WO2017123025A1 - Regulator for respiratory tract immune function disorders, using bacterial-derived extracellular vesicles - Google Patents

Abstract

The present invention relates to an immune regulator for preventing or treating respiratory diseases by using bacteria-derived extracellular vesicles and, specifically, provides: a pharmaceutical composition for immune regulation, which contains bacteria-derived vesicles, as active ingredients, containing surface antigen proteins contained in extracellular vesicles derived from pathogenic bacteria causing respiratory diseases; a preparation method therefor; and the like.

Description

Regulators of Airway Immune Dysfunction by Bacteria-Derived Extracellular Vesicles

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. In addition, chronic obstructive pulmonary disease (COPD) is a disease characterized by irreversible airway obstruction unlike asthma, is a chronic inflammation form of the airway, and includes diseases such as chronic obstructive bronchitis, chronic obstructive bronchiolitis, emphysema. In recent epidemiologic studies, the incidence of increased lung cancer in COPD patients, regardless of smoking, has been recognized as a significant risk factor for lung cancer.

Meanwhile, 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. It is known that the production of inflammatory cytokines is induced in immune cells and lung epidermal cells by endotoxin (lipopolysaccharide, LPS) or peptidoglycan derived from bacteria inhabiting the indoor air [Toxicology. 2005 Nov 5; 215 (1-2): 25-36., Indoor Air. 1999 Dec; 9 (4): 219-25.

Bacterial-derived extracellular vesicles (EVs) 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.

It has been reported that a large amount of extracellular vesicles derived from bacteria exist in indoor dust, and neutrophil airway inflammation with immune dysfunction occurs when the extracellular vesicles in the dust are administered to mice. In addition, neutrophil-inflammation with immune dysfunction occurs in mice due to Gram-positive Staphylococcus aureus-derived extracellular vesicles, and is neutrophils caused by extracellular vesicles derived from Escherichia coli, a gram-negative bacterium. In addition to constitutive airway inflammation, emphysema, a major cause of irreversible airway obstruction, has been reported in COPD.

In immunotherapy, 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.

Bacterial-derived extracellular vesicles as antigen carriers, despite their excellent immuno-inducing effects, have the problem of inducing serious toxicity, such as sepsis. Lipopolysaccharide (LPS), known as endotoxin, exists in the extracellular membrane of Gram-negative bacteria, and it has recently been found to induce sepsis by inducing systemic vascular inflammation through TLR4 receptors present in large amounts in vascular endothelial cells. As a method for solving the toxicity problem caused by Gram-negative bacteria-derived extracellular vesicles, LPS present in extracellular vesicles can be removed or a compound that inhibits LPS activity can be used as an antigen transporter. 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. In addition, drugs such as polymyxin B can be treated to remove the activity of endotoxins present in extracellular vesicles.

However, no antigen-specific immunotherapy has been reported to modulate airway immune dysfunction caused by bacterial extracellular vesicles.

Accordingly, the present invention is to provide 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.

In addition, 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 technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

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.

In one embodiment, the bacteria that produce the extracellular vesicles are characterized in that the Escherichia coli (E. coli) or in Salmonella bacteria (Salmonella spp.).

In another embodiment of the present invention, 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.

In another embodiment of the present invention, 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).

In addition, 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.

In one embodiment of the present invention, 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.

In another embodiment of the present invention, 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).

In another aspect, 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.

In one embodiment of the invention, 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.

In another embodiment of the present invention, the extracellular vesicles may have an average diameter of 10-400nm, preferably 10-200nm.

In addition, the present invention provides a method for preventing or treating respiratory diseases caused by bacteria-derived extracellular vesicles using bacteria-derived extracellular vesicles.

In addition, 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.

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 2, the size of the extracellular vesicles loaded with bacteria-derived extracellular vesicle antigen protein (OMPA) was taken by transmission electron microscope (left) and dynamic light scattering method (size) One result is (right).

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.

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.

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.

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.

8 is an experimental protocol for evaluating the immunogenicity of extracellular vesicles loaded with bacterial derived extracellular vesicle antigen proteins in mice.

IM: intramuscular injection; IN: nasal administration; BAL: Bronchoalveolar Lavage

Figure 9, after administration of the extracellular vesicles loaded with Gram-negative bacteria extracellular vesicle outer membrane protein (OMP) by the method of Figure 8, antigen-specific IgG antibody in the mouse serum and antigen-specific in bronchoalveolar lavage fluid The result of measuring IgA antibody production | generation ability.

Figure 10, after administration of the extracellular vesicles loaded with Gram-negative bacteria-derived vesicle outer membrane protein (OMP) 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.

Figure 11, after administration of the extracellular vesicles loaded with Gram-positive bacteria-derived extracellular vesicle surface protein (Coagulase, CoA) by the method of Figure 8, the antigen-specific IgG antibody in mouse serum and antigen-specific IgA antibody in bronchoalveolar lavage fluid It is the result of measuring the generation ability.

Figure 12, 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.

Figure 13, after administration of extracellular vesicles loaded with Gram-negative bacteria extracellular vesicle outer membrane protein (OMP), inhibits the development of inflammatory airway disease caused by Escherichia coli-derived extracellular vesicles that are pathogenic Gram-negative bacteria This is a protocol for evaluating the preventive effects of

IM: intramuscular injection; IN: Nasal administration

14 is an anti-inflammatory prevention of extracellular vesicles loaded with Gram-negative bacteria-derived extracellular vesicle outer membrane protein (OMP) in inflammatory airway disease mouse disease model caused by Escherichia coli-derived extracellular vesicles, which are pathogenic Gram-negative bacteria. The effect was evaluated by the number of inflammatory cells in bronchoalveolar lavage fluid.

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.

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.

IM: intramuscular injection; IN: nasal administration; p-BNS: extracellular vesicles loaded with outer membrane protein (OMP); EV: Extracellular Vesicles

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.

Mac: macrophages; Neu: neutrophils; Eos: eosinophils; Lymph: Lymphocytes

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. After administration of vesicles, cytokines in bronchoalveolar lavage fluid were measured 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

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

To develop antigen-specific immunomodulators, it is important to select the antigen and the adjuvant that induces the desired immune response. In the present invention, bacteria-derived extracellular vesicle surface antigens present in large quantities in indoor dust were targeted.

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. However, 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. Thus, in the present invention, in order to eliminate the toxicity problem of bacterial vesicles, 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.

Specifically, in the present invention, a representative Gram-negative bacterium, Outer membrane protein A (OMPA) derived from Escherichia coli and Outer membrane protein-derived extracellular vesicles derived from Staphylococcus aureus (Coagulase, CoA) Each was overexpressed in Escherichia coli and cultured, and then treated with lysozyme to prepare protoplasts, and then extracellular vesicles were prepared by extrusion. Subsequently, the immunogenicity of the antigen-loaded extracellular vesicles was evaluated through in vitro and in vitro experiments to determine the route of administration and dose for evaluating efficacy. This suggests that the extracellular vesicles loaded with the antigen are effective immunomodulators.

In addition, in order to evaluate the efficacy of the immunomodulator in a disease model, pathogenic bacteria-derived extracellular vesicles were directly administered to the respiratory tract disease model was used.

In the present invention, '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.

In the present invention, "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.

In the present invention, "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.

In the present invention, there is no particular limitation on bacteria that can overexpress genes, but E. coli or Salmonella spp. Are preferred.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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. Such 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. In addition, 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. As a non-limiting example, 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.

Hereinafter, preferred embodiments of the present invention are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Preparation of Vesicles Loaded with Bacteria-Derived Extracellular Vesicle Surface Antigen (see FIG. 1)

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 obtained by incubating BL21 for 3 hours at 200 rpm, 37 ° C. and 100 ml LB broth (1 mM IPTG) until the OD value was 0.3, and then centrifuged at 3,000 × g and 4 ° C. for 10 minutes was used for Tris. washed with buffer (0.01M Tris HCl, 0.5M sucrose), centrifuged at 4 ° C., 3,000 × g for 10 minutes, and then resuspended with Tris buffer. Resuspended bacteria were treated with 0.01 M EDTA at 37 ° C., 100 rpm for 30 minutes, pelleted at 4 ° C., 3,000 × g for 10 minutes, and then washed with Tris buffer to pellet.

Next, 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 μm, 5 μm, 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.

Example 2: Physical Characterization of Extracellular Vesicles Loaded with OMPA Antigen

The extracellular vesicles loaded with the OMPA antigen obtained in Example 1 were diluted (50 μg / ml) with PBS and loaded 10 μl 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).

In order to measure the size of the extracellular vesicles loaded with the OMPA antigen obtained in Example 1, 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).

Example 3: Innate Immune Response by Extracellular Vesicles Loaded with OMPA Antigen

Since the extracellular vesicles loaded with OMPA antigen must be capable of inducing an immune response in order to be used as an immunomodulator, dendritic cells derived from bone marrow cells to assess the level of innate immune response induced by extracellular vesicles loaded with OMPA antigen In vitro experiments were performed using bone marrow-derived dendritic cells (BMDC).

First, BMDC (5 × 10 ⁵ cells / well) was treated with DMEM containing 10% FBS and antibiotics (100 unit / ml penicillin and 100 μg / 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.

As a result, the expression of MHC class II molecules in antigen presenting cells was greatly increased by extracellular vesicles loaded with OMPA antigen (see FIG. 3). In addition, 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). .

Example 4 Bacterial and Bacterial-Derived Extracellular Vesicle Metagenome Analysis in Apartment and Hospital Dusts

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. In order to separate the extracellular vesicles, about 180 ml of the supernatant was passed through a membrane filter having a pore size of 0.45 μm, and then divided into a 70 ml ultracentrifuge tube and 4 ° C. , Ultracentrifugation (ultracetrifugation) at 100,000 × g for 4 hours. The supernatant was discarded and the extracellular vesicles in the dust were extracted by dissolving the precipitate under the tube with PBS. 100 μl of bacteria and extracellular vesicles were boiled at 100 ° C. for 15 minutes to allow the DNA inside to come out of the lipid and then cooled on ice for 5 minutes. And to remove the remaining suspended solids centrifuged for 30 minutes at 10,000 × g, 4 ℃ and collected only the supernatant. The amount of DNA was quantified using Nanodrop. In order to confirm the presence of bacteria-derived DNA, PCR was performed using 16s rRNA primer [27F (5 '): AGA GTT TGA TCM TGG CTC AG, 1492R (3'): GGT TAC CTT GTT ACG ACT T]. It was confirmed that there is a bacteria-derived gene.

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). For results analysis, 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).

As a result, in the case of the dust present in the apartment, the variety of the bacteria present in the dust collected in the winter compared to the dust collected in the summer, the variety was reduced (see Fig. 5). In addition, in the case of dust collected in the winter in the apartments and hospitals, the variety of bacteria in the dust in the hospital compared to the apartment dust was reduced (see Fig. 5).

The distribution of bacteria in apartments and hospitals showed that Pseudomonas, Acinetobacter, Enterobacteriaceae, and Staphylococcus bacteria were the major bacteria in the dust in apartments, and Pseudomonas in the apartments, and hospitals. Acinetobacter genus bacteria were present at the highest rate regardless of season (see FIG. 6).

Regarding the distribution of 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).

Example 5: Antibody Immune Responses by Extracellular Vesicles Loaded with OMPA Antigen in Mice

In order to confirm the induction of antibody immune response by extracellular vesicles loaded with OMPA antigen in vivo and to determine the effective amount of extracellular vesicles loaded with OMPA antigen, 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).

Specifically, 100 μl of PBS containing 100 ng of OMPA was coated on a 96-well black plate (Greiner Bio-one) at 4 ° C. for 24 hours. The plate coated with OMPA was washed with PBS and treated with 1% BSA solution diluted with PBS for 1 hour to perform a blocking process and washed again with PBS.

Blood, serum and cell layers were separated from mice treated with control (PBS) and extracellular vesicles loaded with OMPA antigen, and serum was separated. Cell layers and supernatants were separated from bronchoalveolar lavage fluid. Supernatants were diluted with 1% BSA solution and reacted for 2 hours on plates coated with OMPA antigen. After incubation, the plate was washed and reacted for 2 hours by adding horseradish peroxidase-conjugated goat anti-mouse IgG and 200 ng / ml of IgA (Santa Cruz Biotechnology). After washing the plate again, ECL substrate (Thermo Scientific) was added and the IgG and sIgA antibody titers were analyzed using Victor Wallac 1420 apparatus (PerkinElmer).

As a result, as shown in FIG. 9, 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. On the other hand, 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).

Example 6: T Cell Immune Responses by Extracellular Vesicles Loaded with OMPA Antigen in Mice

Induced immune responses by administering extracellular vesicles loaded with OMPA antigen in vivo , and cytokines produced in T cells in mice treated with control (PBS) and extracellular vesicles loaded with OMPA antigen The T cell response was assessed by measuring the amount of (see FIG. 8).

That is, the extracted lung tissue was digested by passing through a 100 μm 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 μm cell strainer (BD), followed by 10% FBS, 50 μM 2-ME, 0.01 M HEPES and antibiotics (100 unit / ml penicillin, 100 μg / ml streptomycin). Cultured for 12 hours in 24-well plates using RPMI 1640 medium. At this time, the 24-well plate was coated with 1 μg / ml of anti-CD3 (eBioscience) and 1 μg / ml of anti-CD28 (eBioscience) antibody to re-stimulate T cells.

As a result, as shown in FIG. 10, the amount of IFN-γ, a major cytokine produced by Th1 cells, was increased in the OMPA antigen-loaded extracellular vesicle-administered group compared to the control group, and OMPA antigen-loaded extracellular Dose-dependent nasal vesicles decreased dose-dependently. In the case of IL-17, a major cytokine produced by Th17, the extracellular vesicles loaded with OMPA antigen increased at low concentrations but not at high concentrations. However, IL-4 secreted from Th2 cells did not change regardless of intramuscular injection or nasal administration (see FIG. 10).

Example 7: Antibody Immune Responses by Extracellular Vesicles Loaded with CoA Antigen in Mice

In order to confirm the induction of antibody immune response by extracellular vesicles loaded with CoA antigen in vivo and to determine an effective amount of extracellular vesicles loaded with CoA antigen, 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.

As a result, as shown in FIG. 11, mouse 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. On the other hand, 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).

Example 8: T Cell Immune Responses by Extracellular Vesicles Loaded with CoA Antigen in Mice

Induced immune responses by administering extracellular vesicles loaded with CoA antigen in vivo , and cytokines produced in T cells in mice administered control (PBS) and extracellular vesicles loaded with CoA antigen The T cell response was evaluated by the method of Example 6 by measuring the amount of.

As a result, as shown in FIG. 12, the amount of IFN-γ, 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. In the case of IL-17, a major cytokine produced in Th17, the extracellular vesicles loaded with CoA antigens increased at low concentrations but not at high concentrations. However, 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).

In order to induce an immune response to the extracellular vesicles loaded with the OMPA antigen, 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.

As a result, as shown in FIG. 14, in the positive control mice not administered the extracellular vesicles loaded with the OMPA antigen, the number of inflammatory cells in bronchoalveolar lavage fluid was significantly increased by E. coli-derived extracellular vesicles. In mice that induced an immune response with extracellular vesicles loaded with OMPA antigen before the respiratory disease model was developed, the number of inflammatory cells in bronchoalveolar lavage fluid was significantly reduced to the negative control level. In the mice continuously administered after the preparation, the number of inflammatory cells in bronchoalveolar lavage fluid was similarly reduced (see FIG. 14).

Therefore, it was found that when intracellular vesicles loaded with Gram-negative bacterial envelope membrane protein were administered nasal, respiratory diseases caused by Gram-negative bacteria-derived extracellular vesicles were effectively suppressed.

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. To prepare the model, 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).

To induce an immune response to CoA antigen-loaded extracellular vesicles, 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 After injection and nasal administration, extracellular vesicles loaded with CoA antigens were administered only intranasally (see FIG. 13) during the preparation of the disease model.

As a result, as shown in Fig. 15, in the positive control 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. In mice that induced an immune response with extracellular vesicles loaded with CoA antigen prior to the production of respiratory disease models, the number of inflammatory cells in bronchoalveolar lavage fluid was significantly reduced compared to the positive control. Similarly, the number of inflammatory cells in bronchoalveolar lavage fluid was significantly reduced in mice continuously administered before and after preparation (see FIG. 15).

Therefore, when the extracellular vesicles loaded with Gram-positive bacteria surface protein were administered nasal, it was found that the respiratory disease caused by Gram-positive bacteria-derived vesicles was effectively suppressed.

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

Extracellular vesicles derived from E. coli, a representative Gram-negative bacterium, were administered to the airway to evaluate the therapeutic efficacy of OMPA antigen-loaded extracellular vesicles in an airway inflammation mouse model. To this end, in order to prepare an airway inflammation 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).

In addition, in order to induce an immune response to the extracellular vesicles loaded with OMPA antigen, 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).

As a result, as shown in Fig. 17, in the positive control mice not administered extracellular vesicles loaded with OMPA antigen, the number of inflammatory cells in bronchoalveolar lavage fluid was significantly increased by E. coli-derived extracellular vesicle airway administration. Meanwhile, neutrophil counts in bronchoalveolar lavage fluid were significantly reduced in mice induced with immune responses to extracellular vesicles loaded with OMPA antigen after preparation of the respiratory disease model (see FIG. 17). In addition, it was confirmed that the secretion of gamma interferon, IL-17, and IL-4 in the cytokine secretion in bronchoalveolar lavage fluid was effectively suppressed when the OMPA antigen-loaded extracellular vesicles were administered (see FIG. 18).

After administration of extracellular vesicles loaded with OMPA antigen, 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. As a result, 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).

Therefore, it was found that when intracellular vesicle immunomodulators loaded with Gram-negative bacterial outer membrane protein were administered nasal, airway inflammation caused by Gram-negative bacterial-derived extracellular vesicles could be effectively treated.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

The 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.

Claims (9)

1. A pharmaceutical composition for the prevention or treatment of respiratory diseases containing bacteria-derived extracellular vesicles as an active ingredient, wherein the bacteria are bacteria that overexpress the antigenic protein genes in bacteria-derived extracellular vesicles, and the respiratory disease is directed to bacteria-derived extracellular vesicles. It is a respiratory disease caused by, pharmaceutical composition.
2. The method of claim 1,

   The bacterium producing the extracellular vesicles is E. coli or Salmonella, characterized in that the pharmaceutical composition.
3. The method of claim 1,

   Respiratory disease caused by the bacterial extracellular vesicles is characterized in that selected from the group consisting of asthma, chronic obstructive pulmonary disease, lung cancer, rhinitis, and sinusitis.
4. The method of claim 1,

   The bacterial protein-derived extracellular vesicle antigen protein is characterized in that the Gram-negative bacteria-derived extracellular vesicle outer membrane protein or Gram-positive bacteria-derived extracellular vesicle surface protein.
5. The method of claim 4, wherein

   The Gram-negative bacteria-derived extracellular vesicle outer membrane protein is characterized in that the outer membrane protein A (Outer Membrane Protein A), pharmaceutical composition.
6. The method of claim 4, wherein

   The Gram-positive bacteria-derived extracellular vesicle surface protein is characterized in that the coagulase (Coagulase), pharmaceutical composition.
7. (a) overexpressing an antigenic protein gene in a bacteria-derived extracellular vesicle to bacteria;

   (b) culturing the overexpressed bacteria;

   (c) isolating extracellular vesicles from the bacterial culture; And

   (D) a method for producing bacterial-derived extracellular vesicles for the prevention or treatment of respiratory diseases caused by bacterial-derived extracellular vesicles, comprising the step of removing the outer membrane endotoxin activity of the vesicles, the antigenic protein in the bacterial-derived extracellular vesicles Is a Gram-negative bacterium-derived extracellular vesicle outer membrane protein or Gram-positive bacteria-derived extracellular vesicle surface protein.
8. The method of claim 7, wherein

   The bacterium producing the extracellular vesicles is E. coli or Salmonella, characterized in that the production method.
9. The method of claim 7, wherein

   Respiratory disease caused by the bacterial extracellular vesicles is characterized in that selected from the group consisting of asthma, chronic obstructive pulmonary disease, lung cancer, rhinitis, and sinusitis.

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