KR102122802B1 - Vaccine for treating and preventing tuberculosis comprising B cell loading α-galactosylceramide and ESAT6 - Google Patents

Abstract

The present invention relates to a cell vaccine mediated by B cells loaded with ligands and antigens of natural killer T cells. Specifically, the present invention relates to a cell vaccine having excellent anti-tuberculosis effect by using alpha-galactosylceramide (αGC) as a ligand of natural killer T cells and using ESAT6 as an antigen.
The present invention maximizes antigen delivery by targeting ESAT6 among the antigens of tuberculosis and expressing them in Vaccinia virus, and transducing them into B cells, thereby loading alpha-galactosyl ceramide, a ligand of natural killer T cells, It provides a cell-based vaccine that has an immune response-inducing effect and a CFU-reducing effect on infection.

Description

{Vaccine for treating and preventing tuberculosis comprising B cell loading α-galactosylceramide and ESAT6} with alpha-galactosylceramide and ESAT6 loaded B cells

The present invention relates to a cell vaccine mediated by B cells loaded with ligands and antigens of natural killer T cells. Specifically, the present invention relates to a cell vaccine having excellent anti-tuberculosis effect by using alpha-galactosylceramide (αGC) as a ligand of natural killer T cells and using ESAT6 as an antigen.

Tuberculosis is a chronic infectious disease caused by infection by Mycobacterium tuberculosis ( M. tuberculosis ) and is known as one of the most serious infectious diseases worldwide. In addition, tuberculosis has been known to have the highest mortality rate worldwide since tuberculosis bacteria were discovered (Paulson, T. 2013. Epidemiology: A mortal foe. Nature 502: S2-3). Tuberculosis is known to be caused by poor living conditions due to poverty, overcrowding, malnutrition, alcohol abuse and human immunodeficiency virus (HIV) infection (JAMA 300: 423-430., Lancet 375: 1814-1829 .R, BMC Public Health 9: 450). In addition, patients with chronic renal failure and diabetes are known to have a higher risk of tuberculosis infection (American Journal of Respiratory and Critical Care Medicine 2000;161:S221-S247).

Tuberculosis infection caused by M. tuberculosis is mostly asymptomatic latent tuberculosis, but is known to develop into active tuberculosis at a rate of about 12% (World Health Organization. 2016. Global Tuberculosis Report.World Health Organization, Geneva, Switzerland).

Currently, the BCG (Bacillus-Calmete-Guerin) vaccine is widely used worldwide (Tuberculosis (Edinb) 104: 46-57), and the BCG vaccine functions to prevent tuberculosis meningitis and tuberculosis transmission in childhood, It is known that the protective effect against the initial infection is poor. In addition, the BCG vaccine has no protective effect against latent tuberculosis infection and has been reported to have an efficiency of 0 to 80% against pulmonary tuberculosis. In particular, in adults, the efficiency of the BCG vaccine appears to be very variable (Clin Infect Dis 31 Suppl 3: S64-67). Therefore, it is necessary to develop a new vaccine or a countermeasure to supplement and overcome the limitations and problems of the BCG vaccine.

Numerous studies have been conducted in recent 100 years to develop an effective TB vaccine. There are two types of TB vaccine: preventive vaccine and therapeutic vaccine. Preventive vaccines include priming vaccines and boosting vaccines. The priming vaccine of the preventive vaccine is administered in infancy and is intended for initial exposure to M. tuberculosis. Representatively, attenuated M. tuberculosis vaccines such as recombinant BCG and MTBVAC such as VPM1002 are present (Vaccine 31: 1340-1348. Cell host & microbe 3: 97-103).

In addition, the boosting vaccine of the preventive vaccine is administered to adolescents or adults and is used for the purpose of inducing an increase in the immune response after a priming vaccine or latent tuberculosis infection. Boosting vaccines include subunit vaccines such as Hybrid 1-IC31, Hybrid 1-CAF01, ID93, vaccines using viral vectors such as MVA85A, Crucell Ad35/Aeras 402, Ad5-Ag85A, or cell vaccines such as DAR-901. (Vaccine 32: 7098-7107.Vaccine 33: 4130-4140., Vaccine 34: 2179-2187., Pathog Dis 74: ftw016., Am J Respir Crit Care Med 195: 1171-1180.,PloS one 4: e5856. , Vaccine 33: 3047-3055.).

In the case of therapeutic vaccines, the drug is directly administered in a short period of time, and RUTI, Vaccae (SRL-172), etc. are known (Scandinavian journal of immunology 84: 204-210.J Immune Based Ther Vaccines 9: 3).

Various studies have been conducted to date, but a vaccine for preventing tuberculosis that is superior to the attenuated BCG vaccine has not been developed. In particular, the recent increase in infection due to the emergence of multi-drug resistant tuberculosis bacteria resistant to existing anti-tuberculosis agents and the cross-infection of latent tuberculosis in immunodeficiency patients make the treatment of tuberculosis more difficult. Therefore, it is necessary to develop a tuberculosis vaccine of a new concept different from the existing vaccine, or a tuberculosis vaccine that can complement the existing vaccine.

Vaccine research has been conducted targeting various tuberculosis antigens to develop a new vaccine for prevention and treatment against tuberculosis.

Among antigens, ESAT6 is expressed in M. tuberculosis , whereas it is known that it is not expressed in the commercially available BCG vaccine. However, in inducing an immune response using the ESAT6 antigen, there was a problem in developing an efficient delivery system of ESAT6. In particular, DNA vaccines using ESAT6 and adenovirus vaccines did not reduce CFU for direct tuberculosis infection (Infection and immunity 68: 791-795., Scandinavian journal of immunology 75: 259-265., Molecular medicine reports 14: 1146-1152).

Cell vaccines using dendritic cells have recently shown potential for new vaccines by inducing antigen-specific T cell immunity, but they have few dendritic cells in blood and lymphoid tissues, and have difficulties in proliferation in vitro.

In addition, B cells are present in a large amount in blood and lymphoid tissues and can proliferate in vitro, but there is a need to solve the disadvantages of weak immunogenicity.

The present inventors intend to provide a cell vaccine for the prevention or treatment of tuberculosis, which is mediated by B cells that can proliferate in vitro and have excellent immunogenicity.

Specifically, the present invention provides B cells loaded with ligands and antigens of natural killer T cells. That is, the present invention provides a vaccine having excellent anti-tuberculosis effect by using alpha-galactosylceramide as a ligand of natural killer T cells and ESAT6 as an antigen.

The present inventors target ESAT6 among the antigens of tuberculosis by maximizing the antigen by expressing it in Vaccinia virus, transducing it into B cells, and then loading the alpha-galactosyl ceramide, a ligand of natural killer T cells, to effectively immunize In addition to having a response-inducing effect, a cell-based vaccine having a direct effect of reducing CFU of tuberculosis bacteria was developed.

The present invention relates to a vaccine for the treatment and prevention of tuberculosis, comprising a B cell loaded with a ligand of natural killer T cells and ESAT6.

It is preferred that the ligand is alpha-galactosylceramide.

The ESAT6 can be prepared by introducing the ESAT6 gene sequence of the M. tuberculosis strain into a vaccinia virus vector.

The cell vaccine of the present invention not only increases the content of MHC class II, CD1d and CD86, but also increases the content of TNFα and IFNγ, thereby having an excellent immune response inducing effect.

In addition, the cell vaccine of the present invention can be administered in combination or sequentially with the BCG vaccine. When administered sequentially or in combination with the cytoplasmic vaccine and BCG vaccine, each has a better anti-tuberculosis effect than when administered alone.

The present invention provides a method for preparing a vaccine for the treatment and prevention of tuberculosis comprising the following steps:

(i) expressing ESAT6 of the M. tuberculosis strain in vaccinia virus;

(ii) transfecting the expressed ESAT6 into B cells; And

(iii) Loading alpha-galactosylceramide into B cells.

The cell vaccine of the present invention can not only induce an excellent T cell immune response, but also has an excellent effect of directly reducing the CFU of Mycobacterium tuberculosis.

In addition, since ESAT6, which is not expressed by the BCG vaccine known as the existing tuberculosis vaccine, is used as an antigen, the limitations of BCG can be compensated for when used in combination with BCG, so that it can be developed as a new tuberculosis vaccine.

1 shows the MFI values of MHC class II in each group.
2 shows the MFI value of CD1d in each group.
3 shows the MFI value of CD86 in each group.
4 shows the experimental results of TFNα and IFNγ in each group.
5 shows the amount of IFNγ secretion in each group.
6 shows the results of measuring cytokine through CBA in an in vivo infection experiment.
7 shows the result of confirming the degree of inflammation by staining the lungs by H & E staining method.
8 shows the results of CFU measurement in the lung.
9 shows the results of CFU measurement in the liver.
10 shows the experimental results when BCG and the cell vaccine were administered.

The present invention relates to a vaccine for the treatment and prevention of tuberculosis, comprising a B cell loaded with a ligand of natural killer T cells and ESAT6.

The ligands of the natural killer T cells are derived from alpha-galacturonosylceramide and alpha-glucuronosylceramide from M. tuberculosis of Sphingomonas spp. Phosphatidylinositoltetramannoside, the autoantigen isoglobotrihexosylceramide, ganglioside GD3 (ganglioside GD3), phosphatidylcholine (phosphatidylcholine), beta-galactosyl galactosylyl, Leishmania surface sugar-binding lipophosphoglycan, glycoinositol phospholipids, beta-anomeric galactosylamide, an analog of alpha-galactosyl ceramide, beta-anomeric galcerer, alpha-ano Mer-galactosyl ceramide (alpha-anomeric GalCer), a variant of alpha-galactosyl ceramide (J. Am. Chem. Soc. 126:13602, 2004), and bacterial lipid antigens such as Nocardia falcica ( Nocardia falcinica ); glucose monomycolate (Molucose monomycolate, Moody, DB et al. J. Exp. Med. 192:965, 2000). Preferably, the ligand of natural killer T cells may be alpha-galactosylceramide.

It has been reported that alpha-galactosylceramide does not induce toxicity in rodents and monkeys (Nakata etal., Cancer Res., 58: 1202-1207, 1998). Mice injected with 2200 μg/Kg of alpha-galactosylceramide are reported to have no side effects (Giaccone et al., Clin. Cancer Res., 8: 3702, 200). Even in ongoing clinical trials, some side effects such as mild headaches have been reported by systemic administration of alpha-galactosylceramide (Mie Nieda et al., Blood, 103: 383-389, Giacone et al., Clin. Cancer Res. , 8: 3702, 200), which can be prevented by administration of paracetamol, and does not necessarily show minor systemic side effects (Giaccone et al., Clin. Cancer Res., 8: 3702, 200).

Even in the present invention, alpha-galactosyl ceramide does not cause dose-limiting toxicity (50-4800 µg/m ² ) and shows resistance even in a dose escalation study. Is considered a safe substance.

Viruses that can be introduced into B cells for the expression of tuberculosis antigens include adenovirus, retrovirus, vaccinia virus, pox virus, and sindbis virus And the like, preferably vaccinia virus, but is not limited thereto.

In addition to the method using the virus, methods applicable to antigen gene transfer include (1) DNA binding to liposomes and transduction to protect DNA from enzymatic degradation or absorption into endosomes (2) A method of increasing the efficiency of DNA delivery to a cell by binding a molecular conjugate composed of a protein to a DNA or a synthetic ligand (for example, asialoglycoprotein, transferrin, polymer IgA) )), (3) A new DNA delivery system using PTD (Protein transduction domain), which increases the efficiency of DNA delivery to cells, and delivers antigen genes (e.g., Mph-1), (4) peptides By applying the antigen protein to B cells using a method such as, the B cells can present the antigen.

The vaccine of the present invention can be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In order to formulate a formulation for parenteral administration, the cell vaccine of the present invention is mixed with a stabilizer or a buffer to prepare a solution or suspension and formulated into a unit dosage form of an ampoule or vial. The vaccine can be administered in one to several times in an amount effective to stimulate an immune response in a patient.

"Antigen" of the present invention, when entered into the host's body, any substance that is recognized by the host's immune system and can cause an immune response (eg, protein, peptide, cancer cell, glycoprotein, glycolipid, live virus, dead Virus, DNA, etc.). In the present invention, it means a tuberculosis bacterium and a protein, peptide, glycoprotein, etc. expressed by the tuberculosis bacterium.

Antigens can also be provided in purified or unpurified form, preferably in purified form.

“ESAT6 (Early Secretory Antigen Target-6)” of the present invention is an antigen expressed by Mycobacterium , and the expression level after the initial infection stage of ESAT6 is stabilized at 0.8 transcripts per M. tuberculosis CFU. This level remains stable until at least 100 days after infection (Rogerson, BJ et al. 2006). In addition, transcription data is supported by immune data showing strong T cell recognition of ESAT6 at post-infection stages at the site of infection. This structural expression pattern is an important feature showing that pathogens achieve functions that depend on genes that need to be structurally expressed in order to survive in an immune host.

Hereinafter, examples of the present invention will be described. However, the following examples are only illustrative of the present invention, but the present invention is not limited by the following examples.

[Example 1]

Construction of the transfer vector pVVT1-C7L-tPA-ESAT6

Sequence modification was performed to increase the expression rate in cells using the ESAT6 gene sequence of the M. tuberculosis H37Rv strain. After humanized codon optimization was performed using the M. tuberculosis ESAT6 gene sequence, gene synthesis was performed with the secretory signal peptide tPA (tissue plasmodium activator) sequence upstream of the gene. The synthesized gene was digested using Sfi1 restriction enzyme and ligated to the pVVT1-GFP-C7L plasmid, a vaccinia virus delivery vector, to produce pVVT1-C7L-tPA-ESAT6, which was transformed into E. coli DH5α ( transformation). The produced pVVT1-C7LtPA-ESAT6 confirmed the nucleotide sequence of the inserted gene through gene sequencing using VVTK-F and VVTK-R primers, mass cultured in a medium containing ampicillin, and midiprep kit (midiprep kit) It was separated by mass. Here, the sequencing primer sequence is shown in Table 1 below.

primer order VVTK-F 5`-TTTGAAGCATTGGAAGCAACT-3` VVTK-R 5`-ACGTTGAAATGTCCCATCGACT-3`

[Example 2]

Recombinant virus production

The vaccinia virus strain KVAC103 (Accession No. KCCM11574P) and the cloned transfer vector were co-transfected into Vero cells. Vero cells were seeded at 1×10 ⁵ cells/well using OPTI-MEM (2% FBS) medium in 12-well plates one day prior to transfection. After infecting the parental virus KVAC103 strain at a rate of 0.02 MOI for 2 hours, sprinkle the mixed solution of lipofectin 2000 and the cloned delivery vector 1.5 µg on the pre-infected Vero cells, and after 4 hours of infection, 5% CO CPE (cytopathic effect) was confirmed while incubating for 3 to 4 days in a ₂ incubator.

After 2 to 3 passages of the recombinant virus were cultured using Vero cells cultured in 5% FBS DMEM medium, 2 plaque isolation experiments were performed to perform plaque isolation experiments. It was confirmed whether or not the recombinant virus was successfully produced. As a result of the Western blot experiment, the recombinant virus was confirmed to have protein expression of the antigen gene.

[Example 3]

Recombinant virus culture and concentration

Recombinant virus culture and concentration were confirmed by CPE for 2 to 3 days after infection of the recombinant virus produced in Vero cells (SFM, OptiMEM). When CPE was confirmed, freezing and thawing were repeated 2-3 times to break the cells and recover the medium and cells. After centrifugation at 4000 rpm for 30 minutes to recover only the supernatant from which the cell residue was removed, the recombinant virus was concentrated using an amicon filter of pore size 100,000 NMWL.

[Example 4]

B cell isolation from mouse spleen cells

In the present invention, female C57BL/6, 6-8 weeks of age, was used. The mouse is ORIENTBIO Inc. Purchased from, and all mice were preserved at the laboratory animal center of Kangwon National University.

To purely isolate B cells from mice, spleens were removed from the mice and homogenized. Red blood cells were removed by splenocyte B220 ⁺ using CD45R/B220z biotin (BD bioscience, Cat.553085) and Anti-Biotin Microbeads (Miltenyi Biotec, Cat.130-090-485). 10ul of CD45R/B220 biotin was added to 990ul of PBS+FBS 1% (hereinafter referred to as “PBS+”) and mixed with spleen cells from which red blood cells were removed. Shake at 5°C every 5 minutes and incubate for a total of 15 minutes. After incubation, after washing and centrifugation by adding PBS+, 100ul of Anti-Biotin Microbeads was added to PBS+900ul, mixed with biotin-attached cells, shaken every 5 minutes at 4°C, and incubated for a total of 15 minutes. After incubation, cells were washed and centrifuged, and finally cells were prepared in PBS+1 ml. Thereafter, the LS column (Miltenyi Biotec, Cat.130-042-401) was placed in a magnetic field and pre-washed twice with PBS+ 3 ml each. Then, cells were added and washed twice with PBS+3 ml each. Finally, after the LS column was removed from the magnetic, placed in a new conical tube, 5 ml of PBS+ was added, and cells were separated using a piston.

[Example 5]

T cell isolation from mouse spleen cells

To purely separate T cells from mice, spleens were extracted from the mice and homogenized. After staining the CD8alpha-PE antibody on the spleen cells, the cells were separated using Anti-PE Microbeads (Miltenyi Biotec, Cat.130-097-054). In the cell separation method, homogenized splenocytes were removed by using RBC lysis buffer. 3-5 ul of CD8alpha-PE antibody was mixed with 100 ul of PBS+ and put into spleen cells with red blood cells removed. After incubation at 4°C for 10 minutes, washing and centrifugation were performed. The cells were released in PBS+80ul, and 20ul of Anti-PE Microbeads was added, followed by incubation at 4°C for 15 minutes, followed by washing and centrifugation. Finally, cells were prepared in 1 ml of PBS+. Thereafter, the LS Column (Miltenyi Biotec, Cat.130-042-401) was placed in a magnetic field and pre-washed twice with PBS+3 ml each. Then, cells were added and washed twice with PBS+3 ml each. Finally, after the LS column was removed from the magnetic, placed in a new conical tube, 5 ml of PBS+ was added, and cells were separated using a piston.

Cells selected positively through the LS Column are CD8α ⁺ cells, and cells separated negatively were further separated using a CD4 ⁺ T Cell Isolation Kit mouse (Miltenyi Biotec, Cat. 130-104-454). CD4 ⁺ T cell separation method from negatively separated cells was centrifuged and the cells obtained by negative selection were prepared in PBS + 100ul. In the CD4 ⁺ T Cell Isolation Kit, 30ul of Biotin-Antibody Cocktail was added to the components. After incubation at 4°C for 10 minutes, after washing and centrifugation, cells were prepared in 200ul of PBS+ and 60ul of Anti-Biotin Microbeads was added. After incubation at 4°C for 15 minutes, washing and centrifugation finally prepared in PBS+1 ml. Thereafter, the LS Column (Miltenyi Biotec, Cat.130-042-401) was placed in a magnetic field and pre-washed twice with PBS+3 ml each. Then, after placing a new conical tube under the column, cells were added and washed twice with PBS+3 ml each. Since the CD4 ⁺ T cell isolation kit used is negative selection, the cells received in the conical tube during this process are CD4 ⁺ T cells.

[Example 6]

Dendritic cell isolation from mouse spleen cells

To purely separate dendritic cells from mice, spleens were extracted from the mice and homogenized. After treating collagenase D (Worthington, LS0004186) 1mg/ml to homogenized spleen cells, the reaction was performed at 37°C for 30 minutes, followed by centrifugation to obtain cells. Thereafter, CD11c ⁺ cells were isolated using CD11c Microbeads (Miltenyi Biotec, Cat. 130-108-338). In the cell separation method, homogenized splenocytes were removed by using RBC lysis buffer. Splenocytes from which red blood cells were removed were released in PBS+400ul, and CD11c Microbead 100ul was added, followed by incubation at 4°C for 10 minutes. After washing and centrifugation, cells were finally prepared in 1 ml. Thereafter, the LS Column (Miltenyi Biotec, Cat.130-042-401) was placed in a magnetic field and pre-washed twice with PBS+3 ml each. Then, cells were added and washed twice with PBS+3 ml each. Finally, after the LS column was removed from the magnetic, placed in a new conical tube, 5 ml of PBS+ was added, and cells were separated using a piston.

[Example 7]

Cell vaccine production and T cell response confirmation

After separating the B cells, transfection was performed by co-culturing the B cells and the Vacciniavirus-ESAT6 recombinant virus prepared in Example 2 in RPMI (WELGENE, Cat.LM 011-01) medium without serum in a 6-well plate. Induced.

After co-cultivation for 2 hours in a 37° C. CO ₂ incubator, FBS (Gibco, Cat.26140-079) and RPMI medium were added, followed by alpha-galactosyl ceramide (hereinafter referred to as “αGC”) (Enzo, Cat.BML The well loading -SL232) was added at a concentration of 1 μg/ml. After further incubation for 22 hours in a 37° C. CO ₂ incubator, cells were obtained and washed 3 times with 1X PBS, and then the group administered with B cells alone, B/αGC, and B/αGC/VacESAT6 in C57BL/6 mice, respectively. In the group injected with the BCG strain, the T cell response in vivo was confirmed.

The flow cytometer used a BD Biosciences FACSVerse flow cytometer. Antibodies are PE Mouse Anti-Mouse H-2kb (BD bioscience, Cat.553570), PE Rat Anti-Mouse IA/IE (BD bioscience, Cat.557000), alpha GalCer:CD1d Complex Monoclonal Antibody (L363) PE (ebioscience, Cat.12-2019-82), PE Hamster Anti-Mouse CD80 (BD bioscience, Cat.553769), PE Rat Anti-Mouse CD86 (BD bioscience, Cat.553692), (550954) PerCP-Cy™Rat Anti-Mouse CD4 (BD bioscience, Cat.550954), (553033) PE Rat Anti-Mouse CD8α (BD bioscience, Cat.553033), TNF alpha Antibody, APC (Monoclonal, MP6-XT22) (ebioscience, Cat.17-7321-82 ), IFN gamma Antibody, PE (Monoclonal, XMG1.2) (ebioscience, Cat.12-7311-82), Anti-Mouse CD16/CD32 Purified (ebioscience, 14-0161-85) were used and flow cytometry after cell staining Cell populations were measured using an analyzer.

[Example 8]

M. Kasnsasii Preparation of strain

Mycobactirum Kansasii ( M. Kansasii ) is a strain expressing ESAT6, the strain was stored at 1.5x10 ⁹ CFU/ml, and mouse infection experiments were performed with 10 ⁷ CFU/mouse.

As a medium for measuring CFU of the bacteria, Difco™Middlebrook 7H10 Agar (BD, Cat.262710) and Difco™Middlebrook 7H9 Broth (BD, cat.271310) were used. The 7H10 agar medium was prepared by adding OADC as a supplement, and finally put in a Petri dish to harden it. OADC is Sodium Chloride (Duchefa, Cat.S0520.5000), Dextrose (SHOWA, Cat.0402-2160), Bovine Albumin Fraction V (MPBio, Cat.160069), Catalase (Sigma, Cat.C1345), Oleic acid (Sigma , Cat.O1383) was diluted in tertiary sterile distilled water.

7H9 broth medium was used as supplement by making ADC and finally dispensing it into 50ml conical tube. ADC was prepared by diluting Sodium Chloride, Dextrose, Bovine Albumin Fraction V, Catalase in tertiary sterile distilled water. OADC and ADC were used as filters.

[Example 9]

M. Kasnsasii How to identify CFU in tissues after infection

M. Kansasii diluted stock vial in 1x PBS and proceeded infection through intravenous injection with 10 ⁷ CFU/mouse. Two weeks after infection, mice were sacrificed to obtain lung and liver tissue. Each tissue was homogenized at 125 mg/ml to titrate the medium, homogenized with a buffer solution containing 0.04% Tween80 in 1x PBS for lung tissue, and homogenized with a buffer solution containing 1 mM EDTA in 1x PBS for liver tissue. . Homogenized tissue was diluted to 10 ^-1 -10 ^-5 with 7H9 broth (ADC included). Then, the tissue solution diluted in 7H10 agar was smeared and cultured in a 37°C incubator for 3 weeks, and the number of colonies was counted to calculate CFU.

[Example 10]

How to check cytokine and RNA level in tissue

After the homogenized tissue was used for CFU measurement by the above method, the supernatant was obtained and stored after centrifugation at 13000 rpm, 10 minutes, and 4°C. The cytokine in the tissue was confirmed using a BD™ Cytometric Bead Array (CBA) Mouse Inflammation kit (BD bioscience, Cat.552364) as the tissue supernatant. In addition, RNA extraction was performed using QIAamp Viral RNA Mini Kit (QIAGEN, Cat.52906) as a tissue supernatant, and then qPCR was performed using THUNDERBIRD®Probe qPCR Mix (Toyobo, Cat.QPS-201) to confirm gene levels.

[Test Example 1]

Confirmation of the induction of immune response of a cell vaccine (measurement of MFI values of MHC class II, CD1d and CD86)

After the vacciniaESAT6 was transfected into B cells, an in vitro culture experiment was conducted to confirm whether the experimental method of loading αGC induces an immune response in B cells.

After separating B220 ⁺ cells from the spleen, they were seeded on plates. Control (B cells only), B/BCG (BG introduced into B cells), B/αGC (αGC introduced into B cells), B/vacciniaESAT6 (Introduced vacciniaESAT6 into B cells), B/αGC/vacciniaESAT6 (B cells) B cells were introduced with vacciniaESAT6 and αGC) and cultured for 24 hours, followed by fluorescence staining to confirm with a flow cytometer.

As shown in Figure 1, it was confirmed that the MFI value of MHC class II in B220 ⁺ cells increased in all other groups compared to the control group. In addition, it was confirmed that the B/αGC/vacciniaESAT6 group significantly increased the MFI value compared to other B/BCG, B/αGC and B/vacciniaESAT6 groups.

As shown in FIG. 2, the MFI value of CD1d, a molecule indicative of natural killer T cells (hereinafter referred to as “NKT”), was increased in the B/αGC group, which is a ligand of NKT. In addition, it was confirmed that the B/αGC/vacciniaESAT6 group increased more than the B/αGC group.

As shown in FIG. 3, it was confirmed that the MFI value of CD86, a co-stimulatory molecule of T cell, was increased in all other groups compared to the control group. In addition, it can be seen that the B/αGC/vacciniaESAT6 group showed the greatest increase in MFI value compared to other B/BCG, B/αGC and B/vacciniaESAT6 groups.

Through the above test results, it was confirmed that the cell vaccine of the present invention can sufficiently induce an immune response.

[Test Example 2]

Confirmation of induction of immune response of cell vaccine (animal experiment)

T cell activity, co-stimulatory molecule, and cytokine secreted by T cells, TNFα and IFNγ, are known to be very important for the mechanism of vaccine response and anti-tuberculosis. Since it was confirmed in Test Example 1 that the cell vaccine of the present invention induces an immune response sufficiently, an immune response experiment after administration to the mouse was further performed.

BCG was administered intramuscularly, and cell-based vaccine was administered intravenously. Cell-based vaccines were boosted after 2 weeks. After 2 weeks of boosting, CD4 ⁺ T cells from the spleens of each group and CD11c ⁺ dendritic cells from the spleens of Naive mice (C57BL/6) were isolated and co-cultured.

After co-cultured cells were stimulated with ESAT6-specific CD4 peptide, H37Rv (live) 0.1 MOI, H37Rv (Heat-Killed) 0.1 MOI as a stimulator, and co-cultured in a 37°C CO ₂ incubator for 72 hours. Thereafter, cells were obtained, stained with a fluorescent sample, and secreted amounts of TNFα and IFNγ from CD4 ⁺ T cells were confirmed using a flow cytometer.

As shown in FIG. 4, it was confirmed that the number of individuals positive for both TFNα and IFNγ in CD4 ⁺ T cells increased in the B/αGC/vacESAT6 group regardless of the presence or absence of a stimulus.

As shown in FIG. 5, as a result of confirming the secretion amount of IFNγ through the ELISA technique as a culture supernatant, the IFNγ secretion amount of the B/αGC/vacESAT6 group was significantly increased for the remaining stimulation sources except for the H37RV (live) stimulation source. I could confirm.

Through the above results, it was confirmed that the cell vaccine of the present invention can sufficiently activate T cells even in in vivo conditions.

[Test Example 3]

Comparison of efficacy of cell vaccine and BCG vaccine

Through the test examples 1 and 2, it was sufficiently confirmed that induction of an immune response due to a cell-based vaccine in vitro and in vivo. Therefore, in order to confirm the protective and therapeutic effects against infection, an in vivo infection experiment was performed using the M. kansasii strain expressing ESAT6.

Through in vitro experiments, it was confirmed that the cell vaccine of the present invention induces an immune response against Mycobacterium tuberculosis (MTB), and further verifying whether it is effective in Mycobacterium expressing ESAT6 by verifying the effect in the M. Kansasii infection model Did.

BCG was administered with 10 ⁵ CFU/mouse intramuscular injection, B/αGC/vacESAT6 was administered intravenously, and a week after administration, cell-based vaccine was boosted. Two weeks after the first administration, infection was performed by intravenous administration of 10. ⁷ CFU/mouse M. kansasii , and two weeks after the infection progressed, an experiment was conducted to confirm the degree of infection.

As a result of the test, there was no difference in body weight, but when comparing the sizes of the spleen, lung, and liver, it was confirmed that the size was increased in the infection group. In addition, since the size of the spleen, lung, and liver was known to increase after M. kansasii infection, it was confirmed that the infection progressed normally in the experimental group.

As shown in Figure 6, as a result of measuring the cytokine in the lungs through CBA, it was confirmed that the inflammatory cytokine of TNF, MCP-1 and IL-6 was decreased in the group administered with BCG and cytosolic vaccine compared to the infection group.

As shown in FIG. 7, the lungs were stained by H&E staining to confirm the degree of inflammation. As a result, the difference between the infection group and the administration group was not large.

However, as shown in Figs. 8 and 9, the lungs and liver were homogenized and smeared on the medium, and after 3 weeks, the bacteria were counted. As a result, CFU in the BCG and cell vaccine administration group was compared to the infection group in the lung and liver. It was confirmed that the decrease, CFU reduction in the cell vaccine administration group was found to be greater in the BCG administration group.

From the above results, administration of the cell vaccine of B/αGC/vacESAT6 against M. kansasii infection not only reduces the inflammatory cytokine, but also has the effect of directly reducing the CFU in the lungs and liver, and the effect of reducing the CFU is BCG administration By confirming the larger, it can be seen that it has an excellent anti-tuberculosis effect.

[Test Example 4]

Effect test in the case of second vaccination with cell vaccine after BCG vaccination

When boosting administration of B/αGC/vacESAT6 cell vaccine after administration of BCG, the experiment was conducted as follows to confirm whether the effect of BCG can be further enhanced.

Cell vaccines were administered to the group preemptively administered with BCG two weeks later by boosting, and compared with the single group administered with BCG. After 2 weeks of boosting administration, infection progressed by intravenous injection of M. kansasii and the body weight of the mice was measured for 3 weeks.

As shown in FIG. 10, when the initial weight of the non-infected control group was 100%, the weight of the third week of the control group was 110%, and the weight of the third week of the infection group was about 90%. In addition, the group administered with BCG alone maintained the original body weight at about 100%, and the group administered with boosting with the cell vaccine showed a body weight greater than the group administered with BCG alone at about 105%.

After all, through the test results, it was confirmed that the B/αGC/vacESAT6 cell vaccine can induce a sufficient T cell immune response by itself, and when combined with BCG known as an existing anti-tuberculosis vaccine, the effect of BCG It was confirmed that it can be increased and supplemented.

Claims (7)

A vaccine for the treatment or prevention of tuberculosis, comprising B cells loaded with alpha-galactosylceramide (α-galactosylceramide) and ESAT6.
delete The vaccine according to claim 1, wherein ESAT6 is expressed in vaccinia virus.
The vaccine of claim 1, wherein the vaccine increases the content of MHC class II, CD1d and CD86.
The vaccine of claim 1, wherein the vaccine increases the content of TNFα and IFNγ.
The vaccine of claim 1, wherein the vaccine can be administered in combination or sequentially with the BCG vaccine.
Method for preparing a vaccine for the treatment or prevention of tuberculosis comprising the following steps:
(i) expressing ESAT6 of the Mycobacterium tuberculosis strain in vaccinia virus;
(ii) transfecting the expressed ESAT6 into B cells; And
(iii) Loading alpha-galactosylceramide into B cells.

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