KR20130029946A - Composition for immune enhancement and immunity enhancing method using the same - Google Patents

Abstract

PURPOSE: A composition for enhancing immunity is provided to non-specifically improve animal immunity and to enhance immunogenicity of a vaccine. CONSTITUTION: A composition for enhancing immunity which is prepared from Bordetella pertussis and Staphylococcus aureus Cowan I contains Bacillus megaterium as an active ingredient. A method for enhancing immunity comprises: a step of adding the composition to an inactivated vaccine and vaccinating the mixture to animals exclusive of humans.

Description

Composition for immunity enhancement and immune enhancement method using same {COMPOSITION FOR IMMUNE ENHANCEMENT AND IMMUNITY ENHANCING METHOD USING THE SAME}

The present invention utilizes cells and secreted proteins having immune enhancing activity The present invention relates to a novel animal immunopotentiating composition and a method for enhancing immunity using the same, which non-specifically enhances an animal's immunity, and when used in addition to an inactivated vaccine, can improve the immunogenicity of a vaccine.

Resistance to pathogen infection in animals, i.e., immune capacity, depends on the activity of various immune cells in the body of the animal and the immune status to a particular pathogen. Among the immune cells, phagocytic cells such as neutrophils, macrophages, and dendritic cells serve as the first line of defense against nonspecific phagocytosis and removal of externally invading pathogens, while T cells and B cells are responsible for humoral and cellular immunity. It is responsible for the immune defense, which is responsible for the immune response, which is responsible for the specific immune response, which is not blocked by the primary defense. Therefore, the phagocytic cells responsible for nonspecific immunity and lymphocytes responsible for specific immunity play a pivotal role in immunity and maintain optimal animal health when they are in optimal harmony.

 There are many kinds of immunity enhancing composition for animals developed and used in Korea and abroad, but it shows a big difference in immunity enhancing activity according to the kind of microorganisms used as raw materials. Rather, it is mainly used as a cow's somatic cell count reducing agent. Foreign multinational companies are known to add adjuvants to adjuvant as a company's slander for the purpose of improving the efficacy of the vaccine. However, since the effects vary depending on the type of the composition for enhancing the immune system, the type of the vaccine antigen, the type of the adjuvant, and the target animal, the vaccine may reduce the immune effect of the vaccine or cause side effects. There is a problem with using it.

The present invention has been made to solve the above problems, the problem to be solved in the present invention is to be widely applied to the inactivated vaccine, but also to provide a composition for enhancing immune immunity and excellent immunity enhancing effect using the same It is.

In order to solve the above problems,

The present invention is an immune enhancing composition prepared from Bordetella pertussis and Staphylococcus aureus Cowan I, Bacillus megaterium in the immune enhancing composition. It provides an immune enhancing composition, characterized in that it further comprises as an active ingredient.

The Bordetella pertussis (Bordetella pertussis) is preferably in the secreted protein toxin of Bordetella pertussis (Bordetella pertussis).

The Staphylococcus fluorescein mouse Cowan Ⅰ (Staphylococcus aureus Cowan Ⅰ) is preferably from Staphylococcus fluorescein mouse Cowan Ⅰ secreted protein of Staphylococcus cells and fluorescein-house Cowan Ⅰ of (Staphylococcus aureus Cowan Ⅰ).

The Bacillus megaterium ( Bacillus megaterium ) is preferably a lysate of Bacillus megaterium ( Bacillus megaterium ).

Bacillus megaterium ( Bacillus megaterium ) lysate is preferably contained in 0.01 ~ 10mg / ml.

In addition, the present invention provides a method for enhancing immune immunity of an animal, except for humans, which is inoculated into an animal other than a human after adding the composition for enhancing immune immunity to the inactivated vaccine.

The immune enhancing composition is preferably added 5 to 15 parts by weight per 100 parts by weight of inactivated vaccine.

The present invention improves the efficacy of inactivated vaccines having lower immunogenicity than attenuated live vaccines, thereby providing an immune enhancing composition and an immune enhancing method using the same. There is an effect that can be greatly reduced.

1 is a graph comparing the TNF-alpha generating ability of immune cells by treatment with the composition for enhancing immune immunity of the present invention. # 1 is treated with medium alone, # 2 is treated with NP 1.0mg / ml, # 3 is treated with NP 2.0mg / ml, # 4 is treated with NP 1.5mg / ml, # 5 is treated with NP 3mg / ml , # 6 is a treatment group for immune enhancing composition of the present invention, # 7 is a commercial treatment group for immune enhancing composition, # 8 is a LPS 10ng treatment group, and # 9 represents a LPS 100ng treatment group, respectively.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention is an immune enhancing composition prepared from Bordetella pertussis and Staphylococcus aureus Cowan I, Bacillus megaterium in the immune enhancing composition. It provides an immune enhancing composition, characterized in that it further comprises as an active ingredient.

The Bordetella pertussis (Bordetella pertussis) is preferably in the secreted protein toxin of Bordetella pertussis (Bordetella pertussis).

In a preferred embodiment, the secreted toxin protein of Bordetella pertussis can be prepared by the following method.

First, incubate in BHI broth and add formalin to inactivate strains and secretory toxin proteins. After confirming the inactivation of the strain and secretion toxin protein through a known culture test, such as centrifugation, only the supernatant is collected, and ammonium sulfate is added until supersaturation proceeds. The protein component formed after standing at low temperature is then centrifuged again to collect the precipitate and suspended in PBS. The suspension may be dialyzed in distilled water using a protein purification dialysis membrane to obtain pure secreted toxin protein.

The Staphylococcus fluorescein mouse Cowan Ⅰ (Staphylococcus aureus Cowan Ⅰ) is preferably from Staphylococcus fluorescein mouse Cowan Ⅰ secreted protein of Staphylococcus cells and fluorescein-house Cowan Ⅰ of (Staphylococcus aureus Cowan Ⅰ).

In a preferred embodiment, the cells of Staphylococcus aureus Cowan I and the secreted proteins of Staphylococcus aureus Cowan I can be prepared as follows.

First, the cells of Staphylococcus aureus Cowan I ( Staphylococcus aureus Cowan I) can be prepared by heat-treating and centrifuging the cell culture medium cultured in BHI broth to measure the weight of the precipitate cells and suspended in an appropriate amount of PBS.

Next, the secretion protein of Staphylococcus Oreus Cowan I is heat-treated and cultured the cell culture cultured in BHI broth and added to the centrifuged supernatant until the supersaturation of ammonium sulfate (ammonium sulfate). Thereafter, the precipitate is collected by centrifuging the protein component formed at low temperature, suspended in an appropriate amount of PBS, and then suspended in distilled water using a protein purification dialysis membrane to obtain a pure secreted protein.

The Bacillus megaterium ( Bacillus megaterium ) is preferably a lysate of Bacillus megaterium ( Bacillus megaterium ).

In a preferred embodiment, the lysate of Bacillus megaterium of the present invention can be prepared as follows.

The cells cultured in BHI broth are centrifuged and the precipitated cells are suspended in an appropriate amount of sterile distilled water, and lysed by a sonicator (Branson Digital Sonifier). After confirming the lysis state under a microscope, the precipitated lysates are collected by centrifugation, weighed, and suspended in an appropriate amount of PBS.

The content of each component of the immune enhancing composition of the present invention is 0.01 to 40 mcg / ml of secreted toxin protein of Bordetella pertussis , and 0.01 to 10 mg / cell of Staphylococcus aureus Cowan I. ml, it is preferably contained as 0.01 ~ 40mcg / ml secretion protein of Staphylococcus aureus Cowan I and lysate of 0.01 ~ 10mg / ml of Bacillus megaterium .

In addition, the present invention provides a method for enhancing immune immunity of an animal, except for humans, which is inoculated into an animal other than a human after adding the composition for enhancing immune immunity to the inactivated vaccine.

The immune enhancing composition is preferably added 5 to 15 parts by weight per 100 parts by weight of inactivated vaccine, more preferably 10 parts by weight to be used.

Hereinafter, the present invention will be described in more detail by way of examples.

<Examples>

Example 1 Preparation of a Novel Immune Enhancing Composition

A) Bordetella pertussis Preparation of Secretory Toxin Protein

Bordetella pertussis was incubated for 48 hours in BHI broth and 0.5% formalin was added. After confirming inactivation by performing a culture test and the like, centrifugation was performed at 8,000 xg for 20 minutes to collect only the supernatant. Ammonium sulfate was added to the supernatant until it was supersaturated by dissolving using magnetic stirrer. After standing in the refrigerator overnight, the protein component formed was centrifuged at 10,000 xg for 1 hour to collect the precipitate and suspended in an appropriate amount of PBS. Toxoid suspended in PBS was dialyzed in distilled water using a protein purification dialysis membrane to purify pure Bordetella pertussis toxoid.

B) Preparation of Staphylococcus aureus Cells and Secretory Proteins

The bacterial cultures incubated for 48 hours in BHI broth were heat-treated at 80 ° C. for 10 minutes and centrifuged at 8,000 xg for 20 minutes to weigh the cells as precipitates, which were suspended in an appropriate amount of PBS and stored in the refrigerator until testing and distribution. In addition, ammonium sulfate was added to the supernatant until it was supersaturated while dissolving it using a magnetic stirrer. After standing in the refrigerator overnight, the protein component formed was centrifuged at 10,000 xg for 1 hour to collect the precipitate and suspended in an appropriate amount of PBS. Toxoid suspended in PBS was dialyzed in distilled water using a protein purification dialysis membrane to purify pure Staphylococcus aureus toxoid.

C) Preparation of Bacillus megaterium lysate

The bacterial cultures incubated in BHI broth for 48 hours were centrifuged at 8,000 xg for 20 minutes, and the precipitated cells were suspended in an appropriate amount of sterile distilled water and lysed at 5 ° C., 5 minutes, and 30 cycles with a sonicator (Branson Digital Sonifier). After confirming the lysis state under a microscope, the precipitated lysate was collected by centrifugation at 10,000 xg for 20 minutes, weighed, suspended in an appropriate amount of PBS, and stored in the refrigerator until testing and dispensing.

D) preparation of composition for enhancing immunity

Bordetella pertussis secretion toxin protein (20mcg / ml) of item a), Staphylococcus aureus Cowan I cells (1mg / ml) and Staphylococcus aureus Cowan I secretion protein (20mcg / ml) item Bacillus megaterium lysate (1mg / ml) was mixed, and then added to the protective agent (LPGG) to 20% and prepared by lyophilization.

Example 2: B. megaterium Activating immune cell function of lysates

end) Chemotactic test of neutrophils in B. megaterium lysates

B. megaterium cells to test whether to improve the random and directional migration ability of phagocytic cells such as neutrophils for separating the goat blood neutrophils by treatment with bacterial cells for B. megaterium and migration area using the agarose gel plate (random migration) The results showed that B. megaterium lysates significantly improved the random and directional neutrophil ability of neutrophils compared to untreated controls (Table 1).

sample R-migration (mm ² ) sample  D- migration (mm) B. megaterium lysates 77.74 ± 5.92 B. megaterium lysates 1.10 ± 0.36 Control group 40.70 ± 13.77 Control group 0.65 ± 0.16

B) superoxide (O) by activating Respiratory burst of neutrophils in B. megaterium lysates ^2- ) Measurement of increase in production

B. To test whether increasing the amount generated superoxide (O ^2-) Respiratory burst by the activation of the cells for the megaterium Macrophages Neutrophils, separating the goat blood neutrophils by treatment with bacterial cells for B. megaterium and using a microplate As a result of reading the color development by nitroblue tetrazolium (NBT) pigment with ELISA reader, the OD of neutrophils treated with B. megaterium lysate was 0.4250, which is much higher than that of untreated neutrophils (0.3486). 2).

sample Effective concentration Optical density (O.D) B. megaterium lysates 10mcg / ml 0.4250 ± 0.05 Control group - 0.3486 ± 0.09

C) Enhancement of nonspecific mouse defense of B. megaterium lysates

To test nonspecific mouse defenses of B. megaterium lysates, 0.2 ml of pathogenic staphylococci were inoculated into mice (ICR, 20 g) intraperitoneally with 0.2 ml of lysates (1 mg / ml) and 24 ml of pathogenic staphylococci (10 ml / 0.2 ml). When challenged with intraperitoneal intraperitoneal vaccination for one week, all control mice died, but the mice inoculated with B. megaterium lysate showed 100% protection rate, indicating superior nonspecific protection (Table 3).

sample Mouse defender (S. aureus attack,%) B. megaterium lysate 4/4 (100) Control group 0/4 (0)

Example 3 composition for enhancing immunity ( S. aureus, B. pertussis, B. megaterium  Nonspecific defense ability

A) Experiment for improving nonspecific defense ability

In order to test the non-specific protective effect of the immune enhancing composition prepared according to the item of d) of Example 1, the immune enhancing composition (1 mg / ml) and other control substances were respectively injected into the abdominal cavity of a mouse (ICR, 20 g). After 24 hours of 0.2ml inoculation, 0.2ml of pathogenic staphylococci (10MLD / 0.2ml) were challenged intraperitoneally and examined for survival for 1 week. As a control, the two S. aureus and B. pertussis complexes were 50%, B. While the megaterium lysate showed 75% protective effect, the mice inoculated with S. aureus, B. pertussis, and B. megaterium 3 complexes showed 100% protective effect of the immune enhancing composition ( S. aureus ) . Aureus, B. pertussis, B. megaterium three combinations) showed excellent non-specific defense ability (Table 4).

sample % Defense B. megaterium lysates
S. aureus, B. pertussis complex
S. aureus, B. pertussis, B. megaterium complex 3/4 (75)
2/4 (50)
4/4 (100)

B) Enhancement of cellular immunity of immune enhancing compositions (S. aureus, B. pertussis, B. megaterium)

Tumor cell necrosis factor (TNF-alpha) production ability was tested to investigate the cellular immune enhancing ability of the immune enhancing composition of the present invention. That is, to the L929 cells (100 μl, 1 × 10 ⁵ cells / well), the composition for enhancing immunity (50 μl) and 10 μl of Actinomycin D solution was added and incubated in CO ₂ incubator for 20 hours at 37 ℃, culture supernatant The amount was generated using ELISA kit for TNF-alpha test. As shown in Fig. 1, the amount of TNF-alpha produced in the immune enhancing composition of lane 6 was compared with the other company's immune enhancing composition (lane 7), and immunostimulating protein (NP, 1.0, 1.5, 2.0 and 3mg / ml). ) And LPS (10 or 100 ng) was found to stimulate significantly more TNF-alpha production (Fig. 1).

Example 4 Improvement of Vaccine Efficacy by Addition of Immune Enhancing Composition

end) Mouse Immunogenicity of Don Alone Inactivated Gel Vaccine

After diluting the don solely inactivated gel vaccine 10-fold, the immune enhancing composition to be tested was added to 10% of the vaccine amount, inoculated 0.2 ml subcutaneously into the mouse, and blood was collected from the orbital artery two weeks later to grow antibody titers. The agglutination test (GAT) was used, and the survival rate was examined by challenge vaccination with Don alone toxic wine (T2 strain, 100MLD / 0.2ml). As a result, all the mice in the group without addition of the composition for enhancing immunogenicity were killed and the antibody titer was 4 times or less, whereas the mice with the composition for enhancing the immunogenicity of the present invention showed 50% protection rate and 16 times better antibody titer (Table 1). 5).

vaccine Vaccination Defense effectiveness (%) (100MLD / 0.2ml, S.C) Antibody / GAT
(2 weeks after first vaccination) Vaccine (Immune Enhancing Composition)
Vaccine + Immune Enhancing Composition 0.2ml, S.C 0/4 (0)
2/4 (50) <4
16

B) Immunogenicity of Newcastle Inactivated Gel and Oil Vaccine against Chickens

The New antibody inactivated gel and oil vaccine were added with the composition for enhancing immunity of the present invention, and 6 weeks old chickens were inoculated with 1 ml of muscle and blood collected after 3 weeks to investigate the HI antibody titer in blood. As a result, the HI antibody (log 2) of the gel vaccine to which the immune enhancing composition of the present invention was added was higher than that of the vaccine to which 5.6 was not added, and in the case of the oil vaccine, the oil vaccine containing the immuno enhancing composition of the present invention was added. HI antibody was confirmed to be superior to the oil-free vaccine with 8.1. In addition, the results of measuring antibody titers on the 9th day of pathogenic Newcastle disease virus attack confirmed that the vaccine to which the immune enhancing composition was added showed stable antibody titers (Table 6).

    vaccine     HI titer (log 2) Before and after vaccination 3 weeks after inoculation 9 days of attack Additive-free gel vaccine
No oil vaccine
Gel Vaccine + Immune Enhancing Composition
Oil Vaccine + Immune Enhancing Composition
Control group <1
<1
<1
<1
<1 5.0
7.3
5.6
8.1
<1 6.1
7.8
6.8
8.3
-

C) Protective effect against pig monoclonal vaccination of pig inoculated and pig monoclonal inactivated vaccine (cepha white, green cross)

After diluting pig fold, pig monoclonal inactivated vaccine 10-fold, the composition for enhancing immunity of the present invention was added, and 2 weeks after inoculation with 0.2ml subcutaneously at 2 weeks interval, 2 weeks after inoculation with pig mononuclear bacteria (100MLD ( 8.2x10 ⁸ cfu / head)) and the protective effect was tested, 33.3% of the immune-additive composition-added group was excellent and 8.3% of the vaccine without the immuno-enhancing composition showed 8.3% of protective effect. (Table 7).

vaccine % Defense Seppa Bag
Sepha Bag + Immunity Enhancing Composition
Control group 1/12 (8.3)
5/15 (33.3)
0/15 (0)

D) Immunogenicity in Pigs of the foot-and-mouth inactivating vaccine with the composition for immune enhancement according to the present invention

In order to test the effect on the immunogenicity when the composition for enhancing immune immunity of the present invention is added to the foot-and-mouth inactivating vaccine, the composition for enhancing immune immunity of the present invention is added to the foot-and-mouth inactivating vaccine (Maryal, Lot No. 0-160). 10ml pigs were inoculated twice with 2ml at 4 weeks intervals with or without 10% and the antibody titers of each cycle were examined using the ELISA kit for foot and mouth antibody testing (PrioCHECK, Switzerland). As a result, the average antibody titer of the foot-and-mouth vaccine with the new immune enhancing composition was 72.5 and 92.0 after 4 weeks of inoculation and 4 weeks of inoculation, respectively, much higher than that of 55.5 and 47 of the vaccine. The addition of one immune enhancing composition was 39.5 and 36, lower than that without addition (Table 8).

animal Immune booster Vaccination Public announcement ELISA antibody titers per cycle (average) Before vaccination Four weeks after the first vaccination 4 weeks after the 2nd vaccination pig Immune Enhancing Composition 2 ml, I.M 2 16 80 92 19 65 92 (17.5) (72.5) (92) Third party
Immunity Increasing Composition 2 ml, I.M 2 20 45 41 14 34 31 (17) (39.5) (36) No additives 2 ml, I.M 2 21 58 53 9 53 41 (15) (55.5) (47)

In the above, the present invention has been described with reference to one embodiment, which is merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent embodiments are possible. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

Claims (7)

In the composition for boosting immunity prepared from Bordetella pertussis and Staphylococcus aureus Cowan I, Bacillus megaterium is used as an active ingredient in the composition for boosting immunity. Immunity enhancing composition, characterized in that it further comprises. The method of claim 1,
Bordetella pertussis ( Bordetella pertussis ) is an immune enhancing composition, characterized in that the secreted toxin protein of Bordetella pertussis . The method of claim 1,
Staphylococcus aureus Cowan I ( Staphylococcus aureus Cowan I) is a composition for enhancing immune cells, characterized in that the secretion proteins of Staphylococcus aureus Cowan I ( Staphylococcus aureus Cowan I) and Staphylococcus aureus Cowan (I) . The method of claim 1,
The Bacillus megaterium ( Bacillus megaterium ) is a composition for immune enhancement, characterized in that the lysate of Bacillus megaterium ( Bacillus megaterium ). 5. The method of claim 4,
The bacterium of Bacillus megaterium ( Bacillus megaterium ) is an immune enhancing composition, characterized in that it comprises 0.01 ~ 10mg / ml. An immunity enhancing method comprising inoculating an animal other than a human after adding the composition for enhancing immunity of any one of claims 1 to 5 to an inactivated vaccine. The method according to claim 6,
The immune enhancing composition is a method for adding 5 to 15 parts by weight per 100 parts by weight of inactivated vaccine.

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