Mucosal adjuvant and method for manufacturing mucosal adjuvant

Abstract

[Problem] To provide a highly efficient mucosal adjuvant that can be safely administered to humans. [Solution] A mucosal adjuvant comprises bioapatite and is characterized in that low-sulfate bioapatite using calcined eggshells is included and that the antigenicity of solid membrane vesicles secreted by Streptococcus mutans is enhanced by the low-sulfate bioapatite. With this configuration, the low-sulfate bioapatite, being a component of bones and teeth, can be safely administered to humans and is easily taken up by dendritic cells, which are antigen-presenting cells, due to the very small particle size of approximately 20 nm, thereby providing a highly efficient mucosal adjuvant.

Description

Mucosal adjuvant and method for producing mucosal adjuvant

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

[0002] Vaccines are an effective defense against infectious diseases. Mucosal vaccines, such as those administered intranasally or orally, can build protective immunity against mucosal infections. However, to fully induce mucosal immunity, typically mediated by IgA antibodies, it is necessary to administer an adjuvant together with the vaccine antigen to enhance antigenicity and ensure efficient uptake of the vaccine antigen into antigen-presenting cells.

[0003] Patent Document 1 describes an adjuvant for mucosal immune vaccines that consists of a subunit of verotoxin.

[0004] Patent Document 1: JP 2003-321392 A

[0005] However, the vaccine described in Patent Document 1 has not yet been used as a nasal vaccine for humans due to safety issues such as toxicity.

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

[0007] In order to solve the above problems, the present invention provides a mucosal adjuvant based on bioapatite, which comprises low-sulfate bioapatite made from baked eggshell, and is characterized in that the antigenicity of solid membrane vesicles secreted by Streptococcus mutans is enhanced by the low-sulfate bioapatite.

[0008] With this configuration, low-sulfate bioapatite, a component of bones and teeth, can be safely administered to humans, and its extremely small particle size of approximately 20 nm makes it easily taken up by dendritic cells, which are antigen-presenting cells, providing a highly efficient mucosal adjuvant. The applicant has previously published reports on the adjuvant effect of eggshell-derived apatite and bacterial-derived membrane vesicles (95th Annual Meeting of the Japanese Society for Bacteriology, 20221 ADR (March 29, 2022) and elsewhere), but has now achieved the above-mentioned specific configuration and its remarkable effects.

[0009] The mucosal adjuvant may be configured such that the membrane vesicles are adsorbed around the low sulfate bioapatite.

[0010] With this structure, the low sulfate bioapatite has a protein-adsorbing property on its surface, so it adsorbs membrane vesicles and forms aggregates, thereby increasing its antigenicity and allowing it to be recognized by dendritic cells.

[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] Acidic substances are known to inhibit the regeneration of body tissues. This composition, which is a low-salt, low-sulfate bioapatite, is thought to prevent sulfates from inhibiting the adsorption of solid membrane vesicles secreted by Streptococcus mutans.

[0013] Furthermore, in order to solve the above-mentioned problems, the present invention provides a method for producing a mucosal adjuvant, which comprises: a preparatory step of producing low-sulfate bioapatite by desalting bioapatite from baked eggshells during synthesis, and culturing Streptococcus mutans therein, followed by obtaining 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.

[0014] With this composition, low-sulfate bioapatite is a component of bones and teeth and can be safely administered to humans. In addition, because its particle size is very small, approximately 20 nm, it is easily taken up by dendritic cells, which are antigen-presenting cells, making it possible to produce a highly efficient mucosal adjuvant using a simple method.

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

[0016] FIG. 1 is an electron microscope photograph 1 of a mucosal adjuvant and membrane vesicles in Example 1 of the present invention. FIG. 2 is a graph showing the systemic immune effect of a mucosal adjuvant in Example 1 of the present invention. FIG. 3 is a graph showing the mucosal immune effect of a mucosal adjuvant in Example 1 of the present invention. FIG. 4 is a graph showing cytokines induced by a mucosal adjuvant and membrane vesicles in Example 1 of the present invention. FIG. 5 is a graph showing the biofilm formation inhibitory effect of antibodies induced by a mucosal adjuvant in Example 1 of the present invention. FIG. 6 is a graph showing the specificity of antibodies induced by a mucosal adjuvant and membrane vesicles in Example 1 of the present invention to membrane vesicles by electrophoresis. FIG. 7 is an electron microscope photograph 2 of a mucosal adjuvant and membrane vesicles in Example 1 of the present invention.

[0017] The mucosal adjuvant in Example 1 of the present invention will be described with reference to Figures 1 to 7. Figure 1 is an electron microscope photograph 1 of the mucosal adjuvant and membrane vesicles in Example 1 of the present invention. Figure 2 is a graph showing the systemic immune effect of the mucosal adjuvant in Example 1 of the present invention. Figure 3 is a graph showing the mucosal immune effect of the mucosal adjuvant in Example 1 of the present invention. Figure 4 is a graph showing cytokines induced by the mucosal adjuvant and membrane vesicles in Example 1 of the present invention. Figure 5 is a graph showing the biofilm formation inhibitory effect of antibodies induced by the mucosal adjuvant in Example 1 of the present invention. Figure 6 is a graph showing the specificity of antibodies induced by the mucosal adjuvant and membrane vesicles in Example 1 of the present invention to membrane vesicles by electrophoresis. Figure 7 is an electron microscope photograph 2 of the mucosal adjuvant and membrane vesicles in Example 1 of the present invention.

[0018] The mucosal adjuvant in Example 1 contains low-sulfate bioapatite made from baked eggshells, which enhances the antigenicity of solid membrane vesicles secreted by Streptococcus mutans. Low-sulfate bioapatite has the property of strongly adsorbing proteins, resulting in a structure in which membrane vesicles are adsorbed around it. When proteins are adsorbed onto the low-sulfate bioapatite, the bound proteins are adsorbed onto the plastic surface. This state was imaged using an electron microscope after air-drying and immersion in an acetone solution. (Electron microscope photograph 1 is shown in Figure 1.) Figure 1(a) shows an electron microscope image of low-sulfate bioapatite alone on a plastic surface, and Figure 1(b) shows an electron microscope image of low-sulfate bioapatite with adsorbed membrane vesicles on a plastic surface.

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

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

[0021] (Antibody production effect of mucosal adjuvants) First, the effect on systemic IgG immune responses 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 of the suspension was administered intranasally into one nostril of a 7-week-old BALB / c mouse. A second nasal inoculation was then administered three weeks later, a third nasal inoculation two weeks later, a fourth nasal inoculation two weeks later, and a fifth nasal inoculation two weeks later. The systemic IgG antibody response to the membrane vesicles was evaluated using ELISA. The results of the antibody production effect are shown in Figure 2. The vertical axis of Figure 2 represents absorbance at 450 nm.

[0022] The horizontal axis of Figure 2 (a) shows the antibody production effect of nasally inoculating only low sulfate bioapatite for the first, second, third, fourth, and fifth inoculations from the left. Figure 2 (b) shows the effect of nasally inoculating only membrane vesicles for the first, second, third, fourth, and fifth inoculations from the left. Figure 2 (c) shows the antibody production effect of a combination of the mucosal adjuvant of Example 1 containing low sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans with the membrane vesicles as antigen for the first, second, third, fourth, and fifth inoculations from the left. Figure 2(d) shows the antibody production effect of a combination of polyinosinic polycytidylic acid [poly(IC)] and membrane vesicles used as a positive control group, for the first, second, third, fourth, and fifth inoculations (from the left). Note that polyionosinic polycytidylic acid [poly(IC)] cannot be administered to the human body.

[0023] As shown in Figure 2(c), the combination of the mucosal adjuvant of Example 1, which contains low sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans, and the membrane vesicles as antigens, had a high systemic IgG antibody production effect, and in particular, the antibody production effect at the fifth inoculation was comparable to that of the positive control group.

[0024] Next, the results of evaluating the antibody production effect on mucosal IgA immune responses are shown in Figure 3. The evaluation method was the same as for systemic immunization: mice were given an initial nasal inoculation into one nostril, followed by a second inoculation three weeks later, a third inoculation two weeks later, a fourth inoculation two weeks later, and a fifth inoculation two weeks later. The results of the antigen-specific IgA mucosal immune responses were evaluated using ELISA. The vertical axis of Figure 3 represents absorbance at 450 nm.

[0025] The horizontal axis of Figure 3 (a) shows the antibody production effect of mucosal immunity when low-sulfate bioapatite alone is administered intranasally, for the first, second, third, fourth, and fifth inoculations (from the left). Figure 3 (b) shows the effect when membrane vesicles alone are administered intranasally, for the first, second, third, fourth, and fifth inoculations (from the left). Figure 3 (c) shows the antibody production effect of the combination of the mucosal adjuvant of Example 1 containing low-sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans with the membrane vesicles as antigen, for the first, second, third, fourth, and fifth inoculations (from the left). Figure 3 (d) shows the antibody production effect of the combination of poly:C and membrane vesicles used as a positive control, for the first, second, third, fourth, and fifth inoculations (from the left).

[0026] As shown in Figure 3(c), the mucosal immunity induced by the combination of the mucosal adjuvant of Example 1, which contains low sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans, and the membrane vesicles as antigens, also had a high antibody production effect, and in particular, the antibody production effect at the fifth vaccination was equivalent to that of the positive control group.

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

[0028] As shown in Figure 4, when CD4+ helper T cells derived from the spleens of mice immunized with low-sulfate bioapatite and membrane vesicles were restimulated with membrane vesicles, IFN-γ was produced, but IL-4 was not. In other words, low-sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans induced a predominantly Th1-type immune response.

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

[0030] As shown in Figure 5(d), when IgG antibodies derived from the serum of mice immunized with low sulfate bioapatite and membrane vesicles were added, the serum-derived IgG antibodies significantly inhibited biofilm formation.

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

[0032] Solid membrane vesicles secreted by Streptococcus mutans contain glucosyltransferase (GTF), a caries-causing factor. Figure 6 shows that antibodies against this glucosyltransferase (GTF) were induced in both serum IgG and salivary IgA.

[0033] In the future, it is expected that if a pathogenic factor of an infectious disease is incorporated instead of glucosyltransferase (GTF), which is a pathogenic factor of dental caries, antibodies against that pathogenic factor may be induced.

[0034] (Method for producing a mucosal adjuvant) In the preparation process, large quantities of discarded chicken eggshells are calcined and dissolved in water, and barium chloride is added to precipitate the turbidity. The supernatant is then suction-filtered and desalted to produce low-sulfate bioapatite. Streptococcus mutans is then cultured to obtain solid membrane vesicles. Next, in the stirring process, 2 μg of solid membrane vesicles are added to 100 μg of low-sulfate bioapatite in phosphate-buffered saline (PBS) and stirred. The low-sulfate bioapatite, which has strong protein-adsorbing properties, adsorbs the membrane vesicles, producing aggregates of the antigen membrane vesicles and the mucosal adjuvant low-sulfate bioapatite in the PBS.

[0035] Thus, in Example 1 of the present invention, a mucosal adjuvant using bioapatite is provided, which contains low-sulfate bioapatite made from baked eggshells, and is characterized in that 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 is highly efficient.

[0036] In addition, a method for producing a mucosal adjuvant includes a preparatory step of producing bioapatite from baked eggshells, desalting the bioapatite to produce low-sulfate bioapatite, culturing Streptococcus mutans in the bioapatite, and then obtaining solid membrane vesicles; and a stirring step of adding the low-sulfate bioapatite and the membrane vesicles to phosphate-buffered saline (PBS) and stirring the mixture. This method can provide a highly efficient mucosal adjuvant that can be safely administered to humans.

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

Claims

1. A mucosal adjuvant made of bioapatite, comprising low-sulfate bioapatite made from baked eggshell, wherein the antigenicity of solid membrane vesicles secreted by Streptococcus mutans is enhanced by the low-sulfate bioapatite.

2. The mucosal adjuvant according to claim 1, characterized in that the membrane vesicles are adsorbed around the low sulfate bioapatite.

3. The mucosal adjuvant according to claim 1, characterized in 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.

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

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