Tuberculosis vaccine compositions comprising fusion proteins that induce sterilizing immunity against mycobacterium tuberculosis

Abstract

This invention relates to a tuberculosis vaccine composition comprising a fusion protein that induces sterilization immunity against Mycobacterium tuberculosis. Specifically, the tuberculosis vaccine composition comprising a fusion protein of Rv2299cD2D3-ESAT6-Ag85B (REA) or Rv2299cD2D3-Rv3463-Ag85B (RRA) induces sterilization of Mycobacterium tuberculosis by more than 99%, thereby enabling it to be used not only for the prevention of tuberculosis in adults but also as a vaccine for infants and young children.

Description

Technical Field

[0001] This invention relates to a tuberculosis vaccine composition comprising a fusion protein that induces sterile immunity against Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) and is one of the deadliest infectious diseases in human history. Recently, 1.5 million people worldwide have died from TB. In terms of both TB morbidity and mortality, South Korea is one of the countries with the most severe TB problem among the 32 member countries of the Organization for Economic Cooperation and Development (OECD).

[0003] The most effective strategy to address the tuberculosis problem is to develop a safe and highly effective vaccine. However, BCG (Mycobacterium bovis Bacillus Calmette-Guérin, BCG), as the only tuberculosis vaccine, has a controversial efficacy rate of 0-80% depending on the reported cases, and it has been reported that it does not prevent reactivation of latent tuberculosis or adult tuberculosis. However, because it is effective against severe tuberculosis in children, many countries are implementing vaccination programs; however, to date, no vaccine with superior efficacy to BCG has been developed. Furthermore, the World Health Organization's (WHO) End TB strategy aims to reduce tuberculosis incidence by 90% and mortality by 95% by 2035, based on 2015 targets. South Korea has also formulated a 2030 plan to achieve a tuberculosis incidence rate of less than 10 per 100,000 people and is continuously working towards this goal. To achieve the goal of a tuberculosis-free world, a tuberculosis vaccine with excellent preventative effects and the potential to be a game-changer must be developed.

[0004] Currently, there are more than 10 tuberculosis vaccines in clinical trials worldwide. Based on their function, they can be divided into ① prime vaccines, i.e., BCG replacement vaccines based on live bacteria; ② BCG booster vaccines using immune enhancers or viral vectors; and ③ other immunotherapeutic vaccines aimed at shortening the treatment period or suppressing relapse. The development of BCG replacement vaccines based on live bacteria mainly includes BCG strains with gene recombination and Mycobacterium tuberculosis strains that simultaneously delete two genes. Compared to subunit vaccines, these live bacteria-based vaccines contain more antigens and can induce multiple immune responses, thus promising higher protective efficacy. However, the development of recombination or gene-deleted strains involves issues such as removal of antimicrobial resistance markers and safety verification, resulting in a long development cycle. Strict quality management is also required during vaccine strain preparation after development. Due to these limitations, tuberculosis vaccine development has focused on protein-based subunit vaccines. However, these subunit vaccines have limitations in replacing BCG and are therefore mainly developed as BCG booster vaccines.

[0005] Therefore, a more effective vaccine requires an immune response that can completely eliminate the invading bacteria when vaccinated animals or humans are exposed to Mycobacterium tuberculosis—a sterilizing immunity. In this case, a protective efficacy of over 95% can be expected. However, to date, no vaccine in clinical trials can induce this sterilizing immunity.

[0006] The antigens used in current subunit vaccine development and clinical trials are mainly T-cell stimulating antigens such as Ag85 or ESAT6 antigens, PE / PPE antigens, some pathogenicity-related antigens, and protein antigens associated with inducing latent tuberculosis. H56 and ID93 vaccines are mainly multi-protein vaccines, which fuse proteins associated with inducing latent tuberculosis to proteins that activate T cells or proteins related to virulence. However, considering that even individuals with normal immune responses cannot completely eliminate Mycobacterium tuberculosis, there are limitations to inducing sterilizing immunity solely through T-cell vaccines. Therefore, further research in this area is urgently needed. Summary of the Invention

[0007] The problem the invention aims to solve The present invention aims to provide a tuberculosis vaccine composition comprising a fusion protein that induces sterile immunity against Mycobacterium tuberculosis.

[0008] means for solving problems To address the aforementioned problems, the present invention provides a tuberculosis vaccine composition comprising the Rv2299cD2D3-ESAT6-Ag85B (REA) fusion protein. The Rv2299cD2D3-ESAT6-Ag85B (REA) fusion protein is characterized in that it is encoded by the nucleotide sequence of SEQ NO. 1. Furthermore, the Rv2299cD2D3-ESAT6-Ag85B (REA) fusion protein is characterized in that it comprises the amino acid sequence of SEQ NO. 2.

[0009] Furthermore, the present invention provides a tuberculosis vaccine composition comprising the Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion protein. The Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion protein is characterized in that it is encoded by the nucleotide sequence of SEQ NO. 3. The Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion protein is also characterized in that it comprises the amino acid sequence of SEQ NO. 4. The tuberculosis vaccine composition according to the present invention induces sterilization of Mycobacterium tuberculosis with a sterility rate of over 99%.

[0010] According to the above-described tuberculosis vaccine composition of the present invention, it increases the production of IFN-γ + IL-2 + TNF-α + or IL-2 + TNF-α + antigen-specific CD4T + The proportion of cells can induce sterilization immunity against Mycobacterium tuberculosis.

[0011] This invention provides a recombinant polynucleotide for tuberculosis sterilization immunization, comprising the nucleotide sequence described in SEQ NO. 1 or SEQ NO. 3. Furthermore, this invention provides a recombinant vector comprising the above-mentioned recombinant polynucleotide. The vector can be constructed from pET-22b(+)_Rv2299cD2D3-ESAT6-Ag85B or pET-22b(+)_Rv2299cD2D3-Rv3463-Ag85B, comprising the above-mentioned sequences of antigens containing Rv2299cD2D3, ESAT6, Ag85B and Rv2299cD2D3, Rv3463, Ag85B, and can be constructed using common plasmids and phages. The aforementioned vector can transform host cells containing common Escherichia coli, and in particular, it can obtain the Rv2299cD2D3-ESAT6-Ag85B (REA) or Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion proteins from host cells specifically designed for protein production and secretion.

[0012] This invention provides a recombinant polypeptide for sterilizing and immunizing against tuberculosis, comprising the amino acid sequence shown in SEQ NO. 2 or SEQ NO. 4. The aforementioned recombinant polypeptide for sterilizing and immunizing against tuberculosis comprising the amino acid sequence described in SEQ NO. 2 or SEQ NO. 4 can be an Rv2299cD2D3-ESAT6-Ag85B (REA) fusion protein or an Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion protein. The tuberculosis vaccine composition of this invention, comprising a fusion protein that induces sterilizing immunity against Mycobacterium tuberculosis, can replace BCG; therefore, it can be used not only for the prevention of tuberculosis in adults but also as a vaccine for infants and young children.

[0013] Invention Effects This invention relates to a tuberculosis vaccine composition comprising a fusion protein that induces sterilization immunity against Mycobacterium tuberculosis. Specifically, the tuberculosis vaccine composition comprising a fusion protein of Rv2299cD2D3-ESAT6-Ag85B (REA) or Rv2299cD2D3-Rv3463-Ag85B (RRA) induces sterilization of Mycobacterium tuberculosis by more than 99%, thereby enabling it to be used not only for the prevention of tuberculosis in adults but also as a vaccine for infants and young children. Attached Figure Description

[0014] Figure 1 The charts confirm the purification and endotoxin levels of REA and RRA. (A) shows the results of Western blot analysis (WB) of E. coli extracts transformed with pET plasmids containing REA and RRA, followed by SDS-PAGE, Coomassie Brilliant Blue (CB) staining, and His antibody analysis. (B) shows a comparison of TNF-α production in bone marrow-derived macrophages (BMDM) stimulated with LPS, RRA, or REA, depending on whether polymyxin B pretreatment was performed (****p<0.0001).

[0015] Figure 2This is a graph showing the effects of REA and RRA on antigen-presenting cell activity. (A) shows the changes in the production of IL-12p70, TNFα, and IL-10 when BMDM / BMDCs were stimulated with LPS, Ag85b, ESAT6, Rv2299cD2D3, and different concentrations of RRA. (B) shows the expression of surface markers MHC II, CD80, and CD86 when BMDM / BMDCs were stimulated with LPS, Ag85b, ESAT6, Rv2299cD2D3, and different concentrations of REA (in a bar graph, expressed as a percentage of each surface molecule in Anti-Mo F4 / 80BMDM / CD11c+ BMDC cells). (C) shows the changes in the production of IL-12p70, TNFα, and IL-10 when BMDM / BMDCs were stimulated with Rv3463, Rv2299c, and different concentrations of RRA. (D) shows the expression of surface markers MHC II, CD80 and CD86 when BMDM / BMDC was stimulated with Rv3463, Rv2299c and different concentrations of RRA (data shown are mean ± SD (n = 3); *p<0.05, **p<0.01, ***p<0.001, control group (MC)).

[0016] Figure 3This is a graph showing the Th1 response mediated by dendritic cells (DCs) matured via REA. (A) shows the changes in the production of IFNγ and IL-10 after 72 hours of co-culturing unstimulated DCs (control DCs) and DCs stimulated with Rv2299cD2D3 (5 μg / mL), Ag85B (5 μg / mL), ESAT6 (2 μg / mL), or REA (1 or 2 μg / mL) with naïve T cells (DC:T cells = 1:10). (B) shows that transformed OVA-specific CD4+ T cells isolated from B6.Cg-Tg(TcraTcrb)425Cbn / J mice were stained with CFSE and co-cultured with DCs treated with REA (2 μg) or LPS (100 ng / ml) for 96 hours. Then, T cells were pulsed with OVA323-339 (1 μg / ml) to proliferate (the control group consisted of T cells or T cells co-cultured with untreated DCs). (C) shows the quantitative comparison of IFNγ, IL-2, IL-17 and IL-4 in culture supernatant by enzyme-linked immunosorbent assay (ELISA) under various conditions (data shown are mean ± SD (n = 3); *p<0.05, **p<0.01, ***p<0.001, or ****p<0.0001, ns.: no significant difference compared with appropriate control groups (T cells / DCs treated with OVA257-264 pulse or T cells / DCs treated with OVA323-339 pulse).

[0017] Figure 4This is a graph showing the Th1 response mediated by DCs matured via REA. (A) shows the changes in the production of IFNγ, TNFα, and IL-17 after 72 hours of co-culturing unstimulated DCs (control DCs) and DCs stimulated with Rv2299cD2D3 (5 μg / mL) or RRA (2 μg / mL) with naïve T cells (DC:T cells = 1:10). (B) shows the proliferation of T cells after pulsed treatment with OVA323-339 (1 μg / mL) following 96 hours of co-culturing with DCs treated with RRA (2 μg) or LPS (100 ng / mL) from B6.Cg-Tg(TcraTcrb)425Cbn / J mice using CFSE staining. (Control group consisted of T cells or T cells co-cultured with untreated DCs). (C) shows the quantitative comparison of IL-17, IL-2, IFNγ and IL-4 in culture supernatant by ELISA under various conditions (data shown are mean ± SD; *p<0.05, **p<0.01, ***p<0.001, or ****p<0.0001, ns.: no significant difference compared to appropriate control groups (T cells / DCs treated with OVA257-264 pulse or T cells / DCs treated with OVA323-339 pulse).

[0018] Figure 5 The results show the activation of BMDM by Toll-like receptors 2 and 4 via REA. (A) shows the comparison of TNF-α, IL-12, and IL-6 production in culture supernatants by ELISA after 24 hours of treatment with WT, TLR2- / -, and TLR4- / - mouse-derived BMDM using REA (5 μg / mL), LPS (100 ng / mL), and the TLR2 agonist Pam3CSK4 (100 ng / mL). (One-way ANOVA using Tukey's multiple comparison test; p-values ​​shown are for comparison of cytokines released from BMDM of TLR2- / - and TLR4- / - mice with or without antigen from WT-derived BMDM.) (B, C) show the production of TNF-α, IL-12, and IL-6 in BMDM cells (1 × 10⁻⁶) treated with REA (5 μg / mL) at the time indicated. 5 Immunoblot results of membrane components (B) phospho-p38 (p-p38), p38, phospho-ERK1 / 2 (p-ERK1 / 2), ERK1 / 2, (C) GSK3β, p-PI3K, (D) p-AKT and (E) p-IκBα, IκBα.

[0019] Figure 6The results show the activation of BMDMs by Toll-like receptors 2 and 4 (TLR2 / TLR4) via REA. (A) shows the comparison of TNF-α, IL-12, and IL-6 production in culture supernatants by ELISA after 24 hours of treatment with RRA (5 μg / mL), LPS (100 ng / mL), and the TLR2 agonist Pam3CSK4 (100 ng / mL). (One-way ANOVA using Tukey's multiple comparison test; p-values ​​shown are for comparisons of cytokines released from BMDMs from TLR2- / - and TLR4- / - mice, and from WT-derived BMDMs with or without antigen; ns: no significant difference). (B, C) show the results of RRA (5 μg / mL)-treated BMDMs and BMDM cells (1 × 10⁻⁶ cells) at the time points shown. 5 Immunoblot results of the membrane components (B) phospho-p38 (p-p38), p38, phospho-ERK1 / 2 (p-ERK1 / 2), ERK1 / 2, (C) p-PI3K, p-PI3K, p-AKT, p-IκBα, IκBα, and NFκB p65 in the pores. (D) shows the intracellular localization of the NFκB p65 subunit as detected by immunofluorescence.

[0020] Figure 7 The results show the effects of pharmacological inhibitors on the expression of surface markers and cytokine production in BMDM of REA and RRA. (A, B) show the effects of pharmacological inhibitors of p38 (SB203580, 20 μM), ERK1 / 2 (U0126, 10 μM), and NF-κB (Bay11-7082, 5 μM) or DMSO (solvent control) on BMDM (1×10⁻⁶). 6 After 1 hour of treatment with each well, the cells were treated with REA(A) or RRA(B) (5 μg / mL) for 24 hours. The levels of co-stimulatory surface markers (CD80, CD86) were measured by flow cytometry, and the results of TNF-α and IL-6 in the culture supernatant were measured by ELISA (data shown are mean ± SD (n=3) *p<0.05, **p<0.01, or ***p<0.001).

[0021] Figure 8The results show the effects of REA on inhibiting intracellular growth of Mycobacterium tuberculosis (MTB) in BMDM and on activating infected cells. (A) shows intracellular MTB growth under LPS, Rv2299c, and REA conditions. (B) shows the results of analysis of surface molecule expression related to antigen-presenting capacity during infection (in BMDM infected with H37Rv [MOI:1]) by two-color flow cytometry. (C) shows the ELISA results of cytokine production in the culture supernatant shown in (B). (n=3, expressed as mean ± SD, analyzed using one-way ANOV and Tukey post-hoc test, p-values ​​are comparisons of surface molecule and cytokine levels between MC and antigen-treated BMDM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0022] Figure 9 The results show the effect of RRA-matured T cells on inhibiting intracellular bacterial proliferation. (A) shows the growth of MTB in H37Rv-infected BMDM cells after culturing with LPS, Rv2299c, or RRA. (B) shows the analysis of the expression of surface molecules MHC II, CD80, and IL-12 (P70) associated with antigen-presenting capacity during infection (in H37Rv-infected [MOI:1] BMDM) by two-color flow cytometry. (C) shows the ELISA results of intracellular MTB growth and cytokine production in the culture supernatant after co-culturing BMDM infected with MTB with T cells activated by LPS, Rv2299c or BMDC matured by RRA (n=3, expressed as mean ± SD, analyzed using one-way ANOV and Tukey post-hoc test, p-values ​​are comparisons of surface molecule and cytokine levels between MC and antigen-treated BMDM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0023] Figure 10The results show the effects of REA on early endosome maturation in BMDM infected with MTB. (A) shows BMDM infected with H37Rv Mycobacterium tuberculosis strain (REF-MTB) ([MOI: 1]) (red fluorescent protein labeled) after treatment with LPS, Rv2299c or REA, stained with Ras-associated protein 5 (Rab5), and imaged by confocal microscopy for endosome colocalization. The bar chart shows the proportion of Rab5 colocalization. (B, C) show the staining of cells with anti-phosphatidylinositol 3-kinase vacuole protein sorting 34 (hVPS34) (B) or anti-EEA1 (C) using the same settings as A. (D) shows the time-dependent phosphorylation of class I PI3K and p38 MAPK signaling components and the results of Western blot analysis of total protein levels. (E) shows the degree of co-localization of anti-EEA1 in BMDM pretreated with SB303580 during H37Rv infection after treatment with REA. Phagocytes containing MTB were observed using confocal microscopy to determine the co-localization of the indicated markers. (F) shows intracellular bacterial growth in BMDM infected with REA or LPS-treated MTB after SB303580 pretreatment (*p<0.05 and **p<0.01).

[0024] Figure 11 The results show the effect of REA on phagocyte acidification. (A, B, C) show the effect of Rv2299c or REA (5 μg mL⁻¹) on BMDM (1 x 10⁻¹) infected with RFP-MTB [MOI: 1]. 6 After treatment with RFP-H37Rv [MOI: 1], phagocytes containing RAB7 (A), LAMP1 (B), Lyso Tracker™ Green DND-26 (C), and MTB were stained. (D, E) show the colocalization ratio of LAMP after BMDM were infected with RFP-H37Rv [MOI: 1] for 4 hours and 1 hour before Rv2299c or REA treatment, after treatment with SB203580 p38 inhibitor (D) or LY2940029 PI3K inhibitor (E) (*p<0.05, **p<0.01 and ***p<0.001, comparison of untreated BMDM infected with MTB H37Rv with BMDM infected with Rv2299c or REA).

[0025] Figure 12 The results show that REA induces intracellular Ca2+ co-localization of EEA1 in MTB-infected BMDM. 2+The effects of increased concentrations are shown in (A). (A) illustrates the colocalization of calcium induced by REA treatment over the indicated time intervals after loading BMDM infected with RFP-MTB [MOI: 1] onto Fluo-4 / AM (**p < 0.01, ***p < 0.01, and ****p < 0.001, p values ​​indicating the time dependence of Fluo-4 / AM). (B) illustrates the effect of using BAPTA / AM in (A), Ca... 2+ Colocalization of EEA1 after chelation treatment with REA. (C) shows the growth of Mycobacterium tuberculosis in cells after BAPTA / MA treatment (*p<0.05, **p<0.01, ns: no statistically significant difference).

[0026] Figure 13 The results show the effects of REA on intracellular ROS and NO production. (A) shows the determination of intracellular ROS levels in BMDM infected with RFP-MTB [MOI: 1] by DCDF-based staining after NAC treatment. The bar chart shows the DCF fluorescence intensity calculated using ImagJ.exe. (B) shows the determination of NO levels in culture supernatant 72 hours after treatment with REA or LPS, regardless of infection status. (C) shows the stimulation of BMDM infected with RFP-MTB [MOI: 1] (1x10⁻¹⁰ cells) with LPS or REA after DPI pretreatment. 6 / well), and then, intracellular ROS levels were measured by DCDF-based staining. (D) shows the measurement of intracellular bacterial growth in cells treated with NAC 1 hour before infection (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, ns: no statistically significant difference).

[0027] Figure 14The results show the effects of RRA on RAB5 and its activation of PI3K and p38 MAPK. (A) shows BMDMs cells infected with the red fluorescent protein-labeled MTB H37Rv strain (RFP-MTB) ([MOI:1]) after RRA or Rv2299c treatment, stained with RAB5, and imaged using confocal microscopy for endosome colocalization. The histogram shows the proportion of RAB5 colocalization induced by REA in Rv2299c and the untreated control group. (B) shows the time-dependent phosphorylation and total protein level proteoblotting results of class I PI3K and p38 MAPK signaling components. (C) shows the time-dependent phosphorylation and total protein level proteoblotting results of class I PI3K and p38 MAPK signaling components. (D) Intracellular bacterial growth in BMDMs of MTBs treated with SB303580 followed by RRA or LPS (**p<0.01, ns: no statistically significant difference).

[0028] Figure 15 The results show that RRA induces phagolysosomal fusion by activating PI3K to recruit hVPS34, EEA1, and RAB7. (A, B, D, E, F) show the endosomal colocalization imaging using confocal microscopy after treating BMDM cells infected with red fluorescent protein-labeled MTB H37Rv strain (RFP-MTB) [MOI: 1] with RRA or Rv2299c, along with LY2940029 or SB203580, followed by staining with anti-hVPS34 (A), anti-EEA1 (B), anti-Rab7 (D), or anti-LAMP1 (E) antibodies and Lyso Tracker™ Green DND-26 (F). The bar charts show the proportion of colocalization of each detected protein. (C) shows the growth of Mycobacterium tuberculosis in cells treated with LY2940029 (*p<0.05, **p<0.01, ***p<0.001, ns: no statistically significant difference).

[0029] Figure 16 The results show the effects of RRA on intracellular ROS and NO production. (A, B) show the effects of stimulation with LPS or RRA on BMDM infected with RFP-MTB [MOI: 1] (1x10⁻¹). 6(a) After treatment with DPI (A) or NAC (B), intracellular ROS levels were measured using DCDF-based fluorescence staining. The bar chart shows the DCF fluorescence intensity calculated using ImagJ.exe. (C) shows the NO level in the culture supernatant 72 hours after infection and RRA treatment. (D) shows the measurement of intracellular Mycobacterium tuberculosis in BMDM cells before infection and after pretreatment with RRA or LPS. (E) shows the phosphorylation immunoblotting analysis of p38 and pI3K after DPI pretreatment and infection with MTB and RRA treatment (*p<0.05, **p<0.01, ***p<0.001, ns: no statistically significant difference).

[0030] Figure 17 The results show that REA induces intracellular Ca2+ co-localization of hVPS34 and EEA1 in MTB-infected BMDM. 2+ The effects of the increased concentrations are shown in the following figures. (A) shows the colocalization of calcium in RFP-MTB-infected BMDM after loading Fluo-4 into the cells infected with RFP-MTB [MOI: 1] and followed by REA treatment within the indicated time period, as imaged using confocal microscopy. The bar chart shows the percentage of Fluo-4 / M fluorescence intensity in infected cells. (B) The colocalization of hVPS34 or EEA1 in infected BMDM after RRA treatment with BAPTA / AM was then imaged using confocal microscopy. (C) The growth of Mycobacterium tuberculosis in cells pretreated with BAPTA / MA was measured. (D) The intracellular ROS colocalization induced by RRA in RFP-MTB-infected BMDM after BAPTA / MA pretreatment was shown. (E) The intracellular Ca2+ induced by RRA in RFP-MTB-infected BMDM after DPI pretreatment was shown. 2+ Co-location (**p<0.01).

[0031] Figure 18 The image shows the results of REA-treated DCs and the Th1 / Th17 response of macrophage-activated T cells, as well as the anti-mycobacterial response. (A) shows the spleen CD4 isolated from mice vaccinated with BCG-1 (week 4 post-vaccination). + T cells, compared with REA-treated DCs (1×10⁻⁶) 5 / well) and REA-treated macrophages (1×10⁶) 5 / hole), or with REA-treated DC (5×10) 4 / well) and macrophages (5 × 10 4After 72 hours of treatment with APC:T cells at a ratio of 1:10, the cytokines in the cell lysate supernatant were measured by ELISA. (B, C) show the growth of Mycobacterium tuberculosis in BMDM infected with MTB and the cytokines in the culture supernatant after co-culturing T cells for 3 days with MTB-infected BMDM for 72 hours using the same method as in A (B) (T: T cells, M: macrophages, DC: dendritic cells, mean ± SD (n=3), ns: no significant difference, *P<0.05, **p<0.01, ***p<0.001, ****P<0.0001, control T cells).

[0032] Figure 19 The results show the T cell response in mice immunized with the antigen. (A) shows the vaccination and autopsy schedule for mice (in weeks). (B) shows the cytokine levels in the culture supernatant after stimulation of spleen cells isolated from mice with the labeled antigen (2 μg / ml) at week 6 post-immunization in (A) (**p<0.01, ***p<0.001, ****p<0.0001).

[0033] Figure 20 The results show the vaccine potential comparisons of BCG, Rv2299c-ESAT6, Rv2299cD2D3-ESAT6, and the REA (Rv2299cD2De-ESAT6-Ag85B) of this invention. (A) shows the vaccine administration and necropsy timeline for mice. (B) shows the bacterial load in the lungs and spleen of mice at the start of immunization. (C) shows the stimulation of lung cells (2.0 × 10⁻⁶) with Rv2299c, ESAT-6, and REA (5 μg / ml) in the presence of GolgiStop. 6 Afterwards, IL-2 was produced in cells isolated from the lungs of each group of mice. + IL-2 + TNF-α + IFN-γ + TNF-α + ,IFN-γ + IL-2 + TNF-α + and IFN-γ + IL-2 + antigen-specific CD4 + The proportion of T cells (bar chart), and the production of IFN-γ in each group upon stimulation with each antigen. + IL-2 + TNF-α + or IL-2 +TNF-α + antigen-specific CD4 + Proportion of T cells (bar chart). (n=5). (Mean ± SD, statistical significance determined by unpaired t-test, compared with MPL-DDA alone (unpaired t-test), *p<0.05, **p<0.01, ***p<0.001).

[0034] Figure 21 The results show the antigenic immune responses in mice immunized with BCG, Rv2299c-ESAT6, Rv2299cD2D3-ESAT6, and REA (Rv2299cD2De-ESAT6-Ag85B). In the presence of GolgiStop, spleen cells (2.0 × 10⁻⁶) were stimulated with Rv2299c, ESAT-6, and REA (5 μg / ml), respectively. 6 Afterwards, IL-2 was produced in cells isolated from the lungs of each group of mice. + IL-2 + TNF-α + IFN-γ + TNF-α + IFN-γ + IL-2 + TNF-α + and IFN-γ + IL-2 + antigen-specific CD4 + The proportion of T cells (bar chart), and the production of IFN-γ in each group upon stimulation with each antigen. + IL-2 + TNF-α + or IL-2 + TNF-α + antigen-specific CD4 + Proportion of T cells (bar chart). (n=5). (Mean ± SD, statistical significance determined by unpaired t-test, compared with MPL-DDA alone (unpaired t-test), *p<0.05, **p<0.01, ***p<0.001).

[0035] Figure 22 and Figure 20 Similarly, the data shows the IFN-γ and IL-2 levels in each group after stimulation of lung, spleen, and lymph node cells with 2 μg of Rv2299cD2D3, ESAT6, or REA 14 weeks after mouse immunization.

[0036] Figure 23 What is shown is that, after completion and Figure 22Following the same experiment, the levels of IL-7 and TNF-α in each group after various stimuli were compared.

[0037] Figure 24 The results show the efficacy of vaccines immunized in mice with REA / DDA-MPL and RRA / DDA-MPL against highly virulent MTB H37Rv infection. (A) shows the immunization schedule using REA and RRA. (B) shows the bacterial count in the lungs and spleen of each group of mice at 6 or 16 weeks post-MTB vaccination. (C) shows representative histological images of lung lobes in each group at 6 or 16 weeks post-challenge, using Masson trichrome (MT) and acid-fast bacillus (AFB) staining. (D) shows the granuloma area (%) of lung sections (per 5 mice). (E) The intracellular MTB count in BMDM cells after co-culturing MTB-infected BMDM cells with lung and spleen cells from 6- or 16-week-old mice for 3 days. (n=3, mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns: no significant difference). Detailed Implementation

[0038] Our research team developed a fusion protein that links T-cell antigens to dendritic cell activating proteins or macrophage activating proteins and evaluated its efficacy against tuberculosis vaccines (PMID:34572519, PMID:33105734, PMID:32664238, PMID:32545304, PMID:30862819, PMID:28193909). Recent studies on the preparation and evaluation of the Rv2299c-Ag85B-ESAT6 fusion protein have shown that it can effectively induce and activate dendritic cells. By fusing Rv2299c to the known vaccine composition Ag85-ESAT6, the CD4+ T cell response to Ag85-ESAT6 can be enhanced. In a short-term vaccine evaluation mouse model (evaluated 3 weeks after challenge), enhanced vaccine efficacy of Ag85-ESAT6 was observed. However, there are limitations in maintaining long-term vaccine efficacy, and the large molecular weight also makes purification difficult.

[0039] This research team demonstrated that the N-terminal portion of the dendritic cell activating protein Rv2299c did not exhibit immunogenicity (PMID: 36032588). Furthermore, preliminary experiments showed that when constructing the fusion protein, the fusion protein inserted after Rv229c exhibited better vaccine efficacy compared to attaching the ESAT6 antigen to the end of the fusion protein. Therefore, by modifying the ESAT6 protein, removing the N-terminal portion of Rv2299c, and altering the order of Rv2299cD2D3 (reducing the molecular weight by approximately 25 kDa) and Ag85B-ESAT6, the Rv2299cD2D3-ESAT6-Ag85B fusion protein was constructed. Additionally, the ESAT6 / CFP-10 antigen, which is not included in BCG, was used in the IGRA (IFN-γ release assay) for confirming tuberculosis infection. Therefore, a drawback is that if the vaccine antibody contains ESAT6, it could lead to a positive IGRA test in the vaccinated individual. Therefore, we constructed an Rv2299c-Rv3463-Ag85B fusion protein using the Rv3463 antigen instead of the ESAT6 antigen. Our research team reports that the Rv3463 protein, as a macrophage activating protein, can serve as a vaccine candidate antigen (PMID: 30862819).

[0040] The two fusion proteins, Rv2299cD2D3-ESAT6-Ag85B and Rv2299cD2D3-Rv3463-Ag85B, were named REA and RRA, respectively. These proteins were produced and purified in *E. coli*, and their immunomodulatory activity was analyzed using antigen-presenting cells (APCs). Vaccine efficacy assays in mouse models showed that both fusion proteins exhibited remarkable efficacy as a prime vaccine, with no detectable bacteria in the lungs or spleen. While BCG vaccine efficacy significantly decreased more than 14 weeks after challenge, mice vaccinated three times with each of the two vaccines demonstrated efficacy exceeding that of BCG in clearing the bacteria. To date, no tuberculosis vaccine has been reported that can induce a complete bacterial clearance immune response in mouse models. Therefore, the REA and RRA invented by our research team can be used as tuberculosis vaccines that induce sterile immunity. In addition, although the above two vaccines are subunit vaccines, they can be used to replace BCG. They can be used not only to prevent tuberculosis in adults, but also as vaccines for infants and young children.

[0041] The preferred embodiments of the present invention will now be described in detail. However, the present invention is not limited to the embodiments described herein, and may be embodied in other forms, and the content described herein is intended to fully convey the spirit of the invention.

[0042] <Experimental Materials and Methods> 1. Cloning, production, and purification for recombinant protein generation 1.1 Cloning for recombinant protein generation based on protein domain classification To produce the various recombinant proteins required for the experiment, they were derived from Mycobacterium tuberculosis ( Mycobacterium. tuberculosis Using the genomic DNA of MTB H37Rv (ATCC 27294) as a template, the gene was amplified by PCR using the primers listed in Table 1. The resulting PCR product was inserted into the pET-22b(+) vector (Novagen, Madison, WI, USA) using the inserted restriction endonuclease sequence, and the resulting plasmid sequence was analyzed and confirmed.

[0043] [Table 1]

[0044] 1.2 Cloning for Fusion Protein Production (1) pET-22b(+)_Rv2299cD2D3-ESAT6-Ag85B. Derived from Mycobacterium tuberculosis ( Mycobacterium tuberculosis Using genomic DNA of MTB H37Rv (ATCC27294) as a template, Rv2299c and ESAT6 DNA were obtained by PCR. Overlapping PCR was used to construct the Rv2299cD2D3-ESAT6 DNA fragment containing the nucleotide sequence of SEQ NO. 1 (with NdeI inserted at the 5' end and HindIII restriction endonuclease inserted at the 3' end), and this fragment was inserted into the pET22b vector. The primers used are shown in Table 1 above. The amino acid sequence of the polypeptide decoded from the nucleotide sequence of SEQ NO. 1 is identical to that of SEQ NO. 2.

[0045] (2) pET-22b(+)_Rv2299cD2D3-Rv3463-Ag85B Using Rv2299c, Rv3463, and ESAT6 DNA obtained using the same method described above as templates, a fragment containing the nucleotide sequence of SEQ NO. 3 (with NdeI inserted at the 5' end and XhoI restriction endonuclease inserted at the 3' end) was constructed using PCR and inserted into the pET22b vector. The primers used are shown in Table 1. The amino acid sequence of the polypeptide decoded from the nucleotide sequence of SEQ NO. 1 is identical to that of SEQ NO. 4.

[0046] 1.3 Production of recombinant proteins The recombinant plasmid prepared above was transformed into *E. coli* BL21 cells. *E. coli* cells containing the recombinant plasmid were cultured in a shaker incubator at 37°C. When the predetermined optical density (OD) was reached at 600 nm, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Otae, ELPIS-BIOTECH) was added. After 4 to 6 hours, bacterial cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM imidazole, 6 M urea, and 1 mM benzyl sulfonyl fluoride (Sigma). The same composition was used for purification of the single protein, except for urea. After lysis by sonication, the recombinant protein was purified by nickel-nitrotriacetic acid (Ni-NTA) agarose chromatography according to the manufacturer's instructions (Qiagen, Chatworth, CA, USA). After analysis of each purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie Brilliant Blue staining was performed, followed by Western blotting analysis using an anti-His antibody (Santa Cruze). The purified proteins were concentrated and dialyzed using phosphate-buffered saline (PBS, pH 7.4). PBS was used for dialyzing all individual proteins. To remove endotoxin, the dialyzed proteins were co-cultured with polymyxin B agarose (PMB, Sigma) at 4°C for 2 hours. Finally, the purified endotoxin-free recombinant proteins were filtered, sterilized, and stored at -70°C. Protein concentration was calculated using a quinolinic acid (BCA) protein assay kit (Pierce, Rockford, 1L) based on bovine serum albumin (BSA). The purity of all proteins was assessed using an anti-His antibody, Coomassie Blue (CB) staining, and Western blotting (WB).

[0047] Culture of mouse bone marrow-derived dendritic cells (BMDCs) Mouse bone marrow-derived dendritic cells (BMDCs) were cultured in RPMI 1640 medium (Roswell Park Memorial Institute) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic (Welgene), and 0.1% 2-mercaptoethanol at 37°C and 5% CO2. On day 7 or 8, non-adherent cells and loosely attached proliferative DC aggregates were harvested and treated with 5 mM HEPES buffer, 1% MEM solution, 20 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF), and 2 ng / ml IL-4 for further experiments.

[0048] 3. Culture of mouse bone marrow-derived macrophages (BMDMs) BMDM obtained from the femur and pelvis were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS), 50 ng / ml macrophage colony stimulating factor (M-CSF) (R&D System, USA) and 1% antibiotic (Welgene, South Korea) in a cell culture incubator at 5% CO2 and 37°C.

[0049] 4. Preparation of MTB strains MTB H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were cultured using a medium containing 0.5% glycerol, 0.05% Tween 80, 10% oleic acid, albumin, glucose and catalase.

[0050] 5. Animal preparation In the Biohazard Animal Laboratory of Chungnam National University College of Medicine, South Korea, 5-6 week old female C57BL / 6 mice that were not infected with a specific pathogen were housed. They were kept under 12-hour light / 12-hour darkness conditions and provided with sterilized, standard feed. The mice were monitored daily, and none of them exhibited any clinical symptoms or disease during the experiment.

[0051] 6. Cell infection assay and intracellular Mycobacterium tuberculosis proliferation assay BMDM achieves 1×10 per well 5After high-density plate culture, the bacteria were treated with Mtb H37Rv (MOI=1) for 4 hours to induce infection. Then, to remove uninfected Mycobacterium tuberculosis remaining on the outside of the BMDM, 200 μL of [unspecified substance] was added. The cells were treated with the antibacterial agent amikacin at a concentration of 1 / ml for 2 hours, followed by washing with PBS. Then, a specific antigen was added, and the cells were cultured for 3 or 5 days before the number of Mycobacterium tuberculosis within the cells was determined.

[0052] Alternatively, BMDCs stimulated by various antigens and spleen cells or T cells isolated from spleen cells were cultured at a ratio of 1:10 for 3 days to activate lymphocytes. After co-culturing them with BMDMs infected with Mycobacterium tuberculosis for 3 or 5 days, the number of Mycobacterium tuberculosis in the cells was measured.

[0053] To determine the intracellular bacterial count, BMDMs were collected and treated in distilled water for 30 minutes to lyse the cells and obtain lysates. After serial dilution, the lysates were spread onto 7H10 solid medium and incubated at 37°C. The colony-forming units (CFU) count on the solid medium was then analyzed.

[0054] 7. In vitro T cell proliferation analysis Responder T cells involved in the initial T cell response were isolated from total monocytes extracted from BALB / c mice using a MACS column (Miltenyi Biotec). Responder OVA-specific CD4+ + T cells were obtained from the spleen cells of OT-2 mice. These T cells were stained with 1 μM CFSE (Invitrogen). In the presence of 10... Under the condition of tuberculosis antigen at / ml, DCs treated with OVA peptide (2×10 per well) 5 (cells), with CFSE-stained CD4 + T cells (2×10) 6 The cells were co-cultured for 24 hours at a DC:T cell ratio of 1:10. After 3 or 4 days of co-culture, staining was performed using PerCP-Cy5.5 conjugated with anti-CD4+ monoclonal antibody Ab, and analysis was performed by flow cytometry. The supernatant was collected, and the levels of IFN-γ, IL-2, and IL-4 were analyzed by ELISA.

[0055] 8. Enzyme-linked immunosorbent assay (ELISA) Cytokines produced after antigen stimulation of BMDMs or BMDCs, and cytokines produced under various conditions, were detected in culture medium using a sandwich enzyme-linked immunosorbent assay (ELISA). TNF-α, IL-1β, IFN-γ, IL-2, IL-4, and IL-12p70 were analyzed. Cytokines in the culture medium were analyzed according to the recommendations of the manufacturers (eBioscience and BD Biosciences). The levels of cytokines released into the culture medium were determined by measuring absorbance at 450 nm using a microplate reader. A standard curve of recombinant cytokines was used to calculate cytokine concentrations, and results are expressed in picograms per milliliter (pg / mL).

[0056] 9. Determination of reactive oxygen species (ROS) Intracellular ROS levels were assessed after staining with H2DCFDA (Molecular Probes). BMDMs cells were stimulated with recombinant REA protein and then incubated in a dark chamber at 37°C for 30 minutes with 10 H2DCFDA in PBS, followed by washing with PBS. Samples were then immediately analyzed using a FACS Canto II cell counter, and data were processed using FlowJo.

[0057] 10. Cell surface staining and flow cytometry analysis Regardless of infection status, BMDM or BMDC cells treated with the antigen were collected and washed. For specifically labeled fluorescently conjugated antibodies (CD80(16-10A1), CD86(GL1), MHC class I (34-1-2S), and MHC class II (IA / IE, M5 / 114.15.2), staining was performed using monoclonal antibodies (eBioscience). Staining intensity was measured using a flow cytometer (NovoCyte), and data were analyzed using FlowJo data analysis software (BD Bioscience). To identify cell surface molecules involved in the infection process, BMDM cells were infected with MTB H37Rv (1×10⁻⁶). 5 Four hours after the start of treatment, the patient was treated with an antigen stimulant for 72 hours.

[0058] 11. Immunoblot analysis Activated / inhibited / transfected BMDM cells were collected and lysed using PIPA lysis buffer (pH 7.5) containing 50 mM Tris-HC1, 1 mA EDTA, 150 mM NaCl, 1% Triton X-100, PMSF, 1 mM benzyl sulfonyl fluoride, and 1% (vol / vol) protease inhibitor mixture (Thermo Scientific, Rockford, IL, USA) and phosphatase inhibitor mixture tablets (Sigma-Aldrich, St. Louis). Lysates were centrifuged at 13475 × g for 20 min at 4°C. Total protein concentration was then determined using a Bradford assay (Bio-Rad, Hercules, CA, USA), and equal volumes of protein were separated on SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA, USA), and analyzed by Western blotting. The membrane was blocked for 1 hour at room temperature in 5% (w / v) skim milk and Tris buffer containing 0.1% Tween-20 (TBS / T) buffer, and then incubated with primary antibody at 4°C for 12 hours. It was then co-cultured with a secondary antibody conjugated to the corresponding specific peroxidase. The immunoblot was co-cultured with horseradish peroxidase (HRP) (Millipore) chemiluminescent substrate, and the target protein was detected using a BioRad ChemiDoc imaging system.

[0059] 12. Immunofluorescence and confocal microscopy analysis To track the maturation of MTB-containing phagosomes, co-localization of RAB5, hVPS34, RAB7, and LAMP1 was stained using antibodies labeled with various proteins. A brief description follows: BMDM (2 × 10⁻⁶) was used... 5Cells were seeded overnight on 18 mm diameter circular glass coverslips in 12-well cell culture plates. Then, cells were infected with RFP-MTB H37RV (MOI 1) in antibiotic-free medium and cultured at 37°C in 5% CO2 medium for 4 hours. The cells were then lightly seeded onto standard medium and treated with LPS, Rv2299c, RRA, or REA for the indicated time periods. To determine colocalization, cells were fixed for 10 minutes at room temperature with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA in PBS. Cells were then incubated overnight with rabbit anti-Rab5, anti-hVPS34, anti-EEA1, anti-Rab7, or mouse anti-LAMP1 primary antibodies according to the manufacturer's protocol. Cells stained with Rab5, Hvps34, EEA, and Rab were then co-cultured with the secondary antibody Alexa Flour® 488 conjugate, while LAMP-stained cells were co-cultured with goat anti-Rat Alexa Flour® 488 conjugate. Finally, cellular nucleic acids were stained with 4′,6-diamidinyl-2-phenylindole dihydrochloride (DAPI) and imaged using confocal microscopy. Cells were washed three times with PBS between each step. To quantify intracellular lysosomes, cells were stained with LysoTracker Green (Molecular Rrobes), and LysoTracker Green uptake was analyzed.

[0060] 13. Mixing of protein with DDA / MPL The mixture of protein with dimethyl dioctadecylammonium bromide (DDA) and monophosphoryl lipid A (MPL) was prepared according to the method of Andersen et al. (PMID; 10639447). After mixing 5 μg of protein, 250 μg of DDA, and 25 μg of MPL, 0.2% triethylamine was added to prepare a final volume of 200 μl. The mixture was then heated in a water bath at 70 °C for 30 seconds, followed by sonication for 30 seconds. This process was repeated 2 to 3 times. The protein was mixed with both DDA and MPL before use.

[0061] 14. Vaccine trials The preventive vaccine experiment involves three subcutaneous immunizations of the vaccine composition to be tested. Four or six weeks later, a challenge test is conducted using MTB (H37Ra or H37Rv). After a period of time, the bacterial count in the mouse organs is measured. For the bacterial infection used in the challenge test, mice are first anesthetized with 1.2% 2,2,2-tribromoethanol (Abedin). The trachea is exposed through a small incision in the center, and 50 μl of MTB contained in physiological saline is inoculated into the trachea (IT). To determine bacterial counts in the lungs and spleen, mice were euthanized with CO2, tissues were extracted and homogenized, and then the lung homogenate was serially diluted and inoculated onto Middlebrook 7H10 agar (Difco Laboratories, Detroit, Michigan) supplemented with 10% OADC (Difco Laboratories), amphotericin B (Sigma-Aldrich, St. Louis), and 2 µg / ml 2-thiophenecarbamoylhydrazine (Sigma-Aldrich). After incubation at 37°C for 4 weeks, colony counts were performed. CFU-related data and pneumonia assessment were performed using log10 CFU ± interquartile range (IQR).

[0062] 15. Statistical Analysis All experiments were repeated at least three times. The significance level of sample comparisons was determined using Tukey's multiple comparison test distribution using statistical software (Graphpad Prism Software, version 4.03; GraphPad Software, San Diego, CA). Graph data are expressed as mean ± SEM. *p<0.05, **p<0.01, or ***p<0.001 were considered statistically significant.

[0063] <Experimental Results> 1. Confirmation of endotoxin contamination in purified REA / RRA fusion protein As described above, after transforming *E. coli* with the expression vector pET plasmid containing cloned DNA (encoding REA and RRA fusion proteins), the proteins were purified from the *E. coli* extract, and after SDS-PAGE, Coomassie succinate (CB) staining was performed. The results were analyzed by Western blot (WB) using an anti-His antibody. The results are shown below. Figure 1 A. For example Figure 1As shown in Figure A, recombinant protein expression of REA and RRA was confirmed. Furthermore, to confirm the presence of endotoxin contamination during purification, BMDM was pretreated with polymyxin B for 1 hour or left untreated, then cultured with LPS or RRA / REA for 24 hours. Subsequently, TNF-α production was analyzed in the culture supernatant using ELISA. The results are shown in Figure A. Figure 1 B. For example Figure 1 As shown in Figure B, the recombinant proteins REA and RRA were confirmed to be uncontaminated, and BMDMs did not exhibit significant cytotoxicity at 10 / ml.

[0064] 2. REA and RRA activities in antigen-presenting cells (APCs) Because the proteins that construct REA and RRA contain proteins that activate dendritic cells or macrophages, the activity of APCs for each fusion protein was evaluated. As a control group, single antigenic components (Rv3463, Ag85B, ESAT6, Rv2299c, or Rv2299cD2D3) and LPS were used to construct the fusion proteins.

[0065] First, BMDM or BMDC were stimulated for 24 hours with 100 ng / mL LPS or 1,2,5 or REA and RRA (10 µg / mL), ESAT-6 (2 µg / mL), Ag85B, Rv2299cD2D3, Rv2299c or Rv3463 (5 µg / mL each). The cytokines IL-12p70, TNF-α, and IL-10 in the culture supernatant were then measured. The results are shown below. Figure 2 A and Figure 2 B. For example Figure 2 As shown in Figure A, REA stimulation of BMDMs and BMDCs induced the production of IL-12 and TNF-α in a concentration-dependent manner. In BMDCs, high concentrations of REA promoted increased IL-10 production. Furthermore, as... Figure 2 As shown in Figure C, similar to REA, RRA can also be significantly induced compared to cells that do not produce the inflammatory cytokines IL-12 and TNF-α in BMDMs and BMDCs, and it induces higher IL-10 production compared to REA. In particular, RRA is higher than Rv3463 in BMDMs, while there is no significant difference between RRA and Rv2299c in BMDCs.

[0066] The roles of recombinant REA and RRA proteins in the expression of surface molecules MHC class II, CD80, and CD86 in BMDMs and BMDCs were confirmed. Activated BMDMs or BMDCs were stained with anti-CD80, anti-CD86, or anti-MHC class II antibodies, and the expression of surface markers was confirmed by fluorescence-activated cell sorting (FACS). The results are shown in [Figure / Table / Insert Figure ... Figure 2 B or Figure 2 D. For example Figure 2 As shown in Figure B, in FACS analysis, the expression of surface molecules MHC class II, CD80, and CD86 in BMDMs and BMDCs was also significantly increased upon REA stimulation. Regarding BMDCs, the production and expression of cytokines induced by single antigens Ag85B, ESAT6, and Rv2299cD2D3 were both lower than or similar to those induced by REA. In particular, Ag85B expression induction was the lowest. Furthermore, as... Figure 2 As shown in Figure D, surface molecule expression was significantly increased in both BMDMs and BMDCs compared to the culture medium control group (MC), while there was no significant difference compared to the single antigens Rv3463 or Rv2299c.

[0067] Since both REA and RRA contain Rv2299cD2D3, the activity of dendritic cells matured by each fusion protein was analyzed. Unstimulated DCs (control DCs) and DCs stimulated with Rv2299cD2D3 (5 μg / mL), Ag85 (5 μg / mL), ESAT6 (2 μg / mL), or REA (1 or 2 μg / mL) were co-cultured with naïve T cells (DC:T cell = 1:10) for 72 hours. The production of IFN-γ and IL-10 in the culture supernatant was analyzed by ELISA. The results are shown below. Figure 3 A. For example Figure 3 As shown in Figure A, compared with T cells activated by BMDCs matured with other antigens, T cells activated by BMDCs matured with REA produced significantly higher IFN-γ and lower IL-10.

[0068] In addition, using CFSE, transgenic OVA-specific CD4+ T cells isolated from OVA peptide-specific transgenic mice (B6.Cg-Tg(TcraTcrb)425Cbn / J) were stained, co-cultured with DCs treated with REA (2 μg) or LPS (100 ng / ml) for 96 hours, and then pulsed with OVA323-339 (1 μg / ml). The T cell proliferation results confirmed by T flow cytometry were as follows: Figure 3As shown in Figure B, DCs matured via REA effectively and significantly induced the proliferation of the aforementioned T cells. The concentrations of FNγ, IL-2, IL-17, and IL-14 in the culture supernatant under various conditions, as determined by ELISA, are as follows: Figure 3 As shown in Figure C, the production of IL-17, IL-2, and IFN-γ was also significantly higher.

[0069] Using the same method as described above for REA, unstimulated DCs and DCs stimulated with Rv2299cD2D3 (5 μg / mL) or REA (2 μg / mL) were co-cultured with naïve T cells (DC:T cell = 1:10) for 72 hours. The production levels of IFN-γ, TNF-α, and IL-17 in the culture supernatant were analyzed by ELISA. The results are shown below. Figure 4 A. For example Figure 4 As shown in Figure A, T cells activated by DCs matured via RRA showed higher levels of IFN-γ and TNF-α production. Furthermore, similar results were observed in experiments using transgenic mice. Figure 4 B and Figure 4 As shown in C, DCs differentiated and matured through RRA can effectively induce T cell proliferation and Th17 and Th1 responses, and can induce the production of IL-17, IL-2 and IFNγ.

[0070] 3. Activate APCs via TLR2 and TLR4 pathways The pathways by which the REA and RRA proteins activate antigen-presenting cells (APCs) were analyzed. It has been reported that various Mycobacterium tuberculosis components transmit signals via the TLR2 or TLR4 pathway. Therefore, the signaling pathway was confirmed by the binding of the fusion protein to TLRs present on the surface of tuberculous mycobacteria (BMDMs).

[0071] After treating wild-type (WT), TLR2- / -, and TLR4- / - mouse-derived BMDM mice with REA (5 μg / mL), LPS (100 ng / mL), and the TLR2 agonist Pam3CSK (100 mL) for 24 hours, the production of TNF-α, IL-12, and IL-6 in the culture supernatant was confirmed by ELISA. The results are shown in [Figure / Reference]. Figure 5 A. For example Figure 5As shown in Figure A, the TLR4 ligand LPS failed to induce cytokine secretion in TLR4-deficient BMDM cells, and the TLR2 ligand Pam3 failed to induce cytokine secretion in TLR2-deficient BMDM cells. REA significantly inhibited cytokine secretion in BMDM cells that did not express TLR2 or TLR4, compared to WT cells, but did not completely inhibit it. This implies that at least TLR4 and TLR2 are simultaneously involved in REA-induced macrophage activation. MAPK activity was then confirmed by Western blotting, and the results are shown in Figure A. Figure 5 B to Figure 5 E. For example Figure 5 B to Figure 5 As shown in Figure E, in BEDM stimulated by REA, p38 and ERK are phosphorylated, and glycogen synthase kinase-3β (GSK-3β) PI3K and AKT are also phosphorylated. This means that REA ultimately activates NF-κB- and induces macrophage activation through the PI3K-AKT signaling axis pathway.

[0072] Using the same method described above, wild-type (WT), TLR2- / -, and TLR4- / - mouse-derived BMDM mice were treated with RRA (5 μg / mL), LPS (100 ng / mL), and the TLR2 agonist Pam3CSK (100 mL) for 24 hours. The production of TNF-α, IL-12, and IL-6 in the culture supernatant was then confirmed by ELISA. The results are shown in [Figure / Table / Insert Results Here]. Figure 6 A. For example Figure 6 As shown in Figure A, RRA, like REA, significantly inhibited cytokine secretion in BMDMs that did not express TLR2 and TLR4 compared to WT cells, but not completely. This implies that at least TLR4 and TLR2 are involved in RRA-induced macrophage activation. Furthermore, the immunoblotting results of phosphorylated p38 (p-p38), p38, phospho-ERK1 / 2 (p-ERK1 / 2), ERK1 / 2, p-PI3K, p-PI3K, p-AKT, p-IκBα, IκBα, and NFB p65 of membrane components in BMDMs and BMDCs, and the intracellular localization analysis of the p65 subunit of NF-κB detected by immunofluorescence are shown below. Figure 6 B to Figure 6 As shown in Figure D, it was confirmed that RRA also induced phosphorylation of p38, ERK, AKT, and PI3K in both BMDM and BMDC, and ultimately induced NF-κB activation.

[0073] On the one hand, to confirm the effects of pharmacological inhibitors on the surface marker expression and cytokines of REA and RRA, p38 (SB203580, 20 μM), ERK1 / 2 (U0126, 10 μM), and NF-κB (Bay11-7082, 5 μM) pharmacological inhibitors or DMSO (solvent control) were used to inhibit the expression of BMDM (1×10⁻⁶). 6 After 1 hour of treatment with the culture medium ( / well), followed by 24 hours of treatment with REA or RRA (5 μg / mL), the levels of co-stimulated surface markers (CD80, CD86) and the results of TNF-α and IL-6 in the culture supernatant as measured by ELISA are shown in the figure. Figure 7 A and Figure 7 B. For example Figure 7 A and Figure 7 As shown in B, although there were differences in the degree of inhibition due to MAPK inhibitors [SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), U0126 (ERK1 / 2 inhibitor), BAY11-7082 (NF-κB inhibitor), LY294002 (PI3K inhibitor)], the production of inflammatory cytokines and the increase in surface molecule expression mediated by RRA and REA were both inhibited.

[0074] 4. Antituberculosis activity of REA and RRA Mycobacterium tuberculosis has the ability to survive and proliferate in phagocytes, namely macrophages. Therefore, activating macrophages to induce the killing of phagocytes is an important issue in the anti-tuberculosis immune response. Thus, it was confirmed whether stimulating BMDMs infected with Mycobacterium tuberculosis with REA or RRA could induce the killing of intracellular Mycobacterium tuberculosis.

[0075] Infect BMDM (1×10⁻¹) with H37Rv [Multiple of Infection (MOI) 1]. 5 After 4 hours of treatment with kanamycin, cells / well were treated for another 2 hours. Then, to eradicate extracellular Mycobacterium tuberculosis, infected BMDM cells were washed three times and cultured for 72 hours with Rv229c or REA (5 μg / mL) and LPS (100 ng / mL) to confirm intracellular MTB growth. The results showed that after 72 hours of REA stimulation, the proliferation of Mycobacterium tuberculosis in BMDM cells infected with Mycobacterium tuberculosis was significantly inhibited compared to cells stimulated with LPS or REA. Figure 8 A). The expression of surface molecules related to antigen presentation capacity during infection (in BMDM infected with H37Rv [MOI:1]) was analyzed by two-color flow cytometry. BMDM cells were stained with F4 / 80+, while surface molecules were stained with anti-CD80, anti-CD86, anti-MHC class I, or anti-MHC class II antibodies. Figure 8 The histograms in section B represent five representative experiments, and the bar charts show the percentages of various surface molecules in F4 / 80+ cells (mean ± SD of the five experiments). Furthermore, cytokine production in the culture supernatant at this stage was measured by ELISA, and the results are shown below. Figure 8 C. For example Figure 8 B and Figure 8 As shown in Figure C, BMDMs infected with Mycobacterium tuberculosis showed significantly enhanced expression of surface molecules (MHC molecules, CD80, CD86) and production of inflammatory cytokines and CCL-2 chemokines after REA stimulation compared to infected cells. No significant difference was observed between LPS and REA in these enhancements, but IL-12 levels were significantly increased after REA stimulation compared to LPS. Figure 8 C).

[0076] To confirm the inhibitory effect of T cells matured via RRA and REA on intracellular bacterial proliferation, BMDM (1×10⁻⁶ cells) were infected with H37Rv [multiple of infection (MOI) 1]. 5 After 4 hours of treatment (cells / well), the cells were treated with kanamycin for another 2 hours. Then, to eradicate extracellular mycobacteria, the cells were washed three times and cultured for 72 hours to confirm the growth of intracellular MTB. The results are as follows: Figure 9 As shown in Figure A, treatment of BMDMs infected with Mycobacterium tuberculosis with RRA significantly inhibited intracellular bacterial proliferation compared to the cell culture control group and Rv2299c. The production of IL-12 and expression of surface molecules also increased significantly, but there was no significant difference between Rv2299c and RRA. Figure 9 B). Furthermore, when mature T cells, either RRA-derived or Rv299cD2D3-derived RRA cells, were co-cultured with BMDMs infected with Mycobacterium tuberculosis, intracellular bacterial proliferation was significantly inhibited compared to other conditions. Figure 9 C). Compared to Rv299cD2D3, T cells differentiated from RRA showed better inhibitory effects on bacterial proliferation. At this time, the production of cytokines in the cell culture medium was also significantly higher, but there was no significant difference between LPS, Rv2299cD2D3, and RRA.

[0077] The above results mean that REA and RRA can overcome the inhibitory effect induced by Mycobacterium tuberculosis infection, and can also induce appropriate activation of phagocytes to clear the Mycobacterium tuberculosis they have engulfed.

[0078] 5. Elucidate the mechanism of action of REA in preventing tuberculosis. 5.1 REA induces early endosomal maturation through the p38-PI3K signaling pathway and by recruiting effector molecules. The maturation of phagosomes begins with the recruitment of Rab (Ras-associated protein) molecules into the phagosome membrane. For analysis, *R. phagosome-associated* mycobacteria expressing red fluorescence (RFP-MTB) were stimulated with LPS, Rv2299c, and REA, stained with a green fluorescent Rab5 antibody, and observed using a confocal microscopy.

[0079] BMDM cells were infected with red fluorescent protein-labeled H37Rv strain (RFP-MTB) (MOI-1) for 4 hours. Then, on microscope coverslips, cells were treated with LPS (100 ng / mL), Rv2299c (5 μg / mL), or REA (5 μg / mL) for 1 hour. Cells were then stained with Ras-associated protein 5 (Rab 5), and endosomal colocalization was imaged using confocal microscopy. The results are shown below. Figure 10 A. The bar chart shows the co-location ratio of Rab5. For example... Figure 10 As shown in Figure A, the co-localization of Mycobacterium tuberculosis and Rab5 was significantly increased under REA stimulation compared to other conditions.

[0080] Next, the Vps34 site [vacuolar protein sorting 34, type III PI(3)K], which plays an important role in the production and accumulation of PI(3)P in the phagosome membrane, was analyzed. Using the same settings as above, cells were stained with anti-phosphoinositide 3-kinase vacuolar protein-sorting (hVPS34), and the results are as follows. Figure 10 As shown in Figure B, upon REA stimulation, the co-localization of Mycobacterium tuberculosis with Vps34 was significantly increased compared to other conditions. Since this process is crucial for recruiting early endosomal antigen 1 (EEA1), the location of EEA1 was analyzed, and the results are shown in Figure B. Figure 10 C. The bar chart shows the co-occurrence rate of phagosomes containing hVPS34 or EEA1 and MTB. For example... Figure 10As shown in C, after REA stimulation, the transport of EEA1 into phagocytic corpora containing Mycobacterium tuberculosis increased.

[0081] PI3K and p38 play important roles in early endosome maturation. Therefore, time-dependent phosphorylation and total protein levels of class I PI3K and p38 MAPK signaling components were detected by Western blot analysis. BMDM cells were infected with RFP-MTB [MOI=1] after treatment with REA for 0.25, 0.5, 1, 2, and 6 hours or untreated. Cell lysates were then collected and stained with antibodies against p-P38 MAPK, p38 MARK, p-PI3K, PI3K, and β-actin. The results are shown in [Figure / Table / Insert Table ... Figure 10 D. For example Figure 10 As shown in D, it was confirmed that when infected with Mycobacterium tuberculosis alone, P38 was only temporarily phosphorylated in the initial stage, while P38 was activated for a longer period of time after REA stimulation. PI3K did not show obvious phosphorylation when infected with Mycobacterium tuberculosis, but PI3K was significantly activated after REA stimulation.

[0082] In addition, BMDM cells were pretreated with the SB303580 p38 inhibitor (10 μM) for 1 hour and treated with REA (5 μg mL⁻¹) during H37Rv infection. Cells were then stained with anti-EEA1, and MTB-containing phagocytes were observed using confocal microscopy to confirm the co-localization of the indicated markers. The results are shown in [Figure / Reference]. Figure 10 E. For example Figure 10 As shown in Figure E, pretreatment with P38-specific inhibition significantly suppressed REA-induced co-localization of EEA1 with Mycobacterium tuberculosis. Furthermore, intracellular growth in BMDM cells infected with MTB, pretreated with SB303580 (10 μM) one hour before infection, followed by treatment with REA (5 μg / mL) or LPS (100 ng / mL), was measured, and the results are shown in Figure E. Figure 10 F. For example Figure 10 As shown in F, the inhibitory effect of REA-induced Mycobacterium tuberculosis proliferation is lost.

[0083] 5.2 REA-induced acidification of phagosomes and fusion with lysosomes The next step in endosome maturation involves recruiting Rab, a late endosome-lysosome-associated small GTPase. Inhibiting Rab transport inhibits the fusion of phagosomes with late endosomes. This allows BMDM (1×10⁻⁶) to be produced. 6Cells were infected with RFP-MTB [MOI: 1] for 4 hours, followed by treatment with Rv2299c (5 μg / mL) or REA (5 μg / mL) for 1 hour. Cells were then stained with anti-Ras-associated protein 7 (RAB7), anti-lysosome-associated membrane protein 1 (LAMP1), and Lyso Tracker™ Green DND-26. Confocal microscopy was used to observe MTB-containing phagocytic cells to confirm the co-localization of the indicated markers. Results are shown in [Figure / Video / ]. Figure 11 A to 11C. For example... Figure 11 As shown in Figure A, after REA stimulation, the co-localization of Rab7 with phagosomes containing Mycobacterium tuberculosis was significantly increased compared to other conditions. Furthermore, the co-localization of the lysosomal marker lysosomal-associated membrane protein 1 (LAMP1) with Mycobacterium tuberculosis was also significantly increased after REA treatment. Figure 11 B). The acidification environment of late phagosomes in fused lysosomes was analyzed using a pH-sensitive lysosomal tracker (LysoTracker). Because the LysoTracker, bound to blue fluorophore, is weakly alkaline, it remained persistently within the organelle after binding. As expected, colocalization of the Lysotracker with Mycobacterium tuberculosis was significantly increased in REA treatment compared to Rv2299c treatment. Figure 11 C).

[0084] BMDM was infected with RFP-MTB [MOI:1] for 4 hours, pretreated with SB203580 p38 inhibitor or LY2940029 PI3K inhibitor for 1 hour, and then treated with Rv2299c or REA. Colocalization of LAMP1 with Mycobacterium tuberculosis was shown in Figure 11 D and Figure 11 E. For example Figure 11 D and Figure 11 As shown in Figure E, pretreatment with P38 and PI3K inhibitors significantly inhibited the co-localization of LAMP1 increased by REA. This result suggests that the P38-PI3K signaling pathway is involved in the fusion of phagosomes and lysosomes containing Mycobacterium tuberculosis.

[0085] 5.3REA increases intracellular calcium 2+ Inducing EEA1 localization Intracellular Ca 2+It functions as a multi-signaling molecule. In particular, it plays a crucial role in phagosome-lysosome fusion-related molecules and NADPH oxidase activity. Therefore, Fluo-4 / AM (dynamic single-wavelength fluorescent Ca2+) was used... 2+ Indicator, dynamic single-wavelength fluorescent Ca 2+ (Indicator) of Ca in BMDMs infected with Mycobacterium tuberculosis 2+ The concentration was determined. BMDM (1×10⁻⁶) was applied to an 18 mm coverslip. 6 Four hours after infection with RFP-MTB [MOI:1], calcium ion fluorescent probe Fluo-4 / AM was loaded onto the sample for 30 minutes. Then, after treatment with REA (5 μg / mL) for 0, 1, 5, and 10 minutes, the colocalization of calcium was imaged using confocal microscopy. The results are shown in [Figure / Image / Diagram / Illustration]. Figure 12 A. The bar chart shows the percentage of Fluo-4 / AM fluorescence intensity obtained from infected cells after REA treatment. (See also...) Figure 12 As shown in Figure A, after REA treatment, intracellular Ca... 2+ It increases, reaches its peak at 5 minutes, and then begins to decrease. In the above-mentioned equal amounts of BMDM, BAPTA / AM and Ca are used. 2+ After chelation, the cells were treated with REA for 1 hour. Then, the co-localization of EEA1 was imaged using confocal microscopy, and the results are shown below. Figure 12 B. The bar chart shows that, after Ca... 2+ The percentage of fluorescence intensity (EEA) in infected cells after chelation agent treatment. Figure 12 As shown in Figure B, pretreatment with the calcium chelating agent BAPTA inhibited the co-localization of EEA1 increased by REA. Furthermore, as described above, intracellular bacterial growth was measured 1 hour prior to infection after BAPTA / MA treatment, and the results are shown in... Figure 12 C. For example Figure 12 As shown in Figure C, the inhibitory effect of REA-induced intracellular Mycobacterium tuberculosis proliferation was lost after pretreatment with the calcium chelating agent BAPTA.

[0086] 5.4 REA induces ROS production in cells infected with Mycobacterium tuberculosis. Reactive oxygen species (ROS) and nitric oxide (NO) are substances that mediate the killing of intracellular bacteria. Nicotinamide adenine dinucleotide phosphate (NADPH) and NADPH oxidase (NOX2) transfer electrons to phagosomes within phagocytes via the electron transport chain to produce superoxide. Intracellular calcium... 2+ Increase the production of ROS and NO by initiating NADPH oxidase on the phagosome membrane.

[0087] Apply BMDM (1×10) to an 18mm coverslip. 6 Four hours after infection with RFP-MTB [MOI:1] in one well, the infection was confirmed by fluorescent staining with dihydrodichlorofluorescein (DCDF 10μM). The results are shown in [Figure 1]. Figure 13 A. For example Figure 13 As shown in Figure A, Mycobacterium tuberculosis infection itself does not induce ROS production, but ROS in phagocytosomes increases after Mycobacterium tuberculosis-infected cells are treated with REA. Furthermore, measurements of NO levels in the culture supernatant 72 hours after REA or LPS treatment showed that the concentration of free NO in the phagocytosome cell supernatant also increased after REA treatment of Mycobacterium tuberculosis-infected cells. Figure 13 B). Pretreatment with the NOX enzyme inhibitor diphenyleneiodonium (DPI) significantly inhibited the phagocytic localization of ROS induced by REA. Figure 13 C), after treatment with the ROS scavenger N-acetyl-L-cysteine ​​(NAC, 10 mM), the same effect was also observed. Figure 13 A). Furthermore, one hour before infection, regardless of the presence or absence of NAC, measurements of intracellular bacterial growth showed that the inhibitory effect of REA on intracellular bacterial growth was partially lost due to NAC. Figure 13 D).

[0088] In summary, the inhibitory effect of REA on the proliferation of Mycobacterium tuberculosis in cells is attributed to promoting the fusion and acidification of phagosomes and lysosomes. Furthermore, ROS and NO are also associated. During the induction of the above reactions, P38-PI3K and Ca... 2+The signal transduction pathway is also involved.

[0089] 6. Elucidate the anti-tuberculosis mechanism of RRA. 6.1 RRA induces phagosome maturation via the P38-PI3K signaling pathway. Similar to REA, RRA can also inhibit the proliferation of intracellular Mycobacterium tuberculosis. Therefore, BMDMs infected with Mycobacterium tuberculosis (RFP-MTB) were stimulated with Rv229c or RRA, stained with Rab5 antibody labeled with green fluorescence, and observed by confocal microscopy.

[0090] Four hours after infecting BMDMs cells with red fluorescent protein-labeled MTB H37Rv strain (RFP-MTB) (MOI-1), they were treated with RRA (5 μg / mL) or Rv2299c (5 μg / mL), then stained with Ras-associated protein 5 (Rab 5), and the endosome localization was imaged using confocal microscopy. The results are shown in [Figure / Table / Insert Figure ...Insert Figure / Insert Figure / Insert Figure / Insert Figure / Insert Figure / Insert Figure / Insert Figure / Insert Figure / Insert Figure / Figure 14 A. The bar chart shows the proportion of Rab5 co-location caused by REA. For example... Figure 14 As shown in Figure A, the co-localization of Mycobacterium tuberculosis and Rab5 was significantly increased upon RRA stimulation compared to other conditions.

[0091] In addition, to analyze the associated signaling pathways, Western blotting analysis was used to detect the time-dependent phosphorylation and total protein levels of class I PI3K and p38MAPK signaling components. The results are as follows: Figure 14 As shown in Figure B, RRA significantly increased the phosphorylation of P38 and PI3K in BMDMs. While Mycobacterium tuberculosis infection itself did not lead to significant phosphorylation of PI3K, RRA significantly maintained PI3K activation in BMDMs infected with Mycobacterium tuberculosis. Furthermore, Mycobacterium tuberculosis infection itself only caused temporary phosphorylation of P38, but its activity was maintained after RRA treatment. Moreover, pretreatment with the P38 inhibitor (SB203580) and the PI3K inhibitor (LY294002) significantly inhibited the phosphorylation of P38 and PI3K maintained by RRA. Figure 14 C), after pretreatment with a P38 inhibitor, the intracellular bacterial proliferation inhibition maintained by RRA was lost. Figure 14 D).

[0092] Four hours after infecting BMDMs cells with red fluorescent protein-labeled MTB H37Rv strain (RFP-MTB) (MOI-1), they were treated with RRA (5 μg / mL) or Rv2299c (5 μg / mL) for 1 hour. Then, they were pretreated on microscope coverslips with LY2940029 (20 μM) or SB203580, stained with anti-hVPS34 (A), anti-EEA1 (B), anti-Rab7 (D), or anti-LAMP1 antibodies and Lyso Tracker™ Green DND-26. Endosome colocalization was analyzed using confocal microscopy, and the results are shown below. Figure 15 A to Figure 15 F. For example Figure 15 As shown in A to 15F, the colocalization of the following effector molecules involved in phagosome maturation—class III PI3K hVPS4, EEA—with phagosomes containing Mycobacterium tuberculosis was significantly increased in RRA-stimulated cells compared to untreated cells or cells stimulated only with Rv2299c. Furthermore, the above colocalization was significantly inhibited by pretreatment with the PI3K inhibitor LY294002, and the bacterial proliferation-inhibiting effect of RRA was lost. Figure 15 C). Next, the colocalization between the late endosomal marker Rab and phagosomes containing Mycobacterium tuberculosis was significantly increased in RRA treatment compared to other conditions. Figure 15 D). Based on the above results, it can be concluded that RRA can induce the maturation of phagosomes containing Mycobacterium tuberculosis through the P38 and PI3K signaling pathways.

[0093] Furthermore, the co-localization of the lysosomal marker lysosomal-associated membrane protein 1 (LAMP1) with Mycobacterium tuberculosis was also significantly increased after RRA treatment. Figure 11 B). The acidification environment of late-stage phagosomes in fusion lysosomes was analyzed using a pH-sensitive lysosomal tracker, and the results are as follows: Figure 15 As shown in Figure E, the co-localization of LysoTracker with Mycobacterium tuberculosis was significantly increased after RRA treatment compared to Rv2299c treatment. Furthermore, pretreatment with a P38 inhibitor significantly suppressed the increased LAMP1 co-localization observed after RRA. These results suggest that the signal transduction axis is involved in the fusion of phagosomes and lysosomes containing Mycobacterium tuberculosis.

[0094] 6.2 RRA induces ROS production in cells infected with Mycobacterium tuberculosis. Similar to REA, the analysis also examined whether RRA induced ROS production. Using NAC or DPI (10 μM), BMDM (1×10⁻⁶) in 18 mm coverslips infected with RFP-MTB [MOI:1] for 4 hours was analyzed. 6 After pretreatment ( / well), cells were stimulated with LPS or RRA for 4 hours. Intracellular ROS levels were then measured using a DCDF (10 μM)-based fluorescence assay, and the results are shown in [Figure / Reference]. Figure 14 A and Figure 16 B. For example Figure 16 As shown in Figure A, the results of stimulating BMDMs infected with Mycobacterium tuberculosis with RRA showed that it significantly induced higher ROS production compared to LPS. ROS production was completely inhibited after pretreatment with the NOx inhibitor DPI. Furthermore, the increased ROS production in BMDMs infected with Mycobacterium tuberculosis treated with RRA was also inhibited after NAC pretreatment. Figure 16 B). Furthermore, neither Mycobacterium tuberculosis nor RRA alone leads to an increase in nitrite in cell culture medium, but nitrite production significantly increases after RRA treatment of cells infected with Mycobacterium tuberculosis. Figure 16 C). Furthermore, intracellular bacterial growth in BMDM cells pretreated with NAC one hour before infection, followed by RRA- or LPS-treatment, was measured. The results showed that the RRA-induced inhibition of intracellular Mycobacterium tuberculosis proliferation was lost due to NAC pretreatment. Figure 16 D). In addition, such as Figure 16 As shown in E, it was confirmed that the increased activities of p38 and PI3K after RRA treatment in BMDMs or BMDMs infected with Mycobacterium tuberculosis were inhibited by DPI pretreatment.

[0095] 6.3 Increased intracellular Ca2+ via RRA 2+ Inducing the localization of hVPS34 and EEA1. Similar to REA, it also affects whether RRA induces intracellular Ca2+. 2+ Further analysis was performed. Fluo-4 / AM (dynamic single-wavelength fluorescence Ca2+) was used. 2+ Indicator, dynamic single-wavelength fluorescent Ca 2+ (Indicator) of Ca in BMDMs infected with Mycobacterium tuberculosis 2+ The concentration was determined. BMDM (1×10⁻⁶) was applied to an 18 mm coverslip. 6Four hours after infection with RFP-MTB [MOI:1], calcium ion fluorescent probe Fluo-4 / AM was loaded into the well for 30 minutes. Then, after treatment with REA (5 μg / mL) for the indicated time, colocalization of calcium was imaged using confocal microscopy. The results are shown in [Figure / Table / ]. Figure 17 A. The bar chart shows the percentage of Fluo-4 / AM fluorescence intensity obtained from infected cells. (e.g.) Figure 17 As shown in Figure A, after RRA treatment, intracellular Ca... 2+ It increases at 1 minute, reaches its peak at 5 minutes, and then begins to decrease. Furthermore, pretreatment of infected BMDM with the calcium chelator BAPTA / AM inhibited the co-localization of class III PI3KhVPS34 and EEA1, which were increased by RRA. Figure 17 B). Furthermore, the inhibitory effect of RRA on intracellular Mycobacterium tuberculosis proliferation was also lost; in both the cell culture control group and under LPS-treated conditions, BAPTA pretreatment increased intracellular bacterial proliferation. Figure 17 C).

[0096] Known through Ca 2+ The transduced signal is involved in the ROS generation mechanism. Treatment of BMDM infected with RFP-MTB with BAPTA significantly inhibited the co-localization of ROS generated via RRA with phagosomes containing Mycobacterium tuberculosis. Figure 17 D). Furthermore, after pretreatment with the NOx enzyme inhibitor DPI, the increased Ca2+ levels following RRA... 2+ Co-localization was also significantly suppressed. Figure 17 E). From the above results, we can conclude that Ca... 2+ The signal is associated with RRA-induced phagosome maturation and is also closely related to ROS-mediated signaling pathways.

[0097] 7. Vaccine efficacy assessment for REA and RRA The inventors have demonstrated that dendritic cells matured via the Rv2299c protein can effectively induce T cell activity capable of clearing intracellular Mycobacterium tuberculosis, and the actual Rv2299c-ESAT6 fusion protein exhibits superior BCG-enhanced vaccine efficacy (PMID: 28193909). Furthermore, in a short-term vaccine evaluation mouse model (evaluated 3 weeks after challenge), Rv2299c-Ag85B-ESAT6, incorporating Rv2299c, showed enhanced vaccine efficacy of Ag85-ESAT6 (PMID: 33105734). The inventors' preliminary results indicate that, when constructing various fusion proteins, inserting other proteins after Rv2299c-ESAT6 results in superior vaccine efficacy compared to inserting them between Rv2299c-ESAT6. Based on these results, the Rv2299c-ESAT6-Ag85B fusion protein was constructed by altering the antigen-conjugation sequence. Ultimately, since smaller molecular weights are more conducive to commercialization, the Rv2299cD2D3-ESAT6-Ag85B fusion protein REA was constructed, which conjugates ESAT6-Ag85B to Rv2299cD2D3, which lacks the N-terminus (D1 region) of Rv2299c. Furthermore, the Rv2299cD2D3-Rv3463-Ag85B fusion protein RRA was constructed, using Rv3463 to replace the ESAT6 antigen (which is used as the stimulating antigen in the IGRA assay, widely used as an alternative to the tuberculin skin reaction assay).

[0098] The aforementioned REA and RRA, by activating dendritic cells and macrophages, can effectively enhance Th1 and Th17 responses against epitopes contained in various fusion proteins, potentially possessing vaccine efficacy as a primary immunization vaccine (rather than a BCG booster vaccine). It is known that subunit vaccines have not yet been able to replace BCG; therefore, if REA and RRA, as subunit vaccines, can serve as a viable alternative to BCG for primary immunization, they could be developed for use as vaccines in adults and infants.

[0099] In the above analysis of anti-tuberculosis activity, REA and RRA showed similar effects, simultaneously inducing the activation of dendritic cells and macrophages. Therefore, it was verified whether the simultaneous induction of these cells by REA and RRA could enhance anti-tuberculosis activity.

[0100] First, mice vaccinated with the BCG vaccine were sacrificed four weeks after vaccination, and their spleens were removed from the spleens to isolate CD4. + T cells. The above CD4... + T cells, compared with REA-treated DCs (1×10⁻⁶) 5 / well) and REA-treated macrophages (1×10⁶)5 / hole), or with REA-treated DC (5×10) 4 / well) and macrophages (5 × 10 4 After treatment with APC:T cells at a ratio of 1:10 for 72 hours, the cytokines in the cell supernatant were measured by ELISAE. The results are shown in [Figure number missing]. Figure 18 A. For example Figure 18 As shown in Figure A, T cells activated by both APCs (macrophages and dendritic cells) produced significantly higher concentrations of TNF-α, IFN-γ, IL-2, and IL-17 compared to T cells activated by macrophages or dendritic cells activated by REA. Using the above method, after 3 days of co-culture, T cells were harvested and co-cultured with BMDM infected with MTB for 72 hours. The bacteria within the BMDM were then analyzed, and the results are shown in Figure A. Figure 18 B, and the cytokines in the culture supernatant were measured, and the results are shown in Figure 18 C. For example Figure 18 As shown in Figure B, adding the above cells to macrophages infected with Mycobacterium tuberculosis significantly increased the inhibitory effect on intracellular bacterial proliferation. Furthermore, the production of TNF-α, IFN-γ, and IL-17 was also significantly increased compared to the addition of T cells activated by APC alone. Figure 18 C).

[0101] Then, the fusion of Rv2299cD2D3 in vivo was analyzed to determine whether it could enhance the induction of immune responses to Ag85B and ESAT6. Figure 19 As shown in Figure A, mice were randomly divided into 5 groups and immunized once with each of the following: G0: saline; G1: Rv2299cD2D3_DDA / ML; G2: ESAT6_DDA / MPL; G3: Ag85B_DDA / MPL; and G4: REA_DDA / MPL. Six weeks later, the mice were sacrificed, and spleen cells were isolated. DDA / MPL was used as an immunostimulant. The isolated spleen cells were restimulated with the respective immunoantigens, and the produced cytokines IFN-γ and TNF-α were measured. The results are shown in Figure A. Figure 19 B. For example Figure 19 As shown in Figure B, compared to immunization with a single antigen, REA immunization showed increased IFN-γ and TNF-α levels for all antigens except Rv2299c, and also increased T-cell responses. These results suggest that REA and RRA, which activate two APCs, can effectively induce anti-tuberculosis responses, and that Rv2299cD2D3 can enhance T-cell responses to other antigens constituting REA.

[0102] Since REA and RRA exhibit similar anti-tuberculosis activity, the vaccine potential was evaluated using a mouse infection model with the less pathogenic Mycobacterium tuberculosis strain H37Ra. The vaccine efficacy of the components used to construct the REA fusion protein, Rv2299c-ESAT6 and Rv2299cD2D3-ESAT6 fusion proteins, was also compared with that of REA.

[0103] like Figure 20 As shown in Figure A, five mice were divided into six groups and immunized with the following treatments: G1: saline (PBS), G2: immune enhancer alone (DDA / MPL), G3: BCG alone, G4: Rv2299c-ESAT6_DDA / MPL, G5: Rv2299cD2D3-ESAT6_DDA / MPL, and G6: REA-DDA / MPL. Six weeks after immunization, H37Ra Mycobacterium tuberculosis (1×10⁻⁶) was introduced. 6 CFU (carbohydrate, active ingredient) was directly injected into the bronchus of mice to complete the challenge experiment. Fourteen weeks post-infection, mice were sacrificed, and lungs and spleens were extracted and cells isolated. Then, in the presence of GolgiStop, lung cells (2.0 × 10⁻⁶ cells / ml) were stimulated with each antigen (5 μg / ml) at 37°C. 6 Afterwards, bacterial count and immunoassay are performed. For example... Figure 20 As shown in Figure B, no significant vaccine efficacy was observed with saline, the immune enhancer alone (DDA / MPL), BCG alone, Rv2299c-ESAT6_DDA / MPL, or Rv2299cD2D3-ESAT6_DDA / MPL. However, in mice immunized with REA, Mycobacterium tuberculosis was not detected in any of the mice except for the lungs of one mouse, and no bacteria were detected in any of the spleens. This suggests that it induced sterilizing immunity to eliminate the infection.

[0104] Known immunological factors related to vaccine efficacy and INF-γ + IL-2 + TNF-α + CD4 + T cells and IL-2 + TNF-α + CD4 + The number of T cells is directly proportional to the number of T cells. Therefore, the analysis results of antigen-specific multi-functional T cells are as follows: Figure 20 As shown in Figure C, when lung and spleen cells were restimulated with ESAT6 and REA, INF-γ... + IL-2 + TNF-α + CD4 +The number of T cells was significantly increased in mice immunized with REA compared to other antigen-inoculated groups (e.g., Figure 21 (As shown). When lung cells are stimulated with antigens, INF-γ + IL-2 + TNF-α + CD4 + The number of T cells increases significantly when immunized with the fusion protein. Figure 20 C), but there was no significant difference in the spleen ( Figure 21 Similarly, lung, spleen, and lymph node cells were restimulated with the antigen, and the produced cytokines IFN-γ, IL-2, TNF-α, and IL-17 were measured. The results are shown in [Figure / Reference]. Figure 22 and Figure 23 .like Figure 22 and 23 As shown, it was confirmed that immunization with the fusion antigen containing Rv2299c showed a very high level of activity compared to BCG or the infected control group. In particular, no significant production of IL-2 and IL-17 was observed in mice vaccinated with BCG or those vaccinated with only the immune enhancer. Based on these results, it was confirmed that REA immunization resulted in no detectable bacteria in the lungs and spleen, and the memory immune cell response remained well maintained 14 weeks after challenge. Furthermore, it was also confirmed that INF-γ... + IL-2 + TNF-α + CD4 + The number of T cells is directly proportional to the efficacy of REA vaccine.

[0105] Finally, the defensive efficacy of REA and RRA was evaluated using a mouse model employing a pathogenic H37Rv strain of Mycobacterium tuberculosis. Figure 24 Following immunization (shown in Figure A), a challenge test with Mycobacterium tuberculosis was performed 6 weeks after the final immunization. The number of Mycobacterium tuberculosis in the lungs and spleen was measured at 6 and 16 weeks post-challenge, and the results are shown in... Figure 24 B. At 6 weeks post-challenge, mice immunized with REA or RRA showed a significant reduction in bacterial counts in the lungs and spleen compared to the immune enhancer control group. At 16 weeks post-challenge, neither BCG nor the immune enhancer demonstrated any defensive efficacy. However, surprisingly, no bacteria were detected in the lungs and spleen of all mice immunized with either vaccine, exhibiting over 99% to 100% sterilization immunity inducing Mycobacterium tuberculosis.

[0106] The histopathological findings also support the results of the bacterial count measurements. Six or sixteen weeks after the challenge experiment, lung tissue was stained using Masson trichrome (MT) and acid-fast bacillus (AFB) staining, and the results are shown below. Figure 24 C. For example Figure 24 As shown in Figure C, after 6 and 16 weeks, a large number of Mycobacterium tuberculosis stained with blue dot were detected in the control group. However, in the lungs of mice vaccinated with REA or RRA, only a small number of Mycobacterium tuberculosis were observed after 6 weeks compared to the control group, and almost no bacteria were observed after 16 weeks. Analysis of granulomatous lesions in lung tissue using imaging techniques also showed that the number of lesions in mice vaccinated with REA or RRA was significantly reduced compared to the control group. Figure 22 D). Finally, lung and spleen cells obtained at 6 weeks and week 1 after the challenge experiment were isolated and added to BMDMs infected with Mycobacterium tuberculosis. The inhibitory effect on intracellular Mycobacterium tuberculosis proliferation was measured, and the results are shown in [Figure 1]. Figure 22 E. For example Figure 22 As shown in Figure E, it was confirmed that only lung and spleen cells from mice immunized with REA significantly inhibited the proliferation of intracellular Mycobacterium tuberculosis. This result confirms that the anti-tuberculosis efficacy of immunized mice is maintained.

[0107] In summary, to date, no tuberculosis vaccine, including live attenuated vaccines, has been reported to induce sterilizing immunity in mouse models. Furthermore, it is known that protein-based vaccines cannot yet replace BCG. However, REA and RRA vaccines, as subunit vaccines, can induce an immune response in mouse tissues that eliminates infection with virtually undetectable bacteria. They are effective for preventative use regardless of BCG administration and have been confirmed as subunit vaccines that can replace BCG.

[0108] sequence list An electronic document containing the sequence list is attached.

Claims

1. A tuberculosis vaccine composition comprising the Rv2299cD2D3-ESAT6-Ag85B(REA) fusion protein.

2. The tuberculosis vaccine composition according to claim 1, characterized in that, The Rv2299cD2D3-ESAT6-Ag85B(REA) fusion protein is encoded by the nucleotide sequence of SEQ NO.

1.

3. The tuberculosis vaccine composition according to claim 1, characterized in that, The Rv2299cD2D3-ESAT6-Ag85B(REA) fusion protein contains the amino acid sequence of SEQ NO.

2.

4. A tuberculosis vaccine composition comprising the Rv2299cD2D3-Rv3463-Ag85B (RRA) fusion protein.

5. The tuberculosis vaccine composition according to claim 4, characterized in that, The Rv2299cD2D3-Rv3463-Ag85B(RRA) fusion protein is encoded by the nucleotide sequence of SEQ NO.

3.

6. The tuberculosis vaccine composition according to claim 4, characterized in that, The Rv2299cD2D3-Rv3463-Ag85B(RRA) fusion protein contains the amino acid sequence of SEQ NO.

4.

7. The tuberculosis vaccine composition according to any one of claims 1 to 6, characterized in that, The tuberculosis vaccine composition induces sterilization of more than 90% of Mycobacterium tuberculosis.

8. A recombinant polynucleotide for tuberculosis sterilization and immunization, comprising the nucleotide sequence described in SEQ NO. 1 or 3.

9. A recombinant polynucleotide for tuberculosis sterilization and immunization, comprising the nucleotide sequence described in SEQ NO. 1 or 3.

Images