WO2019216543A1

WO2019216543A1 - CELL-BASED VACCINE FOR TREATING AND PREVENTING TUBERCULOSIS, COMPRISING B CELLS LOADED WITH α-GALACTOSYLCERAMIDE AND ESAT6

Info

Publication number: WO2019216543A1
Application number: PCT/KR2019/003029
Authority: WO
Inventors: 고현정; 권보은; 박은경; 안재희; 정현진; 정혜숙; 신은경; 유정식
Original assignee: 강원대학교 산학협력단
Priority date: 2018-05-11
Filing date: 2019-03-15
Publication date: 2019-11-14
Also published as: KR20190129424A; KR102122802B1

Abstract

The present invention relates to a cell-based vaccine mediated by B cells loaded with a ligand of natural killer T cells and an antigen. Specifically, the present invention relates to a cell-based vaccine exhibiting an excellent antituberculous effect by using α-galactosylceramide (αGC) as a ligand of natural killer T cells, and using ESAT6 as an antigen. The present invention provides a cell-based vaccine whereby, among antigens of tuberculosis, ESAT6 is targeted and expressed in vaccinia virus, and thus antigen transfer is maximized, and after transducing same to B cells, αGC, which is a ligand of natural killer T cells, is loaded, and thus the cell-based vaccine exhibits an effective immune response-inducing effect and a CFU reduction effect against infections.

Description

Cell vaccine for the treatment and prevention of tuberculosis comprising alpha-galactosyl ceramide and B cells loaded with ESAT6

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

Most of the tuberculosis infections caused by M. tuberculosis are asymptomatic tuberculosis, but most of them are known to develop active tuberculosis in about 12% (World Health Organization. 2016. Global Tuberculosis Report.World Health Organization, Geneva, Switzerland).

Although the BCG (Bacillus-Calmete-Guerin) vaccine is now widely used worldwide (Tuberculosis (Edinb) 104: 46-57), the BCG vaccine is responsible for preventing the transmission of tuberculous meningitis and tuberculosis in childhood. The protective effect against early infections is known to be poor. In addition, the BCG vaccine is reported to have no preventive effect against latent tuberculosis infection and have an efficiency of 0 to 80% for pulmonary tuberculosis. Especially 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 for supplementing and overcoming the limitations and problems of the BCG vaccine.

Numerous studies have been conducted in the last 100 years to develop effective TB vaccines. There are two types of tuberculosis vaccines: prophylactic and therapeutic vaccines. Prophylactic vaccines include priming vaccines and boosting vaccines. Priming vaccines of the prophylactic vaccines are administered during infancy and are intended for initial exposure to M. tuberculosis. Typically there are recombinant BCG such as VPM1002 and attenuated M. tuberculosis vaccines such as MTBVAC (Vaccine 31: 1340-1348. Cell host & microbe 3: 97-103).

In addition, the boosting vaccine of the prophylactic vaccine is administered to adolescents or adults and is used for the purpose of inducing an increase in the immune response after priming vaccine or latent tuberculosis infection. Boosting vaccines include subunit vaccines such as Hybrid 1-IC31, Hybrid 1-CAF01, ID93, etc., 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, direct administration of drugs in a short period of time is known, such as RUTI, Vaccae (SRL-172) (Scandinavian journal of immunology 84: 204-210. J Immune Based Ther Vaccines 9: 3).

Various studies have been made so far, but no tuberculosis vaccines have been developed that are far superior to the attenuated BCG vaccine. In particular, the recent increase in infection due to the emergence of multidrug-resistant tuberculosis resistant to the existing anti-tuberculosis agent and cross-infection of latent tuberculosis in immunodeficiency patients make tuberculosis treatment 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 supplement the existing vaccine.

Vaccine studies have been conducted targeting various TB antigens to develop new prophylactic and therapeutic vaccines against tuberculosis.

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

Recently, cell vaccines using dendritic cells have shown the possibility of new vaccines by inducing antigen-specific T cell immunity, but few dendritic cells are present in blood and lymphoid tissues.

In addition, B cells are present in large amounts in blood and lymphoid tissues and can be proliferated in vitro, but there is a need to solve the disadvantage of having weak immunogenicity.

The present inventors intend to provide a cell vaccine for preventing or treating tuberculosis, which is mediated by B cells capable of mass proliferation in vitro and having 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-galactosyl ceramide as a ligand of natural killer T cells and using ESAT6 as an antigen.

The present inventors maximized antigens by expressing them in vaccinia virus by targeting ESAT6 among the antigens of tuberculosis, transducing them to B cells, and loading alpha-galactosyl ceramide, a ligand of natural killer T cells, for effective immunity. In addition to the response inducing effect, a cell-based vaccine was developed which has the effect of directly reducing the CFU of Mycobacterium tuberculosis.

The present invention relates to a vaccine for treating or preventing tuberculosis comprising a ligand of natural killer T cells and B cells loaded with ESAT6.

Preferably, the ligand is alpha-galactosyl ceramide.

The ESAT6 may be prepared by introducing an ESAT6 gene sequence of 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 or CD86, but also has an excellent immune response inducing effect by increasing the content of TNFα or IFNγ.

In addition, the cell vaccine of the present invention may be administered in combination or sequentially with a BCG vaccine. When administered sequentially or in combination with cell vaccines and BCG vaccines, they have a better anti-tuberculosis effect than when administered alone.

The present invention provides a method for preparing a vaccine for treating or preventing tuberculosis, comprising the following steps:

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

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

(iii) loading alpha-galactosylceramide into B cells.

The cell vaccine of the present invention not only induces 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 an existing tuberculosis vaccine, is used as an antigen, when combined with BCG, the limitation of BCG can be compensated for, and a new tuberculosis vaccine can be developed.

1 shows MFI values of MHC class II in each group.

2 shows the MFI value of CD1d in each group.

3 shows MFI values of CD86 in each group.

Figure 4 shows the experimental results of TFNα and IFNγ in each group.

Figure 5 shows the IFNγ secretion in each group.

Figure 6 shows the results of measuring cytokine via CBA in in vivo infection experiments.

Figure 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 measurements in the lungs.

9 shows the results of CFU measurements in the liver.

10 shows the experimental results when BCG and cell vaccines were administered.

Ligands of the natural killer T cells are derived from alpha-galacturonosylceramide, alpha-glucuronosylceramide and M. tuberculosis of Sphingomonas spp. Phosphatidylinositoltetramannoside, its autoantigen isoglobotrihexosylceramide, ganglioside GD3, phosphatidylcholine, beta-galactosyl ceramide Surface glycosylated lipophosphoglycan, glycoinositol phospholipids, beta-anomeric galactosyl ceramides, analogs of alpha-galactosyl ceramides, alpha-anomers Galactosylceramide (alpha-anomeric GalCer), variant of alpha-galactosylceramide (J. Am Som. 126: 13602, 2004), and glucose monomycolate from bacterial lipid antigens such as Nocardia falcinica, Moody, DB et al. J. Exp. Med. 192: 965, 2000). Preferably the ligand of the natural killer T cell may be alpha-galactosyl ceramide.

Alpha-galactosylceramide is reported not to induce toxicity in rodents and monkeys (Nakata et al., 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). In ongoing clinical trials, side effects such as minor headaches have been reported by systemic administration of alpha-galactosyl ceramide (Mie Nieda et al., Blood, 103: 383-389, Giaccone et al., Clin. Cancer Res. , 8: 3702, 200), which can be prevented by administration of paracetamol and not necessarily systemic side effects (Giaccone et al., Clin. Cancer Res., 8: 3702, 200).

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

Viruses that can be introduced into B cells for tuberculosis antigen expression include Adeno virus, Retro virus, Vaccinia virus, Pox virus, Sindbis virus And the like, preferably vaccinia virus, but are not limited thereto.

In addition to using the virus, a method applicable to antigen gene transfer includes (1) a method of binding DNA to a liposome to transduce it to protect the DNA from enzymatic degradation or to absorb it into an endosome, (2) A method of increasing the efficiency of DNA delivery to a cell by binding a molecular conjugate or synthetic ligand composed of protein to DNA (e.g., asialoglycoprotein, transferrin, polymer IgA (polymeric IgA) )), (3) A new DNA delivery system using PTD (Protein transduction domain) to deliver antigen genes by increasing DNA transfer efficiency into cells (eg, Mph-1), (4) using peptides By applying the antigenic protein to the B cells using a method such as, it is possible to allow the B cells to present the antigen.

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

"Antigen" of the present invention is any substance that can be recognized by the host's immune system and trigger an immune response when it enters the host's body (eg, proteins, peptides, cancer cells, glycoproteins, glycolipids, live viruses, dead). Virus, DNA, etc.). In the present invention, it means the tuberculosis bacteria and proteins, peptides, glycoproteins and the like expressed by the tuberculosis bacteria.

The antigen may also be provided in purified or unpurified form, preferably in purified form.

"ESAT6 (Early Secretory Antigen Target-6)" of the present invention is Mycobacterium -expressing antigen, and the expression level after the initial stage of infection of ESAT6 is stabilized at 0.8 transcript per M. tuberculosis CFU. These levels remain stable for at least 100 days after infection (Rogerson, BJ et al. 2006). Transcription data is also supported by immune data showing strong T cell recognition of ESAT6 at the post-infection stage of infection. This structural expression pattern is an important feature that shows that the pathogen achieves a function that depends on the genes that need to be structurally expressed to survive in the immune host.

Hereinafter, examples of the present invention will be described. However, the following examples are merely to illustrate the 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 M. tberculosis H37Rv strain. Humanized codon optimization was performed using the M. tuberculosis ESAT6 gene sequence, followed by gene synthesis with the tPA (tissue plasmodium activator) sequence, an intracellular secreted signaling peptide upstream of the gene. The synthesized gene was ligated to the vaccinia virus transfer vector pVVTT1-GFP-C7L plasmid after cleavage using Sfi1 restriction enzyme to construct pVVT1-C7L-tPA-ESAT6, which was transformed into E. coli DH5α. transformation). The prepared pVVT1-C7LtPA-ESAT6 confirmed the nucleotide sequence of the inserted gene through gene sequencing using the VVTK-F and VVTK-R primers. Mass separation. Here, the sequencing primer sequences are shown in Table 1 below.

프라이머primer	서열order
VVTK-FVVTK-F	5`-TTTGAAGCATTGGAAGCAACT-3`5`-TTTGAAGCATTGGAAGCAACT-3`
VVTK-RVVTK-R	5`-ACGTTGAAATGTCCCATCGACT-3`5`-ACGTTGAAATGTCCCATCGACT-3`

Example 2

Recombinant virus production

The transfer vector cloned with KVAC103 (Accession No. KCCM11574P), a vaccinia virus strain, was 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 infection with the parental virus KVAC103 strain for 2 hours at a rate of 0.02 MOI, 1.5 μg mixture of the lipofectin 2000 and the cloned delivery vector were sprayed onto the pre-infected Vero cells and infected for 4 hours, followed by 5% CO CPE (cytopathic effect) was confirmed by incubating for 3 to 4 days in _two incubators.

Two to three passages of recombinant virus were carried out using Vero cells cultured in 5% FBS DMEM medium, followed by two plaque isolation experiments. Was performed to confirm the success of recombinant virus production. The produced recombinant virus was confirmed by Western blot experiment protein expression of the antigen gene.

Example 3

Recombinant Virus Culture and Concentration

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

Example 4

B cell isolation from mouse splenocytes

In the present invention, female C57BL / 6, 6-8 weeks old, was used. The mouse is ORIENTBIO Inc. And all mice were preserved at Kangwon National University.

In order to purely separate mouse-derived B cells, spleens were removed from the mice and homogenized. Red blood cells were removed by splenic cells 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 splenocytes from which red blood cells were removed. Shake every 5 minutes at 4 ℃ incubated for a total of 15 minutes. After incubation and washing and centrifugation by adding PBS +, 100ul of Anti-Biotin Microbeads was added to 900ul of PBS +, mixed with biotin-coated cells, shaken every 5 minutes at 4 ° C, and incubated for 15 minutes. After incubation, the cells were washed and centrifuged, and finally, the cells were prepared in 1 ml of PBS +. The LS column (Miltenyi Biotec, Cat. 130-042-401) was placed on the magnetic and pre-washed twice with 3 ml of PBS +. Cells were then added and washed twice with 3 ml of PBS +. Finally, the LS column was removed from the magnetic and placed in a new conical tube. Then, 5 ml of PBS + was added and the cells were separated using a piston.

Example 5

T cell isolation from mouse splenocytes

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

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

Example 6

Dendritic Cell Isolation from Mouse Spleen Cells

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

Example 7

Cell Vaccine Preparation and T Cell Response

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

Alpha-galactosyl ceramide (hereinafter referred to as “αGC”) after addition of FBS (Gibco, Cat.26140-079) and RPMI medium after 2 hours co-culture in a 37 ° C. CO ₂ incubator (Enzo, Cat.BML -SL232) wells were added at a concentration of 1μg / ml. After further incubation for 22 hours in a 37 ℃ CO ₂ incubator, the cells were obtained and washed three times with 1X PBS, and C57BL / 6 mice were intravenously administered with B cells alone, B / αGC, and B / αGC / VacESAT6. T cell response in vivo was confirmed in the group injected with the BCG strain intramuscularly.

The flow cytometer was a BD Biosciences FACS Verse flow cytometer. Antibodies include 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), and 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

Preparation of M. Kasnsasii Strains

Mycobactirum Kansasii ( M. Kansasii ) is a strain that expresses ESAT6. The strain was stored at 1.5 × 10 ⁹ CFU / ml and mouse infection experiments were conducted at 10 ⁷ CFU / mouse.

Difco ™ Middlebrook 7H10 Agar (BD, Cat.262710) and Difco ™ Middlebrook 7H9 Broth (BD, cat.271310) were used as media for measuring CFU. 7H10 agar medium was prepared by adding OADC as a supplement and finally solidifying it in a Petri dish. OADCs include 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 prepared by diluting in tertiary sterile distilled water.

7H9 broth medium was prepared by adding ADC and supplementing it with 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 by filtering.

Example 9

How to Determine CFU in Tissues After M. Kasnsasii Infection

M. Kansasii diluted the stock vial in 1x PBS and developed infection by intravenous injection at 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, and titrated in medium. For lung tissue, homogenized with buffer solution containing 0.04% Tween80 in 1x PBS, and liver tissue was homogenized with buffer containing 1 mM EDTA in 1x PBS. . Homogenized tissue was diluted with 7H9 broth (including ADC) to 10 ⁻¹ −10 ⁻⁵ . Then, the tissue solution diluted in 7H10 agar was plated and incubated 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 levels in tissues

The homogenized tissue was used for the CFU measurement by the above method, followed by centrifugation at 13000 rpm, 10 minutes, and 4 ° C., and the supernatant was obtained and stored. As a tissue supernatant, the cytokine in the tissue was identified using a BD ™ Cytometric Bead Array (CBA) Mouse Inflammation kit (BD bioscience, Cat.552364). After also extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Cat.52906) into tissue supernatant RNA were by using the ^{THUNDERBIRD ® Probe qPCR Mix (Toyobo,} Cat.QPS-201) proceeds to check the qPCR gene level.

[Test Example 1]

Confirmation of induction of immune response of cellular vaccine (measuring MFI value of MHC class II, CD1d and CD86)

In order to determine whether the test method of transducing vacciniaESAT6 into B cells and then loading αGC induced an immune response in B cells, an in vitro culture experiment was performed.

B220 ⁺ cells were isolated from the spleen and seeded on plates. In each well control group (B cells only), B / BCG (BC cells introduced into B cells), B / αGC (αGC introduced into B cells), B / vacciniaESAT6 (vacciniaESAT6 introduced into B cells), B / αGC / vacciniaESAT6 ( B cells were introduced with vacciniaESAT6 and αGC), and cultured for 24 hours, and then stained with fluorescence, and confirmed by flow cytometry.

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. In addition, it was confirmed that the B / αGC / vaccinia ESAT6 group significantly increased the MFI value compared to other B / BCG, B / αGC and B / vaccinia ESAT 6 group.

As shown in FIG. 2, the MFI value of CD1d, a molecule indicating 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 was further increased compared to the B / αGC group.

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

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

[Test Example 2]

Induced immune response of cellular vaccine (Animal experiment)

It is known that TNFα and IFNγ, T cell activity, co-stimulatory molecules, and cytokines secreted by T cells, are important for the vaccine response and anti-tuberculosis mechanism. In Test Example 1, it was confirmed that the cell-based vaccine of the present invention sufficiently induces an immune response. Therefore, the immune response experiment after administration to the mouse was further performed.

BCG was administered intramuscularly, and cell-based vaccines were administered intravenously. Cell-based vaccines were boosted after 2 weeks. Two weeks after boosting, CD4 ⁺ T cells were isolated from each group's spleen, and CD11c ⁺ dendritic cells were isolated from the spleen of Naive mice (C57BL / 6), respectively.

Co-cultured cells were stimulated with ESAT6-specific CD4 peptide, H37Rv (live) 0.1 MOI, and H37Rv (Heat-Killed) 0.1 MOI, respectively, and co-cultured in a 37 ° C. CO ₂ incubator for 72 hours. After the cells were obtained and stained with a fluorescent sample and the secretion amount of TNFα and IFNγ in CD4 ⁺ T cells were confirmed by flow cytometry.

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

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

The above results confirmed that the cell vaccine (cell-based 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 Test Examples 1 and 2, the induction of immune responses due to cell-based vaccines in vitro and in vivo was sufficiently confirmed. Thus, in vivo infection experiments were performed using M. kansasii strains expressing ESAT6 to confirm the protective and therapeutic effects against infection.

In vitro experiments confirmed that the cell vaccine of the present invention induces an immune response against Mycobacterium tuberculosis (MTB), and further validates its effectiveness in Mycobacterium expressing ESAT6 by verifying its effectiveness in M. Kansasii infection models. It was.

BCG was administered by 10 ⁵ CFU / mouse intramuscular injection, B / αGC / vacESAT6 was administered intravenously, and 1 week after the administration, the cell-based vaccine was boosted. Two weeks after the initial administration, M. kansasii was infected by intravenous injection of 10 ⁷ CFU / mouse, and two weeks after the infection, sacrifice was performed.

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

As shown in Figure 6, the cytokine was measured in the lung through the CBA, it was confirmed that the inflammatory cytokine of TNF, MCP-1 and IL-6 in the BCG and cell vaccine administration group compared to the infection group.

As shown in Figure 7, the lung was stained by H & E staining to confirm the degree of inflammation, the difference between the infection group and the administration group was not large.

However, as shown in Figs. 8 and 9, homogenization of the lungs and the liver was plated in the medium and the bacteria were counted after 3 weeks. It was confirmed that the decrease in the cell vaccine administration group, the CFU reduction was greater in the BCG administration group.

From the above results, administration of B / αGC / vacESAT6 cell vaccine to M. kansasii infection not only reduced inflammatory cytokine, but also directly reduced CFU in lung and liver. It can be seen that it has a larger antimicrobial effect.

[Test Example 4]

Efficacy of the second dose with cell vaccine after BCG vaccination

In the case of boosting the B / αGC / vacESAT6 cell vaccine after the administration of BCG, the experiment was conducted as follows to determine whether the effect of BCG could be further enhanced.

Two weeks later, the vaccine was preemptively administered to the BCG group, and compared with the BCG single group. Two weeks after boosting administration, the infection progressed by intravenous injection of M. kansasii and the mice were weighed for three weeks.

As shown in FIG. 10, when the initial body weight of the non-infected control group was 100%, the weight of the 3rd week of the control group was 110%, and the weight of the 3rd week of the infected group was about 90%. In addition, the BCG-only group maintained the original body weight at about 100%, and the booster-administered group with the cell vaccine had about 105% more weight than the BCG-only group.

In conclusion, the test results confirmed that the B / αGC / vacESAT6 cell vaccine can induce sufficient T-cell immune response even by its own administration, and when combined with BCG known as an anti-tuberculosis vaccine, It was confirmed that it can increase and supplement.

Claims

A vaccine for treating or preventing tuberculosis comprising a ligand of natural killer T cells and a B cell loaded with ESAT6.
The vaccine for treating or preventing tuberculosis of claim 1, wherein the ligand is alpha-galactosylceramide.
The vaccine of 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 or CD86.
The vaccine of claim 1, wherein the vaccine increases the content of TNFα or IFNγ.
The vaccine of claim 1, wherein the vaccine can be administered in combination or sequentially with a BCG vaccine.
A method for preparing a vaccine for treating or preventing tuberculosis, comprising the following steps:

(i) expressing ESAT6 of Mycobacterium tuberculosis strain in vaccinia virus;

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

(iii) loading alpha-galactosylceramide on B cells.