KR100804507B1 - Adjuvant compositions comprising flagellins useful for immunotherapy of asthma and allergic diseases - Google Patents

Abstract

The present invention relates to an agent for treating asthma and allergic diseases including flagellin, and more specifically, antigen-specific immune response to allergic diseases such as asthma, atopic dermatitis, allergic rhinitis, etc. by containing flagellin as an active ingredient. It relates to an immunotherapeutic that regulates.

Antigen-specific Asthma Therapeutics, Flagellin, Sepsis Vibrio, Immunotherapy

Description

Adjuvant compositions comprising flagellins useful for immunotherapy of asthma and allergic diseases}

FIG. 1 is a graph showing that airway hypersensitivity, a symptom of asthma, is suppressed to a normal level when an asthma-causing antigen and flagellin are administered to an asthma-induced mouse.

FIG. 2 is a graph showing that eosinophilic airway inflammation in bronchial alveolar lavage fluid, a symptom of asthma, is inhibited when an asthma-causing antigen and flagellin are administered to an asthma-induced mouse.

3 is a graph showing that antigen-specific serum immunoglobulin-E decreases when an asthma-causing antigen and flagellin are administered to an asthma-induced mouse.

Figure 4 shows that administration of asthma-causing antigen and flagellin to mice with asthma induced the production of Th2-cytokines, interleukin-4, interleukin-5, and interleukin-10 in splenocyte culture medium and interferon, a Th1-cytokine. The production of gamma is a graph showing an increase.

The present invention relates to an agent for treating asthma and allergic diseases including flagellin, and more specifically, antigen-specific immune response to allergic diseases such as asthma, atopic dermatitis and allergic rhinitis by containing flagellin as an active ingredient. It relates to an immunotherapeutic that regulates.

Flagellin is the basic unit protein that makes up the filament of bacterial flagella and is an important factor in determining the motility of bacteria. The flagella consists of a hook, basal body and filament, which determine the swimming or swimming motility of the bacteria and the taxis of the bacteria. It is known to form a biofilm and determine the adhesion ability of pathogenic microorganisms (McCarter, LL, Microbiol Mol Biol Rev. 65: 445-62, 2001; Kim, YK, McCarter, LL, J Bacteriol. 182 : 3693-704, 2000; McCarter, LL, J Bacteriol., 177: 1595-609, 1995; Boles, BR, McCarter, LL, J Bacteriol. 182: 1035-45, 2000; Prouty, MG, Correa, NE, Klose, KE, Mol Microbiol. 2001 Mar; 39 (6): 1595-609, 2001). Recent studies have shown that mammalian Toll-like receptor-5 (hereinafter, referred to as "TLR-5") recognizes Gram-negative and Gram-positive bacteria flagellin and activates NF-κB. Known (Hayashi, F., Smith, KD, Ozinsky, A., Hawn, TR, Yi, EC, Goodlett, DR, Eng, JK, Akira, S., Underhill, DM, Aderem, A., Nature 410: 1099-1103, 2001). TLRs are receptors that recognize pathogen-associated molecular patterns and act as key constituents of the first innate immune system against many infectious agents, and are effective adaptive immune responses. It is a cellular receptor that is also involved in the stimulation of immune responses (Akira, S., Hemmi, H., Immunol. Lett. 85: 85-95, 2003).

Sepsis Vibrio was first reported in 1976 by bacteriological characteristics of basophilic, pathogenic Vibrio bacteria isolated from humans for 11 years by Hollis et al. Of the Centers for Disease Control (CDC). Lactose-fermenting Vibrio or Lac (+) was named because of its ability to degrade lactose. In 1979, the CDC Blake et al. Analyzed the data of 39 patients reported in the CDC and classified them into primary septicemia and wound infections according to clinical symptoms (Blake, PA). , Merson, MH, Weaver, RE, Hollis, DG, Heublein, PC, N. Engl. J. Med. 300: 1-6, 1979). In the same year, Farmer was named Vibrio vulnificus (vulnus = wound, ficus = forming) as a new species, and is coming today (Farmer, JJ III, Lancet 2: 903, 1979). Sepsis vibrio has one polar flagellum (McCarter, LL, Microbiol Mol Biol Rev. 65: 445-62, 2001), and the flagellin genes are flaA, flaB, flaF, flaC, flaD, and flaE . It consists of six (NCBI microbial genome database; http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html).

Asthma is an allergic disease in which the bronchus narrows due to an allergic inflammatory reaction in the bronchus after repeated exposure to allergens. The development of allergic diseases, including asthma, allergic rhinitis and atopic dermatitis, both genetic and environmental factors are involved. Typical features of asthma include eosinophilia in the airways, increased mucus production from goblet cells, increased Th2-cytokines, increased immunoglobulin-E, morphological changes in the airway wall, sensitivity of the airways to organ narrowing and nonspecific stimulation Increase (Lambrecht, BN, Allergy. 60: 271-282, 2005; Kuipers, H., Lambrecht, BN, Current Opinion in Immunology. 16: 702-708, 2004). In other words, asthma is a chronic inflammatory disease of the bronchus, known as a disease that must be controlled and managed while continuously using asthma medicines according to the severe stage of asthma, and is a disease that is not cured at present.

Antigens, such as bacterial products from experimental sites or proteins injected experimentally, travel through the lymphatic system to local lymph nodes. This antigen sensitizes lymphocytes through dendritic cells, which are representative antigen presenting cells. In other words, antigen presenting cells phage and process antigens and deliver them to naive CD4 + helper T-cells. These T lymphocytes produce clones of activated T cells (Th0 cells), which in turn produce type 1 or type 2 helper T cells (Th1 or Th2 cells). The action of certain cytokines then determines their expression into Th1 or Th2 cells, and each Th cell secretes its own cytokine that regulates a unique immune response (called Th1-cytokines or Th2-cytokines). These two types of cytokines balance the Th1 and Th2 responses through cross-regulation. The balance of Th1 and Th2 is important because it affects immunopathological action and is key to preventing and treating diseases (Abbas A. K., Lichtman, A. H. Cellular and Molecular Immunology 4th edition. Saunders Press. Pp298-344).

A major cause of the development of asthma and allergic diseases is the activation of Th2 cells, which are reversely regulated by the Th1 response (Herrick, C. A., Bottmly, K. Nat. Rev. Immunol. 3: 405-412, 2003). In recent years, many researchers have been interested in the immunotherapy of allergic diseases by controlling the balance of Th1 / Th2 responses. Recently, administration of lipopolysaccharide (LPS), airway infection virus, or CpG together with asthma-causing antigens has been reported to reduce asthma symptoms by controlling the proliferation of antigen-specific T cells (Stumbles, PA, Thomas). , JA, Pimm, CL, Lee, PT, Venaille, TJ, Proksch, S., Holt, PGJ Exp.Med. 188: 2019-2031, 1998; Brimnes, MK, Bonifaz, L., Steinman, RM, Moran, TMJ Exp.Med. 198: 133-44, 2003). The above report shows that allergic incidence in children in developed countries with public health is higher than in children in developing countries because of the high frequency of exposure to children with LPS, viruses, parasites and tuberculosis bacteria (aka “hygiene”). Hypothesis [hygiene hypothesis] "(Wahn, U., von Mutius, EJ Allergy Clin. Immunol. 107: 567-574, 2001; Umetsu, DT, McIntire, JJ, Akbari, O., Macaubas, C. , DeKruyff, RH Nat. Immunol. 3: 715-720, 2002; Yazdanbakhsh, M., Kremsner, PG, van Ree, R. Science. 296: 490-494, 2002) .LPS, Mycobacterium tuberculosis, Virus, and CpG, etc. Is commonly understood to transmit signals to the human body through TLRs (Lambrecht BN Allergy. 60: 271-282, 2005.) Thus, TLR agonists such as flagellin modulate several antigen-specific immune responses. Can be a target of drug development.

Therefore, the present inventors injected with egg albumin intraperitoneally to mice with asthma-induced asthma and egg albumin and TLR5 agonist flagellin at the same time, by suppressing the Th2-immune response characteristic of allergic immune response, airway hypersensitivity characteristic of asthma, The present invention was completed by observing that eosinophilic airway inflammation and antigen specific serum immunoglobulin-E concentrations can be used to treat asthma.

Accordingly, it is an object of the present invention to provide an immunotherapeutic agent through antigen-specific immune response control against allergic diseases such as asthma, atopic dermatitis and allergic rhinitis, including flagellin as an active ingredient.

The present invention provides an asthma and allergic disease immunotherapeutic agent comprising flagellin, a flagella constituent of bacteria.

In the present invention, "asthmatic induction" refers to inducing symptoms of asthma by injecting an asthma inducer into an animal. To study the understanding of the pathogenesis of asthma and the development of therapies, an animal model that induced asthma by administering an asthma-inducing substance to experimental animals was used, and an albumin-containing albumin was used to induce asthma in mice. The method of injecting intraperitoneally by mixing with alum (adjuvant) is typical. To objectively assess asthma symptoms in asthma-induced mice, airway hypersensitivity using methacholine-induced assay, eosinophilic airway inflammation in bronchoalveolar lavage fluid, Th2-cytokine production capacity and serum immunoglobulin-E from splenocytes Measure

Specific examples of allergic diseases according to the present invention include, but are not limited to, asthma, allergic rhinitis, atopic dermatitis, and the like.

In the present invention, the agent for treating asthma and allergic diseases by regulating antigen-specific immune response refers to a substance that exhibits a therapeutic effect by modulating the immune response to the antigen causing the allergic disease to alleviate the allergic reaction, and in the present invention, flagellin Use

In addition, in the present invention, sepsis vibrio flagellin was used, and the sepsis vibrio flagellin has one polar flagellum (McCarter, LL, Microbiol Mol Biol Rev. 65: 445-62, 2001 According to the present study, the flagellin genes of sepsis vibrio are composed of six kinds of flaA, flaB, flaF, flaC, flaD and flaE (NCBI microbial genome database; http: //www.ncbi. nlm.nih.gov/PMGifs/Genomes/micr.html). Flagelins produced from these genes, FlaA, FlaB, FlaF, FlaC, FlaD and FlaE proteins, are particularly preferred as immunotherapy for asthma and allergic diseases of the present invention.

In the present invention, FlaA corresponding to 1-191 amino acid, FlaB corresponding to 1-191 amino acid, FlaF corresponding to 1-191 amino acid, among the flagellin constitutive genes of Vibrio vulnifiucs of SEQ ID NOs: 1 to 12, 1 The N-terminal portion of FlaC corresponding to -191, FlaD corresponding to 1-191, and FlaE corresponding to 1-189; And

FlaA corresponding to 277-376 amino acids, FlaB corresponding to 278-377 amino acids, FlaF corresponding to 278-377, FlaC corresponding to 285-385, FlaD corresponding to 278-377, FlaE corresponding to 276-375 C-terminal portion;

The present invention provides an immunotherapeutic agent for asthma and allergic diseases using an immunogen prepared to have adjuvanticity by flagellin as an active ingredient by substituting a nucleotide sequence of a portion thereof between genes encoding a protein antigen epitope.

The present invention is not limited to the flagellin of sepsis Vibrio, for example, enteritis Vibrio parahaemolyticus , Vibrio cholerae , Vibrio anguillarum , Vibrio fischeri , Vibrio Harvey (Vibrio harveyi), picture tumefaciens Pro peondeom (Photobacterium profundum), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas footage is (Pseudomonas putida), Pseudomonas Sabbath tanoyi (Pseudomonas savastanoi), bacterial heukbanbyeong bacteria (Pseudomonas syringae), Pseudomonas fluorescein sense (Pseudomonas fluorescens), Pseudomonas Men city or (Pseudomonas mendocina), Yersinia pestis (Yersinia pestis), jangtipeuseu bacteria subspecies (Salmonella enterica subspecies), above M. tuberculosis (Yersinia pseudotuberculosis), E. coli (Escherichia coli), janto Pseudomonas Kam pastries (Xanthomonas campestris), bakteo purun di (Citrobacter freundii) into a sheet, Heidi Omar And Roy Hebrews N-Sys (Idiomarina loihiensis), Shigella (赤痢菌, Shigella dysenteriae), Serratia younggyun (Serratia marcescens), soft rot bacterium (Erwinia carotovora), raising Nella succinonitrile's Ness (Wolinella succinogenes), Bordetella pertussis bacteria ( Bordetella pertussis), lime bacteria (Borrelia burgdorferi), borelri Oh, par Kerry (Borrelia parkeri), borelri Oh hereum City (Borrelia hermsii), borelri ah hurt jelly (Borrelia afzelii), borelri Oh japonica (Borrelia japonica), borelri Ah investment Rica Borrelia turicatae , Legionella micdadei , Borrelia garinii , Clostridium tyrobutyricum , Rhodopirellula baltica , Helicobact pylorier Helicobact pylori ), Agrobacterium tumefaciens , Nitrogen-fixed Root Bacteria ( Bradyrhizobium japonicum ), Zymonas mobili Scotland (Zymomonas mobilis), Campylobacter coli (Campylobacter coli), Campylobacter Jeju you (Campylobacter jejuni), Kam Philo raised bakteo up N-Sys (Campylobacter upsaliensis), Listeria sikjungdokgyun (Listeria monocytogenes), Legionella pneumophila (Legionella pneumophila) , Treponema pallidum , Bacillus subtilis , Proteus mirabilis , Chromobacterium violaceum , Listonella anguillarum The same applies to flagellin of motility bacteria having flagella. Among these, flagellin of sepsis vibrio is preferable.

In addition, the asthma and allergic disease immune treatment adjuvant used in the present invention includes an amino acid sequence variant of the flagellin. A variant of flagellin is a protein with alterations in deletion, insertion and substitution of amino acid sequences, as long as the flagellin variant maintains antigen-specific immunomodulatory capacity for allergic reactions, any deletion, insertion and Combinations of substitutions are also possible. In some cases, in order to enhance the function, it may be modified by phosphorylation, glycosylation, methylation and farnesylation.

The amino acid sequence variant of the flagellin according to the present invention is FlaA corresponding to 29-168 amino acids of the flagellin constitutive genes of Vibrio vulnifiucs of SEQ ID NOs: 1 to 12, FlaB corresponding to 29-168 amino acids, 29 The N-terminal portion of FlaF corresponding to -170, FlaC corresponding to 29-168, FlaD corresponding to 29-169, FlaE corresponding to 28-154; And

FlaA corresponding to 284-354 amino acid, FlaB corresponding to 286-350 amino acid, FlaF corresponding to 285-355, FlaC corresponding to 293-362, FlaD corresponding to 286-350, FlaE corresponding to 283-348 C-terminal portion;

As a variant comprising a flagellin or a variant consisting of a polypeptide having a homology of 65% or more to the flagellin constituent amino acid of Vibrio streptococci of SEQ ID NO: 1 to 12.

Flagellin or a portion thereof used as an adjuvant for treating asthma and allergic diseases according to the present invention can be obtained by culturing flagellin-expressing bacteria and separating them from cell or cell culture supernatants or through genetic recombination techniques. When using a recombinant technique, it is common to clone a nucleic acid encoding flagellin or flagellin variant into an appropriate expression vector, and then transform the vector into a host to express flagellin, and then isolate the flagellin from the host. It can be obtained by the recombinant protein separation method of.

Hosts that can be used to obtain recombinant flagellin in the present invention include prokaryotic cells such as Gram-negative E. coli, sepsis Vibrio, Gram-positive Bacillus, and Lactobacillus, but are not limited thereto, fungi including yeasts, Eukaryotic-derived cells, including other lower eukaryotic cells, insect cells, plant cells and mammals, can be used.

Salting out, centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography to separate high purity flagellin proteins Various chromatographic methods such as graphigraphy may be used, and these are commonly used in combination to separate proteins of high purity (Sambrook et al., Molecular cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor laboratory Press ( 2001); Deuscher, M., guide to protein purification methods Enzymology, Vol. 182. Academic press. Inc., San Die, CA (1990)).

The dosage per mouse of the immunotherapeutic agent according to the present invention is preferably about 1 to 15 µg, which is the most optimal concentration in experiments showing the effect of adjuvaticity using tetanus toxin. At doses of 15 μg or more, the antigen specific immunity was rather reduced. The most preferred dose is about 10 μg per mouse and about 1 mg once per person.

The route of administration of the immunotherapeutic agents according to the present invention may be injection, oral administration, intra rectal, intravaginal, transcutaneous using a patch, etc., but is not limited thereto. .

The process of demonstrating the effect of flagellin asthma immunotherapy adjuvant according to the present invention is as follows:

1) preparing and separating a recombinant protein of flagellin FlaB, which is a sepsis Vibrio flagella constitutive factor;

2) administering flagellin to asthma mice induced with egg albumin;

3) measuring airway hypersensitivity in flagellin-administered asthma mice;

4) analyzing inflammatory cells in bronchoalveolar lavage fluid of flagellin-administered asthma mice;

5) measuring antigen specific immunoglobulin-E response in serum of flagellin-administered asthma mice; And

6) measuring cytokines secreted by antigen-stimulation of flagellin-administered asthmatic mouse spleen cells;

It includes.

The present invention will be described in more detail with reference to the following Examples. However, these examples are intended to illustrate the invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

The characteristics of the strains and plasmids used in the present invention are summarized in Table 1. Each manufacturing method and detailed characteristics are indicated in the examples and test examples.

Strain or Plasmid Characteristic source V. vulnificus CMCP6 Clinical separation, strong toxicity inventor MO6-24 / O Clinical separation, strong toxicity Glenn Morris Jr (Maryland University) ATCC29307 Standard strain Purchase from ATCC Escherichia coli ER2566 F- λ- fhuA2 [lon] ompT lacZ :: T7 gene1 gal sulA1 1 ( mcrC -mrr) 114 :: IS10 R (mcr-73 :: miniTn10-TetS) 2 R (zgb-210 :: Tn10) (TetS) endA1 [dcm] New england biolab Plasmids pTYB Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) expression vector, AmpR, 7,417 bp New england biolab

Example 1 Preparation and Separation of Recombinant Flagellin

An open reading frame (ORF) of the flaB gene of sepsis Vibrio ( V. vulnificus MO6-24 / O standard strain obtained from J. Glenn Morris of the University of Maryland School of Medicine. 1.5kb of DNA The fragment was cloned into pTYB12 (New England Biolabs Inc.), an intein fusion expression vector, to prepare a pCMM250 plasmid. This plasmid was put into ER2566 Escherichia coli by electroporation and 0.5 mM of 5-bromo-indole-3-chloro-isopropyl-β-D-galactopyranoside (IPTG) was added to induce expression. Only FlaB protein was isolated and purified from intein fusion protein using chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the manufacturer's (New England Biolabs Inc.) instructions. Endotoxin contained in the isolated FlaB protein was removed using AffinityPak ^™ Detoxi Gel ^™ Endotoxin Removing gel (Pirece).

Example 2 Animal Experiment Schedule Using Asthma-Induced Mice

To produce an asthma-induced mouse model, lyophilized egg albumin (Sigma, Cat. No. A5503) was extracted from egg whites at a concentration of 200 µg / ml using phosphate buffered saline After preparing the same amount of Imject ^(R) alum (Pierce Biotechnology Inc. Cat. No. 77161) was added and stirred for 30 minutes to prepare an egg albumin-alum suspension.

200 μl of the egg albumin-alum suspension was injected three times at two-week intervals into the abdominal cavity of SPB (specific pathogen free) 7-week-old Balb / c female mice. Only the phosphate buffer was administered to the negative control group that did not induce asthma. All the above experiments were carried out in a specific antigen control (SPF) experimental animal room. The SPF is a term meaning "Specific Pathogen Free" means a state free of harmful microorganisms affecting animal experiments. All experimental equipment, feed, and rug are sterilized and brought in. In case of entering or leaving the experimenter, it is a rule to change into sterilized lab suit and enter after an air shower.

In order to observe the asthma treatment effect of flagellin, 12 μl of phosphate buffer containing 20 μg of egg albumin and 10 μg of FlaB was prepared 7 days after the second mouse sensitization. Balb / c mice were anesthetized by injecting 100 μl of a mixture of 20 mg / ml ketamine (Ketamine) and 2.3 mg / ml xylazine (xylazine, Bayer Korea) intraperitoneally. The jellyline mixture was administered three times at weekly intervals. Only 20 μg of egg albumin was administered to the control group. Seven days after the third dose of flagellin-analbumin, mice were confined in an acrylic chamber and challenged with asthma by exposure to a phosphate buffer bubble containing 1% eggalbumin generated by an ultrasonic nebulizer for 30 minutes. The above challenge was conducted for 3 days at daily intervals.

[Test Example 1] Airway hypersensitivity test through methacholine induction test

To measure airway hyperresponsiveness of mice, methacholine induction test was performed 24 hours after asthma induction with 1% egg albumin. 3.125, 6.25, 12.5 and 25mg / ml methacholine solutions were inhaled for 3 minutes at each stage using an ultrasonic nebulizer, and the volumeh recorder was used to measure Penh, an index of airway resistance, for 3 minutes. Shown in

As can be seen in the results of Figure 1, asthma-induced airway resistance of egg albumin-induced asthma significantly increased, and when administered with egg albumin and sepsis vibrio-derived flagellin, airway similar to that of control mice not sensitized with egg albumin at all. Resistance was shown.

Experimental Example 2 Bronchial Alveolar Lavage and Airway Inflammatory Cell Analysis

After the methacholine-induced test, the rats were anesthetized, the catheter was inserted into the trachea, and washed twice with 0.8 ml saline to obtain a bronchoalveolar lavage fluid. After centrifugation of the wash solution, the resulting cell precipitates were prepared using a cell rotator and stained. At least 200 macrophages, lymphocytes, neutrophils and eosinophils were counted under an optical microscope, and the number of cells was expressed as a percentage. Is shown in FIG. 2.

In the results of FIG. 2, bronchoalveolar lavage fluid of mice with asthma induced by egg albumin showed eosinophilia, which is a typical feature of asthma (about 33% eosinophils), but eosinophils when egg albumin and sepsis-derived vibrio-derived administration were administered. The increase was markedly suppressed (about 18%).

Test Example 3 Antigen-Specific Antibody Response in Serum

In order to measure the antigen-specific immune response, blood was collected from the mouse heart after bronchoalveolar lavage, and serum was isolated to measure the egg albumin-specific antibody response in serum by an Enzyme linked immunosorbent assay (ELISA). The results are shown in FIG. 3. As can be seen from the results of Figure 3, the serum of the egg-induced egg albumin-induced asthma was high albumin-specific immunoglobulin-E levels were observed, but immunoglobulin-E when administration of egg albumin and sepsis vibrio-derived flagellin It was confirmed that the value is significantly suppressed.

Test Example 4 Mouse Spleen Cell Culture and Cytokine Measurement

In order to observe the characteristics of immune response to egg albumin of asthma-induced mouse immune cells, spleens of mice were isolated after bronchoalveolar lavage. After preparing spleen cell suspension using RPMI medium to which 10% fetal bovine serum was added, mononuclear cells were isolated by centrifugation using Ficoll Paque PLUS ^(R) (Amersham), and a 24-well plate. After dispensing 1.0x10 ⁶ cells into the incubator, egg albumin was added to 100 µg / ml and incubated for 48 hours. Interleukin-4, Interleukin-5, Interleukin-10 and Interferon gamma liberated in the culture supernatants were measured by ELISA kit (Biosource Co.), and the results are shown in FIG. 4.

As can be seen from the results of FIG. 4, asthma-induced mice have high levels of cytokines, interleukin-4, interleukin-5 and interleukin-10, which are associated with Th2 response, and interferongamma values associated with Th1 response change. However, the levels of cytokines related to Th2 response, interleukin-4, interleukin-5, and interleukin-10, were significantly reduced in the asthma-induced group and were associated with Th1 response. The interferon gamma value was confirmed to increase.

[Test Example 5] Comparison of TLR-related immunomodulators against asthma and superior flagellin

Asthma inhibitors of various types of TLR-related immunomodulators have been reported, but each report has different asthma protocols, and their effects are different because of different mechanisms of action, dosage, timing of administration, and methods of administration. It is difficult to compare quantitatively.

Inhibitory effect of TLR-related immunomodulators on asthma Immunomodulators Related TLR Asthma Features Airway hypersensitivity Eosinophilic inflammation Antigen-specific IgE Th2 immune response Example 1 TLR5 decrease decrease decrease decrease CpG-DNA (1, 2) TLR9 decrease decrease decrease decrease BCG (3, 4) TLR2, 4 decrease decrease no difference decrease LPS (5) TLR4 decrease decrease - decrease Lipoprotein-1 (6) TLR2, 4 - decrease - decrease

※ The numbers in parentheses in the immunomodulator column are references.

Table 2 shows the results of studies on the asthma inhibitory effect of each immunomodulator in the animal asthma model already sensitized to the causative antigen, looking at the asthma inhibitory effect of the flagellin prepared in Example 1 is equal or superior to other immunomodulators It can be seen. Unlike the other immunomodulators, the flagellin prepared in Example 1 is administered simultaneously with the causative antigen to act specifically on the antigen, thereby inhibiting the asthma reaction, and thus the possibility of side effects due to an immune response that may occur nonspecifically in the future. There is an advantage that can be reduced. In addition, this can maximize the effects of flagellin by adding the effect of flagellin to conventional "antigen-specific immunotherapy" used in the treatment of allergic diseases such as asthma, allergic rhinitis and bee venom allergy.

The immunomodulatory effects of CpG-DNA have been reported since 1998 and are currently in phase 2 and 3 clinical studies. Antigen-CpG-DNA conjugates are made and injected into allergy patients to specifically inhibit Th2 response and allergy. It has been reported to increase the effect of existing immunotherapy. Unlike CpG-DNA, the flagellin prepared in Example 1 exhibits an immunomodulatory effect through TLR5, and thus, may simultaneously maximize the immunomodulatory effect by simultaneously using the antigen-CpG-DNA conjugate and flagellin under clinical research. The mechanism of action of CpG-DNA is known to inhibit Th2 response and asthma mainly through a strong Th1 immune response. Recent studies, however, question the mutual antagonism between Th1 and Th2 responses, and report that Th1 may exacerbate asthma inflammation. Therefore, if immunomodulators are developed that can inhibit Th2 responses and asthma by mechanisms other than Th1, it may be safe and effective. In the case of flagellin, there is a possibility of inhibiting the Th2 response by a mechanism other than Th1.

Sepsis Vibrio-derived flagellin according to the present invention significantly reduced airway hypersensitivity, eosinophilic airway inflammation in bronchoalveolar lavage fluid, and serum antigen-specific immunoglobulin-E levels in asthma-induced mice. In addition, the flagellin according to the present invention inhibited the antigen-specific Th2 immune response in asthmatic mice.

Thus, flagellin can be used as an adjuvant to treat various allergic diseases such as atopic dermatitis or allergic rhinitis as well as asthma by regulating antigen specific allergic immune response.

<< Reference >>

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Claims (5)

delete delete FlaA corresponding to 1-191 amino acid, FlaB corresponding to 1-191 amino acid, FlaB corresponding to 1-191 amino acid, FlaF corresponding to 1-191, 1-191 of the flagellin constitutive gene of Vibrio vulnifiucs of SEQ ID NO: 1 to 12 N-terminal part of FlaC corresponding to FlaC, FlaD corresponding to 1-191, and FlaE corresponding to 1-189; And FlaA corresponding to 277-376 amino acids, FlaB corresponding to 278-377 amino acids, FlaF corresponding to 278-377, FlaC corresponding to 285-385, FlaD corresponding to 278-377, FlaE corresponding to 276-375 C-terminal portion; An asthma and allergic disease immunotherapy adjuvant prepared to have adjuvanticity by flagellin by substituting a nucleotide sequence of a portion between the genes encoding a protein antigen epitope. FlaA corresponding to 29-168 amino acids, FlaB corresponding to 29-168 amino acids, FlaB corresponding to 29-168 amino acids, FlaF corresponding to 29-170, and 29-168 of the flagellin constitutive genes of Vibrio vulnifiucs of SEQ ID NO: 1 to 12 N-terminal portion of FlaC corresponding to FlaC, FlaD corresponding to 29-169, FlaE corresponding to 28-154; or FlaA corresponding to 284-354 amino acid, FlaB corresponding to 286-350 amino acid, FlaF corresponding to 285-355, FlaC corresponding to 293-362, FlaD corresponding to 286-350, FlaE corresponding to 283-348 C-terminal portion; Asthma and allergic disease immunotherapy aid comprising a variant consisting of a polypeptide comprising a. The asthma and allergic diseases of claim 4, wherein the polypeptide is modified by a method selected from the group consisting of phosphorylation, glycosylation, methylation, and farnesylation. Immunotherapy aids.

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