative bacteria-derived extracellular vesicle outer membrane protein (Outer membrane protein, OMP) or Gram-positive bacteria-derived extracellular vesicle surface protein.
  • the Gram-negative bacteria-derived extracellular vesicle outer membrane protein may be outer membrane protein A (OMPA), and the Gram-positive bacteria-derived extracellular vesicle surface protein may be coagulase (CoA).
  • the present invention comprises the steps of (a) overexpressing the antigenic protein gene in bacteria-derived extracellular vesicles to bacteria; (b) culturing the overexpressed bacteria; (c) isolating extracellular vesicles from the bacterial culture; And (d) provides a method for producing a bacterial-derived extracellular vesicles for the prevention or treatment of respiratory diseases by bacterial-derived extracellular vesicles, comprising the step of removing the outer membrane endotoxin activity of the extracellular vesicles.
  • the method for separating the extracellular vesicles in the step (c) is to separate the naturally secreted extracellular vesicles by a filter method, and / or ultracentrifugation method, or artificial cells After the outer vesicles are made, they are separated by filtration, and / or ultracentrifugation.
  • the method for removing endotoxin activity in step (d) removes toxins (LPS, peptidoglycan, etc.) in the extracellular vesicles with lysozyme, or the endotoxin (LPS) polymyxin B and methods of inhibiting activity with drugs such as (polymyxin B).
  • toxins LPS, peptidoglycan, etc.
  • LPS endotoxin
  • the present invention provides an immunomodulator for preventing or treating respiratory diseases caused by bacterial-derived extracellular vesicles, comprising the bacterial-derived extracellular vesicles prepared by the above method as an active ingredient.
  • Respiratory diseases caused by bacterial extracellular vesicles include, but are not limited to, asthma, chronic obstructive pulmonary disease, lung cancer, rhinitis, and sinusitis.
  • the immunomodulatory agent is characterized in that the bacteria-derived extracellular vesicle antigen protein is overexpressed in the lumen of the bacteria-derived extracellular vesicles.
  • the extracellular vesicles may have an average diameter of 10-400nm, preferably 10-200nm.
  • the present invention provides a method for preventing or treating respiratory diseases caused by bacteria-derived extracellular vesicles using bacteria-derived extracellular vesicles.
  • the present invention provides a use for the prevention or treatment of respiratory diseases caused by bacteria-derived extracellular vesicles of bacteria-derived extracellular vesicles.
  • the present invention by using a composition containing a bacterial-derived extracellular vesicles loaded with bacteria-derived extracellular vesicle antigen protein as an active ingredient, by controlling the airway immune function adverse reactions caused by bacteria-derived extracellular vesicles, ultimately bacteria-derived cells It is expected to effectively prevent or treat respiratory diseases caused by external vesicles.
  • FIG. 1 is a diagram illustrating a method of making an immunomodulator by cloning a bacterial-derived extracellular vesicle antigen protein, loading it into bacteria, treating it with lysozyme to form a protoplast, and then preparing a vesicle by extrusion method. .
  • Figure 3 shows the expression of co-stimulatory molecules (MHC class II molecules) by treating extracellular vesicles loaded with bacterial derived extracellular vesicle antigen protein (OMPA) to bone marrow-derived dendritic cells. Is the result of measuring.
  • MHC class II molecules co-stimulatory molecules
  • OMPA bacterial derived extracellular vesicle antigen protein
  • cytokine IL 4 is a cytokine IL that induces Th1 and Th17 immune responses by treating extracellular vesicles loaded with bacteria-derived extracellular vesicle antigen protein (OMPA) on bone marrow-derived dendritic cells. This is the result of measuring the secretion amount of -12 and IL-6.
  • OMPA bacteria-derived extracellular vesicle antigen protein
  • FIG. 5 is a diagram illustrating a variety of bacteria present in the dust by separating dust from a bed mattress in an apartment and a hospital in Korea, performing a bacterial metagenome (dielectric) analysis in the dust.
  • FIG. 6 is a diagram representing the distribution of the major bacteria present in the dust in the apartments and hospitals in Korea as a heat map.
  • Figure 7 is a diagram representing the distribution of the major bacteria and bacteria-derived extracellular vesicles (EV) present in the apartment in Korea as a heat map.
  • EV extracellular vesicles
  • IM intramuscular injection
  • IN nasal administration
  • BAL Bronchoalveolar Lavage
  • T cells are isolated from mouse lung tissue and stimulated with anti-CD3 / CD28 After the test, gamma-interferon, IL-17 and IL-4 were measured.
  • OMP Gram-negative bacteria-derived vesicle outer membrane protein
  • T cells after administration of the extracellular vesicles loaded with Gram-positive bacteria-derived extracellular vesicle surface protein (Coagulase, CoA) by the method of Figure 8, T cells are isolated from mouse lung tissue and stimulated with anti-CD3 / CD28 After the test, gamma-interferon, IL-17 and IL-4 were measured.
  • Gram-positive bacteria-derived extracellular vesicle surface protein Coagulase, CoA
  • IM intramuscular injection
  • IN Nasal administration
  • OMP extracellular vesicle outer membrane protein
  • Fig. 15 shows the anti-inflammatory effect of gram-negative bacteria-derived extracellular vesicle outer membrane vesicle protein (OMP) loaded extracellular vesicles in a mouse disease model caused by Staphylococcus aureus-derived extracellular vesicles. The results were evaluated by the number of inflammatory cells in bronchoalveolar lavage fluid.
  • OMP outer membrane vesicle protein
  • Figure 16 shows the effect of inhibiting the inflammatory airway disease inhibited by E. coli-derived extracellular vesicles, which are pathogenic Gram-negative bacteria, after administration of extracellular vesicles loaded with Gram-negative bacteria-derived extracellular vesicle outer membrane protein (OMP). Protocol to evaluate.
  • E. coli-derived extracellular vesicles which are pathogenic Gram-negative bacteria
  • IM intramuscular injection
  • IN nasal administration
  • p-BNS extracellular vesicles loaded with outer membrane protein (OMP)
  • EV Extracellular Vesicles
  • FIG. 17 is an extracellular vesicle loaded with Gram-negative bacteria-derived extracellular vesicle outer membrane protein (OMP) in the method of FIG. 16 in an inflammatory airway disease mouse disease model caused by Escherichia coli-derived extracellular vesicles, which are pathogenic Gram-negative bacteria. After vesicle administration, the anti-inflammatory effect was evaluated by the number of inflammatory cells in bronchoalveolar lavage fluid.
  • OMP extracellular vesicle loaded with Gram-negative bacteria-derived extracellular vesicle outer membrane protein
  • Mac macrophages
  • Neu neutrophils
  • Eos eosinophils
  • Lymph Lymphocytes
  • FIG. 18 is an extracellular vesicle loaded with Gram-negative bacteria-derived extracellular vesicle outer membrane protein (OMP) in the method of FIG. 16 in an inflammatory airway disease mouse disease model caused by Escherichia coli-derived extracellular vesicle which is a pathogenic Gram-negative bacterium.
  • OMP outer membrane protein
  • NC negative control
  • OA p-BNS only group
  • Ec E. coli vesicles alone administration group
  • OE p-BNS group when E. coli vesicles were administered
  • FIG. 19 shows inflammatory airway disease mouse disease model caused by Escherichia coli-derived extracellular vesicles, which are pathogenic Gram-negative bacteria. After administration of vesicles, the secretion of gamma-interferon, IL-17, IL-4, and IL-10 was measured in lung and splenic T cells to evaluate anti-inflammatory effects.
  • NC negative control
  • OA p-BNS only group
  • Ec E. coli vesicles alone administration group
  • OE p-BNS group when E. coli vesicles were administered
  • bacteria-derived extracellular vesicle surface antigens present in large quantities in indoor dust were targeted.
  • bacteria-derived extracellular vesicles As antigen carriers, there are attempts to use bacterially derived extracellular vesicles as an adjuvant based on the fact that extracellular vesicles derived from bacteria can induce various immune responses in the host.
  • bacteria-derived extracellular vesicles contain exotoxins such as LPS, and there is a problem that the toxicity problems caused by LPS contained in bacteria-derived vesicles have to be solved.
  • the protoplasts made by removing the cell wall including the outer membrane and the peptidoglycan of the vesicles (protoplast) After the preparation, artificial extracellular vesicles were prepared and used.
  • OMPA Outer membrane protein A
  • CoA Staphylococcus aureus
  • 'respiratory disease' is not particularly limited as long as it occurs in the respiratory system such as rhinitis, sinusitis, asthma, COPD, lung cancer, but is preferably selected from the group consisting of asthma, COPD, and lung cancer.
  • bacteria-derived extracellular vesicles that cause respiratory disease is not particularly limited as long as the bacteria-derived extracellular vesicles present in the dust, but in the intestinal bacteria, Pseudomonas genus, Acinetobacter genus, or staphylococcus bacteria It is preferably selected from the extracellular vesicles from which they are derived.
  • treatment or prevention of respiratory diseases is meant to include reducing, alleviating and improving symptoms of respiratory diseases, and also includes lowering the likelihood of developing respiratory diseases.
  • bacteria that can overexpress genes, but E. coli or Salmonella spp. Are preferred.
  • the step of separating the extracellular vesicles can be used naturally secreted extracellular vesicles, there is no particular limitation on the method for separating the extracellular vesicles, can be obtained by concentrating with a filter, and then ultracentrifugation, etc. It may include the process of.
  • the extracellular vesicles can be artificially produced, and there is no particular limitation on the method for producing the same, but may be obtained by extruding with a filter, and may further include a process such as centrifugation and ultracentrifugation.
  • the method for separating extracellular vesicles in the present invention is not particularly limited as long as it includes bacterial derived extracellular vesicles, for example, in culture, centrifugation, ultra-fast centrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis
  • Extracellular vesicles can be separated using methods such as polymer addition, capillary electrophoresis, and the like, and combinations thereof. In addition, it may further include a process for washing to remove impurities, concentration of the obtained extracellular vesicles and the like.
  • the extracellular vesicles are naturally secreted or include extracellularly secreted extracellular vesicles.
  • the bacteria-derived extracellular vesicle surface antigen protein is expressed in the lumen of the extracellular vesicles prepared by the above method, wherein the expressed protein is enteric bacteria, Pseudomonas genus, Acinetobacter genus, or staphylococcus. Extracellular vesicle surface antigens derived from aureus bacteria are preferred.
  • the extracellular vesicles loaded with the specific antigen prepared by the method may have an average diameter of 10-400 nm, but preferably 10-200 nm.
  • an immunomodulator for treating or preventing the respiratory disease may be prepared as a pharmaceutical composition. It is possible to administer the bacteria-derived extracellular vesicles of the present invention for use in treatment and prophylaxis, but it is preferred that such vesicles be included as active ingredients of the pharmaceutical composition.
  • the pharmaceutical composition may contain the isolated extracellular vesicles as an active ingredient, and may include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
  • diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.
  • the method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be parenterally or orally administered, such as intramuscular injection, subcutaneous, inhalation, nasal, sublingual, and skin application.
  • Dosage varies depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease.
  • Daily dosage refers to the amount of therapeutic substance of the invention sufficient for treatment for a disease state alleviated by administration to a subject in need thereof.
  • Effective amounts of therapeutic agents depend on the particular compound, disease state and severity thereof, and on the individual in need thereof, and can be routinely determined by one skilled in the art.
  • the dosage of the composition according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient.
  • OMPA and CoA genes were synthesized by cDNA by RT-PCR for the cloning of OMPA, an extracellular vesicle envelope protein derived from E. coli, and CoA, a surface antigen of Staphylococcus aureus.
  • PCR products were inserted into a T-blunt PCR cloning kit (Solgent), and each plasmid was digested with EcoRI (OmpA, ABC transporter) and BglII (FepA). The sections were separated by electrophoresis and inserted into the pET-30a plasmid, transformed into E. coli DH5a by thermal shock, and finally the gene was cloned.
  • the candidate gene clones obtained in the above examples were incubated in Luria Bertani broth (Merch) at 37 ° C., 200 rpm for 12 hours, followed by Exprep plasmid mini prep. Each plasmid was isolated using Kit (GeneAll) and transfected into E. coli BL21 (Real Biotech).
  • the bacterial pellet was resuspended in Tris buffer and treated with 1 mg / ml lysozyme (Sigma) at 37 ° C., 100 rpm, 2 hours to obtain protoplasts, followed by 10 ⁇ m, 5 ⁇ m, and 1 Extruded with LiposoFast extruder (Avestin) in order through the membrane of the pore size. Finally, ultracentrifugation with 10% and 50% opti-prep density gradient medium (OptiPrep) yielded extracellular vesicles loaded with OMPA, CoA antigens.
  • OptiPrep Opti-prep density gradient medium
  • the extracellular vesicles loaded with the OMPA antigen obtained in Example 1 were diluted (50 ⁇ g / ml) with PBS and loaded 10 ⁇ l onto 300-mesh copper grids (EMS). Uranyl acetate (2%) was dropped on the grid for negative staining and observed with a JEM1011 electron microscope (JEOL). As a result, it was confirmed that the extracellular vesicles were spherical and surrounded by a lipid bilayer (see FIG. 2).
  • the extracellular vesicles were diluted with PBS (500 ng / ml), and diameters were determined by dynamic light scattering using Zetasizer Nano ZS (Malvern Instruments). Size distribution was measured and the results were analyzed using Dynamic V6 software. As a result, it was found that the diameter of the extracellular vesicles was about 130 nm (see FIG. 2).
  • BMDC bone marrow-derived dendritic cells
  • BMDC BMDC (5 ⁇ 10 5 cells / well) was treated with DMEM containing 10% FBS and antibiotics (100 unit / ml penicillin and 100 ⁇ g / ml streptomycin) in a 24-well tissue culture plate (TPP) for 24 hours at 37 ° C. Incubated). After removing the medium, extracellular vesicles loaded with OMPA antigen were changed to serum-free DMEM medium added at concentrations of 0.1 and 1 ug / ml. After 15 hours, the expression level of MHC class II molecules in BMDC and ELISA assay were performed on the culture medium to measure the expression level of T-12 cell cytokines IL-12 and IL-6.
  • antigen-presenting cells treated with extracellular vesicles loaded with OMPA antigen significantly increased secretion of IL-6, which is important for differentiation into Th-12 and Th17 cells, which are important for differentiation into Th1 cells (see FIG. 4). .
  • the vacuum cleaner was used to collect dusts from bed mattresses in apartments in Seoul and bed mattresses in the hospitals of major hospitals and intensive care units.
  • the dust present in the filter of the vacuum cleaner was transferred to a clean glass bottle and the mass was measured. 5 g of dust were dissolved in a beaker containing 200 ml PBS for 4 hours at 4 ° C. Afterwards, the large amount of foreign matter is first filtered through a gauze, and the filtered solution is divided into high speed centrifuge tubes, followed by high speed centrifugation for 15 minutes at 10,000 ⁇ g at 4 ° C. ) Was performed twice in succession to collect the bacterial part.
  • Metagenome sequencing extracts genes from the bacterial and extracellular vesicles separated by the above method, amplifies using 16S rDNA primers, and performs sequencing (Roche GS FLX sequencer), and the result is shown in Standard Flowgram Format (SFF). Output the file and convert the SFF file into a sequence file (.fasta) and nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and use the window (20 bps) average base call accuracy. The portion with less than 99% (Phred score ⁇ 20) was removed. After removing the low quality part, only the lead length of 300 bps or more was used (Sickle version 1.33).
  • SFF Standard Flowgram Format
  • clustering is performed according to sequence similarity using UCLUST and USEARCH for analysis of Operational Taxonomy Unit (OUT), gunus 94%, family 90%, order 85%, class 80%, phylum Cluster based on 75% sequence similarity, classify at the phylum, class, order, family, and gunus levels of each OUT, and use BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) to achieve greater than 97% sequence similarity. Bacteria with were analyzed (QIIME).
  • extracellular vesicles derived from bacteria in apartment dust Extracellular vesicles derived from Pseudomonas, Acinetobacter, Enterobacteriaceae, and Staphylococcus bacteria, regardless of season, were mainly derived from bacteria of Pseudomonas. Extracellular vesicles were the most common (see FIG. 7).
  • extracellular vesicles loaded with OMPA antigen were loaded. Immunization was induced by administration to mice. In addition, after inducing an immune response, blood was collected from mice treated with the control group (PBS) and extracellular vesicles loaded with OMPA antigen to measure OMPA-specific IgG antibodies.Bronchoalveolar lavage fluid was collected to determine the concentration of OMPA-specific sIgA antibodies. Was measured (see FIG. 8).
  • mouse serum induced an immune response by intramuscular injection with extracellular vesicles loaded with OMPA antigen was generated in OMPA antigen-specific IgG antibody with or without nasal administration of extracellular vesicles loaded with OMPA antigen. This has been increased.
  • OMPA antigen-specific sIgA production in bronchoalveolar lavage fluid was increased only after intramuscular injection of extracellular vesicles loaded with OMPA antigen (see Fig. 9).
  • the extracted lung tissue was digested by passing through a 100 ⁇ m cell strainer (BD Biosciences) with 5 ml syringe washing buffer (2.5% FBS, DMEM containing 0.01M HEPES).
  • the isolated cells were treated with ammonium chloride solution at 4 ° C. for 10 minutes to allow erythrocyte cells to lyse.
  • the obtained cells were washed with washing buffer and filtered with 40 ⁇ m cell strainer (BD), followed by 10% FBS, 50 ⁇ M 2-ME, 0.01 M HEPES and antibiotics (100 unit / ml penicillin, 100 ⁇ g / ml streptomycin).
  • the amount of IFN- ⁇ a major cytokine produced by Th1 cells
  • OMPA antigen-loaded extracellular vesicle-administered group compared to the control group, and OMPA antigen-loaded extracellular Dose-dependent nasal vesicles decreased dose-dependently.
  • IL-17 a major cytokine produced by Th17
  • the extracellular vesicles loaded with OMPA antigen increased at low concentrations but not at high concentrations.
  • IL-4 secreted from Th2 cells did not change regardless of intramuscular injection or nasal administration (see FIG. 10).
  • extracellular vesicles loaded with CoA antigens were used. Immunization was induced by administration to mice. In addition, after inducing an immune response, blood was collected from a control group (PBS) and mice treated with extracellular vesicles loaded with CoA antigens to measure CoA antigen-specific IgG antibodies, and bronchioalveolar lavage fluid was collected to collect CoA antigen-specific sIgA antibodies. The concentration of was measured by the method of Example 5.
  • mice sera that induced an immune response by intramuscular injection with extracellular vesicles loaded with CoA antigens generated CoA antigen-specific IgG antibodies regardless of the presence or absence of extracoagulants loaded with CoA antigens. This has been increased.
  • CoA antigen-specific sIgA production in bronchoalveolar lavage fluid increased dose-dependently only after intranasal injection of extracellular vesicles loaded with CoA antigen (see FIG. 11).
  • T cell response was evaluated by the method of Example 6 by measuring the amount of.
  • the amount of IFN- ⁇ a major cytokine produced by Th1 cells, was increased in the extracellular vesicle-administered group loaded with CoA antigen compared to the control group, and the extracellular loaded with CoA antigen Increase in vesicle nasal administration.
  • the extracellular vesicles loaded with CoA antigens increased at low concentrations but not at high concentrations.
  • IL-4 secreted from Th2 cells increased upon nasal administration (see FIG. 12).
  • Example 9 Gram-negative bacteria E. coli derived Extracellular By parcel Airway inflammatory response On generation OMPA Antigen Loaded Anti-inflammatory effect of vesicles
  • a representative Gram-negative bacterium is an airway inflammation model, as shown in FIG. 13, to evaluate the efficacy of extracellular vesicles loaded with OMPA antigen in an airway inflammation mouse model induced by administration of extracellular vesicles derived from E. coli into the airways.
  • E. coli-derived extracellular vesicles were administered to the nasal cavity twice a week for a total of three weeks to prepare a respiratory disease model (see FIG. 13).
  • the extracellular vesicles loaded with the OMPA antigen were loaded three times before the respiratory disease model was made, as shown in FIG. 13 at the concentration set in the method of Example 5. After injection and nasal administration, extracellular vesicles loaded with OMPA antigens were administered only intranasally (see FIG. 13) during the construction of the disease model.
  • Example 10 Gram-positive bacteria Derived from Staphylococcus aureus Extracellular By parcel Airway inflammatory response On generation CoA Anti-inflammatory effect of antigen loaded vesicles
  • a representative Gram-positive bacterium is airway inflammation, as shown in FIG. 13, to evaluate the efficacy of extracellular vesicles loaded with CoA antigen in an airway inflammation mouse model induced by airway administration of extracellular vesicles derived from Staphylococcus aureus.
  • Staphylococcus aureus-derived extracellular vesicles were administered to the nasal cavity twice a week for a total of three weeks to prepare a respiratory disease model (see FIG. 13).
  • the intracellular vesicles loaded with CoA antigen three times before the respiratory disease model was created, as shown in FIG. 13 at the concentration set in the method of Example 7
  • extracellular vesicles loaded with CoA antigens were administered only intranasally (see FIG. 13) during the preparation of the disease model.
  • mice that did not receive extracellular vesicles loaded with CoA antigens the number of inflammatory cells in bronchoalveolar lavage fluid was significantly increased by airway administration of vesicles derived from Staphylococcus aureus.
  • the number of inflammatory cells in bronchoalveolar lavage fluid was significantly reduced compared to the positive control.
  • the number of inflammatory cells in bronchoalveolar lavage fluid was significantly reduced in mice continuously administered before and after preparation (see FIG. 15).
  • Example 11 Gram-negative bacteria E. coli derived Extracellular Induced by parcel Airway Inflammation Anti-inflammatory Effect of Vesicles Loaded with OMPA Antigen on Mouse Model
  • E. coli-derived extracellular vesicles were administered to the nasal cavity twice a week for a total of 3 weeks to prepare a respiratory disease model (see FIG. 16).
  • intramuscular injection of OMPA antigen loaded once a week at the concentration set in the method of Example 5 was made on the first week of respiratory disease model. At the 2nd and 3rd week, intramuscular injection was performed simultaneously with the intramuscular injection, and from the 4th week, only nasal administration was performed daily (see FIG. 16).
  • T cells were isolated from lung tissue and spleen, stimulated with anti-CD3 and anti-CD28, and the T cell immune response was assessed by cytokine secretion.
  • the administration of extracellular vesicles loaded with OMPA antigen significantly reduced the amount of IL-4 secreted by T cells in spleen tissues.
  • Modulators decreased cytokine secretion of gamma-interferon, IL-17, IL-4, IL-10 and the like in T cells in lung tissue (see FIG. 19).
  • composition according to the present invention is expected to be able to effectively prevent or treat respiratory diseases caused by bacterial-derived extracellular vesicles, by regulating the adverse effects of airway immune function caused by bacterial-derived extracellular vesicles.

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Abstract

La présente invention concerne un régulateur immunitaire pour prévenir ou traiter des maladies respiratoires au moyen de vésicules extracellulaires dérivées de bactéries et, spécifiquement, concerne : une composition pharmaceutique pour régulation immunitaire, qui contient des vésicules dérivées de bactéries, en tant que substance active, contenant des protéines d'antigène de surface contenues dans des vésicules extracellulaires dérivées de bactéries pathogènes causant des maladies respiratoires ; un procédé de préparation de celle-ci ; et similaire.
PCT/KR2017/000428 2016-01-15 2017-01-12 Régulateur pour troubles de la fonction immunitaire des voies respiratoires, utilisant des vésicules extracellulaires d'origine bactérienne WO2017123025A1 (fr)

Applications Claiming Priority (4)

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KR20160005103 2016-01-15
KR10-2016-0005103 2016-01-15
KR10-2017-0004428 2017-01-11
KR1020170004428A KR101911893B1 (ko) 2017-01-11 2017-01-11 세균유래 세포밖 소포에 의한 기도 면역기능이상 조절제

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WO2017123025A1 true WO2017123025A1 (fr) 2017-07-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4030168A4 (fr) * 2019-09-10 2023-06-14 MD Healthcare Inc. Procédé de diagnostic de maladie pulmonaire basé sur des anticorps dirigés contre des vésicules dérivées de micro-organismes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027990A2 (fr) * 2009-09-04 2011-03-10 주식회사이언메딕스 Vésicules extracellulaires issues de bactéries à gram positif et leur utilisation
WO2011043538A2 (fr) * 2009-10-08 2011-04-14 주식회사이언메딕스 Composition comprenant des vésicules de membranes extracellulaires issues de l'air intérieur, et utilisation de celle-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027990A2 (fr) * 2009-09-04 2011-03-10 주식회사이언메딕스 Vésicules extracellulaires issues de bactéries à gram positif et leur utilisation
WO2011043538A2 (fr) * 2009-10-08 2011-04-14 주식회사이언메딕스 Composition comprenant des vésicules de membranes extracellulaires issues de l'air intérieur, et utilisation de celle-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KLIMENTOVA, J. ET AL.: "Methods of Isolation and Purification of Outer Membrane Vesicles from Gram-negative Bacteria", MICROBIOLOGICAL RESEARCH, vol. 170, 2015, pages 1 - 9, XP029107295 *
PORE, D. ET AL.: "Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model", PLOS ONE, vol. 6, no. 7, 2011, pages e22663, XP055183239 *
ZHANG, Z. ET AL.: "Coagulase-negative Staphylococcus Culture in Chronic Rhinosinusitis", INTERNATIONAL FORUM OF ALLERGY AND RHINOLOGY, vol. 5, no. 3, 2015, pages 204 - 213, XP055399379 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4030168A4 (fr) * 2019-09-10 2023-06-14 MD Healthcare Inc. Procédé de diagnostic de maladie pulmonaire basé sur des anticorps dirigés contre des vésicules dérivées de micro-organismes

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