WO2019216543A1 - VACCIN À BASE DE CELLULES POUR LE TRAITEMENT ET LA PRÉVENTION DE LA TUBERCULOSE, COMPRENANT DES CELLULES B CHARGÉES D'α-GALACTOSYLCÉRAMIDE ET D'ESAT6 - Google Patents

VACCIN À BASE DE CELLULES POUR LE TRAITEMENT ET LA PRÉVENTION DE LA TUBERCULOSE, COMPRENANT DES CELLULES B CHARGÉES D'α-GALACTOSYLCÉRAMIDE ET D'ESAT6 Download PDF

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WO2019216543A1
WO2019216543A1 PCT/KR2019/003029 KR2019003029W WO2019216543A1 WO 2019216543 A1 WO2019216543 A1 WO 2019216543A1 KR 2019003029 W KR2019003029 W KR 2019003029W WO 2019216543 A1 WO2019216543 A1 WO 2019216543A1
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cells
vaccine
cell
tuberculosis
esat6
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고현정
권보은
박은경
안재희
정현진
정혜숙
신은경
유정식
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강원대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4612—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A61K39/464817—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration

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  • 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 ( ⁇ GC) as a ligand of natural killer T cells and using ESAT6 as an antigen.
  • ⁇ GC alpha-galactosylceramide
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • tuberculosis 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.
  • recombinant BCG such as VPM1002
  • attenuated M. tuberculosis vaccines such as MTBVAC (Vaccine 31: 1340-1348. Cell host & microbe 3: 97-103).
  • 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.
  • tuberculosis vaccines have been developed that are far superior to the attenuated BCG vaccine.
  • 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.
  • ESAT6 is expressed in M. tuberculosis , whereas it is not known in the commercially available BCG vaccine.
  • BCG vaccine there is a problem in developing an efficient delivery system of ESAT6 in inducing an immune response using the ESAT6 antigen.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 TNF ⁇ or IFN ⁇ .
  • the cell vaccine of the present invention may be administered in combination or sequentially with a BCG vaccine.
  • 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:
  • 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.
  • ESAT6 which is not expressed by the BCG vaccine known as an existing tuberculosis vaccine, is used as an antigen
  • BCG the limitation of BCG can be compensated for, and a new tuberculosis vaccine can be developed.
  • FIG. 4 shows the experimental results of TFN ⁇ and IFN ⁇ in each group.
  • FIG. 5 shows the IFN ⁇ 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.
  • 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.
  • 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.
  • 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 ⁇ g / 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).
  • alpha-galactosyl ceramide does not cause dose-limiting toxicity (50-4800 ⁇ g / 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.
  • 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
  • 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
  • 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 (poly
  • 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.
  • parenteral administration includes modes such as subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.
  • 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
  • Mycobacterium -expressing antigen 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.
  • 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 DH5 ⁇ . 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.
  • the sequencing primer sequences are shown in Table 1 below.
  • VVTK-F 5 ⁇ -TTTGAAGCATTGGAAGCAACT-3 ⁇
  • VVTK-R 5 ⁇ -ACGTTGAAATGTCCCATCGACT-3 ⁇
  • 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.
  • OPTI-MEM 2% FBS
  • 1.5 ⁇ g 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.
  • 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).
  • Vero cells Vero cell, SFM, OptiMEM
  • freezing and thawing were repeated two or three times to break the cells and recover the medium and the cells.
  • the recombinant virus was concentrated using an amicon filter with a pore size of 100,000 NMWL.
  • mice Female C57BL / 6, 6-8 weeks old, was used.
  • the mouse is ORIENTBIO Inc. And all mice were preserved at Kangwon National University.
  • 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 °C incubated for a total of 15 minutes.
  • PBS + PBS + FBS 1%
  • 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).
  • 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.
  • LS Column Miltenyi Biotec, Cat. 130-042-401
  • 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 +.
  • 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.
  • 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).
  • red blood cells were removed from homogenized splenocytes using RBC lysis buffer.
  • 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.
  • ⁇ GC Alpha-galactosyl ceramide
  • FBS Gibco, Cat.26140-079
  • RPMI medium 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 1 ⁇ g / ml.
  • the cells were obtained and washed three times with 1X PBS, and C57BL / 6 mice were intravenously administered with B cells alone, B / ⁇ GC, and B / ⁇ GC / 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.
  • 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 TM Middlebrook 7H10 Agar (BD, Cat.262710) and Difco TM 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.
  • 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.
  • 7H9 broth including ADC
  • 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.
  • a tissue supernatant the cytokine in the tissue was identified using a BD TM Cytometric Bead Array (CBA) Mouse Inflammation kit (BD bioscience, Cat.552364).
  • CBA Cytometric Bead Array
  • QIAamp Viral RNA Mini Kit QIAGEN, Cat.52906
  • tissue supernatant RNA were by using the THUNDERBIRD ® Probe qPCR Mix (Toyobo, Cat.QPS-201) proceeds to check the qPCR gene level.
  • B220 + cells were isolated from the spleen and seeded on plates.
  • B cells B / BCG (BC cells introduced into B cells), B / ⁇ GC ( ⁇ GC introduced into B cells), B / vacciniaESAT6 (vacciniaESAT6 introduced into B cells), B / ⁇ GC / vacciniaESAT6 ( B cells were introduced with vacciniaESAT6 and ⁇ GC), and cultured for 24 hours, and then stained with fluorescence, and confirmed by flow cytometry.
  • the MFI value of CD1d a molecule indicating natural killer T cells (hereinafter referred to as “NKT”)
  • B / ⁇ GC group which is a ligand of NKT.
  • B / ⁇ GC / vacciniaESAT6 group was further increased compared to the B / ⁇ GC group.
  • the MFI value of CD86 a co-stimulatory molecule of T cells
  • the B / ⁇ GC / vacciniaESAT6 group increased the MFI value the most compared to other B / BCG, B / ⁇ GC and B / vacciniaESAT6 groups.
  • the cell-based vaccine of the present invention can sufficiently induce an immune response.
  • TNF ⁇ and IFN ⁇ , T cell activity, co-stimulatory molecules, and cytokines secreted by T cells are important for the vaccine response and anti-tuberculosis mechanism.
  • 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 TNF ⁇ and IFN ⁇ in CD4 + T cells were confirmed by flow cytometry.
  • IFN ⁇ secretion of the B / ⁇ GC / vacESAT6 group was significantly increased for the remaining stimulus except for H37RV (live) stimulus. I could confirm it.
  • the cell vaccine (cell-based vaccine) of the present invention can sufficiently activate T cells even in in vivo conditions.
  • BCG was administered by 10 5 CFU / mouse intramuscular injection
  • B / ⁇ GC / vacESAT6 was administered intravenously
  • 1 week after the administration the cell-based vaccine was boosted.
  • M. kansasii was infected by intravenous injection of 10 7 CFU / mouse, and two weeks after the infection, sacrifice was performed.
  • 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.
  • 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.
  • the vaccine was preemptively administered to the BCG group, and compared with the BCG single group.
  • the infection progressed by intravenous injection of M. kansasii and the mice were weighed for three weeks.
  • 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%.
  • 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.
  • the test results confirmed that the B / ⁇ GC / 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.

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Abstract

La présente invention concerne un vaccin à base de cellules médié par des cellules B chargées avec un ligand de lymphocytes T tueurs naturels et un antigène. Spécifiquement, la présente invention concerne un vaccin à base de cellules présentant un excellent effet antituberculeux en utilisant l'α-galactosylcéramide (αGC) en tant que ligand de lymphocytes T tueurs naturels, et en utilisant d'ESAT6 en tant qu'antigène. La présente invention concerne un vaccin à base de cellules dans lequel, parmi des antigènes de la tuberculose, ESAT6 est ciblée et exprimée dans le virus de la vaccine, et ainsi le transfert d'antigène est maximisé, et après transduction de celle-ci à des cellules B, αGC, qui est un ligand de lymphocytes T tueurs naturels, est chargée, et ainsi le vaccin à base de cellules présente un effet d'induction de réponse immunitaire efficace et un effet de réduction de CFU contre des infections.
PCT/KR2019/003029 2018-05-11 2019-03-15 VACCIN À BASE DE CELLULES POUR LE TRAITEMENT ET LA PRÉVENTION DE LA TUBERCULOSE, COMPRENANT DES CELLULES B CHARGÉES D'α-GALACTOSYLCÉRAMIDE ET D'ESAT6 WO2019216543A1 (fr)

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WO2023243890A1 (fr) * 2022-06-15 2023-12-21 주식회사 셀리드 Vaccin comprenant des cellules tueuses naturelles chargées de ligands de lymphocytes t tueurs naturels et d'antigènes tumoraux
WO2024058609A1 (fr) * 2022-09-15 2024-03-21 주식회사 셀리드 Vaccin comprenant des cellules mononucléaires du sang périphérique chargées avec un ligand de lymphocyte t tueur naturel et un antigène

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