WO1989000196A1 - Virus proteins having reduced o-linked glycosylation - Google Patents
Virus proteins having reduced o-linked glycosylation Download PDFInfo
- Publication number
- WO1989000196A1 WO1989000196A1 PCT/US1988/002021 US8802021W WO8900196A1 WO 1989000196 A1 WO1989000196 A1 WO 1989000196A1 US 8802021 W US8802021 W US 8802021W WO 8900196 A1 WO8900196 A1 WO 8900196A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glycoprotein
- prv
- insect
- pseudorabies virus
- virus
- Prior art date
Links
- 230000004989 O-glycosylation Effects 0.000 title claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 title description 30
- 241000700605 Viruses Species 0.000 title description 23
- 102000004169 proteins and genes Human genes 0.000 title description 11
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 61
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 61
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims abstract description 44
- 241000238631 Hexapoda Species 0.000 claims abstract description 26
- 241000701447 unidentified baculovirus Species 0.000 claims abstract description 19
- 239000013598 vector Substances 0.000 claims abstract description 14
- 230000003612 virological effect Effects 0.000 claims abstract description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 8
- 102000053602 DNA Human genes 0.000 claims abstract description 7
- 108020004511 Recombinant DNA Proteins 0.000 claims abstract description 7
- 101000686824 Enterobacteria phage N4 Virion DNA-directed RNA polymerase Proteins 0.000 claims description 46
- 101000644628 Escherichia phage Mu Tail fiber assembly protein U Proteins 0.000 claims description 44
- 230000013595 glycosylation Effects 0.000 claims description 21
- 238000006206 glycosylation reaction Methods 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 19
- 229960005486 vaccine Drugs 0.000 claims description 16
- 241000256251 Spodoptera frugiperda Species 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 241000255993 Trichoplusia ni Species 0.000 claims description 3
- 108010062697 pseudorabies virus glycoproteins Proteins 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 241000255789 Bombyx mori Species 0.000 claims 1
- 229940031626 subunit vaccine Drugs 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 39
- 239000012634 fragment Substances 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 23
- 241001465754 Metazoa Species 0.000 description 11
- 230000002163 immunogen Effects 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 239000002671 adjuvant Substances 0.000 description 9
- 241000282898 Sus scrofa Species 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 101710182846 Polyhedrin Proteins 0.000 description 5
- 241000282887 Suidae Species 0.000 description 4
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 4
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 229960000187 tissue plasminogen activator Drugs 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- 101150074155 DHFR gene Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 3
- 108010059485 brain synaptic membrane glycoprotein gp 50 Proteins 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- -1 more specifi¬ cally Proteins 0.000 description 3
- 241001529453 unidentified herpesvirus Species 0.000 description 3
- 241001367049 Autographa Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 241001131785 Escherichia coli HB101 Species 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 230000004988 N-glycosylation Effects 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- KQHXBDOEECKORE-UHFFFAOYSA-L beryllium sulfate Chemical compound [Be+2].[O-]S([O-])(=O)=O KQHXBDOEECKORE-UHFFFAOYSA-L 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229960002442 glucosamine Drugs 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229960005030 other vaccine in atc Drugs 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 1
- 208000020154 Acnes Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 206010006542 Bulbar palsy Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101800000342 Glycoprotein C Proteins 0.000 description 1
- 101710138378 Glycoprotein GX Proteins 0.000 description 1
- 239000004166 Lanolin Substances 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 1
- 241000772415 Neovison vison Species 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010037742 Rabies Diseases 0.000 description 1
- 241000711798 Rabies lyssavirus Species 0.000 description 1
- 101900083372 Rabies virus Glycoprotein Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108010046722 Thrombospondin 1 Proteins 0.000 description 1
- 102100036034 Thrombospondin-1 Human genes 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 229960001614 levamisole Drugs 0.000 description 1
- HEHQDWUWJVPREQ-XQJZMFRCSA-N lipid X Chemical compound CCCCCCCCCCC[C@@H](O)CC(=O)N[C@H]1[C@@H](OP(O)(O)=O)O[C@H](CO)[C@@H](O)[C@@H]1OC(=O)C[C@H](O)CCCCCCCCCCC HEHQDWUWJVPREQ-XQJZMFRCSA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940031348 multivalent vaccine Drugs 0.000 description 1
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 201000002241 progressive bulbar palsy Diseases 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000012770 revaccination Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention relates to- viral glycoproteins having reduced 0- linked glycosylation.
- the invention also relates to methods for producing such glycoproteins. More specifically, the invention relates to a pseudorabies virus glycoprotein having reduced 0-linked glycosylation and methods for producing . said glycoprotein with baculovirus vectors in insect cell hosts. These glycoproteins having reduced 0-linked glycosylation are useful for protecting animals against infection by the viruses they are derived from.
- PRV Pseudorabies virus
- PRV infection are variously called infectious Bulbar paralysis, Aujeszky's disease, and mad itch.
- Infections are known in important domestic animals such as swine, cattle, dogs, cats, sheep, rats and mink.
- the host range is very broad and includes most mammals and, experimentally at least, many kinds of birds (for a detailed list of hosts, see D.P. Gustafson, "Pseudo ⁇ rabies", in Diseases of Swine, 5th ed. , A.D. Leman et al., eds. , (1981)).
- the disease is fatal.
- Adult swine and possibly rats are not killed by the disease and are therefore carriers.
- PRV vaccines have been produced by a variety of techniques and vaccination in endemic areas of Europe has been practiced for more than 15 years. Losses have been reduced by vaccination, but vaccina- tion has maintained the virus in the environment. No vaccine has been produced that will prevent infection. Vaccinated animals that are exposed to virulent virus survive the infection and then shed more virulent virus. Vaccinated animals may therefore harbor a latent infection that can flare up again. (See, D.P. Gustafson, supra) .
- PRV is a herpesvirus.
- the herpesviruses generally are among the most complex of animal viruses. Their genomes encode at least 50 virus specific proteins and contain upwards of 150,000 nucleotides. Among the most immunologically reactive proteins of herpesviruses are the glycoproteins found, among other places, in virion membranes and the membranes of infected cells.
- the literature on PRV glycoproteins refers to at least four viral glycoproteins (T. Ben-Porat and A.S. Kaplan, Virology, 41, pp. 265-73 (1970); A.S. Kaplan and T. Ben-Porat, Proc. Natl. Acad. Sci. USA, 66, pp. 799-806 (1970)).
- European patent publication number 0 127 839 and European patent publication number 0 155 476 refer to methods for producing recom ⁇ binant baculovirus expression vectors for expression of selected genes together with such vectors. They do not disclose the use of such vectors to produce viral glycoproteins having less 0-linked glycosylation than the naturally occurring proteins for use in vaccines. Both of these documents are incorporated herein by reference. S. Alexander and J.H. Elder, "Carbohydrate Dramatically Influ ⁇ ences Immune Reactivity of Antisera to Viral Glycoprotein Antigens", Science, 226, pp. 1328-30 (1984) demonstrated that viral glyco ⁇ proteins with N-linked carbohydrate removed by endoglycosidase F had varying antigenic effects.
- Heteroantisera prepared against virus were virtually unreactive toward the viral glycoproteins after N- linked carbohydrate removal.
- the reactivity of monoclonal antibodies prepared to glycosylated antigen in most cases improved, while in a smaller number of cases it stayed the same, or decreased in reactivity when reacted with the antigen without N-linked carbo- hydrate.
- Most antibodies to synthetic peptide sequences also improved in reactivity after N- inked carbohydrate removal.
- a protein expressed in insect cells will be rich in mannose and lacking sialic acid. At this point, the ramifica ⁇ tions of this are not clear and are likely to be of more or less concern depending on the end use of the product.
- proteins expressed in insect cells are frequently smaller than their native counter ⁇ parts. We attribute this to less complex glycosylation and perhaps substantial hydrolysis of the high mannose complex unit [citation omitted] . This has not been directly determined for heterologous gene products of the baculovirus expression system. "With regard to the possible in vivo physiological effects of this glycosylation difference, much work needs to be done. We have no clear-cut case where the glycosylation difference has altered biological activity of a protein but do consider that this will inevitably be observed.”
- M.W. Wathen and L.K. Wathen, J. Virol., 51, pp. 57-62 (1984) refer to a PRV containing a mutation in a viral glycoprotein (g ⁇ 50)
- the mupant " utilizing neutralizing monoclo ⁇ nal antibody directed against gp50 Iff- and a method for selecting, the mupant " utilizing neutralizing monoclo ⁇ nal antibody directed against gp50. Wathen and Wathen also indicate that a monoclonal antibody directed against gp50 is a strong neutral- izer of PRV, with or without the aid of complement, and that polyval ⁇ ent immune serum is highly reactive against gp50, therefore conclud-
- gp50 may be one of the important PRV immunogens.
- monoclonal antibodies that react with the 98,000 MW envelope glycoprotein neutralize PRV infectivity but that monoclonal antibodies directed against some of the other membrane glycoproteins have very little neutralizing
- BamHI 7 fragment of PRV codes for at least three other viral proteins of 65K, 60K, and 40K MW. They do not disclose or suggest a viral glycoprotein having reduced 0-linked glycosylation of the instant invention or the production of similar polypeptides in insect cells with baculovirus vectors.
- European published patent application 0 133 200 refers to a diagnostic antigenic factor to be used together with certain lectin-- bound PRV glycoprotein subunit vaccines to distinguish carriers and noncarriers of PRV.
- G. E. Smith, et al. "Modification and Secretion of Human Interleukin 2 Produced in Insect Cells by a Baculovirus Expression Vector," Proc. Natl. Acad. Sci. U.S.A., 82, pp. 8404-08 (1985) refer to production of IL-2 by inserting its cDNA into the genome of Autographa califomica nuclear polyhedrosis virus adjacent to the polyhedrin promoter and then using this recombinant virus to infect an insect cell host. They indicated that there was no evidence that the recombinant IL-2 produced was glycosylated. They do not disclose or suggest producing viral glycoproteins having a lower amount of 0- linked glycosylation than their naturally occurring homologs for use as vaccines.
- the present invention relates to viral glycoproteins having a lower amount of 0-linked glycosylation than their naturally occurring glycoprotein, more particularly wherein said glycoprotein is a pseudorabies virus glycoprotein, and even more particularly pseudo ⁇ rabies virus gp50.
- the glycoproteins having a lower amount of glycosylation than the natural glycoproteins are produced by an insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein.
- insect cell host referred to above is selected from the group consisting of Spodoptera frugiperda, Bomhyx mori , Trichoplusia ni , and other lepidopteran cell lines.
- Intact worms can also be used as hosts for example, for AcNPV vectors, cabbage loopers or fall armyworms. See Maeda, et al. , supra.
- the glycoprotein having a lower amount of glycosylation than the natural glycoprotein is produced by a Spodop ⁇ tera frugiperda insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein, more specifi ⁇ cally, gp50.
- the glycoprotein is a pseudo ⁇ rabies virus glycoprotein, more specifically, gp50.
- the insect host cell used in the method set forth above is Spodoptera frugiperda. DETAILED DESCRIPTION OF THE INVENTION
- the glycoprotein encoded by the gene was defined as a glycoprot ⁇ ein that reacted with a particular monoclonal antibody. This glycoprotein did not correspond to any of the previously known PRV glycoproteins.
- Wathen and Wathen mapped a mutation resistant to the monoclonal antibody, which, based on experience with herpes simplex virus (e.g., T.C. Holland et al., J. Virol., 52, pp.566-74 (1984)), indicated that they had mapped the location of the structural gene for gp50.
- Wathen and Wathen mapped the gp50 gene to the smaller Sall/BamHI fragment from within the BamHI 7 fragment of PRV. Rea et al, supra, have mapped the PRV glycoprotein gX gene to the same region.
- gp50 gene under control of the polyhedrin promoter in the baculovirus Autographa califomica nuclear polyhedrosis virus (AcNPV) .
- AcNPV Autographa califomica nuclear polyhedrosis virus
- Two forms of the gene were inserted into AcNPV: (1) the intact gene encoding the complete gp50 amino acid sequence to produce a recombinant virus designated AcNPV-gp50, and (2) the gene with the base pairs encoding the anchor sequence of gp50 deleted to produce a recombinant virus designated AcNPV-gp50T.
- AcNPV-gp50-infected cells produced a cellular form of gp50 and AcNPV-gp50T-infected cells produced a truncated form of g ⁇ 50 which is secreted into the medium. Both of these forms of gp50 produced in insect cells had a sig ⁇ nificantly lower molecular weight than the same proteins produced in mammalian cells because of reduced 0-linked glycosylation.
- the gp50 produced in insect cells is, however, labeled with • ' ⁇ ⁇ 'C-glucosamine. Since the gp50 sequence contains no N-linked glycosylation sites (E.A.
- Insect cells infected with AcNPV- p50 were used to immunize mice. After a series of three immunizations with 10° infected cells, most of the mice survived a challenge with virulent PRV. -
- the gene encoding gp50 mapped to the BamHI 7 fragment of the PRV DNA.
- the BamHI 7 fragment from PRV can be derived from plasmid pPRXhl (also known as pUC1129) and fragments convenient for DNA sequence analysis can be derived by standard subcloning procedures.
- Plasmid pUC1129 is available from E. coli HB101, NRRL B-15772. This culture is available from the permanent collection of the Northern Regional Research Center Fermentation Laboratory (NRRL), U.S. Department of Agriculture, in Peoria, Illi ⁇ nois, U.S.A.
- E. coli HB101 containing pUC1129 can be grown up in L-broth by well known procedures.
- the culture is grown to an optical density of 0.6 after which chloramphenicol is added and the culture is left to shake overnight.
- the culture is then lysed by, e.g., using high salt SDS and the supernatant is subjected to a cesium chloride/ethidium bromide equilibrium density gradient centrifugation to yield the plasmids.
- the a ino acid sequence of PRV glycoprotein gp50 is set forth in Chart A of PCT/US86/01761.
- - Glycoproteins having a smaller amount of 0-linked glycosylation than the naturally occurring glycoproteins produced by the method of the instant invention and displaying PRV glycoprotein antigenicity include the sequence set forth in Chart A and any portion of the polypeptide sequence which is capable of eliciting an immune response in an animal, e.g., a mammal, which has been injected with the polypeptide sequence, and also immunogenically functional analogs of the polypeptides.
- the entire gene coding for the PRV glycoprotein can be employed in constructing the vectors and infecting the insect host cells to express the PRV glycoprotein having reduced 0-lihked glycosylation, or fragments of the gene coding for the PRV glycoprotein can be em ⁇ ployed, whereby the resulting insect host cell will express polypep ⁇ tides displaying PRV antigenicity.
- Any fragment of the PRV glycopro ⁇ tein gene can be employed which results in the expression of a polypeptide which is an immunogenic fragment of the PRV glycoprotein or an analog thereof.
- the degeneracy of the genetic code permits easy substitution of base pairs to produce functionally equivalent genes and fragments thereof encoding polypep ⁇ tides displaying PRV glycoprotein antigenicity. These functional equivalents also are included within the scope of the invention.
- PRV glycoproteins having non-essential amino acid substitutions which are also well-known in the art, and deletions, which do not affect the PRV antigenicity of the polypeptides.
- Charts A and B are set forth to illustrate the constructions of the Examples. Certain conventions are used to illustrate plasmids and DNA fragments as follows:
- the single line figures represent both circular and linear double-stranded DNA.
- pD50 was cut with EcoRI and the ends made blunt by treatment with T4 DNA polymerase. BamHI linkers were then ligated on and the fragment so produced was treated with BamHI.
- the fragment, containing the gp50 gene (about 1300 base pairs), was isolated by agarose gel electrophoresis.
- Plasmid pAc373 (7.1 kb) is a baculovirus expression vector having a unique BamHI site immediately downstream from the polyhedron promoter of AcNPV.
- the plasmid is available from Professor Max Summers of the Department of Entomology, Texas A&M University, College Station, Texas 77843 and is fully described in Mol. and Cell. Biol., 3, pp. 2156-65 (1983) and in European patent publication number 0 127 839. It can also be obtained from the Agricultural Research Culture Collection (NRRL) where it has been assigned accession number NRRL B-15778.
- pAc373 was digested with BamHI, and the ends were dephosphory- lated with bacterial alkaline phosphatase.
- the BamHI fragment containing the gp50 gene from above was ligated into the linearized pAc373 to produce plasmid pAcgp50-4.
- Recombinants having the gp50 gene in the proper orientation with relation to the polyhedron promoter were determined by digestion with Sail which produced a 975 base pair fragment for clones with the correct orientation.
- Example 2 Construction of Plasmid pAcgp50T-5 We constructed this plasmid to put the gene encoding the truncated secreted form of gp50 (see PCT/US86/01761) into the pAc373 vector.
- plasmid pD50T was constructed exactly -li ⁇ the same as plasmid pDIE50T was constructed in PCT/US86/01761, chart L and materials related thereto, except that pD50 was the starting plasmid instead of ⁇ DIE50.
- pD50 is digested with Sail and EcoRI a 4.2 kb Sall/Eco RI fragment is produced instead of the 5.0 kb fragment for pDIE50.
- the only difference between the resulting plasmids is that the cytomegalovirus (CMV) promoter is not in plasmid pD50T and a BamHI site remains.
- CMV cytomegalovirus
- pD50T was manipulated to introduce the gene for the truncated gp5° into the pAc373 vector to produce pAcgp50-T-5.
- the same 975 bp Sail fragment was diagnostic for the correct orientation of the sequence encoding the truncated gp50.
- the plasmids produced above in Examples 1 and 2 were co-trans- fected along with baculovirus AcNPV DNA into Spodoptera frugiperda
- Recombinant viruses containing the gp50 genes were enriched by limited dilution of approximately 10 ⁇ plaque forming units per well in microtiter wells, followed by hybridization with - ⁇ p-iabeled g ⁇ 50 DNA to detect wells where recombinant viruses were growing. Recombinant viruses were isolated by plating out dilute virus and picking individual plaques that lacked occluded virus. The viruses were plaque-purified four times to be sure that occlusion positive virus was eliminated before growing up virus stocks.
- Recombinant viruses were isolated from transfections with both pAcgp50-4 and pAcgp50T-5 and were designated AcNPV-gp50 and AcNPV- gp50T respectively.
- Sf9 cells are infected with one of the recombinant viruses produced in Example 3 at a multiplicity of infection of at least 1.0 plaque forming unit per cell.
- the cells are suspended in Grace's medium, gently centrifuged out, and resuspended in Grace's medium lacking me hionine but to which 100 microcuries per ml of --S-methionine is added.
- Samples are harvested and gp50 expression is analyzed by immunoprecipita ion as described in E.A. Petrovskis, et al, J. Virol., 59, pp. 216-23 (1986).
- AcNPVgpSO-infected cells produced three forms of gp50, having molecular weights of 44, 46 and 47 kd.
- AcNPVgp50T-infected cells produced a form of gp50 having a molecular weight of about 41 kd.
- Two forms of gp50 having molecular weights of 43 and 44 kd were found in the medium of these latter infected cells.
- the lower molecular weight polypeptides correspond to the expected molecular weight of the protein backbone of gp50 molecules with little or no 0-linked glycosylatio .
- Table 1 sets forth the protection of mice from challenge by virulent PRV by immunization with gp50 produced in Sf9 cells or tissue plasminogen activator (tPA) produced in Sf9 cells as a control (AcNPVgp50 or AcNPV-tPA respectively) . Mice were immunized at 28 days, 18 days and 7 days prior to challenge.
- gp50 produced in Sf9 cells
- tPA tissue plasminogen activator
- a vaccine prepared utilizing a gp50 protein of the instant invention or an immunogenic fragment thereof can consist of fixed host cells, a host cell extract, or a partially or completely purified PRV glycoprotein preparation from the host cells.
- the gp50 immunogen prepared in accordance with the present invention is preferably free of PRV virus.
- the vaccine immunogen of the invention is composed substantially entirely of the desired im ⁇ munogenic PRV gp50 polypeptide and/or other PRV polypeptides display ⁇ ing PRV antigenicity.
- the immunogen can be prepared in vaccine dose form by well-known procedures.
- the vaccine can be administered intramuscularly, subcu- taneously or intranasally.
- the immunogen may be combined with a suitable carrier, for example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunomodulating agents including aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emul ⁇ sions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebac- terium parvu (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
- adjuvants or immunomodulating agents including aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (
- adjuvants are available commer ⁇ cially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Another suitable adjuvant is Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich ⁇ igan) .
- the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
- aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (AI2O3 basis).
- the concentration of the immunogen can range from about 1.0 ⁇ g to about 100 mg per animal. In pigs, a preferable range is from about 100 ⁇ g to about 3.0 mg per pig.
- a suitable dose size is about 1-10 ml, preferably about 1.0 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5% aluminum hydroxide. Comparable dose 5 forms can also be prepared for parenteral administration to baby pigs, but the amount of immunogen per dose will be smaller, for example, about 0.25 to about 1.0 mg per dose.
- the first dose can be given from about several months to about 5 to 7
- the second dose of the vaccine then should be administered some weeks after the first dose, for example, about 2 to 4 weeks later, and vaccine can then be administered up to, but prior to, farrowing.
- the vaccine can be administered as a single 2 ml dose, for example, at about 5 to 7 weeks prior to
- the vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments, for example, antibiotics.
- a pharmaceutically acceptable salt for example
- 25 effective amount of the vaccine can be employed with a pharmaceuti ⁇ cally acceptable carrier or diluent to vaccinate animals such as swine, cattle, sheep, goats, and other mammals.
- Plasmid pAc373 is digested with BamHI and BAP to produce fragment 3.
- poly P (d) Fragments 2 and 3 are ligated to produce plasmid pAcgp50-4.
- Plasmid pAcgp50T-5 is produced from plasmid pD50T by the same manipulations as were done in Chart B, but starting with pD50T.
Abstract
The present invention provides viral glycoproteins having a lower amount of O-linked glycosylation than the naturally occurring glycoprotein. These glycoproteins are produced in insect host cells infected with a recombinant DNA molecule comprising a baculovirus vector and the DNA sequence encoding said glycoprotein. The present invention also provides subunit vaccines for pseudorabies virus.
Description
VIRUS PROTEINS HAVING REDUCED O-LINKED GLYCOSYLATION This application is a continuation-in-part of PCT International Application No. PCT/US86/01761, which is incorporated herein by reference. FIELD OF INVENTION
This invention relates to- viral glycoproteins having reduced 0- linked glycosylation. The invention also relates to methods for producing such glycoproteins. More specifically, the invention relates to a pseudorabies virus glycoprotein having reduced 0-linked glycosylation and methods for producing . said glycoprotein with baculovirus vectors in insect cell hosts. These glycoproteins having reduced 0-linked glycosylation are useful for protecting animals against infection by the viruses they are derived from. BACKGROUND OF THE INVENTION Pseudorabies virus (PRV) is a disease which infects many species of animals worldwide. PRV infections are variously called infectious Bulbar paralysis, Aujeszky's disease, and mad itch. Infections are known in important domestic animals such as swine, cattle, dogs, cats, sheep, rats and mink. The host range, is very broad and includes most mammals and, experimentally at least, many kinds of birds (for a detailed list of hosts, see D.P. Gustafson, "Pseudo¬ rabies", in Diseases of Swine, 5th ed. , A.D. Leman et al., eds. , (1981)). For most infected animals the disease is fatal. Adult swine and possibly rats, however, are not killed by the disease and are therefore carriers.
Populations of swine are particularly susceptible to PRV. Although the adult swine rarely show symptoms or die from the disease, piglets become acutely ill when infected and death usually ensues in 24 to 48 hours often without specific clinical signs (T.C. Jones and R.D. Hunt, Veterinary Pathology, 5th ed. , Lea & Febiger (1983)).
PRV vaccines have been produced by a variety of techniques and vaccination in endemic areas of Europe has been practiced for more than 15 years. Losses have been reduced by vaccination, but vaccina- tion has maintained the virus in the environment. No vaccine has been produced that will prevent infection. Vaccinated animals that are exposed to virulent virus survive the infection and then shed more virulent virus. Vaccinated animals may therefore harbor a
latent infection that can flare up again. (See, D.P. Gustafson, supra) .
Live attenuated and inactivated vaccines for PRV are available commercially in the United States and have been approved, by the USDA (See, C.E. Aronson, ed. , Veterinary Pharmaceuticals & Biologicals, (1983)).
PRV is a herpesvirus. The herpesviruses generally are among the most complex of animal viruses. Their genomes encode at least 50 virus specific proteins and contain upwards of 150,000 nucleotides. Among the most immunologically reactive proteins of herpesviruses are the glycoproteins found, among other places, in virion membranes and the membranes of infected cells. The literature on PRV glycoproteins refers to at least four viral glycoproteins (T. Ben-Porat and A.S. Kaplan, Virology, 41, pp. 265-73 (1970); A.S. Kaplan and T. Ben-Porat, Proc. Natl. Acad. Sci. USA, 66, pp. 799-806 (1970)). INFORMATION DISCLOSURE
European patent publication number 0 127 839 and European patent publication number 0 155 476 refer to methods for producing recom¬ binant baculovirus expression vectors for expression of selected genes together with such vectors. They do not disclose the use of such vectors to produce viral glycoproteins having less 0-linked glycosylation than the naturally occurring proteins for use in vaccines. Both of these documents are incorporated herein by reference. S. Alexander and J.H. Elder, "Carbohydrate Dramatically Influ¬ ences Immune Reactivity of Antisera to Viral Glycoprotein Antigens", Science, 226, pp. 1328-30 (1984) demonstrated that viral glyco¬ proteins with N-linked carbohydrate removed by endoglycosidase F had varying antigenic effects. Heteroantisera prepared against virus were virtually unreactive toward the viral glycoproteins after N- linked carbohydrate removal. The reactivity of monoclonal antibodies prepared to glycosylated antigen in most cases improved, while in a smaller number of cases it stayed the same, or decreased in reactivity when reacted with the antigen without N-linked carbo- hydrate. Most antibodies to synthetic peptide sequences also improved in reactivity after N- inked carbohydrate removal.
D.W. Miller, et al, "An Insect Baculovirus Host-Vector System for High-Level Expression of Foreign Genes", Genetic Engineering, 8,
pp. 277-98 (1986) refer to a baculovirus expression system for expressing heterologous genes. They also provide a review of the use of baculovirus expression systems. During this review, they discuss post-translational modifications in baculovirus systems, including glycosylation, as follows:
"Very little is known about the nature of protein glycosylation by insect cells [citations omitted] ....The ability of these cells to carry out N-linked glycosylation appears to end at the addition of the high mannose complex [citation omitted] . 0- linked glycosylation has been examined only indirectly [citation omitted] .
"A protein expressed in insect cells will be rich in mannose and lacking sialic acid. At this point, the ramifica¬ tions of this are not clear and are likely to be of more or less concern depending on the end use of the product. When analyzed by SDS-acrylamide gel electrophoresis, proteins expressed in insect cells are frequently smaller than their native counter¬ parts. We attribute this to less complex glycosylation and perhaps substantial hydrolysis of the high mannose complex unit [citation omitted] . This has not been directly determined for heterologous gene products of the baculovirus expression system. "With regard to the possible in vivo physiological effects of this glycosylation difference, much work needs to be done. We have no clear-cut case where the glycosylation difference has altered biological activity of a protein but do consider that this will inevitably be observed."
T.D. Butters, et al, "Steps in the Biosynthesis of Mosquito Cell Membrane Glycoproteins and the Effects of Tunicamycin" , Bioch. Bioph. Acta, 640, pp. 672-86 (1981) set forth evidence indicating that mosquito cells synthesize N-glycosidically-linked carbohydrate chains and O-glycosidically-linked chains which are attached to glyco¬ proteins.
G.E. Smith, et al, "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Mol. Cell. Biol., 3, pp. 2156-65 (1983) and S. Maeda, et al., "Production of Human α-Interferon in Silkworm Using a Baculovirus Vector," Nature, 315, pp. 592-94 (1985) refer to the production of β- and a- interferon in Spodoptera frugiperda. cells and silkworm using the
Autographs californica and Bonibyx mori nuclear polyhedris virus (AcNPV and BmNPV) as expression vectors, respectively. Smith, et al., indicate that the ^-interferon produced thereby is glycosylated. International patent application PCT/US85/02319 relates to N- 5 linked carbohydrate-free viral polypeptides, and methods for produc¬ ing them by reacting the glycoprotein with an endoglycosidase enzyme, which can be used to induce antibody production in a host animal.
M.W. Wathen and L.K. Wathen, J. Virol., 51, pp. 57-62 (1984) refer to a PRV containing a mutation in a viral glycoprotein (gρ50)
Iff- and a method for selecting, the mupant "utilizing neutralizing monoclo¬ nal antibody directed against gp50. Wathen and Wathen also indicate that a monoclonal antibody directed against gp50 is a strong neutral- izer of PRV, with or without the aid of complement, and that polyval¬ ent immune serum is highly reactive against gp50, therefore conclud-
15 ing that gp50 may be one of the important PRV immunogens. On the other hand, it has been reported that monoclonal antibodies that react with the 98,000 MW envelope glycoprotein neutralize PRV infectivity but that monoclonal antibodies directed against some of the other membrane glycoproteins have very little neutralizing
20 activity (H. Ha pl, et al. , J. Virol., 52, pp. 583-90 (1984); and T. Ben-Porat and A.S. Kaplan, "Molecular Biology of Pseudorabies Virus", in B. Roizman ed. , The Herpesviruses, 3, pp. 105-73 (1984)).
L.M.K. Wathen, et al., Virus Research, 4, pp. 19-29 (1985) refer to the production and characterization of monoclonal antibodies
25 directed against PRV glycoproteins identified as gp50 and gp83 and their use for passively immunizing mice against PRV infection.
A.K. Robbins, et al. , "Localization of a Pseudorabies Virus Glycoprotein Gene Using an E. coli Expression Plasmid Library", in Herpesvirus, pp. 551-61 (1984), refer to the construction of a
30 library of E. coli plasmids containing PRV DNA. They also refer to the identification of a PRV gene that encodes glycoproteins of 74,000 and 92,000 MW. They do not refer to the glycoproteins of the instant invention.
A.K. Robbins, et al., European patent application No. 85400704.4
35 (publication No. 0 162 738) refers to the isolation, cloning and expression of PRV glycoproteins identified as gll and gill. They do not refer to the PRV glycoproteins having reduced 0-linked glyco¬ sylation of the instant invention.
T.C. Mettenleiter, et al., "Mapping of the Structural Gene of Pseudorabies Virus Glycoprotein A and Identification of Two Non- -Glycosylated Precursor Polypeptides", J. Virol., 53, pp. 52-57 (1985), refer to the mapping of the coding region of glycoprotein gA (which they equate with gl) to the BamHI 7 fragment of PRV DNA. They also state that the BamHI 7 fragment of PRV codes for at least three other viral proteins of 65K, 60K, and 40K MW. They do not disclose or suggest a viral glycoprotein having reduced 0-linked glycosylation of the instant invention or the production of similar polypeptides in insect cells with baculovirus vectors.
B. Lomniczi, et al., "Deletions in the Genomes of Pseudorabies Virus Vaccine Strains and Existence of Four Isomers of the Genomes", J. Virol., 49, pp. 970-79 (1984), refer to PRV vaccine strains that have deletions in the unique short sequence between 0.855 and 0.882 map units. This is in the vicinity of the gl gene. T.C. Metten¬ leiter, et al. , "Pseudorabies Virus Avirulent Strains Fail to Express a Major Glycoprotein", J. Virol., 56, pp. 307-11 (1985), demonstrated that three commercial PRV vaccine strains lack glycoprotein gl. We have also found recently that the Bartha vaccine strain contains a deletion for most of the gp63 gene.
T.J. Rea et al., J. Virol., 54, pp. 21-29 (1985), refers to the mapping and the sequencing of the gene for the PRV glycoprotein that accumulates in the medium of infected cells (gX) . Included among the flanking sequences of the gX gene shown therein is a small portion of the gp50 sequence, specifically beginning at base 1682 of Figure 6 therein.
European published patent application 0 133 200 refers to a diagnostic antigenic factor to be used together with certain lectin-- bound PRV glycoprotein subunit vaccines to distinguish carriers and noncarriers of PRV.
G. E. Smith, et al. , "Modification and Secretion of Human Interleukin 2 Produced in Insect Cells by a Baculovirus Expression Vector," Proc. Natl. Acad. Sci. U.S.A., 82, pp. 8404-08 (1985) refer to production of IL-2 by inserting its cDNA into the genome of Autographa califomica nuclear polyhedrosis virus adjacent to the polyhedrin promoter and then using this recombinant virus to infect an insect cell host. They indicated that there was no evidence that the recombinant IL-2 produced was glycosylated. They do not disclose
or suggest producing viral glycoproteins having a lower amount of 0- linked glycosylation than their naturally occurring homologs for use as vaccines.
SUMMARY OF THE INVENTION The present invention relates to viral glycoproteins having a lower amount of 0-linked glycosylation than their naturally occurring glycoprotein, more particularly wherein said glycoprotein is a pseudorabies virus glycoprotein, and even more particularly pseudo¬ rabies virus gp50. The glycoproteins having a lower amount of glycosylation than the natural glycoproteins are produced by an insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein.
More specifically, the insect cell host referred to above is selected from the group consisting of Spodoptera frugiperda, Bomhyx mori , Trichoplusia ni , and other lepidopteran cell lines. Intact worms can also be used as hosts for example, for AcNPV vectors, cabbage loopers or fall armyworms. See Maeda, et al. , supra.
More specifically, the glycoprotein having a lower amount of glycosylation than the natural glycoprotein is produced by a Spodop¬ tera frugiperda insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein, more specifi¬ cally, gp50.
Also provided is a method for producing a glycoprotein having such reduced glycosylation, comprising:
(a) preparing a recombinant DNA molecule, said molecule com¬ prising a DNA sequence coding for a glycoprotein, and a baculovirus vector;
(b) infecting an insect host cell with said recombinant DNA molecule;
(c) culturing said infected insect host cell;
(d) and collecting said glycoprotein.
More specifically in the method, the glycoprotein is a pseudo¬ rabies virus glycoprotein, more specifically, gp50. Also, more specifically, the insect host cell used in the method set forth above is Spodoptera frugiperda. DETAILED DESCRIPTION OF THE INVENTION
The existence and location of the gene encoding glycoprotein
gp50 of PRV was demonstrated by M.W. Wathen and L.M. Wathen, supra.
The glycoprotein encoded by the gene was defined as a glycoprot¬ ein that reacted with a particular monoclonal antibody. This glycoprotein did not correspond to any of the previously known PRV glycoproteins. Wathen and Wathen mapped a mutation resistant to the monoclonal antibody, which, based on experience with herpes simplex virus (e.g., T.C. Holland et al., J. Virol., 52, pp.566-74 (1984)), indicated that they had mapped the location of the structural gene for gp50. Wathen and Wathen mapped the gp50 gene to the smaller Sall/BamHI fragment from within the BamHI 7 fragment of PRV. Rea et al, supra, have mapped the PRV glycoprotein gX gene to the same region.
Many of the details related to production of PRV glycoprotein gp50 and its use as a subunit vaccine for the prevention of PRV infection are set forth in PCT patent application PCT US/01761 and in E.A. Petrovskis, et al, "DNA Sequence of the Gene for Pseudorabies Virus gp50, a Glycoprotein without N-Linked Glycosylation", Virology, 59, pp. 216-23 (1986), which are incorporated herein by reference.
We cloned the gp50 gene under control of the polyhedrin promoter in the baculovirus Autographa califomica nuclear polyhedrosis virus (AcNPV) . Two forms of the gene were inserted into AcNPV: (1) the intact gene encoding the complete gp50 amino acid sequence to produce a recombinant virus designated AcNPV-gp50, and (2) the gene with the base pairs encoding the anchor sequence of gp50 deleted to produce a recombinant virus designated AcNPV-gp50T. AcNPV-gp50-infected cells produced a cellular form of gp50 and AcNPV-gp50T-infected cells produced a truncated form of gρ50 which is secreted into the medium. Both of these forms of gp50 produced in insect cells had a sig¬ nificantly lower molecular weight than the same proteins produced in mammalian cells because of reduced 0-linked glycosylation. The gp50 produced in insect cells is, however, labeled with •'■^'C-glucosamine. Since the gp50 sequence contains no N-linked glycosylation sites (E.A. Petrovskis,et al, supra), this is presumed to be 0-linked glycosylation in insect cells although at a substantially reduced level as compared to that produced in mammalian cells and as compared to the native glycoprotein. As a result, we refer to the glyco¬ protein of the instant invention as "having a lower amount of 0- 1inked glycosylation than its corresponding naturally occurring viral
glycoprotein." It should be clear to those skilled in the art that this does not include such glycoproteins produced in prokaryotic hosts, e.g., E. coli , which would produce glycoproteins having no 0- linked glycosylation. In contrast the polypeptides of the instant invention have a decreased amount of 0-linked glycosylation as compared to the corresponding native polypeptide, but 0-linked carbohydrates are not entirely absent.
Insect cells infected with AcNPV- p50 were used to immunize mice. After a series of three immunizations with 10° infected cells, most of the mice survived a challenge with virulent PRV. -
As mentioned above, the gene encoding gp50, mapped to the BamHI 7 fragment of the PRV DNA. The BamHI 7 fragment from PRV can be derived from plasmid pPRXhl (also known as pUC1129) and fragments convenient for DNA sequence analysis can be derived by standard subcloning procedures. Plasmid pUC1129 is available from E. coli HB101, NRRL B-15772. This culture is available from the permanent collection of the Northern Regional Research Center Fermentation Laboratory (NRRL), U.S. Department of Agriculture, in Peoria, Illi¬ nois, U.S.A. E. coli HB101 containing pUC1129 can be grown up in L-broth by well known procedures. Typically the culture is grown to an optical density of 0.6 after which chloramphenicol is added and the culture is left to shake overnight. The culture is then lysed by, e.g., using high salt SDS and the supernatant is subjected to a cesium chloride/ethidium bromide equilibrium density gradient centrifugation to yield the plasmids.
The a ino acid sequence of PRV glycoprotein gp50 is set forth in Chart A of PCT/US86/01761. - Glycoproteins having a smaller amount of 0-linked glycosylation than the naturally occurring glycoproteins produced by the method of the instant invention and displaying PRV glycoprotein antigenicity, include the sequence set forth in Chart A and any portion of the polypeptide sequence which is capable of eliciting an immune response in an animal, e.g., a mammal, which has been injected with the polypeptide sequence, and also immunogenically functional analogs of the polypeptides.
The entire gene coding for the PRV glycoprotein can be employed in constructing the vectors and infecting the insect host cells to express the PRV glycoprotein having reduced 0-lihked glycosylation,
or fragments of the gene coding for the PRV glycoprotein can be em¬ ployed, whereby the resulting insect host cell will express polypep¬ tides displaying PRV antigenicity. Any fragment of the PRV glycopro¬ tein gene can be employed which results in the expression of a polypeptide which is an immunogenic fragment of the PRV glycoprotein or an analog thereof. As is well known in the art, the degeneracy of the genetic code permits easy substitution of base pairs to produce functionally equivalent genes and fragments thereof encoding polypep¬ tides displaying PRV glycoprotein antigenicity. These functional equivalents also are included within the scope of the invention. Also included are PRV glycoproteins having non-essential amino acid substitutions, which are also well-known in the art, and deletions, which do not affect the PRV antigenicity of the polypeptides.
Charts A and B are set forth to illustrate the constructions of the Examples. Certain conventions are used to illustrate plasmids and DNA fragments as follows:
(1) The single line figures represent both circular and linear double-stranded DNA.
(2) Asterisks (*) indicate that the molecule represented is circular. Lack of an asterisk indicates the molecule is linear.
(3) Endonuclease restriction sites of interest are indicated above the line.
(4) Genes are indicated below the line.
(5) Distances between genes and restriction sites are not to scale. The figures show the relative positions only unless indicated otherwise.
Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail, for example, in Molecu- lar Cloning, T. Maniatis, et al., Cold Spring Harbor Laboratory, (1982) and B. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984), which are incorporated herein by reference. The procedures specifically related to baculoviruses are set forth in M.D. Summers and G.E. Smith, A Manual for Baculovirus Vectors and Insect Cell Culture Procedures, published by the College of Agricul¬ ture, Texas Agricultural Experiment Station, Texas Agricultural Extension Service, College Station, Texas (1986) which is incor¬ porated herein by reference.
While the specific Examples herein relate primarily to PRV, it is clear to those skilled in the art that other viruses and other polypeptides could be used in a similar fashion, for example, respiratory syncitial virus glycoprotein G and herpes simplex virus glycoprotein C.
Example 1 Construction of Plasmid pAcgp50-4
In this example we set forth the construction of a plasmid with the gp50 gene downstream from the polyhedrin promoter, and flanked by baculovirus DNA. The starting material containing the gp50 gene was plasmid pD50 which is disclosed in Chart I of patent application PCT/US86/01761 and textual materials related thereto.
Referring now to Chart A, pD50 was cut with EcoRI and the ends made blunt by treatment with T4 DNA polymerase. BamHI linkers were then ligated on and the fragment so produced was treated with BamHI.
The fragment, containing the gp50 gene (about 1300 base pairs), was isolated by agarose gel electrophoresis.
Plasmid pAc373 (7.1 kb) is a baculovirus expression vector having a unique BamHI site immediately downstream from the polyhedron promoter of AcNPV. The plasmid is available from Professor Max Summers of the Department of Entomology, Texas A&M University, College Station, Texas 77843 and is fully described in Mol. and Cell. Biol., 3, pp. 2156-65 (1983) and in European patent publication number 0 127 839. It can also be obtained from the Agricultural Research Culture Collection (NRRL) where it has been assigned accession number NRRL B-15778. pAc373 was digested with BamHI, and the ends were dephosphory- lated with bacterial alkaline phosphatase. The BamHI fragment containing the gp50 gene from above was ligated into the linearized pAc373 to produce plasmid pAcgp50-4. Recombinants having the gp50 gene in the proper orientation with relation to the polyhedron promoter were determined by digestion with Sail which produced a 975 base pair fragment for clones with the correct orientation. Example 2 Construction of Plasmid pAcgp50T-5 We constructed this plasmid to put the gene encoding the truncated secreted form of gp50 (see PCT/US86/01761) into the pAc373 vector.
Referring now to Chart B, plasmid pD50T was constructed exactly
-li¬ the same as plasmid pDIE50T was constructed in PCT/US86/01761, chart L and materials related thereto, except that pD50 was the starting plasmid instead of ρDIE50. When pD50 is digested with Sail and EcoRI a 4.2 kb Sall/Eco RI fragment is produced instead of the 5.0 kb fragment for pDIE50. The only difference between the resulting plasmids is that the cytomegalovirus (CMV) promoter is not in plasmid pD50T and a BamHI site remains.
By following the techniques set forth above in Example 1 for pD50, pD50T was manipulated to introduce the gene for the truncated gp5° into the pAc373 vector to produce pAcgp50-T-5. The same 975 bp Sail fragment was diagnostic for the correct orientation of the sequence encoding the truncated gp50. Example 3 Construction of Recombinant Baculoviruses
The methods for construction of baculoviruses have been de- scribed in detail in publications by Dr. Max Summers (e.g., Mol. and
Cell Biol., 3, pp. 2156-65 (1983)) and particularly in the manual referred to above (M.D. Summers and G.E. Smith, supra) which are incorporated herein by reference.
The plasmids produced above in Examples 1 and 2 were co-trans- fected along with baculovirus AcNPV DNA into Spodoptera frugiperda
Sf9 cells (available from the American Type Culture Collection,
Rockville Maryland as deposit number ATCC CRL 1711) . Recombinant viruses containing the gp50 genes were enriched by limited dilution of approximately 10^ plaque forming units per well in microtiter wells, followed by hybridization with -^^p-iabeled gρ50 DNA to detect wells where recombinant viruses were growing. Recombinant viruses were isolated by plating out dilute virus and picking individual plaques that lacked occluded virus. The viruses were plaque-purified four times to be sure that occlusion positive virus was eliminated before growing up virus stocks.
Recombinant viruses were isolated from transfections with both pAcgp50-4 and pAcgp50T-5 and were designated AcNPV-gp50 and AcNPV- gp50T respectively.
Example 4 Detection of gp50 Expression from Recombinant Baculo- viruses
Sf9 cells are infected with one of the recombinant viruses produced in Example 3 at a multiplicity of infection of at least 1.0 plaque forming unit per cell. At 24-48 hours post infection the
cells are suspended in Grace's medium, gently centrifuged out, and resuspended in Grace's medium lacking me hionine but to which 100 microcuries per ml of --S-methionine is added. Samples are harvested and gp50 expression is analyzed by immunoprecipita ion as described in E.A. Petrovskis, et al, J. Virol., 59, pp. 216-23 (1986).
AcNPVgpSO-infected cells produced three forms of gp50, having molecular weights of 44, 46 and 47 kd. AcNPVgp50T-infected cells produced a form of gp50 having a molecular weight of about 41 kd. Two forms of gp50 having molecular weights of 43 and 44 kd were found in the medium of these latter infected cells. The lower molecular weight polypeptides correspond to the expected molecular weight of the protein backbone of gp50 molecules with little or no 0-linked glycosylatio .
Similar experiments with -^C-glucosamine indicated that some of these forms of gp50 could be labeled by carbohydrate precursors. Although not wishing to bound by theory, it is likely that insect cells can add 0-linked carbohydrate to glycoproteins, but to a much lesser extent than mammalian cells do and to a much lesser degree than is found for the native glycoproteins, but more than prokaryotic cells.
Example 5 Use of gp50 Produced in Baculovirus as a Vaccine
Table 1 sets forth the protection of mice from challenge by virulent PRV by immunization with gp50 produced in Sf9 cells or tissue plasminogen activator (tPA) produced in Sf9 cells as a control (AcNPVgp50 or AcNPV-tPA respectively) . Mice were immunized at 28 days, 18 days and 7 days prior to challenge.
Table 1 Immunizing Number of %
Agent/Adjuvant Cells Survival5 gp50/CFAb 1 x 105 88 (7/8) tPA/CFA 1 x 106 25 (2/8) gp50/none 1 x 106 100 (8/8) gp50/none 1 x 105 75 (6/8) gp50/none 1 x 104 38 (3/8) unvaccinated 38 (3/8)
aChallenged with 30 LD50 of PRV Rice strain by footpad route. "Complete Freund's adjuvant.
As can be seen from Table 1, cells expressing the gp50 antigen were effective in protecting mice from infection with PRV as compared to both an unvaccinated control and cells expressing a non-PRV antigen, tPA. It also appears that such protection decreases with a decreasing amount of gp50.
A vaccine prepared utilizing a gp50 protein of the instant invention or an immunogenic fragment thereof can consist of fixed host cells, a host cell extract, or a partially or completely purified PRV glycoprotein preparation from the host cells. The gp50 immunogen prepared in accordance with the present invention is preferably free of PRV virus. Thus, the vaccine immunogen of the invention is composed substantially entirely of the desired im¬ munogenic PRV gp50 polypeptide and/or other PRV polypeptides display¬ ing PRV antigenicity. The immunogen can be prepared in vaccine dose form by well-known procedures. The vaccine can be administered intramuscularly, subcu- taneously or intranasally. For parenteral administration, such as intramuscular injection, the immunogen may be combined with a suitable carrier, for example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunomodulating agents including aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emul¬ sions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebac- terium parvu (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commer¬ cially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Another suitable adjuvant is Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich¬ igan) .
The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (AI2O3 basis). On a per dose basis, the concentration of the immunogen can range from about 1.0 μg to about 100 mg per animal. In pigs, a preferable range is from about 100 μg
to about 3.0 mg per pig. A suitable dose size is about 1-10 ml, preferably about 1.0 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5% aluminum hydroxide. Comparable dose 5 forms can also be prepared for parenteral administration to baby pigs, but the amount of immunogen per dose will be smaller, for example, about 0.25 to about 1.0 mg per dose.
For vaccination of sows, a two dose regimen can be used. The first dose can be given from about several months to about 5 to 7
Iff weeks- prior to farrowing. The second dose of the vaccine then should be administered some weeks after the first dose, for example, about 2 to 4 weeks later, and vaccine can then be administered up to, but prior to, farrowing. Alternatively, the vaccine can be administered as a single 2 ml dose, for example, at about 5 to 7 weeks prior to
15 farrowing. However, a 2 dose regimen is considered preferable for the most effective immunization of the baby pigs. Semi-annual revaccination is recommended for breeding animals. Boars may be revaccinated at any time. Also, sows can be revaccinated before breeding. Piglets born to unvaccinated sows may be vaccinated at
20 about 3-10 days, again at 4-6 months and yearly or preferably semi-annually thereafter.
The vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments, for example, antibiotics. A pharmaceutically
25 effective amount of the vaccine can be employed with a pharmaceuti¬ cally acceptable carrier or diluent to vaccinate animals such as swine, cattle, sheep, goats, and other mammals.
Other vaccines may be prepared according to methods well known to those skilled in the art as set forth, for example, in I. Tizard,
30 An Introduction to Veterinary Immunology, 2nd ed. (1982) , which is incorporated herein by reference.
CHART A. Construction of plasmid pAcgp50-4
(a) Plasmid pD50 is cut with EcoRI to produce fragment 1.
BamHI Hindlll PvuII EcoRI PvuII * I I I I I *
dhfr SV40 Amp R 50505050505050 Ori
Fragment 1
EcoRI BamHI EcoRI
505050505050 | | dhfr AmρR (b) Fragment 1 is blunt-ended, BamHI linkers are added and then digested with BamHI to produce fragment 2. BamHI BamHI
505050505050505050505050 (c) Plasmid pAc373 is digested with BamHI and BAP to produce fragment 3. pAc373
BamHI * I *
I I
P poly Fragment 3
BamHI BamHI
BamHI BamHI
* I μ 505050505050 L_| * P poly dhfr -> Dihydrofolate reductase gene
SV40 Ori -*• SV40 promotor and origin of replication Amp*** = Ampicillin resistance gene P — polyhedrin promoter poly - polyhedrin gene
CHART B. Construction of plasmid pAcgp50T-5
(a) Plasmid pAcgp50T-5 is produced from plasmid pD50T by the same manipulations as were done in Chart B, but starting with pD50T.
pD50T
EcoRI BamHI
*
50T50T50T I I dhfr AmpR
pAcgp50T-5
BamHI BamHI *
I 50T50T50T I
P poly
50T = Transacted form of gp50 gene
Claims
1. A viral glycoprotein having a lower amount of 0-linked glycosy¬ lation than its corresponding naturally occurring viral glycoprotein, produced by an insect cell host infected with a recombinant DNA molecule comprising a baculovirus vector and a DNA sequence encoding said glycoprotein.
2. A glycoprotein according to claim 1, wherein the glycoprotein is a pseudorabies virus glycoprotein.
A glycoprotein according to claim 2, pseudorabies virus gp50.
4. A glycoprotein according to claim 1, wherein the insect cell host is selected from the group consisting of Spodoptera frugiperda , Bombyx mori and Trichoplusia ni .
5. A glycoprotein according to claim 1, wherein said glycoprotein is pseudorabies virus gp50 and said insect cell host is Spodoptera frugiperda .
6. A method for producing a viral glycoprotein having reduced 0- linked glycosylation as compared to its naturally occurring glycosyl- ated form comprising:
(a) preparing a recombinant DNA molecule, said molecule com- prising a DNA sequence coding for a glycoprotein and a baculovirus vector;
(b) infecting an insect host cell with said recombinant DNA molecule;
(c) culturing said infected insect host cell; (d) and collecting said glycoprotein.
7. A method according to claim 6 wherein said glycoprotein is pseudorabies virus gp50.
8. A method according to claim 6 wherein said insect host cell is Spodoptera frugiperda .
9. A method according to claim 6 wherein said glycoprotein is pseudorabies virus gp50 and said insect host cell is Spodoptera frugiperda..
10. A pseudorabies virus gp50 polypeptide having a lower amount of O-linked glycosylation than its corresponding naturally occurring form.
11. A vaccine comprising the glycoprotein according to claim 10.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019890700372A KR890701745A (en) | 1987-06-30 | 1988-06-17 | Viral protein with reduced O-linked glycosylation |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6821187A | 1987-06-30 | 1987-06-30 | |
US068,211 | 1987-06-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1989000196A1 true WO1989000196A1 (en) | 1989-01-12 |
Family
ID=22081120
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1988/002021 WO1989000196A1 (en) | 1987-06-30 | 1988-06-17 | Virus proteins having reduced o-linked glycosylation |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0364492A1 (en) |
JP (1) | JPH02504103A (en) |
KR (1) | KR890701745A (en) |
AU (1) | AU2083788A (en) |
WO (1) | WO1989000196A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0405867A1 (en) * | 1989-06-27 | 1991-01-02 | SMITHKLINE BEECHAM BIOLOGICALS s.a. | Novel compounds |
US5298239A (en) * | 1991-10-07 | 1994-03-29 | The Research Foundation Of State University Of New York | Mutations rendering platelet glycoprotein IB α less reactive |
US5317097A (en) * | 1991-10-07 | 1994-05-31 | The Research Foundation Of State University Of New York | Mutations in the gene encoding the α chain on platelet glycoprotein IB |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU616211B2 (en) * | 1987-03-09 | 1991-10-24 | Upjohn Company, The | Transgenic animal cells resistant to viral infection |
FR2631974B1 (en) * | 1988-05-31 | 1992-12-11 | Agronomique Inst Nat Rech | MODIFIED BACULOVIRUS, ITS PREPARATION METHOD AND ITS APPLICATION AS A GENE EXPRESSION VECTOR |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0127839A2 (en) * | 1983-05-27 | 1984-12-12 | THE TEXAS A&M UNIVERSITY SYSTEM | Method for producing a recombinant baculovirus expression vector |
WO1987001287A1 (en) * | 1985-09-06 | 1987-03-12 | Pru-Tech Research And Development Partnership | Pseudorabies virus deletion mutants and vaccines containing same |
WO1987003206A1 (en) * | 1985-11-25 | 1987-06-04 | Quash Gerard A | Modified viral glycoproteins, diagnostic and therapeutic uses therefore |
-
1988
- 1988-06-17 JP JP63506102A patent/JPH02504103A/en active Pending
- 1988-06-17 WO PCT/US1988/002021 patent/WO1989000196A1/en not_active Application Discontinuation
- 1988-06-17 AU AU20837/88A patent/AU2083788A/en not_active Abandoned
- 1988-06-17 EP EP88906390A patent/EP0364492A1/en not_active Withdrawn
- 1988-06-17 KR KR1019890700372A patent/KR890701745A/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0127839A2 (en) * | 1983-05-27 | 1984-12-12 | THE TEXAS A&M UNIVERSITY SYSTEM | Method for producing a recombinant baculovirus expression vector |
WO1987001287A1 (en) * | 1985-09-06 | 1987-03-12 | Pru-Tech Research And Development Partnership | Pseudorabies virus deletion mutants and vaccines containing same |
WO1987003206A1 (en) * | 1985-11-25 | 1987-06-04 | Quash Gerard A | Modified viral glycoproteins, diagnostic and therapeutic uses therefore |
Non-Patent Citations (6)
Title |
---|
CHEMICAL ABSTRACTS, Vol. 102, (1985), Abstract No. 40823b, METTENLEITER THOMAS; & J. VIROL., 1985, 53(1), 52-7 (ENG). * |
CHEMICAL ABSTRACTS, Vol. 105, (1986), Abstract No. 128466k, PETROVSKIS ERIK A.; & J. VIROL., 1986, 59(2), 216-23 (ENG). * |
CHEMICAL ABSTRACTS, Vol. 109, (1988), Abstract No. 70912s, KOZARSKY, KAREN; & PROC. NATL. ACAD. SCI. U.S.A., 1988, 85(12), 4335-9 (Eng). * |
GENE, 50, (1986), CALVIN L. KEELER, "Construction of an Infectous Pseudorabies Virus Recombinant Expressing a Glycoprotein gIII- -Galactosidase Fusion Protein", pages 215-224. * |
GENETIC ENGINEERING, 8, (1986), DAVID W. MILLER, "An Insect Baculovirus Host-Vector System for High-Level Expression of Foreign Genes", pages 277-298. * |
VIROLOGY, 155, (1986), L. MICHELLE BENNETT, "The Processing of Pseudorabies Virus Glycoprotein gX in Infected Cells and in an Uninfected Cell Line", pages 707-715. * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0405867A1 (en) * | 1989-06-27 | 1991-01-02 | SMITHKLINE BEECHAM BIOLOGICALS s.a. | Novel compounds |
US5298239A (en) * | 1991-10-07 | 1994-03-29 | The Research Foundation Of State University Of New York | Mutations rendering platelet glycoprotein IB α less reactive |
US5317097A (en) * | 1991-10-07 | 1994-05-31 | The Research Foundation Of State University Of New York | Mutations in the gene encoding the α chain on platelet glycoprotein IB |
Also Published As
Publication number | Publication date |
---|---|
KR890701745A (en) | 1989-12-21 |
JPH02504103A (en) | 1990-11-29 |
AU2083788A (en) | 1989-01-30 |
EP0364492A1 (en) | 1990-04-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0231209B1 (en) | Virus vaccine | |
EP0732340B1 (en) | Expression of porcine reproductive respiratory syndrome virus polypeptides in the same cell | |
CA2088599A1 (en) | Expression of human cmv glycoprotein-h using the baculovirus-insect cell expression system | |
AU2004269320B2 (en) | Vectors expressing SARS immunogens, compositions containing such vectors or expression products thereof, methods and assays for making and using | |
EP0484382A1 (en) | Feline calicivirus capsid protein and nucleotide sequence. | |
AU601378B2 (en) | Pseudorabies virus protein | |
WO1989000196A1 (en) | Virus proteins having reduced o-linked glycosylation | |
US5674709A (en) | Pseudorabies virus protein | |
US6172186B1 (en) | Pseudorabies virus protein | |
WO1989010965A2 (en) | Glycoprotein h of herpes viruses | |
US6261563B1 (en) | Pseudorabies virus protein | |
DK175114B1 (en) | Pseudo-rabies virus protein | |
JP2810364B2 (en) | Pseudorabies virus protein | |
CA1325610C (en) | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides | |
EP0263207A1 (en) | Virus vaccine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU DK FI JP KR NO US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE FR GB IT LU NL SE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1988906390 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1988906390 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1988906390 Country of ref document: EP |