EP1021545A2 - Dengue virus antigens and treatment of dengue fever - Google Patents
Dengue virus antigens and treatment of dengue feverInfo
- Publication number
- EP1021545A2 EP1021545A2 EP98952614A EP98952614A EP1021545A2 EP 1021545 A2 EP1021545 A2 EP 1021545A2 EP 98952614 A EP98952614 A EP 98952614A EP 98952614 A EP98952614 A EP 98952614A EP 1021545 A2 EP1021545 A2 EP 1021545A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- dengue
- virus
- enhancement
- amino acid
- dengue virus
- 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.)
- Withdrawn
Links
- 241000725619 Dengue virus Species 0.000 title claims abstract description 63
- 239000000427 antigen Substances 0.000 title claims abstract description 53
- 108091007433 antigens Proteins 0.000 title claims abstract description 53
- 102000036639 antigens Human genes 0.000 title claims abstract description 53
- 206010012310 Dengue fever Diseases 0.000 title claims description 82
- 208000025729 dengue disease Diseases 0.000 title claims description 67
- 208000001490 Dengue Diseases 0.000 title claims description 58
- 238000011282 treatment Methods 0.000 title claims description 12
- 229960005486 vaccine Drugs 0.000 claims abstract description 42
- 230000000890 antigenic effect Effects 0.000 claims abstract description 28
- 241000700618 Vaccinia virus Species 0.000 claims abstract description 21
- 206010046865 Vaccinia virus infection Diseases 0.000 claims abstract description 7
- 208000007089 vaccinia Diseases 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 54
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 47
- 108090000623 proteins and genes Proteins 0.000 claims description 42
- 230000003472 neutralizing effect Effects 0.000 claims description 32
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 31
- 230000001419 dependent effect Effects 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 229920001184 polypeptide Polymers 0.000 claims description 23
- 230000000694 effects Effects 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 20
- 108020004414 DNA Proteins 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 15
- 230000002708 enhancing effect Effects 0.000 claims description 11
- 101710137500 T7 RNA polymerase Proteins 0.000 claims description 10
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 claims description 10
- 230000002265 prevention Effects 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 7
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 5
- 108010008038 Synthetic Vaccines Proteins 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 108020004511 Recombinant DNA Proteins 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 12
- 241000700605 Viruses Species 0.000 abstract description 37
- 208000015181 infectious disease Diseases 0.000 abstract description 27
- 239000012634 fragment Substances 0.000 abstract description 7
- 241000710829 Dengue virus group Species 0.000 abstract description 6
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 6
- 239000013604 expression vector Substances 0.000 abstract description 5
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 84
- 210000004027 cell Anatomy 0.000 description 48
- 239000002609 medium Substances 0.000 description 29
- 150000001413 amino acids Chemical group 0.000 description 19
- 238000012423 maintenance Methods 0.000 description 17
- 208000009714 Severe Dengue Diseases 0.000 description 16
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 15
- 201000009892 dengue shock syndrome Diseases 0.000 description 14
- 238000012217 deletion Methods 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 230000037430 deletion Effects 0.000 description 12
- 230000028993 immune response Effects 0.000 description 12
- 230000002458 infectious effect Effects 0.000 description 11
- 238000005119 centrifugation Methods 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
- 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 9
- 239000000178 monomer Substances 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 239000003550 marker Substances 0.000 description 8
- 238000004091 panning Methods 0.000 description 8
- 239000000539 dimer Substances 0.000 description 7
- 210000004408 hybridoma Anatomy 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 241000710815 Dengue virus 2 Species 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 5
- 241000710831 Flavivirus Species 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 241000710772 Yellow fever virus Species 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 239000012894 fetal calf serum Substances 0.000 description 5
- 239000002356 single layer Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- 229940051021 yellow-fever virus Drugs 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 101100289989 Drosophila melanogaster alpha-Man-Ia gene Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 101710121417 Envelope glycoprotein Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101150021286 MAS1 gene Proteins 0.000 description 4
- 108010067902 Peptide Library Proteins 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 238000013207 serial dilution Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 210000002325 somatostatin-secreting cell Anatomy 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 241000255925 Diptera Species 0.000 description 3
- 102000009109 Fc receptors Human genes 0.000 description 3
- 108010087819 Fc receptors Proteins 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 241000710842 Japanese encephalitis virus Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 239000003708 ampul Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 101100098985 Caenorhabditis elegans cct-3 gene Proteins 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 101710204837 Envelope small membrane protein Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 2
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 2
- 101710145006 Lysis protein Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 241000710771 Tick-borne encephalitis virus Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000700647 Variola virus Species 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 2
- 239000013553 cell monolayer Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- UXHQLGLGLZKHTC-CUNXSJBXSA-N 4-[(3s,3ar)-3-cyclopentyl-7-(4-hydroxypiperidine-1-carbonyl)-3,3a,4,5-tetrahydropyrazolo[3,4-f]quinolin-2-yl]-2-chlorobenzonitrile Chemical compound C1CC(O)CCN1C(=O)C1=CC=C(C=2[C@@H]([C@H](C3CCCC3)N(N=2)C=2C=C(Cl)C(C#N)=CC=2)CC2)C2=N1 UXHQLGLGLZKHTC-CUNXSJBXSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 101100275473 Caenorhabditis elegans ctc-3 gene Proteins 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000016918 Complement C3 Human genes 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 208000034657 Convalescence Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 241000710827 Dengue virus 1 Species 0.000 description 1
- 241000710872 Dengue virus 3 Species 0.000 description 1
- 241000710844 Dengue virus 4 Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101150013191 E gene Proteins 0.000 description 1
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 1
- 241000701832 Enterobacteria phage T3 Species 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 241000941423 Grom virus Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 206010021137 Hypovolaemia Diseases 0.000 description 1
- 206010022004 Influenza like illness Diseases 0.000 description 1
- 101710128560 Initiator protein NS1 Proteins 0.000 description 1
- 201000005807 Japanese encephalitis Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 101710144127 Non-structural protein 1 Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101150003725 TK gene Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 108091005948 blue fluorescent proteins Proteins 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229940023605 dengue virus vaccine Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- LWGJTAZLEJHCPA-UHFFFAOYSA-N n-(2-chloroethyl)-n-nitrosomorpholine-4-carboxamide Chemical compound ClCCN(N=O)C(=O)N1CCOCC1 LWGJTAZLEJHCPA-UHFFFAOYSA-N 0.000 description 1
- VOVZXURTCKPRDQ-CQSZACIVSA-N n-[4-[chloro(difluoro)methoxy]phenyl]-6-[(3r)-3-hydroxypyrrolidin-1-yl]-5-(1h-pyrazol-5-yl)pyridine-3-carboxamide Chemical compound C1[C@H](O)CCN1C1=NC=C(C(=O)NC=2C=CC(OC(F)(F)Cl)=CC=2)C=C1C1=CC=NN1 VOVZXURTCKPRDQ-CQSZACIVSA-N 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940021993 prophylactic vaccine Drugs 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 230000006648 viral gene expression Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera 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
-
- 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
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—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
- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to monoclonal antibodies and antigen binding fragments specific for and recognising antigenic epitopes, including synthetic peptides, capable of eliciting antibodies which protect against infection by dengue viruses as well as the said antigenic epitopes and their use for vaccine purposes.
- the present invention also relates to recombinant vaccinia viruses and for example such which are derived from the Modified vaccinia virus Ankara (MVA) encoding for and capable of producing dengue virus antigens or antigenic epitopes recognised by said monoclonal antibodies, and the use of such recombinant viruses as vaccine.
- the invention also relates to pharmaceutical compositions comprising the above monoclonal antibodies, pharmaceutical compositions comprising dengue virus antigens, and pharmaceutical compositions comprising expression vectors 'synthesising dengue virus antigen.
- Dengue viruses are divided into four antigenically related serotypes, called dengue virus serotypes 1, 2, 3, and 4. Complete or partial nucleotide sequences of the dengue 1, 2, 3, and 4 type virus have been published (Chamber, T.J., Hahn, C.S., Galler, R. And Rice, CM. 1990. Annu. Rev. Microbiol., 44, 649, Zhao et al., 1986, Virology 155, 77-88).
- Dengue virus with its four serotypes Den-1 to Den-4, is the most important member of the Flavivirus genus with respect to infections of humans producing diseases that range from flu-like symptoms to severe or fatal illness, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue outbreaks continue to be a major public health problem in densely populated areas of the tropical and subtropical regions, where mosquito vectors are abundant. Therefore, there is a substantial need for the development of prophylactic vaccines. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of immune response.
- DHF dengue haemorrhagic fever
- DSS dengue shock syndrome
- the flavivirus genome consists of a single-stranded positive sense RNA molecule.
- the single encoded open reading frame (ORF) is translated into a polypeptide which is cleaved co- and post-translationally into at least 11 proteins.
- the order of proteins encoded in the ORF is 5'-C-preM(M)-E-NSl- NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' (Venugopal, K., and Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
- the preM protein (18-19 kDa) is a precursor of the structural protein M, which is formed by cleavage and removal of the N-terminal (pre) segment, by a process presumed to be linked to maturation of the envelope glycoprotein and the development of virus infectivity.
- the E glycoprotein (53-54 kDa) is an outer structural protein of the dengue virus. It exhibits a number of biological activities including receptor binding and membrane fusion, and is the target for neutralising antibodies and T-helper cells.
- the E protein is a typical membrane glycoprotein with a C-terminal that spans the membrane.
- the non-structural protein NSl (39-41 kDa) is also modified by glycosylation. It is derived from the ORF by N-terminal signalase cleavage and C-terminal cleavage involving a protease. NSl may be involved in the assembly and release of virions. It is found on the cell surface and in the culture medium of infected cells.
- NSl protein evokes a strong antibody response which protects the host against challenge with flaviviruses, presumably through a complement mediated pathway, although recently it has been suggested that other mechanisms such as antibody-dependent cell cytotoxicity may also be responsible (Venugopal, K., & Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
- a successful vaccine against the prototype yellow fever virus (YFV) has long been in use, as has been the less celebrated vaccines against tick borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV). These successes are unlikely to be easily reproduced for the dengue viruses which are the causative agents for a two important disease syndromes - classical dengue fever (DF) and dengue haemorrhagic fever/ dengue shock syndrome (DHF/DSS). This is because DHF/DSS has been shown to be associated with secondary immune responses to heterologous dengue virus serotypes which may be due to antibody dependent enhancement (ADE) of virus replication in host monocytes via an Fc receptor mediated pathway.
- ADE antibody dependent enhancement
- the ADE phenomenon has been shown to be due to subneutralising concentrations of antibodies to the envelope glycoprotein (E).
- E envelope glycoprotein
- Fc receptors or C3 complement receptors can mediate attachment and uptake in the mechanism termed antibody-dependent enhancement (ADE).
- Modified vaccinia virus Ankara (MVA) is a host range restricted and highly attenuated vaccinia virus strain, which is unable to multiply in human and most other mammalian cell lines tested. But since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses may be used as exceptionally safe and efficient expression vectors.
- the modified vaccinia virus Ankara has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H: ⁇ 1975] Infection 3, 6-14; Swiss Patent No. 568, 392).
- the MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures de Microorganisms, 25, rue du Dondel Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721.
- the MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain.
- MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virio s. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851).
- an effective prevention and therapy of DF, DHF and DSS has been made possible by the identification of dengue virus neutralising monoclonal antibodies which does not cause immune enhancement or is involved in antibody dependent enhancement, and by using one such monoclonal antibody, an antigenic discontinuous epitope in the dengue virus envelope protein which elicits neutralising antibodies without being involved in immune enhancement or antibody dependent enhancement has been identified. Further using this monoclonal antibody to select peptides from a random peptide library, peptides which may be used to elicit dengue neutralising antibodies and which do not cause immune enhancement or is envolved in antibody enhancement have been identified. Further, according to the present invention, an antigen expression system for the in vivo or ex vivo production of a therapeutic agent against DF, DHF or DSS has been prepared.
- a still further object of the present invention is to provide recombinant vaccinia virus based expression systems expressing dengue virus antigens or antigenic epitopes to provide an efficient and safe dengue virus vaccine for animals and humans.
- Still one other object of the present invention is to provide heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same.
- the present invention thus, inter alia, comprises the following, alone or in combination:
- Dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same;
- proteins, polypeptides, peptides and synthetic peptides including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any other amino acid sequence or functional analogues or other equivalents thereof which will elicit anti-dengue antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody that specifically binds to and identifies dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a monoclonal antibody of subclass IgG recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a mouse monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS,APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a vaccine comprising as a first component a recombinant MVA carrying and capable of expressing T7 RNA polymerase and as further components one or more recombinant DNA vectors each carrying at least one dengue virus antigen or synthetic antigen not being involved in immune enhancement or antibody dependent enhancement and being recognised by a dengue virus neutralising antibody when the said antigen or synthetic antigen is under transcriptional control of a T7 RNA polymerase promoter;
- a method for the treatment or prevention of a dengue virus infection comprising inoculating a living animal body, including a human, in need thereof with the first and further components of a vaccine, as above, either simultaneously or with a timelag but using the same inoculation site;
- a recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens not able to effect immune enhancement or antibody dependent enhancement; a recombinant MVA as above, containing and capable of expressing DNA sequences encoding antigens from any of the four dengue virus serotypes (type 1, 2, 3 and 4) not able to effect immune enhancement or antibody dependent enhancement;
- a recombinant MVA as above containing and capable of expressing DNA sequences encoding peptides, polypeptides or proteins being ' recognised by monoclonal antibodies specific to the peptides, polypeptides and/ or proteins including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional or other equivalent thereof which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a recombinant MVA as above, wherein the DNA sequences encoding antigen is under transcriptional control of a vaccinia specific promoter selected from the group of vaccinia virus early/ late promoter P7.5, vaccinia virus synthetic promoter or any other equivalent promoter;
- a method for the treatment or prevention of dengue virus infection comprising administering to a living animal body, including a human, in need thereof a therapeutically effective amount of a recombinant MVA as anyone above, or a vaccine as above.
- Monoclonal antibodies generated to be specifically directed against Dengue virus polypeptides are essential to identify and to target Dengue virus-specific epitopes which enables the rational design of novel prophylactic, therapeutic, and diagnostic tools for the control of human Dengue virus infection.
- the locations in the envelope glycoprotein of dengue virus of a series of protective epitopes are identified.
- neutralising antibodies are screened for immune enhancing properties in order to determine that even at sub-neutralising concentrations the antibodies do not cause antibody dependent enhancement.
- a specific mouse monoclonal antibody identified as MASl which specifically recognises peptides, polypeptides and proteins which contain a sequence functioning as a neutralising but non immune enJiancing epitope for the elicitation of protective immune responses against dengue virus infection is provided.
- the monoclonal antibody MASl can be prepared by anyone skilled in the art and as described generally by Kohler, G. & Milstein, C (1975, Nature, 256: 495-497) and Harlow, E. & Lane, D. (1988, Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratories).
- the monoclonal antibody MASl according to one preferred embodiment of the present invention, is a mouse monoclonal antibody of subclass IgGl which has the property of being able to neutralise dengue virus without causing antibody dependent enhancement in Fc receptor bearing cells at any dilution tested.
- An epitope defined by this monoclonal antibody MASl has the advantage of being an epitope which is neutralizable but not enhanceable and therefore represents an epitope which would be safe and efficacious as a vaccine preparation, being able to elicit protective antibodies which will not cause immune enhancement at any concentration.
- neutralizing monoclonal antibodies which are not immune enhancing are used in the screening of different bacteriophage display libraries, peptide libraries etc. to identify peptide sequences recognised by the said non-immune enhancing dengue virus neutralising monoclonal antibodies.
- the said specific monoclonal antibody MASl has been found to recognise peptides, polypeptides or proteins which contain a sequence which functions as a protective but not enhancing epitope for the elicitation of a protective immune response against dengue virus infection.
- sequences may be derived from the authentic dengue virus proteins or glycoproteins themselves or from synthetic peptides or other mimetic peptides or polypeptides, including but not limited to anti-idiotypic antibodies and their derivatives as well as DNA sequences encoding any of the above-mentioned molecules.
- the monoclonal antibody MASl has been found to recognise polypeptides including the amino acids sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL.
- the monoclonal antibody MASl is further characterised as being a mouse monoclonal antibody within the IgGl family recognising the amino acid sequence LPWYNHS and furthermore by the fact it recognises a discontinuous antigenic epitope within the E gene of dengue 2.
- DNA sequences which code for dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies, and especially by the MASl, capable of eliciting immune responses to dengue virus are thus provided for the treatment of and prevention of dengue virus infection.
- sequences are optionally introduced, with the aid of DNA recombination techniques, into the genome of viral expression systems including for example the MVA vector.
- the newly produced recombinant MVA will be infectious, that is to say able to infect foreign cells and it will express the integrated DNA sequence.
- the recombinant viruses and the recombinant MVA according to the invention will be useful as extremely safe live vaccines for the treatment or prophylactics of dengue infection.
- the recombinant MVA vaccinia viruses can be prepared as set out hereinafter.
- a DNA-construct containing sequences flanking the left and the right side of a naturally occurring deletion, e.g. deletion II or III, within the MVA genome (Altenburger, W., Suter, C.P. and Altenburger J. (1989) Arch. Virol. 105, 15-27) was constructed.
- a DNA-construct which contains a DNA-sequence which codes for a dengue antigen or antigenic epitope flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion II or III, within the MVA genome is introduced into cells infected with MVA, to allow homologous recombination.
- the DNA-construct to be inserted can be linear or circular. A circular DNA is preferred, especially a plasmid.
- the DNA sequence encoding antigenic epitopes is inserted between the sequences flanking the naturally occurring deletion.
- promoters are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198, 328, those of the 7.5 kDa gene (EP-A-110, 385), the vaccinia specific synthetic promoter sP (Sutter et al., 1994, Vaccine 12, 1032-1040) or those of the bacteriophage T7 Polymerase.
- the DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al., Virol. 52, 456-467 [1973]; Wigler et al., Cell 777-785 [1979] by means of electroporation (Neumann et al., EMBO J. 1, 841-845 [1982]), by micro-injection (Graessmann et al., Meth. Enzymology 101, 482-492 (1983)), by means of liposomes (Straubinger et al, Methods in Enzymology 101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 11, 2163-2167 (1980)) or by other methods known to those skilled in the art. Transfection by means of calcium phosphate precipitation is preferred.
- the present invention therefore also relates to recombinant MVA which contains at least one DNA sequence which codes for dengue virus antigen or antigenic epitopes and relates further to vaccines containing such viruses in a physiologically acceptable form.
- the recombinant MVA according to the invention contain and are capable of expressing one or more DNA sequences encoding dengue virus antigens or antigenic epitopes capable of eliciting a protective immune response against dengue viruses.
- the dengue virus antigen includes the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
- the invention therefore in a preferred embodiment relates to a viral expression vector, and preferably a pox virus based expression system and especially a MVA based expression system coding for peptides, polypeptides or proteins including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL including for example the following synthetic expression sequence containing one start codon and two stop codons for translation: 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'-CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3'
- the invention also relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding dengue virus antigens.
- a vaccine according to the invention comprises a recombinant MVA encoding dengue virus type 1 antigen; recombinant MVA encoding dengue virus type 2 antigen; recombinant MVA encoding dengue virus type 3 antigen; recombinant MVA encoding dengue virus type 4 antigen; and/ or includes the amino acid sequence LPWYNHS APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
- the invention further relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding peptides or polypeptides comprising antigenic epitopes recognised by monoclonal antibody MASl which elicit protective immune responses against dengue virus infection.
- the vaccine according to the invention comprises a peptide, polypeptide or protein which contains an epitope recognised by the said monoclonal antibody MASl.
- the epitope can exist within an amino acid sequence derived from random peptide libraries and identified by the said monoclonal antibody MASl, or the epitope can exist within an anti-idiotypic antibody generated against said monoclonal antibody MASl, or can exist within polypeptides and proteins encoded by the envelope genes of dengue viruses and includes but is not limited to nucleic acid sequences which encode for the epitope described by monoclonal antibody MASl.
- a DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter were provided.
- recombinant MVA encoding T7 RNA polymerase (MVA-T7pol) is used in combination with DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter.
- a coding sequence of a given dengue antigen capable of binding to a monoclonal antibody not being involve in immune enhancement is cloned under control of a T7 RNA polymerase promoter preferably in a plasmid vector and the resulting DNA construct is amplified and purified using standard laboratory procedures.
- the vector DNA is inoculated simultaneously or with appropriate timelags together with MVA- T7pol.
- An appropriate timelag allows a cell to take up the inoculated DNA vector, before MVA infection takes place.
- the mode of administration, the dose and number of administration can be optimised by one skilled in the art.
- the recombinant gene of interest is expressed transiently in cells containing both the vector DNA and MVA-T7pol and the corresponding antigen is presented to the host immune system stimulating an antigen-specific immune response.
- the recombinant MVA according to the invention can also carry a marker gene.
- Said marker genes are preferably selected from the group consisting of marker genes which codes for proteins such as ⁇ -galactosidase, neomycin, alcohol dehydrogenase, luciferase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, and green or blue fluorescent proteins.
- marker genes can either facilitate the purification of recombinant MVA or be used as a independent antigenic marker to control status of immune response after a antigen specific vaccination.
- the invention also relates to methods for the preparation of such recombinant MVA and vaccines, and to the use of these vaccines for the prophylactics or treatment of infections caused by dengue virus.
- the recombinant MVA according to the invention are converted into a physiologically acceptable form. This can be done based on the experience in the preparation of MVA vaccines used for vaccination against smallpox (as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392).
- PBS phosphate-buffered saline
- the lyophilisate can contain extenders (such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such as antioxidants, stabilisers, etc.) suitable for parenteral administration.
- extenders such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone
- other aids such as antioxidants, stabilisers, etc.
- the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline, and administered either parenterally, for example by intramuscular inoculation or locally.
- Vaccines or therapeutics according to the invention are preferably injected intramuscularly (Mayr, A. et al. [1978] Zbl. Bakt. Hyg., I. Abt. Orig. B 167, 375-390).
- the mode of administration, the dose and the number of administrations can be optimised by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain appropriate immune responses against the foreign antigen.
- a continuous mosquito cell line, C6/36 was grown to confluency in a 25 mm 2 tissue culture flask in Leibovitz 15 medium supplemented with 3% fetal calf serum (FCS), 10% tryptose phosphate broth, penicillin (100 units/ ml), streptomycin (100 mg/ml). The medium was then replaced with 4.5 ml of maintenance medium (Leibovitz 15 medium supplemented with 1% FCS, 10% tryptose phosphate broth, and antibiotics in the concentrations described above. Half a ml of Dengue infectious virus stock culture supernatant was added and the flask was rocked gently at room temperature overnight.
- the flask was then maintained in a stationary position in an incubator set at 28°C for 4 to 5 days until some signs of a cytopathic effect were seen.
- the medium was then changed to 5 ml of fresh maintenance medium and the cells were incubated at 28°C until syncytia formation occured.
- the supernatant was harvested for infectious virus.
- the supernatant was clarified by centrif ugation for 10 minutes at 4 °C at 5000 rpm in a Beckman Avanti J25 using a JS7.5 rotor.
- the clarified supernatant was the Dengue infectious virus stock and was used for seeding new cultures as well as for all Dengue virus plaque assays including neutralization assays and immune enhancement assays.
- the Dengue infectious virus stock was titrated in a plaque assay in the following manner.
- a confluent monolayer of PS Clone D cells were trypsinized and resuspended at 2 x 10 5 cells/ ml maintenance medium (composition as described in Example 1) and 0.5 ml was dispensed in each well of a 24 well tissue culture cluster plate.
- Ten fold serial dilutions of Dengue infectious virus stock were prepared in maintenance medium and 100 ⁇ l of these dilutions or maintenance medium was added in duplicate to each well containing PS Clone D cells in suspension. The plate was gently tapped to mix the contents of each well and the plate then was incubated at 37 °C for 4 hours until the cells had adhered to the plate.
- the cells were then overlaid with 0.6 ml of maintenance medium containing 1.5% carboxymethylcellulose, and incubated at 37 °C for 4 to 5 days until plaques were visible under an inverted microscope.
- the medium was then tipped out into a container containing 5% chlorox and the monolayers gently washed with phosphate buffered saline, pH 7.4.
- the monolayers were then stained for 15 minutes with naphthalene black and washed with water. The plates were allowed to airdry and the plaques counted.
- Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in four fold serial dilutions in maintenance medium. 100 ⁇ l of undiluted hybridoma supernatant or dilutions were dispensed into 24 well tissue cluster plates in triplicate.
- Control supernatant from hybridomas secreting irrelevant monoclonal antibody (for example to Hepatitis B virus) was also dispensed in triplicate and these wells containing monoclonal antibody or control were then seeded with 100 ⁇ l of virus dilution prepared previously.
- the plate was tapped gently to mix the contents and incubated at 37 °C for one hour.
- a confluent monolayer of PS Clone D cells was resuspended at 3 x 10 5 cells/ ml maintenance medium as described above and dispensed at 0.3 ml per well containing the virus-antibody incubation mixtures or controls. Cells were also added to triplicate wells containing no incubation mixtures. The plate was incubated at 37 °C for 4 hours until the cells had adhered to the plastic and 0.5 ml of maintenance medium containing 1.5% carboxymethylcellulose was overlayed. The plate was incubated at 37 °C for 4 to 5 days until plaques were visible using an inverted microscope. The monolayers were then washed and stained with naphthlene black as described above and plaques counted. Neutralization of Dengue virus was deemed to have occured when the mean number of plaques in wells containing antibody was at least 50% less than in the control wells containing irrelevant antibody or no antibody.
- a dilution of Dengue virus infectious stock which would give approximately 30- 40 plaques per 100 ⁇ l based on the above titration was then selected and prepared by dilution into maintenance medium.
- Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in ten fold serial dilutions in maintenance medium. Similar dilutions were prepared of irrelevant hybridoma supernatants as controls.
- Duplicate wells containing 100 ⁇ l of maintenance medium was used as the negative control.
- 100 ⁇ l of undiluted hybridoma supernatant or dilutions were dispensed into 5 ml round bottomed cell culture tubes.
- 100 ⁇ l of the Dengue virus dilution was added to each tube. The mixture was gently mixed and incubated at 37 °C for 1 hour.
- a confluent monolayer of P388D1 cells were resuspended at 5 x 10 s cells/ml into Leibovitz 15 maintenance medium and dispensed at 0.5 ml per tube.
- the tubes were incubated at 37 °C overnight.
- the cells were then washed by centrifugation for 10 minutes at 800 rpm at room temperature in a Beckman Avanti J25 using a JS7.5 rotor.
- the supernatant was discarded and 1 ml of fresh maintenance medium was added per tube.
- the tubes were centrifuged again as described and replenished with 1 ml of fresh maintenance medium.
- the cells were resuspended and incubated at 37 °C for 5 days.
- Immune enhancement or antibody dependent enhancement is deemed to have occured when the number of plaque forming units (pfu) per ml of culture was double or greater than that of the negative controls containing no antibody.
- a Balb/c mouse was immunised through the ip route with Dengue antigen prepared as follows. C6/36 mosquito cell monolayers wre inoculated with
- Dengue 2 virus as described in Example 1.
- the monolayer was washed once with phosphate buffered saline and lysed with 2 ml of isotonic buffer (1.5 mM magnesium chloride, 5 mM potassium chloride, 10 mM HEPES) containing 1% Nonidet P 40 after gentle rocking at room temperature for 10 minutes, the lysate was harvested and spun for 5 minutes in a microfuge. The supernatant was loaded into a single well comb of a 12% SDS PAGE gel (Biorad Mini Protean II) and run at 100 Volts until the dye front reached the end of the gel. Bands corresponding to Dengue Envelope glycoprotein and pre Membrane protein were excised from the gel, placed in a dialysis bag, electro-eluted in a minimal volume and used as the immunogen.
- the immunised mouse was boosted through the ip route after two weeks with 0.5 ml infectious dengue virus culture supernatant prepared as described in Example 1 and left to rest for a week before the spleen was removed and spleen cells harvested.
- Cell fusion was attained by the method as essentially described by Kohler & Milstein, Nature 256, 495-497 (1975) and Kohler et al., Eur.J.Immunol. 6, 292-295 (1976) and clones were screened for anti Dengue activity by a dot enzyme immunoassay (Cardosa, M.J. & Tio, P.H., 1991.
- Dot enzyme immunoassay Alternative diagnostic aid for dengue fever and dengue haemorrhagic fever, Bulletin of the World Health Organisation, 69, 741-745.
- a mouse immunoglobulin capture ELISA using 96 well ELISA plates coated with anti-mouse immunoglobulin incubated with tissue culture supernatants overnight at 4 °C After washing with phosphate buffered saline containing 0.05% Tween 20, the immunoglobulin bound was identified using dengue virus prepared as described in Example 1 followed by an HRP conjugated anti dengue convalescent human serum (Innis, B.L. Nisalak, A., Nimmannitya, S., et al., 1989. An enzyme-linked immunosorbent assay to characterise dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 40:418- 27) Positive clones were expanded and recloned by limiting dilution twice.
- the hybridoma which yielded MAS 1 was grown to large quantities in 75 mm 2 tissue culture flasks and supernatants harvested every 2 or 3 days.
- the supernatant was clarified by centrifugation at 3000 rpm at 4 °C for 15 minutes in a Beckman Avanti J25 using a JS7.5 rotor, and the clarified supernatant was pushed through a 0.22 micron filter before purification through a HiTrap Protein G column (Pharmacia Biotech, Product Code No. 17-040-01) according to the manufacturer's instructions. Fractions containing protein were pooled and checked for specific activity in a dot enzyme immunoassay (Cardosa & Tio, 1991).
- the affinity purified monoclonal antibody MAS 1 was used in panning experiments to select peptide ligands from a phage display peptide library purchased from New England Biolabs (Catalog number 8100). The experiment was performed according to the instructions of the manufacturer using MAS 1 as the panning antibody bound to the plastic surface of an microtitre ELISA plate. Three pannings were performed and phage selected were purified and DNA sequences of the heptapeptide displayed in each clone was determined. The DNA sequence was translated and concensus peptides were determined using the software Lasergene. The following are the concensus sequences obtaine hy comparison of amino acid sequences.
- First line indicates the concensus sequence. Clones (characterised by a number) from all three pannings are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings only are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings are listed in the first row.
- the MVA virus is a highly attenuated vaccinia virus derived from the vaccinia virus strain Ankara (CVA) by long-term serial passages on primary chicken embryo fibroblast (CEF) cultures.
- CVA vaccinia virus strain Ankara
- CEF primary chicken embryo fibroblast
- CEF cells To prepare CEF cells, 11-days old embryos were isolated from incubated chicken eggs, the extremities are removed, and the embryos are minced and dissociated in a solution composed of 0.25% trypsin at 37 °C for 20 minutes. The resulting cell suspension was filtered and cells were sedimented by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes, resuspended in 10 volumes of medium A (MEM Eagle, for example obtainable from Life Technologies GmbH, Eggenstein, Germany), and sedimented again by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes.
- medium A MEM Eagle, for example obtainable from Life Technologies GmbH, Eggenstein, Germany
- the cell pellet was reconstituted in medium A containing 10% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 mg/ml) and 2 mM glutamine to obtain a cell suspension containing 500 000 cells/ml.
- CEF cells obtained in this way were spread on cell culture dishes. They were left to grow in medium A in a 6% CO2 atmosphere with 95% humidity at 37°C for 1-2 days, depending on the desired cell density, and were used for infection either directly or after one further cell passage.
- FCS fetal calf serum
- penicillin 100 units/ml
- streptomycin 100 mg/ml
- 2 mM glutamine 2 mM glutamine
- MVA viruses were used for infection as follows. CEF cells were cultured in 175 cm ⁇ cell culture bottles. At 90-100% confluence, the medium was removed and the cells were incubated for one hour with an MVA virus suspension (0.01 infectious units (IU) per cell, 0.02 ml/ cm 2 ) in medium A. Then more medium A was added (0.2 ml/ cm 2 ) and the bottles were incubated at 37°C for 2-3 days (until about 90% of the cells show cytopathogenic effect). Crude virus stocks were prepared by scraping cell monolayers into the medium and pelleting the cell material by centrifugation at 3000 rpm in a Sorvall RC-3B centrifuge at 4°C for 5 minutes. The crude virus preparation was stored at -20°C before further processing (e.g. virus purification).
- MVA virus suspension (0.01 infectious units (IU) per cell, 0.02 ml/ cm 2 ) in medium A. Then more medium A was added (0.2 ml/ cm 2 ) and the bottles
- MVA virus obtained from Prof. Anton Mayr was cloned by limiting dilution during three consecutive passages in CEF cultured on 96-well tissue culture plates.
- the MVA clone F6 was selected and amplified in CEF to obtain working stocks of virus that served as starting material for the generation of recombinant MVA viruses described in this patent application as well as for the generation of recombinant MVA viruses described previously (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851; Sutter, G., Wyatt, L., Foley, P., Bennink, J. and Moss, B.
- the gradient was centrifuged in Beckmann SW41 rotor at 13,000 rpm for 50 minutes at 4°C After centrifugation, discrete bands containing virus particles were harvested by pipetting after aspirating volume above band.
- the obtained sucrose solution was diluted with three volumes PBS and the virus particles were sedimented again by centrifugation (Beckmann SW 27/28, 60 minutes at 13,500 rpm, 4° C).
- the pellet which now consisted mostly of pure virus particles, was resuspended in PBS and equilibrated to virus concentrations corresponding on average to l-5x l ⁇ " IU/ml.
- the purified virus stock solution was stored at -80°C and used either directly or diluted with PBS for subsequent experiments.
- the primers for the left 600 bp DNA • flank were 5 X CAG CAG GGT ACC CTC ATC GTA CAG GACGTT CTC-3' and 5'- CAG CAG CCC GGG TAT TCG ATG ATT ATT TTT AAC AAA ATA ACA-3' (sites for restriction enzymes Kpnl and Smal are underlined).
- the primers for the right 550 bp DNA flank were 5'-CAG CAG CTG CAG GAA TCA TCC ATT CCA CTG AAT AGC-3' and 5'-CAG CAG GCA TGC CGA CGA ACA AGG AAC TGT AGC AGA-3' (sites for restriction enzymes PstI and SphI are underlined).
- the E. coli lacZ gene under control of the vaccinia virus late promoter Pll (prepared by restriction digest from pill LZ, Sutter, G. and Moss, B. [1992] PNAS USA 89, 10847-10851) was inserted, using the BarnHI site, to generate the MVA insertion vector pUCII LZ [ Figure 1].
- a 289 bp fragment containing the vaccinia virus early-late promoter P7.5 together with a Smal site for cloning prepared by restriction digest with EcoRI and Xbal from the plasmid vector pSCll [Chakrabarti et al.
- coli LacZ open reading frame was amplified by PCR (primers were 5'-CAG CAG GTC GAC CCC GAC CGC CTT ACT GCC GCC-3' and 5'-GGG GGG CTG CAG ATG GTA GCG ACC GGC GCT CAG-3') and cloned into the Sail and PstI sites of pUC II LZ P7.5 to obtain the MVA vector pUC II LZdel P7.5.
- this vector plasmid can be used to insert DNA sequences encoding a foreign gene under transcriptional control of the vaccinia virus promoter P7.5 into the MVA genome.
- a 147 bp minigene encoding a polypeptide including the N-terminal amino acids LPWYNHS and containing one start codon and two stop codons for translation was prepared by PCR from cloned DNA of a phage M13 library using synthetic oligonucleotides 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'- CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3' and cloned into the Smal restriction of site of the MVA vector plasmid pUCII LZdel P7.5 to create the plasmid pUCII LZdel P7.5- DENminil carrying the minigene under transcriptional control of the vaccinia virus early/ late promoter P7.5.
- the minigene under control of the vaccinia virus early/late promoter P7.5 was inserted into deletion II within the MVA using homologous recombination.
- CEF cells infected with MVA at a multiplicity of 0.05 TCID50 per cell were transfected with DNA of plasmid pUC II LZdel P7.5-DENmini as described previously (Sutter, G, Wyatt, L., Foley, P., Bennink, J. and Moss, B. [1994] Vaccine 12, 1032-1040).
- coli LacZ marker gene were selected by consecutive rounds of plaque purification in CEF cells stained with 5- bromo-4-chloro-3-indolyl ⁇ -D-galactoside (300 mg/ml).
- recombinant MVA viruses containing the E sequence and having deleted the LacZ marker gene were isolated by three additional consecutive rounds of plaque purification screening for non-staining viral foci in CEF cells in the presence of 5-bromo-4-chloro-3-indolyl ⁇ -D-galactoside (300 mg/ml). Subsequently, recombinant viruses were amplified by infection of CEF monolayers.
Abstract
The present invention relates to monoclonal antibodies and antigen binding fragments specific for and recognising antigenic epitopes capable of eliciting antibodies which protect against infection by dengue viruses as well as the said antigenic epitopes and their use for vaccine purposes. The present invention also relates to recombinant vaccinia viruses and for example such which are derived from the Modified Vaccinia Ankara (MVA) encoding for and capable of expressing dengue virus antigen, and the use of the same recombinant viruses encoding dengue virus antigens in vaccines. In addition the invention also relates to pharmaceutical compositions comprising the above monoclonal antibodies, pharmaceutical compositions comprising dengue virus antigens, and pharmaceutical compositions comprising dengue virus antigen expression vectors.
Description
DENGUE VIRUS ANTIGENS AND TREATMENT OF DENGUE FEVER.
The present invention relates to monoclonal antibodies and antigen binding fragments specific for and recognising antigenic epitopes, including synthetic peptides, capable of eliciting antibodies which protect against infection by dengue viruses as well as the said antigenic epitopes and their use for vaccine purposes. The present invention also relates to recombinant vaccinia viruses and for example such which are derived from the Modified vaccinia virus Ankara (MVA) encoding for and capable of producing dengue virus antigens or antigenic epitopes recognised by said monoclonal antibodies, and the use of such recombinant viruses as vaccine. In addition the invention also relates to pharmaceutical compositions comprising the above monoclonal antibodies, pharmaceutical compositions comprising dengue virus antigens, and pharmaceutical compositions comprising expression vectors 'synthesising dengue virus antigen.
BACKGROUND OF THE INVENTION
Dengue viruses are divided into four antigenically related serotypes, called dengue virus serotypes 1, 2, 3, and 4. Complete or partial nucleotide sequences of the dengue 1, 2, 3, and 4 type virus have been published (Chamber, T.J., Hahn, C.S., Galler, R. And Rice, CM. 1990. Annu. Rev. Microbiol., 44, 649, Zhao et al., 1986, Virology 155, 77-88).
Dengue virus, with its four serotypes Den-1 to Den-4, is the most important member of the Flavivirus genus with respect to infections of humans producing diseases that range from flu-like symptoms to severe or fatal illness, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue outbreaks continue to be a major public health problem in densely populated areas of the tropical and subtropical regions, where mosquito vectors are abundant. Therefore, there is a substantial need for the development of prophylactic vaccines. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of immune response. Previous efforts to prepare live candidate dengue vaccines were mainly based on classical attenuation of dengue virus by serial passage in animals or in cultured cells of non-natural hosts. However, this approach has not been consistently successful in producing attenuated vaccine strains. Available
data indicate that recovery and protective immunity after dengue virus infection are correlated to the development of high titres of virus neutralising antibodies. However, this immunity is homotypic mediating resistance to the same virus serotype only. Moreover, individuals immune to one dengue virus serotype may be even at higher risk of developing severe dengue illness if reinfected with another serotype, a phenomenon described as immune enhancement or antibody mediated enhancement. To overcome these problems an ideal vaccine should therefore induce solid immunity against all four dengue virus serotypes without danger of potentiating more severe disease due to the phenomenon of immune enhancement.
Knowledge about the molecular biology of flaviviruses rapidly increased during the last decade, and led to the application of recombinant techniques for the production of new vaccine candidates. These approaches have included E. coli fusion proteins, baculo virus produced recombinant proteins, and live recombinant vaccinia viruses. Notably, the vaccinia approaches have given promising results. In mice, total protection against lethal challenge with dengue virus has been achieved after immunisation with recombinant vaccinia viruses expressing structural and/ or non-structural genes of dengue virus.
The flavivirus genome consists of a single-stranded positive sense RNA molecule. The single encoded open reading frame (ORF) is translated into a polypeptide which is cleaved co- and post-translationally into at least 11 proteins. The order of proteins encoded in the ORF is 5'-C-preM(M)-E-NSl- NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' (Venugopal, K., and Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
Previous results from vaccine trials in animal models indicated that immune responses to dengue structural preM and E proteins, or non-structural NS1 proteins were fully protective against a lethal challenge with homotypic dengue virus (Zhao et al. 1987 J. Virol. 61:4019; Bray et al. 1989 J. Virol. 63:2853; Falgout et al. 1990 J. Virol. 64:4356; Fonseca et al. 1994 Vaccine 12:279; Srivastava et al. 1995 Vaccine 13:1251).
The preM protein (18-19 kDa) is a precursor of the structural protein M, which is formed by cleavage and removal of the N-terminal (pre) segment, by a process presumed to be linked to maturation of the envelope glycoprotein and the development of virus infectivity.
The E glycoprotein (53-54 kDa) is an outer structural protein of the dengue virus. It exhibits a number of biological activities including receptor binding and membrane fusion, and is the target for neutralising antibodies and T-helper cells. The E protein is a typical membrane glycoprotein with a C-terminal that spans the membrane.
The non-structural protein NSl (39-41 kDa) is also modified by glycosylation. It is derived from the ORF by N-terminal signalase cleavage and C-terminal cleavage involving a protease. NSl may be involved in the assembly and release of virions. It is found on the cell surface and in the culture medium of infected cells. During the course of infection, NSl protein evokes a strong antibody response which protects the host against challenge with flaviviruses, presumably through a complement mediated pathway, although recently it has been suggested that other mechanisms such as antibody-dependent cell cytotoxicity may also be responsible (Venugopal, K., & Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
A successful vaccine against the prototype yellow fever virus (YFV) has long been in use, as has been the less celebrated vaccines against tick borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV). These successes are unlikely to be easily reproduced for the dengue viruses which are the causative agents for a two important disease syndromes - classical dengue fever (DF) and dengue haemorrhagic fever/ dengue shock syndrome (DHF/DSS). This is because DHF/DSS has been shown to be associated with secondary immune responses to heterologous dengue virus serotypes which may be due to antibody dependent enhancement (ADE) of virus replication in host monocytes via an Fc receptor mediated pathway. The ADE phenomenon has been shown to be due to subneutralising concentrations of antibodies to the envelope glycoprotein (E). For example in the presence of subneutralising concentrations of antibody bound to virus, Fc receptors or C3 complement receptors can mediate attachment and uptake in the mechanism termed antibody-dependent enhancement (ADE).
In the search for a safe and effective vaccine against DHF/DSS, it became necessary to address the question of the avoidance of ADE, and it was thus a welcome relief when Schlesinger's group reported that passive as well as active immunisation of mice against NSl of Yellow Fever Virus (YFV) conferred solid protection against challenge with YFV (J Immunol, 135:2805-9, 1985; J Virol,
60:1153-55, 1986), a finding that was later confirmed for dengue virus NSl as well (J Gen Virol, 68:853-57, 1987; J Gen Virol, 69:2102-7, 1988).
Since these early experiments however, flavivirus NSl produced as various recombinant proteins in several different expression systems has failed to live up to the early promise of solid protection. Results have been conflicting and although all recombinants generated have elicited a humoral response to NSl, not all have been successful at protection in challenge experiments, with results ranging from solid protection through partial protection through delayed death and no protection at all.
Our studies into the role of antibodies to NSl in patients with DF and DHF/DSS carried out in collaboration with Suchitra Nimmannitya of Children's Hospital, Bangkok, Thailand, show that there is a differential antibody response of DF and DHF/DSS patients to different forms of dengue virus NSl. It has recently been shown that the NSl glycoprotein exists in the natural state as a dimer which on heating degrades into the 46 kDa monomeric form which has been recognised in conventional studies. Our data suggests that the vast majority of patients with DHF (Grades I and II) and DSS (Grades III and IV) show a dominance of antibodies to the dimer form of NSl while those with DF are more likely to have antibodies to both NSl dimer and monomer. The absence of antibodies to NSl monomer in most patients with more severe dengue illness may point to a role for NSl monomer in protection. This, however, appears to be untrue as we have data on the kinetics of appearance of antibodies to NSl dimer and monomer and find that antibodies to NSl monomer do appear early in the infection, may disappear during the period of crisis and then reappear in convalescence. The inverse relationship of antibodies to NSl monomer to disease severity is a critical factor to study in relation to vaccine design. There is a possibility that the clearance of antibodies to NSl monomer may be associated with the marked but transient thrombocytopenia and hypovolemia often observed at the same time. These differences in response to NSl dimer and monomer in DF and DHF/DSS patients may in fact be the key to the conflicting results obtained in the challenge experiments performed after immunisation with recombinant proteins. Not all these studies have addressed the question of dimerization of the recombinant proteins generated, but on examination of the few studies which have addressed this question, it is clear that those recombinant proteins which are expressed primarily as dimers are more convincingly protective unlike those which are primarily expressed as monomers or unstable dimers.
There is still the need for the development of a safe and an effective vaccine with a major goal in the prevention, and perhaps the treatment, of DF and DHF/DSS.
As mentioned above only approaches using recombinant vaccinia virus have so far given promising results. However, occurrence of rare adverse reactions to smallpox vaccination and the increased susceptibility of immunodeficient individuals has made further attenuation and improved safety a priority for human vaccines based on recombinant vaccinia virus.
Modified vaccinia virus Ankara (MVA), is a host range restricted and highly attenuated vaccinia virus strain, which is unable to multiply in human and most other mammalian cell lines tested. But since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses may be used as exceptionally safe and efficient expression vectors.
The modified vaccinia virus Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H: {1975] Infection 3, 6-14; Swiss Patent No. 568, 392). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures de Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721. The MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions of genomic DNA compared with the wild type CVA (deletion I, II, III, IV, V, and VI ) totalling 31,000 base pairs have been identified (Meyer, H., Sutter, G. and Mayr A. [1991] J. Gen. Virol. 72, 1031-1038). MVA is further distinguished by its great attenuation, that is to say by diminished virulence or infectivity while maintaining good immunogenicity. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al, Dtsch. med. Wschr. 99, 2386-2392 [1974]). During these studies in over 120,000 humans, including high risk patients, no side effects were associated with the use of MVA vaccine. MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virio s. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in
non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851). Recently, noλ'el vaccinia vector systems were established on the basis of MVA, having foreign DNA sequences inserted at the site of deletion III within the MVA genome or within the tk gene ( Sutter, G. and Moss, B. [1995] Dev. Biol. Stand. Basel, Karger 84, 195-200 and US patent 5.185.146).
According to the present invention an effective prevention and therapy of DF, DHF and DSS has been made possible by the identification of dengue virus neutralising monoclonal antibodies which does not cause immune enhancement or is involved in antibody dependent enhancement, and by using one such monoclonal antibody, an antigenic discontinuous epitope in the dengue virus envelope protein which elicits neutralising antibodies without being involved in immune enhancement or antibody dependent enhancement has been identified. Further using this monoclonal antibody to select peptides from a random peptide library, peptides which may be used to elicit dengue neutralising antibodies and which do not cause immune enhancement or is envolved in antibody enhancement have been identified. Further, according to the present invention, an antigen expression system for the in vivo or ex vivo production of a therapeutic agent against DF, DHF or DSS has been prepared.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide monoclonal antibodies or antigen binding fragments that specifically bind to and identify dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which are not involved in immune enhancement.
Another object of the present invention is to provide monoclonal antibodies or antigen binding fragments that specifically bind to and identify dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which are not involved in immune enhancement for development of a treatment of and prevention of dengue infection, dengue haemorrhagic fever and dengue shock syndrome.
Still another object of the present invention is to provide antigenic epitopes and antigens which are not involved in immune enhancement and which are useful for vaccination against dengue virus.
A still further object of the present invention is to provide recombinant vaccinia virus based expression systems expressing dengue virus antigens or antigenic epitopes to provide an efficient and safe dengue virus vaccine for animals and humans.
Still one other object of the present invention is to provide heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same.
SUMMARY OF THE INVENTION
The present invention thus, inter alia, comprises the following, alone or in combination:
Dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same;
proteins, polypeptides, peptides and synthetic peptides including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any other amino acid sequence or functional analogues or other equivalents thereof which will elicit anti-dengue antibodies not able to effect immune enhancement or antibody dependent enhancement;
a monoclonal antibody that specifically binds to and identifies dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a monoclonal antibody as above, which is a monoclonal antibody of subclass IgG recognising proteins, polypeptides or peptides including the amino acid
sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a monoclonal antibody as above, which is a monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a monoclonal antibody as above, which is a mouse monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS,APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a DNA construct encoding a dengue virus antigen as above, whereby the antigen is under the transcriptional control of a T7 RNA polymerase promoter;
a vaccine comprising as a first component a recombinant MVA carrying and capable of expressing T7 RNA polymerase and as further components one or more recombinant DNA vectors each carrying at least one dengue virus antigen or synthetic antigen not being involved in immune enhancement or antibody dependent enhancement and being recognised by a dengue virus neutralising antibody when the said antigen or synthetic antigen is under transcriptional control of a T7 RNA polymerase promoter;
a method for the treatment or prevention of a dengue virus infection comprising inoculating a living animal body, including a human, in need thereof with the first and further components of a vaccine, as above, either simultaneously or with a timelag but using the same inoculation site;
a recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens not able to effect immune enhancement or antibody dependent enhancement;
a recombinant MVA as above, containing and capable of expressing DNA sequences encoding antigens from any of the four dengue virus serotypes (type 1, 2, 3 and 4) not able to effect immune enhancement or antibody dependent enhancement;
A recombinant MVA as anyone above wherein the dengue virus antigen includes the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional analogue or other equivalent thereof which will elicit neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a recombinant MVA as above, containing and capable of expressing DNA sequences encoding peptides, polypeptides or proteins being' recognised by monoclonal antibodies specific to the peptides, polypeptides and/ or proteins including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional or other equivalent thereof which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
a recombinant MVA as above, wherein the DNA sequences encoding antigen is under transcriptional control of a vaccinia specific promoter selected from the group of vaccinia virus early/ late promoter P7.5, vaccinia virus synthetic promoter or any other equivalent promoter;
a vaccine containing at least one recombinant MVA as anyone above and a pharmaceutically acceptable carrier or diluent; or
a method for the treatment or prevention of dengue virus infection comprising administering to a living animal body, including a human, in need thereof a therapeutically effective amount of a recombinant MVA as anyone above, or a vaccine as above.
THE PRESENT INVENTION
Monoclonal antibodies generated to be specifically directed against Dengue virus polypeptides are essential to identify and to target Dengue virus-specific
epitopes which enables the rational design of novel prophylactic, therapeutic, and diagnostic tools for the control of human Dengue virus infection.
According to the invention, using panels of dengue virus specific neutralising monoclonal antibodies, the locations in the envelope glycoprotein of dengue virus of a series of protective epitopes are identified. In addition, such neutralising antibodies are screened for immune enhancing properties in order to determine that even at sub-neutralising concentrations the antibodies do not cause antibody dependent enhancement.
In one embodiment of the present invention, a specific mouse monoclonal antibody identified as MASl which specifically recognises peptides, polypeptides and proteins which contain a sequence functioning as a neutralising but non immune enJiancing epitope for the elicitation of protective immune responses against dengue virus infection is provided.
The monoclonal antibody MASl can be prepared by anyone skilled in the art and as described generally by Kohler, G. & Milstein, C (1975, Nature, 256: 495-497) and Harlow, E. & Lane, D. (1988, Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratories). The monoclonal antibody MASl according to one preferred embodiment of the present invention, is a mouse monoclonal antibody of subclass IgGl which has the property of being able to neutralise dengue virus without causing antibody dependent enhancement in Fc receptor bearing cells at any dilution tested. An epitope defined by this monoclonal antibody MASl has the advantage of being an epitope which is neutralizable but not enhanceable and therefore represents an epitope which would be safe and efficacious as a vaccine preparation, being able to elicit protective antibodies which will not cause immune enhancement at any concentration.
According to the invention, neutralizing monoclonal antibodies which are not immune enhancing are used in the screening of different bacteriophage display libraries, peptide libraries etc. to identify peptide sequences recognised by the said non-immune enhancing dengue virus neutralising monoclonal antibodies.
The said specific monoclonal antibody MASl has been found to recognise peptides, polypeptides or proteins which contain a sequence which functions as a protective but not enhancing epitope for the elicitation of a protective immune response against dengue virus infection. Such sequences may be derived from
the authentic dengue virus proteins or glycoproteins themselves or from synthetic peptides or other mimetic peptides or polypeptides, including but not limited to anti-idiotypic antibodies and their derivatives as well as DNA sequences encoding any of the above-mentioned molecules.
The monoclonal antibody MASl has been found to recognise polypeptides including the amino acids sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL.
The monoclonal antibody MASl is further characterised as being a mouse monoclonal antibody within the IgGl family recognising the amino acid sequence LPWYNHS and furthermore by the fact it recognises a discontinuous antigenic epitope within the E gene of dengue 2.
Further, according to the present invention, DNA sequences (genes) which code for dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies, and especially by the MASl, capable of eliciting immune responses to dengue virus are thus provided for the treatment of and prevention of dengue virus infection. Such sequences are optionally introduced, with the aid of DNA recombination techniques, into the genome of viral expression systems including for example the MVA vector. When the DNA sequence encoding the dengue antigen or antigenic epitope is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, e.g. one of the above mentioned deletions, the newly produced recombinant MVA will be infectious, that is to say able to infect foreign cells and it will express the integrated DNA sequence. The recombinant viruses and the recombinant MVA according to the invention will be useful as extremely safe live vaccines for the treatment or prophylactics of dengue infection.
The recombinant MVA vaccinia viruses can be prepared as set out hereinafter.
For homologous recombination of the wild type MVA and a heterologous nucleotide acid sequence in a virus infected cell a DNA-construct containing sequences flanking the left and the right side of a naturally occurring deletion, e.g. deletion II or III, within the MVA genome (Altenburger, W., Suter, C.P. and Altenburger J. (1989) Arch. Virol. 105, 15-27) was constructed.
A DNA-construct which contains a DNA-sequence which codes for a dengue antigen or antigenic epitope flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion II or III, within the MVA genome, is introduced into cells infected with MVA, to allow homologous recombination. The DNA-construct to be inserted can be linear or circular. A circular DNA is preferred, especially a plasmid.
The DNA sequence encoding antigenic epitopes is inserted between the sequences flanking the naturally occurring deletion.
For the expression of a DNA sequence encoding antigenic epitopes, it is necessary for regulatory sequences, which are required for the transcription of the DNA sequence, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198, 328, those of the 7.5 kDa gene (EP-A-110, 385), the vaccinia specific synthetic promoter sP (Sutter et al., 1994, Vaccine 12, 1032-1040) or those of the bacteriophage T7 Polymerase.
The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al., Virol. 52, 456-467 [1973]; Wigler et al., Cell 777-785 [1979] by means of electroporation (Neumann et al., EMBO J. 1, 841-845 [1982]), by micro-injection (Graessmann et al., Meth. Enzymology 101, 482-492 (1983)), by means of liposomes (Straubinger et al, Methods in Enzymology 101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 11, 2163-2167 (1980)) or by other methods known to those skilled in the art. Transfection by means of calcium phosphate precipitation is preferred.
Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker (compare Nakano et al., Proc. Natl. Acad. Sci. USA 79, 1593-1596 [1982], Franke et al., Mol. Cell. Biol. 1918-1924 [1985], Chakrabarti et al., Mol. Cell. Biol. 3403-3409 [1985], Fathi et al., Virology 97-105 [1986]).
The present invention therefore also relates to recombinant MVA which contains at least one DNA sequence which codes for dengue virus antigen or antigenic
epitopes and relates further to vaccines containing such viruses in a physiologically acceptable form.
The recombinant MVA according to the invention contain and are capable of expressing one or more DNA sequences encoding dengue virus antigens or antigenic epitopes capable of eliciting a protective immune response against dengue viruses.
In a preferred embodiment according to the invention the dengue virus antigen includes the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
The invention therefore in a preferred embodiment relates to a viral expression vector, and preferably a pox virus based expression system and especially a MVA based expression system coding for peptides, polypeptides or proteins including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL including for example the following synthetic expression sequence containing one start codon and two stop codons for translation: 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'-CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3'
The invention also relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding dengue virus antigens.
In one such embodiment, a vaccine according to the invention comprises a recombinant MVA encoding dengue virus type 1 antigen; recombinant MVA encoding dengue virus type 2 antigen; recombinant MVA encoding dengue virus type 3 antigen; recombinant MVA encoding dengue virus type 4 antigen; and/ or includes the amino acid sequence LPWYNHS APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
The invention further relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding peptides or polypeptides comprising antigenic epitopes recognised by monoclonal antibody MASl which elicit protective immune responses against dengue virus infection.
In one embodiment therefore, the vaccine according to the invention comprises a peptide, polypeptide or protein which contains an epitope recognised by the said monoclonal antibody MASl. The epitope can exist within an amino acid sequence derived from random peptide libraries and identified by the said monoclonal antibody MASl, or the epitope can exist within an anti-idiotypic antibody generated against said monoclonal antibody MASl, or can exist within polypeptides and proteins encoded by the envelope genes of dengue viruses and includes but is not limited to nucleic acid sequences which encode for the epitope described by monoclonal antibody MASl.
In another embodiment a DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter were provided.
In still another embodiment of the invention recombinant MVA encoding T7 RNA polymerase (MVA-T7pol) is used in combination with DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter.
According to this embodiment, a coding sequence of a given dengue antigen capable of binding to a monoclonal antibody not being involve in immune enhancement is cloned under control of a T7 RNA polymerase promoter preferably in a plasmid vector and the resulting DNA construct is amplified and purified using standard laboratory procedures. Secondly, the vector DNA is inoculated simultaneously or with appropriate timelags together with MVA- T7pol. An appropriate timelag allows a cell to take up the inoculated DNA vector, before MVA infection takes place. The mode of administration, the dose and number of administration can be optimised by one skilled in the art. At the site of inoculation the recombinant gene of interest is expressed transiently in cells containing both the vector DNA and MVA-T7pol and the corresponding antigen is presented to the host immune system stimulating an antigen-specific immune response.
The recombinant MVA according to the invention can also carry a marker gene. Said marker genes are preferably selected from the group consisting of marker genes which codes for proteins such as β-galactosidase, neomycin, alcohol dehydrogenase, luciferase, puromycin, hypoxanthine phosphoribosyl transferase
(HPRT), hygromycin, and green or blue fluorescent proteins. Such a marker gene can either facilitate the purification of recombinant MVA or be used as a independent antigenic marker to control status of immune response after a antigen specific vaccination.
The invention also relates to methods for the preparation of such recombinant MVA and vaccines, and to the use of these vaccines for the prophylactics or treatment of infections caused by dengue virus.
For the preparation of vaccines, the recombinant MVA according to the invention are converted into a physiologically acceptable form. This can be done based on the experience in the preparation of MVA vaccines used for vaccination against smallpox (as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). Typically, about 106-108 particles of the recombinant MVA are freeze-dried in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1 % human albumin in an ampoule, preferably a glass ampoule. The lyophilisate can contain extenders (such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such as antioxidants, stabilisers, etc.) suitable for parenteral administration. The glass ampoule is then sealed and can be stored, preferably at temperatures below -20°C, for several months.
For vaccination or therapy the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline, and administered either parenterally, for example by intramuscular inoculation or locally. Vaccines or therapeutics according to the invention are preferably injected intramuscularly (Mayr, A. et al. [1978] Zbl. Bakt. Hyg., I. Abt. Orig. B 167, 375-390). The mode of administration, the dose and the number of administrations can be optimised by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain appropriate immune responses against the foreign antigen.
The detailed examples which follow are intended to contribute to a better understanding of the present invention. However, the examples are not intended to confine the invention to the subject-matter of the examples. Rather, the invention may be worked according to numerous equivalent or similar procedures all being well known in the art and all of such equivalent or similar procedures to obtain and effect the steps of the present invention, will be appreciated as such by any person of average skill in the art, and should be
considered part of and comprised by the present invention and application and the invention is therefore only to be limited by the full scope of the appended claims. Also it will be appreciated by the person of an average skill in the art, that the MVA vector used herein only constitute an example of an efficient gene expression system and the said MVA vector may be conveniently substituted or exchanged for any suitable gene expression system.
EXAMPLE 1 Growing of dengue 2 virus
A continuous mosquito cell line, C6/36 was grown to confluency in a 25 mm2 tissue culture flask in Leibovitz 15 medium supplemented with 3% fetal calf serum (FCS), 10% tryptose phosphate broth, penicillin (100 units/ ml), streptomycin (100 mg/ml). The medium was then replaced with 4.5 ml of maintenance medium (Leibovitz 15 medium supplemented with 1% FCS, 10% tryptose phosphate broth, and antibiotics in the concentrations described above. Half a ml of Dengue infectious virus stock culture supernatant was added and the flask was rocked gently at room temperature overnight. The flask was then maintained in a stationary position in an incubator set at 28°C for 4 to 5 days until some signs of a cytopathic effect were seen. The medium was then changed to 5 ml of fresh maintenance medium and the cells were incubated at 28°C until syncytia formation occured. When the monolayer was almost completely fused, the supernatant was harvested for infectious virus. The supernatant was clarified by centrif ugation for 10 minutes at 4 °C at 5000 rpm in a Beckman Avanti J25 using a JS7.5 rotor. The clarified supernatant was the Dengue infectious virus stock and was used for seeding new cultures as well as for all Dengue virus plaque assays including neutralization assays and immune enhancement assays.
EXAMPLE 2 Plaque Titration of Dengue 2 Virus
The Dengue infectious virus stock was titrated in a plaque assay in the following manner. A confluent monolayer of PS Clone D cells were trypsinized and resuspended at 2 x 105 cells/ ml maintenance medium (composition as described in Example 1) and 0.5 ml was dispensed in each well of a 24 well tissue culture cluster plate. Ten fold serial dilutions of Dengue infectious virus stock were prepared in maintenance medium and 100 μl of these dilutions or maintenance medium was added in duplicate to each well containing PS Clone D cells in
suspension. The plate was gently tapped to mix the contents of each well and the plate then was incubated at 37 °C for 4 hours until the cells had adhered to the plate. The cells were then overlaid with 0.6 ml of maintenance medium containing 1.5% carboxymethylcellulose, and incubated at 37 °C for 4 to 5 days until plaques were visible under an inverted microscope. The medium was then tipped out into a container containing 5% chlorox and the monolayers gently washed with phosphate buffered saline, pH 7.4. The monolayers were then stained for 15 minutes with naphthalene black and washed with water. The plates were allowed to airdry and the plaques counted.
EXAMPLE 3 Assay for Neutralisation of Dengue 2 Virus
A dilution of Dengue virus infectious stock which would give approximately 30- 40 plaques per well based on the above titration was then selected and prepared by dilution into maintenance medium. The dilution used in this example was 1/500. Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in four fold serial dilutions in maintenance medium. 100 μl of undiluted hybridoma supernatant or dilutions were dispensed into 24 well tissue cluster plates in triplicate. Control supernatant from hybridomas secreting irrelevant monoclonal antibody (for example to Hepatitis B virus) was also dispensed in triplicate and these wells containing monoclonal antibody or control were then seeded with 100 μl of virus dilution prepared previously.
The plate was tapped gently to mix the contents and incubated at 37 °C for one hour.
A confluent monolayer of PS Clone D cells was resuspended at 3 x 105 cells/ ml maintenance medium as described above and dispensed at 0.3 ml per well
containing the virus-antibody incubation mixtures or controls. Cells were also added to triplicate wells containing no incubation mixtures. The plate was incubated at 37 °C for 4 hours until the cells had adhered to the plastic and 0.5 ml of maintenance medium containing 1.5% carboxymethylcellulose was overlayed. The plate was incubated at 37 °C for 4 to 5 days until plaques were visible using an inverted microscope. The monolayers were then washed and stained with naphthlene black as described above and plaques counted. Neutralization of Dengue virus was deemed to have occured when the mean number of plaques in wells containing antibody was at least 50% less than in the control wells containing irrelevant antibody or no antibody.
EXAMPLE 4 Assay for Immune Enhancement of Dengue 2 Virus
A dilution of Dengue virus infectious stock which would give approximately 30- 40 plaques per 100 μl based on the above titration was then selected and prepared by dilution into maintenance medium. Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in ten fold serial dilutions in maintenance medium. Similar dilutions were prepared of irrelevant hybridoma supernatants as controls. Duplicate wells containing 100 μl of maintenance medium was used as the negative control. 100 μl of undiluted hybridoma supernatant or dilutions were dispensed into 5 ml round bottomed cell culture tubes. 100 μl of the Dengue virus dilution was added to each tube. The mixture was gently mixed and incubated at 37 °C for 1 hour.
A confluent monolayer of P388D1 cells were resuspended at 5 x 10s cells/ml into Leibovitz 15 maintenance medium and dispensed at 0.5 ml per tube. The tubes were incubated at 37 °C overnight. The cells were then washed by centrifugation for 10 minutes at 800 rpm at room temperature in a Beckman Avanti J25 using a JS7.5 rotor. The supernatant was discarded and 1 ml of fresh maintenance
medium was added per tube. The tubes were centrifuged again as described and replenished with 1 ml of fresh maintenance medium. The cells were resuspended and incubated at 37 °C for 5 days. On the 5th day, cells were pelleted by centrifugation for 10 minutes at 1000 rpm at 4 °C in a Beckman Avanti J25 using a JS7.5 rotor, and the supernatants were titrated on PS Clone D cells as described in Example 2.
Immune enhancement or antibody dependent enhancement is deemed to have occured when the number of plaque forming units (pfu) per ml of culture was double or greater than that of the negative controls containing no antibody.
EXAMPLE 5 Assay for Non Immune enhancing Properties of Neutralising Antibodies
Ten fold serial dilutions of the monoclonal antibodies (MAS 1, MAS 2, MAS 3) to be tested were prepared in maintenance medium and used simultaneously in an assay for neutralising antibodies and in an assay for immune enhancement as essentially described in Examples 3 and 4. The antibody was considered to be neutralizing and non-enhancing only if there was no enhancing activity as defined in example 4 even at dilutions of monoclonal antibody when neutralizing activity was no longer seen.
EXAMPLE 6 MASl Preparation and Purification
A Balb/c mouse was immunised through the ip route with Dengue antigen prepared as follows. C6/36 mosquito cell monolayers wre inoculated with
Dengue 2 virus as described in Example 1. When massive syncytia formation had occured, the monolayer was washed once with phosphate buffered saline and lysed with 2 ml of isotonic buffer (1.5 mM magnesium chloride, 5 mM potassium chloride, 10 mM HEPES) containing 1% Nonidet P 40 after gentle rocking at room temperature for 10 minutes, the lysate was harvested and spun for 5 minutes in a microfuge. The supernatant was loaded into a single well comb of a 12% SDS PAGE gel (Biorad Mini Protean II) and run at 100 Volts until the dye front reached the end of the gel. Bands corresponding to Dengue Envelope glycoprotein and pre Membrane protein were excised from the gel, placed in a dialysis bag, electro-eluted in a minimal volume and used as the immunogen.
The immunised mouse was boosted through the ip route after two weeks with 0.5 ml infectious dengue virus culture supernatant prepared as described in Example 1 and left to rest for a week before the spleen was removed and spleen cells harvested. Cell fusion was attained by the method as essentially described by Kohler & Milstein, Nature 256, 495-497 (1975) and Kohler et al., Eur.J.Immunol. 6, 292-295 (1976) and clones were screened for anti Dengue activity by a dot enzyme immunoassay (Cardosa, M.J. & Tio, P.H., 1991. Dot enzyme immunoassay: Alternative diagnostic aid for dengue fever and dengue haemorrhagic fever, Bulletin of the World Health Organisation, 69, 741-745.) and by a mouse immunoglobulin capture ELISA using 96 well ELISA plates coated with anti-mouse immunoglobulin incubated with tissue culture supernatants overnight at 4 °C After washing with phosphate buffered saline containing 0.05% Tween 20, the immunoglobulin bound was identified using dengue virus prepared as described in Example 1 followed by an HRP conjugated anti dengue convalescent human serum (Innis, B.L. Nisalak, A., Nimmannitya, S., et al., 1989. An enzyme-linked immunosorbent assay to characterise dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 40:418- 27) Positive clones were expanded and recloned by limiting dilution twice.
The hybridoma which yielded MAS 1 was grown to large quantities in 75 mm2 tissue culture flasks and supernatants harvested every 2 or 3 days. The supernatant was clarified by centrifugation at 3000 rpm at 4 °C for 15 minutes in
a Beckman Avanti J25 using a JS7.5 rotor, and the clarified supernatant was pushed through a 0.22 micron filter before purification through a HiTrap Protein G column (Pharmacia Biotech, Product Code No. 17-040-01) according to the manufacturer's instructions. Fractions containing protein were pooled and checked for specific activity in a dot enzyme immunoassay (Cardosa & Tio, 1991).
EXAMPLE 7 Screening of Bacteriophage Display Library
The affinity purified monoclonal antibody MAS 1 was used in panning experiments to select peptide ligands from a phage display peptide library purchased from New England Biolabs (Catalog number 8100). The experiment was performed according to the instructions of the manufacturer using MAS 1 as the panning antibody bound to the plastic surface of an microtitre ELISA plate. Three pannings were performed and phage selected were purified and DNA sequences of the heptapeptide displayed in each clone was determined. The DNA sequence was translated and concensus peptides were determined using the software Lasergene. The following are the concensus sequences obtaine hy comparison of amino acid sequences.
ALIGNMENT USING TOTUN HEIN METHOD
First line indicates the concensus sequence. Clones (characterised by a number) from all three pannings are listed in the first row.
ALIGλffiNTUSING CLUSTALMETHOD
First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings only are listed in the first row.
ALIGMENT USING CLUSTALMETHOD
First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
ALIGMENT USING TOTUN HEIN METHOD
First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
ALIGMENTUSING CLUSTALMETHOD
First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings are listed in the first row.
EXAMPLE 8 Growing of the MVA virus
The MVA virus is a highly attenuated vaccinia virus derived from the vaccinia virus strain Ankara (CVA) by long-term serial passages on primary chicken embryo fibroblast (CEF) cultures. For a general review of the history of the production, the properties and the use of MVA strain, reference may be made to the summary published by Mayr et al. in Infection 3, 6-14 [1975]. Due to the attenuation in CEF, the MVA virus replicates to high titers in this avain host cell. In mammalian cells, however, MVA is severely growth restricted, and typical plaque formation by the virus is not detectable. Therefore, MVA virus was grown on CEF cells. To prepare CEF cells, 11-days old embryos were isolated
from incubated chicken eggs, the extremities are removed, and the embryos are minced and dissociated in a solution composed of 0.25% trypsin at 37 °C for 20 minutes. The resulting cell suspension was filtered and cells were sedimented by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes, resuspended in 10 volumes of medium A (MEM Eagle, for example obtainable from Life Technologies GmbH, Eggenstein, Germany), and sedimented again by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes. The cell pellet was reconstituted in medium A containing 10% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 mg/ml) and 2 mM glutamine to obtain a cell suspension containing 500 000 cells/ml. CEF cells obtained in this way were spread on cell culture dishes. They were left to grow in medium A in a 6% CO2 atmosphere with 95% humidity at 37°C for 1-2 days, depending on the desired cell density, and were used for infection either directly or after one further cell passage. A detailed description of the preparation of primary cultures can be found in the book by R.I. Freshney, "Culture of animal cell", Alan R. Liss Verlag, New York [1983] Chapter 11, page 99 et seq.
MVA viruses were used for infection as follows. CEF cells were cultured in 175 cm^ cell culture bottles. At 90-100% confluence, the medium was removed and the cells were incubated for one hour with an MVA virus suspension (0.01 infectious units (IU) per cell, 0.02 ml/ cm2) in medium A. Then more medium A was added (0.2 ml/ cm2) and the bottles were incubated at 37°C for 2-3 days (until about 90% of the cells show cytopathogenic effect). Crude virus stocks were prepared by scraping cell monolayers into the medium and pelleting the cell material by centrifugation at 3000 rpm in a Sorvall RC-3B centrifuge at 4°C for 5 minutes. The crude virus preparation was stored at -20°C before further processing (e.g. virus purification).
EXAMPLE9 Cloning of MVA virus
To generate homogeneous stock virus preparations MVA virus obtained from Prof. Anton Mayr was cloned by limiting dilution during three consecutive passages in CEF cultured on 96-well tissue culture plates. The MVA clone F6 was selected and amplified in CEF to obtain working stocks of virus that served as starting material for the generation of recombinant MVA viruses described in this patent application as well as for the generation of recombinant MVA viruses described previously (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89,
10847-10851; Sutter, G., Wyatt, L., Foley, P., Bennink, J. and Moss, B. [1994] Vaccine 12, 1032-1040; Hirsch, V., Fuerst, T., Sutter, G., Carroll, M., Yang, L., Goldstein, S., Piatak, M., Elkins, W., Alvord, G., Montefiori, D., Moss, B. and Lifson, J. [1996] J. Virol. 70, 3741-3752).
EXAMPLElO
Purification of the viruses
The purification steps undertaken to obtain a virus preparation which was as pure as possible and free from components specific to the host cell were similar to those described by (Joklik, Virology 18, 9-18 [1962]). Crude virus stocks which had been stored at -20°C were thawed and suspended once in PBS (10-20 times the volume of the sediment), and the suspension was centrifuged as above. The new sediment was suspended in 10 times the volume of Tris buffer 1 (10 mM Tris-HCl pH 9.0), and the suspension was briefly treated with ultrasound
(Labsonic L, B. Braun Biotech International, Melsungen Germany; 2x 10 seconds at 60 watts and room temperature) in order to further disintegrate cell debris and to liberate the virus particles from the cellular material. The cell nuclei and the larger cell debris were removed in the subsequent brief centrifugation of the suspension (Sorvall GSA rotor obtainable from DuPont Co., D-6353 Bad
Nauheim, FRG; 3 minutes at 3000 rpm and 10°C). The sediment was once again suspended in Tris buffer 1, treated with ultrasound and centrifuged, as described above. The collected supernatants containing the free virus particles were combined and layered over a cushion of 10 ml of 36% sucrose in 10 mM Tris- HCl, pH 9.0, and centrifuged in a Beckman SW 27/ SW 28 rotor for 80 minutes with 13,500 rpm at 4°C The supernatant was discarded, and the sediment containing the virus particles was taken up in 10 ml of 1 mM Tris-HCl, pH 9.0, homogenised by brief treatment with ultrasound (2x 10 seconds at room temperature, apparatus as described above), and applied to a 20-40% sucrose gradient (sucrose in 1 mM Tris-HCl, pH 9.0) for further purification. The gradient was centrifuged in Beckmann SW41 rotor at 13,000 rpm for 50 minutes at 4°C After centrifugation, discrete bands containing virus particles were harvested by pipetting after aspirating volume above band. The obtained sucrose solution was diluted with three volumes PBS and the virus particles were sedimented again by centrifugation (Beckmann SW 27/28, 60 minutes at 13,500 rpm, 4° C). The pellet, which now consisted mostly of pure virus particles, was resuspended in PBS and equilibrated to virus concentrations corresponding on average to l-5x
lθ" IU/ml. The purified virus stock solution was stored at -80°C and used either directly or diluted with PBS for subsequent experiments.
EXAMPLEll Construction of vector plasmids
To allow the generation of recombinant MVA viruses novel vector plasmids were constructed. Insertion of foreign genes into the MVA genome was targeted precisely to the site of the naturally occurring deletion II in the MVA genome. Sequences of MVA DNA flanking the site of a 2500 bp deletion in the Hindlll N fragment of the MVA genome (Altenburger, W., Surer, C.P. and Altenburger, J. [1989], J. Arch. Virol. 105, 15-27) were amplified by PCR and cloned into the multiple cloning site of plasmid pUC18. The primers for the left 600 bp DNA • flank were 5XCAG CAG GGT ACC CTC ATC GTA CAG GACGTT CTC-3' and 5'- CAG CAG CCC GGG TAT TCG ATG ATT ATT TTT AAC AAA ATA ACA-3' (sites for restriction enzymes Kpnl and Smal are underlined). The primers for the right 550 bp DNA flank were 5'-CAG CAG CTG CAG GAA TCA TCC ATT CCA CTG AAT AGC-3' and 5'-CAG CAG GCA TGC CGA CGA ACA AGG AAC TGT AGC AGA-3' (sites for restriction enzymes PstI and SphI are underlined). Between these flanks of MVA DNA inserted in pUC18, the E. coli lacZ gene under control of the vaccinia virus late promoter Pll (prepared by restriction digest from pill LZ, Sutter, G. and Moss, B. [1992] PNAS USA 89, 10847-10851) was inserted, using the BarnHI site, to generate the MVA insertion vector pUCII LZ [Figure 1]. In the following, a 289 bp fragment containing the vaccinia virus early-late promoter P7.5 together with a Smal site for cloning (prepared by restriction digest with EcoRI and Xbal from the plasmid vector pSCll [Chakrabarti et al. 1985, Molecular and Cellular Biology 5, 3403-3409]) was inserted into the Smal site of pUCII LZ to give the MVA vector pUC II LZ P7.5. To construct a vector plasmid that allows isolation of recombinant MVA viruses via transient synthesis of the reporter enzyme b-galactosidase a 330 bp DNA fragment from the 3' end of the E. coli LacZ open reading frame was amplified by PCR (primers were 5'-CAG CAG GTC GAC CCC GAC CGC CTT ACT GCC GCC-3' and 5'-GGG GGG CTG CAG ATG GTA GCG ACC GGC GCT CAG-3') and cloned into the Sail and PstI sites of pUC II LZ P7.5 to obtain the MVA vector pUC II LZdel P7.5. Using the Small site, this vector plasmid can be used to insert DNA sequences encoding a foreign gene under transcriptional control of the vaccinia virus promoter P7.5 into the MVA genome. After the desired recombinant virus has been isolated by screening for expression of β-galactosidase activity further propagation of the
recombinant virus leads to the self-deletion of the reengineered Pll-LacZ expression cassette by homologous recombination.
EXAMPLE12 Construction of recombinant MVA virus expressing a minigene for induction of
Dengue virus-specific immune responses
A 147 bp minigene encoding a polypeptide including the N-terminal amino acids LPWYNHS and containing one start codon and two stop codons for translation was prepared by PCR from cloned DNA of a phage M13 library using synthetic oligonucleotides 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'- CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3' and cloned into the Smal restriction of site of the MVA vector plasmid pUCII LZdel P7.5 to create the plasmid pUCII LZdel P7.5- DENminil carrying the minigene under transcriptional control of the vaccinia virus early/ late promoter P7.5.
The minigene under control of the vaccinia virus early/late promoter P7.5 was inserted into deletion II within the MVA using homologous recombination. CEF cells infected with MVA at a multiplicity of 0.05 TCID50 per cell were transfected with DNA of plasmid pUC II LZdel P7.5-DENmini as described previously (Sutter, G, Wyatt, L., Foley, P., Bennink, J. and Moss, B. [1994] Vaccine 12, 1032-1040). Recombinant MVA viruses containing the Dengue virus cDNA sequence and transiently co-expressing the E. coli LacZ marker gene were selected by consecutive rounds of plaque purification in CEF cells stained with 5- bromo-4-chloro-3-indolyl β-D-galactoside (300 mg/ml). In the following, recombinant MVA viruses containing the E sequence and having deleted the LacZ marker gene were isolated by three additional consecutive rounds of plaque purification screening for non-staining viral foci in CEF cells in the presence of 5-bromo-4-chloro-3-indolyl β-D-galactoside (300 mg/ml). Subsequently, recombinant viruses were amplified by infection of CEF monolayers.
Claims
(1) Dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same.
(2) Proteins, polypeptides, peptides and synthetic peptides including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any other amino acid sequence or functional analogues or other equivalents thereof which will elicit anti-dengue antibodies not able to effect immune enhancement or antibody dependent enhancement.
(3) A monoclonal antibody that specifically binds to and identifies dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(4) A monoclonal antibody according to claim 3, which is a monoclonal antibody of subclass IgG recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(5) A monoclonal antibody according to claim 4, which is a monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(6) A monoclonal antibody according to claim 3, which is a mouse monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or
other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(7) A DNA construct encoding a dengue virus antigen according to anyone of claims 1 to 3, whereby the antigen is under the transcriptional control of a
T7 RNA polymerase promoter.
(8) A vaccine comprising as a first component a recombinant MVA carrying and capable of expressing T7 RNA polymerase and as further components one or more recombinant DNA vectors each carrying at least one dengue virus antigen or synthetic antigen not being involved in immune enhancement or antibody dependent enhancement and being recognised by a dengue virus neutralising antibody when the said antigen or synthetic antigen is under transcriptional control of a T7 RNA polymerase promoter.
(9) A method for the treatment or prevention of a dengue virus infection comprising inoculating a living animal body, including a human, in need thereof with the first and further components of a vaccine according to claim 8 either simultaneously or with a timelag but using the same inoculation site.
(10) A recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens not able to effect immune enhancement or antibody dependent enhancement.
(11) A recombinant MVA according to claim 10 containing and capable of expressing DNA sequences encoding antigens from any of the four dengue virus serotypes (type 1, 2, 3 and 4) not able to effect immune enhancement or antibody dependent enhancement.
(12) A recombinant MVA according to claims 10 to 11 wherein the dengue virus antigen includes the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional analogue or other equivalent thereof which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(13) A recombinant MVA according to claims 10 to 12 containing and capable of expressing DNA sequences encoding peptides, polypeptides or proteins being recognised by monoclonal antibodies specific to the peptides, polypeptides and/ or proteins including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional or other equivalent thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement.
(14) A recombinant MVA according to claims 10 to 13 wherein the DNA sequences encoding antigen is under transcriptional control of a vaccinia specific promoter selected from the group of vaccinia virus early/ late promoter P7.5, vaccinia virus synthetic promoter or any other equivalent promoter.
(15) A vaccine containing at least one recombinant MVA according to anyone of claims 10 to 14 and a pharmaceutically acceptable carrier or diluent.
(16) A method for the treatment or prevention of dengue virus infection comprising administering to a living animal body, including a human, in need thereof a therapeutically effective amount of a recombinant MVA according to claims 10 to 14, or a vaccine according to claim 15.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MY9704411 | 1997-09-23 | ||
| MYPI9704411 | 1997-09-23 | ||
| PCT/EP1998/006009 WO1999015692A2 (en) | 1997-09-23 | 1998-09-21 | Dengue virus antigens and treatment of dengue fever |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1021545A2 true EP1021545A2 (en) | 2000-07-26 |
Family
ID=19749780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP98952614A Withdrawn EP1021545A2 (en) | 1997-09-23 | 1998-09-21 | Dengue virus antigens and treatment of dengue fever |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1021545A2 (en) |
| AU (1) | AU754386B2 (en) |
| ID (1) | ID24941A (en) |
| WO (1) | WO1999015692A2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2345665A3 (en) * | 2001-12-04 | 2012-02-15 | Bavarian Nordic A/S | Flavivirus NS1 subunit vaccine |
| US7473424B2 (en) * | 2003-01-31 | 2009-01-06 | The Scripps Research Institute | Anti-dengue virus antibodies and compositions |
| CU23586A1 (en) | 2005-11-22 | 2010-10-30 | Ct Ingenieria Genetica Biotech | METHODS AND PROTEINS FOR THE PROPHLACTIC AND / OR THERAPEUTIC TREATMENT OF THE FOUR SEROTYPES OF THE DENGUE VIRUS AND OTHER FLAVIVIRUS |
| FR3042121A1 (en) | 2015-10-08 | 2017-04-14 | Jean-Marc Limacher | ANTI-TUMOR COMPOSITION |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY109299A (en) * | 1990-08-15 | 1996-12-31 | Virogenetics Corp | Recombinant pox virus encoding flaviviral structural proteins |
| WO1996040933A1 (en) * | 1995-06-07 | 1996-12-19 | The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services | Infectious dengue 2 virus pdk-53 as quadravalent vaccine |
| MY150893A (en) * | 1996-09-24 | 2014-03-14 | Bavarian Nordic As | Recombinant mva virus expressing dengue virus antigens, and the use thereof in vaccines |
-
1998
- 1998-09-21 EP EP98952614A patent/EP1021545A2/en not_active Withdrawn
- 1998-09-21 AU AU10249/99A patent/AU754386B2/en not_active Ceased
- 1998-09-21 ID IDW20000566A patent/ID24941A/en unknown
- 1998-09-21 WO PCT/EP1998/006009 patent/WO1999015692A2/en not_active Application Discontinuation
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9915692A2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU754386B2 (en) | 2002-11-14 |
| AU1024999A (en) | 1999-04-12 |
| ID24941A (en) | 2000-08-31 |
| WO1999015692A2 (en) | 1999-04-01 |
| WO1999015692A3 (en) | 1999-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6869793B2 (en) | Recombinant MVA virus expressing dengue virus antigens, and the use thereof in vaccines | |
| US8741308B2 (en) | Intergenic regions as insertion sites in the genome of modified vaccinia virus ankara (MVA) | |
| US7198934B2 (en) | Recombinant MVA virus, and the use thereof | |
| US8029800B2 (en) | Recombinant modified vaccinia virus ankara (MVA) comprising human immunodeficiency virus (HIV) genes inserted into one or more intergenic regions (IGRs) | |
| AU754386B2 (en) | Dengue virus antigens and treatment of dengue fever | |
| US20040265324A1 (en) | Recombinant MVA virus expressing dengue virus antigens, and the use thereof in vaccines |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 20000315 |
|
| AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| AX | Request for extension of the european patent |
Free format text: AL PAYMENT 20000315;LT PAYMENT 20000315;LV PAYMENT 20000315;MK PAYMENT 20000315;RO PAYMENT 20000315;SI PAYMENT 20000315 |
|
| 17Q | First examination report despatched |
Effective date: 20030730 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20040525 |