KR20230003945A - Composition for treating superinfection of virus and bacteria - Google Patents

Abstract

The present invention relates to a composition for effectively preventing, alleviating or treating superinfection by viruses and bacteria. The present invention relates to a pharmaceutical composition for preventing or treating superinfection which contains an activity or expression inhibitor of cluster of differentiation 47 (CD47) protein, or an inhibitor of the expression of genes encoding the above proteins as an active component.

Description

Composition for treating superinfection of virus and bacteria {Composition for treating superinfection of virus and bacteria}

The present invention relates to a composition for effectively preventing, improving or treating superinfection by viruses and bacteria or respiratory diseases caused thereby.

Recent studies have shown that between 50 and 90% of severe or fatal cases of influenza virus infection during previous influenza pandemics are associated with bacterial superinfection. In addition, at least 15 types of bacteria that can cause superinfection with influenza viruses have been identified, including Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae. influenzae) and Legionella pneumophila are known to increase the risk of pneumonia and subsequent fatality. Moreover, Pseudomonas aeruginosa has been reported to cause severe pneumonia when co-infected with influenza virus. However, several studies have confirmed that the fatality is highest in superinfection with S. aureus and influenza viruses. As a result of a related study conducted in the Netherlands during the 2009 influenza H1N1 subtype pandemic, S. aureus was identified as a co-pathogen in 59% of influenza-infected patients, which was more than 4 times higher than S. pneumoniae. seemed As a result of the UK study, S. aureus was detected in 27% of patients infected with influenza A in 140 hospitals, which is very high compared to 15% and 4% of S. pneumoniae and Haemophilus, respectively.

One object of the present invention is to provide a composition for preventing, improving or treating superinfection caused by viruses and bacteria.

Another object of the present invention is to provide a composition for preventing, improving or treating respiratory diseases caused by superinfection by viruses and bacteria.

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

According to one embodiment of the present invention, superinfection by viruses and bacteria; Or it relates to a composition for preventing, improving or treating respiratory diseases thereby.

The composition of the present invention is a CD47 (Cluster of Differentiation 47) protein activity or expression inhibitor; Alternatively, an expression inhibitor of the gene encoding the protein may be included as an active ingredient.

In the present invention, the "Cluster of Differentiation 47 (CD47)" is known as integrin associated protein (IAP) and corresponds to a transmembrane protein encoded by the CD47 gene in humans. CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins, and binds thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα) ligands . In the present invention, the CD47 may consist of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, the CD47 protein or the gene encoding the same is used in human respiratory epithelial cells, such as human nasal epithelial cells (HNEC) or human bronchial epithelial cells (HBEC). ), etc. may be present.

In the present invention, the activity or expression inhibitor of the CD47 protein may include at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to these proteins.

In the present invention, the "peptide mimetics" are peptides or non-peptides that inhibit the binding domain of the CD47 protein. Key residues of non-hydrolyzable peptide analogs include the β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemiCal co. Rockland, IL, 1985), azepine (Huffman et al. in Peptides: chemistry and Biology, G.R. Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides; chemistry and Biology, G.R. Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohols (Gordon et al. Biochem Biophys Res. commun 126:419 1985) and a substituted gamma-lactam ring (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

In the present invention, the "Aptamer" is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. Since the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) was first developed (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), aptamers have been developed from low-molecular-weight organic substances to peptides and membrane proteins. Many aptamers capable of binding to various target molecules have been continuously discovered. Aptamers are comparable to single antibodies because of their inherent high affinity (usually pM level) and specificity of being able to bind to target molecules, and in particular, they have high potential as alternative antibodies to the extent that they are called "chemical antibodies."

In the present invention, the "antibodies" prepared by injecting CD47 protein or commercially available antibodies may be used. In addition, the antibody includes polyclonal antibodies, monoclonal antibodies, fragments capable of binding to an epitope, and the like.

Here, the polyclonal antibody may be produced by a conventional method of injecting the CD47 protein into an animal and collecting blood from the animal to obtain antibody-containing serum. Such polyclonal antibodies can be purified by any method known in the art, and can be made from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc.

In addition, the monoclonal antibody can be produced using any technique that provides for the production of antibody molecules through the cultivation of continuous cell lines. Such technologies include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

In addition, antibody fragments containing specific binding sites for the CD47 protein can be prepared. For example, but not limited to, F(ab')2 fragments can be prepared by pepsin digestion of antibody molecules, and Fab fragments can be prepared by reducing disulfide bridges of F(ab')2 fragments. Alternatively, by miniaturizing the Fab expression library, monoclonal Fab fragments having the desired specificity can be quickly and conveniently identified.

In the present invention, the antibody may be bound to a solid substrate to facilitate subsequent steps such as washing or separation of complexes. Solid substrates include, for example, synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resin includes polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, and nylon.

In the present invention, expression inhibitors of the gene encoding the CD47 protein include antisense nucleotides complementary to the gene, short interfering RNA (siRNA), short hairpin RNA (shRNA) and ribozymes ( ribozyme) may include any one or more selected from the group consisting of.

In the present invention, the "antisense nucleotide", as defined in Watson-Crick base pairing, binds (hybridizes) to a complementary nucleotide sequence of DNA, immature-mRNA or mature mRNA to interfere with the flow of genetic information from DNA to protein will be. The specific nature of antisense nucleotides for target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target RNA sequence. Recently, many studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins. Since many recent advances have been made in the field of oligonucleotide chemistry and the synthesis of nucleotides that exhibit improved cell line adsorption, target binding affinity, and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

In the present invention, the "siRNA" and "shRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and can inhibit the expression of a target gene, thereby providing an efficient gene knockdown method or gene therapy. used in a way shRNA is a hairpin structure formed by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by dicer to form small RNA fragments of 21 to 25 nucleotides in size siRNA, which is a double-stranded oligonucleotide, can specifically bind to mRNA with a complementary sequence and suppress its expression. Therefore, which means of shRNA and siRNA to use can be determined by a person skilled in the art, and a similar expression reduction effect can be expected if the mRNA sequences they target are the same. For the purpose of the present invention, the expression of the CD47 gene can be inhibited by specifically acting on the gene encoding the CD47 protein to cleave the CD47 gene (eg, mRNA molecule) to induce RNA interference (RNAi). can siRNA can be synthesized chemically or enzymatically. The method for preparing siRNA is not particularly limited, and methods known in the art may be used. For example, a method for chemically synthesizing siRNA directly, a method for synthesizing siRNA using in vitro transcription, a method for cutting long double-stranded RNA synthesized by in vitro transcription using an enzyme, An expression method through intracellular delivery of shRNA expression plasmids or viral vectors and an expression method through intracellular delivery of polymerase chain reaction (PCR)-induced siRNA expression cassettes, but are not limited thereto.

As an example of the present invention, shRNA specific to the gene encoding the CD47 protein may be represented by SEQ ID NO: 2, but is not limited thereto.

In the present invention, the "ribozyme" refers to an RNA molecule having a catalytic activity. Ribozymes with various activities are known, and the ribozymes of the gene encoding the CD47 protein include known or artificially produced ribozymes, and optionally ribozymes having target-specific RNA cleavage activity are known standard It can be made by the technique.

In the present invention, the "superinfection" is also referred to as "bacterial alternating infection", and refers to a secondary infection by a different pathogen after the primary infection. Usually, when antibiotics are used in large amounts for a long period of time, The normal flora of bacteria, vagina, etc. is changed, and the phenomenon of mycelial alternation occurs. In other words, among the resident bacteria of the normal flora, bacteria resistant to drugs dominate and new infections appear accordingly, and Candida albicans, resistant Staphylococcus aureus, and Pseudomonas aeruginosa are often the causative bacteria.

In the present invention, the superinfection may be simultaneous infection by at least one virus and at least one bacterium.

In the present invention, the virus may be an influenza virus, preferably the type A influenza virus, and the specific type is not particularly limited, but for example, H1N1, H1N2, H2N2, H3N2, H5N1, H5N6 or a subtype of H7N9, preferably a subtype of H1N1.

In the present invention, the bacteria may be pathogenic bacteria, and specific types are not particularly limited, but, for example, Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes , Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium Typhimurium), Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, Campylobacter spp., Salmonella spp., Shigella (Shigella) spp., Yersinia enterocolitica, Vibrio cholerae, Escherichia coli, Staphylococcus aureus, Bacillus cereus ), and Clostridium difficile, etc., preferably Staphylococcus aureus.

In the present invention, the respiratory disease caused by superinfection may include all diseases occurring in the respiratory tract without limitation, and is not particularly limited, but, for example, respiratory inflammatory disease, chronic obstructive pulmonary disease (COPD), sinusitis, It may include allergic rhinitis, lower respiratory tract infection, bronchitis, emphysema, pneumonia, bronchial asthma, sequelae of pulmonary tuberculosis, acute respiratory constriction syndrome, cystic fibrosis, otitis media, and pulmonary fibrosis.

In the present invention, the composition may be used in the form of a pharmaceutical composition or food composition, but is not limited thereto.

In the present invention, "prevention" may include, without limitation, any act of blocking or delaying the symptoms of superinfection or respiratory disease caused by superinfection using the composition of the present invention, or suppressing or delaying the symptoms of superinfection or respiratory disease caused thereby. there is.

In the present invention, "improvement" and "treatment" may include without limitation any action that improves or benefits symptoms of superinfection or respiratory disease caused by administration of the composition of the present invention.

The composition of the present invention can be additionally administered in combination with at least one other active agent, and through this, the prevention, improvement, or treatment effect of superinfection can be further enhanced.

In the present invention, the active agent may be selected from antiviral compounds, antibacterial compounds, antiparasitic compounds, antifungal compounds, and prophylactic or therapeutic vaccines, but is not limited thereto.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.

The pharmaceutical composition of the present invention is not limited to these, but is formulated according to conventional methods into oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, and painless agents for injections. A topical, solubilizing agent, isotonic agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used. The formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing with the above-described pharmaceutically acceptable carrier. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.

The route of administration of the pharmaceutical composition according to the present invention is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intracapsular, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. It can vary widely, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, drug form, administration route and period, but can be appropriately selected by those skilled in the art, and is 0.0001 to 50 mg/day. kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

The food composition of the present invention may be prepared in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread.

When the composition of the present invention is included in the food composition as an active ingredient, the amount may be added in an amount of 0.1 to 50% of the total weight, but is not limited thereto.

When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation except that the food composition is included in the indicated ratio, and as in conventional beverages, various flavoring agents or natural carbohydrates may be included as additional ingredients. . Specifically, as natural carbohydrates, common sugars such as monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, dextrin, and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol, etc. can include Examples of the flavoring agent may include natural flavoring agents (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, A pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like may be further included.

Components included in the food composition of the present invention may be used independently or in combination. The ratio of the additives does not correspond to the core elements of the present invention, but may be selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

In the case of using the composition according to the present invention, co-infection by at least one virus and at least one bacterium, that is, superinfection can be effectively prevented, improved, or treated, and furthermore, respiratory diseases caused by the superinfection can also be prevented. , can be improved or treated.

Figure 1 shows the result of confirming the expression level of the CD47 gene by qRT-PCR after transfecting HNECs with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 1.
FIG. 2 shows the result of confirming the expression level of CD47 protein by Western blot after transfecting HNECs with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 1. FIG.
FIG. 3 shows a photograph of HNECs transfected with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 1, and the expression of surface proteins CD47 and E-cadherin observed by immunofluorescence confocal microscopy.
FIG. 4 shows the results of detection of permeability of FITC-dextran around cells after infecting HNECs with viruses and/or bacteria in the presence of CD47-specific shRNA (CD47 shRNA) or scramble shRNA in Example 1. FIG.
Figure 5 shows the results of transepithelial resistance (TEER) measurement after infecting HNECs with viruses and/or bacteria in the presence of CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 1.
FIG. 6 shows pictures observed under an optical microscope after infecting HNECs with viruses and/or bacteria in the presence of IgG or anti-CD47 antibody in Example 1. FIG.
FIG. 7 shows the results of detecting permeability of FITC-dextran around cells after infecting HNECs with viruses and/or bacteria in the presence of IgG or anti-CD47 antibody in Example 1. FIG.
8 shows the results of transepithelial resistance (TEER) measurement after infecting HNECs with viruses and/or bacteria in the presence of IgG or anti-CD47 antibody in Example 1.
9 shows the result of confirming the expression level of the CD47 gene by qRT-PCR after transfecting HBECs with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 2.
10 shows the result of confirming the expression level of CD47 protein by Western blotting after transfecting HNECs with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA in Example 2.
FIG. 11 shows the results of detecting permeability of FITC-dextran around cells after infecting HBECs with viruses and/or bacteria in the presence of CD47-specific shRNA (CD47 shRNA) or scramble shRNA in Example 2.
FIG. 12 shows the results of detecting permeability of FITC-dextran around cells after infecting HBECs with viruses and/or bacteria in the presence of IgG or anti-CD47 antibody in Example 2.
13 is a schematic diagram of the experimental design of Example 3.
FIG. 14 shows the results of measuring the change in survival rate of mice after injecting anti-mouse CD47 antibody or IgG as a control into the virus-bacteria superinfection mouse model in Example 3. FIG.
15 shows the results of measuring the change in survival rate of mice after injecting anti-mouse CD47 antibody or IgG as a control into the virus-bacteria superinfection mouse model in Example 3.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example

[Example 1] Evaluation of superinfection treatment effect in vitro (1)

1. Cell culture

Human nasal epithelial cells (HNECs) were isolated from nasal polyps and cultured in an air-liquid interface (ALI) culture system. For ALI culture, HNECs of two passages were A 12 mm, 0.45 μm pore transwell-clear culture insert (Costar) was seeded at a density of 2×10 ⁵ cells/cm ² . Cells were cultured in a 1:1 mixture of basic epithelial culture medium and Dulbecco's modified Eagle's medium (Lonza) with the addition of the following growth factors: hydrocortisone (0.5 μg/ml), insulin (5 μg). /ml), transferrin (10 μg/ml), epinephrine (0.5 μg/ml), triiodothyronine (6.5 μg/ml), gentamicin (50 μg/ml) ), amphotericin-B (50 μg/ml), retinoic acid (15 ng/ml), bovine pituitary extract (50 μg/ml), bovine serum albumin (bovine serum albumin) (1.5 μg/ml), and epidermal growth factor (0.5 ng/ml). Once cells filled the transwell membrane, the tip of the transwell was exposed to air. The basal culture medium was changed daily for 14 days. After 14 days of ALI culture, fully differentiated HNECs were used to induce viral and bacterial superinfection.

2. Inoculation of viruses and bacteria

Influenza A virus (H1N1, Korea/01/2009) and Staphylococcus aureus (S. aureus, ATCC® 29213 ^TM ) were used in this experiment. Viral titer was determined by plaque assay, and fully differentiated HNECs were inoculated with the influenza A virus MOI=1 for 2 hours. After inoculation, the cells were washed with PBS three times and then replaced with fresh medium. The next day, superinfection was induced by inoculating the virus-infected cells with S. aureus. S. aureus colonies were cultured overnight at 37° C. on Bacto ^TM Brain Heart Infusion (BHI) (BD Biosciences) agar plates. Colonies were inoculated into 10 ml BHI medium one day before bacterial inoculation and then cultured overnight at 37 °C while stirring at 200 rpm in a shaking incubator. The next day, the culture medium was inoculated into fresh BHI medium and cultured on a shaking incubator until an OD 600 of 1 was reached. The culture medium was centrifuged at 8000 rpm for 10 minutes and washed three times with PBS.

For bacterial infection, i.e. superinfection, HNECs were mock-infected (PBS) or inoculated with S. aureus at MOI=2 and 5 for 3 hours. After inoculation, the cells were washed with PBS three times and then replaced with fresh medium.

Then, neutralization assay was performed using CD47 monoclonal antibody (Invitrogen) for analysis. Briefly, after infection with influenza A virus, HNECs were treated with CD47 antibody (2 μg/ml in 200 μl PBS) for 1 hour, washed three times with PBS, and then infected with S. aureus.

3. shRNA experiments

For human CD47 knockdown, plasmid-based lentiviral shRNA (Lugen Sci Co., Ltd) was used. The sequences of the CD47-specific shRNA are as follows: 5'-GCA TGG CCC TCT TCT GAT TTC-3' (SEQ ID NO: 2); and scrambled shRNA (sc-shRNA) as follows: 5'-GCA CTA CCA GAG CTA ACT CAG ATA GTA CT-3' (SEQ ID NO: 3). At passage 2, HNECs were transfected with MOI=10 shRNAs for 4 hours using polybrene reagent (Lugen Sci Co., Ltd). After transfection, the shRNA treatment medium was replaced with fresh medium.

4. Immunofluorescence Confocal Microscopy

For immunofluorescence staining, cell slides were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 (Amresco) for 5 minutes, and treated with 50 mM ammonium chloride (Sigma Aldrich) for 15 minutes . For blocking, cell slides were incubated for 1 hour at room temperature with 0.1% bovine serum albumin (BSA), 0.1% Triton X-100, and 1% normal donkey serum (NDS). Then, the cell slides were incubated with rabbit polyclonal ZO-1 antibody (Invitrogen) and mouse monoclonal CD47 antibody (Invitrogen) in Dako antibody dilution (Dako) overnight at 4°C temperature. After secondary antibody incubation, the cell slides were cover slipped with DAPI (Sigma Aldrich) with Fluoroshield ^™ and analyzed with a confocal laser-scanning microscope (LSM780, Carl Zeiss MicroImaging GmbH, Jena, Germany).

5. Western blot

For Western blot, HNECs were used in RIPA buffer (Sigma Aldrich) containing Halt ^™ Protease & Phosphatase Inhibitor Single-Use Cocktail, EDTA-free (100X) (Thermo SCIENTIFIC). After separating 25 ug of the protein using an 8% SDS-PAGE gel, it was transferred to a PVDF membrane (Merck Millipore). The membrane was incubated with 5% skim milk in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) containing 0.5% Tween 20 (TTBS) for 1 hour at room temperature and rabbit polyclonal ZO-1 antibody ( Invitrogen) and mouse monoclonal CD47 antibody (Invitrogen) were incubated overnight at 4°C. After incubation with the secondary antibody, target protein bands were detected with Pierce ECL Western blot substrate (Thermo scientific).

6. Pericellular Permeability

After viral and/or bacterial infection of HNECs, tips of HNECs were treated with 4-kd fluorocein isothiocyanate (FITC)-dextran (Sigma Aldrich, 2 μg/ml in 200 μl HBSS). After 2 hours of incubation, the basal medium was recovered and the permeability of FITC-dextran around the cells was detected using a CYTATION 5 imaging reader (BioTEK).

7. Measurement of transepithelial resistance (TEER)

After viral and/or bacterial infection of HNECs, transepithelial resistance was measured using an EVOM voltmeter (World Precision Instruments, Sarasota, FL).

8. Confirmation of the therapeutic effect of virus-bacteria superinfection by knocking down and neutralizing CD47 in HNECs

Previously, after transfecting HNECs with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA, the expression level of CD47 gene or protein was confirmed by qRT-PCR or Western blot. As a result, transfection with CD47-specific shRNA (CD47 shRNA) It was confirmed that the expression level of the CD47 gene and protein was significantly reduced (Figs. 1 and 2), and the results of analyzing the immunofluorescence Z-stack data of the surface protein CD47 showed that the CD47-specific shRNA (CD47 shRNA) transfected In this case, it was confirmed that the expression of CD47 protein decreased (FIG. 3).

Next, in the presence of CD47-specific shRNA (CD47 shRNA) or scramble shRNA, HNECs were infected with viruses and/or bacteria, and then FITC-dextran permeability assay was performed around the cells. When overlapping infection, the permeability significantly increased compared to the case of infection only with virus, but when transfected with CD47-specific shRNA (CD47 shRNA), the level of increase in permeability was insignificant compared to the case of infection only with virus, and scrambled shRNA Compared to the case of injection, it was confirmed that the permeability was remarkably low (FIG. 4). On the other hand, as a result of measuring the change in transepithelial resistance (TEER), when the virus and bacteria were superimposedly infected in the presence of scrambled shRNA, the transepithelial resistance was significantly reduced compared to the case of infection only with the virus, but CD47-specific shRNA (CD47 shRNA ), the level of reduction in transepithelial resistance was insignificant compared to the case of virus-only infection, and it was confirmed that transepithelial resistance was significantly higher than that of scrambled shRNA injection (FIG. 5).

HNECs cells were infected with viruses and/or bacteria, treated with IgG or anti-CD47 antibody, and observed under an optical microscope. The number of was significantly decreased, and it was confirmed that the number of cells was still very low even after treatment with IgG, but it was confirmed that the number of cells increased significantly when treated with anti-CD47 antibody (FIG. 6). In addition, in the presence of anti-CD47 antibody or IgG, HNECs were infected with viruses and/or bacteria, and then FITC-dextran permeability assay was performed around the cells. However, when treated with anti-CD47 antibody, the level of increase in permeability was insignificant compared to the case of infection with only virus, and significantly lower permeability compared to the case of treatment with IgG. could (Fig. 7). As a result of measuring the change in transepithelial resistance (TEER), when the virus and bacteria were superimposedly infected in the presence of IgG, the transepithelial resistance was significantly reduced compared to the case of infection only with the virus, but when treated with the anti-CD47 antibody, the virus Compared to the case of only infection, the level of reduction in transepithelial resistance was insignificant, and it was confirmed that transepithelial resistance was significantly higher than that of IgG treatment (FIG. 8).

[Example 2] Evaluation of superinfection treatment effect in vitro (2)

The experiment was performed in the same manner as in Example 1, except that 'human bronchial epithelial cells (HBECs)' were used instead of 'human nasal epithelial cells (HNECs)'.

After transfection with CD47-specific shRNA (CD47 shRNA) or scrambled shRNA into HBECs, the expression level of CD47 gene or protein was confirmed by qRT-PCR or Western blot. When transfected with CD47-specific shRNA (CD47 shRNA) It was confirmed that the expression levels of the CD47 gene and protein were significantly reduced (FIGS. 9 and 10).

Next, in the presence of CD47-specific shRNA (CD47 shRNA) or scrambled shRNA, HBECs were infected with viruses and/or bacteria, and then FITC-dextran permeability assay was performed around the cells. As a result, viruses and bacteria in the presence of scrambled shRNA When overlapping infection, the permeability significantly increased compared to the case of infection only with virus, but when transfected with CD47-specific shRNA (CD47 shRNA), the level of increase in permeability was insignificant compared to the case of infection only with virus, and scrambled shRNA It was confirmed that the permeability was remarkably low compared to the case of injection (FIG. 11).

In the presence of anti-CD47 antibody or IgG, HBECs were infected with viruses and/or bacteria, followed by pericellular FITC-Dextran permeability assay. As a result, virus and bacteria were superimposed in the presence of IgG, and only virus was infected. However, when treated with anti-CD47 antibody, the level of increase in permeability was insignificant compared to the case of infection with only virus, and it was confirmed that the permeability was significantly lower than that when treated with IgG. (FIG. 12).

[Example 3] Evaluation of superinfection treatment effect in vivo (3)

13 shows the experimental design diagram below.

1. Construction of a virus-bacteria superinfection mouse model

8-week-old C57BL/6 male mice were purchased from Orient Bio Inc. (Seoul, Korea). Mice were anesthetized by intraperitoneal injection of a 1:1:8 mixture (100 μl) of Zoletil:Rompun:PBS, followed by 100 PFU of influenza virus A/Korea/01/2009 H1N1 in 20 μl sterile PBS. nasal infection with 7 days post viral infection, 105 CFU of S. aureus (ATCC® 29213 ^™ ) in 20 μl sterile PBS was slowly instilled through the nasal cavity. The body weight of the mice was measured daily and monitored for signs of other diseases. Mice were euthanized when symptoms of severe disease, such as difficulty in feeding and/or water intake, supine position, unresponsiveness, severe respiratory distress, severe pain or loss of physical condition, etc. occurred. Mortality and weight loss of mice were observed until day 29.

2. In vivo antibody blocking

On days 5 and 7 of viral infection, mice were intranasally injected with 160 μg of anti-mouse CD47 antibody (MIAP301, BioXCell) or 160 μg of anti-mouse CD47 antibody (B6.H12, BioXCell) in 20 μl sterile PBS . On day 7 of viral infection, mice were anesthetized by intraperitoneal injection of a 1:1:8 mixture (100 μl) of Zoletil: Rompun:PBS, and then the anti-mouse CD47 antibody was injected a second time. . After 30 minutes, 105 CFU of S. aureus (ATCC® 29213 ^TM ) was slowly injected through the nasal cavity of the mouse.

3. Confirmation of therapeutic effect by anti-CD47 antibody in virus-bacteria superinfection mouse model

As a result of injecting anti-mouse CD47 antibody or IgG as a control into the virus-bacteria superinfection mouse model prepared above, it was confirmed that the survival rate of mice significantly increased when anti-mouse CD47 antibody was injected (FIGS. 14 and 15) .

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for treating superinfection of viruses and bacteria <130> PDPB214179 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 293 <212> PRT <213> Homo sapiens <400> 1 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Glu 290 <210> 2 <211> 21 <212> RNA <213> artificial sequence <220> <223> CD47 specific shRNA <400> 2 gcatggccct cttctgattt c 21 <210> 3 <211> 29 <212> RNA <213> artificial sequence <220> <223> scrambled shRNA <400> 3 gcactaccag agctaactca gatagtact 29

Claims (16)

activity or expression inhibitors of CD47 (Cluster of Differentiation 47) protein; Or a pharmaceutical composition for preventing or treating superinfection comprising, as an active ingredient, an expression inhibitor of a gene encoding the protein. According to claim 1,
The CD47 protein or the gene encoding it is present in human respiratory epithelial cells, a pharmaceutical composition. According to claim 1,
The activity or expression inhibitor of the CD47 protein comprises at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein, a pharmaceutical composition. According to claim 1,
Expression inhibitors of the gene encoding the CD47 protein include antisense nucleotides complementary to the gene, short interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme. A pharmaceutical composition comprising at least one selected from the group consisting of: According to claim 1,
The superinfection is a simultaneous infection by at least one virus and at least one bacterium, the pharmaceutical composition. According to claim 5,
The virus is an influenza virus (influenza virus), the pharmaceutical composition. According to claim 6,
The virus is a type A influenza virus, pharmaceutical composition. According to claim 5,
The bacterium is a pathogenic bacterium, the pharmaceutical composition. According to claim 8,
The bacteria are Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa , Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Streptococcus pneumoniae, Haemophilus influenzae), Legionella pneumophila, Campylobacter spp., Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae cholerae), Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Clostridium difficile At least one selected from the group consisting of, pharmaceutical composition. activity or expression inhibitors of CD47 (Cluster of Differentiation 47) protein; Or a pharmaceutical composition for preventing or treating respiratory diseases caused by superinfection, comprising as an active ingredient an expression inhibitor of a gene encoding the protein. According to claim 10,
The activity or expression inhibitor of the CD47 protein comprises at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein, a pharmaceutical composition. According to claim 10,
Expression inhibitors of the gene encoding the CD47 protein include antisense nucleotides complementary to the gene, short interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme. A pharmaceutical composition comprising at least one selected from the group consisting of: According to claim 10,
The superinfection is a simultaneous infection by at least one virus and at least one bacterium, the pharmaceutical composition. According to claim 13,
The virus is an influenza virus (influenza virus), the pharmaceutical composition. According to claim 13,
The bacterium is a pathogenic bacterium, the pharmaceutical composition. According to claim 10,
The respiratory diseases include respiratory inflammatory diseases, chronic obstructive pulmonary disease (COPD), sinusitis, allergic rhinitis, lower respiratory tract infections, bronchitis, emphysema, pneumonia, bronchial asthma, pulmonary tuberculosis sequelae, acute respiratory distress syndrome, cystic fibrosis, otitis media and lung A pharmaceutical composition comprising at least one selected from the group consisting of fibrosis.

Images