KR100479735B1 - Non-oral vaccine for preventing dental caries using mannose-coated polymeric micelle-immunoregulator-immunogen complex - Google Patents

Abstract

The present invention relates to a parenteral vaccine for immunologically preventing dental caries, and the parenteral vaccine for immunologically preventing dental caries is a disadvantage of the conventional oral vaccine, that is, a large amount is required and orally administered. It can be destroyed by post-digestive enzymes and has an excellent effect on compensating for disadvantages such as causing an immunological reaction. In addition, the present invention is an immunomodulatory agent for macrophage specific nanoparticles (mannose-coated polymeric micelle) having uniform particle size, large surface area, and easy to disperse or separate in water. It carries retinoic acid and selectively delivers to target organs or cells to reduce side effects of drugs, induces the production of high-efficiency antigen-specific immunoglobulins (IgA), and increases the antigen-specific IgA producing lymphocytes in the blood. Excellent effect.

Description

Non-oral vaccine for preventing dental caries using mannose-coated polymeric micelle-immunoregulator-immunogen complex}

The present invention relates to a parenteral vaccine for immunologically preventing dental caries, and more particularly, the present invention isolates and purifies glucosyltransferase, an immunogen, and provides retinoic acid, an immunomodulator, to macrophage specific nanoparticles. It relates to a mucosal anti-teeth immune vaccine of a parenteral new mechanism that is loaded to induce a large amount of high potency antigen-specific immunoglobulin (IgA) production and increase antigen-specific IgA producing lymphocytes in the blood.

Infectious diseases are mainly transmitted through the gastro-intestinal system or the respiratory system, so mucosal immunity is important. Therefore, a vaccine for mucus immunity according to the path of infection should be developed. Among the immunoglobulins, IgA is the most homologous type of immunoglobulin secreted in the oral cavity or intestinal tract. In particular, secretory IgA (sIgA) blocks the passage of mucosal tissues of pathogenic microorganisms such as bacteria, viruses, and fungi that invade the intestines. It appears to be secreted in large quantities to make. Therefore, the production of antigen-specific IgA should be effectively induced for effective prevention using the immune mechanism of infectious diseases through oral cavity and intestine.

On the other hand, the most common disease that occurs in teeth, 'Caries caries' in our country, the tooth decay rate of permanent teeth reaches about 80%, one person has been reported to have an average of 2-3 tooth decay. Human dental caries is caused by bacteria and the main causative agents are Sterptococcus mutans ( SM) and Streptococcus sobrinus, which are commonly found in dental bacterial membranes. Among these antigens, Ag I / II, PAc and glucosyltransferase (GTF) have been mainly dealt with in studies related to anti-caries vaccines. In particular, antigen-specific secreted immunoglobulins (sIgA) produced by oral immunization with GTF or Ag I / II have been reported to inhibit colonization by caries-forming Streptococcus mutans and reduce the formation of dental caries. . Generally, the oral route of administration of antigens to induce the production of mucous IgA requires a large amount of antigens for oral administration, and these antigens are easily destroyed by digestive enzymes and cause immunological tolerance. There is a disadvantage. Therefore, in order to compensate for these shortcomings, cholera toxin B subunits or liposomes may be bound to the antigen, or a combination of supplements may be used. have.

Usually, IgA production in B cells undergoes 1) switch differentiation of B cells and 2) terminal differentiation of IgA B cells. In particular, the first stage is known to be regulated by cytokines in relation to the Th1 / Th2 balance of the peripheral immune system. In fact, the immune system maintains a close relationship with the neurological and endocrine systems (Neuroendocrinoimmunology) and various hormones are known to be involved in the immune response. From these facts, the addition of immunomodulatory hormones to immunogens can alter the pattern of immune responses and reveal the possibility of parenteral vaccination to compensate for the shortcomings of oral immunity.

Drug delivery systems (DDS) using nanoparticles, which are in the spotlight recently, use liposomes, microspheres, polymeric micelles, and the like. In particular, the polymer microparticles generally refer to microparticles having a size of 10 nm to 150 nm, and are widely applied to various aspects of drug release because of their uniform particle size, large surface area, and easy dispersion or separation in water. have. These microparticles can contain relatively high amounts of drugs, enable continuous drug release, concentrate on only the areas where drugs are needed in the body according to the characteristics of each polymer microparticles, and minimize the drug concentration in other areas. It is also used. In particular, the polymeric micelle method has been used in a target drug release system as a method that can be applied to a wide range of target organs compared to active targeting such as a monoantibody antibody method. In vivo, it has the advantage of reducing the side effects of drugs by selectively delivered to the target organs or cells through intravenous injection. In addition, even with a small amount of antigen, long-term retention and stimulation of immune cells results in high efficacy immunity with a single administration. They can alter the surface structure of the microparticles and promote the reaction with macrophages to increase the immunity to specific antigens, thus inferring new applicability as vaccines.

Accordingly, an object of the present invention is to prevent dental caries, which is one of the most frequently occurring bacterial diseases in Korea, to compensate for the shortcomings of conventional oral vaccines and to provide a more powerful new concept of parenteral mucolytic drugs. To provide a dental caries vaccine.

It is an object of the present invention to produce and purify glucosyltransferase (GTF), which is a dental caries antigen from Streptococcus mutan, synthesize new PLA nanoparticles and coat the PLA particles with sugar chain PV-Man groups and then physically Investigate the chemical properties, analyze the affinity and interaction of the nanoparticles and macrophages, investigate the stability and drug release of nanoparticles, and the activity of lymphocytes, macrophages and cytokine production of nanoparticle-immunomodulators This was achieved by investigating and examining the production capacity of immunoglobulin (IgA) and IgA producing B lymphocytes in vivo of micelle-antigen-immunomodulators and investigating immunity and side effects in dental caries models.

Hereinafter, the specific configuration of the present invention will be described in detail.

The present invention comprises the steps of producing and purifying from Streptococcus mutan GTF, one of the major factors of dental caries; Synthesizing new PLA nanoparticles, coating and preparing sugar chain PV-Man groups, and then examining the physicochemical properties; Analyzing the affinity and interaction between the nanoparticles and macrophages and investigating the stability and drug release of the nanoparticles; Testing the lymphocytes, macrophage activity and cytokine production capacity of the nanoparticle-immunomodulators; Examining IgA producing ability and IgA producing B lymphocytes in vivo of the nanoparticle-antigen-immune modulator; This study consists of developing new parenteral anti-dental vaccines by analyzing immunity effects and side effects in dental caries.

The present invention is not limited to specific infectious diseases and may be applied to the development of vaccines for all other infectious diseases.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1 Purification of Immunogen Glucosyltransferase (GTF)

In order to develop a parenteral vaccine for preventing dental caries of the present invention, the method described by Smith et al . From the Streptococcus mutans SJ32 (Smith DJ et al, Infect. Immun. 64: 3069-3073, 1996). Streptococus sobrinus B13 strains were cultured on glucose-containing BHI broth for 2 days and centrifuged to remove cells and supernatant. The supernatant was chromatographed using Sephadex G-100 (from Pharmacia) to purify GTF, and the GTF-rich solution was diluted in 6M guanidine (FPLC) using Superose 6 (from Pharmacia). After the analysis, the activity was analyzed and SDS-PAGE was performed.

As shown in FIG. 1, glucosyltransferase isolated from Streptococcus sobrinus was identified as a single band.

Example 2 Synthesis of Nanoparticles Specific to Macrophages of the Invention

reagent

Complete Freund's adjuvant, ovalbumin (OVA), 3-amino-9-ethylcarbazole, 2-mercaptoethanol, retinoic acid (RA), pilocarpine, sodium pyrophosphate, 3- [4 , 5-dimethylthiazol-2-yl] -2,5 diphenyl tetrazolium bromide (MTT), anti-IgM (μchain) antibody labeled with peroxides, anti-IgG (γ labeled with peroxidase Chain) antibody, anti-IgA (α chain) antibody labeled with peroxidase, avidin-peroxidase, o-phenyldiamine dihydrochloride (OPD), diaminobenzidine (DAB), PKH26 / PKH67 kit, LPS ( E. coli O111 ; B4) and carbacol are used by Sigma, and mouse monoantigens against rat α chains and goat's anti-mouse IgG-alkaline phosphatase are manufactured by Tago. Fatal Calf Serum (FCS), L-Glutamine, RNase Inhibitor (RNasin), Random Primer, Reverse Transcriptase, dNTP, Dithiothreitol TT) and RPMI 1640 medium were manufactured by Gibco, Grand Island, NY, and Poly (L-lactic acid, PLA, Mn = 10,000) was used by Wako pure Chemicals Inc. Poly [Np-vinylbenzyl-oD-mannopyranosyl (1-4) -D-gluconaamide] (PV-Man, Mn = 40,000) was purchased from Seikagaku Co., Ltd. Chitosan (high molecular weight) And tripolyphosphate (TPP, pentasodium salt) and Tween ^® 80 were purchased from Sigma-Aldrich Korea.

Experimental Example 1 Synthesis of Nanoparticles Specific to Macrophages

In order to synthesize PLA nanoparticles specifically binding to macrophages, the monomer L-lactic acid was polymerized using triethylamine as an initiator. At this time, poly [Np-vinylbenzyl-oD-mannopyranosyl (1-4) -D-gluconamide] (PV-Man) including mannose in sugar chains recognizing predetermined PLA and macrophages was dissolved in DMSO. This was dialyzed to prepare nanoparticles. After dialysis uncoated PV-Man was removed by centrifugation and lyophilized. The size distribution of the synthesized microparticles was measured using dynamic light scattering (DLS). The shape of the fine particles was observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). FITC was attached to the nanoparticles, and splenocytes were used to analyze selective binding with macrophages using FACS.

As shown in Figure 2, as a result of confirming the size of the PLA nanoparticles coated with PV-Man of the present invention by TEM, it has a size of 250-500nm. Figure 2b shows the results of the size distribution of the nanoparticles by performing photon correlative spectroscopy.

In addition, as shown in FIG. 3, the cell affinity of the FITC-PLA-PV-Man labeled FITC showed a specific affinity for macrophages.

Experimental Example 2 Preparation of Chitosan Fine Particles

A chitosan solution (0.25%) was prepared using a mixture of 2% (v / v) acetic acid and 1% (v / v) Tween ^® -80. Then, 20 ml of 15% (w / v) TPP was added dropwise to 100 ml of the chitosan solution, mixed for 5 minutes, and continuously sonicated (5W, Vibra cellTM, Sonics & Materials, Inc., USA). . The chinosan particulate suspension was centrifuged at 3,000 rpm for 30 minutes, and then the pellet was suspended in distilled water to wash the particulates, and the washed fine particles were lyophilized.

Experimental Example 3 Loading of Ovalbumin

Ovalbumin loading of chitosan microspheres was incubated for 24 hours by adding 1.5% (w / v) chitosan microspheres and 0.5% (w / v) of ovalbumin in phosphate buffer (PBS, pH 7.4) and shaking at 25 ° C. Was done. After 24 hours, the suspension was centrifuged at 3,000 rpm for 30 minutes to remove unloaded albumin. The degree of loading was determined by quantifying unbound ovalbumin in the supernatant using BCA protein analysis (Pierce), and the average loading rate of the ovalbumin was 55-60%.

Experimental Example 4 Loading of Retinoic Acid and In Vitro Glass Test

In order to determine the degree of release of the supported drug of the macrophage specific nanoparticles of the present invention, after dialysis, the dose of the retinoic acid supported on the PLA core-shell type nanoparticles was 10.8 wt .-% And 15.7 wt .-%.

As shown in Figure 4, the release of the drug supported on the macrophage specific nanoparticles of the present invention was slowed down at a higher concentration, that is 15.7%. In addition, the initial release of the supported drug appeared before the first day and then released slowly at over 8 days.

Example 3 Effects on the Cytotoxicity and Cytokine Production of PV-Man-RA Nanoparticles of the Present Invention

In order to measure the cytotoxicity of macrophage specific nanoparticles of the present invention and the activity of immune cells in vitro, lymphocytes, macrophages and fibroblasts (L929) of the mouse spleen were used. At this time, 10% FCS, L-glutamine, 2-mercaptoethanol (2-ME), and RPMI 1640 and DMEM medium to which antibiotics were added were used. In order to stimulate these splenocytes in culture, a certain concentration of anti-CD3 antibody and PHA / Con-A were used as a promoter for division.

Experimental Example 1 Effects of PV-Man-RA Complex on Cytotoxicity and Cell Division Cycle

In order to investigate the cytotoxicity of the synthetic complex carrying the retinoic acid of the present invention, the mouse fibroblast line L929 and mouse spleen lymphocytes were examined by MTT method.

As shown in Figure 5a, the PV-Man-RA complex of the present invention showed little toxicity in L929 cells and lymphocytes until 5 ug / ml in the culture medium.

The cell division cycle of the synthetic complex of the present invention was investigated using the PKH26 staining method. Cells cultured over time were analyzed using FACScan software from a flow cytometer (Becton-Dickinson, Oxnard, Calif.). In order to measure the cell division cycle, the cell cycle analysis software (Verity S / W House Inc, Maine, U.S.A) was used for final analysis and the proliferation index (PI) was calculated using the following formula.

(Ak, Gaussian region of each generation; k, number of generations)

The results are shown in Figure 5b.

Experimental Example 2: Effect on the cytokine production capacity of the PV-Man-RA complex of the present invention

In order to investigate the effects of the PV-Man-RA complex of the present invention on the production capacity of IL-2, IL-4 and IFN-γ, the experimental group and the control group administered the macrophage specific nanoparticles carrying the retinoic acid of the present invention Spleens were removed from the mice and cell solutions were prepared. The cell solution was incubated for 24 hours by administering a constant concentration of anti-CD3 or PHA, and then the supernatant of the culture solution was collected and centrifuged. Each lymphokine was measured by the method described below.

Assays for IL-2 and IL-4

The amount of IL-2 and IL-4 in the supernatant of T-cell culture media stimulated with anti-CD3 monoclonal antibody was observed by activating IL-2 and IL-4 by observing the growth-promoting capacity of CTLL-2 cells. Was examined. Anti-IL-2 antibody (S4B6) and anti-IL-2 receptor antibody (7D4) were used, and anti-IL-4 antibody (11B11) was used to determine the titer of IL-2. Finally it was read using an ELISA reader (Molecular Device Co.) at 570nm wavelength using 3- [4,5-Dimethylthiazole-2-yl] -2,5 diphenyl tetrazolium bromide (MTT).

Test method of IFN-γ

In order to quantify IFN-γ present in the culture supernatant, a small modification of the cytopathic effect reduction assay by encephalomyelo-carditis (EMC) virus was used. The final titer of IFN-γ was at the highest dilution rate at which L929 cells lived more than 50%.

Intracellular Cytokine Staining

Cytokines produced by activating mouse splenocytes with anti-CD3 and adding protein transfer inhibitor minensin to 2M were accumulated in the cells and dissolved in fixed buffer (4% paraformaldehyde dissolved in PBS) and permeation buffer (PBS). Dissolved in 0.1% saponin, 0.1% sodium azide, 1% FBS). Anti-IFN-γ (XMG1.2), anti-IL-4 (11B11) was added and analyzed by flow cytometer.

As shown in Figure 6, when stimulated with anti-CD3 antibody while culturing splenocytes extracted from mice pretreated with retinoic acid (2.5 mg pellet injected 3 days before) from the splenocytes activated in the retinoic acid treated group The amount of IL-2 produced in vitro was decreased compared to the control, but the production of IL-4 was increased, and the amount of IFN-γ produced from splenocytes of the retinoic acid treated group was significantly suppressed compared to the control.

Example 4 Immunological Effects Using a Mouse Experimental Model of Micelle-Antigen-Immune Modulator of the Present Invention

In order to investigate the in vivo immune effects of micelle-antigen-immunomodulators of the present invention, immunoglobulin production was examined from mouse experimental models.

To this end, GTF and OVA, etc. were used as an immunogen and as an adjuvant, alum (aluminum hydroxide) was prepared and used. Each antigen was mixed with alum or with alum and retinoic acid or the micelle-antigen-retinoic acid was brought to a final concentration of 60 μg, 5 μg and 10 μg of Alum, OVA / GTF and RA in a volume of 0.25 ml. . Mice were divided into 5 groups according to the composition of the antigen and the route of administration, and 5-6 mice in each group. That is, the first group is an oral administration of the antigen and alum using a vinyl tube, the second group is the antigen and alum, and the third group is the subcutaneous administration of the antigen, alum and retinoic acid to the mouse abdomen. In the fourth group, antigen, alum, micelles, and retinoic acid were subcutaneously administered to the mouse abdomen, and the fifth group was classified as a control group without antigen. Seven days after the final administration, spleen and blood were collected from the mice and used for the experiment. As the experimental animals, normal BALB / c female mice 8 to 10 weeks of age were used. During the experiment, all mice were placed in an animal breeding facility maintained at constant temperature and humidity and aseptic condition by putting 6 or less per cage of 18cm x 28cm in size. Breeding, tap water and commercial animal feed were supplied.

Experimental Example 1: Coagulation activity of antibody in mice immunized with GTF

Agglutination titer was measured by reacting Intlett strain with LM-7 and OMZ-175 while injecting Streptococcus mutan cells together with Complete Freund's adjuvant (CFA).

As shown in FIG. 7, it was hardly detected in the oral administration group, but showed a high aggregation titer of 1: 2560 in the subcutaneous administration group, and when the retinoic acid was added thereto, the aggregation activity was rather decreased. In addition, antibodies produced by immunization with Streptococcus mutans (Ingritt strain) showed strong cross-reaction with LM-7 but rarely cross-reaction with OMZ-175. As a result of the absorption reaction using GTF and Ag I / II, the aggregation reaction was observed again, and after the absorption reaction by Ag I / II, the aggregation titer was considerably decreased, but after the absorption reaction by GTF, It didn't change at all.

Experimental Example 2: Effect of cytokines on mRNA expression of immunomodulators

To investigate the effects of the immunomodulator, retinoic acid, on mRNA expression of IL-10, IL-12 and TGFβ1, mouse peritoneal macrophages with 3 × 10 ⁶ cells / well in a 12-well culture plate were 0.5 μg / ml RA (10 ^-7 M) or End (10 ^-12 M) was also administered while stimulating with LPS, and total RNA was extracted after a certain time to perform reverse transcription polymerase chain reaction (RT-PCR).

Polymerization with reverse transcription kit (promega) to analyze mRNA expression of various cytokines in immune cells activated by antigen or schizophrase (anti-CD3, PHA, Con-A) in the presence of immunomodulators Reverse transcription polymerization reaction was carried out using a chain reaction kit (premix). That is, 1 μg of total RNA extracted by the rapid guanidium isothiocyanate method was subjected to reverse transcription reaction solution (0.1 μg; oligo (dT) 15, 1 × reverse transcription buffer, 5 mM MgCl ₂ , 1 mM dNTP mixture, 10 unit; rRNase ribonuclease inhibitor, 10 units; AMV reverse transcriptase), and cDNA was synthesized by reacting for 30 minutes at 42 ℃. 2.5 μl of the cDNA synthesized above was mixed with a polymer chain reaction solution (1 unit; heat stable DNA polymerase, 0.25 mM dNTP, 50 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl ₂ ), and a ThermoCycler (Molecular). Biology Technique Center, Thermojet, THE-NEW.V 1.0) was used for 30 minutes at 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 42 seconds to amplify the cDNA, followed by 1.2% agarose gel. Electrophoresis. Table 1 shows the primers of the cytokines used.

Primers of Cytokines Used in Reverse Transcription Polymerization Chain Reaction Cytokines and Primers Oligonucleotides (5 ---> 3) PCR product (bp) β-actin Antisense TGG AAT CCT GTG GCA TCC ATG AAA CTAA AAC GCA GCT CAG TAA CAG TCC G 348 IL-4 Antisense CAG TTT GTA TCT CTG AGA ACC TCTCTC ACT ACC TGT GAT GAG TTT T 515 TGF-β Antisense CTG GTG ACA ACC ACG GCC TTC CCT AATG CTT AGG CAT AAC GCA CTA GGT T 600 IFN-γ Antisense TGA ACG CTA CAC ACT GCA TCT TGGCGA CTC CTT TTC CGC TTC CTG AG 460

As shown in FIG. 8A, as a result of investigating the effects of the immunomodulator retinoic acid on the mRNA expression of IL-10, IL-12 and TGFβ1, HPRT is known to be always expressed in a constant amount without being affected by external stimuli. It can be seen that it is constantly expressed. IL-12 p40 was expressed at 2 hours after LPS stimulation, peaked at 4-8 hours, did not return to pre-stimulation state until 24 hours, and little p40 mRNA was expressed without LPS stimulation (not shown). After 8 hours of culture, RA decreased IL-12 mRNA expression, and End increased IL-12 mRNA expression for IL-12 p40 mRNA expression of peritoneal macrophages.

On the other hand, as shown in Figure 8b, after treatment of RA after 1 or 2 days after LPS stimulation, TGFβ1 expression did not show a difference compared to the control group reacted only LPS.

As shown in FIG. 8C, IL-12 was not expressed in splenocytes measured 2 hours after LPS stimulation, whereas IL-10 was LPS + RA treated group compared to PBS (lane 4) and LPS (lane 5) treated groups. Increased expression was seen at (lane 6).

Experimental Example 3: Effect of immunomodulators on IgA production and IgA production B cell number in splenocytes

To examine IgA production and IgA production B cell numbers in splenocytes after each or combination of immunomodulators, mouse spleen macrophages were cultured in the presence of 5 μg / ml LPS, retinoic acid (10 ⁻⁷ M), VD3 (10 ^-9 M) and DHEA were administered together and incubated for 5 days, and then the amount of IgA in the supernatant was measured and compared by ELISA.

As shown in FIG. 9, as a result of treatment with immunomodulators, IgA production of splenocytes was significantly increased by the addition of retinoic acid. Especially, DHEA had no effect on IgA production but added VD3 alone and VD3 and DHEA. The group added together significantly increased (P <0.05) compared to the group administered with LPS alone.

Experimental Example 4 Effect of Retinoic Acid on GTF-Specific Antibody Production in GTF Immunized Mice

In order to investigate the effect of retinoic acid, an immunomodulator, on the production of GTF-specific antibodies in GTF immunized mice, serum anti-GTF antibodies produced by administration of GTF with Alum were examined by ELISA. To test the antibodies, antigen-specific antibodies IgG, IgM, IgA and all secreted IgA (sIgA) were detected in immune serum and splenocyte cultures and saliva. In other words, the antigen and anti-mouse IgA were dissolved in carbonate buffer at a concentration of 5 μl / ml, respectively, and 50 μl were aliquoted into 96-well culture plates. Reaction was performed at 1 week. Subsequently, the antibody attached to the antigen was reacted with peroxidase-attached anti mouse IgA (1: 1000 diluent), followed by reaction with an enzyme pigment substrate orthophenylenediamine (OPD), and the reaction was stopped with 5N sulfate solution. The finally produced pigment was read using an ELISA reader (Molecular Devices Inc.) at 490 nm wavelength.

As shown in Figure 10, IgM of the anti-GTF antibody was detected in a significant amount in the subcutaneous group and the production was increased by the addition of RA, the oral group showed a slight increase compared to the control group, but was administered subcutaneously Was less than the military. GTF-specific IgG was not detected at all in the orally administered group, and was significantly increased only in the subcutaneously administered group. Anti-GTF IgA was not detected at all in the subcutaneous group. However, RA production increased the antibody production and the degree of increase exceeded that of the orally administered group.

Example 5 Investigation of Immune Effect Using Mouse Caries Experimental Model of PV-Man / RA Antigen Complex of the Present Invention

An SPF Sprague-Dawley white paper that was not infected from pathogens was used in this example. Mice used in this experiment were administered a high concentration of sucrose diet 2000 at 20 days of age, each experimental group was configured as follows. 1) non-immunized and uninfected, 2) orally administered GTF + Alum, 3) subcutaneously administered GTF + Alum, 4) subcutaneously administered GTF + Alum + RA, 5) GTF + Alum + Subcutaneous administration of PLA-Pv-Man-RA. Thirteen days after the second injection, blood was collected from the posterior eye and blood vessel of the white paper, and saliva was obtained after injection of pilocarpine (1.0 mg / 100 g body weight). All experiments were terminated 71 days after infection with S. mutans , and the composition of oral Streptococcus mutan was observed to determine the recovery of bacteria. The bacteria obtained by washing the whole tooth and sonication were properly diluted and inoculated into MS medium and MS medium containing 1 mg of bacitracin, and Streptococcus mutans were distinguished by their colony shape and expressed as a percentage of the total Streptococcus. . Tooth decay analysis was carried out under the microscope of the depth and extent of caries according to the modified Keyes method.

Experimental Example 1: Effect of PV-Man / RA Antigen Complex on IgA-producing B Cell Number in Spleen Cells

The effect of PV-Man / RA / antigen complex administration on the number of IgA-producing B cells in splenocytes was investigated by administering free retinoic acid with ALUM or PV-Man-RA during challenge with mice. To determine the number of splenocytes secreting IgA, affinity-purified goat anti-mouse isotype specific antibody (150 ng / ml) and antigen (50 ug / ml) After coating on a polystyrene 96-well plate (Millipoa Co., Ltd.) attached to it was attached overnight at 4 ℃. Each well of the plate was then blocked by reacting with 200 μl of 1% BSA-PBS-0.05% Tween (PBST) at 37 ° C. for 1 hour. After the appropriate number of splenocytes were put in each well and incubated for 2.5 hours at 37 ℃, CO ₂ incubator. After washing the plate again with PBST, 150 μl of biotin-labeled goat's anti-mouse isotype, in particular antibodies, were diluted in PBST containing 1% BSA, reacted at room temperature for 2 hours, then washed with PBST and 150 Μl of avidin-peroxidase was reacted for 2 hours at room temperature. Spots were developed with 3-amino-9-ethylcarbazole to determine the number and size on a stereomicroscope.

As shown in FIG. 11, as a result of investigating the effect of the PV-Man / RA / antigen complex of the present invention on the number of IgA-producing B cells in splenocytes, the PV-Man / RA / antigen complex was examined subcutaneously. When administered, the number of IgA-producing B cells in splenocytes was significantly increased compared to the group treated with alum and antigens orally and the group treated with alum + antigen + free retinoic acid subcutaneously.

Experimental Example 2 Effect of Saliva Anti-GTF Antibody on Saliva of PV-Man / RA Antigen Complex of the Present Invention

To investigate the effect of PV-Man / RA / antigen complex administration on anti-GTF antibody changes in saliva by administration of free retinoic acid along with alum or PV-Man-RA during challenge.

As shown in FIG. 12, when the PV-Man / RA / antigen complex was administered subcutaneously, the anti-GTF IgA together with the group administered with oral and alum + antigen + free retinoic acid subcutaneously All increased, but significantly increased in the subcutaneous group of PV-Man / RA / antigen complex.

As described above, the present invention, as described through Examples and Experimental Examples, the parenteral vaccine for immunologically preventing dental caries of the present invention is a disadvantage of the conventional oral vaccine, that is, a large amount is required and digested after oral administration It can be destroyed by enzymes and has an excellent effect on resolving disadvantages such as causing immunological tolerance. In addition, the present invention is an immunomodulatory agent for macrophage specific nanoparticles (mannose-coated polymeric micelle) having a uniform particle size, large surface area, and easy dispersion or separation in water. Supports retinoic acid to selectively deliver to target organs or cells to reduce side effects of drugs, to induce high-efficiency antigen-specific immunoglobulin (IgA) production, and to increase antigen-specific IgA-producing lymphocytes in the blood Because of its effectiveness, the present invention is a very useful invention in the pharmaceutical industry because it can be prepared with a mucosal anti-teeth immune vaccine of a novel parenteral mechanism.

Figure 1 shows the results of FPLC chromatography and SDS-PAGE of glucosyltransferase (GTF), a dental caries antigen isolated from Streptococcus mutan.

Figure 2 shows the results of the size and stability of the macrophages specific nanoparticles of the present invention by TEM and photon correlative spectroscopy. a) shows the size of macrophages specific nanoparticles, b) shows the size distribution of nanoparticles.

Figure 3 shows the affinity of macrophages specific nanoparticles of the present invention to macrophages.

Figure 4 shows the degree of in vitro free macrophages specific nanoparticles of the present invention.

Figure 5 shows the effect on the cytotoxicity of the macrophage specific nanoparticles of the present invention.

Figure 6 shows the effect on the cytokine of the macrophages, in particular the nanoparticles of the present invention, a complex containing a retinoic acid as an immunomodulator. a) shows the production capacity of IL-2 and IL-4, b) shows the production capacity of IFN-γ.

Figure 7 shows the aggregation titers of antibodies in mice immunized with glucosyltransferase (GTF), a dental caries antigen isolated from Streptococcus mutan.

Figure 8 shows the effect on the cytokine mRNA expression of the macrophage specific nanoparticles of the present invention. a) mRNA of IL-2, b) mRNA of TGFβ1, c) effect on mRNA expression of IL-10.

Figure 9 shows the effect of ELISA on the IgA and IgA producing B cell number of splenocytes of immunomodulators. a) shows the effect of immunomodulators on immunoglobulin and immunoglobulin-producing B cell number, and b) shows the effect on immunoglobulin production after administration of LPS, a cleavage promoter.

10 shows the effect of retinoic acid on GTF specific antibodies of mice immunized with GTF, a dental caries antigen.

Figure 11 shows the effect on the number of IgA producing B cells in mouse splenocytes of the macrophage specific nanoparticles of the present invention.

Figure 12 shows the effect of the macrophage-specific nanoparticles of the present invention on the production of GTF specific antibodies in the saliva of mice.

Claims (3)

PV-Man is obtained by dissolving poly L-lactic acid (PLA) and poly [Np-vinylbenzyl-oD-mannopyranosyl (1-4) -D-gluconamide] (PV-Man) in DMSO followed by dialysis. PLA nanoparticles; Retinoic acid; And glucosyltransferase (glucosyltransferase, GTF) complex comprising a parenteral vaccine for preventing dental caries, characterized in that it comprises a complex. delete delete