CA2274900A1 - Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine - Google Patents
Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine Download PDFInfo
- Publication number
- CA2274900A1 CA2274900A1 CA 2274900 CA2274900A CA2274900A1 CA 2274900 A1 CA2274900 A1 CA 2274900A1 CA 2274900 CA2274900 CA 2274900 CA 2274900 A CA2274900 A CA 2274900A CA 2274900 A1 CA2274900 A1 CA 2274900A1
- Authority
- CA
- Canada
- Prior art keywords
- ehv
- gene
- virus
- recombinant
- deletion
- 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.)
- Abandoned
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 101
- 241000701081 Equid alphaherpesvirus 1 Species 0.000 title claims abstract description 98
- 229960005486 vaccine Drugs 0.000 title claims abstract description 41
- 239000000203 mixture Substances 0.000 claims abstract description 22
- 238000009472 formulation Methods 0.000 claims abstract description 16
- 241000700605 Viruses Species 0.000 claims description 86
- 238000012217 deletion Methods 0.000 claims description 54
- 230000037430 deletion Effects 0.000 claims description 50
- 241001465754 Metazoa Species 0.000 claims description 23
- 239000002773 nucleotide Substances 0.000 claims description 20
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- 208000015181 infectious disease Diseases 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 230000002238 attenuated effect Effects 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 230000002163 immunogen Effects 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 8
- 241000283073 Equus caballus Species 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000011321 prophylaxis Methods 0.000 claims description 3
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 28
- 108090000765 processed proteins & peptides Proteins 0.000 description 20
- 238000002255 vaccination Methods 0.000 description 16
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 239000012634 fragment Substances 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 108700026244 Open Reading Frames Proteins 0.000 description 11
- 101150038678 0.7 gene Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 101150066555 lacZ gene Proteins 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000002245 particle Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 244000309466 calf Species 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 239000007758 minimum essential medium Substances 0.000 description 6
- 210000002345 respiratory system Anatomy 0.000 description 6
- 210000002845 virion Anatomy 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 230000003472 neutralizing effect Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 210000001944 turbinate Anatomy 0.000 description 5
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 108020005202 Viral DNA Proteins 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 210000000956 olfactory bulb Anatomy 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000029812 viral genome replication Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000283086 Equidae Species 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 206010058874 Viraemia Diseases 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002480 immunoprotective effect Effects 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000004927 skin cell Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101100442582 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) spe-1 gene Proteins 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-N sodium;5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,6-trione Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)NC1=O QGMRQYFBGABWDR-UHFFFAOYSA-N 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 229960004854 viral vaccine Drugs 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000700587 Alphaherpesvirinae Species 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241000272194 Ciconiiformes Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000698776 Duma Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101150017040 I gene Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 208000035999 Recurrence Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000024932 T cell mediated immunity Effects 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
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002941 microtiter virus yield reduction assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000000427 trigeminal ganglion Anatomy 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- C12N2710/16711—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
-
- 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
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16761—Methods of inactivation or attenuation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Vaccine formulation comprising EHV-1 gene 71 dysfunctional mutant and uses thereof.
Description
RECOMBINANT EQUINE HERPESVIRUS TYPE 1 (EHV-1 ) COMPRISING A DYSFUNCTIONAL GENE
AND USE THEREOF AS A VACCINE
The present invention relates to a viral vaccine containing an attenuated EHV-1 virus comprising a gene deletion in the genome thereof, uses thereof and methods of treating EHV-1 related disease. In particular, the invention relates to a viral vaccine composition for use against Equine herpesvirus type 1 ( EHV-1 ) .
EHV-1 is a member of the subfamily alphaherpesvirinae and is a significant viral pathogen of horses. Clinical problems caused by EHV-1 include respiratory disease, abortion and neurological disorders (Bryans J.T., and Allen, G.P., Kluwer Academic Publishers, Norwell MA, 1989). As such, EHV-1 is responsible for significant economic losses within the equine industry.
The EHV-1 genome is a linear double-stranded DNA molecule of approximately 150 kbp in size which can be divided into two covalently linked components: the long and short regions. The long region consists of an unique sequence (UL) flanked by a small inverted repeat (IRS and TRL). The short region comprises an unique sequence (Us) flanked by a large inverted repeat (IRS
and TRs) .
EHV-1 occurs as pathogenic and non-pathogenic strains and recently, the complete DNA sequence of a pathogenic strain, Ab4, has been determined and the sequence has been deposited with the GenBank Library under Accession No. M 86664 (Telford, E.A.R. et al., Virology 189, pp. 304-316 (1990)).
AND USE THEREOF AS A VACCINE
The present invention relates to a viral vaccine containing an attenuated EHV-1 virus comprising a gene deletion in the genome thereof, uses thereof and methods of treating EHV-1 related disease. In particular, the invention relates to a viral vaccine composition for use against Equine herpesvirus type 1 ( EHV-1 ) .
EHV-1 is a member of the subfamily alphaherpesvirinae and is a significant viral pathogen of horses. Clinical problems caused by EHV-1 include respiratory disease, abortion and neurological disorders (Bryans J.T., and Allen, G.P., Kluwer Academic Publishers, Norwell MA, 1989). As such, EHV-1 is responsible for significant economic losses within the equine industry.
The EHV-1 genome is a linear double-stranded DNA molecule of approximately 150 kbp in size which can be divided into two covalently linked components: the long and short regions. The long region consists of an unique sequence (UL) flanked by a small inverted repeat (IRS and TRL). The short region comprises an unique sequence (Us) flanked by a large inverted repeat (IRS
and TRs) .
EHV-1 occurs as pathogenic and non-pathogenic strains and recently, the complete DNA sequence of a pathogenic strain, Ab4, has been determined and the sequence has been deposited with the GenBank Library under Accession No. M 86664 (Telford, E.A.R. et al., Virology 189, pp. 304-316 (1990)).
The genome is 150,223 by in size and contains 81 open reading frames predicted to encode polypeptides. The sizes of its components are UL, 112,870 bp; TRL/IR~, 32 bp; U5, 11,861 bp;
and IRS/TR~, 12,714 bp. Interestingly, there are five genes, 1, 2, 67, 71, and 75, which have no homologues in any of the herpesviruses sequenced to date; i.e. they are unique to EHV-1.
Each of the genes 1, 2, 67, 71 and 75 is believed to encode a protein, however, the function of the individual proteins is unclear. Recently it has been demonstrated that the EHV-1 gene 71 product is involved in adsorption/penetration of virus and egress of virus from infected cell nuclei (Sun Y. et al. , Journal of General Virology 77 pp. 493-500 (1996)).
The prior art does not teach or suggest the use of EHV-1 gene 71 deletion mutants comprising a dysfunctional gene 71 region in the manufacture and use of vaccines against EHV-1 related disease.
Control by vaccination of EHV-1 infection has been a long-sought goal. Current EHV-1 vaccines comprise chemically inactivated virus vaccines and modified live virus vaccines.
Inactivated vaccines generally induce a low level of immunity and require additional immunisations and are expensive to produce.
The use of such vaccines carries with it the risk that some infectious viral particles may survive the inactivation process and cause disease after administration to the animal.
In general, attenuated live virus vaccines are preferred because they evoke a longer-lasting immune response (often both humoral and cellular) and are easier to produce. Live attenuated EHV-1 vaccines are available which are based on live EHV-1 virus attenuated by serial passage of virulent strains in tissue culture. However, serial passaging of virulent strains can give rise to uncontrolled mutations of the viral genome, resulting in a population of virus particles heterogeneous in their virulence and immunising properties. It is also known that such EHV-1 attenuated live virus vaccines can revert to virulence resulting in disease of the inoculated animals and the possible spread of pathogen to other animals.
The present inventors have now identified a suitable strain of live EHV-1 mutant virus comprising a dysfunctional region of the EHV-1 genome located within the short unique region thereof, which mutant may be used in a live EHV-1 vaccine formulation.
Specifically, the inventors have found that EHV-1 mutants dysfunctional for production of a protein encoded by gene 71 can be used in a live EHV-1 vaccine formulation. Such mutants are shown to be substantially less virulent than wild type EHV-1 viruses. Furthermore, gene 71 has been found to be non-essential for EHV-1 growth in cell culture (Sun Y. and Brown S.M. , Virology 199 pp. 448-452 (1994)). The inventors have also found that EHV-1 viruses comprising dysfunctional gene 71 regions of their genome are immunogenic. Such viruses are indicated for use as components in vaccine formulations or therapeutic compositions against EHV-1 infection. Accordingly, it is with EHV-1 viruses w comprising a dysfunctional region located in the gene 71 protein coding region, and in particular between nucleotides 129,096 and 131,489 of the native genome which the present invention is concerned.
WO 98!26049 PCT/GB97/03327 Statement of Invention A first aspect of the present invention provides a vaccine formulation comprising a live recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the US region of the virus genome and a pharmaceutically acceptable carrier.
A "dysfunctional gene 71 region" is one which is substantially incapable of coding for the native polypeptide or a functional equivalent. Thus, a "dysfunctional gene 71 regi9n"
means that the gene 71 region has been modified by deletion, insertion or substitution (or other change in the DNA sequence such as by rearrangement) such that the gene 71 region does not express a native EHV-1 gene 71 polypeptide or a functionally equivalent product thereof. It is known that EHV-1 gene 71 encodes a 797 amino acid polypeptide and that the peptide is an O-linked 192 kDa glycoprotein (Sun, Y. et al., Journal of General Virology 75, pp. 3117-3126 (1994)). Thus, vaccine formulations comprising modified EHV-1 viruses of the invention may include viruses modified in one or more ways via recombinant DNA
technology. Examples of the types of modifications which may be made include:
(i) A deletion of the entire gene 71 from the genome of an EHV-1 wild type virus. For example, a deletion of the nucleotide sequence from the wild type EHV-1 genome between about nucleotide 129,096 to about nucleotide 131,489:
(ii) A deletion of a portion of gene 71 from the genome of an EHV-1 wild type virus. A "portion of the gene 71" means a deletion which is sufficient to render any polypeptide encoded by the gene 71 deletion mutant and expressed thereby substantially incapable of a physiological activity similar to that of the native polypeptide produced by wild type EHV-1. The deletion may be between 50% and 1000 of the nucleotide sequence located between about nucleotides 129,096 and 131,489 of the wild type EHV-1 genome. The deletion may be from 70% to 1000 of the gene 71 nucleotide sequence, or the deletion may be from about 70 o to 90% of the gene 71 nucleotide sequence, for example, about 80% of the gene 71 nucleotide sequence.
(iii) The deletion of the or a portion of gene 71 as described in {i) and (ii) above will leave a "gap" in the EHV-1 genome corresponding to the gene 71 open reading frame (ORF) or a portion thereof . A suitable gene or genes may be inserted into the "gap" such as a marker gene. Suitable marker genes include but are not restricted to enzyme marker genes, for example the lac-Z gene from E.coli, antibiotic marker genes such as hygromycin, neomycin and the like. Such marker genes are commonly employed in the art. Generally, marker genes, if any, which may be employed in a gene 71 deletion mutant of the invention should be such so as to not cause substantial deleterious or long lasting side-effects to a recipient animal.
In a preferment, the "gap" made by the deletion of the or a portion of the gene 71 from a wild type EHV-1 virus is not filled with a gene insert, the cut ends of the two pieces of the genome being ligated together using conventional recombinant DNA
technology. The skilled addressee will appreciate that the term "deletion mutant" encompasses those situations wherein the "gap"
left by the partial or total deletion of gene 71 may be filled with a gene insert, for example a marker gene or nonsense nucleotide sequence (i.e. a sequence incapable of giving rise to a protein or polypeptide product) or those situations wherein the gap is not filled by a heterologous or other nucleotide sequence.
In such a case, the appropriate free ends of the two pieces of the genome are ligated together.
(iv) The deletion within the gene 71 region may comprise a deletion of a small number of nucleotides, for example 1, 2 or more nucleotides. Such deletions can be achieved using recombinant DNA technology. Thus, the translational ORF can be altered resulting in the production of a protein which lacks the physiological function of the gene 71 native polypeptide. The skilled addressee will also appreciate that such deletions in the translational ORF of gene 71 may also give rise to a dysfunctional gene 71 which is incapable of coding for a whole polypeptide, truncated peptide or even any peptide. Such proteins, if produced, generally lack the physiological functionality of the protein product of a normal gene 71 ORF.
(v) Nucleotide insertions can also be made in the EH~I-1 gene 71 region using recombinant DNA technology which gives rise to dysfunctional gene 71 polypeptides substantially incapable of functional activity. For example, stop codons may be inserted into the gene 71 region, resulting in the production of non-functional fragments of the polypeptide encoded by native gene 71.
The skilled addressee will appreciate that such nucleotide insertions can be of any length from 1 or more nucleotides to a number of nucleotides making up, for example, nonsense nucleotide sequences which can have the effect of altering the translational ORF resulting in the non-production of a polypeptide or indeed, the production of a protein lacking the physiological function of the gene 71 native polypeptide. The skilled addressee will also appreciate that such insertions in the translational ORF of gene 71 may also give rise to a dysfunctional gene 71 which is incapable of coding for a whole polypeptide, truncated peptide or even any peptide. Such proteins, if produced, generally lack physiological functionality.
Naturally, the skilled addressee will appreciate that gene 71 deletions and insertions from nvn-wild type EHV-1 viruses as outlined above are encompassed by the present invention.
In a preferment there is provided a vaccine formulation comprising a live recombinant attenuated immunogenic EHV-1 gene 71 deletion mutant virus and a pharmaceutically acceptable carrier.
In a second aspect of the invention there is provided a live, recombinant EHV-1 comprising a dysfunctional gene 71 region for use as a vaccinating agent or in a vaccine formulation.
Preferably, there is provided a live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus for use as a vaccinating agent or in a vaccine formulation.
The live, recombinant EHV-1 may optionally include an inserted gene positioned at the gene 71 locus in lieu of a substantial portion of gene 71 or the whole of gene 71.
Generally, the vaccine or vaccine formulation is not used on non-pregnant animals because it can give rise to abortigenesis.
WO 98!26049 PCT/GB97103327 In a third aspect of the invention there is provided the use of a live, recombinant EHV-1 virus for producing antibodies or cell mediated immunity to EHV-1 which comprises a dysfunctional gene 71 region located within the US region of the virus genome for the manufacture of an EHV-1 vaccine for the prophylaxis and/or treatment of EHV-1 infection. Preferably, there is provided use of a live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus for the manufacture of an EHV-1 vaccine for the prophylaxis and/or treatment of EHV-1 infecti9n.
Most preferably, the use is in horses.
In a fourth aspect of the invention there is provided a method of treating animals which comprises administering thereto a vaccine composition comprising a live, recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the US region of the virus genome to animals in need thereof. Preferably, the animals are horses. Preferably still, the method of treating animals comprises administering a vaccine composition comprising a recombinant, live, attenuated, immunogenic EHV-1 gene 71 deletion mutant virus to animals in need thereof. Naturally, the vaccine formulation may be formulated for administration by oral dosage (e. g. as an enteric coated tablet), by parenteral injection or otherwise.
The invention also provides a process for preparing a live modified EHV-1 virus vaccine, which process comprises admixing a virus according to the invention with a suitable carrier or adjuvant.
For the preparation of a live attenuated vaccine, standard methodology may be used.
The mode of administration of the vaccine of the invention may be by any suitable route which delivers an immunoprotective amount of the virus of the invention to the subject. However, the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
Generally, the vaccine will usually be presented as a pharmaceutical formulation including a carrier or excipient, for example an injectable carrier such as saline or apyrogenic water.
The formulation may be prepared by conventional means.
The appropriate immunoprotective and non-toxic dose of such a vaccine can be determined readily by those skilled in the art, i.e., the appropriate immunoprotective and non-toxic amount of the virus contained in the vaccine of this invention may be in the range of the effective amounts of antigen in conventional whole virus vaccines. It will be understood, however, that the specific dose level for any particular recipient animal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration;
synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
Embodiments of the invention will now be illustrated by way of the following Figures and Examples.
FiQUre 1:
Schematic representation of the sequence arrangement of EHV-1 DNA and plasmids constructed for gene 71: deletion and substitution. Line 1, EHV-1 genome consisting of UL and US and inverted repeat regions (IRS and TRs). Expanded cloned fragment:
and IRS/TR~, 12,714 bp. Interestingly, there are five genes, 1, 2, 67, 71, and 75, which have no homologues in any of the herpesviruses sequenced to date; i.e. they are unique to EHV-1.
Each of the genes 1, 2, 67, 71 and 75 is believed to encode a protein, however, the function of the individual proteins is unclear. Recently it has been demonstrated that the EHV-1 gene 71 product is involved in adsorption/penetration of virus and egress of virus from infected cell nuclei (Sun Y. et al. , Journal of General Virology 77 pp. 493-500 (1996)).
The prior art does not teach or suggest the use of EHV-1 gene 71 deletion mutants comprising a dysfunctional gene 71 region in the manufacture and use of vaccines against EHV-1 related disease.
Control by vaccination of EHV-1 infection has been a long-sought goal. Current EHV-1 vaccines comprise chemically inactivated virus vaccines and modified live virus vaccines.
Inactivated vaccines generally induce a low level of immunity and require additional immunisations and are expensive to produce.
The use of such vaccines carries with it the risk that some infectious viral particles may survive the inactivation process and cause disease after administration to the animal.
In general, attenuated live virus vaccines are preferred because they evoke a longer-lasting immune response (often both humoral and cellular) and are easier to produce. Live attenuated EHV-1 vaccines are available which are based on live EHV-1 virus attenuated by serial passage of virulent strains in tissue culture. However, serial passaging of virulent strains can give rise to uncontrolled mutations of the viral genome, resulting in a population of virus particles heterogeneous in their virulence and immunising properties. It is also known that such EHV-1 attenuated live virus vaccines can revert to virulence resulting in disease of the inoculated animals and the possible spread of pathogen to other animals.
The present inventors have now identified a suitable strain of live EHV-1 mutant virus comprising a dysfunctional region of the EHV-1 genome located within the short unique region thereof, which mutant may be used in a live EHV-1 vaccine formulation.
Specifically, the inventors have found that EHV-1 mutants dysfunctional for production of a protein encoded by gene 71 can be used in a live EHV-1 vaccine formulation. Such mutants are shown to be substantially less virulent than wild type EHV-1 viruses. Furthermore, gene 71 has been found to be non-essential for EHV-1 growth in cell culture (Sun Y. and Brown S.M. , Virology 199 pp. 448-452 (1994)). The inventors have also found that EHV-1 viruses comprising dysfunctional gene 71 regions of their genome are immunogenic. Such viruses are indicated for use as components in vaccine formulations or therapeutic compositions against EHV-1 infection. Accordingly, it is with EHV-1 viruses w comprising a dysfunctional region located in the gene 71 protein coding region, and in particular between nucleotides 129,096 and 131,489 of the native genome which the present invention is concerned.
WO 98!26049 PCT/GB97/03327 Statement of Invention A first aspect of the present invention provides a vaccine formulation comprising a live recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the US region of the virus genome and a pharmaceutically acceptable carrier.
A "dysfunctional gene 71 region" is one which is substantially incapable of coding for the native polypeptide or a functional equivalent. Thus, a "dysfunctional gene 71 regi9n"
means that the gene 71 region has been modified by deletion, insertion or substitution (or other change in the DNA sequence such as by rearrangement) such that the gene 71 region does not express a native EHV-1 gene 71 polypeptide or a functionally equivalent product thereof. It is known that EHV-1 gene 71 encodes a 797 amino acid polypeptide and that the peptide is an O-linked 192 kDa glycoprotein (Sun, Y. et al., Journal of General Virology 75, pp. 3117-3126 (1994)). Thus, vaccine formulations comprising modified EHV-1 viruses of the invention may include viruses modified in one or more ways via recombinant DNA
technology. Examples of the types of modifications which may be made include:
(i) A deletion of the entire gene 71 from the genome of an EHV-1 wild type virus. For example, a deletion of the nucleotide sequence from the wild type EHV-1 genome between about nucleotide 129,096 to about nucleotide 131,489:
(ii) A deletion of a portion of gene 71 from the genome of an EHV-1 wild type virus. A "portion of the gene 71" means a deletion which is sufficient to render any polypeptide encoded by the gene 71 deletion mutant and expressed thereby substantially incapable of a physiological activity similar to that of the native polypeptide produced by wild type EHV-1. The deletion may be between 50% and 1000 of the nucleotide sequence located between about nucleotides 129,096 and 131,489 of the wild type EHV-1 genome. The deletion may be from 70% to 1000 of the gene 71 nucleotide sequence, or the deletion may be from about 70 o to 90% of the gene 71 nucleotide sequence, for example, about 80% of the gene 71 nucleotide sequence.
(iii) The deletion of the or a portion of gene 71 as described in {i) and (ii) above will leave a "gap" in the EHV-1 genome corresponding to the gene 71 open reading frame (ORF) or a portion thereof . A suitable gene or genes may be inserted into the "gap" such as a marker gene. Suitable marker genes include but are not restricted to enzyme marker genes, for example the lac-Z gene from E.coli, antibiotic marker genes such as hygromycin, neomycin and the like. Such marker genes are commonly employed in the art. Generally, marker genes, if any, which may be employed in a gene 71 deletion mutant of the invention should be such so as to not cause substantial deleterious or long lasting side-effects to a recipient animal.
In a preferment, the "gap" made by the deletion of the or a portion of the gene 71 from a wild type EHV-1 virus is not filled with a gene insert, the cut ends of the two pieces of the genome being ligated together using conventional recombinant DNA
technology. The skilled addressee will appreciate that the term "deletion mutant" encompasses those situations wherein the "gap"
left by the partial or total deletion of gene 71 may be filled with a gene insert, for example a marker gene or nonsense nucleotide sequence (i.e. a sequence incapable of giving rise to a protein or polypeptide product) or those situations wherein the gap is not filled by a heterologous or other nucleotide sequence.
In such a case, the appropriate free ends of the two pieces of the genome are ligated together.
(iv) The deletion within the gene 71 region may comprise a deletion of a small number of nucleotides, for example 1, 2 or more nucleotides. Such deletions can be achieved using recombinant DNA technology. Thus, the translational ORF can be altered resulting in the production of a protein which lacks the physiological function of the gene 71 native polypeptide. The skilled addressee will also appreciate that such deletions in the translational ORF of gene 71 may also give rise to a dysfunctional gene 71 which is incapable of coding for a whole polypeptide, truncated peptide or even any peptide. Such proteins, if produced, generally lack the physiological functionality of the protein product of a normal gene 71 ORF.
(v) Nucleotide insertions can also be made in the EH~I-1 gene 71 region using recombinant DNA technology which gives rise to dysfunctional gene 71 polypeptides substantially incapable of functional activity. For example, stop codons may be inserted into the gene 71 region, resulting in the production of non-functional fragments of the polypeptide encoded by native gene 71.
The skilled addressee will appreciate that such nucleotide insertions can be of any length from 1 or more nucleotides to a number of nucleotides making up, for example, nonsense nucleotide sequences which can have the effect of altering the translational ORF resulting in the non-production of a polypeptide or indeed, the production of a protein lacking the physiological function of the gene 71 native polypeptide. The skilled addressee will also appreciate that such insertions in the translational ORF of gene 71 may also give rise to a dysfunctional gene 71 which is incapable of coding for a whole polypeptide, truncated peptide or even any peptide. Such proteins, if produced, generally lack physiological functionality.
Naturally, the skilled addressee will appreciate that gene 71 deletions and insertions from nvn-wild type EHV-1 viruses as outlined above are encompassed by the present invention.
In a preferment there is provided a vaccine formulation comprising a live recombinant attenuated immunogenic EHV-1 gene 71 deletion mutant virus and a pharmaceutically acceptable carrier.
In a second aspect of the invention there is provided a live, recombinant EHV-1 comprising a dysfunctional gene 71 region for use as a vaccinating agent or in a vaccine formulation.
Preferably, there is provided a live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus for use as a vaccinating agent or in a vaccine formulation.
The live, recombinant EHV-1 may optionally include an inserted gene positioned at the gene 71 locus in lieu of a substantial portion of gene 71 or the whole of gene 71.
Generally, the vaccine or vaccine formulation is not used on non-pregnant animals because it can give rise to abortigenesis.
WO 98!26049 PCT/GB97103327 In a third aspect of the invention there is provided the use of a live, recombinant EHV-1 virus for producing antibodies or cell mediated immunity to EHV-1 which comprises a dysfunctional gene 71 region located within the US region of the virus genome for the manufacture of an EHV-1 vaccine for the prophylaxis and/or treatment of EHV-1 infection. Preferably, there is provided use of a live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus for the manufacture of an EHV-1 vaccine for the prophylaxis and/or treatment of EHV-1 infecti9n.
Most preferably, the use is in horses.
In a fourth aspect of the invention there is provided a method of treating animals which comprises administering thereto a vaccine composition comprising a live, recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the US region of the virus genome to animals in need thereof. Preferably, the animals are horses. Preferably still, the method of treating animals comprises administering a vaccine composition comprising a recombinant, live, attenuated, immunogenic EHV-1 gene 71 deletion mutant virus to animals in need thereof. Naturally, the vaccine formulation may be formulated for administration by oral dosage (e. g. as an enteric coated tablet), by parenteral injection or otherwise.
The invention also provides a process for preparing a live modified EHV-1 virus vaccine, which process comprises admixing a virus according to the invention with a suitable carrier or adjuvant.
For the preparation of a live attenuated vaccine, standard methodology may be used.
The mode of administration of the vaccine of the invention may be by any suitable route which delivers an immunoprotective amount of the virus of the invention to the subject. However, the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
Generally, the vaccine will usually be presented as a pharmaceutical formulation including a carrier or excipient, for example an injectable carrier such as saline or apyrogenic water.
The formulation may be prepared by conventional means.
The appropriate immunoprotective and non-toxic dose of such a vaccine can be determined readily by those skilled in the art, i.e., the appropriate immunoprotective and non-toxic amount of the virus contained in the vaccine of this invention may be in the range of the effective amounts of antigen in conventional whole virus vaccines. It will be understood, however, that the specific dose level for any particular recipient animal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration;
synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
Embodiments of the invention will now be illustrated by way of the following Figures and Examples.
FiQUre 1:
Schematic representation of the sequence arrangement of EHV-1 DNA and plasmids constructed for gene 71: deletion and substitution. Line 1, EHV-1 genome consisting of UL and US and inverted repeat regions (IRS and TRs). Expanded cloned fragment:
5.8-kb BamHI/EcoRI fragment in pU71 (line 2). Line 3, location and direction of genes. Line 4, sequence arrangement of constructed deletion and substitution plasmid pD71 of gene 71 (line 4). Gaps flanked by solid lines represent deleted regi9ns substituted by lacZ (solid boxes). Pertinent restriction sites:
Ba, Ec, EcoRI; Ms, Sg and Bam HI.
FiQUre 2:
Genome structure of the deletion and substitution mutant.
Restriction enzyme sites within the region of the genome encompassing gene 71 are shown. The wild-type virus genome is represented by line 1, and the deletion and substitution by line 2. Relevant fragments generated following digestion with Smal are shown. Fragment sizes are given in kb. Pertinent retriction sites Ms, Sm, Sg.
Figure 3:
Virus titres far mice inoculated with Ab4p.
FicTUre 4:
Virus titres for mice inoculated with ED 71.
Figwre 5:
Virus titres for mice inoculated with ED 71 revertant.
Figwre 6:
Mean virus titres observed in challenged mice previously immunised with RK cell lysate.
Fiqure 7:
Mean virus titres observed in challenged mice previously immunised with Ab4p.
Figure 8:
Mean virus titres observed in challenged mice previously immunised with ED 71.
Standard methods are as described in "Molecular Cloning -A Laboratory Manual", Second Edition, Sambrook J. et al. Cold Spring Harbor Laboratory Press 1989.
Methods Cells and Virus Baby hamster kidney clone 13 (BHK-21/C13; Macpherson I &
Stoker M.G. (1962) Virology 16 pp. 147-151) were grown as previously described (Brown et al., 1973 J. Gen. Virol. 18 pp.
32-346). EHV-1 strain Ab4 was used as the wild-type strain in this study. Stock preparation of virus at passage 13 was made by low multiplicity infection in equine dermal NBL-6 cells maintained in MEM with 1% fetal calf cerum. Mutant ED71 in which the gene 71 ORF was removed and replaced by the E.coli lacZ gene and the revertant Re71 in which the deletion in ED71 was restored have been previously described (Sun Y. and Brown S.M. (1994) Virology 199 pp. 448-452; Sun Y. et al. (1994) J. Gen. Virol. 75 pp. 3117-3126).
Purification and Quantification of Virions The procedure used was essentially as described by Szilagyi J.F. and Cunningham C. (1992) J. Gen. Virol. 27 pp. 661-668 and Sun et al. (1994) supra. BHK-21/C13 monolayers in roller bottles were infected with virus at a multiplicity of infection (m.o.i.) of 0.01 or 5 p.f.u. per cell. At either 72 hours post-infection (p.i.) or 20 hours p.i. the supernatant was harvested and centrifuged at 2500 r.p.m. for 20 minutes to remove the cell debris. Supernatant virus was pelleted for 2 hours at 12000 r.p.m, and the pellet gently resuspended in 1 ml Eagle's medium without phenol red and laid onto a 5 to 15o Ficoll gradient before centrifuging at 12000 r.p.m. for 2 hours at 4°C. The virion band collected by side puncture was diluted and pelleted at 21000 r.p.m. for 2 hours at 4°C. The virion pellet was gently resuspended in 200 ~,1 of Eagle's medium and stored at -70°C.
Infectivity was determined by titration on BHK-21/C13 cells. The number of particles was determined by eletron microscopy. The specific infectivities (particle p.f.u. ratio) of the purified mutant and wild-type virus are presented in Table 1.
Table 1: The Specific Infectivity (particle/p.f.u. ratio) of ED71, and Wild-Type Virus EHV-1).
Virus Particle/p.f.u.ratio* Particle/p.f.u.
ratio ~
EHV-1 101.7/1 63.8/1 Re71 103.5/1 107.7/1 * Virus from 4x10 BHK-21/C13 cells infected with virus at an m.o.i. of 0.01 p.f.u. per cell and harvested at 72 h.p.i.
.~ Purified virions from 4x10A BHK-21/C13 cells infected with virus at an m.o.i. of 5 p.f.u./cell and harvested at 20 h.p.i.
Suitable gene 71 deletion mutant viruses of the invention were prepared according to the teaching of Sun Y. and Brown S.M.
(supra).
Example I
Briefly, to clone the fragment which contains the gene 71, equine dermal cells (NBL-6) were infected with EHV-1 strain Ab4 (Gibson J.S. et al. Arch. Virol. 124 pp. 351-366 (1992)) at 0.1 pfu/cell and the progeny virions were purified by centrifugation on 5-55% (w/v) sucrose gradients as described by Dumas et a1. J.
Gen. Virol. 47 pp. 233-235 {1980)). EHV-1 Ab4 genomic DNA was extracted from the purified virions and digested with a range of restriction enzymes. A relevant fragment, for example, the 5.8-kb BamHI/EcoRI fragment (residues 126,517 to 132,305) was cloned into the vector pUCl9 so that a plasmid, pU71 containing the 5.8-kb BamHI/EcoRI fragment inserted at BamHI/EcoRI sites, was constructed (Fig. 1). To construct a deletion plasmid, the cloned plasmid was digested by restriction enzymes which cut at unique sites to remove most of the coding sequence of gene 71.
The flanking sequences were relegated with complementary synthetic oligonucleotides containing an unique Spe1 site to allow insertion of the lacZ gene and an upstream in-frame stop codon to prevent synthesis of a lacZ fusion protein. The lacZ
gene on a 4.1-kb Xba1 fragment from pFJ3 (Rixon F.J. and McLauchlan J., J. Gen Virol. 71 pp. 2931-2939 (1990)) raas inserted into the Spe1 site. lacZ was in the same orientation as the gene transcript. The construct could encode only a very short polypeptide of the remaining N-terminal amino acids of the deleted gene. In this way, deletion plasmid pD71 with a deletion from the Msc1 to the SgraAl site (residues 129,211 - 131,022) in pU7l, was generated (Fig. 1).
Example 2 For generation of virus mutants, 1-2 ~.g of EHV-1 Ab4 DNA was cotransfected into BHK21/C13 cells (MacPherson I. and Stoker M.G.
Virology 16 pp. 147-151 (1962)) with varying amounts of the linearized deletion plasmid pD7l, (0.2 to 4 ~,g, an approximately 2- to 20-fold molar excess) in the presence of carrier calf thymus DNA using the calcium phosphate precipitation/DMSO method described by Stow N.D., and Wilkie N.M., J. Gen. Virol. 33 pp.
447-458 (1976). The cells were incubated at 37° in Eagle's medium containing 5a newborn calf serum. When the c.p.e. was widespread, the virus was harvested and titrated on BHK21/CI3 cells under methylcellulose. Two days after the infection, a WO 98!26049 PCT/GB97/03327 further 2 ml of methylcellulose medium containing 0.7 mg/ml X-gal was added to each plate. Individual blue plaques were isolated for further rounds of plaque purification. A mutant with a lacZ
substitution was isolated: ED71 with a deletion of 1811 by from the 2393 by gene 71 ORF. The deleted region of the mutant was confirmed by Southern blotting with a probe of the 32P-labelled deleted sequence. The structure of the virus mutant was confirmed by Southern blotting and restriction enzyme digestion of 3zP-labelled viral DNA prior to the preparation of virus stork.
The restriction enzyme digestion of 32P-labelled viral DNA is represented diagrammatically in Fig. 2. Gene 71 lies within the 3.8-kb Smal fragment of wild-type viral DNA. Deletion of gene 71 and substitution by the lacZ gene resulted in the loss of the 3.8-kb fragment and the generation of a new larger fragment of 6.2-kb. The mutant had the expected genome structure, with no other detectable differences from wild-type viral DNA.
Example 3 Growth characteristics of the mutant in tissue culture was also investigated. Monolayers of BHK21/C13 cells were separately infected at a multiplicity of infection (m.o. i. ) of 5 pfu/cell and 0.01 pfu/cell with wild-type virus and the deletion and substitution mutant ED71. The culture was harvested and virus was released by sonication at intervals throughout a 72 hour period. Virus titers were measured by plaque assay and the growth patterns were compared with those of wild-type virus. The plaque morphology of the mutants was not obviously different from the wild-type virus plaques.
Mutant ED71 grew more slowly and the final yield was reduced by about 5-fold compared with that of wild-type virus. Similar results were seen at high multiplicity (data not shown) , although the reduction in the yield of the ED71 mutant was less than that at low multiplicity. To determine whether the mutant was temperature sensitive or had a host-range phenotype, they were grown at a high m.o.i. of 5 pfu/cell in BHK21/C13 cells at different temperatures ( 31 ° , 37 ° , and 38 . 5 °C) and at 37 ° C in NBL-6, Vero, HFL, and 3T6 cells. The cultures were harvested at 24 hour post-infection and progeny virus was titrated in BHK21/C13 cells. The ED71 mutant at 38°C grew 10-fold less well than at 31° and compared to wild-type virus at 38.5°C (data not shown).
The slightly impaired growth of the ED71 mutant was apparent in NBL-6, Vero, HFL, and 3T6 as well as in BHK21/C13 cells. Thus it is concluded that gene 71 is nonessential for EHV-1 growth in cell culture.
Example 4: Infection Experiments: Mortality and Clinical Signs Materials and Methods Virus Strains Wild-type and mutant viruses were grown either in RK cells at the Department of Clinical Veterinary Medicine, Cambridge or in BHK cells at the Institute of Virology, Glasgow. Wild-type for primary infection experiments was EHV-1 strain Ab4p. Virus used to challenge previously immunised mice was EHV-1 strain Ab4.
Mouse Model Female Balb/c mice were obtained at 3-4 weeks of age (Bantin and Kingman, UK). Mice were inoculated intranasally under isofluorane/oxygen anaesthesia.
Tissue Culture RK cell monolayers were cultured in Eagle s Minimum Essential Medium (EMEM) with Earle~s Salts with loo newborn calf serum.
Virus Titration Tissue samples obtained from three mice per group were homogenised using an Ultraturrax motorised homogenises. Samples were then sonicated in an ice-cold waterbath and centrifuged at low speed to separate cellular debris. Ten-fold serial dilutions of the supernatant were made and 100,1 of each dilution inoculated onto confluent monolayers of RK cells, in duplicate.
Virus was allowed to adsorb to the cell sheet for 45 minutes before all samples were overlayed with medium containing 4%
foetal calf serum and 2o carboxymethylcellulose. Plates were incubated at 37 °C for about 3 days and then washed in sterile phosphate buffered saline prior to fixing and staining with crystal violet in 20% ethanol.
Experimental Protocol At days 1, 3 and 5 post-infection groups of three mice were euthanased with 0.15 ml of pentobarbitone sodium (Sagatal, Rhone Merieux), tissues removed, placed in 1 ml of virus isolation medium, frozen at -70°C and then titrated for virus growth.
Tissue samples taken were lung, turbinates, olfactory bulb and trigeminal ganglia. Clinical signs were monitored in a separate group of mice from day 0 to day 8 post-infection. Blood samples were taken on days 8, 16, 23 and 30 post-infection for immunological tests. A group of surviving animals were then challenged with a dose of 5x106pfu/mouse of EHV-1 strain Ab4.
Tissue samples were taken as above and clinical signs monitored.
Mortality and clinical results are shown in Table 2. Virus titre results are shown in Figures 3 to 8 and Tables 4(a)-4(d) inclusive.
Mortality and Clinical Signs Virus Mortality Clinical Signs*
Ab4 77~ Severe ED71 8~ Mild ED71 Rev 60~ Severe * Clinical signs observed between Day 1 and Day 8 post-infection.
Example 5: Immunology - ELISA
The protocol of Tewari D., et al (1994) Journal of Gen.
Virol. 75 pp. 1735-1741 was followed. Results are shown in Table 3.
ELISA
Acute Phase Virus Day 8 p.i. Day 16 p.i. Day 23 p.i. Day 30 p.i.
ur t (C) 1:25 1:125 1:125 1:125 ED71 1:25 1:125 1:125 1:625 Post Challenge Virus Day 3 Day 5 Day g w/t (C) 1:125 1:625 1:3125 ED71 1:625 1:625 1:3125 TABLE 4(a) Day +1 Post Challenge Lung .., MEAN RANGE No. of +ve Log~oReduc-Mice tion MEAN
Negative 4.3 4.6 3/3 -control 4.0 Positive 2.6 3.3 3/3 1.7 control 2.0 Gene 2.9 4.2 2/3 1.4 Deletion <0.7 Turbinates Negative 4.3 4.8 3/3 -control 3.9 Positive 3.1 3.3 3/3 1.2 control 3.0 Gene 3.0 4.3 2/3 1.3 Deletion <0.7 Olfactorybulb Negative 2.3 2.5 3/3 -Control 2.0 Positive 1.3 1.6 3/3 1.0 Control 1.0 ~
I
Gene 2.0 2.2 3/3 0.3 Deletion 1.7 TABLE 4(b) Day +3 Post Challenge Lung MEAN RANGE No. of +ve Log,QReduc-Mice tion MEAN
Negative 4.9 5.2 3/3 -Control 4.6 Positive 0.9 1.0 2/3 4.0 Control <0.7 Gene 0.8 0.9 1/3 4.1 Deletion 71 <0.7 Turbinates Negative 4.6 5.2 3/3 -Control 4.1 Positive <0.7 <0.7 0/3 >3.9 Control <0.7 Gene <0.7 <0.7 0/3 >3.9 Deletion 71 <0.7 Olfactor bulb Negative 1.8 2.2 3/3 -Control 1.4 Positive 0.8 0.9 1/3 1.0 Control <0.7 Gene 0.8 0.9 1/3 1.0 Deletion 71 <0.7 TABLE 4(c) Day +5 Post Challenge Lung MEAN RANGE No. of +ve Log~QReduc-Mice tion MEAN
Negative <0.7 <0.? 0/3 -Control <0.7 Positive <0.7 <0.'7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Turbinates Negative 2.9 5.2 3/3 -Control 4.1 Positive <0.7 <0.7 0/3 >2.2 Control <0.7 Gene <0.7 <0.7 0/3 >2.2 Deletion 71 <0.7 Olfactorybulb Negative 0.75 2.2 1/3 -Control 1.4 Positive <0.7 0.9 0/3 <0.05 Control <0.7 Gene <0.7 0.9 0/3 <0.05 Deletion 71 <0.7 TABLE 4(d) Day +8 Post Challenge Lung MEAN RANGE No. of +ve Log,oReduc-Mice tion MEAN
Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Turbinates Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Olfactorybulb Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 1. Experimental details The trial was performed in pony colts using 3 animals per group and two groups, one vaccinated and one not (control group) .
The trial animals were selected on the basis that they had no or low EHV-1 neutralising and EHV-1 complement fixing (CF) antibodies. The experimental groups were kept in separate rooms in isolation with filtered air in and out. Colts, 7, 15 and 20 were each vaccinated intranasally with 6.0 1og10 TCIDS~ of gene 71 deleted EHV-1 (ED71), in 2.Omls of MEM (Gibco) containing neomycin (100~,g/ml), 2% y-irradiated foetal calf serum (FCS) (Tissue Culture Services), giving l.Oml into each nostril.
Following vaccination both vaccinated test colts (internal numbering 7, 15 and 20) and control colts (internal numbering 5, 8 and 16) were tested for virus replication in the upper respiratory tract by taking nasal swabs daily for 2 weeks. All six animals were bled at intervals and their sera tested for EHV-1 neutralising and CF antibodies (Table 7). Intranasal challenge infection with wild type strain AB-4 was conducted 51 days after vaccination when colts in both groups were each given 6.0 log, TCIDSO of AB-4 in 2.Omls of MEM medium supplemented with 2o FCS.
Following challenge the procedures performed were the same as those after vaccination, ie. assessment of virus growth in the upper respiratory tract (Table 5).
Experimental groups and procedures Group Colt Vaccination Procedures afterChallenge &
No vaccination Procedures Test 7,15,20 Intranasally All 6 colts All 6 colts Group 6.0 log~p of ED71 in 2.Omls(i) Nasal swabs (i) Nasal swabs day 1-14 day 1-14 Intranasally with 6.0 log, TCID~ of wild ' type strain AB-4 and (ii) Leukocyte viraemia on days 0,1,3, 5,7,9,11 &
13.
Control 5,8,16 None (control) Group 2. Results 2.1 Replication of ED71 virus in the upper respiratory tract Viruses were isolated from nasal swabs in MEM medium supplemented as described above, following standard procedures.
Results of virus isolation from daily nasal swabs following intranasal vaccination are given in Table 6. ED71 virus at low titre (mostly below 3.0 log,, TCIDS~,/ml) was isolated from 2 of 3 vaccinated colts, on days 2 and 3 from colt 7, and days 1 to 5 from colt 15. No EHV-1 was recovered from control colts from daily nasal swab samples over 14 days.
2.2 Serological responses following vaccination Sera were titrated fro virus neutralising (VN) and complement fixing (CF) antibodies. Results of VN tests performed according to the method of Thompson G.R., et al Equine Vet.
Journal Vol. 8 pp 58-65, for both post vaccination and challenge are given in Table 7 and those for CF test performed according to the method of Thompson et al supra (vaccination only) are given in Table 8.
In the VN test against two different strains of EHV-1 namely ED71 virus (parent strain AB-4) and M8 no significant differences in titres were recorded. In the vaccinated group all three colts were just detectably VN antibody positive at intranasal vaccination. All three animals responded with significant (>_4-fold rise) antibody response, Nos 7 & 20 by week four and No 15 by week two.
There was no VN antibody rise in the control animals until after challenge. By the CF test against EHV-1 two (15 and 20) of three colts showed a significant rise (>_4-fold rise) by week two after vaccination; colt No 7 had high activity at vaccination (Table 8). Control animals (5, 8 and 16) did not show significant change in CF antibody titres.
In keeping with the virus isolation results, there was no seroconversion in control animals indicating the absence of a field infection or EHV-1 recrudescence.
WO 98/2b049 PCT/GB97103327 3. Challenge findings 3.1 Challenge virus replication in the upper respiratory tract Virus isolation results from nasal swabs are given in Table 9. Virus at low titre (2.0 loge, TCIDSO/ml) isolated from only one (no 7) of three colts on two occasions (day 1 and 2). This was in marked contrast to the control colts (5, 8 and 16) from which virus was recovered for 3 (no 5) to 5 to 6 days (Nos 8 and 16) at much higher titres.
3.2 Viraemia due to the challenge virus Challenge EHV-1 isolation from leukocytes is given in Table 10. There was no challenge virus detected in ED71 vaccinated colts. In contrast all three control colts became viraemic yielding, at peak between 12 to 200 infected leukocytes/2 x 10' cells.
Vaccine virus replication in upper respiratory tract Group Colt Virus No isolated (log,o TCID~/ml) from nasal swabs following intranasal vaccination (days) TEST 7 - - 2.7 s.5_ _ _ _ _ _ _ _ _ _ _ ( ) 1S - 1.54.3 l.72.0 1.5_ _ _ _ _ _ _ _ _ 20 - _ _ _ _ _ _ _ _ _ _ _ _ _ _ CONTROL 5 - - - - - - - - - - - _ _ _ _ a denotes no EHV-1 isolated in equine dermal cells from 4x200,1 of the lowest (10'') dilution of the nasal swab titration.
Virus neutralising (VN) antibody responses Group Colt Circulating VN and EHV-MBantibody' to EHV-1-ED71 No week week week weak week +7 week +
-1 0 +2 +4 challenge'10 vacb TEST 7 8) 8 4) 8 l6, 16 32) 32 16, 32 32) 32 15 4) 4 4, 8 32) 64 64, 64 64) 64 64) 128 20 8, 8 8) 8 16, 16 32) 64 32, 32 32, 32 CONTROL S 4) 4 4, 4 4) 4 4, 4 4, 4 32, 32 8 <4, <4 <4, <4 <4, <4 <4) <4 <4) <4 32, 32 16 4, 4 4) 4 < 4) 4) 4 4, 4 32, 64 a VN tests were performed against ED71 virus (lefthand figures) and EHV-1 M8 (righthand figures). Titres denote reciprocal of serum dilution completely neutralising. 200 (ED71) to 316 (M8 ) TCIDS~ of EHV-1 .
b Vac denotes vaccination on week 0.
c Challenge on week +7.
Complement fixing (CF) antibody responses to EHV-1 Group Colt Circulating CF antibody to EHV-1 (AB-4) No week -1 week 0 week +2 week +4 vac"
a vac denotes vaccination on week 0.
t Challenge virus replication in the upper respiratory tract Group Colt Virus No isolated (log,a TCID~/ml) from nasal swabs following intranasal challenge (days) TEST 7 -' - 2.0 2.0 _ _ _ _ _ _ _ _ _ _ (ED71) 15 - _ _ _ _ _ _ _ _ . _ _ _ _ 20 - _ _ _ _ _ _ _ . _ _ _ _ _ CONTROL 5 - 3.72.0 _ _ 2.7_ _ _ _ _ _ $ - 2.73.7 3.5 2.53.72.0 - _ _ _ _ _ 16 - 4.54.3 3.~t3.72.5_ _ _ _ _ _ _ _ Leukocyte viraemia following intranasal EHV-1 challenge Group Colt Number No of virus infected leukocytes"/2X10' cells days after challenge TEST 7 -b - _ _ _ _ _ _ (ED71) 15 - _ _ _ _ _ _ _ 20 - _ _ _ _ _ _ _ CONTROL 5 - - - 12 5 2.5 - -g _ _ _ 200 1.3 - _ _ 16 - - - 20 2.5 2.5 - -a Buffy coat cell layer from lOmls of citrated blood was separated on a percoll cushion washed in phosphate buffered saline and titrated in monolayers of equine dermal cells (Animal Health Trust, Newmarket, Suffolk) using 8 monolayers/dilution and 3 tenfold dilutions (MEM, 10% 'y-irradiated FCS supplemented with neomycin).
b No EHV-1 isolated after two serial passages at 37°C.
Ba, Ec, EcoRI; Ms, Sg and Bam HI.
FiQUre 2:
Genome structure of the deletion and substitution mutant.
Restriction enzyme sites within the region of the genome encompassing gene 71 are shown. The wild-type virus genome is represented by line 1, and the deletion and substitution by line 2. Relevant fragments generated following digestion with Smal are shown. Fragment sizes are given in kb. Pertinent retriction sites Ms, Sm, Sg.
Figure 3:
Virus titres far mice inoculated with Ab4p.
FicTUre 4:
Virus titres for mice inoculated with ED 71.
Figwre 5:
Virus titres for mice inoculated with ED 71 revertant.
Figwre 6:
Mean virus titres observed in challenged mice previously immunised with RK cell lysate.
Fiqure 7:
Mean virus titres observed in challenged mice previously immunised with Ab4p.
Figure 8:
Mean virus titres observed in challenged mice previously immunised with ED 71.
Standard methods are as described in "Molecular Cloning -A Laboratory Manual", Second Edition, Sambrook J. et al. Cold Spring Harbor Laboratory Press 1989.
Methods Cells and Virus Baby hamster kidney clone 13 (BHK-21/C13; Macpherson I &
Stoker M.G. (1962) Virology 16 pp. 147-151) were grown as previously described (Brown et al., 1973 J. Gen. Virol. 18 pp.
32-346). EHV-1 strain Ab4 was used as the wild-type strain in this study. Stock preparation of virus at passage 13 was made by low multiplicity infection in equine dermal NBL-6 cells maintained in MEM with 1% fetal calf cerum. Mutant ED71 in which the gene 71 ORF was removed and replaced by the E.coli lacZ gene and the revertant Re71 in which the deletion in ED71 was restored have been previously described (Sun Y. and Brown S.M. (1994) Virology 199 pp. 448-452; Sun Y. et al. (1994) J. Gen. Virol. 75 pp. 3117-3126).
Purification and Quantification of Virions The procedure used was essentially as described by Szilagyi J.F. and Cunningham C. (1992) J. Gen. Virol. 27 pp. 661-668 and Sun et al. (1994) supra. BHK-21/C13 monolayers in roller bottles were infected with virus at a multiplicity of infection (m.o.i.) of 0.01 or 5 p.f.u. per cell. At either 72 hours post-infection (p.i.) or 20 hours p.i. the supernatant was harvested and centrifuged at 2500 r.p.m. for 20 minutes to remove the cell debris. Supernatant virus was pelleted for 2 hours at 12000 r.p.m, and the pellet gently resuspended in 1 ml Eagle's medium without phenol red and laid onto a 5 to 15o Ficoll gradient before centrifuging at 12000 r.p.m. for 2 hours at 4°C. The virion band collected by side puncture was diluted and pelleted at 21000 r.p.m. for 2 hours at 4°C. The virion pellet was gently resuspended in 200 ~,1 of Eagle's medium and stored at -70°C.
Infectivity was determined by titration on BHK-21/C13 cells. The number of particles was determined by eletron microscopy. The specific infectivities (particle p.f.u. ratio) of the purified mutant and wild-type virus are presented in Table 1.
Table 1: The Specific Infectivity (particle/p.f.u. ratio) of ED71, and Wild-Type Virus EHV-1).
Virus Particle/p.f.u.ratio* Particle/p.f.u.
ratio ~
EHV-1 101.7/1 63.8/1 Re71 103.5/1 107.7/1 * Virus from 4x10 BHK-21/C13 cells infected with virus at an m.o.i. of 0.01 p.f.u. per cell and harvested at 72 h.p.i.
.~ Purified virions from 4x10A BHK-21/C13 cells infected with virus at an m.o.i. of 5 p.f.u./cell and harvested at 20 h.p.i.
Suitable gene 71 deletion mutant viruses of the invention were prepared according to the teaching of Sun Y. and Brown S.M.
(supra).
Example I
Briefly, to clone the fragment which contains the gene 71, equine dermal cells (NBL-6) were infected with EHV-1 strain Ab4 (Gibson J.S. et al. Arch. Virol. 124 pp. 351-366 (1992)) at 0.1 pfu/cell and the progeny virions were purified by centrifugation on 5-55% (w/v) sucrose gradients as described by Dumas et a1. J.
Gen. Virol. 47 pp. 233-235 {1980)). EHV-1 Ab4 genomic DNA was extracted from the purified virions and digested with a range of restriction enzymes. A relevant fragment, for example, the 5.8-kb BamHI/EcoRI fragment (residues 126,517 to 132,305) was cloned into the vector pUCl9 so that a plasmid, pU71 containing the 5.8-kb BamHI/EcoRI fragment inserted at BamHI/EcoRI sites, was constructed (Fig. 1). To construct a deletion plasmid, the cloned plasmid was digested by restriction enzymes which cut at unique sites to remove most of the coding sequence of gene 71.
The flanking sequences were relegated with complementary synthetic oligonucleotides containing an unique Spe1 site to allow insertion of the lacZ gene and an upstream in-frame stop codon to prevent synthesis of a lacZ fusion protein. The lacZ
gene on a 4.1-kb Xba1 fragment from pFJ3 (Rixon F.J. and McLauchlan J., J. Gen Virol. 71 pp. 2931-2939 (1990)) raas inserted into the Spe1 site. lacZ was in the same orientation as the gene transcript. The construct could encode only a very short polypeptide of the remaining N-terminal amino acids of the deleted gene. In this way, deletion plasmid pD71 with a deletion from the Msc1 to the SgraAl site (residues 129,211 - 131,022) in pU7l, was generated (Fig. 1).
Example 2 For generation of virus mutants, 1-2 ~.g of EHV-1 Ab4 DNA was cotransfected into BHK21/C13 cells (MacPherson I. and Stoker M.G.
Virology 16 pp. 147-151 (1962)) with varying amounts of the linearized deletion plasmid pD7l, (0.2 to 4 ~,g, an approximately 2- to 20-fold molar excess) in the presence of carrier calf thymus DNA using the calcium phosphate precipitation/DMSO method described by Stow N.D., and Wilkie N.M., J. Gen. Virol. 33 pp.
447-458 (1976). The cells were incubated at 37° in Eagle's medium containing 5a newborn calf serum. When the c.p.e. was widespread, the virus was harvested and titrated on BHK21/CI3 cells under methylcellulose. Two days after the infection, a WO 98!26049 PCT/GB97/03327 further 2 ml of methylcellulose medium containing 0.7 mg/ml X-gal was added to each plate. Individual blue plaques were isolated for further rounds of plaque purification. A mutant with a lacZ
substitution was isolated: ED71 with a deletion of 1811 by from the 2393 by gene 71 ORF. The deleted region of the mutant was confirmed by Southern blotting with a probe of the 32P-labelled deleted sequence. The structure of the virus mutant was confirmed by Southern blotting and restriction enzyme digestion of 3zP-labelled viral DNA prior to the preparation of virus stork.
The restriction enzyme digestion of 32P-labelled viral DNA is represented diagrammatically in Fig. 2. Gene 71 lies within the 3.8-kb Smal fragment of wild-type viral DNA. Deletion of gene 71 and substitution by the lacZ gene resulted in the loss of the 3.8-kb fragment and the generation of a new larger fragment of 6.2-kb. The mutant had the expected genome structure, with no other detectable differences from wild-type viral DNA.
Example 3 Growth characteristics of the mutant in tissue culture was also investigated. Monolayers of BHK21/C13 cells were separately infected at a multiplicity of infection (m.o. i. ) of 5 pfu/cell and 0.01 pfu/cell with wild-type virus and the deletion and substitution mutant ED71. The culture was harvested and virus was released by sonication at intervals throughout a 72 hour period. Virus titers were measured by plaque assay and the growth patterns were compared with those of wild-type virus. The plaque morphology of the mutants was not obviously different from the wild-type virus plaques.
Mutant ED71 grew more slowly and the final yield was reduced by about 5-fold compared with that of wild-type virus. Similar results were seen at high multiplicity (data not shown) , although the reduction in the yield of the ED71 mutant was less than that at low multiplicity. To determine whether the mutant was temperature sensitive or had a host-range phenotype, they were grown at a high m.o.i. of 5 pfu/cell in BHK21/C13 cells at different temperatures ( 31 ° , 37 ° , and 38 . 5 °C) and at 37 ° C in NBL-6, Vero, HFL, and 3T6 cells. The cultures were harvested at 24 hour post-infection and progeny virus was titrated in BHK21/C13 cells. The ED71 mutant at 38°C grew 10-fold less well than at 31° and compared to wild-type virus at 38.5°C (data not shown).
The slightly impaired growth of the ED71 mutant was apparent in NBL-6, Vero, HFL, and 3T6 as well as in BHK21/C13 cells. Thus it is concluded that gene 71 is nonessential for EHV-1 growth in cell culture.
Example 4: Infection Experiments: Mortality and Clinical Signs Materials and Methods Virus Strains Wild-type and mutant viruses were grown either in RK cells at the Department of Clinical Veterinary Medicine, Cambridge or in BHK cells at the Institute of Virology, Glasgow. Wild-type for primary infection experiments was EHV-1 strain Ab4p. Virus used to challenge previously immunised mice was EHV-1 strain Ab4.
Mouse Model Female Balb/c mice were obtained at 3-4 weeks of age (Bantin and Kingman, UK). Mice were inoculated intranasally under isofluorane/oxygen anaesthesia.
Tissue Culture RK cell monolayers were cultured in Eagle s Minimum Essential Medium (EMEM) with Earle~s Salts with loo newborn calf serum.
Virus Titration Tissue samples obtained from three mice per group were homogenised using an Ultraturrax motorised homogenises. Samples were then sonicated in an ice-cold waterbath and centrifuged at low speed to separate cellular debris. Ten-fold serial dilutions of the supernatant were made and 100,1 of each dilution inoculated onto confluent monolayers of RK cells, in duplicate.
Virus was allowed to adsorb to the cell sheet for 45 minutes before all samples were overlayed with medium containing 4%
foetal calf serum and 2o carboxymethylcellulose. Plates were incubated at 37 °C for about 3 days and then washed in sterile phosphate buffered saline prior to fixing and staining with crystal violet in 20% ethanol.
Experimental Protocol At days 1, 3 and 5 post-infection groups of three mice were euthanased with 0.15 ml of pentobarbitone sodium (Sagatal, Rhone Merieux), tissues removed, placed in 1 ml of virus isolation medium, frozen at -70°C and then titrated for virus growth.
Tissue samples taken were lung, turbinates, olfactory bulb and trigeminal ganglia. Clinical signs were monitored in a separate group of mice from day 0 to day 8 post-infection. Blood samples were taken on days 8, 16, 23 and 30 post-infection for immunological tests. A group of surviving animals were then challenged with a dose of 5x106pfu/mouse of EHV-1 strain Ab4.
Tissue samples were taken as above and clinical signs monitored.
Mortality and clinical results are shown in Table 2. Virus titre results are shown in Figures 3 to 8 and Tables 4(a)-4(d) inclusive.
Mortality and Clinical Signs Virus Mortality Clinical Signs*
Ab4 77~ Severe ED71 8~ Mild ED71 Rev 60~ Severe * Clinical signs observed between Day 1 and Day 8 post-infection.
Example 5: Immunology - ELISA
The protocol of Tewari D., et al (1994) Journal of Gen.
Virol. 75 pp. 1735-1741 was followed. Results are shown in Table 3.
ELISA
Acute Phase Virus Day 8 p.i. Day 16 p.i. Day 23 p.i. Day 30 p.i.
ur t (C) 1:25 1:125 1:125 1:125 ED71 1:25 1:125 1:125 1:625 Post Challenge Virus Day 3 Day 5 Day g w/t (C) 1:125 1:625 1:3125 ED71 1:625 1:625 1:3125 TABLE 4(a) Day +1 Post Challenge Lung .., MEAN RANGE No. of +ve Log~oReduc-Mice tion MEAN
Negative 4.3 4.6 3/3 -control 4.0 Positive 2.6 3.3 3/3 1.7 control 2.0 Gene 2.9 4.2 2/3 1.4 Deletion <0.7 Turbinates Negative 4.3 4.8 3/3 -control 3.9 Positive 3.1 3.3 3/3 1.2 control 3.0 Gene 3.0 4.3 2/3 1.3 Deletion <0.7 Olfactorybulb Negative 2.3 2.5 3/3 -Control 2.0 Positive 1.3 1.6 3/3 1.0 Control 1.0 ~
I
Gene 2.0 2.2 3/3 0.3 Deletion 1.7 TABLE 4(b) Day +3 Post Challenge Lung MEAN RANGE No. of +ve Log,QReduc-Mice tion MEAN
Negative 4.9 5.2 3/3 -Control 4.6 Positive 0.9 1.0 2/3 4.0 Control <0.7 Gene 0.8 0.9 1/3 4.1 Deletion 71 <0.7 Turbinates Negative 4.6 5.2 3/3 -Control 4.1 Positive <0.7 <0.7 0/3 >3.9 Control <0.7 Gene <0.7 <0.7 0/3 >3.9 Deletion 71 <0.7 Olfactor bulb Negative 1.8 2.2 3/3 -Control 1.4 Positive 0.8 0.9 1/3 1.0 Control <0.7 Gene 0.8 0.9 1/3 1.0 Deletion 71 <0.7 TABLE 4(c) Day +5 Post Challenge Lung MEAN RANGE No. of +ve Log~QReduc-Mice tion MEAN
Negative <0.7 <0.? 0/3 -Control <0.7 Positive <0.7 <0.'7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Turbinates Negative 2.9 5.2 3/3 -Control 4.1 Positive <0.7 <0.7 0/3 >2.2 Control <0.7 Gene <0.7 <0.7 0/3 >2.2 Deletion 71 <0.7 Olfactorybulb Negative 0.75 2.2 1/3 -Control 1.4 Positive <0.7 0.9 0/3 <0.05 Control <0.7 Gene <0.7 0.9 0/3 <0.05 Deletion 71 <0.7 TABLE 4(d) Day +8 Post Challenge Lung MEAN RANGE No. of +ve Log,oReduc-Mice tion MEAN
Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Turbinates Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 Olfactorybulb Negative <0.7 <0.7 0/3 -Control <0.7 Positive <0.7 <0.7 0/3 -Control <0.7 Gene <0.7 <0.7 0/3 -Deletion 71 <0.7 1. Experimental details The trial was performed in pony colts using 3 animals per group and two groups, one vaccinated and one not (control group) .
The trial animals were selected on the basis that they had no or low EHV-1 neutralising and EHV-1 complement fixing (CF) antibodies. The experimental groups were kept in separate rooms in isolation with filtered air in and out. Colts, 7, 15 and 20 were each vaccinated intranasally with 6.0 1og10 TCIDS~ of gene 71 deleted EHV-1 (ED71), in 2.Omls of MEM (Gibco) containing neomycin (100~,g/ml), 2% y-irradiated foetal calf serum (FCS) (Tissue Culture Services), giving l.Oml into each nostril.
Following vaccination both vaccinated test colts (internal numbering 7, 15 and 20) and control colts (internal numbering 5, 8 and 16) were tested for virus replication in the upper respiratory tract by taking nasal swabs daily for 2 weeks. All six animals were bled at intervals and their sera tested for EHV-1 neutralising and CF antibodies (Table 7). Intranasal challenge infection with wild type strain AB-4 was conducted 51 days after vaccination when colts in both groups were each given 6.0 log, TCIDSO of AB-4 in 2.Omls of MEM medium supplemented with 2o FCS.
Following challenge the procedures performed were the same as those after vaccination, ie. assessment of virus growth in the upper respiratory tract (Table 5).
Experimental groups and procedures Group Colt Vaccination Procedures afterChallenge &
No vaccination Procedures Test 7,15,20 Intranasally All 6 colts All 6 colts Group 6.0 log~p of ED71 in 2.Omls(i) Nasal swabs (i) Nasal swabs day 1-14 day 1-14 Intranasally with 6.0 log, TCID~ of wild ' type strain AB-4 and (ii) Leukocyte viraemia on days 0,1,3, 5,7,9,11 &
13.
Control 5,8,16 None (control) Group 2. Results 2.1 Replication of ED71 virus in the upper respiratory tract Viruses were isolated from nasal swabs in MEM medium supplemented as described above, following standard procedures.
Results of virus isolation from daily nasal swabs following intranasal vaccination are given in Table 6. ED71 virus at low titre (mostly below 3.0 log,, TCIDS~,/ml) was isolated from 2 of 3 vaccinated colts, on days 2 and 3 from colt 7, and days 1 to 5 from colt 15. No EHV-1 was recovered from control colts from daily nasal swab samples over 14 days.
2.2 Serological responses following vaccination Sera were titrated fro virus neutralising (VN) and complement fixing (CF) antibodies. Results of VN tests performed according to the method of Thompson G.R., et al Equine Vet.
Journal Vol. 8 pp 58-65, for both post vaccination and challenge are given in Table 7 and those for CF test performed according to the method of Thompson et al supra (vaccination only) are given in Table 8.
In the VN test against two different strains of EHV-1 namely ED71 virus (parent strain AB-4) and M8 no significant differences in titres were recorded. In the vaccinated group all three colts were just detectably VN antibody positive at intranasal vaccination. All three animals responded with significant (>_4-fold rise) antibody response, Nos 7 & 20 by week four and No 15 by week two.
There was no VN antibody rise in the control animals until after challenge. By the CF test against EHV-1 two (15 and 20) of three colts showed a significant rise (>_4-fold rise) by week two after vaccination; colt No 7 had high activity at vaccination (Table 8). Control animals (5, 8 and 16) did not show significant change in CF antibody titres.
In keeping with the virus isolation results, there was no seroconversion in control animals indicating the absence of a field infection or EHV-1 recrudescence.
WO 98/2b049 PCT/GB97103327 3. Challenge findings 3.1 Challenge virus replication in the upper respiratory tract Virus isolation results from nasal swabs are given in Table 9. Virus at low titre (2.0 loge, TCIDSO/ml) isolated from only one (no 7) of three colts on two occasions (day 1 and 2). This was in marked contrast to the control colts (5, 8 and 16) from which virus was recovered for 3 (no 5) to 5 to 6 days (Nos 8 and 16) at much higher titres.
3.2 Viraemia due to the challenge virus Challenge EHV-1 isolation from leukocytes is given in Table 10. There was no challenge virus detected in ED71 vaccinated colts. In contrast all three control colts became viraemic yielding, at peak between 12 to 200 infected leukocytes/2 x 10' cells.
Vaccine virus replication in upper respiratory tract Group Colt Virus No isolated (log,o TCID~/ml) from nasal swabs following intranasal vaccination (days) TEST 7 - - 2.7 s.5_ _ _ _ _ _ _ _ _ _ _ ( ) 1S - 1.54.3 l.72.0 1.5_ _ _ _ _ _ _ _ _ 20 - _ _ _ _ _ _ _ _ _ _ _ _ _ _ CONTROL 5 - - - - - - - - - - - _ _ _ _ a denotes no EHV-1 isolated in equine dermal cells from 4x200,1 of the lowest (10'') dilution of the nasal swab titration.
Virus neutralising (VN) antibody responses Group Colt Circulating VN and EHV-MBantibody' to EHV-1-ED71 No week week week weak week +7 week +
-1 0 +2 +4 challenge'10 vacb TEST 7 8) 8 4) 8 l6, 16 32) 32 16, 32 32) 32 15 4) 4 4, 8 32) 64 64, 64 64) 64 64) 128 20 8, 8 8) 8 16, 16 32) 64 32, 32 32, 32 CONTROL S 4) 4 4, 4 4) 4 4, 4 4, 4 32, 32 8 <4, <4 <4, <4 <4, <4 <4) <4 <4) <4 32, 32 16 4, 4 4) 4 < 4) 4) 4 4, 4 32, 64 a VN tests were performed against ED71 virus (lefthand figures) and EHV-1 M8 (righthand figures). Titres denote reciprocal of serum dilution completely neutralising. 200 (ED71) to 316 (M8 ) TCIDS~ of EHV-1 .
b Vac denotes vaccination on week 0.
c Challenge on week +7.
Complement fixing (CF) antibody responses to EHV-1 Group Colt Circulating CF antibody to EHV-1 (AB-4) No week -1 week 0 week +2 week +4 vac"
a vac denotes vaccination on week 0.
t Challenge virus replication in the upper respiratory tract Group Colt Virus No isolated (log,a TCID~/ml) from nasal swabs following intranasal challenge (days) TEST 7 -' - 2.0 2.0 _ _ _ _ _ _ _ _ _ _ (ED71) 15 - _ _ _ _ _ _ _ _ . _ _ _ _ 20 - _ _ _ _ _ _ _ . _ _ _ _ _ CONTROL 5 - 3.72.0 _ _ 2.7_ _ _ _ _ _ $ - 2.73.7 3.5 2.53.72.0 - _ _ _ _ _ 16 - 4.54.3 3.~t3.72.5_ _ _ _ _ _ _ _ Leukocyte viraemia following intranasal EHV-1 challenge Group Colt Number No of virus infected leukocytes"/2X10' cells days after challenge TEST 7 -b - _ _ _ _ _ _ (ED71) 15 - _ _ _ _ _ _ _ 20 - _ _ _ _ _ _ _ CONTROL 5 - - - 12 5 2.5 - -g _ _ _ 200 1.3 - _ _ 16 - - - 20 2.5 2.5 - -a Buffy coat cell layer from lOmls of citrated blood was separated on a percoll cushion washed in phosphate buffered saline and titrated in monolayers of equine dermal cells (Animal Health Trust, Newmarket, Suffolk) using 8 monolayers/dilution and 3 tenfold dilutions (MEM, 10% 'y-irradiated FCS supplemented with neomycin).
b No EHV-1 isolated after two serial passages at 37°C.
Claims (10)
1. Vaccine formulation comprising a live, recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the U S region of the virus genome and a pharmaceutically acceptable carrier.
2. A vaccine formulation according to claim 1 comprising a live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus and a pharmaceutically acceptable carrier.
3. A vaccine formulation according to claim 1 or claim 2 wherein the dysfunctional gene 71 region of the recombinant EHV-1 virus comprises a deletion of at least one nucleotide between nucleotide 129,096 and nucleotide 131,489 of a wild type EHV-1 genome.
4. A vaccine formulation according to any one of claims 1 to 3 wherein the recombinant EHV-1 comprises a marker gene.
5. A live, recombinant EHV-1 comprising a dysfunctional gene 71 region for use as a vaccinating agent.
6. A live, recombinant, attenuated immunogenic EHV-1 gene 71 deletion mutant virus for use as a vaccinating agent.
7. Use of a live, recombinant, EHV-1 gene 71 deletion mutant virus in the manufacture of an EHV-1 vaccine for the prophylaxis and/or therapy of EHV-1 infection.
8. A method of treating an animal which comprises administering to an animal a vaccine composition comprising a live, recombinant EHV-1 virus modified so as to contain a dysfunctional gene 71 region located within the U S region of the virus genome.
9. A method according to claim 8 wherein the animal is a horse.
10. A method according to claim 8 or claim 9 wherein the vaccine composition comprises a recombinant, live, attenuated, immunogenic EHV-1 gene 71 deletion mutant virus.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9625968A GB9625968D0 (en) | 1996-12-13 | 1996-12-13 | Vaccine |
GB9625968.4 | 1996-12-13 | ||
PCT/GB1997/003327 WO1998026049A1 (en) | 1996-12-13 | 1997-12-10 | Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2274900A1 true CA2274900A1 (en) | 1998-06-18 |
Family
ID=10804436
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2274900 Abandoned CA2274900A1 (en) | 1996-12-13 | 1997-12-10 | Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0946714A1 (en) |
JP (1) | JP2002512504A (en) |
AU (1) | AU738684B2 (en) |
CA (1) | CA2274900A1 (en) |
GB (1) | GB9625968D0 (en) |
HU (1) | HUP0002374A3 (en) |
WO (1) | WO1998026049A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1129722A1 (en) | 2000-02-17 | 2001-09-05 | Boehringer Ingelheim Vetmedica Gmbh | gM-negative EHV-mutants |
AR040601A1 (en) | 2002-07-19 | 2005-04-13 | Boehringer Ingelheim Vetmed | GM NEGATIVE EHV MUTANTS WITHOUT HETEROLOGICAL ELEMENTS |
WO2004011677A2 (en) * | 2002-07-26 | 2004-02-05 | Animal Health Trust | Viral marker |
WO2013142371A1 (en) * | 2012-03-20 | 2013-09-26 | Merial Limited | Recombinant equine herpesvirus-1 vaccine containing mutated glycoprotein c and uses thereof |
-
1996
- 1996-12-13 GB GB9625968A patent/GB9625968D0/en active Pending
-
1997
- 1997-12-10 EP EP19970947773 patent/EP0946714A1/en not_active Ceased
- 1997-12-10 WO PCT/GB1997/003327 patent/WO1998026049A1/en not_active Application Discontinuation
- 1997-12-10 HU HU0002374A patent/HUP0002374A3/en unknown
- 1997-12-10 JP JP52634398A patent/JP2002512504A/en active Pending
- 1997-12-10 AU AU54018/98A patent/AU738684B2/en not_active Ceased
- 1997-12-10 CA CA 2274900 patent/CA2274900A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
HUP0002374A3 (en) | 2001-12-28 |
WO1998026049A1 (en) | 1998-06-18 |
JP2002512504A (en) | 2002-04-23 |
HUP0002374A2 (en) | 2000-11-28 |
AU738684B2 (en) | 2001-09-27 |
GB9625968D0 (en) | 1997-01-29 |
AU5401898A (en) | 1998-07-03 |
EP0946714A1 (en) | 1999-10-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP3826055B2 (en) | Immunization with recombinant avipoxvirus | |
AU633663B2 (en) | Recombinant herpesvirus of turkeys and live vector vaccines derived thereof | |
KR101028937B1 (en) | Modified vaccinia virus ankara for the vaccination of neonates | |
KR100746524B1 (en) | Recombinant porcine adenovirus vector | |
NZ254267A (en) | Animal vaccines comprising a pseudorabies virus having a mutation in its gp50 gene | |
EP1100925B1 (en) | Attenuated equine herpesvirus | |
JPH07500972A (en) | Non-released herpesvirus live vaccine | |
AU738684B2 (en) | Recombinant equine herpesvirus type 1 (EHV-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine | |
EP0606452B1 (en) | Vector vaccines of recombinant feline herpesvirus | |
US5674499A (en) | Equine herpesvirus gene 15 mutants | |
EP0668355B1 (en) | Vaccine for the protection of horses against equine herpesvirus infection | |
JP5177927B2 (en) | gM negative EHV mutant | |
KR101111998B1 (en) | gM-Negative EHV-mutants without heterologous elements | |
US20020155131A1 (en) | Recombinant equine herpesvirus type 1 (EHV-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine | |
EP0119025B1 (en) | Thymidine kinase-negative temperature resistant bovine herpesvirus-1 mutant as a vaccine against infectious bovine rhinotracheitis | |
AU778157B2 (en) | Equine herpes virus temperature sensitive mutant and live vaccine thereof | |
MXPA99005452A (en) | Recombinant equine herpesvirus type 1 (ehv-1) comprising a dysfunctional gene 71 region and use thereof as a vaccine | |
Visek | Generation, in vitro characterization and in vivo assessment of recombinant equine herpesvirus type 1 expressing glycoproteins gD and gB of feline herpesvirus type 1 | |
US20040120972A1 (en) | Equine herpes virus temperature sensitive mutant and live vaccine thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDC | Discontinued application reinstated | ||
FZDE | Discontinued |