CN104491852A - Tumor DNA vaccine and virus vector vaccine taking mucoprotein 1 and surviving as target spots - Google Patents
Tumor DNA vaccine and virus vector vaccine taking mucoprotein 1 and surviving as target spots Download PDFInfo
- Publication number
- CN104491852A CN104491852A CN201410788971.7A CN201410788971A CN104491852A CN 104491852 A CN104491852 A CN 104491852A CN 201410788971 A CN201410788971 A CN 201410788971A CN 104491852 A CN104491852 A CN 104491852A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- tumor
- group
- mice
- survivin
- 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
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 96
- 241000700605 Viruses Species 0.000 title claims abstract description 21
- 229940126580 vector vaccine Drugs 0.000 title abstract description 7
- 108010041986 DNA Vaccines Proteins 0.000 title abstract description 6
- 229940021995 DNA vaccine Drugs 0.000 title abstract description 6
- 102000001621 Mucoproteins Human genes 0.000 title 1
- 108010093825 Mucoproteins Proteins 0.000 title 1
- 229960005486 vaccine Drugs 0.000 claims abstract description 128
- 238000002649 immunization Methods 0.000 claims abstract description 51
- 230000003053 immunization Effects 0.000 claims abstract description 49
- 241000701161 unidentified adenovirus Species 0.000 claims abstract description 42
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 108010002687 Survivin Proteins 0.000 claims description 80
- 102000000763 Survivin Human genes 0.000 claims description 77
- 102100034256 Mucin-1 Human genes 0.000 claims description 57
- 210000004027 cell Anatomy 0.000 claims description 49
- 239000012634 fragment Substances 0.000 claims description 43
- 239000013612 plasmid Substances 0.000 claims description 43
- 190000008236 carboplatin Chemical compound 0.000 claims description 38
- 229960004562 carboplatin Drugs 0.000 claims description 33
- 108020004414 DNA Proteins 0.000 claims description 24
- 238000011238 DNA vaccination Methods 0.000 claims description 16
- 239000000568 immunological adjuvant Substances 0.000 claims description 14
- 229960004854 viral vaccine Drugs 0.000 claims description 8
- 238000013467 fragmentation Methods 0.000 claims description 6
- 238000006062 fragmentation reaction Methods 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 108010071942 Colony-Stimulating Factors Proteins 0.000 claims description 3
- 102000007644 Colony-Stimulating Factors Human genes 0.000 claims description 3
- 102000004127 Cytokines Human genes 0.000 claims description 3
- 108090000695 Cytokines Proteins 0.000 claims description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 3
- 102000015696 Interleukins Human genes 0.000 claims description 3
- 108010063738 Interleukins Proteins 0.000 claims description 3
- 229930012538 Paclitaxel Natural products 0.000 claims description 3
- 206010046865 Vaccinia virus infection Diseases 0.000 claims description 3
- 230000000118 anti-neoplastic effect Effects 0.000 claims description 3
- 229960004316 cisplatin Drugs 0.000 claims description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 3
- 229960001756 oxaliplatin Drugs 0.000 claims description 3
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 3
- 229960001592 paclitaxel Drugs 0.000 claims description 3
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 3
- 208000007089 vaccinia Diseases 0.000 claims description 3
- 229940029030 dendritic cell vaccine Drugs 0.000 claims description 2
- 230000006872 improvement Effects 0.000 claims description 2
- 229940023143 protein vaccine Drugs 0.000 claims description 2
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims 2
- 230000000259 anti-tumor effect Effects 0.000 abstract description 8
- 239000013598 vector Substances 0.000 abstract description 5
- 108020004511 Recombinant DNA Proteins 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 83
- 108010008707 Mucin-1 Proteins 0.000 description 55
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 54
- 241000699666 Mus <mouse, genus> Species 0.000 description 46
- 230000004083 survival effect Effects 0.000 description 42
- 108090000623 proteins and genes Proteins 0.000 description 38
- 238000002474 experimental method Methods 0.000 description 27
- 230000014509 gene expression Effects 0.000 description 27
- 230000001900 immune effect Effects 0.000 description 25
- 238000010172 mouse model Methods 0.000 description 25
- 210000002966 serum Anatomy 0.000 description 23
- 230000004044 response Effects 0.000 description 22
- 230000004614 tumor growth Effects 0.000 description 22
- 210000004881 tumor cell Anatomy 0.000 description 21
- 230000029087 digestion Effects 0.000 description 19
- 108090000765 processed proteins & peptides Proteins 0.000 description 19
- 230000036039 immunity Effects 0.000 description 18
- 210000004698 lymphocyte Anatomy 0.000 description 18
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 17
- 230000000694 effects Effects 0.000 description 16
- 239000000427 antigen Substances 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 230000002401 inhibitory effect Effects 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- 238000011160 research Methods 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 239000002671 adjuvant Substances 0.000 description 10
- 230000008859 change Effects 0.000 description 10
- 239000013605 shuttle vector Substances 0.000 description 10
- 230000009182 swimming Effects 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 230000009471 action Effects 0.000 description 9
- 229940021704 adenovirus vaccine Drugs 0.000 description 9
- 230000001413 cellular effect Effects 0.000 description 9
- 238000012797 qualification Methods 0.000 description 9
- 238000011740 C57BL/6 mouse Methods 0.000 description 8
- 238000005267 amalgamation Methods 0.000 description 8
- 239000012636 effector Substances 0.000 description 8
- 230000005847 immunogenicity Effects 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000002560 therapeutic procedure Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 101150036876 cre gene Proteins 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 241001529936 Murinae Species 0.000 description 6
- 230000002424 anti-apoptotic effect Effects 0.000 description 6
- 239000002585 base Substances 0.000 description 6
- 230000034994 death Effects 0.000 description 6
- 230000001976 improved effect Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000003252 repetitive effect Effects 0.000 description 6
- 210000004989 spleen cell Anatomy 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 108010091086 Recombinases Proteins 0.000 description 5
- 102000018120 Recombinases Human genes 0.000 description 5
- 238000012300 Sequence Analysis Methods 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 102100031780 Endonuclease Human genes 0.000 description 4
- 108010042407 Endonucleases Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 4
- 108090000364 Ligases Proteins 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000012447 hatching Effects 0.000 description 4
- 238000009169 immunotherapy Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 3
- 101150060716 S8 gene Proteins 0.000 description 3
- 108010052160 Site-specific recombinase Proteins 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000003449 preventive effect Effects 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 108010051219 Cre recombinase Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 101150107184 MS gene Proteins 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 101150003725 TK gene Proteins 0.000 description 2
- 108020004440 Thymidine kinase Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000000240 adjuvant effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000002434 immunopotentiative effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000005760 tumorsuppression Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 101150029662 E1 gene Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 1
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 101150114927 MUC1 gene Proteins 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010076039 Polyproteins Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000001218 Rec A Recombinases Human genes 0.000 description 1
- 108010055016 Rec A Recombinases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 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
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008088 immune pathway Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001759 immunoprophylactic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229940065638 intron a Drugs 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000000627 locus coeruleus Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229940024231 poxvirus vaccine Drugs 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of tumor DNA vaccines and virus vector vaccines. Specifically, the invention relates to a recombinant DNA vector VR-S8 vaccine, a VR-MS vaccine, a recombinant adenovirus Ad-S8 vaccine, an Ad-MS vaccine and a recombinant poxvirus MVA-MS vaccine, and application of the DNA vaccines and the virus vector vaccines as well as the application of optimal combination of immunization ways of the virus vector vaccines in preparation of antitumor vaccines.
Description
The application is divisional application, and applying date of its original application is December in 2009 4 days, and application number is 200910252427.X, and name is called " tumor DNA vaccine being target spot with MUC-1 and survivin and vector-viral vaccine ".
Technical field
The present invention relates to tumor DNA vaccine and vector-viral vaccine field.Specifically, the present invention relates to recombinant DNA carrier VR-S8 and VR-MS, recombinant adenovirus Ad-S8 and Ad-MS and recombinant poxvirus MVA-MS vaccine, and between DNA vaccination and vector-viral vaccine and vector-viral vaccine, the purposes in anti-tumor vaccine is being prepared in the optimum organization of immunization ways.
Background technology
Survivin (Survivin) is apoptosis inhibit albumen (IAP) family member, there is the dual-use function of anti-apoptotic and regulating cell division, wide expression in various embryonal tissue and cancerous cell, but is not expressed in normal terminally differentiated cells.These features make survivin become tumor generation, a novel targets in the fields such as development and treatment.
Current research shows that survivin has potential value in therapy of tumor: its tumor specific expression, and is proportionate with prognosis mala, drug resistance and patient's short survival etc.The immunotherapy of tumors carried out for survivin and mutant etc. thereof has become the focus that people pay close attention to.Current strategy both at home and abroad has (see Li, F.et al., 2006; Li, F., 2003; Altieri, D.C., 2008): the tumor target gene therapy that (1) utilizes the tumor specific expression of survivin promoter to carry out; (2) for the therapeutic strategy of survivin itself: a) antisense of survivin or the cDNA of total length or antisense nucleotide expression vector strategy, relatively be applicable to the functional study of survivin and front clinical research, but potential danger may be had with the survivin of total length; B) survivin function negative mutant (dominant negative mutant, DNM) strategy, mainly SurvT34A and SurvC84A, but this scheme may not be suitable for direct treatment of cancer; C) survivin ribozyme scheme, namely shears Survivin mRNA to reduce the expression of survivin with Survivin mRNA specific ribozyme, and this scheme is also a kind of effective research tool, but is not suitable for cancer clinical treatment; D) survivin RNAi technology, current research shows: RNAi can effectively make mammalian gene expression reticent, can be used for gene functional research, but the expense of costliness limits its clinical practice; E) suppress the expression of survivin with small organic molecule or little peptide or disturb its function; F) with the immunization therapy of survivin specificity epitope, this is a very attractive field, but only uses the little peptide of survivin, and immunogenicity may not be high.
Because survivin has the function of anti-apoptotic, if design vaccine by its total length may there is potential safety hazard, intend adopting survivin anti-apoptotic afunction spliced body to design vaccine according to domestic and international tactful advantage and drawback, preservation length of trying one's best when ensureing its anti-apoptotic afunction is to improve immunogenicity.Therefore the present invention adopts the anti-apoptotic afunction spliced body of survivin to design vaccine.Survivin works with dimeric forms in vivo, and three aminoacid of its 6th, the 7th and the 10th, N-end form dimeric key amino acid (see Shi, Y.et al., 2000); The 5th of survivin is that the specific antigen epitope of HLA-A2 is (see Andersen to the 14th amino acids, M.H.et al., 2001), spliced body S8 (namely cut survivin N-and hold 7 aminoacid) is devised first based on these 2 the present invention, wish to break its dimeric structure of ring, thus make it lack the function of anti-apoptotic.Therefore be that target spot is to design the safety that vaccine may improve vaccine with S8.
MUC-1 (MUC1) is mucin family member, is transmembrane glycoprotein, is present in the tumor cell surface in normal glandular tube epithelial cell and source thereof, is made up of polypeptide core and side shoot sugar chain.Its core peptide extracellular fragment contains the Tandem repeat (VNTR) that number does not wait.In normal structure, MUC1 is distributed in glandular tube epithelial cells pole, relatively with immunocyte isolates, and glycosylation is enriched; And at tumor tissues, it extensively distributes and is extremely expressed in cell surface galore, glycosylation is incomplete, therefore epi-position hidden is under normal circumstances exposed, become the target spot of immune cells attack, and express and strengthen, can reach Normocellular more than 100 times, therefore MUC1 is comparatively ideal anti-tumor target molecule.
PDTRP sequence in MUC1 VNTR is the epi-position of B cell and T cell common identification, can combine with MUC1 specific antibody and cytotoxic T lymphocyte (CTL), therefore claim the immunogenic position of this most to be immuno-dominant domain, therefore, what current major part utilized MUC1 to study immunization therapy is all concentrate on VNTR region (see Singh, R.etal., 2007; Persson, J.et al., 2006; Xing, P.X.et al., 1992; Tang, C.K.etal., 2008; Tang, C.K.et al., 2008).A lot of vaccine research work has been carried out with it till now from discovery MUC1, and have some vaccines to enter Human clinical's conceptual phase, but great majority are all traditional polypeptide class, protide or dendritic cell (DC) class vaccines, all there is the problem such as immunogenicity difference or preparation difficulty in these vaccines mostly; Enter clinical gene vaccine mainly recombinant poxvirus vaccine, although toxicity do not detected, exempt from the beginning of direct recombinant poxvirus vaccine-strengthen or there is larger potential safety hazard, and the clinical effectiveness produced neither be very desirable; DNA vector vaccine is also in the preclinical study stage at present substantially due to less immunogenic.
The early-stage Study of inventor shows that the number increasing VNTR can strengthen the immune effect of MUC1 to a certain extent (see Zhang S et al., 2008), the immunne response that the MUC1 VNTR tandem repetitive sequence that the immunne response that MUC1 VNTR tandem repetitive sequence as 33 copies causes obviously is better than 2 copies causes, therefore the present invention to select containing the tandem repetitive sequence MUC1 VNTR of 33 as antigen to design vaccine, and itself and S8 amalgamation and expression are built amalgamation and expression vaccine to strengthen broad spectrum activity and the effectiveness of vaccine immunity, survivin and MUC1 VNTR use in conjunction are carried out oncotherapy yet there are no report at present.
Summary of the invention
The invention provides a kind of DNA fragmentation S8, the sequence of described S8 fragment is SEQ IDNO:8.
The invention provides a kind of recombiant plasmid VR-S8, the skeleton carrier of wherein said recombiant plasmid is the VR1012 as Figure 1A, in the S8 fragment described in the multiple clone site of described VR1012.Preferably, the S8 fragment inserted is between the multiple clone site SalI and BamHI of VR1012.
The invention provides the DNA fragmentation MS shown in a kind of SEQ ID NO:9, described sequence MS comprises the DNA fragmentation 33M of a MUC1 VNTR containing 33 copies and described S8 fragment.
The invention provides a kind of recombiant plasmid VR-MS, it is characterized in that the skeleton carrier of described recombiant plasmid is VR1012, in the multiple clone site of described VR1012, insert described MS fragment.Preferably, the MS fragment inserted is between the multiple clone site SalI and BamHI of VR1012.
The invention provides a kind of recombinant adenovirus Ad-S8, it is characterized in that in adenovirus, insert described S8 fragment.Preferably, the skeleton carrier of described recombinant adenovirus is pBHGlox Δ E1,3Cre, and the S8 fragment inserted is between the multiple clone site EcoRI and BglII of described recombinant adenovirus
The invention provides a kind of recombinant adenovirus Ad-MS, it is characterized in that in adenovirus, insert described MS fragment.Preferably, the skeleton carrier of described recombinant adenovirus is pBHGlox Δ E1,3Cre, and the MS fragment inserted is positioned at the multiple clone site BglII place of described recombinant adenovirus.
The invention provides a kind of recombinant mva virus MVA-MS, it is characterized in that in described recombinant mva virus, insert described MS fragment.Preferably, the MS fragment inserted is between the multiple clone site SalI and KpnI of described recombinant mva virus.
The invention provides described S8 fragment or described MS fragment for the preparation of the purposes of carrying out in the protein vaccine of antineoplastic immune, DNA vaccination, vector-viral vaccine or dendritic cell vaccine.
In a preferred embodiment, immunological adjuvant and/or chemotherapeutics is comprised in above-mentioned vaccine, wherein said immunological adjuvant is selected from cytokine interleukin element 2 (IL-2), cytokine grain-giant cell colony stimulating factor (GM-CSF) and non-methylated CpG motif immune stimulates DNA sequence, and wherein said chemotherapeutics is selected from carboplatin, cisplatin, oxaliplatin and paclitaxel.
First the present invention is the research that target spot carries out tumor gene vaccine with S8, constructed dna carrier bacterin (VR-S8) and adenovirus carrier vaccine (Ad-S8) respectively, adopt at the beginning of DNA and exempt from-recombinant adenovirus booster immunization (DNA prime-rAd boost) immunization strategy, adopt the plasmid (VR-IL2) of expressing interleukin II (IL2) as immunological adjuvant simultaneously, detected immunogenicity and the anti-tumor activity of S8 gene vaccine by immune mouse, result shows: mice can produce specific antibody (see Fig. 4) for S8 and CTL (see Fig. 5); This vaccine has certain anti-tumor activity, in preventive experiment, compares with PBS group, VR-S8/VR-IL2/Ad-S8 vaccine immunity group model mice tumors grew suppression ratio is 68.54%, life span extension 35.6% (see Fig. 6).In curative test, compare with PBS group, VR-S8/VR-IL2/Ad-S8 vaccine immunity group model small mouse tumor growth receives obvious suppression (suppression ratio is 23.42%), but does not have the life cycle (see Fig. 7) of obvious lengthening model mice.
The present invention adopts survivin and MUC1 to be shot design amalgamation and expression gene vaccine.Because MUC1 gene polynorphisms determines the difference of VNTR number between Different Individual, modal for containing 30-90 repetitive sequence continuously, feature according to building MUC1 VNTR gene is voluntarily selected with 33 duplicate block sequences (referred to as 33M, this sequence contains the tandem repetitive sequence of 33 VNTR, 6 are had owing to building the base sequence of the restriction enzyme site introduced between each repetitive sequence,) build MUC1 DNA vector vaccine (VR-33M), build the DNA vector vaccine (VR-MS) of 33M and S8 amalgamation and expression simultaneously.Whether be significantly improved compared with the vaccine of the two single expression with anti-tumor activity by the immune effect of immune model mice detection fusion expressing gene vaccine, result shows that the immune effect of survivin and MUC1 amalgamation and expression gene vaccine is obviously better than the immune effect of the vaccine of the two single expression, such as: in the preventative research of tumor, amalgamation and expression vaccine immunity group tumour inhibiting rate compared with the independent immune group of survivin vaccine improves 134%, life span extension 166%; Compare with the independent immune group of MUC1, tumour inhibiting rate improves 25%, life span extension 73% (see Figure 12).In order to the immune effect strengthening MS vaccine further constructs again its vector-viral vaccine (Ad-MS and MVA-MS), adopt at the beginning of DNA and exempt from-the immunization strategy of recombinant adenovirus booster immunization, adopt the plasmid of expressing IL2 as immunological adjuvant simultaneously, by immune effect and the anti-tumor activity of immune model mice detection fusion expressing gene vaccine, result shows to exempt from the beginning of DNA-and the immunization strategy strengthened of recombinant adenovirus can the growth of obvious inhibition mouse tumor, and effectively extend the life cycle of mice; IL2 also show obvious adjuvant potentiation (see Figure 13).
Accompanying drawing explanation
Fig. 1 .DNA carrier and recombinant DNA carrier vaccine collection of illustrative plates.A is VR1012 Vector map; B is recombiant plasmid VR-S8 collection of illustrative plates; C is recombiant plasmid VR-33M collection of illustrative plates; D is recombiant plasmid VR-MS collection of illustrative plates.
Fig. 2. recombinant adenoviral vector collection of illustrative plates and preparation flow.A is AdMax
tMadenovirus vector and recombinant virus preparation flow; B is shuttle vector pDC316 collection of illustrative plates; C is recombinant shuttle vector pDC316-S8 collection of illustrative plates; D is recombinant shuttle vector pDC316-MS collection of illustrative plates.
Fig. 3 .MVA restructuring principle and relevant carriers collection of illustrative plates.A is MVA restructuring schematic diagram; B is shuttle vector pSC11 collection of illustrative plates; C is recombinant shuttle vector pSC11-MS collection of illustrative plates, and MS expression of structural gene framework is recombinated in the TK gene of MVA, and the promoter of gene expression adopts P7.5.
Fig. 4. the detection of the anti-S8 antibody of mouse immune serum.C57/BL mice is through PBS (2), VR-IL2 (3), VR-S8 (4), VR-S8/VR-IL2 (5), after VR-S8/VR-IL2/Ad-S8 (6) immunity, using the serum of mice as primary antibodie, to express the COS-7 lysis supernatant of S8 as the antigen detecting mice serum, using S8 monoclonal antibody as positive control (1), carry out the result that Western blotting (Western-blot) detects.
Fig. 5. in different forms after S8 DNA gene vaccine, the specificity CTL killing ability of mouse spleen lymphocyte detects.From being effector lymphocyte by the lymphocyte that is separated immune mouse spleen cell, and with the murine lung cancer cell strain B16 of specific antigen peptide (H-2d is restricted) labelling as target cell, the CTL adopting on-radiation LDH method for releasing to detect immunized mice replys.
Fig. 6. multi-form S8 gene vaccine is to MS
f +the immunoprophylactic effect of B16 (purchased from ATCC) tumor cell tumor growth.C57BL/6 mice through PBS matched group, VR-IL2, VR-S8, VR-S8/VR-IL2 and VR-S8/VR-IL2/Ad-S8 through every other week immunity four times after one week, inoculate MS
f +b16 tumor cell, measure tumor size (A) to 26 days, Survival is observed to tumor inoculation 50 days (B).
Fig. 7. multi-form S8 gene vaccine is to MS
f +the Immunotherapy of B16 tumor cell tumor growth.Giving C57BL/6 mouse hypodermic inoculation MS
f +after B16 tumor cell, give PBS matched group, carboplatin (Carboplatin), VR-S8/VR-IL2/Ad-S8 and VR-S8/VR-IL2/Ad-S8/ carboplatin vaccine respectively, measure tumor size (A) to 27 days, Survival is observed to tumor inoculation 50 days (B).
Fig. 8. the detection of anti-S8,33M and MS antibody of mouse immune serum.C57/BL mice is after PBS (1), VR-S8 (2), VR-33M (3), VR-MS (4) immunity, using the serum of mice as primary antibodie, to express the COS-7 cell lysate of MS as antigen, carry out the result of Western blotting detection.
Fig. 9 .MS vaccine produces comparing of specificity CTL killing ability with the vaccine-induced mice of single expression survivin and MUC1.From being effector lymphocyte by the lymphocyte that is separated immune mouse spleen cell, and with the murine lung cancer cell B16 of survivin (A) and MUC1 (B) specific antigen peptide (H-2d is restricted) labelling as target cell, adopt on-radiation LDH method for releasing to detect the CTL response of immunized mice.
Figure 10. the detection of the anti-MS antibody of mouse immune serum.C57/BL mice is after PBS (1), VR-IL2 (2), VR-MS (3), VR-MS/VR-IL2 (4), VR-MS/VR-IL2/Ad-MS (5) immunity, using the serum of mice as primary antibodie, to express the COS-7 cell lysate of MS as antigen, carry out the result of Western blotting detection.
Figure 11. after the multi-form immunity of MS gene vaccine, the specificity CTL killing ability of mouse spleen lymphocyte detects.From being effector lymphocyte by the lymphocyte that is separated immune mouse spleen cell, and with the murine lung cancer cell B16 of survivin (A) and MUC1 (B) specific antigen peptide (H-2d is restricted) labelling as target cell, adopt on-radiation LDH method for releasing to detect the CTL response of immunized mice.
The vaccine of Figure 12 .MS vaccine and single expression survivin and MUC1 is to model mice immunoprophylaxis effectiveness comparison.C57BL/6 mice through PBS matched group, VR-S8, VR-33M and VR-MS through every other week immunity three times after one week, inoculate MS
f +b16 tumor cell, measure tumor size (A) to 26 days, Survival is observed to tumor inoculation 50 days (B).
Figure 13. multi-form MS gene vaccine is to MS
f +the Immunotherapy of B16 tumor cell tumor growth.Giving C57BL/6 mouse hypodermic inoculation MS
f +after B16 tumor cell, give PBS matched group, carboplatin, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS and VR-MS/VR-IL2/Ad-MS/ carboplatin group respectively, measure tumor size (A) to 27 days, Survival is observed to tumor inoculation 50 days (B).
Detailed description of the invention
One. the preparation method of above-mentioned fragment 33M and S8 and condition
1.33M preparation
MUC1VNTR is a repeated fragment containing 60 bases, and No. GenBank is NM_002456 (SEQ ID NO:1).Two pairs of primers are devised according to the base sequence of a VNTR, overlap extension pcr (SOE PCR) is adopted to synthesize 1 VNTR (1m) duplicate block fragment, then restricted enzyme SalI and XhoI enzyme action is utilized to produce the characteristic of identical sticky end, connect successively, build 33M genetic fragment.Concrete grammar is as follows:
1) overlap extension pcr
PCR reaction condition is: 95 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 30s, carry out 30 circulations, the 1 copy MUC1VNTR utilizing primer P1 (SEQ ID NO:2) and P2 (SEQ ID NO:3) to amplify without ATG (is abbreviated as m), copies MUC1VNTR (being abbreviated as Am) fragment with P2 and P3 (SEQ ID NO:4) for primer amplification goes out containing 1 of ATG.
2) pGEM-T-33M structure, enzyme action qualification and sequence analysis
Above-mentioned PCR primer m is connected with pGEM-T-easy (Promega company) carrier respectively with Am, obtain pGEM-T-m and pGEM-T-Am two plasmids containing described m and Am fragment respectively, then restricted enzyme SalI and XhoI enzyme action is utilized to produce the characteristic of identical sticky end, carry out successively connecting the pGEM-T-33M plasmid obtained containing described 33M fragment, identify laggard row sequence analysis through SalI and XhoI enzyme action, sequencing result is correct.Namely pGEM-T-m SalI and XhoI double digestion are first obtained object segment m by detailed process, and then are inserted into the XhoI site of pGEM-T-m, namely obtain pGEM-T-2m.And then pGEM-T-2m SalI and XhoI double digestion are obtained object segment 2m, and then be inserted into the XhoI site of pGEM-T-2m, namely pGEM-T-4m is obtained, in like manner obtain pGEM-T-32m, and then pGEM-T-Am SalI and XhoI double digestion are obtained object segment Am, and then be inserted into the SalI site of pGEM-T-32m, namely obtain plasmid pGEM-T-33m.
2.S8 preparation
Survivin, No. GenBank is NM_001168, and two pairs of primers according to the sequential design of survivin, adopt RT-PCR technology to amplify the genetic fragment of S8 from the total serum IgE of 293 cells, sequence is the base sequence shown in SEQ NO:8.Concrete grammar is as follows:
1) RT-PCR technology
TRIzol RNA extraction test kit (Gibco company) is adopted to extract the total serum IgE of 293 cells, Super-Script Reverse Transcriptase Reagents kit (Gibco company) is adopted therefrom to obtain the cDNA of 293 cells, then with it for template, with P4 (SEQ ID NO:5) and P5 (SEQ IDNO:6) for primer carries out PCR (reaction condition is: 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, carries out 30 circulations) amplification survivin cDNA.
2) pGEM-T-S8 structure, enzyme action qualification and sequence analysis
Carry out the PCR primer of survivin cDNA and pGEM-T-easy carrier to be connected the pGEM-T-Surv plasmid obtained containing survivin cDNA.Confirm correctly through sequence analysis, take pGEM-T-Surv as template, be that primer is by pcr amplification S8 fragment (SEQ ID NO:8) with P6 (SEQ ID NO:7) and P5, forward primer P6 introduces the restriction enzyme site of EcoRI, XbaI and SalI, and reverse primer P5 introduces the restriction enzyme site of BamHI.S8PCR product is connected into pGEM-T-easy carrier and obtains the pGEM-T-S8 plasmid containing described S8 fragment, identify laggard row sequence analysis through SalI and BamHI enzyme action, sequencing result is correct.
Two. the preparation method of recombiant vaccine and corresponding condition
1.DNA carrier bacterin
1) structure of VR-33M
(VR1012 is the carrier that may be used for the test of human body gene vaccine clinical through U.S. FDA official approval of Vical company exploitation to VR1012 carrier, the clinical trial completed shows that it is safe in the application of human body, the building process of this carrier is see document (Human GeneTherapy 1996,7:1205-1217)) containing CMV promoter, kalamycin resistance gene, the conventional components compositions such as intron A and BGH PolyA translation termination signal, 4913bp altogether, Figure 1A is shown in by collection of illustrative plates.
Above-mentioned obtained pGEM-T-33M plasmid is reclaimed test kit (Beijing Tian Gen biochemical technology company limited) with glue after SalI/XhoI double digestion and reclaims 33M genetic fragment, carrier for expression of eukaryon VR1012 carrier is reclaimed test kit (Beijing Tian Gen biochemical technology company limited) with glue after SalI single endonuclease digestion and reclaims carrier segments, utilize and there is this character of identical sticky end after SalI and XhoI enzyme action by reclaimed genes of interest fragment and carrier segments, with T4 DNA ligase, at 16 DEG C, connect 3h.Product conversion competence escherichia coli Top10 (invitrogen company) is connected with gained, the LB of coating kalamycin resistance is dull and stereotyped, and at 37 DEG C overnight incubation, select positive bacteria to fall within 5mlLB culture medium and cultivate 16h at 37 DEG C, 1,2000rpm harvested by centrifugation thalline, plasmid is extracted with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), double digestion and order-checking qualification is carried out with SalI/BamHI, result is correct, namely obtain recombiant plasmid VR-33M, Fig. 1 C is shown in by collection of illustrative plates.
2) structure of VR-S8
By pGEM-T-S8 and carrier for expression of eukaryon VR1012 carrier respectively through SalI/BamHI double digestion, reclaim test kit (Beijing Tian Gen biochemical technology company limited) with glue and reclaim genes of interest fragment S8 and VR1012 carrier segments, use T
4dNA ligase, 16 DEG C connect 3h.Product conversion competence escherichia coli Top10 (invitrogen company) is connected with gained, the LB of coating kalamycin resistance is dull and stereotyped, and at 37 DEG C overnight incubation, select positive bacteria to fall within 5mlLB culture medium and cultivate 16h at 37 DEG C, 1,2000rpm harvested by centrifugation thalline, plasmid is extracted with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), double digestion and order-checking qualification is carried out with SalI/BamHI, result is correct, namely obtain recombiant plasmid VR-33M, Figure 1B is shown in by collection of illustrative plates.
3) structure of VR-MS
PGEM-T-33M is reclaimed genes of interest fragment 33M after SalI/XhoI double digestion, VR-S8 carrier is reclaimed carrier segments after SalI single endonuclease digestion, utilizes and there is this character of identical sticky end by recovery genes of interest fragment and carrier segments T after SalI and XhoI enzyme action
4dNA ligase, 16 DEG C connect 3h.Obtain VR-33M-S8, be called for short VR-MS plasmid, carry out double digestion and order-checking qualification with SalI/BamHI, result is correct, and namely obtain recombiant plasmid VR-MS, Fig. 1 D is shown in by collection of illustrative plates.
MS sequence is the such as base sequence shown in SEQ ID NO:9.
2. the acquisition of recombinant adenovirus vaccine (Ad-S8, Ad-MS)
Adenovirus is a kind of uncanned double-stranded DNA virus, and genome is about 36kb, and capsid is in 20 body structures of rule, and diameter is about 80-110nm.Respectively there is the inverted terminal repeat (ITR) of one section of 100bp at the two ends of adenoviral gene group, are the initiation sites copied.The packaging signal (ψ) having a segment length to be about 300bp in the 3 ' side of left end ITR mediates adenoviral gene group and is packaged into viral capsid.For adenovirus, the sequence only comprising the ITR at two ends and the about 0.5kb of packaging signal (ψ) is cis acting element, namely must be self-contained by adenovirus vector, and other more than 30 plant albumen and can supply by helper virus (or cell) is trans.
The adenovirus system adopted in this experiment is AdMax
tMadenovirus carrier system (Microbix company), this system is site-specific recombination system, Site-specific recombinase occurs in the restructuring on two DNA chain specific sites, and the generation of restructuring needs one section of homologous sequence and specific position and site-specific protein factor and recombinase to participate in catalysis.Recombinase can only restructuring between catalysis specific position, can not restructuring between other any two homologies of catalysis or nonhomologous sequence, and thus restructuring has specificity and well-conserved.Accordingly, Site-specific recombinase is also known as conservative restructuring, and this regrouping process does not need RecA enzyme to participate in.What current application was more has Cre/loxP, FLP/FRT and BP/attBP system, wherein Cre, FLP and BP are specific recombinase, belong to recombinase lambda integrase family, the response type of their catalysis, target site and recombination mechanism are quite similar, loxP, FRT and attBP are specific position, also have similar structure.What AdMaxTM Adenovirus carrier was applied is Cre/loxP system, Cre gene expression product is 343 amino acid whose single glycoprotein polyprotein precursors, molecular weight is 38kDa, it is the specific recombinase needed for Site-specific recombinase occurs, its recognition site is called as loxP (locus of crossing-over P1), be the DNA sequence of one section of 34bp, form 5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3 ' by the inverted repeat of two 13bp and the asymmetric intervening sequence (being also called core sequence) of a 8bp.LoxP site base forms criss-cross structure after resetting, and core sequence forms a single-stranded loop, and it is the substrate of Cre recombinase cutting, and be also that DNA identifies the site with restructuring, symmetrical 13bp inverted repeat is the sequence that Cre recombinase combines.
AdMaxTM Adenovirus carrier system is made up of shuttle vector and skeleton carrier (pBHGlox Δ E1,3Cre), and respectively containing a LoxP site arranged in the same way, wherein skeleton carrier is also containing the Cre gene regulated and controled by CMV promoter.By shuttle vector and skeleton carrier cotransfection packaging cell line 293, Cre gene entrained by skeleton carrier starts to express, mediate the restructuring between two LoxP sites, cut off reproduction element in the antibacterial on skeleton carrier, resistant gene and Cre gene, complete the insertion of genes of interest simultaneously.Although but lack be formed with the necessary packaging signal of infection ability adenovirus particles (ψ) because skeleton carrier carry most adenovirus genomic dna, shuttle vector is then containing ψ, therefore only between two carriers, there occurs restructuring, just can complete adenovirus biocycle, be formed with the recombinant adenovirus granule of infection ability; In addition, skeleton carrier disappearance E1 gene, therefore needs 293 cells of transfection constitutive expression E1 albumen.By skeleton carrier and shuttle vector, (shuttle vector selected of this research is pDC316, Fig. 2 B is shown in by collection of illustrative plates) cotransfection 293 cell, utilize the E1 albumen in 293 cells, about 10 skies just can produce the recombinant adenovirus plaque (see Fig. 2 A) containing exogenous gene, picking plaque, carry out PCR (reaction condition is: 95 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 1min, carries out 30 circulations) qualification, choose that correct clone increases in a large number, purification and titer determination.
1) to shuttle back and forth the acquisition of recombiant plasmid pDC316-S8 and pDC316-MS
S8 object fragment is obtained by reclaiming after pGEM-T-S8 EcoRI/BamHI double digestion, to reclaim as carrier after pDC316 EcoRI/Bgl П double digestion, BamHI and Bgl П is utilized to have the character of identical sticky end, by recovery genes of interest fragment and carrier segments T
4dNA ligase, 16 DEG C connect 3h.Product conversion competence escherichia coli Top10 (Invitrogen company) is connected with gained, the LB of coating kalamycin resistance is dull and stereotyped, and at 37 DEG C overnight incubation, select positive bacteria to fall within 5mlLB culture medium and cultivate 16h at 37 DEG C, 1,2000rpm harvested by centrifugation thalline, plasmid is extracted with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), double digestion and order-checking qualification is carried out with EcoRI/Bgl П, result is correct, namely obtains recombiant plasmid pDC316-S8 (Fig. 2 C is shown in by collection of illustrative plates) plasmid.MS object fragment is obtained by reclaiming after VR-MS Bgl П single endonuclease digestion, to reclaim as carrier after pDC316 Bgl П single endonuclease digestion, recovery genes of interest fragment is connected with carrier segments, correct with order-checking qualification result through double digestion, namely obtain pDC316-MS (Fig. 2 D is shown in by collection of illustrative plates) plasmid.
2) acquisition of Ad-S8 and Ad-MS recombinant virus
After pBHGlox Δ E1,3Cre and pDC316-S8 or pDC316-MS cotransfection 293 cell, after screening, amplification, purification, obtain recombinant adenovirus Ad-S8 or Ad-MS respectively containing S8 and MS fragment.
3. the acquisition of recombinant MVA vaccine (MVA-MS)
Improvement Vaccinia Ankara (MVA, Modified Vaccinia Ankara) has the advantages such as safety is high, exogenous gene capacity is large makes it be widely used in immunotherapy of tumors.MVA contains thymidine kinase gene (TK) (see Fig. 3 A); PSC11 (see Fig. 3 B) is its shuttle plasmid, there is polyclone restriction enzyme site can insert exogenous gene, there is the left arm of thymidine kinase and right arm (TKL, TKR) can carry out homologous recombination with MVA, simultaneously containing lacZ gene, may be used for the locus coeruleus screening of recombinant MVA (rMVA).Described MVA system (containing shuttle plasmid pSC 11) is purchased from ATCC.
1) to shuttle back and forth the acquisition of recombiant plasmid pSC11-MS
VR-MS and pSC11 is reclaimed after SalI/KpnI double digestion, by recovery genes of interest fragment MS and pSC11 carrier segments T
4dNA ligase, 16 DEG C connect 3h.Product conversion competence escherichia coli Top10 (invitrogen company) is connected with gained, the LB of coating kalamycin resistance is dull and stereotyped, and at 37 DEG C overnight incubation, select positive bacteria to fall within 5mlLB culture medium and cultivate 16h at 37 DEG C, 12000rpm harvested by centrifugation thalline, plasmid is extracted with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), double digestion and order-checking qualification is carried out with SalI/KpnI, result is correct, namely obtains recombiant plasmid pSC11-MS (see Fig. 3 C).
2) acquisition of MVA-MS recombinant virus
First (purchased from ATCC, No. ATCC is CRL-1632 to infect tk-ts13 cell with the MVA of titre 0.05pfu/cell
tM, be the mutant of the BHK-21 not containing TK gene).Infect after 2 hours, utilize the method for Lipofection2000 (INVITROGEN) to be transfected into by pSC11-MS and infected in the tk-ts13 cell of MVA.MVA is in the process of tk-ts13 time multiplexed cell, because pSC11-MS has TKL and TKR with the TK DNA homolog of MVA, so there is a certain proportion of MVA virus to recombinate with pSC11-MS, result MS and LacZ reading frame are recombined in MVA viral genome.Form MVA-MS recombinant virus as shown in Figure 3 C.
Infected cell culture is after three days above, collecting cell, cell lysis, ultrasonic Treatment, and the centrifugal 10min of 2000rpm removes cell residue and retains supernatant.Get a certain amount of supernatant, as kind of a poison, in 6 orifice plates, virus is diluted to 10 successively
-2, 10
-3, 10
-4, 10
-5, 10
-6, infect tk-ts13 cell 2h.By 2% low melting-point agarose of isopyknic pre-temperature at 45 DEG C and 2 × DMEM-10/BrdU (BrdU, 5-bromine uracil, can by TK phosphorylation, the BrdU of phosphorylation can be incorporated in wild type MVA, produce lethal mutation, thus wild type MVA can be made to reduce gradually) culture medium mixing, every hole adds 3ml culture medium, at room temperature solidify, in containing 5%CO at 37 DEG C
2incubator in cultivate 48h.Spread layer-selective culture medium, the 4%X-gal of composition is more than bottom agar 1/120 volume.Overnight incubation, selects blue single speckle, multigelation three breakup releasing virus, and carry out the screening of next round, step is identical, so carries out 6 and takes turns above screening, last until obtain only containing the clone strain of recombinant virus MVA-MS.
Three. immune effect of vaccine
1.S8 immune effect of vaccine
1.1S8 the immunogenicity of vaccine
This part experiment inquires into survivin DNA vaccinations and recombinant adenovirus vaccine from humoral immunization and cellular immunization two aspects, and its immunne response with the common immune induction mice of IL-2 plasmid.C57/BL/6 mice divides into groups by table 1 by experiment, and 1 group at the 0th, 2,4,6 week injection PBS; 2-4 group is at the 0th, 2,4,6 week injection VR-S8 vaccine and VR-IL2 adjuvant; At the 0th, 2,4 week injection VR-S8DNA vaccine and VR-IL2 adjuvant, injected recombinant adenovirus vaccine at the 6th week for 5 groups.Investigate the survivin DNA single solely immune effect of immunity, the adjuvant effect of IL-2 plasmid, and the immunological enhancement of recombinant adenovirus.
Table 1 S8 vaccine immunogenicity
1.1.1 the Western blotting of humoral immunization-antibody detects
After final immunization 1 week, tail venous blood sampling is carried out to mice, institute's separation of serum is used for detection of specific antibody.Collect the cracking supernatant of the COS-7 cell of expressing S8, after SDS-PAGE is separated, be transferred on nitrocellulose filter, as the antigen detecting mice serum.The immune serum of 1: 20 dilution (diluent is the milk of 1% of PBS preparation) is got as first antibody from each experimental group, using anti-survivin monoclonal antibody as positive control, using the horse against murine IgG (immuno research company) with alkali phosphatase enzyme mark as second antibody, detect the specificity of each immune serum survivin antibody through western blotting.
Result as shown in Figure 4, PBS (swimming lane 2) group and IL2 (swimming lane 3) group do not dye band, and other experimental group testing results are all positive, show that C57/BL/6 mice is after survivin gene vaccine immunity, creates the specific antibody of anti-survivin antigen in its serum.The S8 separately immune antibody titer produced is more weak, and the antibody titer produced after adding IL-2 strengthens to some extent, and after recombinant adenovirus Ad-S8 booster immunization, antibody titer obviously strengthens.
1.1.2 cellular immunization-cytotoxic T lymphocyte (CTL) is active
Within after final immunization two weeks, put to death mice, get spleen and be separated lymphocyte.With from by the lymphocyte that is separated in immune mouse spleen cell for effector lymphocyte, and with survivin specific antigen peptide (S
88-96) the B16 cell (purchased from ADCC) of labelling, mix above-mentioned effector lymphocyte and target cell by a certain percentage, adopt inactive lactic acid dehydrogenase (LDH) method for releasing detection kit Cyto Tox96 (Promega) to detect lymphocyte and situation is killed and wounded to target cell, be used for characterizing the specific CTL response strength of described vaccine at mice Immune inducing in vivo.
The present invention predicts the H-2D of survivin according to SYFPEITHI algorithm (www.syf-peithi.de)
bmHCI restricted epitope peptide, selects and H-2D
bantigenic peptides (the S that the binding constant value of MHCI is higher
88-96) carry out above-mentioned experiment as antigenic peptides.
As shown in Figure 5, as seen from Figure 5, compared with PBS matched group, VR-IL2 group does not detect survivin specific CTL (p > 0.05) to experimental result; When effect target ratio is 100: 1, VR-S8 group only can detect that CTL replys, but do not have statistical significance; VR-S8/VR-IL2 combined immunization group all detects specific CTL response (p < 0.05) when effect target ratio is 100: 1 and 33: 1, and the CTL response of immune group more independent than VR-S8 increases but do not have statistical significance (p > 0.05); VR-S8/VR-IL2/Ad-S8 group has injected again a pin recombinant adenovirus vaccine Ad-S8 booster immunization after having injected three pin DNA vaccinations, result all detects specific CTL response (p < 0.01) when to imitate target ratio be 100: 1,33: 1 and 10: 1, compares CTL reply and also significantly improve (p < 0.05) with VR-S8/VR-IL2 group.
Can find out that independent application VR-S8 gene vaccine can produce specific antibody for survivin and CTL immunne response by inducing mouse by above-mentioned humoral immunization and cellular immunization result.After immunological adjuvant VR-IL2 plasmid and VR-S8DNA vaccine are injected altogether, the specific humoral immune that inducing mouse produces and cell immune response strengthen all to some extent, show that VR-IL2 can strengthen the immunne response of VR-S8 gene vaccine, serve the effect of immunological adjuvant; Recombinant adenovirus vaccine carries out antibody titer and the CTL response level that booster immunization can significantly improve VR-S8/VR-IL2 combined immunization group after three pin DNA vaccination immunity, serves the effect of booster immunization.
1.2S8 vaccine is tumor protecting
1.2.1 preventative
Specific humoral immunity and cellular immunization can be produced by inducing mouse according to the known survivin gene vaccine of result above; this section adopts the DNA vaccination of survivin and recombinant adenovirus vaccine and IL2 to carry out the prevention experiment of model mice tumor; whether can produce protectiveness anti-tumor immune response at mice Immune inducing in vivo to investigate survivin gene vaccine, and whether VR-IL2 plasmid adjuvant and prime-boost immunization ways can promote this protective effect.
Table 2 survivin vaccine prevention effect
C57BL/6 mice is divided into five groups (15/group) at random by table 3.1st, 2,3,4 groups respectively at 0,2,4,6 week immune DNA vaccination; Recombinant adenovirus vaccine booster immunization was injected at the 6th week for 5th group at 0,2,4 week immune DNA vaccination.All mices all carry on the back bottom subcutaneous vaccination MS in right side of mice at final immunization after one week
f +b16 tumor cell, investigates into the change of tumor time and tumor size, other five groups of changes simultaneously observing mouse survival rate.Experiment grouping and immunization time as shown in table 4.
1.2.1.1 the inhibitory action of tumor growth
Experimental result shows, compared with PBS immune group, the model mice tumor growth of VR-S8, VR-S8/VR-IL2 and VR-S8/VR-IL2/Ad-S8 immune group obviously slows down, and slows down successively with the reinforcement of recombinant adenovirus Ad-S8 along with adding of IL2 adjuvant.Fig. 6 A is prevention experiment each group of gross tumor volume situation of change, and as seen from the figure: VR-IL2 immune group compares with PBS matched group, tumor growth is without significant change; VR-S8 immune group compares with PBS group, the poor growth of mouse tumor, and there were significant differences for the two (after inoculated tumour 23 to 26 days, p<0.05); Compare with the independent immune group of VR-S8, altogether immune group tumor growth is obviously slowly (after inoculated tumour 20 to 26 days, p<0.05) for VR-S8/VR-IL2; Compared with VR-S8/VR-IL2 altogether immune group, VR-S8/VR-IL2/Ad-S8 group tumor growth obviously slows down (after inoculated tumour 20 to 23 days, p<0.05; 24 to 26 days, p<0.01).
1.2.1.2 model mice life extends situation
PBS group mean survival time (MST) is 33.92 ± 1.36 days, and to inoculated tumour cell, when 40 days, mice is all dead, VR-IL2 group the average survival time 35.40 ± 1.70 days, and to inoculated tumour cell, 48 days mices are all dead, with PBS group without obviously distinguishing; VR-S8 group the average survival time 37.47 ± 1.60 days, to inoculated tumour cell 50 days, have the mouse survival of 6.7%, increase in life span was 10.5%, but compared with PBS group and do not have significant difference; And VR-S8/VR-IL2 group the average survival time 39.67 ± 1.70 days, have the mouse survival of 6.7% to the observation period, increase in life span is 17%, compares with PBS group, life obviously extends (p<0.05), but compares with VR-S8 group and do not have notable difference; And VR-S8/VR-IL2/Ad-S8 group the average survival time 46.01 ± 1.60 days, still have the mouse survival of 53.3% to the observation period, increase in life span is 35.6%, compares with other groups, and life obviously extends (p<0.01), sees Fig. 6 B.
As can be seen from above-mentioned preventive experiment result, VR-S8 vaccine can the growth of obvious inhibition mouse tumor, but can not effective lengthening model survival time of mice; But after adding IL2 adjuvant (VR-S8/VR-IL2 immune group), not only the growth of remarkable inhibition mouse tumor can effectively can also improve life cycle of model mice, therefore VR-IL2 serves the effect of immunological adjuvant; After VR-S8/VR-IL2 immunity three times, with can the growth of inhibition mouse tumor of highly significant the life cycle of very significant lengthening model mice after recombinant adenovirus booster immunization (VR-S8/VR-IL2/Ad-S8 immune group), illustrate that prime-boost strategy obviously can strengthen the immune effect of vaccine.
1.2.2 therapeutic
According to the result of model mouse prevention experiment; S8DNA vaccine is under IL-2 adjuvant is auxiliary; antitumor protective effect can be very effectively played with recombinant adenovirus vaccine Ad-S8 booster immunization; therefore in the Experiment on therapy of model mice tumor; use recombinant adenovirus Ad-S8 booster immunization after selecting VR-S8 and VR-IL2 to be total to immune three pins, investigate VR-S8/VR-IL2/Ad-S8 gene vaccine to the inhibitory action of model mice tumor growth in vivo and the prolongation effect to mice with tumor life.Meanwhile, do positive control with chemotherapeutics carboplatin, whether by the mode of VR-S8/VR-IL2/Ad-S8 vaccine and carboplatin administration use in conjunction, inquire into the two has synergism in the treatment of model mice tumor.
Table 3 survivin vaccine therapy effect
(note: the administration time of the carboplatin in table 3 is the 1st, 6,11,16 day)
C57BL/6 mice is divided into VR-S8/VR-IL2/Ad-S8, VR-S8/VR-IL2/Ad-S8 and carboplatin synergy group (VR-S8/VR-IL2/Ad-S8/Carb), carboplatin positive controls (carboplatin) and matched group (PBS) at random by table 3, often organizes 15.Subcutaneous vaccination MS in 0.7cm place is about in right side of mice back of the body bottom near root of the tail portion at the 0th day
f +b16 tumor cell, within the 1st, 8,15 day, give gene vaccine and PBS respectively, administering mode is intramuscular injection; The administration time of carboplatin is the 1st, 6,11,16 day, and administering mode is lumbar injection.The growth of tumor and the change of size is observed, other four groups of changes simultaneously observing mouse survival rate from about the 10th day.Tumour inhibiting rate group experiment mice starts death about the 28th day control group mice, puts to death whole mice, peels off tumor, measures tumor weight, calculates tumour inhibiting rate; Life cycle, group experiment mice observed the Survival of mice, until mice is all dead, the time is about 50 days.
1.2.2.1 the inhibitory action of tumor growth
Fig. 7 A is that Experiment on therapy respectively organizes gross tumor volume situation of change.Compare with PBS matched group, VR-S8/VR-IL2/Ad-S8, carboplatin and VR-S8/VR-IL2/Ad-S8/ carboplatin group tumor growth obviously slow down, after inoculated tumour 21 to 27 days, are p<0.05); VR-S8/VR-IL2/Ad-S8 compares with the two independent set of applications with carboplatin use in conjunction group, tumor growth rate obviously slows down (after inoculated tumour 21 to 27 days, be p<0.05), result shows that carboplatin and vaccine use in conjunction can significantly improve the inhibitory action of vaccine to mice with tumor tumor growth.
1.2.2.2 model mice life extends situation
PBS group mean survival time (MST) is 31.21 days, and to inoculated tumour cell, when 38 days, mice is all dead; VR-S8/VR-IL2/Ad-S8 group the average survival time 33.64 days, to inoculated tumour cell, when 44 days, mice is all dead, and comparing with PBS group does not have significant difference; Carboplatin group the average survival time natural law is respectively 35.29, and to the mouse survival having 7.1% during the observation period, increase in life span is 13.07%, compares Bearing Mice Life all obviously extend (being p<0.05) with matched group; VR-S8/VR-IL2/Ad-S8 and carboplatin use in conjunction group the average survival time natural law are 39.29 days, to the mouse survival having 21.4% during the observation period, increase in life span is 25.89%, compares Bearing Mice Life all obviously extend (being p<0.01) with matched group; Carboplatin compares with the independent set of applications of vaccine with vaccine use in conjunction group, and Bearing Mice Life obviously extends, and shows that carboplatin can significantly improve the prolongation effect of vaccine to mice with tumor life cycle, as Fig. 7 B.
2.MS immune effect
The immunogenicity of 2.1 MS vaccines
2.1.1 the comparing of immune effect of MS vaccine and survivin vaccine and MUC1 vaccine
Whether this part experiment inquires into survivin from humoral immunization and cellular immunization two aspects has obvious enhancing with the immunogenicity of MUC1 fusion gene vaccine compared with the vaccine of both single expression.C57/BL/6 mice divides into groups by table 4 by experiment, often organizes 5, gives respectively and PBS for first group at 0,2,4 weeks, the 2nd, 3,4 groups at 0,2,4 weeks the respectively corresponding DNA vaccination of immunity, all mices drew neck execution at the 6th week, get spleen is CTL.Whether investigation survivin and MUC1 fusion gene immune effect of vaccine are better than the immune effect both applying separately.
Comparing of the immune effect of table 4 MS vaccine and survivin vaccine and MUC1 vaccine
2.1.1.1 the Western blotting of humoral immunization-antibody detects
After final immunization 1 week, carry out tail venous blood sampling to mice, separation of serum is used for detection of specific antibody.Collect the cracking supernatant of the COS-7 cell of expressing MS, after SDS-PAGE is separated, be transferred on nitrocellulose filter, as the antigen detecting mice serum.The immune serum of 1: 20 dilution (diluent is the milk of 1% of PBS preparation) is got as first antibody from each experimental group.With the horse against murine IgG (immuno research company) of alkali phosphatase enzyme mark as second antibody, detect the titre of survivin and MUC1 specific antibody in each immune group mice serum through western blotting.
As shown in Figure 8, for each group of immune mouse produces the situation of antibody, VR-S8 (swimming lane 2), VR-33M (swimming lane 3) and VR-MS (swimming lane 4) organize the specific antibody that mice all creates anti-survivin and MUC1 as seen from the figure, wherein the antibody titer of VR-33M group generation is the strongest, and the antibody titer that VR-MS group produces is weaker than the antibody titer that VR-33M group produces.This is unexpected result, infer its reason may be MUC1 this as transmembrane protein and major part outside born of the same parents, therefore producible antibody titer is higher; And survivin is intracellular protein, MUC1 may be hindered to cell membrane transporter after survivin and MUC1 merge, extend the holdup time of described fusion rotein in born of the same parents, the probability that protease is hydrolyzed it may be improved, thus reduce described fusion rotein (MS) stability in vivo, make it be degraded rapidly, and then antibody titer is declined.But this process may provide a large amount of little peptides that can be combined with MHC molecule for cellular immune pathway, and then promote cellullar immunologic response.
2.1.1.2 cellular immunization-cytotoxic T lymphocyte (CTL) is active
With from by the lymphocyte that is separated in immune mouse spleen cell for effector lymphocyte, and with survivin or the peptide-labeled B16 cell of MUC1 specific antigen, mix above-mentioned effector lymphocyte and target cell by a certain percentage, adopt inactive lactic acid dehydrogenase (LDH) method for releasing detection kit Cyto Tox96 (Promega) to measure the cleaved ratio of target cell B16, be used for characterizing the specific CTL response strength of this vaccine at mice Immune inducing in vivo.Result as shown in Figure 9.
Fig. 9 is each immune group (VR-S8, VR-33M and VR-MS) the mice generation survivin of table 4 correspondence and the situation of MUC1 specific CTL.A figure be the B16 cell of hatching using the CTL restricted epitope peptide special with survivin as target cell, detect the situation producing survivin specific CTL with different vaccine-induced mices.As seen from the figure, compared with PBS matched group, VR-S8 group does not detect survivin specific CTL (p > 0.05); VR-MS group detects survivin specific CTL (p < 0.05), but the difference compared with VR-S8 group on not statistically significant; And VR-33M group does not detect that CTL replys, illustrate that the CTL produced is plain specific for survival.
B figure be the B16 cell of hatching using the CTL restricted epitope peptide special with MUC1 as target cell, detect the situation producing MUC1 specific CTL with different vaccine-induced mices.As seen from the figure, compared with PBS matched group, VR-33M and VR-MS group all creates the specific CTL of MUC1 (p < 0.05), and the CTL that VR-MS group produces replys level, a little more than the response level that VR-33M group produces, but the difference on not statistically significant; And VR-S8 group does not detect that CTL replys, illustrate that the CTL produced is that MUC1 is specific.
Visible, VR-33M, VR-MS gene vaccine all can produce special CTL immunne response by inducing mouse.
2.1.2 just exempt from-booster immunization (Prime-boost) immunization strategy and IL2 adjuvant to the immunological enhancement of MS vaccine
This part experiment adopts at the beginning of DNA exempts from the-immunization strategy of recombinant adenovirus booster immunization, and IL2 immunological adjuvant inquires into from humoral immunization and cellular immunization two aspects the situation that survivins and the vaccine-induced mice of MUC1 fusion gene produce immunne response, investigation prime-boost immunization strategy is to the adjuvant effect of the potentiation of immune effect of vaccine and IL2 plasmid.
Exempt from the beginning of table 5-booster immunization immunization strategy and IL2 adjuvant be to the immunological enhancement of MS vaccine
C57/BL/6 mice is divided into groups by table five, often organize 5,1st, DNA vaccination was injected at the 0th, 2,4,6 week respectively for 2,3 groups, 4th, DNA vaccination was injected at the 0th, 2,4 week respectively for 5 groups, 6th week injection recombinant adenovirus vaccine, within 8th week, put to death all mices, get spleen, detect survivin and MUC1 specific CTL.
2.1.2.1 the Western blotting of humoral immunization-antibody detects
After final immunization 1 week, carry out tail venous blood sampling to mice, separation of serum is used for detection of specific antibody.Collect the cracking supernatant of the COS-7 cell of expressing MS, after SDS-PAGE is separated, be transferred on nitrocellulose filter, as the antigen detecting mice serum.The immune serum of 1: 20 dilution (diluent is the milk of 1% of PBS preparation) is got as first antibody from each experimental group.Using the horse against murine IgG (immuno research company) with alkali phosphatase enzyme mark as second antibody, detect the titre of survivin and MUC1 specific antibody in each immune group mice serum through western blotting.
Figure 10 is the production of survivin and MUC1 specific antibody in each immune group mice serum of table 5 correspondence.As seen from Figure 10, mice through PBS (swimming lane 1) and IL2 (swimming lane 2) immunity does not produce survivin and MUC1 specific antibody, and the mice of VR-MS (swimming lane 3), VR-MS/Ad-MS (swimming lane 4) and VR-MS/IL2/Ad-MS (swimming lane 5) immunity all creates survivin and MUC1 specific antibody, and antibody titer strengthens successively, show that recombinant adenovirus Ad-MS and IL2 all serves the effect strengthening immune effect.
2.1.2.2 cellular immunization---cytotoxic T lymphocyte (CTL) is active
The present invention with from by the lymphocyte that is separated in immune mouse spleen cell for effector lymphocyte, and with survivin or the peptide-labeled B16 cell of MUC1 specific antigen or stable expression cell line MS
f +b16 (purchased from ATCC), as target cell, detects in Mice Body and produces specific CTL response situation.Result as shown in figure 11.
Figure 11 is each immune group (PBS, VR-IL2, VR-MS, VR-MS/Ad-MS and VR-MS/VR-IL2/Ad-MS) the mice generation survivin of table 5 correspondence and the situation of MUC1 specific CTL.A figure be the B16 cell of hatching using the CTL restricted epitope peptide special with survivin as target cell, detect the situation that different vaccine-induced mices produces survivin specific CTL.As seen from the figure, compared with PBS matched group, VR-MS, VR-MS/Ad-MS and VR-MS/VR-IL2/Ad-MS group all detects survivin specific CTL (p < 0.05), the CTL response level produced raises successively, and all has statistical significance (being followed successively by p < 0.05); And VR-IL2 group does not detect that CTL replys, illustrate that the CTL produced is plain specific for survival.
B figure be the B16 cell of hatching using the CTL restricted epitope peptide special with MUC1 as target cell, detect the situation that different vaccine-induced mices produces MUC1 specific CTL.As seen from the figure compared with PBS matched group, VR-MS, VR-MS/Ad-MS, VR-MS/VR-IL2/Ad-MS group all creates the specific CTL of MUC1 (p < 0.05), the CTL response level produced raises successively, and all has statistical significance (being followed successively by p < 0.05); And VR-IL2 group does not detect that CTL replys, illustrate that the CTL produced is that MUC1 is specific.
In sum, VR-MS, VR-MS/Ad-MS, VR-MS/VR-IL2/Ad-MS gene vaccine all can produce for survivin or the special CTL immunne response of MUC1 by inducing mouse, and the CTL level produced obviously raises successively, illustrate exempt from the beginning of DNA-immunization strategy of recombinant adenovirus booster immunization obviously can strengthen the immune effect of vaccine; IL2 also serves the potentiation of immunological adjuvant.
2.2MS vaccine is to the protective effect of model mice
2.2.1 prophylactic action
Adopt DNA vaccination VR-S8, VR-33M and VR-MS to carry out the prevention experiment of Model Tumor, whether the protection investigating survivin and MUC1 amalgamation and expression vaccine on mouse tumor is significantly improved compared with the protection of the vaccine of both single expression.
C57BL/6 mice is divided into four groups (10/group).PBS, VR-S8, VR-33M and VR-MS group is respectively at 0,2,4,6 week immune DNA vaccination and PBS, and all mices all inoculate MS in the right leg outer side of mice at final immunization after one week
f +b16 tumor cell, investigates into the change of tumor time and tumor size, the change of other four groups of observation mouse survival rates.Experiment grouping and immunization time as follows:
Preventative to model mice tumor of table 6 MS vaccine
2.2.1.1 the inhibitory action of tumor growth
Figure 12 A is group gross tumor volume situation of change each in prevention experiment.VR-S8, VR-33M immune group is compared with PBS matched group, and the poor growth of mouse tumor, has significant difference (from after inoculated tumour 22 days, p<0.05); And VR-MS immune group is compared with PBS matched group, the growth of mouse tumor is obviously slowed down, and has significant differences (from after inoculated tumour 20 days, p<0.05, beginning in 22 days, p<0.01); VR-MS immune group is compared with the independent immune group of VR-S8, and gross tumor volume obviously reduces (after inoculated tumour, 22 start, p<0.05, and 24 days start, p<0.01), but compares without significant difference with VR-33M group.
2.2.1.2 the Survival of mice
PBS group mean survival time (MST) is 31.6 days, and to inoculated tumour cell, when 38 days, mice is all dead; VR-S8 group the average survival time 35.7 days, to inoculated tumour cell, 45 days mices are all dead, with PBS group without obviously distinguishing; VR-33M group the average survival time 37.89 days, to inoculated tumour cell 50 days, have the mouse survival of 11.1%, comparing with PBS group had significant difference (p<0.05); And VR-MS group the average survival time 42.5 days, the mouse survival still having 37.5% is terminated to the observation period, increase in life span is 34.49% (p<0.01), compares with VR-S8 group and VR-33M group, and life obviously extends (p<0.05).See Figure 12 B.
As can be seen from above-mentioned preventive experiment result, VR-MS vaccine is obviously better than VR-S8 vaccine to the growth inhibited effect of model mice tumor with to the model mice life situation that extends; Compared with VR-33M vaccine, although do not significantly improve in Tumor growth inhibition, can obvious lengthening model Bearing Mice Life.Started to tend to the prolongation to tumor patient life and the raising of quality of life to the evaluation of tumor vaccine at present, even disappearing with tumor regression becomes the mindset of people already as treatment of solid tumors evaluation criterion.And when being indiscriminately imitated to tumor vaccine and evaluating, people but find that it can not reflect the true curative effect of vaccine.Although because some tumor vaccine effectively extends patient vitals, improve quality of life, gross tumor volume may not obviously reduce.Therefore, we think that the protection of VR-MS vaccine to tumor is significantly improved.
2.2.2 therapeutic effect
The immune effect of vaccine and the protection to mouse tumor can be significantly improved according to the known IL2 adjuvant of experimental result above; DNA just exempts from-and the immunization strategy of recombinant adenovirus booster immunization significantly can strengthen the immune effect of DNA vaccination and the protection to mouse tumor; The immune effect of survivin and MUC1 amalgamation and expression vaccine and tumor protection power significantly improve when being comparatively used alone the two.Therefore this section is according to above-mentioned conclusion, in the Experiment on therapy of model mice tumor, selection VR-MS and VR-IL2 uses recombinant adenovirus Ad-MS booster immunization after being total to immune three pins, investigates VR-MS/VR-IL2/Ad-MS gene vaccine to the inhibitory action of model mice tumor growth in vivo and the prolongation effect to mice with tumor life.Meanwhile, do positive control with chemotherapeutics carboplatin, by VR-MS/VR-IL2/Ad-MS immunity and the mode of carboplatin administration use in conjunction, both inquiring into, in the treatment of model mice tumor, whether have synergism.Simultaneously in order to investigate the immunopotentiation effect of MVA-MS, devising again VR-MS/VR-IL2/MVA-MS immune group in an experiment, and then comparing the power of Ad-MS and MVA-MS immunopotentiation effect.
C57BL/6 mice is divided into VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS, VR-MS/VR-IL2/Ad-MS and carboplatin synergy group (MS/IL2/Ad-MS/ carboplatin), carboplatin positive controls and matched group (PBS) at random by table 7, often organizes 15.At the 0th day in right side of mice back of the body bottom subcutaneous vaccination MS
f +b16 tumor cell, gave gene vaccine and PBS respectively at the 1st, 8,15 day, and administering mode is intramuscular injection; The administration time of carboplatin is the 1st, 6,11,16 day, and administering mode is lumbar injection.The growth of tumor and the change of size is observed, other four groups of changes simultaneously observing mouse survival rate from about the 10th day.Tumour inhibiting rate group experiment mice starts death about the 28th day control group mice, puts to death whole mice, peels off tumor, measures tumor weight, calculates tumour inhibiting rate; Life cycle, group experiment mice observed the Survival of mice, until mice is all dead, the time is about 50 days.
Table 7 MS vaccine is to the therapeutic (tumour inhibiting rate experiment) of model mice tumor
(note: the administration time of the carboplatin in table 7 is the 1st, 6,11,16 day)
2.2.2.1 the inhibitory action of tumor growth
Figure 13 A is that Experiment on therapy respectively organizes gross tumor volume situation of change.Compare with PBS matched group, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS, carboplatin and VR-MS/VR-IL2/Ad-MS/ carboplatin group all can the growths (after inoculated tumour 21 to 27 days, be p<0.05) of obvious Tumor suppression; VR-MS/VR-IL2/Ad-MS compares with the group both applying separately with carboplatin use in conjunction group, and tumor growth rate slightly slows down, but does not have statistical significance, and the two use in conjunction fails to play collaborative or cumulative effect; VR-MS/VR-IL2/Ad-MS group compares with VR-MS/VR-IL2/MVA-MS group does not have significant difference.
2.2.2.2 the Survival of mice
PBS group mean survival time (MST) is 31.21 days, and to inoculated tumour cell, when 38 days, mice is all dead, VR-MS/VR-IL2/MVA-MS and VR-MS/VR-IL2/Ad-MS group the average survival time is respectively 34.21 and 35.28 days, and comparing with PBS group does not have significant difference; Carboplatin group the average survival time natural law is 35.29, and to the mouse survival having 7.1% during the observation period, increase in life span is respectively 13.07%, compares with matched group, and Bearing Mice Life all obviously extends (p<0.05); VR-MS/VR-IL2/Ad-MS and carboplatin use in conjunction group the average survival time natural law are 38.36, to the mouse survival having 28.6% during the observation period, increase in life span is 22.91%, compare Bearing Mice Life all obviously to extend (being p<0.01) with matched group, compare Bearing Mice Life with the set of applications of the two single expression vaccine and obviously extend (p<0.05); VR-MS/VR-IL2/Ad-MS group does not have notable difference compared with VR-MS/VR-IL2/MVA-MS group, as shown in Figure 13 B.
As can be seen from above-mentioned curative test result, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS can obviously inhibition mouse tumor growth but can not life cycle of effective lengthening model mice; VR-MS/VR-IL2/Ad-MS vaccine and carboplatin use in conjunction not only can the growth of remarkable inhibition mouse tumor can also life cycle of effective lengthening model mice; Compared with vaccine is applied separately with carboplatin combined immunization and the two, although do not embody obvious advantage in Tumor suppression growth, the life cycle of energy significant prolongation model mice.Traditional tumor vaccine evaluation criterion tends to reducing of gross tumor volume, but the clinical practice of oncotherapy proves that some tumor vaccine effectively can extend patient vitals, improves the quality of living, but obviously may not necessarily reduce gross tumor volume.Therefore, we think that carboplatin and vaccine use in conjunction and the two are applied the protection comparing model mouse tumor separately and be significantly improved.
List of references
1.Li,F.and X.Ling,Survivin study:an update of"what is thenext wave"？J Cell Physiol,2006.208(3):p.476-86.
2.Li,F.,Survivin study:what is the next wave？J Cell Physiol,2003.197(1):p.8-29.
3.Altieri,D.C.,Survivin,cancer networks and pathway-directeddrug discovery.Nat Rev Cancer,2008.8(1):p.61-70.
4.Shi,Y.,Survivin structure:crystal unclear.Nat Struct Biol,2000.7(8):p.620-3.
5.Andersen,M.H.,et al.,Spontaneous cytotoxic T-cell responsesagainst survivin-derived MHC class I-restricted T-cell epitopes in situas well as ex vivo in cancer patients.Cancer Res,2001.61(16):p.5964-8.
6.Singh,R.and D.Bandyopadhyay,MUC1:a target molecule forcancer therapy.Cancer Biol Ther,2007.6(4):p.481-6.
7.Persson,J.and M.Ohlin,Antigens for the selection ofpan-variable number of tandem repeats motif-specific humanantibodies against Mucin-1.J Immunol Methods,2006.316(1-2):p.116-24.
8.Xing,P.X.,J.Prenzoska,and I.F.McKenzie,Epitope mapping ofanti-breast and anti-ovarian mucin monoclonal antibodies.MolImmunol,1992.29(5):p.641-50.
9.Tang,C.K.,M.Katsara,and V.Apostolopoulos,Strategies usedfor MUC1 immunotherapy:human clinical studies.Expert RevVaccines,2008.7(7):p.963-75.
10.Tang,C.K.and V.Apostolopoulos,Strategies used for MUC1immunotherapy:preclinical studies.Expert Rev Vaccines,2008.7(7):p.951-62.
11.Zhang S,Zhang H,Shi H,Yu X,Kong W,Li W.Induction ofimmune response and anti-tumor activities in mice with a DNA vaccineencoding human mucin 1 variable-number tandem repeats.Hum.Immunol.69(4–5),250–258(2008).
Claims (10)
1. the DNA fragmentation MS shown in SEQ ID NO:9 is for the preparation of a purposes of carrying out in the protein vaccine of antineoplastic immune, DNA vaccination, vector-viral vaccine or dendritic cell vaccine, and wherein said fragment MS comprises the DNA fragmentation S8 fragment that the DNA fragmentation 33M of the MUC1 VNTR containing 33 copies and a kind of sequence are SEQ ID NO:8.
2. the purposes described in claim 1, also comprises immunological adjuvant in wherein said vaccine; Preferably, described immunological adjuvant is selected from cytokine interleukin element 2 (IL-2), cytokine grain-giant cell colony stimulating factor (GM-CSF) and non-methylated CpG motif immune stimulates DNA sequence.
3. the purposes described in claim 1 or 2, also comprises chemotherapeutics in wherein said vaccine; Preferably, described chemotherapeutics is selected from carboplatin, cisplatin, oxaliplatin and paclitaxel.
4. a recombiant plasmid VR-MS is for the preparation of the purposes of carrying out in the DNA vaccination of antineoplastic immune, the feature of wherein said recombiant plasmid VR-MS is that the skeleton carrier of described recombiant plasmid is carrier VR1012, in the multiple clone site of described VR1012, insert the MS fragment shown in a SEQ ID NO:9; Preferably, the MS fragment inserted is between the multiple clone site SalI and BamHI of described VR1012.
5. the purposes described in claim 4, a kind of recombinant adenovirus Ad-MS or a kind of recombinant mva virus MVA-MS is also comprised in wherein said vaccine, and carry out just exempting from VR-MS, carry out booster immunization with Ad-MS or MVA-MS, the feature of wherein said recombinant adenovirus Ad-MS is in adenovirus, insert the MS fragment shown in SEQ ID NO:9; Preferably, the feature of described recombinant adenovirus Ad-MS is that the skeleton carrier of described recombinant adenovirus is pBHGlox Δ E1,3Cre, the MS fragment inserted is positioned at the multiple clone site BglII place of described recombinant adenovirus, and the feature of wherein said recombinant mva virus MVA-MS is to insert the MS fragment shown in SEQ ID NO:9 in improvement vaccinia ankara virus; Preferably, the feature of described recombinant mva virus MVA-MS is that inserted MS fragment is between the multiple clone site SalI and KpnI of described recombinant mva virus.
6. the purposes described in claim 4 or 5, also comprises immunological adjuvant in wherein said vaccine, and wherein said immunological adjuvant and described VR-MS together give.
7. the purposes described in claim 6, wherein said immunological adjuvant is selected from cytokine interleukin element 2 (IL-2), cytokine grain-giant cell colony stimulating factor (GM-CSF) and non-methylated CpG motif immune stimulates DNA sequence.
8. the purposes according to any one of claim 4-6, also comprises chemotherapeutics in wherein said vaccine.
9. purposes according to claim 8, wherein said chemotherapeutics is selected from carboplatin, cisplatin, oxaliplatin and paclitaxel.
10. purposes in any one of the preceding claims wherein, wherein said tumor is express the tumor of survivin and MUC1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410788971.7A CN104491852B (en) | 2009-12-04 | 2009-12-04 | Tumor DNA vaccine with MUC-1 and survivin as target spot and vector-viral vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410788971.7A CN104491852B (en) | 2009-12-04 | 2009-12-04 | Tumor DNA vaccine with MUC-1 and survivin as target spot and vector-viral vaccine |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910252427.XA Division CN102086453B (en) | 2009-12-04 | 2009-12-04 | Tumor DNA vaccine taking mucin 1 and survivin as targets and viral vector vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104491852A true CN104491852A (en) | 2015-04-08 |
| CN104491852B CN104491852B (en) | 2016-08-03 |
Family
ID=52933365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410788971.7A Active CN104491852B (en) | 2009-12-04 | 2009-12-04 | Tumor DNA vaccine with MUC-1 and survivin as target spot and vector-viral vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104491852B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174469B (en) * | 2011-01-26 | 2012-11-21 | 宋鑫 | Method for effectively culturing tumor infiltrating lymphocytes (TILs) |
-
2009
- 2009-12-04 CN CN201410788971.7A patent/CN104491852B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104491852B (en) | 2016-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4805511B2 (en) | Improvement in immune response to HIV or improvement in immune response | |
| EP2402451B1 (en) | Method for stabilizing an DNA insert in a recombinant vaccinia vector | |
| KR20220154114A (en) | Vaccines and their use for inducing an immune response against SARS-CoV2 | |
| EP2488649B1 (en) | Recombinant modified vaccinia ankara (mva) vaccinia virus containing restructured insertion sites | |
| KR20170048396A (en) | Recombinant modified vaccinia virus ankara(mva) filovirus vaccine | |
| WO2012053646A1 (en) | Virus vector for prime/boost vaccines, which comprises vaccinia virus vector and sendai virus vector | |
| US20100080827A1 (en) | Recombinant influenza viruses expressing tumor-associated antigens as antitumor agents | |
| CN102782136A (en) | Vectors expressing HIV antigens and GM-CSF and related methods for generating an immune response | |
| JP2010138201A (en) | Nucleic acid mucosal immunization | |
| US10695417B2 (en) | Human adenovirus serotype 5 vectors containing E1 and E2B deletions encoding the ebola virus glycoprotein | |
| JP2001512308A (en) | Synthetic HIV GAG gene | |
| KR20220016137A (en) | modified adenovirus | |
| KR20210090650A (en) | Alphavirus neoantigen vectors and interferon inhibitors | |
| CN102086453B (en) | Tumor DNA vaccine taking mucin 1 and survivin as targets and viral vector vaccine | |
| KR20220041844A (en) | HIV antigen and MHC complex | |
| CN101142319B (en) | Vaccines based on the use of modified vaccinia Ankara virus | |
| CN102732543B (en) | With the oncogene engineered vaccine that MUC-1 and survivin are target spot | |
| KR20230006825A (en) | Infectious disease antigens and vaccines | |
| CN104013973A (en) | Tumor DNA (Deoxyribose Nucleic Acid) vaccine and virus vector vaccine taking mucoprotein 1 and surviving as targets | |
| CN113832115A (en) | Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs | |
| CN105879060B (en) | Tumor DNA vaccine and virus vector vaccine with fibroblast activation protein α as target spot | |
| US7312202B2 (en) | Rationally designed and chemically synthesized promoter for genetic vaccine and gene therapy | |
| WO2005026316A2 (en) | Alphavirus vaccines | |
| CN104491852A (en) | Tumor DNA vaccine and virus vector vaccine taking mucoprotein 1 and surviving as target spots | |
| US20230059344A1 (en) | Medical Uses of 4-1BBL Adjuvanted Recombinant Modified Vaccinia Virus Ankara (MVA) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: JILIN UNIVERSITY Effective date: 20150416 |
|
| C10 | Entry into substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20150416 Address after: 130012 Torch Road 1260, hi tech Development Zone, Jilin, Changchun Applicant after: CHANGCHUN BCHT BIOTECHNOLOGY Co. Applicant after: Jilin University Address before: 130012 Torch Road 1260, hi tech Development Zone, Jilin, Changchun Applicant before: CHANGCHUN BCHT BIOTECHNOLOGY Co. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |