US20200216516A1 - Fusion peptides of cd4 helper t cell epitopes and vaccines thereof - Google Patents
Fusion peptides of cd4 helper t cell epitopes and vaccines thereof Download PDFInfo
- Publication number
- US20200216516A1 US20200216516A1 US16/650,318 US201816650318A US2020216516A1 US 20200216516 A1 US20200216516 A1 US 20200216516A1 US 201816650318 A US201816650318 A US 201816650318A US 2020216516 A1 US2020216516 A1 US 2020216516A1
- Authority
- US
- United States
- Prior art keywords
- antigen
- epitope
- seq
- epitopes
- fusion peptide
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 69
- 229960005486 vaccine Drugs 0.000 title claims abstract description 62
- 230000004927 fusion Effects 0.000 title claims abstract description 53
- 210000002443 helper t lymphocyte Anatomy 0.000 title claims abstract description 16
- 102000004196 processed proteins & peptides Human genes 0.000 title description 3
- 108091007433 antigens Proteins 0.000 claims abstract description 87
- 102000036639 antigens Human genes 0.000 claims abstract description 87
- 239000000427 antigen Substances 0.000 claims abstract description 86
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 61
- 241000701022 Cytomegalovirus Species 0.000 claims abstract description 50
- 230000002163 immunogen Effects 0.000 claims abstract description 41
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 15
- 230000024932 T cell mediated immunity Effects 0.000 claims abstract description 13
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 208000015181 infectious disease Diseases 0.000 claims abstract description 9
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 3
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 3
- 241000700605 Viruses Species 0.000 claims description 33
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 claims description 28
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 claims description 28
- 108020001507 fusion proteins Proteins 0.000 claims description 27
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 26
- SSOORFWOBGFTHL-OTEJMHTDSA-N (4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-5-carbamimidamido-1-[[(2S)-5-carbamimidamido-1-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-diaminohexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SSOORFWOBGFTHL-OTEJMHTDSA-N 0.000 claims description 23
- 102000037865 fusion proteins Human genes 0.000 claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 102100039510 Cancer/testis antigen 2 Human genes 0.000 claims description 20
- 101000889345 Homo sapiens Cancer/testis antigen 2 Proteins 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 20
- 230000005847 immunogenicity Effects 0.000 claims description 11
- 108091033319 polynucleotide Proteins 0.000 claims description 11
- 102000040430 polynucleotide Human genes 0.000 claims description 11
- 239000002157 polynucleotide Substances 0.000 claims description 11
- 230000001965 increasing effect Effects 0.000 claims description 10
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 102000000440 Melanoma-associated antigen Human genes 0.000 claims description 4
- 108050008953 Melanoma-associated antigen Proteins 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 238000012737 microarray-based gene expression Methods 0.000 claims description 4
- 238000012243 multiplex automated genomic engineering Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 244000045947 parasite Species 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 208000037581 Persistent Infection Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 claims description 2
- 206010057644 Testis cancer Diseases 0.000 claims description 2
- 239000013566 allergen Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 208000029742 colonic neoplasm Diseases 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000028996 humoral immune response Effects 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 201000003120 testicular cancer Diseases 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 claims description 2
- 210000000987 immune system Anatomy 0.000 abstract description 7
- 230000002708 enhancing effect Effects 0.000 abstract description 3
- 230000006058 immune tolerance Effects 0.000 abstract description 2
- 238000001976 enzyme digestion Methods 0.000 description 84
- 239000013598 vector Substances 0.000 description 53
- 241000699670 Mus sp. Species 0.000 description 44
- 239000013612 plasmid Substances 0.000 description 43
- 238000000034 method Methods 0.000 description 37
- 206010022000 influenza Diseases 0.000 description 28
- 108010041986 DNA Vaccines Proteins 0.000 description 27
- 229940021995 DNA vaccine Drugs 0.000 description 27
- 241001465754 Metazoa Species 0.000 description 21
- 230000003053 immunization Effects 0.000 description 20
- 238000002649 immunization Methods 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 19
- 210000001744 T-lymphocyte Anatomy 0.000 description 18
- 229940023143 protein vaccine Drugs 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 18
- 230000004614 tumor growth Effects 0.000 description 17
- 230000009465 prokaryotic expression Effects 0.000 description 16
- 239000013604 expression vector Substances 0.000 description 15
- 239000002671 adjuvant Substances 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- 230000004083 survival effect Effects 0.000 description 14
- 102000009016 Cholera Toxin Human genes 0.000 description 13
- 108010049048 Cholera Toxin Proteins 0.000 description 13
- 101100235725 Homo sapiens LMNB1 gene Proteins 0.000 description 13
- 102100026517 Lamin-B1 Human genes 0.000 description 13
- 108091008146 restriction endonucleases Proteins 0.000 description 13
- 230000000890 antigenic effect Effects 0.000 description 12
- 239000012154 double-distilled water Substances 0.000 description 12
- 238000012795 verification Methods 0.000 description 12
- 238000010276 construction Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 230000028993 immune response Effects 0.000 description 10
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 9
- 238000002255 vaccination Methods 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 108020004705 Codon Proteins 0.000 description 7
- 102100024739 E3 ubiquitin-protein ligase UHRF1 Human genes 0.000 description 7
- 101100483728 Homo sapiens UHRF1 gene Proteins 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 108091008874 T cell receptors Proteins 0.000 description 5
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 244000309466 calf Species 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- PHEDXBVPIONUQT-UHFFFAOYSA-N Cocarcinogen A1 Natural products CCCCCCCCCCCCCC(=O)OC1C(C)C2(O)C3C=C(C)C(=O)C3(O)CC(CO)=CC2C2C1(OC(C)=O)C2(C)C PHEDXBVPIONUQT-UHFFFAOYSA-N 0.000 description 4
- 108700010070 Codon Usage Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 108010032595 Antibody Binding Sites Proteins 0.000 description 3
- 241000724252 Cucumber mosaic virus Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 108091054438 MHC class II family Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000001994 activation Methods 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- ZBQCCTCQUCOXBO-UHFFFAOYSA-N 4-(4-aminophenyl)-2,2,6,6-tetramethylcyclohex-3-en-1-amine Chemical compound CC1(C)C(N)C(C)(C)CC(C=2C=CC(N)=CC=2)=C1 ZBQCCTCQUCOXBO-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 241001529572 Chaceon affinis Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 241000305071 Enterobacterales Species 0.000 description 2
- 241001529459 Enterovirus A71 Species 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 241000239221 Tachypleus gigas Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229940028617 conventional vaccine Drugs 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 2
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229940023041 peptide vaccine Drugs 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229940126577 synthetic vaccine Drugs 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 108010060123 Conjugate Vaccines Proteins 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010093013 HLA-DR1 Antigen Proteins 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010025327 Lymphopenia Diseases 0.000 description 1
- 108700005092 MHC Class II Genes Proteins 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000223960 Plasmodium falciparum Species 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 108010055044 Tetanus Toxin Proteins 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940031670 conjugate vaccine Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 101150005152 ctb gene Proteins 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 108700010900 influenza virus proteins Proteins 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 231100001023 lymphopenia Toxicity 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003046 sporozoite Anatomy 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940118376 tetanus toxin Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
- A61K39/001186—MAGE
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
- A61K39/001188—NY-ESO
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/107—Vibrio
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
- C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention belongs to the fields of molecular biology and immunology.
- the present invention relates to a fusion peptide of CD4 helper T cell epitopes, especially to a vaccine comprising the epitope fusion peptide, and use thereof.
- T helper cells are the cells that play an important role in the immune system, particularly in the adaptive immune system. They promote the activities of other immune cells by releasing T cell cytokines. These cells help to inhibit or modulate immune responses. They are essential in the conversion of B cell antibody classes, the activation and growth of cytotoxic T cells, and the maximization of bactericidal activity of phagocytic cells, such as macrophages.
- CD4 + T cells Mature Th cells expressing the protein CD4 are known as CD4 + T cells.
- helper T cells such CD4 + T cells are typically subjected to a pre-defined process within the immune system. For example, when antigen presenting cells express an antigen on MHC class II, CD4 + cells would assist these cells by a combination of cell-to-cell interactions (e.g., CD40 (protein) and CD40L) and cytokines.
- helper T cells can be seen with respect to HIV, a virus that primarily infects CD4 + T cells.
- HIV acquired immune deficiency syndrome
- AIDS acquired immune deficiency syndrome
- a continuous treatment may delay its occurrence. If AIDS occurs, the treatment can also better manage the course of AIDS.
- Other rare diseases, such as lymphopenia, result in loss or dysfunction of CD4 + T cells. These diseases produce similar symptoms, many of which are thtal.
- Antigenic epitope for short, also known as “antigenic determinant”, refers to chemical groups on the surface of an antigen that determines the antigen specificity.
- An antigenic epitope can be recognized by the immune system, especially by antibodies, B cells or cells.
- a site of an antibody capable of recognizing an antigenic epitope is referred to as a “paratope” or an “antibody determinant”.
- an antigenic epitope usually refers to a part of foreign protein or the like, an epitope that can be recognized by the autoimmune system is also classified as an antigenic epitope.
- the epitopes of protein antigen are divided into conformational epitopes and linear epitopes according to their structures and interactions with a paratope. Since a conformational epitope consists of discrete portions in the amino acid sequence of an antigen, the interaction of a paratope with the antigenic epitope is based on the three-dimensional characteristics and shape of the surface or tertiary structure of the antigen. Most antigenic epitopes belong to conformational epitopes. In contrast, a linear epitope consists of a continuous amino acid sequence of an antigen, and the interaction with the antigen is based on its primary structure.
- a T cell epitope consists mainly of a short peptide of 8-17 amino acids and exists on antigen-presenting cells (APC), which as an antigen epitope would bind to major histocompatibility complex (MHC) to form a complex and bind to the corresponding T cell epitope receptors, thereby activating T cells and generating a corresponding cellular immune response (Shimonikevitz et al., 1984; Babbitt et al., 1985; Buus et al., 1986; Townsend and Bodmer, 1989).
- MHC major histocompatibility complex
- major histocompatibility complex class I usually presents a T cell antigenic epitope consisting of a polypeptide of 8 to 11 amino acids in length, while major histocompatibility complex class 11 presents a relatively longer I cell antigenic epitope consisting of 13-1.7 amino acids.
- helper T cell epitopes refer to a class of T cell epitopes which bind to MHC molecules and form complexes that can be recognized by CD4 helper T cell receptors. Th epitopes bind primarily to the molecules present on the surfaces of antigen-presenting cells (APC) encoded by MHC class II genes. The complexes of class II molecule and peptide epitope are then recognized by the specific T cell receptors (TCR) on the surfaces of T helper lymphocytes. In this way, the T cells presenting an antigenic epitope in the context of MHC molecules can be activated and provide the essential signal for B lymphocyte differentiation.
- APC antigen-presenting cells
- TCR specific T cell receptors
- the source of T-helper epitope of peptide immunogen has been a carrier protein covalently coupled to a peptide, but the coupling process may involve other issues, such as modification of antigenic determinants during the coupling process and induction of an antibody against the carrier at the expense of an antibody against the peptide (Schutze, M. P., Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol. 1985, 135, 2319-2322; DiJohn, D., Torrese, J. R. Murillo, J. Herrington, D. A. et al. Effect of priming with carrier on response to conjugate vaccine.
- a suitable carrier protein which is important in the design of peptide vaccine, is limited by the factors, such as toxicity and feasibility in a large-scale production. There are other limitations to this method, including the load size of peptide that can be coupled and the dose of carrier that can be safely administered (Audibert, F. a. C., L. 1984. Modern approaches to vaccines. Molecular and chemical basis of virus virulence and immunogenicity., Cold Spring Harbor Laboratory, New York.).
- carrier molecules allow to induce a strong immune response, they are also associated with the adverse effects, such as inhibition of anti-peptide antibody response (Herzenberg, L. A.
- an immunogen must contain a helper T cell epitope in addition to an epitope to be recognized by a surface Ig or the receptors present on the cytotoxic T cells. It will be appreciated that these types of epitopes may be very different.
- the conformation is important because B-cell receptors bind directly to native immunogens.
- an epitope to be recognized by T cells is independent of the conformational integrity of the epitope, and consists of a short sequence of about 9 amino acids against CTL and a slightly longer sequence (having less length restriction) against helper T cells.
- a Th epitope can stimulate and activate helper ‘I’ cells, and accordingly promote activation of CD8 T cells and B cells, ultimately increasing the immune response.
- a Th epitope in addition to being able to activate an immune response against itself, is also effective in aiding the immune response to other antigens or epitopes associated therewith.
- a heterologous strong Th epitope can be fused to a target immunogen, thereby increasing the immunogenicity of the target immunogen.
- PADRE pan HLA DR-binding Epitope
- Th epitope derived from tetanus toxin P2 is also commonly used in coupling with a target immunogen to enhance the immunogenicity (Panina-Bordignon P et al., Eur J Immunol, 1989, 19: 2237-42; La Rosa, Corinna et al., The Journal of infectious diseases, 2012, 205: 1294-304).
- Th epitope used to increase the immunogenicity is usually heterologous.
- a high level of immune response against the Th epitope itself will not be produced in a vaccine subject.
- the immune system of vaccine subject is initially exposed to such a Th epitope, the activations against both such a Th epitope and a target immunogen in the recipient immune system are substantially synchronized, and the generation time and numbers of T cells against such a Th epitope are similar to those against the target immunogen. In this way, the effect on assisting the target immunogen will be limited, accordingly.
- Th epitope Especially for a weakly immunogenic tumor antigen, the helping effect of such a Th epitope is more difficult to exert. Indeed, the direct use of a strong Th epitope, although being capable of activating a tumor antigen, elicits a lower level of cellular immune response that do not meet the needs of a tumor vaccine (Ghaffari-Nazari H et al., PLoS ONE, 2015, 10 (11): e0142563).
- Th epitope strategies are needed to enhance the immunogenicities of target immunogens, particularly some weak immunogens, such as tumor antigens.
- the present invention utilizes a strong Th epitope derived from cytomegalovirus (CMV) and influenza (Flu) virus (influvirus) to obtain an epitope fusion peptide for enhancing the immunogenicity of a target immunogen.
- CMV cytomegalovirus
- Flu influenza virus
- Epitope fusion peptide refers to a peptide formed by joining together several epitopes.
- Target immunogen refers to an immunogen used for achieving a certain immune response, including a substance having an immunological activity, such as an antigen, preferably a protein.
- the present invention provides a fusion peptide of CD4 helper T cell epitopes, comprising a CMV epitope and/or an influenza virus epitope.
- the epitope fusion peptide comprises one or more of CMV epitopes selected from those shown in SEQ ID NOs: 1-10, and/or one or more of influenza virus epitopes selected from those shown in SEQ ID NOs: 11-23.
- the epitope fusion peptide consists of one or more of CMV epitopes selected from those shown in SEQ ID NOs: 1-10, and/or one or more of influenza virus epitopes selected from those shown in SEQ ID NOs: 11-23.
- the epitope fusion peptide consists of 5 or 10 CMV epitopes, and/or 8 or 13 influenza virus epitopes, such as the epitope fusion peptide shown in SEQ ID NO: 34 or 44.
- the epitope fusion peptide consists of 13 influenza virus epitopes, such as the epitope fusion peptide shown in SEQ ID NO: 48.
- the epitope fusion peptide induces a humoral or cellular immune response.
- the present invention also provides a fusion protein of the epitope fusion peptide and a target immunogen.
- the present invention also provides a polynucleotide encoding the epitope fusion peptide and/or the fusion protein.
- the target immunogen is any one or more immunogens.
- the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other whole microorganism.
- the antigen is a tumor antigen or an infection-related antigen.
- the tumor antigen is one or more tumor antigens selected from lung cancer antigen, testicular cancer antigen, melanoma antigen, liver cancer antigen, breast cancer antigen or prostate cancer antigen.
- the tumor antigen is one or more tumor antigens selected from LAGE antigen, MAGE antigen or NY-ESO-1 antigen.
- the LAGE antigen is LAGE-1
- the MAGE antigen is MAGE-A3.
- the tumor antigen comprises LAGE-MAGE-A3 and NY-ESO-1.
- the amino acid sequence of LAGE-1 is shown in SEQ ID NO: 24, the amino acid sequence of MAGE-A3 is shown in SEQ ID NO: 25, and the amino acid sequence of NY-ESO-1 is shown in SEQ ID NO: 26.
- the infection-related antigen is one or more infection-related antigen selected from an HIV antigen, a Flu virus antigen or an HBV antigen.
- the fusion protein is as shown in one of SEQ ID NOs: 55-58.
- Another object of the present invention is to provide an immunogenic composition
- an immunogenic composition comprising a therapeutically effective amount of the epitope fusion peptide, the fusion protein and/or the polynucleotide according to the present invention, and a pharmaceutically acceptable carrier.
- the immunogenic composition is a vaccine.
- the present invention also provides use of the epitope fusion peptide, the fusion protein, the polynucleotide and/or the immunogenic composition according to the present invention in the manufacture of a medicament or a vaccine for increasing the immunogenicity of a target immunogen.
- the present invention also provides a method for increasing the immunogenicity of a target immunogen using the epitope fusion peptide according to the present invention, comprising using a CD4 helper T cell epitope having a stronger immune response in a vaccine subject or population with a target immunogen to form a fusion protein.
- the method specifically comprises the following steps of:
- CD4 helper T cell epitopes selected one or more CD4 helper T cell epitopes, wherein a complex formed by combining the epitopes with MHC molecules can be recognized by CD4 helper T cell receptors, and a T cell immune response has been generated against at least one of the epitopes in a vaccine subject before vaccination;
- the expression vector can be in the form of a DNA vaccine vector, a protein vaccine vector or a virus vaccine vector;
- vaccinating the vaccine subject with the above vaccine and a suitable adjuvant, such as incomplete Freund's adjuvant, complete Freund's adjuvant, or aluminum hydroxide adjuvant and the like that can be selected for vaccination.
- a suitable adjuvant such as incomplete Freund's adjuvant, complete Freund's adjuvant, or aluminum hydroxide adjuvant and the like that can be selected for vaccination.
- step (1) in the method further comprises a step of examining the MHC phenotype of the vaccine subject.
- examining the MHC phenotype of the vaccine subject comprises examining the MHC class II gene subtype of the vaccine subject.
- the present invention also provides a method for treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of the epitope fusion peptide, the fusion protein, the immunogenic composition and/or the polynucleotide of the present invention.
- the condition is one or more conditions selected from malignant tumors, and bacterial and viral chronic infections.
- the malignant tumor is breast cancer or colon cancer.
- the DNA vaccine vector is used for the prime immunization
- a protein vaccine vector is used for the boost immunization.
- the pVKD1.0-C1-hMNB DNA vaccine is used for the prime immunization
- the LMNB-I13 protein is used for the boost immunization.
- the epitope fusion peptide provided by the present invention can substantially improve the level of cellular immune response to a target immunogen, particularly a weak immunogen, and is an effective means for overcoming the immune tolerance of immune system to an antigen, particularly to a tumor antigen or an infection-related antigen, and is suitable for efficiently enhancing the efficacy of a vaccine.
- FIGS. 1 and 2 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-hLMN carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, respectively.
- FIGS. 3 and 4 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-hLMN-CTB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin subunit B, respectively.
- FIGS. 5 and 6 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-C1 carrying the encoding sequences of CMV and Flu virus-derived CD4 epitopes, respectively.
- FIGS. 7 and 8 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-C1-LMNB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, cholera toxin subunit B, and CMV and Flu virus-derived CD4 epitopes, respectively.
- FIGS. 9 and 10 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-LMN carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, respectively.
- FIGS. 11 and 12 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-LMN-CTB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin subunit B, respectively.
- FIGS. 13 and 14 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV Th carrying the encoding sequence of a CMV-derived epitope, respectively.
- FIGS. 15 and 16 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV10-LMNB carrying the encoding sequences of a CMV-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively.
- FIGS. 17 and 18 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV Th carrying the encoding sequences of a Flu virus-derived epitope, respectively.
- FIGS. 19 and 20 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-Influ8-LMNB carrying the encoding sequences of a Flu virus-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively.
- FIGS. 21 and 22 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-Influ13-LMNB carrying the encoding sequences of a Flu virus-derived epitope, LAGS-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively.
- FIG. 23 shows the detection results of cellular immune responses in the animal immunization experiment.
- FIGS. 24 and 25 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV5-LMNB carrying the encoding sequences of a CMV-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively.
- FIG. 26 shows the detection results of cellular immune responses in the animal immunization experiment in Example 12.
- FIG. 27 shows the tumor growth of mice in Example 13.
- FIGS. 28 and 29 show the tumor-free survival and overall survival of mice in Example 13, respectively.
- FIG. 30 shows the tumor growth of mice in each treatment group for the 4T1-hNY-ESO-1 mouse tumor model.
- FIG. 31 shows the tumor growth of mice in each treatment group for the CT26-hLAGE-1 mouse tumor model.
- amino acid sequences of LAGE-I, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively.
- SEQ ID NOs: 24-26 amino acid sequences of LAGE-I, MAGE-A3 and NY-ESO-1.
- the nucleotide sequences were synthesized by Shanghai Generay Biotech Co., Ltd., and then cloned between the multiple cloning sites Sal I and BamH I on the DNA vaccine vector pVKD1.0 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a method well known in the art to construct the DNA vaccine vector pVKD1.0-hLMN capable of expressing the fusion protein as an antigen (the plasmid map is shown in FIG. 1 ), which was stored after being sequenced for identification.
- the vector pVKD1.0-hLMN was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 1), and its enzyme digestion map for verification is shown in FIG. 2 .
- Enzyme digestion system for identification of the plasmid pVKD1.0-hLMN (enzyme digestion at 37° C., 2 h)
- Enzyme digestion system Volume Plasmid pVKD1.0-hLMN 3 ⁇ L, about 1 ⁇ g Sal I (Takara, Cat. No. 1080A) 1 ⁇ L BamH I (Takara, Cat. No. 1010A) 1 ⁇ L
- the mammalian codon optimized sequence (SEQ ID NO: 31) of the amino acid sequence (SEQ ID NO: 30) of cholera toxin subunit B (CTB) and its eukaryotic expression vector pVKD1.0-CTB were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- the primers were designed by using pVKD1.0-CTB as a template (see Table 2).
- the CTB gene fragment was amplified by PCR, and the corresponding fragment was then recovered from the gel.
- the CTB fragment was inserted into a corresponding position on the linearized vector pVKD1.0-hLMN by a homologous recombination method, to construct the DNA vaccine vector pVKD1.0-hLMN-CTB (the plasmid map is shown in FIG. 3 ), which was stored after being sequenced for identification.
- the vector pVKD1.0-hLMN-CTB was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 3), and its enzyme digestion map for verification is shown in FIG. 4 .
- Enzyme digestion system for identification of plasmid pVKD1.0-hLMN-CTB (enzyme digestion at 37° C., 2 h)
- Enzyme digestion system Volume Plasmid pVKD1.0-hLMN-CTB 3 ⁇ L, about 1 ⁇ g Sal I (Takara, Cat. No. 1080A) 1 ⁇ L BamH I (Takara, Cat. No. 1010A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the strong Th epitopes derived from cytomegalovirus (CMV) and influenza (Flu) virus were obtained from an immune epitope database (IEDB, http://wwwiedb.org), wherein the strong Th epitopes of CMV include pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104, and the strong Th epitopes of Flu virus include HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210.
- the selected epitopes in Table 4 cover most subtypes of MHC class II molecules in both human and mouse.
- the selected epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221 were then linked together in tandem to form an fusion peptide of CMV virus epitopes and Flu virus epitopes having the amino acid sequence shown in SEQ ID NO: 34.
- the epitope fusion peptide was subjected to mammal codon optimization to give the nucleic acid sequence shown in SEQ ID NO: 35, which was sent to Suzhou Synbio Technologies Co., Ltd for synthesis, and then inserted into the DNA vaccine vector pVKD1.0 (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a molecular biology method well known in the art to form the vector pVKD1.0-CI (the plasmid map is shown in FIG. 5 ), and stored after being sequenced fbr identification.
- the vector pVKD1.0-CI was identified by the restriction endonucleases Pst I and Bgl II (the enzyme digestion system is shown in Table 5) and its enzyme digestion map for verification is shown in FIG. 6 .
- Enzyme digestion system for identification of plasmid pVKD1.0-CI (enzyme digestion at 37° C., 2 h)
- Enzyme digestion system Volume Plasmid pVKD1.0-CI 3 ⁇ L, about 1 ⁇ g Pst I (Takara, Cat. No. 1073A) 1 ⁇ L Bgl II (Takara, Cat. No. 1021A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the primers were designed by using the vector pVKD1.0-hLMN-CTB in Example 2 as a template (see table 6).
- the target gene fragment hLMN-CTB was amplified by PCR, and was then inserted between the restriction sites Not I and Bam HI on the pVKD1.0-CI vector by a molecular biology method well known in the art to construct the DNA vaccine vector pVKD1.0-CI-LNINB (the plasmid map is shown in FIG. 7 ), which was stored after being sequenced for identification.
- the vector pVKD1.0-CI-LMNB was identified by the restriction endonucleases Barn HI and EcoR V (the enzyme digestion system is shown in Table 7), and its enzyme digestion map for verification is shown in FIG. 8 .
- Enzyme digestion system for identification of plasmid pVKD1.0-CI-LMNB (enzyme digestion at 37° C., 2 h)
- Enzyme digestion system Volume Plasmid pVKD1.0-CI-LMNB 3 ⁇ L, about 1 ⁇ g Bam HI (Takara, Cat, No. 1010A) 1 ⁇ L EcoR V (Takara, Cat. No. 1042A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively.
- SEQ ID NOs: 24-26 amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1.
- an online codon optimization software http://www.jcat.de/
- the nucleotide sequences for E. coli codon usage preference shown in SEQ ID NOs: 38-40 respectively were obtained through optimization based on the antigen amino acid sequences.
- the nucleotide sequences were synthesized by Suzhou Synbio Technologies Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No.
- the vector pET-30a(+)-LMN was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 8), and its enzyme digestion map for verification is shown in FIG. 10 .
- Enzyme digestion system for identification of plasmid pET-30a(+)-LMN (enzyme digestion at 37° C., overnight)
- Enzyme digestion system Volume Plasmid pET-30a(+)-LMN 3 ⁇ L, about 1 ⁇ g Nco I (Takara, Cat. No. 1160A) 1 ⁇ L Xho I (Takara, Cat. No. 1094A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the amino acid sequence of cholera toxin subunit B (CTB) (SEQ ID NO: 30) and its prokaryotic codon optimized nucleic acid sequence (SEQ ID NO: 41) were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- the primers were designed (see table 9), and a nucleic acid fragment containing the CTB encoding sequence was amplified by a PCR method using the pET-30a(+)-CTB (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method.
- the nucleic acid fragment was then inserted into the pET-30a(+)-LMN vector by means of homologous recombination to construct the pET-30a(+)-LMN-CTB vector (the plasmid map is shown in FIG. 11 ), which was stored after being sequenced for identification.
- the vector pET-30a(+)-LMN-CTB was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 10), and its enzyme digestion map for verification is shown in FIG. 12 .
- coli codon usage preference (SEQ ID NO: 45) was obtained through optimization based on the amino acid sequence of Th epitopes.
- the nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a(+)-CMV Th (the plasmid map is shown in FIG. 13 ) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification.
- the vector pET-30a (+)-CMV Th was identified by the restriction endonucleases Mlu I and Xho I (the enzyme digestion system is shown in Table 11), and its enzyme digestion map for verification is shown in FIG. 14 .
- CMV Th1 contains 5 CMV Th epitopes consisting of pp65-1L pp65-71, pp65-92, pp65-123 and pp65-128 in tandem
- CMV Th2 contains 5 CMV Th epitopes consisting of pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104.
- Three restriction enzyme sites such as EcoR I, Sac I and Sal I were introduced between CMV Th1 and CMV Th2.
- Enzyme digestion system for identification of plasmid pET-30a(+)-CMV Th (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-CMV Th 3 ⁇ L, about 1 ⁇ g Mlu I (Takara, Cat. No. 1071A) 1 ⁇ L Xho I (Takara, Cat. No. 1094A) 1 ⁇ L Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the Primers were designed (see Table 12), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method.
- the nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV10-LMNB vector (the plasmid map is shown in FIG.
- pET-30a(+)-CMV10-LMNB contains CMV Th1 and CMV Th2 fragments, i.e. all 10 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104.
- Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight)
- Enzyme digestion system Volume Plasmid pET-30a(+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Takara, Cat. No. 1010A) 1 ⁇ L Xho I (Takara, Cat. No. 1094A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- Th Epitopes derived from Flu virus HA203, NP438, NSI-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210 were selected from Table 4, and linked together in tandem to form the amino acid sequence shown in SEQ ID NO: 48.
- SEQ ID NO: 49 the nucleotide sequence for E. coli codon usage preference was obtained through optimization based on the amino acid sequence containing Flu virus Th epitopes.
- the nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a(+)-Influ Th (the plasmid map is shown in FIG. 17 ) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification.
- the vector pET-30a(+)-Influ Th was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 14), and its enzyme digestion map for verification is shown in FIG. 18 .
- Influ Th1 contains 8 Flu virus Th epitopes consisting of HA203, NP438, HA375, NP24, NP95 and NP221 in tandem
- Influ Th2 contains 5 Flu virus Th epitopes consisting of RA434, HA440, NP324, M1-127 and M1-210.
- Three restriction sites such as EcoR 1, Sac I and Sal I were introduced between Influ Th1 and Influ Th2.
- the primers were designed (see Table 15), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method.
- the nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-Influ Th vector in Example 7 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ8-LMNB vector (containing 8 Flu virus Th epitopes; the plasmid map is shown in FIG.
- the vector pET-30a(+)-Influ8-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 16) and its enzyme digestion map for verification is shown in FIG. 20 .
- the pET-30a(+)-Influ8-LMNB vector contains the Influ Th1 segment, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4.
- Enzyme digestion system for identification of plasmid pET-30a(+)-Influ8-LMNB (enzyme digestion at 37° C., overnight)
- Enzyme digestion system Volume Plasmid pET-30a(+)-Influ8-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Takara, Cat. No. 1010A) 1 ⁇ L Xho I (Takara, Cat. No. 1094A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the primers were designed (see Table 17), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) was referred to for the specific method.
- This nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-Influ Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ13-LMNB vector (containing 13 Flu virus Th epitopes; the plasmid map is shown in FIG.
- the vector pET-30a(+)-CMV10-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 18) and its enzyme digestion map for verification is shown in FIG. 22 .
- the pET-30a(+)-Influ13-LMNB vector contains both the Influ Th1 and Influ Th2 segments, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4, and 5 Flu virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4,
- the vector includes all 13 Flu virus Th epitopes in total in Table 4
- the prokaryotic expression vector pET-30a(+)-LMN constructed in Example 4 the prokaryotic expression vector pET-30a(+)-LMN-CTB constructed in Example 5
- the prokaryotic expression vectors pET-30a(+)-Influ8-LMNB and pET-30a(+)-Influ13-LMNB constructed in Example 7 were respectively transformed into BL21 (DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., Cat. No.
- LMN (its amino acid sequence is shown in SEQ ID NO: 59)
- LMNB its amino acid sequence is shown in SEQ ID NO: 54
- LMNB-C10 its amino acid sequence is shown in SEQ ID NO: 58
- LMNB-18 its amino acid sequence is shown in SEQ ID NO: 55
- LMNB-13 its amino acid sequence is shown in SEQ ID NO: 56
- the concentrations of the recombinant proteins prepared are 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method.
- the contents of endotoxin in the prepared recombinant proteins were less than IEU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which meet the requirements of an animal experiment, and the instructions of horseshoe crab agent were referred to for the detection method.
- the information of the vaccines prepared in Examples 2, 3 and 8 is shown in Table 19.
- the DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VP1 (VP1 protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by the Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- mice Sixteen (16) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 20. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 ⁇ g/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 ⁇ g/animal.
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- mice Two weeks after the last immunization, the mice were sacrificed, and their serum and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively.
- the mouse IFN- ⁇ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN- ⁇ ELISPOT kit from BD were referred to for the method.
- the stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with the final concentration of 10 ⁇ g/mL.
- the positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.
- the 96-Well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both the recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- the plates were coated with the proteins in NaHCO 3 buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 ⁇ g/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5% Tween 20 in phosphate buffered saline (PBST).
- BSA bovine serum albumin
- PBS phosphate buffered saline
- a value which is 2.1 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer.
- a titer was defined as 50 when it was less than the initial dilution of 1:100.
- the results of cellular immune response assay are shown in FIG. 23 .
- the group primed with pVKD1.0-CI-LMNB DNA vaccine and boosted with LMNB-I13 protein i.e., group D in Example 8
- group D the group primed with pVKD1.0-CI-LMNB DNA vaccine and boosted with LMNB-I13 protein
- group C the group boosted with LMNB-18
- group C the level of cellular immune response in the group boosted with LMNB-I13 protein was nearly 3-fold higher than that in the parallel control group (group B).
- the results showed that a load of 13 Flu virus Th epitopes (group D) could significantly increase the cellular immune response level of weak immunogens.
- the primers were designed (see Table 21), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method.
- the nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV5-LMNB vector (the plasmid map is shown in FIG. 24 ), which was stored after being sequenced for identification.
- the vector pET-30a(+)-CMV5-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 22) and its enzyme digestion map for verification is shown in FIG. 25 .
- pET-30a(+)-CMV5-LMNB contains a CMV Th1 fragment, i.e. the first 5 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123 and pp65-128.
- Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight)
- Enzyme digestion system Volume Plasmid pET-30a(+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Takara, Cat. No. 1010A) 1 ⁇ L Xho I (Takara, Cat. No. 1094A) 1 ⁇ L
- Enzyme digestion buffer 1 ⁇ L ddH 2 O q.s. to 10 ⁇ L
- the prokaryotic expression vector pET-30a(+)-CMV5-LMNB constructed in Example 10 was transformed into BL21 (DE3) competent cells ('Tangen Biotech (Beijing) Co., Ltd., Cat. No. CB105; the instructions of competent cells were referred to for the transformation method) to prepare the recombinant protein LMNB-05 (its amino acid sequence is shown in SEQ ID NO: 57) according to the pET System Manual (TB055 8th Edition February 1999, Novagen), which was stored at ⁇ 80° C. after subpackage.
- the concentration of the recombinant protein prepared was 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method.
- the content of endotoxin in the prepared recombinant protein was less than IEU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which met the requirements of an animal experiment, and the instructions of Horseshoe Crab agent were referred to for the detection method.
- the vaccine information is shown in Table 19.
- the DNA vaccine pVKD1.0-CI (Example 3) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- mice Twenty (20) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 23. All DNA vaccines were injected into the tibial anterior muscle of the calf at 1.00 ⁇ g/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 ⁇ g/animal.
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- mice Two weeks after the last immunization, the mice were sacrificed, and serum and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively.
- the mouse IFN- ⁇ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN- ⁇ ELISPOT kit from BD were referred to for the method.
- the stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with a final stimulating concentration of 10 ⁇ g/mL.
- the positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.
- 96-Well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- the plates were coated with the proteins in NaHCO 3 buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 ⁇ g/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5% Tween 20 in phosphate buffered saline (PBST).
- BSA bovine serum albumin
- PBS phosphate buffered saline
- a value which is 2 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer.
- a titer was defined as 50 when it was less than the initial dilution of 1:100.
- the results of cellular immune response assay are shown in FIG. 26 .
- the group primed with the pVKD1.0-CI-LMNB DNA vaccine, boosted with the LMNB-C5 protein (i.e. group C in Example 11) and boosted with the LMNB-C10 protein (i.e. group D in Example 11) had the best immune effect, which was significantly higher than that in the parallel control (group B).
- the results showed that a load of 5 CMV virus Th epitopes (group C) and 10 CMV virus Th epitopes (group D) could significantly improve the cellular immune response of weak immunogens.
- the information of vaccines prepared in Examples 2, 3 and 8 is shown in Table 19.
- the DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn. Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VPI (VPI protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- mice Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by 4T1-hNY-ESO-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1 ⁇ 10 5 cells/mouse, and the tumor growth was continuously observed and measured after the inoculation.
- mice in each group The tumor growth of immunized mice in each group is shown in FIG. 27 .
- group A all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor innoculation), and the tumors grew rapidly.
- the tumor growth of mice in each immunization group lagged behind that in the control group, wherein the mice in the group boosted with LMNB-I13 (group D) and the nice in the group boosted with a mixture of LMNB-I13 and LMNB-C10 (group E) had the slowest tumor growth, so these two groups of vaccines had the best effects.
- mice were 14 days.
- the tumor-free survival of the mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the tumor-free survival of immunized mice.
- group D with the I13 epitope fusion peptide, and group E and F with the I13 and C10 epitope fusion peptide had the best effects, and the tumor-free survival of mice was doubled at the most.
- the tumor-free survival was significantly increased by about 40% compared to the conventional vaccine group (group B), showing that a load of 13 Th epitopes of Flu virus or 10 Th epitopes of CMV could significantly improve the protection effect of tumor vaccine against tumor.
- mice overall survival was also performed and the results are shown in FIG. 29 .
- OS median overall survival
- the overall survival of mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the survival of mice after immunization.
- group D with I13 epitope fusion peptide and groups E and F with I13 and C10 epitope fusion peptides had the best effects, and the overall survival rate was increased by 83%.
- the vaccine group with the I13 epitope fusion peptide significantly increased the tumor-free survival by 28% at the most, indicating that a load of thirteen (13) Flu virus Th epitopes or ten (10) CMV Th epitopes could greatly improve the tumor protection effect of tumor vaccine.
- Example 9 The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 25. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 ⁇ g/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 ⁇ g/animal.
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- mice in each group The tumor growth of immunized mice in each group is shown in FIG. 30 .
- all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor innoculation), and the tumors grew rapidly.
- the mice in the LMNB-I13 protein vaccine treated group (group C) had the slowest tumor growth compared to the untreated control group (group A).
- the tumor size of mice in the LMNB-I13 protein vaccine treated group was significantly smaller than that in the control group (group A) on day 22 after the tumor challenge, and there was still a significant difference in tumor size between the two groups until day 30.
- the tumor growth of mice began to accelerate in group C, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine.
- Example 9 The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 26. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 ⁇ g/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 ⁇ g/animal.
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- mice in each group The tumor growth of immunized mice in each group is shown in FIG. 31 . Due to the failure of tumor innoculation of some mice in the untreated control group (group A) after the tumor challenge (i.e. after the tumor innoculation), such mice were not included for the analysis, and the parallel controlled vaccine group (group B) and the LMNB-I13 treated group (group C) were compared. Compared with group B, the tumor growth of mice in group C was slower, and the tumor size of mice in the LMNB-I13 protein vaccine treatment group was significantly smaller than that in parallel controlled vaccine group (group B) on day 22 after the tumor challenge. There was still a significant difference in tumor size between the two groups until day 30.
- mice in group C were also observed at a later stage in the CT26 mouse model, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine, These results indicated that the LMNB-I13 protein vaccine could inhibit tumor growth in mice.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Pulmonology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- The present invention belongs to the fields of molecular biology and immunology. In particular, the present invention relates to a fusion peptide of CD4 helper T cell epitopes, especially to a vaccine comprising the epitope fusion peptide, and use thereof.
- T helper cells (Th cells) are the cells that play an important role in the immune system, particularly in the adaptive immune system. They promote the activities of other immune cells by releasing T cell cytokines. These cells help to inhibit or modulate immune responses. They are essential in the conversion of B cell antibody classes, the activation and growth of cytotoxic T cells, and the maximization of bactericidal activity of phagocytic cells, such as macrophages.
- Mature Th cells expressing the protein CD4 are known as CD4+ T cells. As helper T cells, such CD4+ T cells are typically subjected to a pre-defined process within the immune system. For example, when antigen presenting cells express an antigen on MHC class II, CD4+ cells would assist these cells by a combination of cell-to-cell interactions (e.g., CD40 (protein) and CD40L) and cytokines.
- The importance of helper T cells can be seen with respect to HIV, a virus that primarily infects CD4+ T cells. In the late stage of HIV infection, loss of functional CD4+ T cells leads to a stage of infectious symptoms known as acquired immune deficiency syndrome (AIDS). When HIV virus is found early in blood or other body fluids, a continuous treatment may delay its occurrence. If AIDS occurs, the treatment can also better manage the course of AIDS. Other rare diseases, such as lymphopenia, result in loss or dysfunction of CD4+ T cells. These diseases produce similar symptoms, many of which are thtal.
- Antigenic epitope, “epitope” for short, also known as “antigenic determinant”, refers to chemical groups on the surface of an antigen that determines the antigen specificity. An antigenic epitope can be recognized by the immune system, especially by antibodies, B cells or cells. A site of an antibody capable of recognizing an antigenic epitope is referred to as a “paratope” or an “antibody determinant”. Although an antigenic epitope usually refers to a part of foreign protein or the like, an epitope that can be recognized by the autoimmune system is also classified as an antigenic epitope.
- The epitopes of protein antigen are divided into conformational epitopes and linear epitopes according to their structures and interactions with a paratope. Since a conformational epitope consists of discrete portions in the amino acid sequence of an antigen, the interaction of a paratope with the antigenic epitope is based on the three-dimensional characteristics and shape of the surface or tertiary structure of the antigen. Most antigenic epitopes belong to conformational epitopes. In contrast, a linear epitope consists of a continuous amino acid sequence of an antigen, and the interaction with the antigen is based on its primary structure.
- A T cell epitope consists mainly of a short peptide of 8-17 amino acids and exists on antigen-presenting cells (APC), which as an antigen epitope would bind to major histocompatibility complex (MHC) to form a complex and bind to the corresponding T cell epitope receptors, thereby activating T cells and generating a corresponding cellular immune response (Shimonikevitz et al., 1984; Babbitt et al., 1985; Buus et al., 1986; Townsend and Bodmer, 1989). There are two major classes of MHC molecules which bind to an epitope. Among them, major histocompatibility complex class I usually presents a T cell antigenic epitope consisting of a polypeptide of 8 to 11 amino acids in length, while major histocompatibility complex class 11 presents a relatively longer I cell antigenic epitope consisting of 13-1.7 amino acids.
- Among T cell epitopes, helper T cell epitopes (Th epitopes) refer to a class of T cell epitopes which bind to MHC molecules and form complexes that can be recognized by CD4 helper T cell receptors. Th epitopes bind primarily to the molecules present on the surfaces of antigen-presenting cells (APC) encoded by MHC class II genes. The complexes of class II molecule and peptide epitope are then recognized by the specific T cell receptors (TCR) on the surfaces of T helper lymphocytes. In this way, the T cells presenting an antigenic epitope in the context of MHC molecules can be activated and provide the essential signal for B lymphocyte differentiation. Traditionally, the source of T-helper epitope of peptide immunogen has been a carrier protein covalently coupled to a peptide, but the coupling process may involve other issues, such as modification of antigenic determinants during the coupling process and induction of an antibody against the carrier at the expense of an antibody against the peptide (Schutze, M. P., Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol. 1985, 135, 2319-2322; DiJohn, D., Torrese, J. R. Murillo, J. Herrington, D. A. et al. Effect of priming with carrier on response to conjugate vaccine. The Lancet. 1989, 2, 1415-1416). In addition, the use of an irrelevant protein in the preparation may involve quality control problems. The choice of a suitable carrier protein, which is important in the design of peptide vaccine, is limited by the factors, such as toxicity and feasibility in a large-scale production. There are other limitations to this method, including the load size of peptide that can be coupled and the dose of carrier that can be safely administered (Audibert, F. a. C., L. 1984. Modern approaches to vaccines. Molecular and chemical basis of virus virulence and immunogenicity., Cold Spring Harbor Laboratory, New York.). Although carrier molecules allow to induce a strong immune response, they are also associated with the adverse effects, such as inhibition of anti-peptide antibody response (Herzenberg, L. A. and Tokuhisa, T. 1980. Carrier-priming leads to hapten-specific suppression. Nature 285: 664; Schutze, M. P., Leclerc, C., Jolivet, M. Audibert, F., and Chedid, L. 1985. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol 135: 2319; Edinger, H. M., Felix, A. M., Gillessen, D., Heimer, E. P., Just, M., Pink, J. R., Sinigaglia, F., Sturchler, D., Takacs, B., Trzeciak, A., and et al., 1988. Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite, Plasmodium falciparum. J Immunol 140: 626).
- In general, an immunogen must contain a helper T cell epitope in addition to an epitope to be recognized by a surface Ig or the receptors present on the cytotoxic T cells. It will be appreciated that these types of epitopes may be very different. For B-cell epitopes, the conformation is important because B-cell receptors bind directly to native immunogens. In contrast, an epitope to be recognized by T cells is independent of the conformational integrity of the epitope, and consists of a short sequence of about 9 amino acids against CTL and a slightly longer sequence (having less length restriction) against helper T cells. The only requirement for these epitopes is that they can be accommodated in the binding clefts of class I or class II molecules, respectively, and the complexes can then bind to T cell receptors. The binding sites of class II molecules are open at both ends, allowing a greater variation in the length of a peptide (Brown, J. H., T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger and D. C. Wiley. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364: 33) that binds to a reported epitope of as short as 8 amino acid residues (Fahrer, A. M., Geysen, H. M., White, D. O., Jackson, D. C. and Brown, L. E. Analysis of the requirements for class IT-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to HEd J. Immunol. 1995. 155: 2849-2857).
- A Th epitope can stimulate and activate helper ‘I’ cells, and accordingly promote activation of CD8 T cells and B cells, ultimately increasing the immune response. In essence, a Th epitope, in addition to being able to activate an immune response against itself, is also effective in aiding the immune response to other antigens or epitopes associated therewith. Thus, a heterologous strong Th epitope can be fused to a target immunogen, thereby increasing the immunogenicity of the target immunogen. An artificial strong Th epitope called PADRE (pan HLA DR-binding Epitope) has been used in the fusion construction of multiple vaccines to increase the levels of immune responses to the relevant immunogens (del Guercio et al,, Vaccine, 1997, 15: 441.; Franke, E. D. et al., Vaccine, 1999, 17:1201; Jeff Alexander et al., J Immunol, 2000, 164 (3) 1625-1633; Jeff Alexander et al., Vaccine, 2004, 22: 2362.; La Rosa, Corinna et al., The Journal of infectious diseases, 2012, 205: 1294-304). In addition, as a strong Th epitope derived from tetanus toxin, P2 is also commonly used in coupling with a target immunogen to enhance the immunogenicity (Panina-Bordignon P et al., Eur J Immunol, 1989, 19: 2237-42; La Rosa, Corinna et al., The Journal of infectious diseases, 2012, 205: 1294-304).
- In general, however, a Th epitope used to increase the immunogenicity is usually heterologous. In other words, a high level of immune response against the Th epitope itself will not be produced in a vaccine subject. Thus, when a vaccine subject is vaccinated with a strong Th epitope as described above, it is likely that the immune system of vaccine subject is initially exposed to such a Th epitope, the activations against both such a Th epitope and a target immunogen in the recipient immune system are substantially synchronized, and the generation time and numbers of T cells against such a Th epitope are similar to those against the target immunogen. In this way, the effect on assisting the target immunogen will be limited, accordingly. Especially for a weakly immunogenic tumor antigen, the helping effect of such a Th epitope is more difficult to exert. Indeed, the direct use of a strong Th epitope, although being capable of activating a tumor antigen, elicits a lower level of cellular immune response that do not meet the needs of a tumor vaccine (Ghaffari-Nazari H et al., PLoS ONE, 2015, 10 (11): e0142563).
- Thus, new Th epitope strategies are needed to enhance the immunogenicities of target immunogens, particularly some weak immunogens, such as tumor antigens.
- It is an object of the present invention to provide a fusion peptide of CD4 helper T cell epitopes, by which the immunogenicity of a target immunogen is enhanced.
- Further, the present invention utilizes a strong Th epitope derived from cytomegalovirus (CMV) and influenza (Flu) virus (influvirus) to obtain an epitope fusion peptide for enhancing the immunogenicity of a target immunogen.
- For the purposes of the present invention, the following terms are defined below.
- “Epitope fusion peptide” refers to a peptide formed by joining together several epitopes.
- “Target immunogen” refers to an immunogen used for achieving a certain immune response, including a substance having an immunological activity, such as an antigen, preferably a protein.
- It is another object of the present invention to provide a fusion protein of the epitope fusion peptide and the target immunogen.
- To achieve the above object, the present invention provides a fusion peptide of CD4 helper T cell epitopes, comprising a CMV epitope and/or an influenza virus epitope.
- In one embodiment of the present invention, the epitope fusion peptide comprises one or more of CMV epitopes selected from those shown in SEQ ID NOs: 1-10, and/or one or more of influenza virus epitopes selected from those shown in SEQ ID NOs: 11-23.
- In one embodiment of the present invention, the epitope fusion peptide consists of one or more of CMV epitopes selected from those shown in SEQ ID NOs: 1-10, and/or one or more of influenza virus epitopes selected from those shown in SEQ ID NOs: 11-23. Preferably, the epitope fusion peptide consists of 5 or 10 CMV epitopes, and/or 8 or 13 influenza virus epitopes, such as the epitope fusion peptide shown in SEQ ID NO: 34 or 44. Most preferably, the epitope fusion peptide consists of 13 influenza virus epitopes, such as the epitope fusion peptide shown in SEQ ID NO: 48.
- Preferably, the epitope fusion peptide induces a humoral or cellular immune response.
- The present invention also provides a fusion protein of the epitope fusion peptide and a target immunogen.
- The present invention also provides a polynucleotide encoding the epitope fusion peptide and/or the fusion protein.
- In one embodiment of the present invention, the target immunogen is any one or more immunogens. Preferably, the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other whole microorganism. In one embodiment of the present invention, the antigen is a tumor antigen or an infection-related antigen.
- In one embodiment of the present invention, the tumor antigen is one or more tumor antigens selected from lung cancer antigen, testicular cancer antigen, melanoma antigen, liver cancer antigen, breast cancer antigen or prostate cancer antigen.
- In one embodiment of the present invention, the tumor antigen is one or more tumor antigens selected from LAGE antigen, MAGE antigen or NY-ESO-1 antigen. Preferably, the LAGE antigen is LAGE-1, and the MAGE antigen is MAGE-A3. Further preferably, the tumor antigen comprises LAGE-MAGE-A3 and NY-ESO-1. Preferably, the amino acid sequence of LAGE-1 is shown in SEQ ID NO: 24, the amino acid sequence of MAGE-A3 is shown in SEQ ID NO: 25, and the amino acid sequence of NY-ESO-1 is shown in SEQ ID NO: 26.
- In one embodiment of the present invention, the infection-related antigen is one or more infection-related antigen selected from an HIV antigen, a Flu virus antigen or an HBV antigen.
- Preferably, the fusion protein is as shown in one of SEQ ID NOs: 55-58.
- Another object of the present invention is to provide an immunogenic composition comprising a therapeutically effective amount of the epitope fusion peptide, the fusion protein and/or the polynucleotide according to the present invention, and a pharmaceutically acceptable carrier. Preferably, the immunogenic composition is a vaccine.
- It is another object of the present invention to provide a kit comprising the epitope fusion peptide, the fusion protein, the polynucleotide and/or the immunogenic composition according to the present invention, and instructions for use thereof.
- The present invention also provides use of the epitope fusion peptide, the fusion protein, the polynucleotide and/or the immunogenic composition according to the present invention in the manufacture of a medicament or a vaccine for increasing the immunogenicity of a target immunogen.
- The present invention also provides a method for increasing the immunogenicity of a target immunogen using the epitope fusion peptide according to the present invention, comprising using a CD4 helper T cell epitope having a stronger immune response in a vaccine subject or population with a target immunogen to form a fusion protein. The method specifically comprises the following steps of:
- (1) selecting one or more CD4 helper T cell epitopes, wherein a complex formed by combining the epitopes with MHC molecules can be recognized by CD4 helper T cell receptors, and a T cell immune response has been generated against at least one of the epitopes in a vaccine subject before vaccination;
- (2) fusing the epitopes to prepare an epitope fusion peptide, fusing the epitope fusion peptide with a target immunogen to prepare a fusion protein, expressing the fusion protein and preparing it into a vaccine, wherein the expression vector can be in the form of a DNA vaccine vector, a protein vaccine vector or a virus vaccine vector; and
- (3) vaccinating the vaccine subject with the above vaccine, and a suitable adjuvant, such as incomplete Freund's adjuvant, complete Freund's adjuvant, or aluminum hydroxide adjuvant and the like that can be selected for vaccination.
- Further, step (1) in the method further comprises a step of examining the MHC phenotype of the vaccine subject. Preferably, examining the MHC phenotype of the vaccine subject comprises examining the MHC class II gene subtype of the vaccine subject.
- The present invention also provides a method for treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of the epitope fusion peptide, the fusion protein, the immunogenic composition and/or the polynucleotide of the present invention. Preferably, the condition is one or more conditions selected from malignant tumors, and bacterial and viral chronic infections. Preferably, the malignant tumor is breast cancer or colon cancer. Preferably, in the method, the DNA vaccine vector is used for the prime immunization, and a protein vaccine vector is used for the boost immunization. More preferably, the pVKD1.0-C1-hMNB DNA vaccine is used for the prime immunization, and the LMNB-I13 protein is used for the boost immunization. The epitope fusion peptide provided by the present invention can substantially improve the level of cellular immune response to a target immunogen, particularly a weak immunogen, and is an effective means for overcoming the immune tolerance of immune system to an antigen, particularly to a tumor antigen or an infection-related antigen, and is suitable for efficiently enhancing the efficacy of a vaccine.
- Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:
-
FIGS. 1 and 2 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-hLMN carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, respectively. -
FIGS. 3 and 4 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-hLMN-CTB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin subunit B, respectively. -
FIGS. 5 and 6 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-C1 carrying the encoding sequences of CMV and Flu virus-derived CD4 epitopes, respectively. -
FIGS. 7 and 8 are a plasmid map and a map of double enzyme digestion for identification of the DNA vaccine vector pVKD1.0-C1-LMNB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, cholera toxin subunit B, and CMV and Flu virus-derived CD4 epitopes, respectively. -
FIGS. 9 and 10 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-LMN carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens, respectively. -
FIGS. 11 and 12 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-LMN-CTB carrying the encoding sequences of LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin subunit B, respectively. -
FIGS. 13 and 14 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV Th carrying the encoding sequence of a CMV-derived epitope, respectively. -
FIGS. 15 and 16 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV10-LMNB carrying the encoding sequences of a CMV-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively. -
FIGS. 17 and 18 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV Th carrying the encoding sequences of a Flu virus-derived epitope, respectively. -
FIGS. 19 and 20 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-Influ8-LMNB carrying the encoding sequences of a Flu virus-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively. -
FIGS. 21 and 22 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-Influ13-LMNB carrying the encoding sequences of a Flu virus-derived epitope, LAGS-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively. -
FIG. 23 shows the detection results of cellular immune responses in the animal immunization experiment. -
FIGS. 24 and 25 are a plasmid map and a map of double enzyme digestion for identification of the prokaryotic vector pET-30a(+)-CMV5-LMNB carrying the encoding sequences of a CMV-derived epitope, LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin subunit B, respectively. -
FIG. 26 shows the detection results of cellular immune responses in the animal immunization experiment in Example 12. -
FIG. 27 shows the tumor growth of mice in Example 13. -
FIGS. 28 and 29 show the tumor-free survival and overall survival of mice in Example 13, respectively. -
FIG. 30 shows the tumor growth of mice in each treatment group for the 4T1-hNY-ESO-1 mouse tumor model. -
FIG. 31 shows the tumor growth of mice in each treatment group for the CT26-hLAGE-1 mouse tumor model. - The present invention is described in further detail below with reference to the specific embodiments. The examples are given for the purpose of illustration of the present invention only, and are not intended to limit the scope of the present invention.
- The amino acid sequences of LAGE-I, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively. By means of an online codon optimization software (http://www.jcatdel), the nucleotide sequences for mammalian codon usage preference as shown in SEQ ID NOs: 27-29 respectively were obtained by optimization based on the above antigen amino acid sequences. The nucleotide sequences were synthesized by Shanghai Generay Biotech Co., Ltd., and then cloned between the multiple cloning sites Sal I and BamH I on the DNA vaccine vector pVKD1.0 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a method well known in the art to construct the DNA vaccine vector pVKD1.0-hLMN capable of expressing the fusion protein as an antigen (the plasmid map is shown in
FIG. 1 ), which was stored after being sequenced for identification. The vector pVKD1.0-hLMN was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 1), and its enzyme digestion map for verification is shown inFIG. 2 . -
TABLE 1 Enzyme digestion system for identification of the plasmid pVKD1.0-hLMN (enzyme digestion at 37° C., 2 h) Enzyme digestion system Volume Plasmid pVKD1.0- hLMN 3 μL, about 1 μg Sal I (Takara, Cat. No. 1080A) 1 μL BamH I (Takara, Cat. No. 1010A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The mammalian codon optimized sequence (SEQ ID NO: 31) of the amino acid sequence (SEQ ID NO: 30) of cholera toxin subunit B (CTB) and its eukaryotic expression vector pVKD1.0-CTB were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The primers were designed by using pVKD1.0-CTB as a template (see Table 2). The CTB gene fragment was amplified by PCR, and the corresponding fragment was then recovered from the gel. The CTB fragment was inserted into a corresponding position on the linearized vector pVKD1.0-hLMN by a homologous recombination method, to construct the DNA vaccine vector pVKD1.0-hLMN-CTB (the plasmid map is shown in
FIG. 3 ), which was stored after being sequenced for identification. The vector pVKD1.0-hLMN-CTB was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 3), and its enzyme digestion map for verification is shown inFIG. 4 . -
TABLE 2 Primers in Example 2 Primer Sequence 1F (SEQ ID TCCCTCAGGGCAGAGGCGCATCAAGCTGAAGTTCGG NO: 32) CGTG IR (SEQ ID GAAGGCACAGCAGATCTGGATCCTCAGTTGGCCATG NO: 33) CTGATGGC -
TABLE 3 Enzyme digestion system for identification of plasmid pVKD1.0-hLMN-CTB (enzyme digestion at 37° C., 2 h) Enzyme digestion system Volume Plasmid pVKD1.0-hLMN- CTB 3 μL, about 1 μg Sal I (Takara, Cat. No. 1080A) 1 μL BamH I (Takara, Cat. No. 1010A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The strong Th epitopes derived from cytomegalovirus (CMV) and influenza (Flu) virus (see Table 4) were obtained from an immune epitope database (IEDB, http://wwwiedb.org), wherein the strong Th epitopes of CMV include pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104, and the strong Th epitopes of Flu virus include HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210. The selected epitopes in Table 4 cover most subtypes of MHC class II molecules in both human and mouse. The selected epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221 were then linked together in tandem to form an fusion peptide of CMV virus epitopes and Flu virus epitopes having the amino acid sequence shown in SEQ ID NO: 34. The epitope fusion peptide was subjected to mammal codon optimization to give the nucleic acid sequence shown in SEQ ID NO: 35, which was sent to Suzhou Synbio Technologies Co., Ltd for synthesis, and then inserted into the DNA vaccine vector pVKD1.0 (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a molecular biology method well known in the art to form the vector pVKD1.0-CI (the plasmid map is shown in
FIG. 5 ), and stored after being sequenced fbr identification. The vector pVKD1.0-CI was identified by the restriction endonucleases Pst I and Bgl II (the enzyme digestion system is shown in Table 5) and its enzyme digestion map for verification is shown inFIG. 6 . -
TABLE 4 Th Epitopes in Example 3 Epitope Name Source Amino acid sequence pp65-11 CMV LLQTGIHVRVSQPSL (SEQ ID NO: 1) pp65-71 CMV IIKPGKISHIMLDVA (SEQ ID NO: 2) pp65-92 CMV EHPTFTSQYRIQGKL (SEQ ID NO: 3) pp65-123 CMV AGILARNLNPMVATV (SEQ ID NO: 4 pp65-128 CMV KYQEFFWDANDIYRI (SEQ ID NO: 5) pp65-57 CMV KVYLESFCEDVPSGK (SEQ ID NO: 6) pp65-62 CMV TLGSDVEEDLTMTRN (SEQ ID NO: 7) pp65-30 CMV PLKMLNIPSINVHHY (SEQ ID NO: 8) pp65-112 CMV ACTSGVMTRGRLKAE (SEQ ID NO: 9) pp65-104 CMV TERKTPRVTGGGAMA (SEQ ID NO: 10) HA203 Influ NQRALYHTENAYVSVVS (SEQ ID NO: 11) NP438 Influ SDMRAEIIKMMESARPE (SEQ ID NO: 12) NS1-84 Influ ALASRYLTDMTIEEMSR (SEQ ID NO: 13) M1-181 Influ LASTTAKAMEQMAGSSE (SEQ ID NO: 14) HA375 Influ SGYAADQKSTQNAINGITNKVN (SEQ ID NO: 15) NP24 influ EIRASVGKMIDGIGRFYI (SEQ ID NO: 16) NP95 influ PIYRRVDGKWMRELVLY (SEQ ID NO: 17) NP221 Influ RMCNILKGKFQTAAQRAM (SEQ ID NO: 18) HA434 Influ IWTYNAELLVLLENERT (SEQ ID NO: 19) HA440 Influ ELLVLLENERTLDFHDS (SEQ ID NO: 20) NP324 Influ HKSQLVWMACNSAAFED (SEQ ID NO: 21) M1-127 Influ CMGLIYNRMGAVTTESA SEQ ID NO: 22) M1-210 Influ RQMVQAMRAIGTHPSSSTGLKND SEQ ID NO: 23) -
TABLE 5 Enzyme digestion system for identification of plasmid pVKD1.0-CI (enzyme digestion at 37° C., 2 h) Enzyme digestion system Volume Plasmid pVKD1.0- CI 3 μL, about 1 μg Pst I (Takara, Cat. No. 1073A) 1 μL Bgl II (Takara, Cat. No. 1021A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - Finally, the primers were designed by using the vector pVKD1.0-hLMN-CTB in Example 2 as a template (see table 6). The target gene fragment hLMN-CTB was amplified by PCR, and was then inserted between the restriction sites Not I and Bam HI on the pVKD1.0-CI vector by a molecular biology method well known in the art to construct the DNA vaccine vector pVKD1.0-CI-LNINB (the plasmid map is shown in
FIG. 7 ), which was stored after being sequenced for identification. The vector pVKD1.0-CI-LMNB was identified by the restriction endonucleases Barn HI and EcoR V (the enzyme digestion system is shown in Table 7), and its enzyme digestion map for verification is shown inFIG. 8 . -
TABLE 6 Primers in Example 3 Primer Sequence 3F (SEQ ID GCGCGGCCGCTGTCACCGTCGTCGACATGCAGGCCG NO: 36) AA 3R (SEQ ID GCGATCCTCAGTTGGCCATGCTGATGGCGGCGATG NO: 3 -
TABLE 7 Enzyme digestion system for identification of plasmid pVKD1.0-CI-LMNB (enzyme digestion at 37° C., 2 h) Enzyme digestion system Volume Plasmid pVKD1.0-CI- LMNB 3 μL, about 1 μg Bam HI (Takara, Cat, No. 1010A) 1 μL EcoR V (Takara, Cat. No. 1042A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively. By means of an online codon optimization software (http://www.jcat.de/), the nucleotide sequences for E. coli codon usage preference shown in SEQ ID NOs: 38-40 respectively were obtained through optimization based on the antigen amino acid sequences. The nucleotide sequences were synthesized by Suzhou Synbio Technologies Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a(+)-LMN (the plasmid map is shown in
FIG. 9 ), which was stored after being sequenced for identification. The vector pET-30a(+)-LMN was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 8), and its enzyme digestion map for verification is shown inFIG. 10 . -
TABLE 8 Enzyme digestion system for identification of plasmid pET-30a(+)-LMN (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)- LMN 3 μL, about 1 μg Nco I (Takara, Cat. No. 1160A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The amino acid sequence of cholera toxin subunit B (CTB) (SEQ ID NO: 30) and its prokaryotic codon optimized nucleic acid sequence (SEQ ID NO: 41) were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The primers were designed (see table 9), and a nucleic acid fragment containing the CTB encoding sequence was amplified by a PCR method using the pET-30a(+)-CTB (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted into the pET-30a(+)-LMN vector by means of homologous recombination to construct the pET-30a(+)-LMN-CTB vector (the plasmid map is shown in
FIG. 11 ), which was stored after being sequenced for identification. The vector pET-30a(+)-LMN-CTB was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 10), and its enzyme digestion map for verification is shown inFIG. 12 . -
TABLE 9 Primers in Example 5 Primer Sequence 5F (SEQ ID GGTGGTGGTGGTGCTCGAGTTAGTTAGCCATAGAGA NO: 42) 5R (SEQ ID TCTGCGTGAAGGTGAAGAAGCTCAGGCTGAAGGTCG NO: 43) TGG -
TABLE 10 Enzyme digestion system for identification Example 5 (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-LMN- CTB 3 μL, about 1 μg Nco I (Takara, Cat. No. 1160A) 1 μL Xho 1 (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - Ten (10) CMV-derived Th epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104 were selected from Table 4, and linked together in tandem to form the amino acid sequence shown in SEQ ID NO: 44, wherein the sequence segment “EFELRRQ” in SEQ ID NO: 44 is formed due to the introduction of enzyme restriction site, which belongs to a common technique for fusion and construction. By means of an online codon optimization software (http://www.jcat.del), the nucleotide sequence for E. coli codon usage preference (SEQ ID NO: 45) was obtained through optimization based on the amino acid sequence of Th epitopes. The nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a(+)-CMV Th (the plasmid map is shown in
FIG. 13 ) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification. The vector pET-30a (+)-CMV Th was identified by the restriction endonucleases Mlu I and Xho I (the enzyme digestion system is shown in Table 11), and its enzyme digestion map for verification is shown inFIG. 14 . - As shown in
FIG. 13 , CMV Th1 contains 5 CMV Th epitopes consisting of pp65-1L pp65-71, pp65-92, pp65-123 and pp65-128 in tandem, and CMV Th2 contains 5 CMV Th epitopes consisting of pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104. Three restriction enzyme sites such as EcoR I, Sac I and Sal I were introduced between CMV Th1 and CMV Th2. -
TABLE 11 Enzyme digestion system for identification of plasmid pET-30a(+)-CMV Th (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)- CMV Th 3 μL, about 1 μg Mlu I (Takara, Cat. No. 1071A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The Primers were designed (see Table 12), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV10-LMNB vector (the plasmid map is shown in
FIG. 15 ), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV10-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 13) and its enzyme digestion map for verification is shown in FIG. 16. As shown inFIG. 15 , pET-30a(+)-CMV10-LMNB contains CMV Th1 and CMV Th2 fragments, i.e. all 10 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104. -
TABLE 12 Primer design in Example 6 Primer Sequence 6F (SEQ ID NO: 46 GCGCGGCCGCGACGACAAGGCCATGGCT 6R (SEQ ID NO: 47) GCCTCGAGGTTAGCCATAGAGATAGC -
TABLE 13 Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-CMV10- LMNB 3 μL, about 1 μg BamH I (Takara, Cat. No. 1010A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - Thirteen (13) Th Epitopes derived from Flu virus, HA203, NP438, NSI-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210 were selected from Table 4, and linked together in tandem to form the amino acid sequence shown in SEQ ID NO: 48. By means of an online codon optimization software (http://www.jcat.de/), the nucleotide sequence for E. coli codon usage preference (SEQ ID NO: 49) was obtained through optimization based on the amino acid sequence containing Flu virus Th epitopes. The nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a(+)-Influ Th (the plasmid map is shown in
FIG. 17 ) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification. The vector pET-30a(+)-Influ Th was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 14), and its enzyme digestion map for verification is shown inFIG. 18 . - As shown in
FIG. 17 , Influ Th1 contains 8 Flu virus Th epitopes consisting of HA203, NP438, HA375, NP24, NP95 and NP221 in tandem, and Influ Th2 contains 5 Flu virus Th epitopes consisting of RA434, HA440, NP324, M1-127 and M1-210. Three restriction sites such asEcoR 1, Sac I and Sal I were introduced between Influ Th1 and Influ Th2. -
TABLE 14 Enzyme digestion system for identification in Example 7 (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)- Influ Th 3 μL, about 1 μg Nco I (Takara, Cat. No. 1160A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The primers were designed (see Table 15), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-Influ Th vector in Example 7 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ8-LMNB vector (containing 8 Flu virus Th epitopes; the plasmid map is shown in
FIG. 19 ), which was stored after being sequenced for identification. The vector pET-30a(+)-Influ8-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 16) and its enzyme digestion map for verification is shown inFIG. 20 . As shown inFIG. 19 , the pET-30a(+)-Influ8-LMNB vector contains the Influ Th1 segment, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4. -
TABLE 15 Primers in Example 7 Primer Sequence 7F1 (SEQ ID NO; 50) GGCGGCCGCGTTAGCCATAGAGATAGC 7R1 (SEQ ID NO: 51) GCGTCGACAAGACGACAAGGCCATGGC TATGC -
TABLE 16 Enzyme digestion system for identification of plasmid pET-30a(+)-Influ8-LMNB (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-Influ8- LMNB 3 μL, about 1 μg BamH I (Takara, Cat. No. 1010A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The primers were designed (see Table 17), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) was referred to for the specific method. This nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-Influ Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ13-LMNB vector (containing 13 Flu virus Th epitopes; the plasmid map is shown in
FIG. 21 ), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV10-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 18) and its enzyme digestion map for verification is shown inFIG. 22 . - As shown in
FIG. 21 , the pET-30a(+)-Influ13-LMNB vector contains both the Influ Th1 and Influ Th2 segments, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4, and 5 Flu virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4, The vector includes all 13 Flu virus Th epitopes in total in Table 4 -
TABLE 17 Primer design in Example 7 Primer Sequence 7F2 (SEQ ID NO: 52) GCCTCGAGGTTAGCCATAGAGATAGCA 7R2 (SEQ ID NO: 53) GCGCGGCCGCGACGACAAGGCCATGGC TATG -
TABLE 18 Enzyme digestion system for identification in Example 7 (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-Influ13- LMNB 3 μL, about 1 μg BamH I (Takara, Cat. No. 1010A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - The prokaryotic expression vector pET-30a(+)-LMN constructed in Example 4, the prokaryotic expression vector pET-30a(+)-LMN-CTB constructed in Example 5, the prokaryotic expression vectors pET-30a(+)-CMV5-LMNB and pET-30a(+)-CMV10-LMNB constructed in Example 6, the prokaryotic expression vectors pET-30a(+)-Influ8-LMNB and pET-30a(+)-Influ13-LMNB constructed in Example 7 were respectively transformed into BL21 (DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., Cat. No. CB105; the instructions of competent cells were referred to for the transformation method) to prepare the recombinant proteins LMN (its amino acid sequence is shown in SEQ ID NO: 59), LMNB (its amino acid sequence is shown in SEQ ID NO: 54), LMNB-C10 (its amino acid sequence is shown in SEQ ID NO: 58), LMNB-18 (its amino acid sequence is shown in SEQ ID NO: 55), and LMNB-13 (its amino acid sequence is shown in SEQ ID NO: 56) according to the pET System Manual (TB055 8th Edition February 1999, Novagen respectively, which were stored at −80° C. after subpackage.
- The concentrations of the recombinant proteins prepared are 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method. The contents of endotoxin in the prepared recombinant proteins were less than IEU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which meet the requirements of an animal experiment, and the instructions of horseshoe crab agent were referred to for the detection method.
- The information of the vaccines prepared in Examples 2, 3 and 8 is shown in Table 19. The DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VP1 (VP1 protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by the Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- Sixteen (16) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 20. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were sacrificed, and their serum and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. The mouse IFN-γ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN-γ ELISPOT kit from BD were referred to for the method. The stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with the final concentration of 10 μg/mL. The positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.
- An ELISA method is well known for a person skilled in the art, and is briefly described below. The 96-Well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both the recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The plates were coated with the proteins in NaHCO3 buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 μg/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5
% Tween 20 in phosphate buffered saline (PBST). An incubation with the mouse serum at room temperature was carried out for 1 hour at an initial dilution of 1:100 and washed 5 times with PBST, and another incubation with goat anti-mouse HRP secondary antibody (Santacruz, USA) was carried out at 1:5000 at 37° C. for 30 min. After beingwashing 5 times with PBST, the substrate was developed with 3,3,5,5-tetramethylbenzidine (TMB) at 37° C. for 15 min and stopped with 2M dilute sulfuric acid, and then the absorbance (A) values were read at 450 nm using a microplate reader (Thermo, USA). A value which is 2.1 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer. A titer was defined as 50 when it was less than the initial dilution of 1:100. -
TABLE 19 Vaccine information Vaccine Attribute Antigen CD4 Th epitope pVKD1.0-LMN-CTB DNA LAGE-1, Epitope-free MAGE-A3, NY-ESO-1, pVKD1.0-CI-LMNB DNA LAGE-1, 5 CMV epitopes, MAGE-A3, 8 influenza virus NY-ESO-1 epitopes pVKD1.0- CI DNA None 5 CMV epitopes, 8 influenza virus epitopes pVKD1.0- NP DNA NP 5 influenza virus epitopes LMNB Recombinant LACE-1, Epitope-free protein MAGE-A3, NY-ESO-1 LMNB-18 Recombinant LAGE-1, First 8 influenza protein MAGE-A3, virus epitopes NY-ESO-1 LMNB-I13 Recombinant LAGE-1, 13 influenza virus protein MAGE-A3, epitopes NY-ESO-1 LMNB-C5 Recombinant LAGE-1, First 5 CMVprotein MAGE-A3, epitopes NY-ESO-1 LMNB-C10 Recombinant LAGE-1, 10 CMV epitopes protein MAGE-A3, NY-ESO-1 VP1 Recombinant VP1 Epitope-free protein -
TABLE 20 Grouping and immunization schemes Week 0, 4, 8 Week 12, 16, 20Week 24 Week 28 Grouping Vaccine Dose Vaccine Dose Vaccine Dose Vaccine Dose A(n = 4) pVKD1.0- NP 100 μg pVKD1.0 100 μg VP1/ CFA 100 μg VPI/ IFA 100 μg B(n = 4) pVKD1.0- NP 100 μg pVKD 1.0- 100 μg LMNB/ CFA 100 μg LMNB/ IFA 100 μg LMNB C(n = 4) pVKD1.0- NP 100 μg pVKD 1.0- 100 μg LMNB- 100 μg LMNB- 100 μg CI-LMNB I8/CFA I8/IFA D(n = 4) pVKD1.0- NP 100 μg pVKD 1.0- 100 μg LMNB- 100 μg LMNB- 100 μg CI-LMNB I13/CFA I13/IFA - The results of cellular immune response assay are shown in
FIG. 23 . Among them, the group primed with pVKD1.0-CI-LMNB DNA vaccine and boosted with LMNB-I13 protein (i.e., group D in Example 8) had the best immune effect, which was significantly higher than those of the parallel control group (group B) and the group boosted with LMNB-18 (group C). Moreover, the level of cellular immune response in the group boosted with LMNB-I13 protein was nearly 3-fold higher than that in the parallel control group (group B). The results showed that a load of 13 Flu virus Th epitopes (group D) could significantly increase the cellular immune response level of weak immunogens. - The primers were designed (see Table 21), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV5-LMNB vector (the plasmid map is shown in
FIG. 24 ), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV5-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 22) and its enzyme digestion map for verification is shown inFIG. 25 . As shown inFIG. 24 , pET-30a(+)-CMV5-LMNB contains a CMV Th1 fragment, i.e. the first 5 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123 and pp65-128. -
TABLE 21 Primer design in Example 10 Primer Sequence 7F1 (SEQ ID NO: 50) GCGCGGCCGCGTTAGCCATAGAGATAGC 7R1 (SEQ ID NO: 51) GCGTCGACAAGACGACAAGGCCATGGCT ATGC -
TABLE 22 Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight) Enzyme digestion system Volume Plasmid pET-30a(+)-CMV10- LMNB 3 μL, about 1 μg BamH I (Takara, Cat. No. 1010A) 1 μL Xho I (Takara, Cat. No. 1094A) 1 μL Enzyme digestion buffer 1 μL ddH2O q.s. to 10 μL - As described in Example 8, the prokaryotic expression vector pET-30a(+)-CMV5-LMNB constructed in Example 10 was transformed into BL21 (DE3) competent cells ('Tangen Biotech (Beijing) Co., Ltd., Cat. No. CB105; the instructions of competent cells were referred to for the transformation method) to prepare the recombinant protein LMNB-05 (its amino acid sequence is shown in SEQ ID NO: 57) according to the pET System Manual (TB055 8th Edition February 1999, Novagen), which was stored at −80° C. after subpackage.
- The concentration of the recombinant protein prepared was 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method. The content of endotoxin in the prepared recombinant protein was less than IEU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which met the requirements of an animal experiment, and the instructions of Horseshoe Crab agent were referred to for the detection method.
- The vaccine information is shown in Table 19. The DNA vaccine pVKD1.0-CI (Example 3) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- Twenty (20) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 23. All DNA vaccines were injected into the tibial anterior muscle of the calf at 1.00 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were sacrificed, and serum and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. The mouse IFN-γ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN-γ ELISPOT kit from BD were referred to for the method. The stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with a final stimulating concentration of 10 μg/mL. The positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.
- An ELISA method is well known in the art and briefly described below. 96-Well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The plates were coated with the proteins in NaHCO3 buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 μg/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5
% Tween 20 in phosphate buffered saline (PBST). An incubation with mouse serum was carried out at room temperature for 1 hour at an initial dilution of 1:100 and washed 5 times with PBST. Another incubation with goat anti-mouse HRP secondary antibody (Santacruz, USA) was carried out at 1:5000 at 37° C. for 30 min, and washed 5 times with PBST. The substrate was then developed with 3,3,5,5-tetramethylbenzidine (TMB) at 37° C. for 15 min and stopped with 2M dilute sulfuric acid, and the absorbance (A) values were read at 450 nm using a microplate reader (Thermo, USA). A value which is 2 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer. A titer was defined as 50 when it was less than the initial dilution of 1:100. -
TABLE 23 Grouping and immunization schemes Week 0, 4, 8 Week 12, 16, 20Week 24 Week 28 Grouping Vaccine Dose Vaccine Dose Vaccine Dose Vaccine Dose A (n = 5) pVKD1.0- CI 100 μg pVKD1.0 100 μg VP1/ CFA 100 μg VPI/ IFA 100 μg B (n = 5) pVKD1.0- CI 100 μg pVKD1.0- LMNB 100 μg LMNB/ CFA 100 μg LMNB/ IFA 100 μg C (n = 5) pVKD1.0- CI 100 μg pVKD1.0-CI- LMNB 100 μg LMNB-C5/ CFA 100 μg LMNB-C5/ CFA 100 μg D (n = 5) PVKD1.0- CI 100 μg pVKD1.0-CI- LMNB 100 μg LMNB-C10/ CFA 100 μg LMNB-C10/ CFA 100 μg - The results of cellular immune response assay are shown in
FIG. 26 . Among them, the group primed with the pVKD1.0-CI-LMNB DNA vaccine, boosted with the LMNB-C5 protein (i.e. group C in Example 11) and boosted with the LMNB-C10 protein (i.e. group D in Example 11) had the best immune effect, which was significantly higher than that in the parallel control (group B). The results showed that a load of 5 CMV virus Th epitopes (group C) and 10 CMV virus Th epitopes (group D) could significantly improve the cellular immune response of weak immunogens. - The information of vaccines prepared in Examples 2, 3 and 8 is shown in Table 19. The DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn. Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VPI (VPI protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.
- Sixty (60) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 24. All DNA vaccines were injected into the tibials anterior muscle of the calf at 100 μg/animal protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by 4T1-hNY-ESO-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1×105 cells/mouse, and the tumor growth was continuously observed and measured after the inoculation. The tumor volume was calculated according to the following equation: tumor volume (mm3)=length×width2/2. The mice were sacrificed when the tumor volume exceeded 2000 mm3.
-
TABLE 24 Grouping and immunization schemes Week 0, 4, 8 Week 12, 16, 20 Week 24 Week 28 Grouping Vaccine Dose Vaccine Dose Vaccine Dose Vaccine Dose A (n = 10) pVKD1.0-NP 50 μg pVKD1.0 100 μg VP1/CFA 10 μg VP1/IFA 10 μg pVKD1.0-CI 50 μg B (n = 10) pVKD1.0-NP 50 μg pVKD1.0-LMNB 100 μg LMNB/CFA 10 μg LMNB/IFA 10 μg pVKD1.0-CI 50 μg C (n = 10) pVKD1.0-NP 50 μg pVKD1.0-CI-LMNB 100 μg LMNB-I8/CFA 10 μg LMNB-I8/IFA 10 μg pVKD1.0-CI 50 μg D (n = 10) pVKD1.0-NP 50 μg pVKD1.0-CI-LMNB 100 μg LMNB-I13/CFA 10 μg LMNB-I13/IFA 10 μg pVKD1.0-CI 50 μg E (n = 10) pVKD1.0-NP 50 μg pVKD1.0-CI-LMNB 100 μg LMNB-I13/CFA 10 μg LMNB-C10/IFA 10 μg pVKD1.0-CI 50 μg F (n = 10) pVKD1.0-NP 50 μg pVKD1.0-CI-LMNB 100 μg LMNB-C10/CFA 5 μg LMNB-C10/IFA 5 μg pVKD1.0-CI 50 μg LMNB-I13/CFA 5 μg LMNB-I13/IFA 5 μg - The tumor growth of immunized mice in each group is shown in
FIG. 27 . Among them, all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor innoculation), and the tumors grew rapidly. The tumor growth of mice in each immunization group lagged behind that in the control group, wherein the mice in the group boosted with LMNB-I13 (group D) and the nice in the group boosted with a mixture of LMNB-I13 and LMNB-C10 (group E) had the slowest tumor growth, so these two groups of vaccines had the best effects. - In addition, an analysis of tumor-free survival was performed for the mice, and the results are shown in
FIG. 28 . The median tumor-free survival (TFS) of the mice in the control group A was 14 days. The tumor-free survival of the mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the tumor-free survival of immunized mice. Among them, group D with the I13 epitope fusion peptide, and group E and F with the I13 and C10 epitope fusion peptide had the best effects, and the tumor-free survival of mice was doubled at the most. In the vaccine group with the I13 epitope fusion peptide (group D), the tumor-free survival was significantly increased by about 40% compared to the conventional vaccine group (group B), showing that a load of 13 Th epitopes of Flu virus or 10 Th epitopes of CMV could significantly improve the protection effect of tumor vaccine against tumor. - Finally, an analysis of mouse overall survival was also performed and the results are shown in
FIG. 29 . Among them, the median overall survival (OS) of mice in control group A was 35 days. The overall survival of mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the survival of mice after immunization. Among them, group D with I13 epitope fusion peptide and groups E and F with I13 and C10 epitope fusion peptides had the best effects, and the overall survival rate was increased by 83%. Compared with the conventional vaccine group (group B), the vaccine group with the I13 epitope fusion peptide (groups D and group F) significantly increased the tumor-free survival by 28% at the most, indicating that a load of thirteen (13) Flu virus Th epitopes or ten (10) CMV Th epitopes could greatly improve the tumor protection effect of tumor vaccine. - The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 25. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by the tumor cells 4T1-hNY-ESO-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at a dose of 1×105 cells/mouse, and the corresponding mice were inoculated subcutaneously with the protein vaccine on
day -
TABLE 25 Grouping and immunization schemes Week 0, 4, 8 Week 10Week 11, 12 Grouping Vaccine Dose Vaccine Dose Vaccines Dose A (n = 10) pVKD1.0- NP 100 μg pVKD1.0 100 μg VP1/ CFA 10 μg B (n = 10) pVKD1.0- NP 100 μg LMNB/ CFA 10 μg LMNB/ IFA 10 μg C (n = 10) pVKD1.0- NP 100 μg LMNB-I13/ CFA 10 μg LMNB-I13/ IFA 10 μg - The tumor growth of immunized mice in each group is shown in
FIG. 30 . Among them, all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor innoculation), and the tumors grew rapidly. The mice in the LMNB-I13 protein vaccine treated group (group C) had the slowest tumor growth compared to the untreated control group (group A). Furthermore, the tumor size of mice in the LMNB-I13 protein vaccine treated group was significantly smaller than that in the control group (group A) onday 22 after the tumor challenge, and there was still a significant difference in tumor size between the two groups untilday 30. Byday 35, the tumor growth of mice began to accelerate in group C, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine. The results indicated that the LMNB-I13 protein vaccine could inhibit tumor growth in mice. - The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 26. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by the tumor cells CT26-hLAGE-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1×105 cells/mouse, and the corresponding mice were inoculated subcutaneously with the protein vaccine on
day -
TABLE 26 Grouping and immunization schemes Week 0, 4, 8 Week 10Week 11, 12 Grouping Vaccine Dose Vaccine Dose Vaccine Dose A (n = 10) pVKD1.0- NP 100 μg pVKD1.0 100 μg VP1/ CFA 10 μg B (n = 10) pVKD1.0- NP 100 μg LMNB/ CFA 10 μg LMNB/ IFA 10 μg C (n = 10) pVKD1.0- NP 100 μg LMNB-I13/ CFA 10 μg LMNB-I13/ IFA 10 μg - The tumor growth of immunized mice in each group is shown in
FIG. 31 . Due to the failure of tumor innoculation of some mice in the untreated control group (group A) after the tumor challenge (i.e. after the tumor innoculation), such mice were not included for the analysis, and the parallel controlled vaccine group (group B) and the LMNB-I13 treated group (group C) were compared. Compared with group B, the tumor growth of mice in group C was slower, and the tumor size of mice in the LMNB-I13 protein vaccine treatment group was significantly smaller than that in parallel controlled vaccine group (group B) onday 22 after the tumor challenge. There was still a significant difference in tumor size between the two groups untilday 30. Similarly, the increased tumor growth of mice in group C was also observed at a later stage in the CT26 mouse model, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine, These results indicated that the LMNB-I13 protein vaccine could inhibit tumor growth in mice.
Claims (14)
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNPCT/CN2017/104401 | 2017-09-29 | ||
WOPCT/CN2017/104401 | 2017-09-29 | ||
PCT/CN2017/104401 WO2019061297A1 (en) | 2017-09-29 | 2017-09-29 | Cd4 helper t-cell epitope fusion peptide and vaccine thereof |
PCT/CN2018/108331 WO2019062853A1 (en) | 2017-09-29 | 2018-09-28 | Cd4 helper t cell epitope fusion peptide and vaccine therewith |
Publications (2)
Publication Number | Publication Date |
---|---|
US20200216516A1 true US20200216516A1 (en) | 2020-07-09 |
US11352411B2 US11352411B2 (en) | 2022-06-07 |
Family
ID=65900409
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/650,318 Active US11352411B2 (en) | 2017-09-29 | 2018-09-28 | Fusion peptides of CD4 helper T cell epitopes and vaccines thereof |
US17/280,803 Pending US20220002750A1 (en) | 2017-09-29 | 2019-06-12 | Recombinant viral vector, immune composition containing same, and use thereof |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/280,803 Pending US20220002750A1 (en) | 2017-09-29 | 2019-06-12 | Recombinant viral vector, immune composition containing same, and use thereof |
Country Status (5)
Country | Link |
---|---|
US (2) | US11352411B2 (en) |
EP (1) | EP3715359A4 (en) |
JP (1) | JP7146926B2 (en) |
CN (4) | CN115850506A (en) |
WO (3) | WO2019061297A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019061297A1 (en) * | 2017-09-29 | 2019-04-04 | 苏州工业园区唯可达生物科技有限公司 | Cd4 helper t-cell epitope fusion peptide and vaccine thereof |
GB201804468D0 (en) * | 2018-03-21 | 2018-05-02 | Valo Therapeutics Oy | PeptiCRAd Cancer Therapy |
WO2021087851A1 (en) * | 2019-11-07 | 2021-05-14 | 苏州工业园区唯可达生物科技有限公司 | Recombinant viral vector, immunogenic composition comprising same and use thereof |
CN110923266A (en) * | 2019-11-07 | 2020-03-27 | 苏州工业园区唯可达生物科技有限公司 | Recombinant virus vector, immune composition containing same and application |
Family Cites Families (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9808932D0 (en) * | 1998-04-27 | 1998-06-24 | Chiron Spa | Polyepitope carrier protein |
TWI229679B (en) * | 1998-06-20 | 2005-03-21 | United Biomedical Inc | Artificial T helper cell epitopes as immune stimulators for synthetic peptide immunogens |
CN101260152A (en) * | 1998-09-15 | 2008-09-10 | 法麦克萨有限公司 | Method for down-regulating Osteoprotegerin ligand activity |
EA003634B1 (en) * | 1998-10-05 | 2003-08-28 | Фармэкса А/С | Novel methods for therapeutic vaccination |
AU2003203140A1 (en) * | 2002-01-17 | 2003-07-30 | Pharmexa A/S | Immunogenic carcinoembryonic antigen (cea) |
JP2005527506A (en) * | 2002-02-28 | 2005-09-15 | インターツェル・アクチェンゲゼルシャフト | Ligand E. G. T cell epitope isolation method |
EP1485122A2 (en) * | 2002-03-11 | 2004-12-15 | Pharmexa A/S | Novel application of vaccination against tnf-alpha |
CA2490449A1 (en) | 2002-06-25 | 2003-12-31 | City Of Hope | Adjuvant-free peptide vaccine |
AR041086A1 (en) * | 2002-08-30 | 2005-04-27 | Glaxo Group Ltd | VACCINE |
CN1517437B (en) * | 2003-01-15 | 2010-06-09 | 张聪慧 | Vaccine for specificity treating tumour or endocellular infection and application |
ITMI20031201A1 (en) * | 2003-06-13 | 2004-12-14 | Istituto Giannina Gaslini | EPITOPES OF CITOMEGALOVIRUS PP65 PROTEIN |
GB0409940D0 (en) * | 2004-05-04 | 2004-06-09 | Glaxosmithkline Biolog Sa | Vaccine |
AU2006228872A1 (en) * | 2005-03-31 | 2006-10-05 | Pharmexa A/S | Immunogenic EGFR peptides comprising foreign Tcell stimulating epitope |
AU2006312847A1 (en) * | 2005-11-09 | 2007-05-18 | Pharmexa A/S | Therapeutic vaccines targeting HMGB1 |
AU2007319669A1 (en) * | 2006-05-30 | 2008-05-22 | Elenore S. Bogoch | Replikin peptides and uses thereof |
NZ619576A (en) * | 2006-12-27 | 2014-07-25 | Harvard College | Compositions and methods for the treatment of infections and tumors |
MY153679A (en) * | 2007-01-15 | 2015-03-13 | Glaxosmithkline Biolog Sa | Vaccine |
WO2008157419A2 (en) | 2007-06-13 | 2008-12-24 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immunogenic peptides of influenza virus |
EA017887B1 (en) * | 2007-08-02 | 2013-03-29 | Байондвакс Фармасьютикалз Лтд. | Multimeric multiepitope influenza vaccines |
US8580276B2 (en) * | 2009-06-05 | 2013-11-12 | City Of Hope | Genetically stable recombinant modified vaccinia ankara (rMVA) vaccines and methods of preparation thereof |
CN101579527B (en) * | 2009-06-24 | 2010-10-27 | 山东大学齐鲁医院 | HLA Specific human cytomegalovirus Multi-epitope adenovirus DNA Vaccine of Chinese population |
JP5933437B2 (en) * | 2009-08-26 | 2016-06-08 | セレクタ バイオサイエンシーズ インコーポレーテッドSelecta Biosciences,Inc. | Composition to induce T cell help |
WO2011046996A2 (en) * | 2009-10-13 | 2011-04-21 | The Johns Hopkins University | Consensus sequence for influenza a virus |
WO2013093514A2 (en) * | 2011-12-23 | 2013-06-27 | Retroscreen Virology Ltd | Vaccines - peptides |
WO2013093512A2 (en) * | 2011-12-23 | 2013-06-27 | Retroscreen Virology Ltd | Vaccine - screening method |
CN102600466B (en) * | 2012-03-23 | 2014-06-11 | 河南农业大学 | Anti-influenza A virus and novel universal epitope vaccine and preparing method thereof |
CA3005127C (en) * | 2015-11-18 | 2023-01-24 | Immunovaccine Technologies Inc. | Adjuvanting systems and water-free vaccine compositions comprising a polyi:c polynucleotide adjuvant and a lipid-based adjuvant |
WO2019061297A1 (en) * | 2017-09-29 | 2019-04-04 | 苏州工业园区唯可达生物科技有限公司 | Cd4 helper t-cell epitope fusion peptide and vaccine thereof |
CN110923266A (en) * | 2019-11-07 | 2020-03-27 | 苏州工业园区唯可达生物科技有限公司 | Recombinant virus vector, immune composition containing same and application |
-
2017
- 2017-09-29 WO PCT/CN2017/104401 patent/WO2019061297A1/en active Application Filing
-
2018
- 2018-09-28 EP EP18861153.7A patent/EP3715359A4/en active Pending
- 2018-09-28 CN CN202210955241.6A patent/CN115850506A/en active Pending
- 2018-09-28 WO PCT/CN2018/108331 patent/WO2019062853A1/en unknown
- 2018-09-28 US US16/650,318 patent/US11352411B2/en active Active
- 2018-09-28 CN CN201811138035.6A patent/CN109575142B/en active Active
- 2018-09-28 JP JP2020539127A patent/JP7146926B2/en active Active
-
2019
- 2019-06-12 US US17/280,803 patent/US20220002750A1/en active Pending
- 2019-06-12 CN CN201910505212.8A patent/CN110205340A/en active Pending
- 2019-06-12 CN CN202410006185.0A patent/CN117987471A/en active Pending
- 2019-06-12 WO PCT/CN2019/090877 patent/WO2020062930A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
CN109575142A (en) | 2019-04-05 |
WO2019061297A1 (en) | 2019-04-04 |
WO2019062853A1 (en) | 2019-04-04 |
WO2020062930A1 (en) | 2020-04-02 |
CN117987471A (en) | 2024-05-07 |
EP3715359A4 (en) | 2021-06-23 |
CN115850506A (en) | 2023-03-28 |
JP2020534874A (en) | 2020-12-03 |
US20220002750A1 (en) | 2022-01-06 |
CN109575142B (en) | 2022-07-12 |
JP7146926B2 (en) | 2022-10-04 |
CN110205340A (en) | 2019-09-06 |
US11352411B2 (en) | 2022-06-07 |
EP3715359A1 (en) | 2020-09-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11352411B2 (en) | Fusion peptides of CD4 helper T cell epitopes and vaccines thereof | |
Ellis | New technologies for making vaccines | |
JP2023524054A (en) | Betacoronavirus prevention and treatment | |
US11352416B2 (en) | Mosaic chimeric viral vaccine particle | |
CN102892431A (en) | Peptides, conjugates and method for increasing immunogenicity of a vaccine | |
Ellis | Technologies for the design, discovery, formulation and administration of vaccines | |
US11866463B2 (en) | Immunogenic compositions to treat and prevent microbial infections | |
Patel et al. | Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses | |
WO2022127825A1 (en) | Vaccine composition for novel coronavirus infection | |
KR101832610B1 (en) | Soluble recombinant antigen protein of porcine epidemic diarrhea virus and vaccine composition for preventing or treating porcine epidemic diarrhea comprising the same | |
Sani et al. | Genetically engineered fusion of allergen and viral-like particle induces a more effective allergen-specific immune response than a combination of them | |
CN109575141A (en) | A kind of CD4 helper T lymphocyte epitope fusogenic peptide and its vaccine | |
JP7410018B2 (en) | Immunogenic peptides against group A streptococci | |
Ohtake et al. | Recombinant therapeutic protein vaccines | |
JP2023550004A (en) | Fusion genes, recombinant novel coronavirus high-efficiency immune DNA vaccines, their construction methods and uses | |
CN102584943A (en) | Vascular smooth muscle L type calcium ion channel immunogenic peptide and application thereof | |
KR101953374B1 (en) | Protein nanoparticle based multivalent vaccines | |
Cárdenas-Vargas et al. | Evaluation of the Immunogenicity of a Potyvirus-Like Particle as an Adjuvant of a Synthetic Peptide | |
Fan et al. | Fusion of DsbA to the N-terminus of CTL chimeric epitope, F/M2: 81-95, of respiratory syncytial virus prolongs protein-and virus-specific CTL responses in Balb/c mice | |
CN105198982B (en) | IL-6-based epitope and application thereof | |
Gandhapudi et al. | Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice., 2023, 15, 432 | |
Smerdova et al. | Features of population immunity against measles and rubella viruses. Why do adults suffer? | |
Shuklina et al. | Immunization with universal influenza vaccine enhances immune response to subsequent infection | |
Grimaldi et al. | Stimulation of pneumovirus-specific CD8+ T-cells using a non-toxic recombinant ricin delivery system | |
CN105294857A (en) | FIX-based epitope and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
AS | Assignment |
Owner name: VACDIAGN BIOTECHNOLOGY CO., LTD, CHINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:XU, JIANQING;HUANG, YANG;ZHANG, XIAOYAN;REEL/FRAME:052626/0001 Effective date: 20200425 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |