CN108743931A - 抗结核病疫苗及其制备方法和用途 - Google Patents
抗结核病疫苗及其制备方法和用途 Download PDFInfo
- Publication number
- CN108743931A CN108743931A CN201810410653.5A CN201810410653A CN108743931A CN 108743931 A CN108743931 A CN 108743931A CN 201810410653 A CN201810410653 A CN 201810410653A CN 108743931 A CN108743931 A CN 108743931A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- preparation
- antituberculosis
- ray
- tuberculosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 79
- 230000002365 anti-tubercular Effects 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 47
- 230000005855 radiation Effects 0.000 claims abstract description 16
- 230000002285 radioactive effect Effects 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 27
- 230000001580 bacterial effect Effects 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 12
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 208000035473 Communicable disease Diseases 0.000 claims description 5
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 5
- 238000000265 homogenisation Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 230000002052 anaphylactic effect Effects 0.000 claims description 3
- 239000002552 dosage form Substances 0.000 claims description 3
- 239000007927 intramuscular injection Substances 0.000 claims description 3
- 238000010255 intramuscular injection Methods 0.000 claims description 3
- 210000003928 nasal cavity Anatomy 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 208000008128 pulmonary tuberculosis Diseases 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 206010056377 Bone tuberculosis Diseases 0.000 claims description 2
- 208000009360 Osteoarticular Tuberculosis Diseases 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 201000008267 intestinal tuberculosis Diseases 0.000 claims description 2
- 210000000214 mouth Anatomy 0.000 claims description 2
- 206010053583 Peritoneal tuberculosis Diseases 0.000 claims 1
- 208000008744 Tuberculous Peritonitis Diseases 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 229960002109 tuberculosis vaccine Drugs 0.000 abstract description 21
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 abstract description 15
- 201000008827 tuberculosis Diseases 0.000 abstract description 15
- 230000002265 prevention Effects 0.000 abstract description 7
- 208000033065 inborn errors of immunity Diseases 0.000 abstract description 5
- 208000028529 primary immunodeficiency disease Diseases 0.000 abstract description 5
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 abstract description 3
- 230000001900 immune effect Effects 0.000 abstract description 3
- 230000028993 immune response Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 230000000890 antigenic effect Effects 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 230000002045 lasting effect Effects 0.000 abstract description 2
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 30
- 241000699666 Mus <mouse, genus> Species 0.000 description 29
- 210000000952 spleen Anatomy 0.000 description 12
- 239000002574 poison Substances 0.000 description 11
- 231100000614 poison Toxicity 0.000 description 11
- 230000004083 survival effect Effects 0.000 description 11
- 239000000725 suspension Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical group NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 238000007920 subcutaneous administration Methods 0.000 description 7
- 238000011740 C57BL/6 mouse Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000002062 proliferating effect Effects 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 229960001212 bacterial vaccine Drugs 0.000 description 4
- 230000007969 cellular immunity Effects 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 238000011579 SCID mouse model Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000008004 immune attack Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229960003350 isoniazid Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 206010062049 Lymphocytic infiltration Diseases 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000187644 Mycobacterium vaccae Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000001223 meningeal tuberculosis Diseases 0.000 description 2
- 230000036457 multidrug resistance Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002633 protecting effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000003393 splenic effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000816 toxic dose Toxicity 0.000 description 2
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010013453 Disseminated tuberculosis Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 240000007015 Melilotus indicus Species 0.000 description 1
- 206010027259 Meningitis tuberculous Diseases 0.000 description 1
- 201000006836 Miliary Tuberculosis Diseases 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 206010062207 Mycobacterial infection Diseases 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000022971 Tuberculous meningitis Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- UPAZUDUZKTYFBG-HNPUZVNISA-N azane [(2S,3R,4R,5S,6R)-2,5-dihydroxy-6-[[(2R,3R,4R,5S,6R)-6-(hydroxymethyl)-5-phosphonooxy-3-[[(3R)-3-tetradecanoyloxytetradecanoyl]amino]-4-[(3R)-3-tetradecanoyloxytetradecanoyl]oxyoxan-2-yl]oxymethyl]-3-[[(3R)-3-hydroxytetradecanoyl]amino]oxan-4-yl] (3R)-3-hydroxytetradecanoate Chemical compound [NH4+].CCCCCCCCCCCCCC(=O)O[C@H](CCCCCCCCCCC)CC(=O)N[C@H]1[C@H](OC[C@H]2O[C@H](O)[C@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H]2O)O[C@H](CO)[C@@H](OP(O)([O-])=O)[C@@H]1OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC UPAZUDUZKTYFBG-HNPUZVNISA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011354 first-line chemotherapy Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- LWYXLXAMDLNBFQ-UHFFFAOYSA-N iso-6-Carnavalin Natural products CC(O)CCCCCCCCCCC1CCC(O)C(C)N1 LWYXLXAMDLNBFQ-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- 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
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/544—Mucosal route to the airways
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pulmonology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Communicable Diseases (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Physical Education & Sports Medicine (AREA)
- Dermatology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Otolaryngology (AREA)
- Physiology (AREA)
- Nutrition Science (AREA)
- Neurosurgery (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
Abstract
本发明属于结核病疫苗领域,具体涉及一种抗结核病疫苗及其制备方法和用途。针对现有疫苗对成人效果不佳;或不适合免疫力低下病人等问题,本发明提供一种抗结核病疫苗的制备方法:先获取分枝杆菌单细胞菌体,再采用射线低剂量、间断、循环辐照单细胞菌体,得到分枝杆菌疫苗,射线为X射线、γ射线或同位素放射源Co60产生的射线,辐射剂量率采用10~20Gy/min,每照射20min间隔5~10min,照射8~10次。本发明不仅完整地保留菌体的所有抗原特性,还可更迅速地激发更强的特异性免疫应答,获得有效持久的免疫力。制备的疫苗毒性低,起效快,更安全可以用于免疫不全者的预防及治疗结核病。
Description
技术领域
本发明属于结核病疫苗领域,具体涉及一种抗结核病疫苗及其制备方法和用途。
背景技术
结核病流行现状:结核病(Tuberculosis,TB)是全球范围内危害严重的传染病。据2016年WHO报告,全球新发TB病例1040万,儿童占10%。其中多药耐药结核(MDR)48万例,另有10万例耐利福平结核患者。我国是TB高负担国家,WHO预测:2016-2020年结核病高负担国家的结核感染及发病特点将是多药耐药结核(MDR-TB)和艾滋病合并结核病(TB/HIV)。
结核病防治现状:卡介苗(Bacillus Calmette-Guérin vaccine,BCG)是目前唯一的预防结核病的手段,但其免疫原性不足,对结核保护效果为0-80%,差异大,只能预防儿童的严重结核病(粟粒性结核、结核性脑膜炎),保护时限仅5-10年;对已感染人群无效,对降低暴露后感染风险及感染病例无效,对青少年和成年人肺结核无效;由于BCG为减毒活疫苗,故不能用于免疫低下人群的接种。
近年来关于抗结核疫苗的研究很多,但目前尚无一例上市。在研抗结核疫苗可分为以下四类:1)病毒载体疫苗:如Ad5Ag85A、ChAdOx1.85A/MVA85A、MVA85A/MVA85A、TB/FLU-04L等;2)重组蛋白/佐剂疫苗:如H1/H56:IC31、H4:IC31、ID93+GLA-SE、M72+AS01E等;3)分枝杆菌-全菌或提取物疫苗:如M.Vaccae、DAR-901、VPM 1002、RUTI等;4)减毒结核分枝杆菌和重组BCG疫苗:MTBVAC、VPM1002。除M.Vaccae、M.Indicus pran11已进入III期临床试验以外,其它均处于I、II期。
目前还没有一种抗结核疫苗能够较好的作用于成人和儿童,尤其是免疫力低下的病人,亟待开发一种效果更好的抗结核疫苗。
发明内容
本发明要解决的技术问题为:现有疫苗只对儿童有效,对成人效果不佳;或不适合免疫力低下病人等问题。
本发明解决上述技术问题的技术方案为:提供一种抗结核病疫苗及其制备方法和用途。
本发明提供一种抗结核病疫苗的制备方法,包括以下步骤:
先获取分枝杆菌单细胞菌体,再采用射线低剂量、间断、均匀、循环辐照单细胞菌体,得到分枝杆菌疫苗。
进一步的,上述抗结核病疫苗的制备方法,包括以下步骤:
a、获取分枝杆菌细菌株,接种培养至对数增长期,加入重悬介质,均质处理,过筛,获得分枝杆菌单细胞菌体;
b、采用射线低剂量、间断、均匀、循环辐照分枝杆菌单细胞菌体,得到抗结核病疫苗。
其中,上述抗结核病疫苗的制备方法中,步骤a中所述的重悬介质为加吐温的磷酸盐缓冲液(PBST)。进一步的,所述重悬介质为含有0.05%~0.1%吐温80的PBS。
其中,上述抗结核病疫苗的制备方法中,步骤a中所述分枝杆菌单细胞菌体的浓度为106/ml~108/ml。
其中,上述抗结核病疫苗的制备方法中,步骤b所述的射线为X射线、γ射线或同位素放射源Co60产生的射线。
其中,上述抗结核病疫苗的制备方法中,步骤b所述的低剂量是指辐照总剂量为:3000~6000Gy。
其中,上述抗结核病疫苗的制备方法中,步骤b所述的间断、均匀、循环辐照的具体操作方式为:辐射剂量率采用10~20Gy/min,每照射20min间隔5~10min,均匀照射8~10次。进一步的,所述的均匀照射是指辐照剂量率恒定不变。
其中,上述抗结核病疫苗的制备方法中,步骤b所述辐照剂量的均一性比率≤1.6。
其中,上述抗结核病疫苗的制备方法中,步骤b所述辐照的采用X射线时,能量范围为40~160KVP,峰值为70~90KVP。
其中,上述抗结核病疫苗的制备方法中,为了去除培养基及相应细菌胞外分泌物,步骤b所述辐照步骤进行前或进行后,还包括对分枝杆菌单细胞菌体进行纯化的步骤。
进一步的,所述的纯化步骤为采用不引起机体过敏反应的溶剂进行纯化。优选溶剂为含0.05%~0.1%吐温80的PBS。
本发明还提供了一种由上述方法制备得到的抗结核病疫苗。
进一步的,上述疫苗还含有佐剂。
其中,上述疫苗的剂型为皮下注射制剂、肌注注射制剂、口服或鼻腔吸入制剂等常用的剂型。
同时,本发明还提供了上述的抗结核病疫苗在制备预防或治疗结核分枝杆菌引起的感染性疾病药物中的用途。
进一步的,上述用途中,所述的感染性疾病包括:肺结核、脑膜结核、女性盆腔结核、骨结核或肠结核中的至少一种。
本发明所述的抗结核病疫苗,可用于儿童或免疫力低下的成人,包括HIV合并结核分枝杆菌感染患者。
本发明的有益效果在于:
本发明提供一种经射线辐照处理的分枝杆菌全菌体疫苗,通过采用低剂量、间断、循环照射,以低剂量反复刺激细菌,通过循环照射,累加剂量,使之获得更佳的免疫原性,达到灭活增效的目的。本发明不仅完整地保留菌体的所有抗原特性,还可更迅速地激发更强的特异性免疫应答,获得有效持久的免疫力。本发明制备的疫苗为灭活全菌疫苗,毒性低、起效快、更安全,能够产生足够的抗体及细胞免疫分子对抗潜在的感染病菌,可以用于免疫不全者的预防及治疗结核病。
附图说明
图1、不同辐照方式抗结核疫苗免疫小鼠的生存曲线图;
图2、不同辐照方式抗结核疫苗降低小鼠攻毒后脾组织CFU计数;
图3、透射电镜下菌体形态图;
图4、小鼠免疫攻毒后体重图;
图5、小鼠免疫攻毒后4周肺组织病理图;
图6、小鼠免疫攻毒后4周脾、肺组织CFU计数;A为脾组织CFU计数,B为肺组织CFU计数;
图7、小鼠免疫后第3~8周脾淋巴细胞CD4+/CD8+;
图8、小鼠免疫后第6周脾淋巴细胞增殖率;
图9、SCID小鼠累积免疫后生存曲线图。
具体实施方式
本发明提供了一种抗结核病疫苗。
该疫苗由经射线辐照处理的分枝杆菌全菌体组成,射线辐照使细菌丧失增殖能力,但结构仍然完整,因此保留了绝大部分免疫原性,机体可针对免疫原刺激产生强有效的免疫反应,即足够的体液及细胞免疫预防及治疗由结核分枝杆菌引起的感染。同时也具有足够的安全性。
本发明提供了一种抗结核病疫苗的制备方法,包括以下步骤:
a、获取分枝杆菌细菌株,接种培养至对数增长期,加入重悬介质,均质处理,过筛,获得分枝杆菌单细胞菌体;
b、采用射线低剂量、间断、均匀、循环辐照分枝杆菌单细胞菌体,得到抗结核病疫苗。
其中,上述抗结核病疫苗的制备方法中,步骤a中所述的重悬介质为加吐温的磷酸盐缓冲液(PBST)。进一步的,所述重悬介质为含有0.05%~0.1%吐温80的PBS。
其中,上述抗结核病疫苗的制备方法中,步骤a中所述分枝杆菌单细胞菌体的浓度为106/ml~108/ml。
其中,上述抗结核病疫苗的制备方法中,所述的射线为X射线、γ射线或同位素放射源Co60产生的射线。
其中,上述抗结核病疫苗的制备方法中,所述的低剂量是指辐照总剂量为:3000~6000Gy。
其中,上述抗结核病疫苗的制备方法中,所述的间断、均匀、循环辐照的具体操作方式为:辐射剂量率采用10~20Gy/min,每照射20min间隔5~10min,均匀照射8~10次。进一步的,所述的均匀照射是指辐照剂量率恒定不变。
其中,上述抗结核病疫苗的制备方法中,步骤b所述辐照剂量的均一性比率≤1.6。
其中,上述抗结核病疫苗的制备方法中,步骤b所述辐照的采用X射线时,能量范围为40~160KVP,峰值为70~90KVP。
本发明所述的抗结核病疫苗的具体制备方法为:
(1)获取分枝杆菌细菌株;此细菌株包括但不限于临床样品菌株、商品化菌株、实验室改造的菌株;
(2)增殖细菌:将步骤(1)获取的细菌株接种在适宜的液体培养基中培养,使细菌处于对数增长期;所述的培养基、培养时间、培养温度等条件可采用本领域常规的条件,培养基只要是适合菌体增殖的种类即可,如实施例中使用的培养基;培养条件一般是在35~37℃,优选为37℃,优选培养时间为3周;
(3)制备单细胞菌体悬液:收集步骤(2)得到的对数增长期菌体,加入重悬介质,经均质机处理、过筛获得细菌悬液;细菌悬液中菌体浓度为106/ml~108/ml。
(4)纯化菌体:将细菌悬液再次离心收集离心沉淀物。重复此步骤至少2次,保证离心沉淀物中全为菌体,而不含有培养基及相应细菌分泌物,减少疫苗受体的过敏反应;
(5)射线照射:将上述净化处理的离心沉淀物重悬后移置非金属容器中,辐照细菌悬液;所述辐照为低剂量、长时间、间断、均匀循环照射,采用剂量为10~20Gy/min,每照射20min间隔5~10min,均匀照射8~10次,总射线剂量为3000Gy~6000Gy;
(6)半成品检验:检验细菌增殖能力和代谢活性;
(7)冷冻干燥:将步骤(5)得到的细菌悬液分装,冷冻干燥,即得到预防及治疗性的抗结核病疫苗;
(8)疫苗储存:将上述步骤(7)得到的细菌疫苗保存备用,优选方案为-20℃或者-80℃;
(9)成品检验:上述步骤(7)得到的细菌疫苗按《中华人民共和国药典》第三部检测其装量差异、菌数、鉴别试验、内毒素、纯菌检查、效力等指标。
本发明对菌体进行增菌时,根据需要测定细菌数量,以确认培养进度。菌体数量可以以吸光度OD或者浊度进行反映,也可以使用PCR的DNA复制数确定,也可以使用其他的本领域常用方法确定。
本发明细菌培养后,要对收集得到的菌体进行纯化。纯化步骤可以使用各种不引起机体过敏反应的溶剂进行洗涤。优选方案为PBST洗涤。收集细菌和纯化步骤也可以使用如浓缩、过柱收集法等本领域常用方法。而纯化步骤的实施,可以在辐照前,也可以在辐照后。或者在辐照前后均实施。
本发明射线辐照所用的射线,可以使用如X射线,γ射线或同位素放射源射线Co60的射线等种类进行,也可以使用本领域常用的其它辐照源进行。
细菌接受的辐射总剂量以满足以下要求为准:使辐照处理后的细菌无增殖能力即可。检测处理后的菌体是否具有增殖能力的方法采用本领域常用的方法即可,优选使用下述方法:
取出辐照处理后的细菌用无菌生理盐水稀释,然后用细菌推棒均匀涂布在TH10琼脂平板上,37℃孵箱培养3周,观察没有菌落产生,即确定细菌无增殖能力。
本发明还提供了一种上述方法制备得到的抗结核病疫苗,可含有常用的免疫佐剂以增强疫苗的免疫效果。
本发明提供的结核病疫苗制剂可以有多种免疫方式,如皮下、肌注、口服、鼻腔吸入等,或者是上述方式的组合。
本发明提供的结核病疫苗制剂,为一次免疫,但具体实施过程不局限于此,可以根据实际情况改变或者调整免疫次数和免疫时间点。
本发明提供的结核病疫苗制剂的剂量可根据受体情况调整,优选剂量为每次(1~5)×106个细菌体。具体免疫数量也可以根据受体情况调整。
本发明提供的疫苗可以用来制备预防或治疗疾病的药物。针对正常的动物或者人体,使用本发明方法制备的疫苗可以增强机体的预防和抗病能力,达到预防疾病的目的;对于患有相应疾病或者感染的动物和人体,特别是免疫力低下或已感染过结核杆菌的成人,包括HIV合并结核分枝杆菌感染患者,本疫苗可以诱导机体产生特定的针对致病因素的反应,有效地消灭病原体,达到治疗疾病和病患的目的。
下面将通过实施例对本发明的具体实施方式做进一步的解释说明,但不表示将本发明的保护范围限制在实施例所述范围内。
实施例中所述的分枝杆菌菌株均来源于中国生物制品检定所,TH10购自西格玛奥德里奇(上海)贸易有限公司,C57BL/6小鼠、SCID小鼠均购买自北京华阜康生物科技股份有限公司,X射线辐照仪为美国Rad Source公司产品,型号:RS 2000(RS2000-BiologicalIrradiator)。其余试剂和设备为常规市售。
实施例1 抗结核疫苗的辐照方式筛选
选用C57BL/6小鼠,分为模型组(PBS)、XBCGa组(均匀辐照;剂量率恒定为19Gy/min,每次20min,共9次,辐照总量3420Gy)、XBCGd组(递减辐照;剂量率从35Gy/min依次递减为19Gy/min和12Gy/min,每个剂量率均辐照3次,每次20min,共9次,辐照总量3960Gy)、XBCGi组(递增辐照;剂量率从12Gy/min依次递增为19Gy/min和35Gy/min,每个剂量率均辐照3次,每次20min,共9次,辐照总量3960Gy)、BCG组(卡介菌,市售),每组10只,皮下免疫1次,注射量0.1mL(约106CFU/mL)。免疫4周后,以新鲜培养的BCG静脉攻毒,攻毒剂量为107CFU,观察小鼠攻毒后8周的生存情况。
结果如图1所示:
除模型组外,其余免疫组均在1周内出现死亡。其中BCG组动物生存率为80%,死亡率为20%;XBCGa组动物生存率为90%,死亡率为10%;XBCGd组动物生存率为50%,死亡率为50%;XBCGi组动物生存率为60%,死亡率为40%。
由实验结果可知:采用均匀辐照方式制备的抗结核疫苗(XBCGa)免疫小鼠后,当机体再次受到结核杆菌攻击后更安全。
实施例2 本发明抗结核疫苗的辐照方式免疫保护观察
选用C57BL/6小鼠,分为模型组(PBS)、XBCGa组(均匀辐照)、XBCGd组(递减辐照)、XBCGi组(递增辐照)、BCG组,每组10只,皮下免疫1次,注射量0.1mL约105CFU。免疫4周后,以新鲜培养的BCG静脉攻毒,攻毒剂量为106CFU,于攻毒后第4周采集脾组织匀浆涂7H10计数。
结果如图2所示:
实验显示:第4周XBCGa组和BCG组均相对于模型组CFU有较显著下降,而XBCGd组和XBCGi组较模型组没有显著变化。结果提示采用均匀辐照方式制备的抗结核疫苗(XBCGa)免疫小鼠保护效果优于递减或递增辐照方式制备的疫苗。
实施例3 本发明抗结核病疫苗的制备方法
选用卡介苗(BCG)菌种D2BP302S11株,用改良苏通培养基(配方为:L-天冬氨酸4.00g,柠檬酸2.00g,K2HPO4 0.50g,MgSO4 0.50g,柠檬酸铁0.05g,甘油35ml,吐温80 2ml,蒸馏水900ml)在37℃培养增殖2~3周,收集菌膜于离心管中,3000g(4120rpm)离心10min,去上清,加入适量生理盐水或PBS重悬,3000g(4120rpm)离心10min,去上清,加入适量PBST重悬,经均质机均质处理,调整菌液至1×107CFU/mL。将细菌悬液装入50ml BD管中,置于X射线辐照仪放射源中心位置下辐照,辐照条件:160KV,25mA,第5层,剂量率19Gy/min,20min/次,每次间隔5~10min,辐照9次(约4.5h),总剂量约3.4KGy。
将完成照射处理的细菌悬液与未照射的细菌悬液做透射电镜观察细胞形态变化,可见与未照射的菌体相比,本疫苗(XBCG)菌体仍保持完整,外膜致密度减低,出现发射状丝样结构(箭头所示),包绕菌体。菌体内容物的分布和密度改变,提示核酸的结构和完整性变化(见图3)。
将完成照射处理的细菌0.1ml用细菌涂棒均匀涂布在TH10琼脂平板上,37℃培养3周,观察无菌落生长,表明细菌无增殖能力。
将细菌悬液分装,经冷冻干燥后保存在-20℃备用。
实施例4 本发明抗结核病疫苗的功效验证1
选用C57BL/6小鼠皮下免疫,分为疫苗(XBCG)免疫组、BCG组和模型组,每组10只。实验组小鼠于腋下免疫0.1ml(约106CFU/ml)抗结核疫苗(XBCG)、BCG疫苗,模型组小鼠腋下免疫0.1ml无菌PBS,观察小鼠生存状态。1周后将BCG活菌2×106个菌体尾静脉注射入小鼠体内攻毒,图4为体重结果,结果显示:疫苗(XBCG)免疫组小鼠的体重缓慢增长,与模型组无显著差异,而BCG组小鼠体重在活菌攻毒后4周内连续下降,与其它两组比有非常显著性差异;图5为4周后各组小鼠的肺组织病理检查,结果显示模型组动物肺泡间隔增厚,肺内血管扩张,肺泡腔内和周围有大量的淋巴细胞浸润,BCG组未见改善,而XBCG组则未见间隔增厚,未见淋巴细胞浸润。
实施例5 本发明抗结核病疫苗的功效验证2
选用C57BL/6小鼠皮下免疫,分为疫苗(XBCG)免疫组、异烟肼(INH)组、XBCG+INH组、BCG组和模型组,每组10只。实验组小鼠于腋下免疫0.1ml(约106CFU/ml)抗结核疫苗(XBCG)、BCG疫苗,XBCG+INH组除皮下免疫XBCG外,另灌胃异烟肼,异烟肼组仅灌胃异烟肼,模型组小鼠腋下免疫0.1ml无菌PBS,观察小鼠生存状态。1周后将BCG活菌2×106个菌体尾静脉注射入小鼠体内攻毒,第4周取小鼠脾、肺组织匀浆,生理盐水10倍系列稀释后,各取0.1ml样涂7H10平板,37℃培养3周,进行CFU计数。结果如图6所示,结果显示:XBCG组的脾、肺细菌荷载量显著低于BCG组,提示本发明的疫苗与治疗结核病的一线化疗药联用效果可能具有协同效应(本实施例在异烟肼上进行了研究,显示有协同效应,由此推测,对于利福平、链霉素等一线抗结核药物,可能同样具有协同效应)。
实施例6 本发明结核病疫苗的功效验证3
选用C57BL/6小鼠皮下免疫,分为疫苗(XBCG)免疫组、BCG组和模型组,每组24只。实验组小鼠分别在腋下免疫0.1ml(约106CFU/mL)抗结核疫苗(XBCG)和BCG,模型组小鼠腋下免疫0.1ml PBS,观察小鼠生存状态。免疫后第1、2、3、4、5、6、8周,断颈法处死小鼠,无菌取脾、碾磨脾脏,用小鼠脾细胞分离液分离淋巴细胞,流式细胞仪检测CD3+、CD4+、CD8+细胞比例;另将细胞接种至96孔板,并用结核菌素PPD或刀豆蛋白ConA进行刺激,37℃、5%CO2全湿润培养72h后,用CCK8检测细胞增殖情况。实验结果如图7和图8所示。图7显示:各组小鼠脾淋巴细胞CD4+/CD8+,XBCG组小鼠脾淋巴细胞CD4+/CD8+比值在第3~8周高于BCG组和对照组,表明细胞免疫增强。图8显示:各组小鼠脾淋巴细胞在PPD或ConA刺激下的增殖率,第6周的XBCG组小鼠脾淋巴细胞在PPD刺激下,细胞增殖率高于BCG组和模型组,表明特异性细胞免疫更强。
实施例7 本发明结核病疫苗的安全性试验
选用SCID小鼠,分为XBCG组、BCG组和PBS组,每组8只。每天分别给小鼠腋下免疫0.1ml(约107CFU/mL)抗结核疫苗(XBCG)、BCG和PBS,连续28天,观察小鼠生存状态。结果除PBS组外,小鼠体重持续下降,BCG组动物从第23天起出现死亡,至第28天全部死亡,存活率为0;XBCG组动物从第25天起出现死亡,至第28天仅死亡3只,存活率为62.5%(见图9)。实验结果提示XBCG较BCG更安全。
由上述实验结果可知:本发明提供了一种抗结核病疫苗,该疫苗为灭活全菌疫苗,毒性低,起效快,更安全,能够产生足够的抗体及细胞免疫分子对抗潜在的感染病菌,可以用于免疫不全者的预防及治疗结核病,为预防或治疗结核病提供了一种新的选择。
Claims (16)
1.抗结核病疫苗的制备方法,其特征在于,包括以下步骤:
先获取分枝杆菌单细胞菌体,再采用射线低剂量、间断、均匀、循环辐照单细胞菌体,得到分枝杆菌疫苗。
2.根据权利要求1所述的抗结核病疫苗的制备方法,其特征在于,包括以下步骤:
a、获取分枝杆菌细菌株,接种培养至对数增长期,加入重悬介质,均质处理,过筛,获得分枝杆菌单细胞菌体;
b、采用射线低剂量、间断、均匀、循环辐照分枝杆菌单细胞菌体,得到抗结核病疫苗。
3.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤a中所述的重悬介质为加吐温的磷酸盐缓冲液PBST。
4.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤a中所述分枝杆菌单细胞菌体的浓度为106/ml~108/ml。
5.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述的射线为X射线、γ射线或同位素放射源Co60产生的射线。
6.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述的剂量是指辐照总剂量为:3000~6000Gy。
7.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述的间断、均匀、循环辐照的具体操作方式为:辐射剂量率采用10~20Gy/min,每照射20min间隔5~10min,均匀照射8~10次。
8.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述辐照剂量的均一性比率≤1.6。
9.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述辐照的采用X射线时,X射线能量范围为40~160KVP,能量峰值为70~90KVP。
10.根据权利要求2所述的抗结核病疫苗的制备方法,其特征在于:步骤b所述辐照步骤进行前或进行后,还包括对分枝杆菌单细胞菌体进行纯化的步骤。
11.根据权利要求10所述的抗结核病疫苗的制备方法,其特征在于:所述的纯化步骤为采用不引起机体过敏反应的溶剂进行纯化。
12.权利要求1~11任一项所述的方法制备得到的抗结核病疫苗。
13.根据权利要求12所述的抗结核病疫苗,其特征在于:所述疫苗还含有佐剂。
14.根据权利要求12所述的抗结核病疫苗,其特征在于:所述疫苗的剂型为皮下注射制剂、肌注注射制剂、口服或鼻腔吸入制剂。
15.权利要求12~14任一项所述的抗结核病疫苗在制备预防或治疗结核分枝杆菌引起的感染性疾病药物中的用途。
16.根据权利要求15所述的用途,其特征在于:所述的感染性疾病包括:肺结核、脑膜结核、女性盆腔结核、骨结核或肠结核中的至少一种。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810410653.5A CN108743931B (zh) | 2018-05-02 | 2018-05-02 | 抗结核病疫苗及其制备方法和用途 |
US17/051,993 US11426454B2 (en) | 2018-05-02 | 2019-07-01 | Tuberculosis vaccine, preparation method therefor, and use thereof |
PCT/CN2019/094201 WO2019210888A2 (zh) | 2018-05-02 | 2019-07-01 | 抗结核病疫苗及其制备方法和用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810410653.5A CN108743931B (zh) | 2018-05-02 | 2018-05-02 | 抗结核病疫苗及其制备方法和用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108743931A true CN108743931A (zh) | 2018-11-06 |
CN108743931B CN108743931B (zh) | 2022-08-16 |
Family
ID=64008761
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810410653.5A Active CN108743931B (zh) | 2018-05-02 | 2018-05-02 | 抗结核病疫苗及其制备方法和用途 |
Country Status (3)
Country | Link |
---|---|
US (1) | US11426454B2 (zh) |
CN (1) | CN108743931B (zh) |
WO (1) | WO2019210888A2 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019210888A3 (zh) * | 2018-05-02 | 2019-12-19 | 成都卫可信生物科技有限公司 | 抗结核病疫苗及其制备方法和用途 |
CN114364787A (zh) * | 2019-08-22 | 2022-04-15 | 四川大学 | 铜绿假单胞菌疫苗在呼吸系统疾病中的应用 |
CN114845732A (zh) * | 2019-09-26 | 2022-08-02 | 萨拉戈萨大学 | 通过肺部递送减毒活分枝杆菌的治疗效果 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6264952B1 (en) * | 1993-11-05 | 2001-07-24 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Method for protecting a mammalian host against infection by Brucella |
CN1420784A (zh) * | 2001-03-23 | 2003-05-28 | 科学与工业研究会 | 一种治疗结核病和其他细胞内感染疾病的疫苗及其制备方法 |
CN101721693A (zh) * | 2009-12-15 | 2010-06-09 | 四川大学 | 一种改良的重组卡介苗 |
CN104189898A (zh) * | 2014-06-27 | 2014-12-10 | 四川大学 | 铜绿假单胞菌疫苗及其制备方法 |
CN107802831A (zh) * | 2016-09-08 | 2018-03-16 | 潍坊华英生物科技有限公司 | 一种灭活乳酸菌疫苗佐剂 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6284255B1 (en) | 1996-08-29 | 2001-09-04 | Genesis Research & Development Corporation Limited | Compounds and methods for treatment and diagnosis of mycobacterial infections |
PT2144626E (pt) | 2007-04-12 | 2015-03-03 | Lighter Jennifer | Vacina para a tuberculose e processo para o seu uso |
WO2018006939A1 (en) | 2016-07-05 | 2018-01-11 | Universidad De Zaragoza | Inactivated tuberculosis vaccine |
CN108743931B (zh) | 2018-05-02 | 2022-08-16 | 成都威斯克生物医药有限公司 | 抗结核病疫苗及其制备方法和用途 |
-
2018
- 2018-05-02 CN CN201810410653.5A patent/CN108743931B/zh active Active
-
2019
- 2019-07-01 WO PCT/CN2019/094201 patent/WO2019210888A2/zh active Application Filing
- 2019-07-01 US US17/051,993 patent/US11426454B2/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6264952B1 (en) * | 1993-11-05 | 2001-07-24 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Method for protecting a mammalian host against infection by Brucella |
CN1420784A (zh) * | 2001-03-23 | 2003-05-28 | 科学与工业研究会 | 一种治疗结核病和其他细胞内感染疾病的疫苗及其制备方法 |
CN101721693A (zh) * | 2009-12-15 | 2010-06-09 | 四川大学 | 一种改良的重组卡介苗 |
CN104189898A (zh) * | 2014-06-27 | 2014-12-10 | 四川大学 | 铜绿假单胞菌疫苗及其制备方法 |
CN107802831A (zh) * | 2016-09-08 | 2018-03-16 | 潍坊华英生物科技有限公司 | 一种灭活乳酸菌疫苗佐剂 |
Non-Patent Citations (3)
Title |
---|
CHA 等: "Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice", 《PLOS ONE》 * |
NISHIHARA 等: "Immunogenicity of gamma-irradiated Mycobacterium tuberculosis H37Hv (GIV) in mice", 《AM REV RESPIR DIS》 * |
育巍: "紫外线与细菌免疫性", 《国外医学情报》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019210888A3 (zh) * | 2018-05-02 | 2019-12-19 | 成都卫可信生物科技有限公司 | 抗结核病疫苗及其制备方法和用途 |
US11426454B2 (en) | 2018-05-02 | 2022-08-30 | Sichuan University | Tuberculosis vaccine, preparation method therefor, and use thereof |
CN114364787A (zh) * | 2019-08-22 | 2022-04-15 | 四川大学 | 铜绿假单胞菌疫苗在呼吸系统疾病中的应用 |
CN114364787B (zh) * | 2019-08-22 | 2024-03-01 | 四川大学 | 铜绿假单胞菌疫苗在呼吸系统疾病中的应用 |
CN114845732A (zh) * | 2019-09-26 | 2022-08-02 | 萨拉戈萨大学 | 通过肺部递送减毒活分枝杆菌的治疗效果 |
Also Published As
Publication number | Publication date |
---|---|
US20210052714A1 (en) | 2021-02-25 |
WO2019210888A2 (zh) | 2019-11-07 |
US11426454B2 (en) | 2022-08-30 |
CN108743931B (zh) | 2022-08-16 |
WO2019210888A3 (zh) | 2019-12-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Mackaness et al. | Host-parasite relations in mouse typhoid | |
JP4892296B2 (ja) | 魚類のエドワジェラ症及び連鎖球菌症用ワクチン | |
CN104189898B (zh) | 铜绿假单胞菌疫苗及其制备方法 | |
Hobson | Resistance to reinfection in experimental mouse typhoid | |
CN108743931A (zh) | 抗结核病疫苗及其制备方法和用途 | |
JP4135190B2 (ja) | 免疫治療剤とその使用 | |
US7955601B2 (en) | Immunogenic agents against Burkholderia psudomallei and/or Burkholderia mallei, comprising lipopolysaccharide, capsular polysaccharide and/or proteins from Burkholderia pseudomallei | |
Shepard et al. | Heat stability of Mycobacterium leprae immunogenicity | |
Shepard | The first decade in experimental leprosy | |
Rodriguez et al. | Induction of resistance by Listeria monocytogenes cell wall fraction | |
WO2010031883A1 (es) | Agente inmunoterapéutico apropiado para la profilaxis primaria de la tuberculosis | |
CN112940987B (zh) | 一株兔金黄色葡萄球菌及其在制备灭活疫苗中的应用 | |
Baldridge et al. | Induction of immunity with avirulent Listeria monocytogenes 19113 depends on bacterial replication | |
Weiss | Antituberculous vaccination in the guinea pig with non-living vaccines | |
US6019981A (en) | Modified live Edwardsiella ictaluri against enteric septicemia in channel catfish | |
Khan et al. | Clinical management of cutaneous bovine papillomatosis in a cow calf: a case report | |
CN108703977A (zh) | 蓝细菌及其膜外囊泡在制备治疗疾病药物中的应用 | |
CN112449604A (zh) | 用作有结核感染风险的患者的预防剂或用作治疗结核感染患者的辅助剂的组合物 | |
US6596282B1 (en) | Treatment of chronic viral infections with M. vaccae | |
RU2468816C1 (ru) | Пробиотический бактериальный препарат корпускулярных антигенов коринебактерий липопептидополисахаридной природы для профилактики и лечения туберкулеза, способ его получения | |
Yarkoni et al. | Active specific immunotherapy of guinea pigs with visceral tumor implants | |
CN114870005A (zh) | 一种单核细胞增生李斯特菌灭活疫苗及其制备方法和用途 | |
Klesius et al. | Modified live Edwardsiella ictaluri against enteric septicemia in channel catfish | |
RU2581255C1 (ru) | Способ получения лабораторной модели артифициального флегмонозного воспаления мягких тканей у крыс на фоне искусственной иммуносупрессии | |
JP2024512039A (ja) | 黄色ブドウ球菌ワクチンの工業的製造方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
TA01 | Transfer of patent application right | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20210514 Address after: 610000 Chengdu Tianfu international biological city, Chengdu, Sichuan Province (No. 552, Fenghuang Road, Shuangliu District) Applicant after: Chengdu weisk biomedical Co.,Ltd. Address before: 610065, No. 24, south section of first ring road, Chengdu, Sichuan, Wuhou District Applicant before: SICHUAN University |
|
GR01 | Patent grant | ||
GR01 | Patent grant |