AU592076B2 - Process for the preparation of an antigen specific to herpes simplex virus type 2, agents suitable for this purpose, and the use of this antigen - Google Patents
Process for the preparation of an antigen specific to herpes simplex virus type 2, agents suitable for this purpose, and the use of this antigen Download PDFInfo
- Publication number
- AU592076B2 AU592076B2 AU55077/86A AU5507786A AU592076B2 AU 592076 B2 AU592076 B2 AU 592076B2 AU 55077/86 A AU55077/86 A AU 55077/86A AU 5507786 A AU5507786 A AU 5507786A AU 592076 B2 AU592076 B2 AU 592076B2
- Authority
- AU
- Australia
- Prior art keywords
- section
- genome
- deoxyribonucleic acid
- polypeptide
- vector
- 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.)
- Ceased
Links
- 241000701074 Human alphaherpesvirus 2 Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000000427 antigen Substances 0.000 title description 10
- 102000036639 antigens Human genes 0.000 title description 10
- 108091007433 antigens Proteins 0.000 title description 10
- 239000003795 chemical substances by application Substances 0.000 title description 3
- 108020004414 DNA Proteins 0.000 claims abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 17
- 102000053602 DNA Human genes 0.000 claims abstract description 15
- 239000012634 fragment Substances 0.000 claims abstract description 15
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 15
- 230000000890 antigenic effect Effects 0.000 claims abstract description 14
- 229920001184 polypeptide Polymers 0.000 claims abstract description 13
- 239000013598 vector Substances 0.000 claims abstract description 13
- 241000700605 Viruses Species 0.000 claims abstract description 7
- 238000002955 isolation Methods 0.000 claims abstract description 6
- 241000700584 Simplexvirus Species 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 229960005486 vaccine Drugs 0.000 claims description 8
- 241000701822 Bovine papillomavirus Species 0.000 claims description 5
- 210000004102 animal cell Anatomy 0.000 claims description 4
- 101150066555 lacZ gene Proteins 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 210000005260 human cell Anatomy 0.000 claims description 3
- 241000186046 Actinomyces Species 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 241000235649 Kluyveromyces Species 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 241000235070 Saccharomyces Species 0.000 claims description 2
- 241000187747 Streptomyces Species 0.000 claims description 2
- 241000235648 Pichia Species 0.000 claims 1
- 229940039227 diagnostic agent Drugs 0.000 claims 1
- 239000000032 diagnostic agent Substances 0.000 claims 1
- 210000002966 serum Anatomy 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 description 29
- 102000004169 proteins and genes Human genes 0.000 description 15
- 102000037865 fusion proteins Human genes 0.000 description 13
- 108020001507 fusion proteins Proteins 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 6
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000010367 cloning Methods 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000004873 anchoring Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 241000511343 Chondrostoma nasus Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000016917 Complement C1 Human genes 0.000 description 1
- 108010028774 Complement C1 Proteins 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010000196 Factor XIIIa Proteins 0.000 description 1
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 description 1
- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- GLUYKHMBGKQBHE-JYJNAYRXSA-N Phe-Val-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 GLUYKHMBGKQBHE-JYJNAYRXSA-N 0.000 description 1
- 101710155259 Repressor protein CI Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- JTWIMNMUYLQNPI-WPRPVWTQSA-N Val-Gly-Arg Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N JTWIMNMUYLQNPI-WPRPVWTQSA-N 0.000 description 1
- 241001672648 Vieira Species 0.000 description 1
- 108020005202 Viral DNA 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
- 239000002671 adjuvant Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000003643 water by type Substances 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
- 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
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Treatments Of Macromolecular Shaped Articles (AREA)
- Materials For Medical Uses (AREA)
Abstract
1. A process for the preparation of a polypeptide having antigenic determinants of the herpes simplex virus, by isolation of a section of the genome, which codes for this type of polypeptide, from the deoxyribonucleic acid of this virus, binding of the section of the genome enzymatically to the deoxyribonucleic acid of a vector, introduction of this combined deoxyribonucleic acid into a cell in which the combined deoxyribonucleic acid brings about the production of this polypeptide, and obtaining of this polypeptide, which process comprises the section of the genome being the Sall fragment (section of about 0.62 - 0.64 map units of the genome) of herpes simplex virus type 2.
Description
rl_ 592o7 Farm COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPEC' IFICATION
(ORIGINAL)
Class I nt. Class Application Number: 5 sol Lodged: Complete Specification Lodged: Accepted: Published: Priority: 1 q Related Art: t,' a t0 0 0 0 1 Name of Applicant: BEHRINGWERKE AKTIENGESELLSCHAFT QO t ad0 D I Address of Applicant. D-3550 Marburg 1, Federal Republic of Germany 00 0 Actual Inventor: 0 Alidress for Service: MICHAEL BROKER and EGON AMANN EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled: PROCESS FOR THE PREPARATION OF AN ANTIGEN SPECIFIC TO HERPES SIMPLEX VIRUS TYPE 2, AGENTS SUITABLE FOR THIS PURPOSE, AND THE USE OF THIS ANTIGEN The following statement is a full description of this invention, including the best method of performing it known to us -la- BEHRINGWERKE AKTIENGESELLSCHAFT 85/B 005 Ma 523 Dr. Ha/hy Process for the preparation of an antigen specific to herpes simplex virus type 2, agents suitable for this purpose, and the use of this antigen The invention relates to a recombinant deoxyribonucleic acid which contains the genetic code for an antigenic protein from herpes simplex virus type 2 (HSV-2), to a cell which contains the said recombinant DNA and produces this antigenic protein or parts thereof, to the use of the said recombinant DNA or of this cell for the preparation of this protein, and to a process for the preparation of the protein and to the use of this antigenic protein.
'A protein which is able to induce the formation of antibodies against HSV-2 is of great interest for the prep-aration of vaccines for the prophylaxis and treatment of dis- 15 eases caused by HSV-2. Investigations in recent years lead to the assumption that certain tumors may be caused by herpes viruses (HV).
i For this reason, a requirement to be made of a vaccine against HSV-2 is that it must be free of biologically 20 active viral DNA.
The conventional preparation of a vaccine which meets these requirements would demand elaborate purification steps, .which can be avoided by the synthesis of antigenic components of HSV-2. The antigenic components of HSV which have been described hitherto are a few glycoproteins from the coat of the virus. It has been possible to assign their gene loci on the HSV DNA and to determine their DNA sequehce, for example gD from HSV-1 (gD-1) and from HSV-2 gD-1 prepared by genetic engineering can induce ,V-specific antibodies in mice and confer protection against HSV-1 and HSV-2.
k 2 2 In contrast to gD, which predominantly carries type-conmnon antigenic determinants, gC from HSV-1 (gC-1) and HSV-2 (gC-2) have been described as glycoproteins with predominantly type-specific antigenic determinants. The gene Loci and DNA sequences of gC-1 and gC-2 are Likewise known.
It has been possibLe to synthesize antigenic determinants from gC-1 in E. coli. The process for the preparation, and the use, of this antigen is described in European Patent 0 100 521 A2.
It can be assumed, in analogy to gC-1, that gC-2 is of importance for the formation of protective antibodies against HSV-2 infections.
It has been found that gC-2 can be prepared by isoLation of the section of the HSV-2 genome which codes for gC-2, and j 15 transfer this DNA into a microorganism in which it is re- Sti plicated and expressed. The foLLowing text describes a rlrr process for the expression of antigenic determinants which are specific to gC-2 but which can be not onLy used as vaccines against HSV-2 but also empLoyed in diagnosis.
t S 20 Thus the invention relates to a process for the preparation of a polypeptide having an antigenic determinant of the herpes simplex virus, by isolation of a section of the genome, which codes for this type of polypeptide, from the deoxyribonucleic acid of this virus, binding of the section i. 25 of the genome enzymatically to a vector -DNA- introduction of this combined deoxyribonucleic S| acid into a cell in which the combined deoxyribonucleic acid brings about the production of this polypeptide, and obtaining of this polypeptide, which process comprises the section of the genome being the SaLI fragment (section of about 0.62-0.64 map units of the genome) of herpes simplex virus type 2.
The section of the genome is preferably a part of the SaLI fragment of the HSV-2 DNA which codes for gC.
In::r- iiara~rl-;?~ jllC__ 3 The vector is preferably plasmid pBR322, a plasmid of the pUC series, or a derivative thereof.
In an advantageous manner, the vector can be an SV40 vector or a bovine papillomavirus vectbr.
The combined deoxyribonucleic acid can advantageously contain the entire sequence coding for gC, or parts thereof, and LacZ or parts of lacZ, so that a gC-2 and P-gal fusion protein is obtained.
The cell which is used for expression can be an animal or human cell or a microorganism.
The cells are preferably those of Escherichia coli. They can also be cells of a microorganism of the genus Bacillus, Pseudomonas, Streptomyces or Actinomyces, or a yeast of the genus Saccharomyces, KLuyveromyces, SchizosaccharomycesorHansenula.
13 The process according to the invention is described in detail below.
1. Isolation of virus-specific nucleic acid, and cloning of the gC-2 gene The virological methods (propagation and isolation of 20 the virus) and molecular-biological methods (cloning of DNA fragments into plasmids) are generally known and are described in textbooks (Maniatis et al., 1982, Molecular Laboratory, Cold Spring Harbor, and have been applied to the cloning of the gC-1 gene in European Patent 0 100 521 A2.
SI
9 9
I
I
9 99 t9 I 99 It 99 9 9 9999 The SalI DNA fragment of HSV-2 coding for gC-2 was cloned into pUC 18 (Vieira and Messing, 1982, Gene, 19, 259-268) (Fig. 1, pUC 18.gC-2).
4- -4-1 2. Cloning of portions coding for gC-2 into pUC 18 The BalI/EcoRI fragment of pUC 18.gC-2, which is about 1200 bp in size and codes for about 82% of the gC-2, was, after cleavage of the plasmid with SmaI and EcnR,I, cloned into pUC 18 (Fig. The new plasmid, called pOB.C21, contains the section of the genome coding for gC-2 (amino acids 25-418) without the amino-terminal leader sequence and carboxyl-terminal membrane-anchoring 1 sequence. The absence of these hydrophobic portions of j 10 the protein ought to facilitate the expression of gC-2 in bacteria, since experience has shown that sequences I of this type may have andetrirrental effect on the growth Sof bacteria. The SmaI/EcoRI fragment which codes for S gC-2 is flanked in the pOB.C21 by various recognition I 15 sequences for endonucleases, and can be cut out of the plasmid with suitable enzymes and inserted into other I t plasmids so that it can bring about the synthesis of the appropriate protein.
3. Expression of gC-2 The preparation of proteins which comprise gC-2 and a portion, of various sizes, of -gal is described below (Kalnins et al., 1983, EMBO J. 4, 593-597) (Fig. 3).
SpOB.C23 was obtained by ligation into pUC 19, which had been digested with BamHI and EcoRI, of the BamHI/EcoRI 25 fragment of pOB.C21 which codes for gC-2.
"4 t pOB.C23 codes for a -gal:gC-2 fusion protein containing only 4 amino acids of P-gal. In order to obtain pOBC33, the same BamHI/EcoRI fragment from DOB.C21 which codes for gC-2 was cloned into pBD2 which has been cleaved with BamHI and HindIII. The EcoRI and the HindIII sites had been "filled" with polymerase I (large fragment) and dNTP before the ligation. The portion of A -gal coded for by pBD2 is 375 amino acids. pBD2 had been produced by ligation of the lacZ portion, coding for the amino terminal end, on the EcoRI/EcoRV fragment
I
of pUK 230 (Koenen et al., 1982, EMBO 1, 509-512) into pUC8 after hydrolysis of the plasmid with SmaI and 'i EcoRI. pOB.C33 thus codes for the fusion protein -gal:: gC-2, in which the portion of 3-gal comprises 375 amino acids. pOB.C43, which codes for a portion of 583 amino acids of 3 -gal in the 3-gaL::gC-2 fusion protein, was prepared as follows: pWR 590 (Guo et al., 1984, Gene, 29, 251-254 was digested with SalI, and treated with polymerase I and with BamHI. The fragment of pOB.C21 coding for gC-2 was now, after hydrolysis with EcoRI, polymerase I treatment and BamHI hydrolysis, inserted into this plasmid.
SIn order to obtain pOB.C53, the DNA fragment coding for V t gC-2 was likewise modified as for the formation of 15 pOB.C43, and cloned into pUR 278 (Ruther and MLLer-HiLL, S 1983, EMBO 2, 1791-1794) which had been subjected Sto pretreatment with XbaI, polymerase I and BamHI. Thus, pOB.C53 codes for a protein which comprises all cff/-gal and gC-2.
E. coli cells which contain the plasmids pOB.C23, pOB.C33, pOB.C43 or pOB.C53 were Labeled with S-methionine, and the synthesized products were identified.
S; Apart from pOB.C23, all the plasmids are abLe to bring Sabout the synthesis in E. coli of a fusion protein of 25 the -gal::gC-2 type which is controlled by the lac ,;promoter and is recognized by goat anti-HSV-2 antiserum but not by goat anti-HSV-1 antiserum. Thus, in E. coli, gC-2 is protected from proteolysis, and is immunoreactive, owing to fusion with 3 -gal portions of various sizes.
Other possibilities of stable expression of gC-2 in E. coli comprise the synthesis of a fusion protein which is composed of qC-2 and P-gal and in which the gC-2 portion is not -rfsed to the carboxyterminal end but to the amino terminal end of/ -gal or in which is gC-2 flanked on both sides by P-gal (-gal::gC-2:: 6 P i-gal). It is possible for not only -gaL but also other proteins, such as, for example, the bacteriophage repressor protein cI or parts thereof, as a constituent of fusion proteins to protect heterologous proteins in E. coli from proteoLysis Tripartite fusion proteins of the type cI::gene product -gaL Shave proved to be very stable, so that, for example, synthesis of gC-2 as a fusion protein of the type cI::gC-2:: -gal also appears to be suitable.
The fusion proteins containing P-gal can be purified by i affinity chromatography or on immunoabsorption columns I and, if the -gal portion is desired, can be subjected to cleavage with cyanogen bromide or by proteoLysis, so I that gC-2 peptides which are free of other constituents I 15 can be isolated. Compared withy -gal in its entirety, S' shortened portions of /-gal have the advantage as a con- .I stituent of fusion proteins that, in the purification T processes, fewer peptides are produced after the cleavage with cyanogen bromide, and the gC-2-specific peptide is more straightforward to purify.
i Specific proteolytic cleavage to remove undesired antii t genic portions of the gC--2 fusion proteins can also be t achieved by incorporation into the expression plasmids of oligonucleotides which contain the recognition set 25 quences of specific proteases. Specific recognition rej gions of this type may be as follows: Ile-GLu-GLy-Arg, inter alia for factor XIIIa; Phe-Val- Arg or Gly-Pro-Arg, inter alia for thrombin, Val-GLy-Arg or Glu-Gly-Arg, inter alia for urokinase, or Lys-Gly-Arg for C1 esterase. Following the treatment of the gC-2 fusion proteins with the particular protease, the desired gC-2 antigens can then be obtained in the pure form from the resulting peptide mixture by affinity chromato graphy.
It is also possible to use eukaryotic cells (yeasts, or L~ayuraauJP-=r.a~ d ii ii j
U::
44 1) #i /1c ft 2 4. r 4 roo ti iFT 0 7 animal or human celL Lines) for the preparation of immunorelevant gC-2 antigens. For this purpose, the gC-2 gene is modified such that the hydrophobic amino acid sequences at the COOH terminal end, which serve to anchor the protein in the cell membrane (or virus coat) are removed. This takes place by elimination of the appropriate coding DNA sections of the gC-2 gene using restriction endonucleases and by introduction of a synthetic stop codon (TAG, TGA, TAA). The gene thus modified is now integrated into an expression vector, for example SV40 vectors or bovine papilloma virus (BPV) vectors, under the control of efficient promoters, some of which can be regulated (for example metallothionein promoters) The resulting recombinant plasmid is now intro- 15 duced into suitable host cells by transfection. As a rule, a selectable marker gene is used for this purpose, for example that for dihydrofolate reductase in the case of dhfr cells (for example CHO cells), followed by amplification using methotrexate, HSV-1 or thymidine ki- 20 nase in the case of tk cells (for example mouse fibroblast cells, "L cells"), antibiotic-resistance genes, for example against G418, or even transforming genes (oncogenes), for example in the case of focus-formation following BPV transfection. The marker gene can be lo- 25 cated together with the gC-2 gene on one plasmid, but can also be located on a second plasmid and then introduced into the host cell by cotransfection. It is possible in this way to establish cell lines which secrete a gC-2 antigen which is shortened by the anchoring sequence and which can then be obtained from the culture supernatants. The expression of gC-2 in animal cells has the advantage of correct processing, for example glycosylation, and thus a possibly improved antigenicity, for example by increased stability in the circulation following vaccination.
Because of its antigenic properties, a protein of this type is suitable for obtaining antisera and/or vaccines, where appropriate using adjuvants and customary auxilif c; t F*.
I t r E -e -111 a +rrarrr=--- .4 i i i -8 aries. The prepared recombinant gC-2 can be used alone as a HSV-2-specific vaccine or be mixed with other gLycoproteins such as, for example, gB or gD, so that the vaccine is active against both HSV-1 and HSV-2.
It can also be used for diagnostic purposes, for example for detection of antibodies against HSV-2. Antisera against a protein of this type can be used for the detection of HSV-2 itself.
a sa r r~, 9
S
C a a a a
Claims (9)
1. A process for the preparation of a polypeptide having antigenic determinants of the herpes simplex virus, by isolation of a section of the genon'e, which codes for this type of polypeptide, from the deoxyribonucleic acid of this virus, binding of the section of the genome enzymatically to the deoxyribonucleic acid of a vector, introduction of this combined deoxyribonucleic acid into a cell in which the combined deoxyribonucleic acid brings about the production of this polypeptide, in a transformed :host, characterised in that the section of the genome being j the SalI fragment (section of about 0.62-0.64 map units of v tne genome) of herpes simplex virus type 2. t t
2. The process as claimed in claim 1, wherein the section of the genome is a part of the SalI fragment (section of about 0.62-0.64 map units of the genome) of the HSV-2 DNA which codes for gC.
3. The process as claimed in claim 1, wherein the 'vector is the plasmid pBR322, a pUC or a derivative thereof.
4. The process as claimed in claim 1, wherein the combined deoxyribonucleic acid is composed of the section of 0.62-0.64 map units of the HSV-2 genome and of a part of the j vectors pBR322 or pUC. The process as claimed in claim 1, wherein the combined deoxyribonucleic acid contains the entire sequence coding for gC, or parts thereof, and lacZ or parts of lacZ.
6. The process as claimed in claim 1, wherein the cell is Escherichia coli. *v I I J I: I 10
7. The process as claimed in claim 1, wherein the cell is a microorganism of the genus Bacillus, Pseudomonas, Streptomyces or Actinomyces, or a yeast of the genus Saccharomyces, Kluyveromyces, Schizosacchamyces or Hansenula.
8. A process as claimed in claim 1, wherein the cell is an animal or human cell.
9. The process as claimed in claim 1, wherein the vector is an SV40 vector or a bovine papilloma virus vector. A polypeptide obtained by the method of claim 1.
11. The use of a polypeptide as claimed in claim 10 for obtaining an antigenic serum and/or for the preparation of a vaccine or of a diagnostic agent. ft I I t1 I I iii C II t *f I Ii I C -rl C: *Ii I: Ii *r 1 Ie ii C II C DATED this 10th day of August, 1989. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS Queen Street, MELBOURNE. VIC. 3000. AUSTRALIA. DBM:JMW:JZ (8.43)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853510734 DE3510734A1 (en) | 1985-03-25 | 1985-03-25 | METHOD FOR PRODUCING A HERPES SIMPLEX VIRUS TYPE 2-SPECIFIC ANTIQUE, MEANS THAT ARE SUITABLE FOR THIS, AND USE OF THIS ANTIQUE |
| DE3510734 | 1985-03-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU5507786A AU5507786A (en) | 1986-10-02 |
| AU592076B2 true AU592076B2 (en) | 1990-01-04 |
Family
ID=6266239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU55077/86A Ceased AU592076B2 (en) | 1985-03-25 | 1986-03-24 | Process for the preparation of an antigen specific to herpes simplex virus type 2, agents suitable for this purpose, and the use of this antigen |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0199091B1 (en) |
| JP (1) | JPS61227794A (en) |
| AT (1) | ATE49999T1 (en) |
| AU (1) | AU592076B2 (en) |
| DE (2) | DE3510734A1 (en) |
| DK (1) | DK135686A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1678783A (en) * | 1982-07-20 | 1984-01-26 | Molecular Genetics, Inc. | Production of herpes simplex viral proteins |
| AU1795283A (en) * | 1982-09-30 | 1984-04-05 | Up-Right Inc. | Scaffold propulsion unit |
| AU4725885A (en) * | 1984-08-24 | 1986-03-24 | University Patents Inc. | Herpes virus specific immunological materials and methods |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT77014B (en) * | 1982-07-20 | 1986-01-24 | Molecular Genetics Inc | Production of herpes simplex viral proteins |
| DE3228501A1 (en) * | 1982-07-30 | 1984-02-02 | Behringwerke Ag, 3550 Marburg | METHOD FOR PRODUCING A HERPES ANTIGENT, MEANS THAT ARE SUITABLE FOR ITS PRODUCTION, AND METHOD FOR THE PRODUCTION THEREOF AND THE USE OF THIS ANTIQUE |
-
1985
- 1985-03-25 DE DE19853510734 patent/DE3510734A1/en not_active Withdrawn
-
1986
- 1986-03-19 DE DE8686103754T patent/DE3668642D1/en not_active Expired - Fee Related
- 1986-03-19 JP JP61061927A patent/JPS61227794A/en active Pending
- 1986-03-19 EP EP86103754A patent/EP0199091B1/en not_active Expired - Lifetime
- 1986-03-19 AT AT86103754T patent/ATE49999T1/en active
- 1986-03-24 DK DK135686A patent/DK135686A/en not_active Application Discontinuation
- 1986-03-24 AU AU55077/86A patent/AU592076B2/en not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1678783A (en) * | 1982-07-20 | 1984-01-26 | Molecular Genetics, Inc. | Production of herpes simplex viral proteins |
| AU1795283A (en) * | 1982-09-30 | 1984-04-05 | Up-Right Inc. | Scaffold propulsion unit |
| AU4725885A (en) * | 1984-08-24 | 1986-03-24 | University Patents Inc. | Herpes virus specific immunological materials and methods |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0199091B1 (en) | 1990-01-31 |
| DE3668642D1 (en) | 1990-03-08 |
| ATE49999T1 (en) | 1990-02-15 |
| DK135686D0 (en) | 1986-03-24 |
| DK135686A (en) | 1986-09-26 |
| DE3510734A1 (en) | 1986-09-25 |
| EP0199091A1 (en) | 1986-10-29 |
| AU5507786A (en) | 1986-10-02 |
| JPS61227794A (en) | 1986-10-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0127839B1 (en) | Method for producing a recombinant baculovirus expression vector | |
| US4879236A (en) | Method for producing a recombinant baculovirus expression vector | |
| US5851533A (en) | Vaccine based on membrane bound proteins and process for making them | |
| Lasky et al. | Protection of mice from lethal herpes simplex virus infection by vaccination with a secreted form of cloned glycoprotein D | |
| EP0236145B1 (en) | Processes for the production of hcmv glycoproteins, antibodies thereto and hcmv vaccines, and recombinant vectors therefor | |
| EP0192902B1 (en) | Dna sequences from varicella-zoster virus, vectors and hosts containing them, the corresponding polypeptides and the compositions containing them | |
| EP0486170B1 (en) | Baculoviral expression system comprising procaryotic leader sequence | |
| CA1273884A (en) | Process for the preparation of a herpes antigen, an agent suitable for this and a process for its preparation, and the use of this antigen | |
| US5788962A (en) | DNA sequences coding for mycoplasma hyopneumoniae surface antigens, corresponding proteins and use in vaccines and diagnostic procedures | |
| JP3435574B2 (en) | Equine herpesvirus (EHV) containing foreign DNA, methods for its preparation and its use in vaccines | |
| EP0538341B1 (en) | Ehv-4 glycoprotein vaccine | |
| US7264817B1 (en) | Immunogenic composition based on a truncated derivative of a membrane bound protein and process for making it | |
| US6162620A (en) | Processes for the production of HCMV glycoproteins, antibodies thereto and HCMV vaccines, and recombinant vectors therefor | |
| EP0769056B1 (en) | NON-SPLICING VARIANTS OF gp350/220 | |
| AU592076B2 (en) | Process for the preparation of an antigen specific to herpes simplex virus type 2, agents suitable for this purpose, and the use of this antigen | |
| Battista et al. | Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris | |
| Chikateru et al. | Expression of herpes simplex virus glycoprotein B gene in yeast | |
| Jacquet et al. | Purification and characterization of recombinant varicella-zoster virus glycoprotein gpII, secreted by Chinese hamster ovary cells | |
| US5674735A (en) | DNA encoding the EHV-4 gH or gC glycoprotein | |
| Watson et al. | Expression of Herpes simplex virus type 1 and type 2 glycoprotein D genes using the Escherichia coli lac promoter | |
| RU2151801C1 (en) | Recombinant plasmid dna pul 32 hcmv determining expression of human cytomegalovirus gene ul 32 fragment encoding hydrophilic site of basic matrix protein pp 150 in escherichia coli bacterium cells |