JP5242855B2 - Immune adjuvant - Google Patents

Description

  The present invention relates to an immune adjuvant that is administered together with an immunogenic substance to enhance immunity by the immunogenic substance.

  β-glucan is a polysaccharide composed of D-glucose as a constituent sugar. For example, β-1,3-glucan has β-pyranose type cyclic D-glucose bonded to carbon at the 1-position of glucose. A polysaccharide which is condensed by a β-1,3 glucoside bond between a hydroxyl group which is bonded to a carbon at the 3-position of glucose, and a cyclic β-pyranose type D-glucose is polymerized It is. Polysaccharides such as β-glucan are found in a wide variety of natural organisms as structural molecules such as sugar storage molecules and cell walls that are energy sources, but mushrooms such as Agaricus moth, Reishi, Mai Β-glucan contained in maitake mushrooms, shiitake mushrooms, etc. is known to have various physiological activities for maintaining and enhancing health, immune enhancing action, antitumor activity, cancer cell proliferation Attempts to use it as functional materials and pharmaceuticals for the purpose of suppressing, antiallergic, anti-inflammatory, cholesterol-lowering, antithrombotic, dietary fiber, hypotensive, hypoglycemic, and liver Many have been made. In addition, since it is indigestible, it is expected as a functional material for polysaccharides that can be expected to have a wide range of applications, such as intestinal preparations for the prevention and improvement of constipation, and cosmetics that utilize its moisturizing properties.

  As β-glucan having beneficial activity, in addition to the main chain consisting of β-1,3 glucoside bond, β-1,3-1,6- having a side chain of D-glucose from carbon at the 6-position of glucose Although glucans are well known and branching structures are thought to be necessary for activity, the mechanism of action is not always clear. In addition, since β-glucan is a high molecular polymer obtained from natural products, the structure such as the degree of branching, the length of the main chain, the length of the side chain, amination, phosphorylation, methylation, The presence / absence and degree of modification of the D-glucose hydroxyl group by acetylation and the like are not uniform, and the principle regarding the effect of the structure and chemical modification on the activity is not shown. Thus, empirically, mushroom-derived β-glucan is only favorably used, and there have been few attempts to use β-glucan derived from other species.

  Recently, mushrooms are also derived from β-1,3-1,6-glucan produced by microorganisms (commonly known as black yeast) belonging to the genus Aureobasidium sp. It has been clarified that functions equivalent to or better than those of β-glucan can be obtained.

  For example, in Patent Document 1 below, an aureobasidium culture solution mainly composed of β-1,3-1,6 glucan has a high oral antitumor activity and immunostimulatory activity, and is a drug for various diseases. It is described that it can be applied.

  On the other hand, in the field of vaccine and antibody production, those that have the effect of enhancing the immunity effect by immunogenic substances are called immunoadjuvants, and those that have the action of such immunoadjuvants include inorganic aluminum hydroxide, calcium phosphate In addition, Freund's complete adjuvant in which dead bacteria of M. tuberculosis are suspended in a micelle of mineral oil or liquid paraffin is known.

Japanese Patent Laid-Open No. 2002-204687

  However, immunoadjuvants may not be able to exert sufficient effects depending on the type of vaccine, etc., and may themselves cause side effects that worsen allergic symptoms, and it was expected to provide new immune adjuvants. .

  Accordingly, an object of the present invention is to provide an immunoadjuvant that uses an active ingredient derived from a natural product and has an excellent effect.

The inventors of the present invention have intensively studied to achieve the above object and have completed the present invention. That is, the present invention is as follows.
[1] An immunoadjuvant that is administered nasally together with the immunogenic substance to enhance immunity by the immunogenic substance, and is a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp. A nasal immunoadjuvant characterized by containing a product as an active ingredient .
[2 ] The nasal immunoadjuvant according to the above [ 1 ], wherein the immunogenic substance is in the form of a vaccine, and the vaccine is an influenza virus vaccine.
[3] The nasal immunoadjuvant according to the above [2], which is applied to a form in which Poly (I: C) is further administered nasally in combination.
[4] The nasal immune adjuvant according to any one of [1] to [3], wherein the culture composition is a culture solution obtained by culturing the microorganism or a powder obtained by drying the culture solution.

  According to the immunoadjuvant of the present invention, the composition contains a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium (Aureobasidium sp.) As an active ingredient. Strengthen the immunostimulatory effect of the immunogenic substance.

It is a graph which shows collectively the average result of each test group of the test groups 1-4 about the IgA antibody titer in a nasal cavity washing | cleaning liquid. It is the graph which compared the result according to the solid of the test group 1 and the test group 2 about the IgA antibody titer in a nasal cavity washing | cleaning liquid. It is the graph which compared the result according to the solid of the test group 2 and the test group 4 about the IgA antibody titer in a nasal cavity washing | cleaning liquid. It is the graph which compared the result according to the solid of the test group 2 and the test group 3 about the IgA antibody titer in a nasal cavity washing | cleaning liquid. It is a graph which shows collectively the average result of each test group of the test groups 1-4 about the IgG antibody titer in serum. It is the table | surface which compared the result according to individual of the test group 1 and the test group 2 about the IgG antibody titer in serum. It is the graph which compared the result according to the solid of the test group 2 and the test group 4 about the IgG antibody titer in serum. It is the graph which compared the result according to the solid of the test group 2 and the test group 3 about the IgG antibody titer in serum. It is a graph which shows collectively the average result of each test group of the test groups 1-4 about the IgM antibody titer in serum. It is the graph which compared the result according to the solid of the test group 1 and the test group 2 about the IgM antibody titer in serum. It is the graph which compared the result according to the individual of the test group 2 and the test group 4 about the IgM antibody titer in serum. It is the graph which compared the result according to the solid of the test group 2 and the test group 3 about the IgM antibody titer in serum. It is a graph which shows the average result of each test group of the test groups 5-12 about IgG antibody titer in serum. It is a graph which shows the average result of each test group of the test groups 5-12 about the IgM antibody titer in serum. It is a graph which shows the average result of each test group of the test groups 5-12 about the IgA antibody titer in a nasal cavity washing | cleaning liquid by 1-8 times dilution.

  The immune adjuvant of the present invention contains a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp. (Hereinafter referred to as “Aureobasidium-derived culture composition”) as an active ingredient. The aureobasidium-derived culture composition includes a culture solution in which microorganisms belonging to the genus Aureobasidium sp. (Hereinafter referred to as “Aureobasidium microorganisms”) are cultured, and the cells are separated by centrifugation, etc. Not only the removed culture solution, the concentrate of the culture solution, the diluted solution of the culture solution, or the solids obtained by removing water from the culture solution, but also desalting them to specific components such as β-glucan Those with a higher content of are also included.

  The Aureobasidium microorganism used in the present invention may be any microorganism that belongs to the genus Aureobasidium and has the ability to produce β-glucan. For example, Aureobasidium pullulans M-1 (Aureobasidium) pullulans M-1, AIST (National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Accession Number FERM BP-08615), Aureobasidium pullulans M-2, National Institute of Advanced Industrial Science and Technology, Patent Biology Deposit center accession number FERM BP-10014) is preferably used. In addition, β-glucan produced by these strains is obtained from the main chain in which glucose is β-1,3 linked by NMR analysis (13CNMR: Varian UNITY INOVA500 type, 1HNMR: Varian UNITY INOVA600 type). It has been clarified that it is β-1,3-1,6-glucan having a structure in which glucose is branched by -1,6 bonds.

  The aureobasidium microorganism can be cultured according to a known method (see Japanese Patent Application Laid-Open No. 57-149301). That is, a medium (pH 5.2 to 2) containing 0.5 to 5.0% by mass of a carbon source (sucrose), 0.1 to 5.0% by mass of an N source, and other trace substances (for example, vitamins and inorganic substances). 6.0) is inoculated with bacteria, and aeration culture is performed at a temperature of 20 to 30 ° C. for 2 to 14 days, preferably aeration and agitation culture. As β-glucan is produced, the viscosity of the culture solution increases and becomes a highly viscous gel. The culture solution thus obtained usually contains 0.6 to 10% by mass of solid content, and the solid content contains 5 to 80% by mass of β-glucan.

  In the present invention, it is preferable to use a culture containing β-glucan obtained by the above culture after sterilization by heating or pressure heating. Alternatively, the cells may be sterilized after being separated and removed by centrifugation or the like. Moreover, what was concentrated or dried as needed can also be used. Furthermore, what extracted the component rich in (beta) -glucan, and what desalted and refine | purified it can also be used. A culture of microorganisms belonging to the genus Aureobasidium sp. Is used as a food additive such as a thickening stabilizer and has high safety.

  The immune adjuvant of the present invention is used so as to be administered together with the immunogenic substance in order to enhance the effect of immunity by the immunogenic substance. In other words, for the purpose of preventing infection with pathogenic bacteria or viruses, or preventing or treating their seriousness, it can be used to enhance the effect when immunization by a vaccine is expressed. For example, influenza virus vaccine etc. are mentioned. Alternatively, for the purpose of producing antiserum in animals such as mice, rats, rabbits, goats, cows, monkeys, etc., it can be used to enhance the effect when immunization with a predetermined antigen is expressed.

  As an application form of the present invention, when an immunogenic substance such as a vaccine or a predetermined antigen is inoculated into a human or animal to develop immunity by this, the immune adjuvant of the present invention is administered together to effect the immunity. Can be increased. There is no particular limitation on the administration route, and it is possible to select a known preparation form as appropriate and adapt it to oral administration, intramuscular administration, intranasal administration and the like.

  The immunoadjuvant of the present invention is preferably administered in combination in the form of a mixture with an immunogenic substance. According to this, a synergistic action with an immunogenic substance for enhancing the effect of immunity can be expected more.

  The dose of the immunoadjuvant of the present invention and the use amount relative to the use amount of the immunogenic substance are different from the immunogenic substance, the health state, symptoms, age, administration method / number of administration / administration of the recipient or animal. It can be determined appropriately depending on the time. For example, when taking a general dose, for example, when taken orally, it is taken in an amount of 0.025 to 4000 mg / kg (body weight) in terms of solid content of the aureobasidium-derived culture composition. In addition, in the case of intranasal administration, ingested in an amount of 0.05 to 5 mg / kg (body weight) in terms of solid content of the aureobasidium-derived culture composition.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

[Test Example 1]
Using a culture solution of a microorganism belonging to the genus Aureobasidium sp., Its effectiveness as an immune adjuvant was examined. Specifically, the effectiveness as an immune adjuvant when immunizing with an influenza virus vaccine was examined.

  As a culture solution of microorganisms belonging to the genus Aureobasidium (Aureobasidium sp.), Aureobasidium pullulans M-2 (FERM BP-10014) is cultured, and the culture solution is incubated at 121 ° C. for 15 minutes. A culture solution prepared by sterilization and having a β-glucan content of 0.65 g / 100 g was used. Hereinafter, this is referred to as “Aureo broth”.

  H1N1 subtype influenza virus A / PR / 8 was used as an influenza virus to infect mice. In addition, as a vaccine against it, an inactivated HA vaccine (A / PR8 split vaccine, total protein amount 2.1 mg / day) prepared by treating virus particles with diethyl ether and extracting into the ether phase according to a conventional method. ml) was used.

  As experimental animals, mice (C57BL / 6NJcl strain, male, 6 weeks old) were prepared and acclimated for 1 week. As shown in Table 1 below, (1) Aureo culture solution and vaccine were passed through. Test group 1 for nasal administration, (2) Test group 2 for nasal administration of vaccine alone, (3) Test group 3 for nasal administration of PBS, (4) Oral culture solution administered orally and nasal administration of vaccine The test group 4 was divided into a total of 4 groups.

  Nasal administration of vaccine to mice (in Test Group 3 PBS administration) was performed as follows. That is, the mouse was placed in a container filled with isoflurane, taken out after confirming slow respiration, and 5 μl was inoculated into the nasal cavity of the mouse with a micropipette. At that time, in test group 1, the aureo culture was mixed with the vaccine and inoculated. After that, it was confirmed that it was awakened from anesthesia and returned to the breeding gauge. In Test Group 4, oral administration of Aureo broth was started on the same day as the first vaccine nasal administration, and 200 μL was orally administered once a day during the test period by the sonde method.

Immunization with the vaccine was performed twice, a first time for mice after acclimation rearing and a second time at 4-day intervals. Influenza virus was infected 4 days after the final immunization. Mice were infected with influenza virus as follows. That is, a diluted virus solution was prepared by serial dilution with PBS so that the virus titer was 5 × 10 ⁵ pfu / ml, and 2 μl (1000 pfu / animal) of the diluted nasal cavity of anesthetized mice was used in the same manner as described above. Inoculated inside.

  Three days after the virus infection, nasal lavage fluid and serum were collected from the mice, and by ELISA, IgA antibody titer in nasal lavage fluid reactive to inactivated influenza virus, IgG antibody titer in serum, and IgM Each antibody titer was measured.

  In addition, the inactivated influenza virus used for the antibody titer measurement by ELISA method was prepared as follows. That is, MDCK cells were cultured as a host to increase influenza virus, and the virus was concentrated from the culture solution by ultracentrifugation. This was suspended in PBS, suspended, and inactivated by adding 10 times the amount of Disruption buffer (0.1% Triton-X / PBS). The inactivated virus was diluted with PBS to a virus concentration of 5 μg / mL.

Moreover, the antibody titer measurement by ELISA method was performed as follows. That is, the inactivated influenza virus adjusted to the above virus concentration was added to a 96-well plate by 50 μL / well, and allowed to stand overnight at 4 ° C. to solidify the virus. After discarding the virus solution from the well, it was blocked with 3% skim milk / PBS-T. Thereafter, serially diluted nasal lavage fluid or serum collected from the mice was added to each well, and a secondary antibody was further added to react. As secondary antibodies, Anti-Mouse IgA HRP was used for IgA antibody titers, Anti-Mouse IgG HRP for IgG antibody titers, and Anti-Mouse IgM HRP for IgM antibody titers. According to a conventional method, the color was developed with a TMB reagent, the reaction was stopped with 2N H ₂ SO ₄ , and the absorbance at 450 nm was measured. And the value of the light absorbency obtained according to the dilution rate of the sample used for ELISA was plotted, and each antibody titer was compared.

  The results are shown in FIGS. In addition, FIG. 1, FIG. 5 and FIG. 9 are graphs collectively showing the average results of each test group for each of the IgA antibody titer, serum IgG antibody titer, and serum IgM antibody titer in the nasal lavage fluid. is there. In addition, FIG. 2, FIG. 6, and FIG. 10 are diagrams comparing the results of each test group 1 and test group 2 for the IgA antibody titer, serum IgG antibody titer, and serum IgM antibody titer in the nasal lavage fluid. It is. In addition, FIG. 3, FIG. 7 and FIG. 11 are graphs comparing the results of test group 2 and test group 4 for each of the IgA antibody titer, serum IgG antibody titer, and serum IgM antibody titer in nasal lavage fluid. It is. In addition, FIG. 4, FIG. 8, and FIG. 12 are diagrams comparing the results of each test group 2 and test group 3 for the IgA antibody titer, serum IgG antibody titer, and serum IgM antibody titer in the nasal washing solution, respectively. It is.

  As a result, as shown in FIG. 4, there was almost no difference between the test group 2 inoculated with the vaccine and the test group 3 inoculated with PBS in terms of the IgA antibody titer in the nasal lavage fluid. Moreover, as can be seen from FIGS. 8 and 12, the serum IgG antibody titer and IgM antibody titer were also not significantly increased in the test group 2 inoculated with the vaccine. Therefore, in this test example, the effect of vaccination was relatively limited. This was considered to be due to the short vaccination period in this test example.

  On the other hand, as seen in FIGS. 2 and 3, for the IgA antibody titer in the nasal wash, test group 1 in which the aureo culture solution was mixed with the vaccine and administered nasally rather than test group 2 inoculated with the vaccine alone. In the test group 4 in which the aureo broth was orally administered together with nasal administration of the vaccine, the antibody titer was higher. Therefore, it was revealed that the aureo culture medium functions as an immune adjuvant that enhances nasal cavity immunity against influenza virus.

  Similarly, as shown in FIGS. 6 and 7 for the serum IgG antibody titer and FIGS. 10 and 11 for the serum IgM antibody titer, the aureo culture solution was administered to the vaccine more than the test group 2 inoculated with the vaccine alone. The antibody titer was higher in Test Group 1 administered nasally after mixing in Test Group 4 and Test Group 4 administered orally with the aureo broth along with nasal administration of the vaccine. Therefore, it was revealed that the aureo culture medium functions as an immune adjuvant that enhances systemic immunity against influenza virus.

  From the above, it was revealed that the aureo culture solution is effective as an immune adjuvant when immunizing with an influenza virus vaccine.

[Test Example 2]
In Test Example 1 described above, it became clear that the Aureo broth was effective as an immune adjuvant. Therefore, synthetic double-stranded RNA Poly (I: C) (manufactured by Amersham Biosciences), which is a ligand of TLR3 (Toll-Like Receptor 3), which is known as an immunoadjuvant of a nasal mucosal vaccine against influenza virus, and yeast Using Zymosan (manufactured by Sigma-Aldrich) which is a cell wall fraction, the effectiveness as an immune adjuvant was further examined.

  Aureo broth, influenza virus, vaccine, and mouse were the same as in Test Example 1 above. Also, Aureobasidium pullulans M-2 (FERM BP-10014) was cultured, and the culture was dried to prepare a powder having a β-glucan content of 0.39 g / 100 g. . Hereinafter, this is referred to as “Aureo powder”.

  As an experimental animal, a mouse (C57BL / 6NJcl strain, male, 6 weeks old) was prepared and acclimated for 2 weeks. As shown in Table 2 below, (5) Aureo culture solution and vaccine were applied nasally. Test group 5 for administration, (6) Test group 6 for nasal administration of aureo powder and vaccine, (7) Test group 7 for nasal administration of aureo culture solution, Poly (I: C) and vaccine, (8) Zymosan (Zymosan), Poly (I: C) and vaccine group 8 (9) Poly (I: C) and vaccine group 9 (10) Nasal vaccine alone The test group 10 was divided into 8 groups: (11) test group 11 in which PBS was administered nasally, (12) test group 12 in which aureo culture solution was orally administered, and vaccine was administered nasally.

  For nasal administration to mice, in the same manner as in Test Example 1, the test substance prepared in 5 μl of PBS with a micropipette was inoculated into the nasal cavity of the mouse under anesthesia. In test group 12, oral administration of the aureo broth was started one week before the first nasal administration of vaccine, and 200 μL was orally administered once a day during the test period by the sonde method.

Immunization with the vaccine was performed twice, a first time for mice after acclimation rearing and a second time at 3-week intervals. Influenza virus was infected 2 weeks after the final immunization. Mice were infected with influenza virus as follows. That is, a diluted virus solution was prepared by serial dilution with PBS so that the virus titer would be 1 × 10 ⁵ pfu / ml, and 2 μl (200 pfu / animal) of the diluted nasal cavity of anesthetized mice was prepared in the same manner as described above. Inoculated inside.

  Three days after virus infection, nasal lavage fluid and serum were collected from the mice, and in the same manner as in Test Example 1 above, the IgG antibody titer in the serum reactive to inactivated influenza virus and IgM by ELISA method. The antibody titer and the IgA antibody titer in the nasal wash were measured.

  The results are shown in FIGS. FIG. 15 shows the IgA antibody titer in the nasal wash when diluted 1 to 8 times.

  As shown in FIG. 13, the serum IgG antibody titer was obtained by using the aureo broth as a vaccine, rather than the test group 10 inoculated with the vaccine alone or the test group 12 in which the aureo broth was orally administered and nasally administered. Test group 5 mixed and administered nasally, Test group 6 mixed nasally with aureo powder mixed with vaccine, and Test group 9 mixed nasally with Poly (I: C) mixed with vaccine The antibody titer increased. However, the effect was limited. This was considered to be because the amount of influenza virus infection in this test example was less than 1/5 (200 pfu / animal) compared to test example 1. On the other hand, in the test group 7 which used aureo culture solution and Poly (I: C) in combination, Zymosan and Poly (I: The same effects as those of the test group 8 using C) were obtained.

  Similarly, as shown in FIG. 15, in regard to IgA antibody titer in nasal lavage fluid, in test group 7 in which Aureo culture solution and Poly (I: C) were used in combination, an immunizing adjuvant of a nasal mucosal administration vaccine against influenza virus As a result, an effect comparable to that of the test group 8 in which Zymosan and Poly (I: C) were used in combination was obtained. Therefore, it was revealed that the aureo culture solution has high effectiveness as an immunological adjuvant that enhances immunity against influenza virus.

  On the other hand, as seen in FIG. 14, the difference between the test groups was not significant for the serum IgM antibody titer. As described above, in this test example, it was considered that the amount of influenza virus infection was less than 1/5 (200 pfu / animal) compared to test example 1.

[Test Example 3]
It was investigated whether the aureo culture solution contributed to the improvement of the antibody titer when thyroglobulin (Tg) was used as an immunogenic substance.

  As experimental animals, BALB / c mice (6 weeks old, female) were prepared and acclimated for 1 week, and then (i) a control group that only performed normal antigen immunization, and (ii) antigen immunization. The group was divided into a total of 3 groups (8 mice each): a group to which 200 μL of aureo culture solution was administered, and a group to which (iii) 400 μL was administered. Aureo broth or lactic acid bacteria beverage was orally administered by the sonde method once a day continuously during the test period.

  For antigen immunization, thyroglobulin (Tg) purified and purified from thyroid tissue by a conventional method was used, and an emulsion was prepared at a ratio of 1: 9 with Freund's complete adjuvant (Wako Pure Chemical Industries, Ltd.) and then administered intraperitoneally to mice (80 μg). /). Immunization was performed at 0, 2, 4, and 6 weeks after the start of the test.

  For antibody titer measurement, blood was collected on the administration start date and on the third, fifth, and seventh weeks after immunization, and the antibody titer of the anti-Tg antibody (IgG) was measured by home-made ELISA using the above thyroglobulin (Tg). The antibody titer measurement by ELISA method was performed by serially diluting mouse serum 1000 to 10,000 times using enzyme-labeled IgG secondary antibody according to a conventional method, and measuring the absorbance within a range of its quantitativeness.

  As a result, at the seventh week of immunization, (i) control group was 0.781 ± 0.226, (ii) aureo culture solution 200 μL group was 1.341 ± 0.304, and (iii) aureo culture solution 400 μL group was 1 The absorbance was 314 ± 0.322, and the antibody titer was significantly (P ≦ 0.05) increased by administration of 200 μL or 400 μL of the aureo broth compared to the control group.

  From the above, it has been clarified that the antibody titer is increased by administration of aureo medium even when thyroglobulin (Tg) is used as an immunogenic substance.

Claims (4)

An immunoadjuvant administered nasally with the immunogenic substance for the purpose of enhancing immunity by the immunogenic substance, and an active ingredient comprising a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp. A nasal immunoadjuvant characterized by comprising: The immunogenic substance is in the form of a vaccine the vaccine, nasal immunoadjuvant according to claim 1, wherein the influenza virus vaccine.   The nasal immune adjuvant according to claim 2, which is further applied to a form in which Poly (I: C) is used for nasal administration in combination.   The nasal immune adjuvant according to any one of claims 1 to 3, wherein the culture composition is a culture solution obtained by culturing the microorganism or a powder obtained by drying the culture solution.