Mucosa 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 technology]

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

[0003] Patent Document 1 describes an adjuvant for mucosal immunity vaccines that consists of a subunit of verotoxin. [Prior art documents] [Patent documents]

[0004] Patent Document 1: JP 2003-321392 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, the one 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. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention provides a mucosal adjuvant using bioapatite, comprising: The present invention provides a mucosal adjuvant, 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.

[0008] With this composition, low-sulfate bioapatite is a component of bones and teeth and can be safely administered to humans. Furthermore, because its particle size is extremely small at approximately 20 nm, it is easily taken up by dendritic cells, which are antigen-presenting cells, making it possible to provide a highly efficient mucosal adjuvant. The applicant has previously published a report on the adjuvant effect of using eggshell-derived apatite and bacterial-derived membrane vesicles (95th General Meeting of the Japanese Society for Bacteriology 20221ADR (March 29, 2022) and others), but has now been able to obtain the specific composition and its remarkable effects as described above.

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

[0010] With this configuration, the low sulfate bioapatite has a surface with protein-adsorbing properties, and thus adsorbs membrane vesicles and forms aggregates, thereby enhancing antigenicity and allowing recognition 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] It is known that acidic substances inhibit the regeneration of body tissues. This composition, which is a low-salt, low-sulfate bioapatite, is believed to prevent sulfate from inhibiting the adsorption of solid membrane vesicles secreted by Streptococcus mutans.

[0013] In order to solve the above problems, the present invention provides a method for producing a mucosal adjuvant, comprising the steps of: desalting bioapatite from baked eggshells during synthesis to produce low-sulfate bioapatite; culturing Streptococcus mutans in the bioapatite; and obtaining solid membrane vesicles. The present invention provides a method for producing a mucosal adjuvant, which comprises 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 the mixture.

[0014] With this composition, low-sulfate bioapatite is a component of bones and teeth and can be safely administered to humans. Furthermore, because its particle size is extremely small at 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 in a simple manner. Effect of the Invention

[0015] INDUSTRIAL APPLICABILITY 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. [Brief description of the drawings]

[0016] [Figure 1] 1 is an electron microscope photograph 1 of a mucosal adjuvant and membrane vesicles in Example 1 of the present invention. [Diagram 2] 1 is a graph showing the systemic immune effect of a mucosal adjuvant in Example 1 of the present invention. [Diagram 3] 1 is a graph showing the mucosal immune effect of the mucosal adjuvant in Example 1 of the present invention. [Figure 4] 1 is a graph showing cytokines induced by mucosal adjuvants and membrane vesicles in Example 1 of the present invention. [Diagram 5] 1 is a graph showing the biofilm formation inhibitory effect of antibodies induced by a mucosal adjuvant in Example 1 of the present invention. [Figure 6]1 is a graph showing the specificity of antibodies induced by the mucosal adjuvant and membrane vesicles to membrane vesicles in Example 1 of the present invention by electrophoresis. [Figure 7] 2 is an electron microscope photograph 2 of a mucosal adjuvant and membrane vesicles in Example 1 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[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 the antibody induced by the mucosal adjuvant in Example 1 of the present invention. Figure 6 is a graph showing the specificity of the antibody induced by the mucosal adjuvant and membrane vesicles in Example 1 of the present invention to the 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 eggshell, and enhances the antigenicity of solid membrane vesicles secreted by Streptococcus mutans. Low sulfate bioapatite has a property of strongly adsorbing proteins, and therefore has a structure in which membrane vesicles are adsorbed around it. When proteins are adsorbed to the low sulfate bioapatite, the bound proteins are adsorbed to the plastic surface. This state was naturally dried and then immersed in an acetone solution, and photographed with an electron microscope. (Electron microscope photograph 1 is shown in Figure 1.) Figure 1(a) shows an electron microscope image when only low sulfate bioapatite is placed on the plastic surface, and Figure 1(b) shows an electron microscope image when low sulfate bioapatite with membrane vesicles adsorbed is placed on the 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 the low sulfate bioapatite can be clearly observed. When membrane vesicles are adsorbed onto the 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, so that it is easily taken up by dendritic cells when administered to a human.

[0021] (Antibody-producing effect of mucosal adjuvants) First, the effect on the systemic IgG immune response will be explained. 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 to one nostril of a 7-week-old BALB / c mouse. The second administration was then performed three weeks later, the third administration two weeks later, the fourth administration two weeks later, and the fifth administration two weeks later. The systemic IgG antibody response to the membrane vesicles was evaluated by ELISA. The results of the antibody production effect are shown in Figure 2. The vertical axis of Figure 2 shows the absorbance at 450 nm.

[0022] The horizontal axis of Fig. 2 (a) shows the antibody production effect when only low sulfate bioapatite is administered intranasally for the first, second, third, fourth, and fifth inoculations from the left. Fig. 2 (b) shows the effect when only membrane vesicles are administered intranasally for the first, second, third, fourth, and fifth inoculations from the left. Fig. 2 (c) shows the antibody production effect when the mucosal adjuvant of Example 1 containing low sulfate bioapatite and solid membrane vesicles secreted by Streptococcus mutans is combined with the membrane vesicles as the antigen for the first, second, third, fourth, and fifth inoculations from the left. Figure 2(d) shows the antibody production effect of the 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 in the fifth vaccination 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, with the first intranasal inoculation into one nostril of the mouse, followed by a second intranasal 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 response were evaluated by ELISA. The vertical axis of Figure 3 shows the absorbance at 450 nm.

[0025] The horizontal axis of FIG. 3 (a) shows the antibody production effect of mucosal immunity when only low sulfate bioapatite is inoculated nasally for the first, second, third, fourth, and fifth inoculations from the left. FIG. 3(b) shows the effect when only membrane vesicles are inoculated nasally for the first, second, third, fourth, and fifth inoculations from the left. FIG. 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 and the membrane vesicles as the antigen for the first, second, third, fourth, and fifth inoculations from the left. FIG. 3(d) shows the antibody production effect of the combination of polyl:C and membrane vesicles used as a positive control group for the first, second, third, fourth, and fifth inoculations from the left.

[0026] As shown in Figure 3(c), the antibody production effect was also high in 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. In particular, the antibody production effect in the fifth vaccination was equivalent to that of the positive control group.

[0027] Next, the cytokine production effect by 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 antigens will be described with reference to FIG. 4. The vertical axis of FIG. 4 shows the amount of cytokines (IL-4, IFN-g) produced. On the horizontal axis of FIG. 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 are 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 are cultured together with antigen presenting cells and membrane vesicles.

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

[0029] Next, the biofilm formation inhibitory effect of the induced antibody will be described with reference to FIG. 5. The vertical axis of FIG. 5 shows the absorbance at 492 nm. In the horizontal axis of FIG. 5, (a) shows the case where no IgG antibody was added, but membrane vesicles and Streptococcus mutans gtfB, C-deficient mutant strains were added, (b) shows the case where IgG antibody from a mouse immunized with low sulfate bioapatite, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutant strains were added, (c) shows the case where IgG antibody from a mouse immunized with membrane vesicles, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutant strains were added, (d) shows the case where IgG antibody from a mouse immunized with low sulfate bioapatite and membrane vesicles, and membrane vesicles and Streptococcus mutans gtfB, C-deficient mutant strains were added, and (e) shows the case where IgG antibody and membrane vesicles were not added, but Streptococcus mutans gtfB, C-deficient mutant strains were 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 formation of biofilm was significantly inhibited by the IgG antibodies derived from the serum.

[0031] Next, analysis of the specificity of induced antibodies against glucosyltransferase (GTF) will be described with reference to Fig. 6. In Fig. 6, serum or saliva samples collected 2 weeks after the last immunization were applied as primary antibodies to Western blotting to evaluate reactivity against membrane vesicles, where (a) shows the reactivity of the serum sample against membrane vesicles, and (b) shows the reactivity of the saliva sample against membrane vesicles.

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

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

[0034] (Method of producing mucosal adjuvant) In the preparation process, eggshells, which are discarded in large quantities, are calcined and dissolved in water, barium chloride is added to precipitate the turbidity, the supernatant is suction filtered, and desalted to produce low-sulfate bioapatite. Streptococcus mutans is also cultivated, and solid membrane vesicles are obtained. Next, in a stirring step, 2 μg of solid membrane vesicles are added to 100 μg of low sulfate bioapatite in phosphate buffered saline (PBS) and stirred. Low-sulfate bioapatite, which has strong protein-adsorbing properties, adsorbs membrane vesicles, making it possible to form aggregates of membrane vesicles (antigens) and low-sulfate bioapatite (mucosal adjuvant) in PBS.

[0035] Thus, in Example 1 of the present invention, a mucosal adjuvant made of bioapatite is A mucosal adjuvant containing low sulfate bioapatite made from baked eggshells, characterized in that the antigenicity of solid membrane vesicles secreted by Streptococcus mutans is enhanced by the low sulfate bioapatite, can be safely administered to humans and provides a highly efficient mucosal adjuvant.

[0036] Also, a method for producing a mucosal adjuvant includes a preparation step of producing bioapatite from baked eggshells, desalting the bioapatite to produce low sulfate bioapatite, culturing Streptococcus mutans, and obtaining solid membrane vesicles; The method for producing a mucosal adjuvant, which is characterized by including a stirring step of adding the low sulfate bioapatite and the membrane vesicles to phosphate buffered saline (PBS) and stirring the mixture, can provide a highly efficient mucosal adjuvant that can be safely administered to humans. [Industrial Applicability]

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

Claims

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

2. 2. The mucosal adjuvant according to claim 1, wherein 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 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.

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