KR20120082529A - Vaccine for preventing and treating inflammatory bowel disease comprising mycobacterium paratuberculosis - Google Patents

Abstract

PURPOSE: A vaccine containing Mycobacterium paratuberculosis and a vaccine composition containing the same are provided to effectively prevent inflammatory bowel disease. CONSTITUTION: A vaccine for preventing and treating inflammatory bowel disease contains Mycobacterium paratuberculosis as an active ingredient. Mycobacterium paratuberculosis is live bacteria or killed bacteria. The inflammatory bowel disease includes ulcerative colitis or Crohn's disease. A vaccine composition for preventing and treating inflammatory bowel disease contains the vaccine and pharmaceutically acceptable carrier or immunoadjuvant.

Description

Vaccine for preventing and treating inflammatory bowel disease comprising Mycobacterium paratuberculosis

The present invention relates to a vaccine for the prevention and treatment of inflammatory bowel disease, including Mycobacterium paratuberculosis as an active ingredient, a vaccine composition comprising the same, and a method for treating or preventing inflammatory bowel disease using the same. .

Inflammatory bowel disease (IBD) is a serious chronic inflammation of the digestive system that is known to be intractable and difficult to treat. Representatives include ulcerative colitis (UC) and Crohn's disease. , CD). Inflammatory bowel disease has occurred a lot in Westerners such as Caucasians or Jews, but recently it is increasing rapidly in Korea. The prevalence varies from country to country, but there are reports of one out of every 750 people in the United States and one out of every 500 people in Canada.

As the prevalence of inflammatory bowel disease increased, international interest increased. In 2008, the American Society for Microbiology (ASM) formed a consortium for the study and eradication of diseases. Unexplained and concluded that environmental factors, rather than the above, are more important for the pathogenesis of inflammatory bowel disease. The possibility of various pathogenesis mechanisms has been suggested based on environmental factors. However, the exact pathogenesis mechanisms are very few.

The hypothesis that Mycobacterium avium subsp. Paratuberculosis (M. paratuberculosis) is involved in the pathogenesis of Crohn's disease, one of the inflammatory bowel diseases, has recently been in the spotlight. . M. paratuberculosis causes Johne's disease, characterized by chronic granulomatous inflammation, in animals such as cattle, sheep, goats, and deer, and has been reported to be closely associated with Crohn's disease in humans. Since 2000, it has been reported mainly in developed countries. The researches reported to date reveal the significance of mycobacteria as the cause of Crohn's disease and the association between Crohn's disease and microorganisms by comparing Leprosy, tuberuculosis, paratuberculosis and Crohn's disease with human Crohn's disease. Identification of public health risks of M. paratuberculosis as a presumed cause, isolation of M. paratuberculosis from people with Crohn's disease using in situ hybridization method, p35 antigen of M. paratuberculosis Serological studies of human Crohn's disease using and p36 protein have been performed. However, despite these studies, M. paratuberculosis is not convinced to be the cause of Crohn's disease because of the extremely slow growth of the pathogen and the difficulty of cell culture. Thus, there is no research on the potential of M. paratuberculosis as a vaccine.

On the other hand, the most clinical feature of inflammatory bowel disease is the loss of normal cells present in the intestinal system due to constant inflammation, the change in the intestinal structure and normal function. Secondly, it is known that the inflammation is aggravated by endogenous infection of the herds. Therefore, in recent clinical studies, there is increasing interest in the treatment of inflammatory bowel disease, but until now the primary treatment of inflammatory bowel disease is dependent on immunosuppressive agents such as 5-ASA, 6-MP, Sulfasalazine, steroids, and TNF-alpha blockers. It is true. This prevents the loss of normal cells and restores the function of the intestinal tract by continually relieving inflammation, but there is a problem that only secondary concerns and temporary effects. Therefore, in the situation in which the treatment of inflammatory bowel disease is difficult and there is also a lack of effective treatment for it, there is a need for the development of an effective vaccine that can prevent and worsen inflammatory bowel disease.

Therefore, the present inventors have made efforts to develop a vaccine that can prevent and treat inflammatory bowel disease. As a result, M. para in the most representative model of inflammatory bowel disease using dextran sulfate sodium salt (DSS). It has been confirmed that tuberculosis has an effect as an excellent vaccine, such as showing an immunosuppressive effect and an inflammatory protective effect when administered before or simultaneously with inflammation, thereby completing the present invention.

One object of the present invention to provide a vaccine for the prevention and treatment of inflammatory bowel disease, including Mycobacterium paratuberculosis ( Mycobacterium paratuberculosis ) as an active ingredient.

Another object of the present invention to provide a vaccine composition for the prevention and treatment of inflammatory bowel disease comprising the vaccine, a pharmaceutically acceptable carrier or an adjuvant.

It is another object of the present invention to provide a method for preventing or treating inflammatory bowel disease by administering the vaccine composition to a subject.

In order to achieve the above object, the present invention provides a vaccine for the prevention and treatment of inflammatory bowel disease, including Mycobacterium paratuberculosis ( Mycobacterium paratuberculosis ) as an active ingredient.

In the present invention, the mycobacterium paratuberculosis may be live, dead, or attenuated form, but the live or dead form is preferred, and is most preferred when administered in live form.

In the present invention, the inflammatory bowel disease may be ulcerative colitis or Crohn's disease, the inflammatory bowel disease may be an inflammatory bowel disease caused by a bacterium, and the bacterium is Mycobacterium paratuberculus. It can be a rosis.

In the present invention, the ulcerative colitis is a chronic inflammatory disease occurring in the mucous membrane of the large intestine in the digestive tract, the inflammation of the entire intestine from the rectum to the transverse colon or the right colon, and Crohn's disease, unlike ulcerative colitis It is a disease that can occur in any part of the entire digestive tract and mainly shows lesions at the distal end of the ileum, and represents a representative refractory disease among the digestive diseases.

In a specific embodiment of the present invention, in order to determine the protective effect of inflammatory bowel disease caused by M. paratuberculosis, M. para live in mice induced by inflammation of DSS known as the best inflammatory bowel disease model. An experiment was conducted in which the tuberculosis was intraperitoneally infected two weeks before the induction of inflammation. As a result, the DSS-ingested group died all on the 29th day of the experiment, whereas the M. paratuberculosis-infected group survived 100% as well as the negative control group. In addition, in the group treated with DSS, a rapid weight loss was observed after DSS ingestion, whereas in M. paratuberculosis-infected group, a sudden change in body weight was not observed until 35 days, and thus M. paratuberculosis was administered as a vaccine. When it was confirmed that there is an immune defense effect against inflammatory bowel disease (see Figure 1).

In addition, based on the above experiment, in order to prove the vaccine effect of M. paratuberculosis, live or sterile forms, intraperitoneal or oral administration, two weeks before or concurrently with DSS, DSS daily In the ingested group, all mice died on the 14th day of the experiment, while the survival rate was improved by 55% -80% in the M. paratuberculosis group, and the rate of weight change was also rapid in the DSS group. In contrast, in the group treated with M. paratuberculosis, no further weight loss was observed from day 14 and it was found that a constant weight was maintained (see FIG. 2).

In addition, we investigated the expression production of cytokines to investigate the immune suppression of M paratuberculosis against inflammatory-associated cytokines in the intestinal tissues of mice induced by inflammation by DSS. While cytokines were observed, there was a significant decrease in cytokines in the group infected with live M. paratuberculosis intraperitoneally 2 weeks prior to DSS ingestion (see FIG. 3).

In addition, LPS treatment was performed on dendritic cells with the highest levels of inflammatory cytokines to investigate the specific mechanisms of inflammation-mediated cytokines. As a result, LPS-treated dendritic cells produced significantly higher cytokines. On the other hand, after LPS treatment, dendritic cells treated with M. paratuberculosis inhibit the activation of dendritic cells by LPS by reducing the expression of surface molecules involved in the activation of dendritic cells and reducing the secretion of inflammatory cytokines. It was confirmed that (see Figure 5).

These results suggest that M. paratuberculosis may be administered before or concurrently with inflammation to reduce inflammatory cytokines in the intestinal tract and inhibit activation of dendritic cells, thereby inhibiting inflammation and increasing individual survival. As a vaccine for treatment and prevention, it can be seen that there is an excellent protective effect.

As another aspect, the present invention relates to a vaccine composition for the prevention and treatment of inflammatory bowel disease comprising the vaccine.

As used herein, the term "prevention" means any action that inhibits or delays the development of inflammatory bowel disease by administration of a vaccine composition. As used herein, the term "treatment" refers to any action that improves or benefits the symptoms of inflammatory bowel disease already caused by administration of the vaccine composition.The symptoms of inflammatory bowel disease include bloody stool, mucus, abdominal pain, vomiting, High fever, edema, etc. are mentioned.

In addition, the vaccine composition may further comprise a pharmaceutically acceptable carrier or adjuvant (adjuvant). Pharmaceutically acceptable carriers are known in the art and include stabilizers, diluents and buffers. Suitable stabilizers include carbohydrates such as sorbitol, lactose, mannitol, starch, sugars, dextran and glucose; Proteins such as albumin or casein and the like. Suitable diluents include salts, Hanks balanced salts and Ringer's solution. Suitable buffers include alkali metal phosphates, alkali metal carbonates and alkaline earth metal carbonates, and the like. In addition, the vaccine composition of the present invention may include one or more immunoadjuvant and the like to improve or enhance an immune response. Suitable surface modifiers include peptides; Aluminum hydroxide; Aluminum phosphate; Aluminum oxide; Compositions comprising mineral or vegetable oils such as Marcol 52 and one or more emulsifiers or surface actives such as lysolecetin, polyvalent cations, polyvalent anions, and the like. The formulation method of the vaccine composition may use methods known to those skilled in the art.

As another aspect, the present invention relates to a method for preventing or treating inflammatory bowel disease by administering the vaccine composition to a subject.

The subject refers to a mammal including an animal such as a mouse, a mouse, a cow, a sheep, a goat, a deer, and a human, and preferably a human.

The route of administration of the composition may be administered via any general route as long as it can reach the desired tissue. As used herein, the term "administration" means introducing a given substance into an individual in any suitable manner and can be formulated for human or veterinary administration and administered by various routes. The vaccine composition may be administered by parenteral routes such as intraperitoneal, vascular, intravenous, intraarterial, intramuscular or subcutaneous. It may also be administered by inhalation route via oral, nasal, rectal, transdermal or aerosol. It may preferably be administered intraperitoneally or orally.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to exhibit a vaccine effect and an amount not to cause side effects or serious or excessive immune responses, and an effective dose level refers to a disorder or disorder to be treated. The severity, activity of a particular compound, route of administration, duration of treatment, drugs used in combination or concurrent with the vaccine, age, weight, sex, diet, general health of the individual, and factors known in the pharmaceutical and medical arts. It will depend on a variety of factors. Generally, the amount effective for immunotherapy or for immunoprevention will range from about 0.1 to 10 mg / kg body weight. The unit dose may be boosted two or more times at about two month intervals after the initial administration. However, lower or higher doses and other dosing schedules may be applied.

As described in detail above, the vaccine and vaccine composition comprising the mycobacterium paratuberculosis of the present invention as an active ingredient has an excellent protective effect against inflammatory bowel disease known as refractory diseases.

FIG. 1 shows 1 × 10 ⁶ CFU / mouse of live M. paratuberculosis ATCC19698 two weeks before ingesting 2.5% DSS in mice to investigate the protective effect of M. paratuberculosis against inflammatory bowel disease. After the infection by intraperitoneal injection, the survival rate (1a), body weight (1b), and the weight change rate (1c) is shown the results of the investigation.
2 is 1 x 10 ⁶ CFU / mouse of M. paratuberculosis ATCC19698 by varying the above conditions in mice in order to examine the vaccine effect according to viable bacteria, dead bacteria, infection route, and infection time of M. paraparatuberculosis. After infection, the survival rate (2a), body weight (2b), and weight change rate (2c) were examined.
FIG. 3 shows inflammatory activity in the ileal tip of mice 7 days after DSS intake for the control group treated with nothing, the DSS-only group, and the group infected with live M. paratuberculosis intraperitoneally 2 weeks prior to DSS ingestion. Cytokines (NF-a, IL-1 beta, IL-6, IL-8) are shown (* P <0.05. ** P <0.005).
4 is a control group treated with nothing in the dendritic cells, LPS-only group, and M. paratuberculosis in order to investigate the inhibitory effect of dendritic cell activation by LPS of M. paratuberculosis MOI 1: In the group infected with 1 and treated with LPS 100ng / ml after 4 hours, the expression of co-stimulatory factors (CD80, CD86) and MHC class I and II molecules was examined.
FIG. 5 shows the control of M. paratuberculosis on cytokine secretion of dendritic cells by LPS stimulation. The results of the investigation of changes in secretion of inflammatory cytokines TNF-alpha, IL-6, IL-12, and IL-1 beta in the group infected with tuberculosis and LPS treatment (* P <0.05. ** P < 0.005).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

In order to examine the effect of the vaccine of the present invention, in vivo experiments, M. paratuberculosis first, in order to see the protective effect of the inflammatory bowel disease as a vaccine, M. live screening before inducing intestinal inflammation with DSS in mice An experiment was conducted to intraperitoneally infect paraparauculosis two weeks ago. In addition, based on the results of the experiment, the mice were administered with various routes and live or heat-killed M. paratuberculosis two weeks before the DSS or simultaneously with the DSS.

Next, an experiment was conducted to determine whether M. paratuberculosis has an inhibitory effect on inflammation by inhibiting cellular activity by LPS of dendritic cells.

Hereinafter, the experimental materials and experimental methods used in the above experiments and the experimental results according thereto are as follows.

Materials and Experiments

1. Preparation of the experimental mouse

Female C57BL / 6 mice (7weeks old) were purchased from Charles River Laboratories Korea (Orient bio, Korea) and administered to Chungnam National University Ecology Research Building to stabilize mice for a week. The experiment was conducted in 8-week old mice.

2. M. paratuberculosis strains used

M. paratuberculosis ATCC19698 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and 10% OADC (BD Biosciences, SanJose, CA, USA) and 2 μg / ml of MycobactinJ (AlliedMonitor, Fayette, MO, USA) containing one month of incubation in 7H9 broth, washed three times with PBS and stored in the -80 degrees refrigerator. After one week, CFU was measured.

3. Mouse group

20 mice were assigned to each group and tested.

(1) Negative Control: Normal control group that received no treatment.

(2) Positive control, 2.5% DSS group: Dextran sulfate sodium salt (M.W: 36,000 ~ 50,000 Da; MP Biomedicals) diluted in sterile tap water at 2.5% for 8 weeks.

(3) Group 1: Demonstrating the vaccine effect of M. paratuberculosis Group 1: A group infected with live M. paratuberculosis ATCC19698 by intraperitoneal injection 2 weeks prior to DSS administration with 1 × 10 ⁶ CFU / mouse.

(4) Group 2: Demonstrating the vaccine effect of M. paratuberculosis Group 2: Group infected orally infected with live M. paratuberculosis ATCC19698 2 weeks prior to DSS administration with 1 × 10 ⁶ CFU / mouse.

(3) Group 3: M. paratuberculosis to demonstrate the vaccine effect: M. paratuberculosis ATCC19698, dead by heat treatment at 100 ° C. for 30 minutes, intraperitoneally 2 weeks before DSS administration at 1 × 10 ⁶ CFU / mouse The group treated with my injection.

(6) Group 4: Group demonstrating the vaccine effect of M. paratuberculosis A live M. paratuberculosis ATCC19698 was infected by intraperitoneal injection concurrently with DSS at 1 × 10 ⁶ CFU / mouse.

3. Induction of Inflammatory Bowel Disease

Sterilized tap water with 2.5% dextran sulfate sodium salt (MW: 36,000 to 50,000 Da; MP Biomedicals) using the method described in Nature protocols (Chemically induced mouse models of intestinal inflammation Nature protocols, Vol. 2, No. 3, 2007). Diluted in, daily free intake.

4. Weight measurement and status measurement

Body weight and bloody stool are the most representative indicators of inflammatory bowel disease. All mice 'body weights, bloody stools, and activity were measured daily from one week before the experiment until the end of the experiment.

5. Measurement of cytokines in tissues

After inducing inflammatory bowel disease with DSS, TNF-a, IL-1 beta, IL-6, IL were analyzed using ELISA kit (BD bioscience) to analyze the pattern of inflammatory cytokine inhibition by M. paratuberculosis. Inflammatory cytokines of -8 were analyzed. Tissues of the same intestinal part (terminal ileum) of each group of mice were cut to 3 cm, 1 mL of sterile PBS was added, the tissues were crushed using a bead beater and centrifuged at 13,000 xg for 10 minutes to collect only the supernatant. The harvested supernatants were normalized to a protein concentration of 1 mg / mL and used for the experiment.

6. Analysis of Inflammatory Inhibitory Mechanism by M. paratuberculosis

(1) Dendritic cells are representative cells that mass produce cytokines involved in inflammation, and form various inflammatory mediators such as TNF-alpha, IL-1 beta, and IL-6 in response to LPS. Therefore, after dendritic cells were infected with M. paratuberculosis with MOI 1: 1, LPS was treated to measure cytokines related to inflammation.

(2) Dendritic cells were derived from mouse bone marrow cells as previously known methods. First, bone marrow was harvested using a bone marrow harvesting injection from C57BL / 6 mice. After the collected bone marrow was washed, red blood cells were removed using ammonium chloride. The isolated cells were placed in RPMI 1640 (10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 5 × 10 ⁻⁵ M 2-ME, 10 mM in a 6-well plate). HEPES (pH 7.4), 10 ng / ml GM-CSF, 10 ng / ml IL-4) was added to incubate. On days 3 and 5 after incubation, suspended cells were removed and fresh medium was added. In the experiments of the present invention, unattached dendritic cells on day 6 of culture were used. In some experiments CD11c ⁺ dendritic cells were used after separation using anti-CD11c (N418) microbeads and magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). LPS ( E. coli 055: B type, Sigma) was used as a positive control for activation of dendritic cells.

(3) An experiment was conducted to investigate the activation inhibition of dendritic cells by M. paratuberculosis. After dendritic cells were infected with M. paratuberculosis, 4 hours after treatment with LPS at a concentration of 100 ng / ml for 20 hours, the dendritic cells were recovered. Untreated controls were treated with medium only. In order to inhibit nonspecific binding, 1 μg / ml of Fcγ I / III (BD bioscience) was treated at 4 ° C. for 20 minutes and then stained with CD11c-FITC (BD bioscience) for 20 minutes at 4 ° C. for dendritic cell analysis. . In addition, 1 μg of anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience) were used to measure the expression patterns of co-stimulatory factors and MHC molecules. / ml each treated for 30 minutes at 4 ℃. Wash twice with PBS buffer (containing 2% FBS and 0.01% sodium azide) and analyze with FACScallibur (Beckson Dikinson, USA) after fixation of 1% paraformaldehyde (FIG. 2A).

(4) Cytokines were measured in the same manner as described above in "5. Measurement of cytokines in tissues".

Experiment result

1. To examine the protective effect of M. paratuberculosis as a vaccine against inflammatory bowel disease, a screening experiment was conducted. Two weeks before the mice received 2.5% DSS, live M. paratuberculus Survival and body weight were examined after infection with Rossis ATCC19698 by intraperitoneal injection of 1 × 10 ⁶ CFU / mouse.

As a result, as shown in FIG. 1A, a graph showing survival rate, all of the groups ingested with 2.5% DSS died on the 29th day of the experiment, whereas in the group infected with M. paratuberculosis, 100 as in the negative control group treated with nothing. % Survived.

In addition, Figure 1b (wt) and Figure 1c (% body weight) shows the rate of change in body weight, in the group administered 2.5% DSS was observed a sharp weight loss after DSS ingestion, while infected with M. paratuberculosis The group did not observe a sharp change in body weight until 35 days.

2. Next, as a result of investigating the effects of M. paratuberculosis according to the live bacteria, dead bacteria, root of infection, and the timing of infection, as shown in the graph showing the survival rate of FIG. While all mice died on the first day, the survival rate was improved by 55% -80% in the M. paratuberculosis group. The group with the highest survival up to 56 days was treated with live M. paratuberculosis bacteria from day 0 when DSS began.

2B and 2C are graphs showing the rate of change in body weight. In the group treated with 2.5% DSS, the body weight was rapidly decreased until 14 days after DSS ingestion, while in the group treated with M. paratuberculosis, From day one, no further weight loss was seen and it was found that constant weight was maintained.

3. Investigation of the pattern of inflammatory cytokines 7 days after DSS ingestion in the mouse ileum, as shown in Fig. 3, significantly higher inflammatory cytokines (NF-a, IL-1 beta, IL-6, IL-8) were observed, whereas a significant decrease in cytokines was observed in the group intraperitoneally infected 2 weeks prior to ingestion of live M. paratuberculosis DSS (* P <0.05). * P <0.005).

4. When dendritic cells are activated, the expression of co-stimulatory factors (CD80, CD86) and molecules such as MHC class I, II is significantly increased. Therefore, the inhibition of activation by LPS after infection of M. paratuberculosis in dendritic cells is suppressed. These molecules were investigated. M. paratuberculosis was first infected with dendritic cells with MOI 1: 1, and after 4 hours, LPS, which induces an inflammatory response through activation of dendritic cells, was treated at a concentration of 100 ng / ml and incubated at 37 ° C. for 20 hours. did. As a result, as shown in Figure 4, M. paratuberculosis reduced the expression of surface molecules of dendritic cells by LPS. Therefore, it was found that M. paratuberculosis inhibits the activation of dendritic cells by LPS.

5. In addition, an experiment was performed to investigate the effect of M. paratuberculosis on cytokine secretion of dendritic cells by LPS stimulation. In other words, when dendritic cells are activated by LPS treatment, the secreted cytokines are also changed. Therefore, we tested whether dendritic cells were infected with M. paratuberculosis and when LPS treatment affected the change in inflammatory cytokine secretion. Dendritic cells infected with M. paratuberculosis significantly reduced all cytokines of inflammatory cytokines TNF-alpha, IL-6, IL-12, IL-1 beta secreted by LPS stimulation (* P <0.05. ** P <0.005).

6. In conclusion, M. paratuberculosis mitigates inflammatory cytokine reduction and rapid weight loss by intestinal DSS in inflammatory bowel disease model in mice, and stimulates activation of dendritic cells with the most active secretion of inflammatory cytokines. By suppressing, it was confirmed that the protective effect as an excellent therapeutic and prophylactic vaccine to suppress inflammation.

Claims (8)

Mycobacterium tuberculosis Para-to-beokyul (Mycobacterium A vaccine for the prevention and treatment of inflammatory bowel disease, including paratuberculosis ) as an active ingredient. The vaccine of claim 1, wherein the mycobacterium paratuberculosis is in the form of live or dead bacteria. The vaccine of claim 1, wherein the vaccine has an activity of reducing inflammatory cytokines in intestinal tissue. The vaccine of claim 1, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. A vaccine composition for preventing and treating inflammatory bowel disease, comprising the vaccine of any one of claims 1 to 4, a pharmaceutically acceptable carrier or an adjuvant. A method of preventing or treating inflammatory bowel disease by administering the composition of claim 5 to a subject. The method of claim 6, wherein the subject is a subject other than a human. The method of claim 6, wherein said administration is administered orally or intraperitoneally to the subject two weeks before or at the time of inflammation.

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