KR20190050752A - Adjuvant and vaccine composition comprising STING agonist - Google Patents

Abstract

The present invention relates to an immunological antigen adjuvant composition and a vaccine composition containing a stimulator of interferon genes (STING) agonist. According to the present invention, the immunological antigen adjuvant composition and the vaccine composition containing the STING agent can effectively activate various in-vivo immune responses and also remarkably reduce infection by Mycobacterium tuberculosis. Therefore, when using the same along with vaccine of other pathogens other than Mycobacterium tuberculosis, it is possible to effectively reduce infection by various pathogens by remarkably increasing an effect of existing vaccines for preventing infections.

Description

[0001] The present invention relates to an adjuvant and a vaccine composition comprising a STING agonist,

The present invention relates to immunoadaginant compositions and vaccine compositions comprising STING agents.

Various strategies have been used in vaccine development. Vaccines made from live or attenuated bacteria are very effective, but stability problems are often reported due to problems with regression, and sometimes self-replication (Neutra MR et al. , 2006). Although vaccines using dead cells are effective for treatment, there is a possibility that toxic substances such as LPS may be contained and living bacteria may be present (Ryan EJ et al ., 2001). In order to overcome such limitations The developed DNA vaccine is also inserted into the genome of the host to increase mutagenicity and the possibility of oncogenic (Schalk JA et al ., 2006). Subunit vaccines, on the other hand, are more stable than other vaccines because they use purified antigens. However, because these purified antigens are often poorly immunogenic, strong immunogen antagonist (adjuvant) is required to enhance this immunogenicity.

Meanwhile, c-di-GMP (cyclic diguanylate) is Acetobacter xylinum (Ross P et al ., 1987). Such signaling through c-di-GMP is not present in eukaryotes and has been reported to be characteristic only in bacteria (Galperin MY et al ., 2004). C-di-GMP in bacteria has also been reported to play a role as a second messenger of signaling (Romling U et al ., 2006; Schirmer T et al ., 2009), particularly in the bacterial survival of bacteria It is known to regulate physiological mechanisms such as motility, adhesion, intercellular communication, biofilm formation, exopolysaccharide synthesis (Tischler AD et al ., 2004; Romlingi et al ., 2005; Wangxue Chen et al ., 2010). Importantly, this bacterial-derived c-di-GMP acts as a ligand of the host's cytosolic sensor, Stimulator of IFN gene (STING), to produce Type I IFN through TBK1-IRF3 and NF- (Burundi et al ., 2010; Burdette et al ., 2011; McWhirter et al ., 2009), recently reported that a STING agonist such as c-di-GMP has been shown to induce cytokine production Has been shown to enhance antigen-specific T cells and humoral immune responses (Burcu Temizoz et al ., 2015; Li et al ., 2013).

Therefore, the present inventors conducted this experiment to confirm that the STING agent can be used as a vaccine immunoadjuvant.

DISCLOSURE Technical Problem The present invention has been conceived to solve the above problems of the prior art, and it is an object of the present invention to provide a vaccine composition comprising an immunogenicity enhancer composition and an antigen in the composition, do.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to " one embodiment " or " embodiment " means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment " or " an embodiment " in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention provides an adjuvant composition comprising an immunogen antagonist and an STING activator (Stimulator of interferon genes agonist) as an active ingredient, and a vaccine composition containing an antigen as an active ingredient .

In one embodiment of the present invention, the STING operator is preferably a DNA, an RNA, a protein, a peptide fragment, or a compound capable of activating STING signaling, more preferably a c-di CGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'-cGAMP, 10- (carboxymethyl) 9 (10H) acridone (10- (carboxymethyl) 9 (10H) acridone (CMA)), 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone , 6 ', 4'-dimethoxyflavone, 4'-methoxyflavone, 3', 6'-dihydroxyflavone, , 7, 2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, and xanthone xanthone), and the like, or substances capable of binding to STING to activate signal transduction .

In another embodiment of the present invention, the immunoassay enhancer composition may further include other known immunoassay enhancers. Monophosphoryl lipid A (MPL), preferably monophosphoryl lipid A ) And GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil-in-water).

In another embodiment of the present invention, the immunoconjugants may be encapsulated in liposomes, but are not limited thereto, as long as they are easy to inject into the body.

In another embodiment of the present invention, the antigen is a pathogen-specific antigen, preferably an M. tuberculosis-specific antigen, more preferably an ESAT-6 antigen or an antigen used in a subunit vaccine Do not.

In another embodiment of the present invention, the vaccine composition is characterized by preventing infection of Mycobacterium tuberculosis.

The composition of the present invention may contain at least one known active ingredient having a pathogenic bactericidal effect.

In addition, the composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, sucrose solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, intramuscularly or topically) depending on the intended method, and the dose may vary depending on the weight, Sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease.

Since the immunoadjuvant composition and the vaccine composition containing the STING agent according to the present invention not only effectively activate various immune responses in the body but also have an effect of significantly reducing the infection of Mycobacterium tuberculosis, , It is anticipated that it will significantly reduce the infection effect of various pathogens by significantly increasing the existing vaccine for infection prevention.

Figure 1A shows an experimental design to validate the efficacy of c-di-GMP as an adjuvant for immunity. Figure IB shows the effect of ESAT-6 on MPL or c-di-GMP immunized spleen cells Figure 6 shows the results of confirming the production ability of IFN-y after ESAT-6 protein stimulation. FIG. 1C is a graph showing the results of confirming ESAT-6 antigen-specific antibody production in immunized mice, FIG. 1D is a graph showing the results of analysis of memory T cells infiltrated into spleen and lung tissue of immunized mice to be.
FIG. 2A shows a method for analyzing multifunctional T cells, and FIG. 2B is a graph showing the results of ESAT-6 protein stimulation in lung and splenocytes of mice immunized with MPL or c-di-GMP based on ESAT-6 antigen Antigen-specific multifunctional T cells.
FIGS. 3A and 3B are histopathological analyzes of lung tissues after 16 weeks of infecting M. tuberculosis with mice immunized with MPL or c-di-GMP based on ESAT-6 antigen, and FIG. 3C Is a diagram showing the results of measurement of the number of tuberculosis bacteria in the lung and spleen.
FIG. 4A is a graph showing an experimental design for confirming the synergy effect of c-di-GMP and conventional MPL immuno-antigen adjuvant, and FIG. 4B is a graph showing the results of analysis of T cell activity in immunized mice. FIG. 4C is a graph showing the results of the generation of ESAT-6 antigen-specific antibodies in immunized mice by time, FIG. 4D is a graph showing the ratio of germinal center-related T cells to B cells in immunized mice Fig.
Fig. 5A is a diagram showing the results of histopathological analysis of lung tissue after infecting M. tuberculosis bacteria with mice immunized with MPL or c-di-GMP / MPL based on ESAT-6 antigen 4 weeks later, Is a graph showing the results of measurement of the number of mycobacteria growing in the lungs. FIG. 5C is a graph showing the results of analyzing antigen-specific multifunctional T cells through ESAT-6 protein stimulation by in vitro isolation of cells from the lungs and spleen 4 weeks after infection with Mycobacterium sp.
Figure 6 shows the ratio of antigen-specific multifunctional T cells induced by ESAT-6 protein stimulation in lung and spleen cells of mice immunized with c-di-GMP and / or GLA-SE based on ESAT-6 antigen FIG.
FIG. 7 shows the results of analysis of T cell activity in immunized mice. FIG.
Figure 8 shows the results of measurement of the number of Mycobacterium tuberculosis in lungs and spleens of mice immunized with GLA-SE alone or with c-di-GMP and GLA-SE based on ESAT-6 antigen.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Identification of the role of STING agonists as adjuvants

The following experiment was conducted to confirm that a STING agent (STING) agonist can be used as an adjuvant for Mycobacterium tuberculosis.

1.1. Experimental animal

6-week-old female C57BL / 6 mice (Japan SLC, Inc. Shijuoka, Japan) without specific pathogens were used. The mice were housed in the restricted space of the ABSL-3 biohazard animal room in Yonsei University Clinical Research Center Sterilized commercial mouse feed and water.

1.2. Experimental design

Animal experiments were performed to determine whether c-di-GMP (invivogen), a type of STING agonist, could be used alone as an adjuvant. A specific experimental method is shown in Fig. 1A. As a comparative control for c-di-GMP, monophosphoryl lipid A (MPL), which is a TLR4 agonist that has been used as an adjuvant for immunotherapy, was used . Each immunostimulant was formulated with Dimethyldioctadecylammonium (DDA) liposomes either alone or in combination with ESAT-6 protein.

As shown in Figure 1A, MPL / DDA, ESAT-6 + MPL / DDA or ESAT-6 + c-di-GMP / DDA were administered intramuscularly intramuscular injection), and some mice were euthanized prior to infection with a mouse of Mycobacterium tuberculosis, and ex vivo experiments were performed by isolating spleen cells and lung cells from the spleen and lungs. Each separated cell was subcultured and cultured in the usual manner. The immunization method is described in detail in Example 1.3 below. Thereafter, IFN-γ production by antigen stimulation and ESAT-6 protein immunization were observed to increase memory T cells infiltrating the spleen and lungs. In addition, we investigated the ability of multifunctional CD4 ⁺ T cells to develop in the recent defense of tuberculosis. We analyzed the number of bacteria in the spleen and lungs and the degree of inflammation in the lungs at 16 weeks after the infection.

1.3. Immunization

Each of the mice used in the experiment was immunized by intramuscular injection of the experimental vaccine composition three times at intervals of 3 weeks. The experimental vaccine composition was prepared by diluting the DDA liposome to a final concentration of 5 mg / mL, heating it to 65 ° C. and sonicating it in parallel and dissolving it, and mixing it with each immunoantigen adjuvant so that the volume became 2: 1 . The volume injected per mouse was adjusted to a final volume of 200 [mu] L, so that 1 [mu] g of ESAT-6 protein per week was included as an antigen. As a negative control, MPL / DDA compositions without antigen were injected intramuscularly.

1.4. Identification of IFN-γ production by ESAT-6 antigen

In order to examine the effect of c-di-GMP / DDL immunogen adjuvant on the immunogenicity of the immunized antigen compared with the MPL / DDA immunoadapagent used previously as an adjuvant, The antigen-specific IFN-y production ability was analyzed. For experiments, mouse spleen cells obtained in the same manner as in Example 1.2 were stimulated ex vivo with ESAT-6 protein, and antigen-specific IFN-gamma production was measured in stimulated cells by ELISA (enzyme-linked immunosorbent assay). The results are shown in Fig. 1B.

As shown in Figure IB, antigen-specific IFN- [gamma] production in both experimental groups immunized with ESAT-6 + MPL / DDA vaccine composition and ESAT-6 + c-di- GMP / DDA vaccine composition against ESAT- gamma < / RTI > The IFN-γ production was significantly increased in the experimental group immunized with ESAT-6 + c-di-GMP / DDA vaccine composition using c-di-GMP as an adjuvant.

1.5. Identification of antigen-specific antibody production

To verify the effect of the c-di-GMP / DDL immunoadjuvant on the antigen-specific antibody production ability, the serum of each mouse was isolated at the time when the immunization was completed three times in total Antibody production was confirmed. ESAT-6 protein at a concentration of 1 μg / ml was added to a 96-well plate and reacted at room temperature for 2 hours to coat each well. Mouse serum according to each experimental group was then added to the coated wells . IgGG (Sigma), anti-IgG1 (BD Bioscience), or anti-IgG2c (Southern Biotech) to which horseradish peroxidase (HRP) is bound are added to each well and reacted with ELISA The specificity of antibody production was confirmed. The results are shown in Figure 1C.

As shown in FIG. 1C, IgG1 associated with Th2 immunity and IgG2c associated with Th1 immunity, which is reported to be important for defense against tuberculosis, were confirmed to be enhanced over total immunoglobulin .

1.6. c- di - GMP As an immunogen antagonist  Identify the type and number of T cells induced when used

In general, when infected with Mycobacterium tuberculosis, mycobacterial antigen-specific T cells are cloned and differentiated to become effector T cells. These effector T cells are mostly killed when the immune response disappears, and some form long-lasting memory T cells do. These memory T cells are known to play an important role in maintaining rapid acquired defense immunity and T cell memory. Therefore, the experiment was conducted to verify whether MPL or c-di-GMP affects memory T cells when immunized with ESAT-6 protein as an adjuvant. For experiments, spleen cells and lung cells obtained in the same manner as in Example 1.2 were washed twice with PBS and centrifuged. To the centrifuged cells, anti-CD3-BV421 (BD Bioscience), anti-CD4- CD86-FITC (ebioscience), anti-CD127-APC (ebioscience), and the like, as well as the anti-CD57-PE5 (BD Bioscience), anti-CD8-APC-Cy7 The antibodies were treated and reacted at 4 ° C for 30 minutes. Completion of the reaction cells are effector T cells using flow cytometry (FACS verse, BD) after removal of the unbound antibody is washed several times with ^{PBS (Teff; CD3 + CD4 +} CD62L - CD44 + CD127 -), effector / memory T cells ^{(Tem; CD3 + CD4 + CD62L} - CD44 + CD127 +), central memory T cells ^{(Tcm; CD3 + CD4 + CD62L} + CD44 + CD127 +), and naive T cells (Naive; CD3 ⁺ CD4 ⁺ CD62L ⁺ CD44 ^- CD127 ⁺ ) were analyzed. The results are shown in Figure 1D.

As shown in FIG. 1D, when MPL was immunized with c-di-GMP as an adjuvant, the effects of CD4 ⁺ / CD8 ^{+ on} CD4 ⁺ / CD8 ⁺ memory T cells were significantly increased.

1.7. c- di - GMP Immune antigen adjuvant  T cell activation induced when used

Since multifunctional T cells (T cells that secrete IFN-y, TNF-a and / or IL-2) are reported to be essential immunologic indicators to defend against Mycobacterium tuberculosis, c-di- When used as a reinforcing agent, it was confirmed whether multifunctional T cells were produced. The lung cells and spleen cells of the mice isolated in the same manner as in Example 1.2 were stimulated with ESAT-6 protein. The cells were treated with GolgiStop (BD Bioscience) to prevent the secretion of cytokines, and the cells were incubated at 37 ° C for 12 hours and washed twice with PBS. The washed cells were treated with anti-CD4-PerCP-Cy5.5 (BD Bioscience), anti-CD8-APC-Cy7 (BD Bioscience) And unbound antibody was removed by washing with PBS. Cytofix / Cytoperm (BD Bioscience) was added to the cells, followed by reaction at 4 ° C for 30 minutes, followed by washing with PBS. To the washed cells, anti-IFN-γ-PE (BD Bioscience), anti-IL-2-PE-Cy7 (BD Bioscience) and anti-TNF-α-APC (BD Bioscience) were further treated and reacted at 4 ° C for 30 minutes. After completion of the reaction, cells were washed several times with Perm / Wash solution (BD Bioscience) and analyzed using a flow cytometer. The results are shown in FIG. 2A. The results analyzed by flow cytometry were further analyzed by gating strategy using FlowJo software, and the results are shown in FIG. 2B.

As shown in FIG. 2B, T-cells secreting all three types of IFN-y, TNF-alpha and IL-2 cytokines were increased in all cases where c-di-GMP or MPL was used as an adjuvant In particular, it was confirmed that the generation of antigen-specific multifunctional T cells in the lung cells was significantly increased in the group immunized with ESAT-6 + c-di-GMP / DDA composition than in the case of using MPL as an adjuvant. The results showed that STING agonists can be effectively used for vaccination against Mycobacterium tuberculosis as compared to conventional immunoadjuvants by increasing the production of multifunctional T cells essential for the prevention and treatment of Mycobacterium tuberculosis.

1.8. Mycobacterium tuberculosis culture and air infection

Mycobacterium tuberculosis strain HN878, a highly pathogenic Mycobacterium tuberculosis, was cultivated in 7H9-OADC broth for 15 days. The cultured bacteria were collected, and the bacteria were disrupted by gentle vortexing with 6 mm glass beads. The crushed bacteria were centrifuged to settle the cell aggregates, and the supernatant was collected. The collected supernatant was divided and stored at -70 ° C. The stored bacteria were thawed at the time of experiment and then diluted and cultured in Middlebrook 7H11 Agar (Difco, Detroit, MI, USA) in order to confirm the number of bacteria. To infect mice, And then diluted with PBS (pH 7.2) to obtain a desired number of Mycobacterium tuberculosis. Infections of tubercle bacillus were inhaled and air infected with 200-250 mycobacteria per mouse using a Glas-Col inhalation device (Terre Haute, IN).

1.9. Histopathological analysis and confirmation of the inhibitory effect of Mycobacterium tuberculosis infection

In order to confirm whether the immunization using c-di-GMP as an immunopotentiator has an inhibitory effect against Mycobacterium tuberculosis infection, mice were infected with Mycobacterium tuberculosis in the same manner as in Example 1.8, and mice infected after 16 weeks were euthanized, Lung tissue was removed from the mouse experimental group. And stored in 10% neutral-buffered formalin and fixed in paraffin. The fixed lung tissue was cut to a thickness of 4 to 5 mm and hematoxylin and eosin (H & E) staining was performed. The results are shown in Fig. 3A. The area of the affected area was quantified using the ImageJ software program (National Institute of Health, MD, USA). The result is shown in Fig. 3B. In addition, a homogenous suspension was obtained from the lungs of the euthanized mouse and the spleen attached to the spleen, and a homogeneous suspension was obtained. Each homogenate suspension was diluted stepwise and cultured in Middlebrook 7H11 Agar (Difco, Detroit, MI, USA) The number of mycobacteria was counted and the number of infected M. tuberculosis was expressed as an average log ₁₀ CFU ± standard deviation per total lung or spleen tissue. The result is shown in Fig. 3C.

As shown in FIG. 3B, inflammation was reduced in the lung tissues of the mice immunized with the immunoprecipitant agent as compared with the negative control using only the immunoantigen adjuvant. In particular, the ESAT-6 + c-di-GMP / DDA vaccine composition , The inflammation was reduced in the experimental group immunized with the ESAT-6 + MPL / DDA vaccine composition than in the experimental group immunized with the ESAT-6 + MPL / DDA vaccine composition.

In addition, as shown in FIG. 3C, the number of infected M. tuberculosis bacteria in the experimental group immunized with the ESAT-6 + c-di-GMP / DDA vaccine composition as compared to the negative control immunized with only the immunoadapagent was significantly reduced Respectively. It was confirmed that c-di-GMP alone could be used as an adjuvant to prevent infection of the highly pathogenic M. tuberculosis strain HN878.

Based on the above results, STING agents such as c-di-GMP (cyclic diguanylate) have not only an adjuvantity but also an effect as compared with MPL, which is a conventional immunoadjuvant, there was. In addition, it was confirmed that infection of various mycobacteria can be effectively prevented by using STING agent alone as an adjuvant.

Example 2: Confirmation of synergistic effect between STING agent and immunogen antagonist

In order to confirm the synergistic effect when the STING agonist is used in combination with MPL, which is a known immunoadjuvant, adjuvant, the following experiment was carried out.

2.1. Experimental animal

6-week-old female C57BL / 6 mice (Japan SLC, Inc. Shijuoka, Japan) without specific pathogens were used. The mice were housed in the restricted space of the ABSL-3 biohazard animal room in Yonsei University Clinical Research Center Sterilized commercial mouse feed and water.

2.2. Experimental design

Animal experiments were conducted to confirm whether synergy was observed when c-di-GMP (invivogen) was used in combination with a previously known immunostimulatory adjuvant. A specific experimental method is shown in Fig. 4A. The experimental group used c-di-GMP and MPL together in DDA liposomes.

As shown in FIG. 4A, mice were immunized by intramuscular injection three times a total of three times at 3-week intervals, 10 weeks before the mice were infected with Mycobacterium tuberculosis, and mice were infected with Mycobacterium tuberculosis Were euthanized and splenocytes and lung cells were separated from the spleen and lungs, and ex vivo experiments were also performed. Each separated cell was subcultured and cultured in the usual manner. The immunization method is described in detail in Example 2.3 below. Thereafter, IFN-γ production by antigen stimulation and ESAT-6 protein immunization were observed to increase memory T cells infiltrating the spleen and lungs. In addition, we investigated the ability of multifunctional CD4 ⁺ T cells to develop in the recent defense of tuberculosis. The number of bacteria in the spleen and lungs and the degree of inflammation in the lungs were analyzed at 4 weeks after the infection.

2.3. Immunization

Each of the mice used in the experiment was immunized by intramuscular injection of the experimental vaccine composition three times at intervals of 3 weeks. The experimental vaccine composition was prepared by diluting the DDA liposome to a final concentration of 5 mg / mL, heating it to 65 ° C. and sonicating it in parallel and then mixing it with 5 to a volume of 2: 1. The volume injected per mouse was adjusted to a final volume of 200 [mu] L, so that 1 [mu] g of ESAT-6 protein per week was included as an antigen. As a negative control, MPL / DDA compositions without antigen were injected intramuscularly.

2.4. CD4 ⁺ / CD8 ⁺  T cell activation

In order to confirm whether the combined immunoadjuvant affects T cell activity, the activity of T cells was confirmed in the same manner as in Example 1.11 using CD44, PD-1, CD62L, and CD127, which are active markers of T cells. The result is shown in Fig. 4B.

As shown in FIG. 4B, when c-di-GMP and MPL were used in combination, the number of CD8 ⁺ T cells was increased in the spleen compared with the case of using a single immunostimulant, and in the case of CD4 ⁺ T cells, The numbers of CD44 ⁺ PD-1 ⁺ and CD62L ^- CD127 ^- T cells were significantly increased in all lungs.

2.5. Identification of antigen-specific antibody production

In order to examine the effect of the combined immunoadjuvant on the antigen-specific antibody production ability, serum of each mouse was separated at the time of completion of the immunization three times in total, Respectively. ESAT-6 protein at a concentration of 1 μg / ml was added to a 96-well plate and reacted at room temperature for 2 hours to coat each well. Mouse serum according to each experimental group was then added to the coated wells . IgGG (Sigma), anti-IgG1 (BD Bioscience), or anti-IgG2c (Southern Biotech) in which horseradish peroxidase (HRP) is bound are added to each well, And the ability of the antigen-specific antibody produced was confirmed. The result is shown in Fig. 4C.

As shown in Fig. 4C, in both IgG1 associated with Th2 immunity and IgG2c associated with Th1 immunity, which is reported to be important for defense against tuberculosis, total IgG, c-di - GMP and MPL were used in combination with the adjuvant, and it was confirmed that the antibody production was increased and balanced immune response was induced.

2.6. Identification of embryo-related cells

The germinal center (GC) is located in the follicle of the secondary lymphoid tissue B cell, and the embryo-related B cell has the somatic hypermutation and the memory of the B cell. It is known that the cells undergo a functional maturation process which is important for controlling defensive humoral immunity such as formation of the cells. Further, as one kinds of follicular helper T cell (T _FH) is an important cell which can enhance the antibody response of B cells and expression of T _FH cells are B-cell areas of CXCR5 gene of T _FH cells of the CD4 ⁺ T cell Have been reported to play an important role in the interaction with cognate B cells. Therefore, it is important to enhance the activity of _TFH cells, which can induce immune responses in the center of the embryo, for the sustained fixation of effector with high affinity through humoral immunity. Therefore, in order to confirm whether the multiple immunoelectinic adjuvant of the present invention activates T _FH cells, spleen cells were finally isolated from the immunized mice, and the embryonic center-related cells were identified using a flow cytometer. The result is shown in Fig. 4D.

As shown in FIG. 4D, in comparison with the experimental group in which MPL alone was used as an adjuvant, CD4 ⁺ CXCR5 ⁺ PD-1 ⁺ T _FH cells and B220 ⁺ CD138 ⁺ plasma cells were significantly increased. It appears to be mediated by STING signaling by c-di-GMP. On the other hand times the center-related cells such as CD4 ⁺ CD44 ^hi CXCR5 ⁺ GL7 ⁺ T _FH cells but has slightly increased in the case of (GC T _FH cell) and CD19 ⁺ B220 ⁺ Fas ⁺ GL7 ⁺ B cells (GC B cell), a large difference is not Respectively.

2.7. Mycobacterium tuberculosis culture and air infection

Mycobacterium tuberculosis strain HN878, a highly pathogenic M. tuberculosis, was cultured in 7H9-OADC broth for 15 days. The cultured bacteria were collected, and the bacteria were disrupted by gentle vortexing with 6 mm glass beads. The crushed bacteria were centrifuged to settle the cell aggregates, and the supernatant was collected. The collected supernatant was divided and stored at -70 ° C. The stored bacteria were thawed at the time of experiment and then diluted and cultured in Middlebrook 7H11 Agar (Difco, Detroit, MI, USA) in order to confirm the number of bacteria. To infect mice, And then diluted with PBS (pH 7.2) to obtain a desired number of Mycobacterium tuberculosis. Infections of tubercle bacillus were inhaled and air infected with 200-250 mycobacteria per mouse using a Glas-Col inhalation device (Terre Haute, IN).

2.8. Histopathological analysis and confirmation of the inhibitory effect of Mycobacterium tuberculosis infection

In order to confirm whether the immunization using c-di-GMP as an immunopotentiator has an inhibitory effect against Mycobacterium tuberculosis infection, mice were infected with Mycobacterium tuberculosis in the same manner as in Example 2.7, and mice infected after 4 weeks were euthanized, Lung tissue was removed from the mouse experimental group. And stored in 10% neutral-buffered formalin and fixed in paraffin. The fixed lung tissue was cut to a thickness of 4 to 5 mm and hematoxylin and eosin (H & E) staining was performed. The results are shown in FIG. 5A. In addition, a homogenous suspension was obtained by extracting the Mycobacterium tuberculosis attached to the lung of euthanized mouse with PBS, and each homogeneous suspension was diluted stepwise and cultured in Middlebrook 7H11 Agar (Difco, Detroit, MI, USA) And the number of infected M. tuberculosis was expressed as mean log ₁₀ CFU ± standard deviation per total lung or spleen tissue. The result is shown in Fig. 5B.

As shown in FIG. 5A, it was confirmed that inflammation was reduced in the lung tissue of the mice immunized with the combined immunoadjuvant, as compared with the experimental group using only the MPL immunoreactive adjuvant.

In addition, as shown in FIG. 5B, the number of infected Mycobacterium tuberculosis was significantly reduced in the experimental group immunized with the combined immunoadjuvant, compared to the experimental group using only the MPL immunostimulant, and c-di-GMP MPL as an adjuvant to the immune system can effectively prevent the infection of the HN878 strain of the highly pathogenic M. tuberculosis.

2.9. Identification of T cell activity induced by combined immunoadjuvant

Multifunctional T cells (T cells that secrete IFN-γ, TNF-α and / or IL-2) are reported to be important immunological indicators for defense against Mycobacterium tuberculosis. Therefore, multifunctional T cells Was generated. The immunized mice were infected with Mycobacterium tuberculosis. After 4 weeks, lung cells and spleen cells of the mice isolated in the same manner as in Example 2.2 were stimulated with ESAT-6 protein. The cells were treated with GolgiStop (BD Bioscience) to prevent the secretion of cytokines, and the cells were incubated at 37 ° C for 12 hours and washed twice with PBS. The washed cells were treated with anti-CD4-PerCP-Cy5.5 (BD Bioscience), anti-CD8-APC-Cy7 (BD Bioscience) And unbound antibody was removed by washing with PBS. Cytofix / Cytoperm (BD Bioscience) was added to the cells, followed by reaction at 4 ° C for 30 minutes, followed by washing with PBS. To the washed cells, anti-IFN-γ-PE (BD Bioscience), anti-IL-2-PE-Cy7 (BD Bioscience) and anti-TNF-α-APC (BD Bioscience) were further treated and reacted at 4 ° C for 30 minutes. After completion of the reaction, cells were washed several times with Perm / Wash solution (BD Bioscience) and analyzed by flow cytometry. The results were further analyzed by gating strategy using FlowJo software. The results are shown in FIG. 5C .

As shown in FIG. 5C, when c-di-GMP and MPL were used as a combination of immunoadapine, CD4 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ , CD4 ⁺ IFN -γ ⁺ TNF-α ⁺ and CD4 ⁺ IFN-γ ⁺ IL-2 ⁺ were significantly increased.

These results indicate that the effect of the combination of conventional immune antigen adjuvant such as MPL together with the STING agent such as c-di-GMP was remarkably increased. In addition, through the use of STING agents, it was confirmed that infection of various Mycobacterium tuberculosis can be effectively prevented by using the STING agent in the existing adjuvant.

Example 3: Confirmation of synergy between STING agent and existing immunoadjuvant

(STING) agonist and a previously known adjuvant GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil-in-water) In order to confirm the synergy effect, the following experiment was carried out.

3.1. STING Operator  And GLA Identification of T-cell activity induced by -SE

To determine whether the combined immunoadjuvant affects the activity of T cells, the spleen and lung were isolated before the mice immunized with a single immunoprecipitator or multiple immunoantigen adjuvant were infected with M. tuberculosis. The production of antigen and T cells induced after treatment of ESAT-6 peptide pool or protein in vitro (ex vivo) was confirmed by flow cytometry. Detailed experimental procedures were carried out in the same manner as described in Example 1.7. The results are shown in Fig.

As shown in FIG. 6, the CD4 ⁺ IFN-.gamma. ⁺ Immunoreactivity was significantly increased in the immunized group with c-di-GMP and GLA-SE conjugated immunotherapy adjuvant, compared with the group immunized with GLA- It was confirmed that the production of TNF-α ⁺ multifunctional T cells was significantly increased.

(3) BCG (bacille de Calmette-Guerin vaccine) alone treatment group, (4) BCG prime, ESAT-6, and GLA-SE (6) ESAT-6 and GLA-SE treatment groups, (7) ESAT-6, GATA-6 and GATA treatment groups, STING agonist and GLA-SE treated group, the mice were euthanized at the time when the immunization was completed three times in total, and then the mice were euthanized in the same manner as in Examples 1.5, 1.7 and 1.11 T cell activity. Immunization was performed by intramuscular injection. The results are shown in Fig.

As shown in FIG. 7, when a combination of c-di-GMP and GLA-SE was used in combination with a cytokine such as IFN-y, TNF-a and IL-2, Secretion was increased.

From these results, it was confirmed that infection of various Mycobacterium tuberculosis can be effectively prevented by using GLA-SE and STING agents as adjuvant.

3.2. STING agonist and GLA-SE inhibited the inhibition of M. tuberculosis infection

(1) BCG (bacille de Calmette-Guerin vaccine) alone, (2) ESAT-6 and GLA (2) were administered 10 times before the mice were infected with Mycobacterium tuberculosis (3) The vaccine composition of ESAT-6, STING agonist and GLA-SE was immunized by intramuscular injection injection three times at intervals of 3 weeks, and mice were immunized by the same method as in Example 2.7 After 4 weeks of infection with the Mycobacterium tuberculosis infection, the infected mice were euthanized. Mycobacteria attached to the lungs of each mouse were extracted with PBS to obtain a homogenous suspension, each of the homogenous suspensions was diluted stepwise, and then diluted with Middlebrook 7H11 Agar , Detroit, MI, USA), and the number of infected M. tuberculosis was expressed as mean log ₁₀ CFU ± standard deviation per total lung or spleen tissue. The results are shown in Fig.

As shown in FIG. 8, when c-di-GMP and GLA-SE were simultaneously used as adjuvant, the number of infected Mycobacterium tuberculosis was significantly decreased as compared with the group using GLA-SE alone as an adjuvant Respectively. It was confirmed that the combination of c-di-GMP and GLA-SE as an adjuvant can effectively prevent infection with the highly pathogenic M. tuberculosis strain HN878.

From the above results, it was confirmed that the effect of using a conventional immune antigen adjuvant such as MPL and GLA-SE together with a STING agent such as c-di-GMP was remarkably increased. In particular, recently, it has been shown that the generation of multifunctional T cells of CD4 ⁺ IFN-γ ⁺ TNF-α ⁺ is essential for the prophylaxis and treatment of Mycobacterium tuberculosis. The STING agent of the present invention is more effective than the conventional immunoadjuvant alone It was confirmed that the production of multifunctional T cells in the spleen and / or lung was significantly increased. These results suggest that the use of STING agents in addition to existing immunoadhesin agents can significantly increase the effectiveness of conventional immunoadapine adjuvants that are less effective in preventing and treating M. tuberculosis. It was confirmed that the combined immunoadjuvant comprising STING agent can effectively prevent infection of various Mycobacterium tuberculosis.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (13)

An adjuvant composition comprising an STING activator (Stimulator of interferon genes agonist) as an active ingredient. The method according to claim 1,
The STING agonist is any one or more selected from the group consisting of c-di-GMP (cyclic diguanylate), cGAMP, 3'3'-cGAMP, c-di-GAMP, c- , An immunoadjuvant composition. The method according to claim 1,
The STING agonist may be selected from the group consisting of c-di-GMP (cyclic diguanylate), cGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'- 10H) acridone (CMA) (10- (carboxymethyl) 9 (10H) acridone (CMA)), 5,6-dimethylxanthenone-4-acetic acid (DMXAA) , Methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3 ', 6'-dihydroxyflavone (3 Dihydroxyflavone, 7'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, methyl ether, and xanthone. < RTI ID = 0.0 > 18. < / RTI > The method according to claim 1,
Wherein the composition further comprises at least one immunoprecipitant selected from the group consisting of MPL (Monophosphoryl Lipid A) and GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil-in-water) . 5. The method of claim 4,
Wherein the immunoassay enhancer is encapsulated in a liposome. STING activator (Stimulator of interferon genes agonist) and an antigen as an active ingredient. The method according to claim 6,
The STING agonist is any one or more selected from the group consisting of c-di-GMP (cyclic diguanylate), cGAMP, 3'3'-cGAMP, c-di-GAMP, c- , Vaccine composition. The method according to claim 6,
The STING agonist may be selected from the group consisting of c-di-GMP (cyclic diguanylate), cGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'- 10H) acridone (CMA) (10- (carboxymethyl) 9 (10H) acridone (CMA)), 5,6-dimethylxanthenone-4-acetic acid (DMXAA) , Methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3 ', 6'-dihydroxyflavone (3 Dihydroxyflavone, 7'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, methyl ether, and xanthone. The method according to claim 6,
Wherein the composition further comprises at least one immunoprecipitant selected from the group consisting of MPL (monophosphoryl lipid A) and GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil-in-water). 10. The method of claim 9,
Wherein the immunoassay enhancer is encapsulated in a liposome. The method according to claim 6,
Wherein the antigen is an M. tuberculosis-specific antigen. 12. The method of claim 11,
Wherein said M. tuberculosis-specific antigen is ESAT-6. The method according to claim 6,
Wherein said vaccine composition prevents infection of Mycobacterium tuberculosis.

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