Mucosal adjuvant and method for producing mucosal adjuvant

Abstract

To provide a highly efficient mucosal adjuvant that enables safe administration to humans.SOLUTION: The present invention provides a bioapatite-based mucosal adjuvant, comprising low-sulfate bioapatite derived from calcined eggshells, characterized in that the low-sulfate bioapatite enhances the antigenicity of solid membrane vesicles secreted by Streptococcus mutans. This structure enables the low-sulfate bioapatite, a bone and tooth component, to be safely administered to humans, and allows the low-sulfate bioapatite, with a very small particle size of about 20 nm, to be easily taken up by dendritic cells, which are antigen-presenting cells. This can provide a highly efficient mucosal adjuvant.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a mucosal adjuvant using bioapatite derived from eggshells and a method for producing the mucosal adjuvant.

Background Art

[0002] Vaccines are effective means of defense against infectious diseases. For pathogens that infect through mucosal surfaces, constructing a defensive immunity on the mucosal surface using mucosal vaccines such as nasal immunization and oral immunization methods can lead to infection prevention. However, in order to sufficiently induce mucosal immunity typified by IgA antibodies, it is necessary to administer an adjuvant agent together with the vaccine antigen to enhance antigenicity and efficiently incorporate the vaccine antigen into antigen-presenting cells.

[0003] Patent Document 1 describes an adjuvant for a mucosal immunization vaccine composed of subunits of verotoxin.

Prior Art Documents

Patent Documents

[0004] Patent Document 1: JP 2003 - 321392

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, what is described in Patent Document 1 has not been used in nasal vaccines for humans due to safety problems such as toxicity.

[0006] An object of the present invention is to solve the above problems and provide a highly efficient mucosal adjuvant that can be safely administered to humans.

Means for Solving the Problems

[0007] In order to solve the above problems, the present invention provides a mucosal adjuvant (Registered Trademark) by Y bioapatite Twhich is characterized in that using calcined eggshells with a sulfate content of 0.048% or less low-sulfate bioapatite around the antigenicity of the solid vesicles secreted by Streptococcus mutans is adsorbed, and the said membrane vesicle is enhanced by the low-sulfate bioapatite, and provides a mucosal adjuvant.

[0008] With this configuration, low-sulfate bioapatite is a component of bone and teeth and can be safely administered to humans. Moreover, since its particle size is very small at about 20 nm, it is easily taken up by dendritic cells, which are antigen-presenting cells, and can provide a highly efficient mucosal adjuvant. Since low-sulfate bioapatite has protein adsorptivity on its surface, it adsorbs membrane vesicles, forms aggregates, enhances antigenicity, and is recognized by dendritic cells. It is known that acidic substances inhibit the regeneration of body tissues. By using low-sulfate bioapatite with a low sulfate content of 0.048% or less, it is considered that the inhibition of the adsorption of solid membrane vesicles (vesicles) secreted by Streptococcus mutans by sulfate can be prevented. The applicant has previously published on the adjuvant effect using apatite derived from eggshells and bacterial-derived membranous vesicles (95th General Meeting of the Japanese Society for Bacteriology 20221ADR (March 29, 2022), etc.). However, this time, the applicant was able to obtain the specific configuration and its remarkable effects as described above.

[0011] The mucosal adjuvant may be configured such that the salt concentration of the low-sulfate bioapatite is 0.048% or less, preferably 0.0048% or less, and more preferably 0.0001% or less.

[0012] It is known that acidic substances inhibit the regeneration of body tissues. With this configuration, by using low-sulfate bioapatite with a low salt concentration, it is considered that the inhibition of the adsorption of solid vesicles secreted by Streptococcus mutans by sulfates can be prevented.

[0013] Also, to solve the above problems, the present invention provides a method for producing a mucosal adjuvant, which comprises a preparation step of synthesizing bioapatite from calcined eggshells and desalting during synthesis to produce low-sulfate bioapatite, and culturing Streptococcus mutans to obtain solid membrane vesicles, and a stirring step of adding 100 μg or more of the low-sulfate bioapatite and the membrane vesicles to phosphate-buffered saline (PBS) and stirring them, characterized in that it provides a method for producing a mucosal adjuvant.

[0014] With this configuration, low-sulfate bioapatite is a component of bone and teeth and can be safely administered to humans. Moreover, since its particle size is very small, about 20 nm, it is easily taken up by dendritic cells, which are antigen-presenting cells. Therefore, a highly efficient mucosal adjuvant can be produced by a simple method.

Advantages of the Invention

[0015] The mucosal adjuvant and the method for producing a mucosal adjuvant of the present invention can be safely administered to humans and can provide a highly efficient mucosal adjuvant.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

Examples

[0017] The mucosal adjuvant in Example 1 of the present invention will be described with reference to FIGS. 1-7. FIG. 1 is Electron Micrograph 1 of the mucosal adjuvant and the membrane vesicles in Example 1 of the present invention. FIG. 2 is a graph showing the systemic immune effect of the mucosal adjuvant in Example 1 of the present invention. FIG. 3 is a graph showing the mucosal immune effect of the mucosal adjuvant in Example 1 of the present invention. FIG. 4 is a graph showing the cytokines induced by the mucosal adjuvant and the membrane vesicles in Example 1 of the present invention. FIG. 5 is a graph showing the inhibitory effect on biofilm formation by the antibody induced by the mucosal adjuvant in Example 1 of the present invention. FIG. 6 is a graph showing the specificity of the antibody induced by the mucosal adjuvant and the membrane vesicles in Example 1 of the present invention against the membrane vesicles by electrophoresis. FIG. 7 is Electron Micrograph 2 of the mucosal adjuvant and the membrane vesicles in Example 1 of the present invention.

[0018] The mucosal adjuvant in Example 1 contains low-sulfate bioapatite using calcined eggshells, and enhances the antigenicity of solid membrane vesicles secreted by Streptococcus mutans. Since low-sulfate bioapatite has the property of strongly adsorbing proteins, it has a structure in which membrane vesicles are adsorbed around it. When proteins are adsorbed onto low-sulfate bioapatite, they are adsorbed onto the plastic surface by the bound proteins. The state after natural drying and immersion in an acetone solution was imaged with an electron microscope. (Electron micrograph 1 is shown in Fig. 1.) Fig. 1(a) shows the electron micrograph when only low-sulfate bioapatite is placed on the plastic surface, and Fig. 1(b) shows the electron micrograph when low-sulfate bioapatite adsorbed with membrane vesicles is placed on the plastic surface.

[0019] Also, another electron micrograph 2 is shown in Fig. 7. Fig. 7(a) is an electron micrograph of membrane vesicles, Fig. 7(b) is an electron micrograph of low-sulfate bioapatite, and Fig. 7(c) is an electron micrograph of the state where membrane vesicles are adsorbed onto low-sulfate bioapatite. As shown in Fig. 7(c), the state where membrane vesicles are adsorbed onto low-sulfate bioapatite can be clearly observed. When membrane vesicles are adsorbed onto low-sulfate bioapatite, they form aggregates to enhance antigenicity and are recognized by dendritic cells.

[0020] The mucosal adjuvant in Example 1 has a very small particle size of about 20 nm for low-sulfate bioapatite, and due to the structure in which nano-sized membrane vesicles are adsorbed around it, it is easily taken up by dendritic cells when administered to humans.

[0021] (Antibody production effect of mucosal adjuvant) First, the effects on the systemic IgG immune response will be described. 100 μg of low-sulfate bioapatite was suspended in 10 μl of PBS for 2 μg of membrane vesicles, and 5 μl each was nasally inoculated for the first time into one nostril of 7-week-old BALB / c mice. Then, the second inoculation was performed 3 weeks later, the third inoculation was performed 2 weeks after that, the fourth inoculation was performed 2 weeks after that, and the fifth inoculation was performed 2 weeks after that. The systemic IgG antibody response against the membrane vesicles was evaluated by ELISA. The results of the antibody production effect are shown in Figure 2. The vertical axis in Figure 2 indicates the absorbance at 450 nm.

[0022] In Figure 2, (a) on the horizontal axis shows the antibody production effect when only low-sulfate bioapatite was nasally inoculated, from the left, for the first inoculation, second inoculation, third inoculation, fourth inoculation, and fifth inoculation. Figure 2(b) shows the effect when only membrane vesicles were nasally inoculated, from the left, for the first inoculation, second inoculation, third inoculation, fourth inoculation, and fifth inoculation. Also, Figure 2(c) shows the antibody production effect of the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and the solid membrane vesicles secreted by Streptococcus mutans and the membrane vesicles as the antigen, from the left, for the first inoculation, second inoculation, third inoculation, fourth inoculation, and fifth inoculation. Figure 2(d) shows the antibody production effect of the combination of polyinosinic polycytidylic acid [poly(I-C)] used as a positive control group and membrane vesicles, from the left, for the first inoculation, second inoculation, third inoculation, fourth inoculation, and fifth inoculation. Note that polyionosinic polycytidylic acid [poly(I-C)] cannot be administered to humans.

[0023] As shown in Fig. 2(c), the effect of producing systemic IgG antibodies in the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and the solid vesicles secreted by Streptococcus mutans and the vesicles as the antigen is high. In particular, the antibody production effect at the fifth vaccination is comparable to that of the positive control group.

[0024] Next, Fig. 3 shows the evaluation results of the antibody production effect on the mucosal IgA immune response. The evaluation method is the same as that for systemic immunity. After the first intranasal inoculation into one nostril of the mouse, the second inoculation was performed 3 weeks later, the third inoculation was performed 2 weeks after that, the fourth inoculation was performed 2 weeks after that, and the fifth inoculation was performed 2 weeks after that. Then, the results for the antigen-specific IgA mucosal immune response were evaluated by the ELISA method. The vertical axis in Fig. 3 indicates the absorbance at 450 nm.

[0025] In Fig. 3, (a) on the horizontal axis shows the antibody production effect of mucosal immunity when only low-sulfate bioapatite was intranasally inoculated, from the left for the first inoculation, the second inoculation, the third inoculation, the fourth inoculation, and the fifth inoculation. Fig. 3(b) shows the effect when only vesicles were intranasally inoculated, from the left for the first inoculation, the second inoculation, the third inoculation, the fourth inoculation, and the fifth inoculation. Also, Fig. 3(c) shows the antibody production effect in the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and the solid vesicles secreted by Streptococcus mutans and the vesicles as the antigen, from the left for the first inoculation, the second inoculation, the third inoculation, the fourth inoculation, and the fifth inoculation. Fig. 3(d) shows the antibody production effect of the combination of polyI:C used as the positive control group and vesicles, from the left for the first inoculation, the second inoculation, the third inoculation, the fourth inoculation, and the fifth inoculation.

[0026] As shown in Fig. 3(c), in the mucosal immunity induced by the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and the solid membrane vesicles (vesicles) secreted by Streptococcus mutans and the membrane vesicles as the antigen, the antibody production effect is also high. In particular, the antibody production effect at the fifth vaccination is equivalent even when compared with the positive control group.

[0027] Next, with reference to Fig. 4, the cytokine production effect by the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and the solid membrane vesicles (vesicles) secreted by Streptococcus mutans and the membrane vesicles as the antigen will be described. The vertical axis of Fig. 4 indicates the production amount of cytokines (IL-4, IFN-γ). In the horizontal axis of Fig. 4, (a) shows the case where CD4-positive helper T cells derived from the spleen of mice immunized with low-sulfate bioapatite and membrane vesicles were cultured together with antigen-presenting cells, and (b) shows the case where CD4-positive helper T cells derived from the spleen of mice immunized with low-sulfate bioapatite and membrane vesicles were cultured together with antigen-presenting cells and membrane vesicles.

[0028] As shown in Fig. 4, when CD4-positive helper T cells derived from the spleen of mice immunized with low-sulfate bioapatite and membrane vesicles were restimulated with membrane vesicles, IFN-γ was produced. IL-4 was not produced. That is, low-sulfate bioapatite and the solid membrane vesicles (vesicles) secreted by Streptococcus mutans mainly induced a Th1-type immune response.

[0029] Next, the inhibitory effect of the induced antibody on biofilm formation will be described with reference to Fig. 5. The vertical axis of Fig. 5 indicates the absorbance at 492 nm. On the horizontal axis of Fig. 5, (a) shows the case where no IgG antibody was added, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutants were added; (b) shows the case where IgG antibody derived from a mouse immunized with low-sulfate bioapatite, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutants were added; (c) shows the case where IgG antibody derived from a mouse immunized with membrane vesicles, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutants were added; (d) shows the case where IgG antibody derived from a mouse immunized with low-sulfate bioapatite and membrane vesicles, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutants were added; (e) shows the case where no IgG antibody and membrane vesicles were added, and Streptococcus mutans gtfB, C-deficient mutants were added.

[0030] As shown in Fig. 5(d), when the IgG antibody derived from the serum of a mouse immunized with low-sulfate bioapatite and membrane vesicles was added, the formation of biofilm was significantly inhibited by the serum-derived IgG antibody.

[0031] Next, the analysis of the specificity of the induced antibody against glucosyltransferase (GTF) will be described with reference to Fig. 6. Fig. 6 shows the application of serum or saliva samples collected two weeks after the last immunization as the primary antibody to Western blotting to evaluate the reactivity against membrane vesicles. (a) shows the reactivity of the serum sample against membrane vesicles, and (b) shows the reactivity of the saliva sample against membrane vesicles.

[0032] The solid membrane vesicles (vesicles) secreted by Streptococcus mutans contain glucosyltransferase (GTF), which is a causative agent of dental caries. According to Fig. 6, it shows that antibodies against this glucosyltransferase (GTF) were induced both in IgG in serum and IgA in saliva.

[0033] In the future, it is expected that by incorporating the causative agent of infectious diseases instead of glucosyltransferase (GTF), which is the causative agent of this caries, antibodies against it can be induced.

[0034] (Method for producing mucosal adjuvant) In the preparation step, chicken eggshells that are discarded in large quantities are baked, dissolved in water, barium chloride is added, the turbidity is allowed to precipitate, the supernatant is suction filtered, and then desalted to produce low-sulfate bioapatite. Also, after culturing Streptococcus mutans, solid membrane vesicles are obtained. Next, in the stirring step, 2 μg of solid membrane vesicles are added to 100 μg of low-sulfate bioapatite in phosphate buffered saline (PBS) and stirred. Due to the adsorption of the low-sulfate bioapatite having strong protein adsorption properties to the membrane vesicles, aggregates of the membrane vesicles as antigens and the low-sulfate bioapatite as a mucosal adjuvant can be produced in PBS.

[0035] Thus, in Example 1 of the present invention, there is provided a mucosal adjuvant using bioapatite, which contains low-sulfate bioapatite using calcined eggshells, and the antigenicity of solid membrane vesicles secreted by Streptococcus mutans is enhanced by the low-sulfate bioapatite. This mucosal adjuvant can be safely administered to humans and can provide a highly efficient mucosal adjuvant.

[0036] Also, a method for producing a mucosal adjuvant, which includes a preparation step of producing bioapatite from calcined eggshells, desalting to produce low-sulfate bioapatite, culturing Streptococcus mutans, and obtaining solid membrane vesicles, and By the method for producing a mucosal adjuvant, which comprises a stirring step of adding the low-sulfate bioapatite and the membrane vesicles into phosphate buffered saline (PBS) and stirring them, it is possible to safely administer to humans and provide a highly efficient mucosal adjuvant.

Industrial Applicability

[0037] The mucosal adjuvant and the method for producing a mucosal adjuvant in the present invention can be widely used for various vaccines by inserting various protein antigens contained in membrane vesicles (vesicles) by genetic recombination.

Claims

1. A mucosal adjuvant based on Bioapatite (registered trademark), comprising: A mucosal adjuvant comprising solid membrane vesicles secreted by Streptococcus mutans adsorbed around low-sulfate bioapatite made from baked eggshells and having a sulfate content of 0.048% or less, and wherein the antigenicity of the membrane vesicles is enhanced by the low-sulfate bioapatite.

2. A method for producing a mucosal adjuvant, comprising the steps of: producing bioapatite from baked eggshells, desalting the bioapatite to produce low-sulfate bioapatite; culturing Streptococcus mutans in the bioapatite; and obtaining solid membrane vesicles. A method for producing a mucosal adjuvant, comprising a stirring step of adding the low sulfate bioapatite and the membrane vesicles to phosphate buffered saline (PBS) and stirring the mixture.

Images