WO2022111021A1 - 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 - Google Patents
一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 Download PDFInfo
- Publication number
- WO2022111021A1 WO2022111021A1 PCT/CN2021/120517 CN2021120517W WO2022111021A1 WO 2022111021 A1 WO2022111021 A1 WO 2022111021A1 CN 2021120517 W CN2021120517 W CN 2021120517W WO 2022111021 A1 WO2022111021 A1 WO 2022111021A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- protein
- hpv11
- vaccine
- virus
- Prior art date
Links
- 101000642125 Human papillomavirus 11 Major capsid protein L1 Proteins 0.000 title abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 71
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 51
- 239000002245 particle Substances 0.000 claims abstract description 43
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 208000009608 Papillomavirus Infections Diseases 0.000 claims abstract description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 239000013598 vector Substances 0.000 claims abstract description 7
- 230000002265 prevention Effects 0.000 claims abstract 2
- 235000018102 proteins Nutrition 0.000 claims description 47
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 46
- 210000004899 c-terminal region Anatomy 0.000 claims description 40
- 150000001413 amino acids Chemical class 0.000 claims description 39
- 210000004027 cell Anatomy 0.000 claims description 36
- 235000001014 amino acid Nutrition 0.000 claims description 29
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 27
- 239000004472 Lysine Substances 0.000 claims description 27
- 239000004471 Glycine Substances 0.000 claims description 23
- 235000009697 arginine Nutrition 0.000 claims description 21
- 239000004475 Arginine Substances 0.000 claims description 20
- 241000238631 Hexapoda Species 0.000 claims description 20
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 20
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 17
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 12
- 235000003704 aspartic acid Nutrition 0.000 claims description 12
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 12
- 239000002671 adjuvant Substances 0.000 claims description 11
- 108091033319 polynucleotide Proteins 0.000 claims description 8
- 239000002157 polynucleotide Substances 0.000 claims description 8
- 102000040430 polynucleotide Human genes 0.000 claims description 8
- 241000701447 unidentified baculovirus Species 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000002378 acidificating effect Effects 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004473 Threonine Substances 0.000 claims description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 2
- 235000004279 alanine Nutrition 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
- 239000004220 glutamic acid Substances 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 claims description 2
- 239000004474 valine Substances 0.000 claims description 2
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims 1
- 108020004705 Codon Proteins 0.000 claims 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims 1
- 239000000969 carrier Substances 0.000 claims 1
- 230000009466 transformation Effects 0.000 claims 1
- 241000701806 Human papillomavirus Species 0.000 abstract description 18
- 239000002773 nucleotide Substances 0.000 abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 abstract description 6
- 239000012646 vaccine adjuvant Substances 0.000 abstract description 2
- 229940124931 vaccine adjuvant Drugs 0.000 abstract description 2
- 230000014509 gene expression Effects 0.000 description 32
- 238000000034 method Methods 0.000 description 16
- 238000000746 purification Methods 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 241000341655 Human papillomavirus type 16 Species 0.000 description 9
- 230000028993 immune response Effects 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
- 230000003472 neutralizing effect Effects 0.000 description 7
- 230000001681 protective effect Effects 0.000 description 7
- 206010059313 Anogenital warts Diseases 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000003053 immunization Effects 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 208000000907 Condylomata Acuminata Diseases 0.000 description 3
- 229940124897 Gardasil Drugs 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 241001112090 Pseudovirus Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 208000025009 anogenital human papillomavirus infection Diseases 0.000 description 3
- 201000004201 anogenital venereal wart Diseases 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 231100000590 oncogenic Toxicity 0.000 description 3
- 230000002246 oncogenic effect Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 229940124957 Cervarix Drugs 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000701828 Human papillomavirus type 11 Species 0.000 description 2
- WTDPSUUWWKMZLE-UHFFFAOYSA-K P(=O)([O-])([O-])[O-].S(=O)(=O)(O)O.[Al+3] Chemical compound P(=O)([O-])([O-])[O-].S(=O)(=O)(O)O.[Al+3] WTDPSUUWWKMZLE-UHFFFAOYSA-K 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 238000003921 particle size analysis Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- UEEJHVSXFDXPFK-UHFFFAOYSA-N N-dimethylaminoethanol Chemical compound CN(C)CCO UEEJHVSXFDXPFK-UHFFFAOYSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- 208000037581 Persistent 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
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000000260 Warts Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 150000001484 arginines Chemical class 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940031416 bivalent vaccine Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229940102767 gardasil 9 Drugs 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000003300 oropharynx Anatomy 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940031351 tetravalent vaccine Drugs 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 208000013139 vaginal neoplasm Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- A61K39/12—Viral antigens
-
- 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
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/01—DNA viruses
- C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
- C12N7/04—Inactivation or attenuation; Producing viral sub-units
-
- 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
- A61K2039/525—Virus
-
- 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
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- 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
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- 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
- A61K2039/70—Multivalent vaccine
-
- 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
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/866—Baculoviral vectors
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20023—Virus like particles [VLP]
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本申请涉及一种C端改造的人乳头瘤病毒11型L1蛋白及其用途。具体地,本申请涉及C端改造的人乳头瘤病毒(HPV)11型L1蛋白、其编码的核苷酸、包含所述核苷酸的载体、包含所述载体的细胞、由所述HPV11L1蛋白组成的五聚体或病毒样颗粒、含该五聚体或病毒样颗粒和疫苗佐剂的疫苗、以及其在预防HPV感染和HPV感染相关疾病中的用途。
Description
本申请涉及生物技术领域,具体涉及一种改造的人乳头瘤病毒蛋白、及由其形成的五聚体或病毒样颗粒、以及人乳头瘤病毒蛋白、五聚体或人乳头瘤病毒病毒样颗粒在制备预防乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
人乳头瘤病毒(human papillomavirus,HPV)是一类感染上皮组织的无包膜小DNA病毒,病毒基因组为双链闭环DNA,大小约7.2-7.9kb,有8个开放读码框,分别编码E1、E2、E4、E5、E6、E7合计6个早期基因,及L1、L2合计2个晚期基因;此外,还含有长调控区。病毒颗粒的直径约为45-44nm,外壳为T=7的正二十面体,由72个L1五聚体及72个L2蛋白构成。
目前已鉴定200余型,其中40余型主要感染肛周、泌尿生殖器及口咽部的粘膜及附近皮肤;根据感染诱发病变性质不同,分为诱发恶性肿瘤的致癌型(HPV16、-18、-31、-33、-45、-52、-58等)和诱发疣状增生的低危型(HPV6、-11等)。目前致癌型HPV约有20多型,其中12种常见的致癌型为高危型。分子流行病学研究发现,致癌型HPV持续感染可诱发约100%的宫颈癌、88%的肛门癌、70%的阴道癌、50%的阴茎癌、43%的阴户癌及72%的头颈癌。低危型HPV目前鉴定出了12种型别,分别是HPV6、-7、-11、-13、-32、-40、-42、-43、-44、-54、-74、-91。其中,HPV6型和HPV11型是世界范围内肛周生殖器及咽部黏膜疣的主要流行型别。对我国自1990年1月至2013年12期间发表的67篇文献,累计10757例尖锐湿疣(CA)患者的HPV分析发现,CA患者的HPV感染阳性率为86.7%(9328),HPV6和/或HPV 11阳性率为81.2%(3671/4623)、HPV6的为43.8%(2445/6134)、HPV11的为38.3%(2155/6134),HPV16、HPV 18和/或HPV 11阳性率为21.7%(870/3781)。对哥伦比亚地区的261例经活检组织学确诊的生殖器疣(GW)患者(其中女性155例,男性106例)的HPV分析显示,女性GW患者的HPV检出率为87.7%,男性为90.6%;其中HPV6的检出率最高,达59.7%(155.8/261),在女性和男性的检出率分别为62%及56%;其次是HPV11,检出率为29.8%(77.8/261);HPV16的检出率位居第三,检出率为16%;HPV6、HPV 11感染阳性率合计为80.3%。
L1蛋白体外表达后,可组装成VLP。表达体系主要有酵母表达体系、昆虫细胞表达体系、大肠杆菌表达体系等。采用昆虫细胞表达体系生产的L1VLP疫苗的优势在于:蛋白可溶性表达水平高、细胞容易破碎、不含内毒素。目前上市的3种L1VLP疫苗分别是Merck公司采用酵母表达体系生产的Gardasil四价苗(HPV16/18/6/11 L1VLP,磷酸铝硫酸盐佐剂)、Gardasil-9九价苗 (HPV16/18/6/11/31/33/45/52/58 L1VLP,磷酸铝硫酸盐佐剂)及GSK公司采用昆虫细胞表达体系生产的Cervarix二价苗(HPV16/18 L1VLP,AS04佐剂)。
临床研究发现,Cervarix中,较低剂量的16L1VLP(20μg/剂)及相同剂量的18L1VLP(20μg/剂)分别诱发产生的HPV16及HPV18特异性中和抗体的滴度、Th细胞反应、及记忆性B细胞数目均较Gardasil诱发的高(单剂Gardasil中16L1VLP的含量为40μg,18L1VLP的为20μg)。
提高HPV L1VLP在昆虫细胞内的表达水平,可显著提高L1VLP的纯化得率,降低疫苗的生产成本。在原核表达体系中,采用N端截短法对HPV16、-18、-31、-33、-45、-52、-58、-6、-11型别的L1进行改造,发现可上调L1表达水平的N端截短的氨基酸数目因型别不同而异,且没有规律。在昆虫表达系统中,采用C端截短的方法对BPV1 L1进行改造,发现截短型的BPV L1的组装效率提高3倍;对HPV58型的截短型L1的VLP虽有报道,但未见C端截短对蛋白表达量的影响的报道。利用酵母表达体系,生产11型L1VLP均是采用密码子优化的全长基因。
本申请发现,通过对L1的C端改造,可以显著提高11L1VLP表达水平及产量,而且生产获得的HPV11 L1VLP可诱发高滴度的型别特异性中和抗体。
发明内容
本申请提供了一种新的C端改造的HPV11 L1蛋白、由其组成的五聚体或病毒样颗粒,及含该五聚体或病毒样颗粒的疫苗,并研究该疫苗在预防HPV感染和感染相关疾病中的用途。
本发明人经研究出人意料地发现,适当地对HPV11 L1蛋白的C端碱性氨基酸进行置换,可提高HPV11 L1蛋白在昆虫细胞表达系统中的表达量,该截短蛋白可组装成VLP,并可诱发针对HPV11的保护性免疫反应。
因此,根据本申请的一些实施方案,涉及一种与野生型HPV11 L1蛋白(例如NCBI数据库P04012.1序列对应的氨基酸序列)相比,其C端31个氨基酸内的1个或多个碱性氨基酸被置换为极性不带电荷氨基酸、非极性氨基酸和/或酸性氨基酸。
具体地,本申请提供了一种C端改造的HPV11 L1蛋白,其中所述改造的HPV11 L1蛋白与野生型HPV11 L1蛋白相比,其C端31个氨基酸内的1个或多个精氨酸(R)和/或赖氨酸(K)被置换为极性不带电荷氨基酸、非极性氨基酸和/或酸性氨基酸。优选地,所述极性不带电荷氨基酸选自甘氨酸(G)、丝氨酸(S)或苏氨酸(T),所述非极性氨基酸选自丙氨酸(A)或缬氨酸(V),所述酸性氨基酸为天冬氨酸(D)或谷氨酸(E)。
在具体的实施方案中,本申请所述C端改造的HPV11 L1蛋白在SEQ ID No.1所示序列(NCBI数据库P04012.1序列对应的氨基酸序列)基础上进行改造;特别优选地,所述C端改造的HPV11 L1蛋白选自11L1CS1、11L1CS2、11L1CS3、 11L1CS4、11L1CS5、11L1CS6,其氨基酸序列如SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7所示。
野生型HPV11 L1蛋白也可来自但不限于NCBI数据库CBM41728.1、QEE83877.1、CRF31180.1、CRF31054.1、CRF31153.1、CBM41721.1、ACL12350.1、QDH43412.1、AAF25684.1等来自HPV11变异株的L1蛋白,相应变异株的C端改造L1蛋白,其特征为C端31个氨基酸与上述C端改造的HPV11 L1蛋白相同,如通过序列比较来评价。
根据本申请的一些实施方案,涉及编码本申请C端改造的HPV11 L1蛋白的多核苷酸。优选的,所述多核苷酸采用常用的表达系统的密码子优化,如大肠杆菌表达系统、酵母表达系统、昆虫细胞表达系统等。特别优选地,所述多核苷酸采用昆虫细胞密码子优化。
根据本申请的一些实施方案,涉及含有上述所述多核苷酸的载体,优选地,所述载体选自质粒、重组Bacmid和重组杆状病毒。
根据本申请的一些实施方案,涉及包含上述载体的细胞,优选地,所述细胞为大肠杆菌细胞、酵母细胞或昆虫细胞,特别优选地,所述细胞为昆虫细胞。
根据本申请的一些实施方案,涉及一种HPV11 L1多聚体或病毒样颗粒,该多聚体(例如五聚体)或病毒样颗粒含有上述C端改造的HPV11 L1蛋白,或由上述C端改造的HPV11 L1蛋白组成。
根据本申请的一些实施方案,涉及一种预防HPV感染或HPV感染相关疾病的疫苗,该疫苗含有上述HPV11 L1多聚体或病毒样颗粒,其中HPV11 L1病毒样颗粒的含量为能诱发保护性免疫反应的有效量。优选地,该疫苗还可包含至少一种选自其他嗜黏膜组和/或嗜皮肤组的HPV的五聚体或病毒样颗粒,这些五聚体或病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。上述疫苗通常还包含疫苗用赋形剂或载体。
优选地,所述疫苗含有上述的HPV11 L1多聚体或病毒样颗粒,以及至少一种选自HPV2、-5、-6、-7、-8、-16、-18、-26、-27、-28、-29、-30、-31、-32、-33、-34、-35、-38、-39、-40、-43、-44、-45、-51、-52、-53、-56、-57、-58、-59、-61、-66、-67、-68、-69、-70、-73、-74、-77、-81、-82、-83、-85、-91的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
进一步优选地,所述疫苗含有上述的HPV11 L1多聚体或病毒样颗粒,以及HPV6、-16、-18、-26、-31、-33、-35、-39、-45、-51、-52、-56、-58、-59、-68及-73的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
进一步优选地,所述疫苗含有上述HPV11 L1多聚体或病毒样颗粒,以及HPV6、-16、-18、-31、-33、-35、-39、-45、-52及-58的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
进一步优选地,所述疫苗含有上述HPV11 L1多聚体或病毒样颗粒,以及HPV6、-16、-18、-52及-58的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
进一步优选地,所述疫苗含有上述HPV11 L1多聚体或病毒样颗粒,以及HPV16、-18及-58的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
特别优选地,所述疫苗含有上述HPV11 L1多聚体或病毒样颗粒,以及HPV6的L1病毒样颗粒,这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
根据本申请的一些实施方案,涉及一种新型疫苗,其包含上述HPV11 L1多聚体或病毒样颗粒及佐剂,可进一步提高免疫反应。优选地,所使用的佐剂为人用疫苗佐剂。
根据本申请的一些实施方案,涉及上述疫苗在预防HPV感染或HPV感染相关疾病中的用途。
相关术语的说明及解释
根据本申请,术语“昆虫细胞表达系统”包括昆虫细胞、重组杆状病毒、重组Bacmid及表达载体。其中昆虫细胞来源于市场上可得到的细胞,在此举例但不限于:Sf9、Sf21、High Five。
根据本申请,术语“野生型HPV11 L1蛋白”的例子包括但不限于NCBI数据库中编号为P04012.1的序列对应的L1蛋白。
根据本申请,术语“赋形剂或载体”是指选自一种或多种,包括但不限于:pH调节剂、表面活性剂、离子强度增强剂。例如,pH调节剂举例但不限于磷酸盐缓冲液,表面活性剂包括阳离子、阴离子或非离子型表面活性剂,举例但不限于聚山梨酯80(Tween-80),离子强度增强剂举例但不限于氯化钠。
根据本申请,术语“佐剂”是指在临床上可应用于人体的佐剂,包括当前已获得批准的和将来可能获得批准的各种佐剂。
根据本申请,本申请的疫苗可采用患者可接受的形式,包括但不限于口服或者注射,优选注射。
根据本申请,本申请疫苗优选单位剂型使用,其中单位剂型中C端改造的HPV11 L1蛋白病毒样颗粒的剂量为5μg-80μg,优选20μg-40μg。
图1显示了本申请实施例4中C端改造的HPV11 L1在昆虫细胞中的表达鉴定。结果显示,6种C端改造的HPV11 L1均可在昆虫细胞中表达。泳道1至7分别表示野生型HPV11L1、11L1CS1、11L1CS3、11L1CS4、11L1CS5、11L1CS6、11L1CS2。
图2A至图2E显示了本申请实施例6中纯化后获得的野生型HPV11L1、 11L1CS1、11L1CS3、11L1CS4及11L1CS5突变体蛋白的动态光散射分析结果。结果显示野生型HPV11L1、11L1CS1、11L1CS3、11L1CS4及11L1CS5重组蛋白形成的病毒样颗粒水化动力学直径分别为117nm、143nm、95.2nm、121nm和119nm,颗粒组装的百分比均为100%。图2A表示野生型HPV11L1;图2B表示11L1CS1;图2C表示11L1CS3;图2D表示11L1CS4;图2E表示11L1CS5。
图3A至图3C显示了本申请实施例7中纯化后获得的野生型HPV11L1、11L1CS4及11L1CS5的VLP的透射电镜观察结果。视野中可见大量的直径约35-55nm的病毒样颗粒,颗粒的大小与理论值相符,均一度好。Bar=50nm。图3A、图3B、及图3C分别表示野生型HPV11L1、11L1CS4及11L1CS5。
图4显示了本申请实施例8中的野生型HPV11L1、11L1CS1、11L1CS4、11L1CS5 VLP接种小鼠后免疫血清HPV11中和抗体滴度的分析。
下面将通过非限制性实施例进一步说明本申请,本领域技术人员公知,在不背离本申请精神的情况下,可以对本申请做出许多修改,这样的修改也落入本申请的范围。下面的实施例仅用于说明本申请,而不应视为限定本申请的范围,因为实施方案必然是多样的。本说明书中使用的用语仅是为了阐述特定的实施方案,而非作为限制,本申请的范围已界定在所附的权利要求中。
除非特别说明,本说明书中所使用的所有技术和科学用语均和本案所属技术领域的技术人员所普遍明了的意义相同。下面就本申请的优选方法和材料加以叙述,但是与本说明书中所述方法和材料类似或等效的任何方法和材料均可用以实施或测试本申请。下述实验方法如无特别说明,均为常规方法或产品说明书所描述的方法,所使用的实验材料如无特别说明,均可容易地从商业公司获取。本说明书中所提到的所有公开文献均被并入于此作为参考,以揭示并说明所述公开文献中的方法和/或材料。
实施例1:C端改造的HPV11L1基因的合成及表达载体构建
野生型HPV11L1的氨基酸序列为SEQ ID No.1所示的序列,其对应的昆虫细胞密码子优化的全长基因由上海生工生物工程技术服务有限公司全基因合成(SEQ ID No.8所示)。
C端改造的HPV11L1基因在SEQ ID No.8所示基因的基础上进行改造,由上海生工生物工程技术服务有限公司合成,具体如下:
1)11L1CS1基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为甘氨酸(G)、第481位赖氨酸(K)突变为甘氨酸(G)、第482位精氨酸(R)突变为丝氨酸(S)、第493位赖氨酸(K)突变为天冬氨酸(D)、第494位精氨酸(R)突变为甘氨酸(G)、第495位赖氨酸(K)突变为丝氨酸(S)、 第498位赖氨酸(K)突变为天冬氨酸(D),其氨基酸序列如SEQ ID No.2所示,对应的核苷酸序列如SEQ ID No.9所示;
2)11L1CS2基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为甘氨酸(G)、第481位赖氨酸(K)突变为甘氨酸(G)、第482位精氨酸(R)突变为丝氨酸(S)、第493位赖氨酸(K)突变为甘氨酸(G)、第494位精氨酸(R)突变为天冬氨酸(D)、第495位赖氨酸(K)突变为丝氨酸(S)、第498位赖氨酸(K)突变为天冬氨酸(D),其氨基酸序列如SEQ ID No.3所示,对应的核苷酸序列如SEQ ID No.10所示;
3)11L1CS3基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为甘氨酸(G)、第481位赖氨酸(K)突变为甘氨酸(G)、第482位精氨酸(R)突变为丝氨酸(S)、第487位赖氨酸(K)突变为丝氨酸(S)、第493位赖氨酸(K)突变为丝氨酸(S)、第494位精氨酸(R)突变为甘氨酸(G)、第495位赖氨酸(K)突变为丝氨酸(S)、第496位精氨酸(R)突变为甘氨酸(G)、第498位赖氨酸(K)突变为甘氨酸(G),其氨基酸序列如SEQ ID No.4所示,对应的核苷酸序列如SEQ ID No.11所示;
4)11L1CS4基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为甘氨酸(G)、第481位赖氨酸(K)突变为甘氨酸(G)、第482位精氨酸(R)突变为丝氨酸(S)、第493位赖氨酸(K)突变为丝氨酸(S)、第494位精氨酸(R)突变为甘氨酸(G)、第495位赖氨酸(K)突变为丝氨酸(S)、第496位精氨酸(R)突变为甘氨酸(G)、第498位赖氨酸(K)突变为甘氨酸(G),其氨基酸序列如SEQ ID No.5所示,对应的核苷酸序列如SEQ ID No.12所示;
5)11L1CS5基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为天冬氨酸(D)、第481位赖氨酸(K)突变为甘氨酸(G)、第482位精氨酸(R)突变为丝氨酸(S)、第493位赖氨酸(K)突变为天冬氨酸(D)、第494位精氨酸(R)突变为甘氨酸(G)、第495位赖氨酸(K)突变为丝氨酸(S)、第498位赖氨酸(K)突变为天冬氨酸(D),其氨基酸序列如SEQ ID No.6所示,对应的核苷酸序列如SEQ ID No.13所示;
6)11L1CS6基因:将SEQ ID No.1所示的HPV11L1的第473位精氨酸(R)突变为甘氨酸(G)、第481位赖氨酸(K)突变为天冬氨酸(D)、第482位精氨酸(R)突变为丝氨酸(S)、第493位赖氨酸(K)突变为天冬氨酸(D)、第494位精氨酸(R)突变为甘氨酸(G)、第495位赖氨酸(K)突变为丝氨酸(S)、第498位赖氨酸(K)突变为天冬氨酸(D),其氨基酸序列如SEQ ID No.7所示,对应的核苷酸序列如SEQ ID No.14所示。
采用EcoRI/XbaI酶切位点,分别对上述PCR扩增得到的基因进行酶切,并分别插入商业化表达载体pFastBac1(Invitrogen公司生产)中,得到包含C端改造 的HPV11L1基因的重组表达载体:pFastBac1-11L1CS1、pFastBac1-11L1CS2、pFastBac1-11L1CS3、pFastBac1-11L1CS4、pFastBac1-11L1CS5、pFastBac1-11L1CS6。上述酶切、连接及克隆构建的方法都是公知的,例如专利CN101293918B。
实施例2:C端改造的HPV11L1基因的重组Bacmid及重组杆状病毒的构建体
分别使用C端改造的HPV11L1基因的重组表达载体pFastBac1-11L1CS1、pFastBac1-11L1CS2、pFastBac1-11L1CS3、pFastBac1-11L1CS4、pFastBac1-11L1CS5、pFastBac1-11L1CS6转化大肠杆菌DH10Bac感受态,筛选获得重组Bacmid,然后用重组Bacmid转染昆虫细胞Sf9,在Sf9内扩增重组杆状病毒。重组Bacmid的筛选及重组杆状病毒的扩增方法都是公知的,例如专利CN101148661B。
实施例3:C端改造的HPV11L1基因在Sf9细胞中的表达
Sf9细胞分别接种野生型HPV11L1的优化基因及6种C端改造的HPV11L1基因的重组杆状病毒,进行C端改造的HPV11L1蛋白的表达,27℃培养约88h后收发酵液,3000rpm离心15min,弃上清,用PBS洗涤细胞后,用于表达鉴定及纯化。感染表达的方法是公开的,例如专利CN 101148661 B。
实施例4:C端改造的HPV11L1的表达鉴定
取实施例3中所述表达不同C端改造的HPV11L1的细胞各1×10
6个,重悬于200μl PBS溶液中,加入6×上样缓冲液50μl,75℃变性8分钟,分别取10μl进行SDS-PAGE电泳及Western印记鉴定。结果如图1所示,6种C端改造的HPV11L1蛋白均可在昆虫细胞中表达,其大小约55kDa。SDS-PAGE电泳及Western印记鉴定的方法是公开的,例如专利CN 101148661 B。
实施例5:C端改造的HPV11L1蛋白与野生型HPV11L1蛋白的表达量比较
取实施例3中所述表达C端改造的HPV11L1蛋白及野生型HPV11L1的细胞各1×10
6个,重悬于200μl PBS溶液中,采用超声破碎法(宁波新芝超声破碎仪,2#探头,100W,超声5s,间隔7s,总时间3min)破碎细胞,12000rpm高速离心10分钟。收取裂解上清,采用夹心ELISA法检测上清中的L1含量,该方法是公知的,例如专利CN104513826A。
使用本发明人制备的HPV11L1单克隆抗体包被酶标板,80ng/孔,4℃孵育过夜;使用5%BSA-PBST室温封闭2h,再用PBST洗板3次。用PBS将裂解上清进行连续2倍稀释,并且将HPV11L1 VLP标准品也进行梯度稀释,浓度从2 μg/ml-0.0625μg/ml,分别加入酶标板,每孔100μl,37℃孵育1h。用PBST洗板3次,加入1:3000稀释的HPV6L1兔多抗,每孔100μl,37℃孵育1h。用PBST洗板3次,加入1:3000稀释的HRP标记的山羊抗小鼠IgG(1:3000稀释,中杉金桥公司),37℃孵育45分钟。用PBST洗板5次,每孔加入100μl OPD底物(Sigma公司),37℃显色5分钟,用50μl 2M硫酸终止反应,在490nm处测定吸光值。依据标准曲线计算裂解上清中C端改造的HPV11L1蛋白及野生型HPV11L1蛋白的浓度。
结果如表1所示,本申请的C端改造的HPV11L1蛋白中,11L1CS1、11L1CS3、11L1CS4、11L1CS5的表达量高于野生型HPV11L1蛋白,而11L1CS2、11L1CS6的表达水平与野生型11L1相当。
表1.HPV11L1蛋白表达量分析
实施例6:C端改造的HPV11L1蛋白的纯化及动态光散射粒径分析
取野生型HPV11L1或C端改造的HPV11L1的细胞发酵液50ml,使用10ml PBS重悬细胞,加PMSF至终浓度1mg/ml,超声破碎(宁波新芝超声破碎仪,6#探头,100W,超声5s,间隔7s,总时间5min),取破碎上清进行纯化,纯化步骤在室温进行。在裂解液中加入4%β-巯基乙醇(w/w)对VLP进行解聚,然后使用0.22μm滤器过滤样品,依次使用DMAE阴离子交换层析(20mM Tris,180mM NaCl,4%β-ME,pH7.9洗脱)、TMAE阴离子交换层析(20mM Tris,180mM NaCl,4%β-ME,pH7.9洗脱)及羟基磷灰石层析(100mM NaH2PO4,30mM NaCl,4%β-ME,pH 6.0洗脱)纯化。纯化产物采用Planova超滤系统进行浓缩,并更换缓冲液(20mM NaH2PO4,500mM NaCl,pH6.0)促使VLP组装。纯化结果显示,6种C端改造的HPV11L1蛋白的纯化得率均很高,值得注意的是,虽然11L1CS2、11L1CS6的表达水平与野生型11L1相当,但纯化得率约是野生型11L1的1.5倍。以上纯化方法均是公开的,例如专利CN101293918B、CN1976718A等。
取纯化后的野生型HPV11L1蛋白或C端改造的HPV11L1蛋白溶液进行DLS粒径分析(Zetasizer Nano ZS 90动态光散射仪,Malvern公司),结果如表2所示,其中野生型HPV11L1、11L1CS1、11L1CS3、11L1CS4及11L1CS5的DLS分析图如图2A至图2E所示。
表2.HPV11L1蛋白DLS分析
实施例7:C端改造的HPV11L1 VLP的透射电镜观察
按实施例6所述的层析纯化方法,对C端改造的HPV11L1 VLP分别进行纯化,使用透析后的VLP制备铜网,并用1%醋酸铀进行染色,充分干燥后使用JEM-1400电镜(奥林巴斯)进行观察。部分结果如图3A至图3C所示,C端改造的HPV11L1VLP直径约为35-55nm,形状规则。铜网制备及电镜观察的方法均是公开的,例如专利CN 101148661 B。
实施例8:C端改造的HPV11L1 VLP的小鼠免疫及中和抗体滴度测定
取4-6周龄的BALB/c小鼠,随机分组,每组5只,分别用野生型HPV11L1 VLP及C端改造的HPV11L1 VLP免疫小鼠。肌肉注射,L1 VLP的免疫剂量为0.1μg,于第0,2周免疫,共2次。第2次免疫后2周尾静脉采血,分离血清。
使用HPV11假病毒对免疫血清的HPV11中和抗体滴度进行检测,结果如图4所示,野生型HPV11L1 VLP、11L1CS1 VLP、11L1CS4 VLP及11L1CS5 VLP免疫小鼠后均可有效诱发中和抗体,且C端改造的HPV11L1 VLP诱发的中和抗体滴度与野生型HPV11L1 VLP诱发的无统计学差异。
假病毒制备及假病毒中和实验的方法均是公开的,例如专利CN 104418942A。
综上所述,发明人发现,对HPV11L1的C端进行氨基酸置换改造,获得的突变体表达水平各不相同,且没有明显的规律。因此,采用C端置换改造的方法获得表达水平高、可有效组装、免疫活性好的HPV11L1突变体,具有不可预见性。本申请中筛选获得的C端氨基酸置换改造的HPV11L1突变体可用于多价HPV预防性疫苗的配制及广谱HPV预防性疫苗的构建,具有很好的研发前景。
序列说明
SEQ ID No.1:HPV11L1
SEQ ID No.2:11L1CS1
SEQ ID No.3:11L1CS2
SEQ ID No.4:11L1CS3
SEQ ID No.5:11L1CS4
SEQ ID No.6:11L1CS5
SEQ ID No.7:11L1CS6
SEQ ID No.8:HPV11L1nt
SEQ ID No.9:11L1CS1nt
SEQ ID No.10:11L1CS2nt
SEQ ID No.11:11L1CS3nt
SEQ ID No.12:11L1CS4nt
SEQ ID No.13:11L1CS5nt
SEQ ID No.14:11L1CS6nt
Claims (10)
- 一种C端改造的HPV11 L1蛋白,与野生型HPV11 L1蛋白相比,其C端31个氨基酸中的1个或多个碱性氨基酸置换为选自以下的氨基酸:极性不带电荷氨基酸、非极性氨基酸或酸性氨基酸;优选地,所述野生型HPV11 L1蛋白是选自以下的序列所示;NCBI登录号;P04012.1、CBM41728.1、QEE83877.1、CRF31180.1、CRF31054.1、CRF31153.1、CBM41721.1、ACL12350.1、QDH43412.1、AAF25684.1;更优选地,所述野生型HPV11 L1蛋白是SEQ ID No.1所示;优选地,所述碱性氨基酸选自:精氨酸、赖氨酸;优选地,所述极性不带电荷氨基酸选自:甘氨酸、丝氨酸、苏氨酸;优选地,所述非极性氨基酸选自:丙氨酸、缬氨酸;优选地,所述酸性氨基酸选自:天冬氨酸、谷氨酸。
- 根据权利要求1所述的C端改造的HPV11 L1蛋白,其是选自以下的序列所示:SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7。
- 一种多核苷酸,其编码权利要求1或2所述的C端改造的HPV11 L1蛋白;优选地,所述多核苷酸的序列是采用昆虫细胞密码子进行全基因优化的。
- 根据权利要求3所述的多核苷酸,其是选自以下的序列所示:SEQ ID No.9、SEQ ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14。
- 一种载体,其包含权利要求3或4所述的多核苷酸;优选地,所述载体选自:质粒、重组Bacmid和重组杆状病毒。
- 一种宿主细胞,其包含权利要求5所述的载体;优选地,所述宿主细胞选自:大肠杆菌、酵母细胞、昆虫细胞。
- 一种多聚物,其中:所述多聚物为五聚体或病毒样颗粒;所述多聚物含有权利要求1或2所述的C端改造的HPV11 L1蛋白、或由权利要求1或2所述的C端改造的HPV11 L1蛋白形成。
- 一种用于预防乳头瘤病毒感染或其相关疾病的疫苗,其包含:权利要求7所述的多聚物、佐剂、以及疫苗用赋形剂或载体;优选地,所述佐剂是人用佐剂;任选地,所述用于预防乳头瘤病毒感染或其相关疾病的疫苗还包含选自以下的一种或组合:嗜黏膜组HPV病毒样颗粒或嵌合病毒样颗粒、嗜皮肤组HPV病毒样颗粒或嵌合病毒样颗粒。
- 权利要求1或2所述的C端改造的HPV11 L1蛋白在制备疫苗中的用途;优选地,所述疫苗用于预防乳头瘤病毒感染或其相关疾病。
- 权利要求7所述的多聚物在制备疫苗中的用途;优选地,所述疫苗用于预防乳头瘤病毒感染或其相关疾病。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/254,157 US20240000915A1 (en) | 2020-11-26 | 2021-09-26 | C-terminally modified human papillomavirus type 11 l1 protein and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011346631.0A CN114539363B (zh) | 2020-11-26 | 2020-11-26 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
CN202011346631.0 | 2020-11-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022111021A1 true WO2022111021A1 (zh) | 2022-06-02 |
Family
ID=81659474
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/120517 WO2022111021A1 (zh) | 2020-11-26 | 2021-09-26 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20240000915A1 (zh) |
CN (1) | CN114539363B (zh) |
WO (1) | WO2022111021A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114539363A (zh) * | 2020-11-26 | 2022-05-27 | 中国医学科学院基础医学研究所 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021013069A1 (zh) * | 2019-07-19 | 2021-01-28 | 神州细胞工程有限公司 | 嵌合的人乳头瘤病毒11型l1蛋白 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997018301A1 (en) * | 1995-11-15 | 1997-05-22 | Merck & Co., Inc. | Synthetic hpv11 virus-like particles |
WO2000035479A1 (en) * | 1998-12-17 | 2000-06-22 | Merck & Co., Inc. | Synthetic virus-like particles with heterologous epitopes |
US6165471A (en) * | 1997-07-03 | 2000-12-26 | University Of Colorado, University Technology Corporation | Homogeneous human papillomavirus capsomere containing compositions, methods for manufacture, and use thereof as diagnostic, prophylactic or therapeutic agents |
CN1578787A (zh) * | 2001-08-31 | 2005-02-09 | 开普敦大学 | 关于hpv-11和hpv-16l1衣壳蛋白的载体,构建体,和转基因植物 |
CN104418942A (zh) * | 2013-08-30 | 2015-03-18 | 长春百克生物科技股份公司 | 截短的人乳头瘤病毒的l1蛋白、其类病毒颗粒及其制备方法和应用 |
CN104710515A (zh) * | 2013-12-17 | 2015-06-17 | 北京康乐卫士生物技术股份有限公司 | 人乳头瘤病毒l1蛋白突变体及其制备方法 |
CN106831958A (zh) * | 2015-12-04 | 2017-06-13 | 厦门大学 | 一种人乳头瘤病毒11型l1蛋白的突变体 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2264563T3 (es) * | 1994-10-07 | 2007-01-01 | Loyola University Of Chicago | Particulas semejantes al virus del papiloma, proteinas de fusion y procedimiento para su preparacion. |
EP2907821B1 (en) * | 2007-05-29 | 2016-12-28 | Xiamen University | Method for producing a N-terminaly truncated L1 protein of human papillomavirus (HPV) |
CN105039358B (zh) * | 2013-12-03 | 2020-02-28 | 北京康乐卫士生物技术股份有限公司 | 58型重组人乳头瘤病毒病毒样颗粒及其制备方法 |
CN107188932B (zh) * | 2016-03-15 | 2020-02-11 | 中国医学科学院基础医学研究所 | 截短型人乳头瘤病毒16型l1蛋白及其应用 |
CN114539363B (zh) * | 2020-11-26 | 2023-12-01 | 中国医学科学院基础医学研究所 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
-
2020
- 2020-11-26 CN CN202011346631.0A patent/CN114539363B/zh active Active
-
2021
- 2021-09-26 US US18/254,157 patent/US20240000915A1/en active Pending
- 2021-09-26 WO PCT/CN2021/120517 patent/WO2022111021A1/zh active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997018301A1 (en) * | 1995-11-15 | 1997-05-22 | Merck & Co., Inc. | Synthetic hpv11 virus-like particles |
US6165471A (en) * | 1997-07-03 | 2000-12-26 | University Of Colorado, University Technology Corporation | Homogeneous human papillomavirus capsomere containing compositions, methods for manufacture, and use thereof as diagnostic, prophylactic or therapeutic agents |
WO2000035479A1 (en) * | 1998-12-17 | 2000-06-22 | Merck & Co., Inc. | Synthetic virus-like particles with heterologous epitopes |
CN1578787A (zh) * | 2001-08-31 | 2005-02-09 | 开普敦大学 | 关于hpv-11和hpv-16l1衣壳蛋白的载体,构建体,和转基因植物 |
CN104418942A (zh) * | 2013-08-30 | 2015-03-18 | 长春百克生物科技股份公司 | 截短的人乳头瘤病毒的l1蛋白、其类病毒颗粒及其制备方法和应用 |
CN104710515A (zh) * | 2013-12-17 | 2015-06-17 | 北京康乐卫士生物技术股份有限公司 | 人乳头瘤病毒l1蛋白突变体及其制备方法 |
CN106831958A (zh) * | 2015-12-04 | 2017-06-13 | 厦门大学 | 一种人乳头瘤病毒11型l1蛋白的突变体 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114539363A (zh) * | 2020-11-26 | 2022-05-27 | 中国医学科学院基础医学研究所 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
CN114539363B (zh) * | 2020-11-26 | 2023-12-01 | 中国医学科学院基础医学研究所 | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 |
Also Published As
Publication number | Publication date |
---|---|
US20240000915A1 (en) | 2024-01-04 |
CN114539363B (zh) | 2023-12-01 |
CN114539363A (zh) | 2022-05-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7976848B2 (en) | Optimized expression of HPV 58 L1 in yeast | |
US10882887B2 (en) | Papillomavirus chimeric protein and application thereof | |
JP4799789B2 (ja) | ヒト細胞中での発現用に最適化された合成ヒトパピローマウイルス遺伝子 | |
US20080138361A1 (en) | Optimized expression of HPV 45 L1 in yeast | |
US20120087937A1 (en) | Novel compositions | |
HU225893B1 (en) | Papilloma virus capsomere vaccine formulations and methods of use | |
US10940194B2 (en) | Mutant of L1 protein of human papillomavirus type 58 | |
WO2017157172A1 (zh) | 一种乳头瘤病毒嵌合蛋白及其用途 | |
WO2022111021A1 (zh) | 一种c端改造的人乳头瘤病毒11型l1蛋白及其用途 | |
US8039001B2 (en) | Therapeutic and prophylactic vaccine for the treatment and prevention of papillomavirus infection | |
WO2022142525A1 (zh) | 一种人乳头瘤病毒58型嵌合蛋白及其用途 | |
WO2022111020A1 (zh) | 一种c端改造的人乳头瘤病毒6型l1蛋白及其用途 | |
WO2022142524A1 (zh) | 一种人乳头瘤病毒31型嵌合蛋白及其用途 | |
WO2022111022A1 (zh) | 一种改造的人乳头瘤病毒52型l1蛋白及其用途 | |
WO2022142523A1 (zh) | 一种人乳头瘤病毒18型嵌合蛋白及其用途 | |
US8715681B2 (en) | Minimal motifs of linear B-cell epitopes in L1 protein from human papillomavirus type 58 and their applications | |
JP2002509863A (ja) | パピローマウイルス特異的腫瘍の回避用または治療用医薬 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21896524 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 18254157 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21896524 Country of ref document: EP Kind code of ref document: A1 |