GB2283021A - Morbillivirus recombinant poxvirus vaccines - Google Patents
Morbillivirus recombinant poxvirus vaccines Download PDFInfo
- Publication number
- GB2283021A GB2283021A GB9500214A GB9500214A GB2283021A GB 2283021 A GB2283021 A GB 2283021A GB 9500214 A GB9500214 A GB 9500214A GB 9500214 A GB9500214 A GB 9500214A GB 2283021 A GB2283021 A GB 2283021A
- Authority
- GB
- United Kingdom
- Prior art keywords
- virus
- recombinant
- poxvirus
- gene
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000712045 Morbillivirus Species 0.000 title claims abstract description 27
- 229940024231 poxvirus vaccine Drugs 0.000 title description 3
- 241000712079 Measles morbillivirus Species 0.000 claims abstract description 98
- 241000700618 Vaccinia virus Species 0.000 claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 230000004927 fusion Effects 0.000 claims abstract description 29
- 241000178270 Canarypox virus Species 0.000 claims abstract description 27
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 23
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 23
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 13
- 208000000655 Distemper Diseases 0.000 claims abstract description 9
- 108020004440 Thymidine kinase Proteins 0.000 claims abstract description 8
- 208000014058 canine distemper Diseases 0.000 claims abstract description 8
- 230000002008 hemorrhagic effect Effects 0.000 claims abstract description 8
- 108010041388 Ribonucleotide Reductases Proteins 0.000 claims abstract description 6
- 108700026220 vif Genes Proteins 0.000 claims abstract description 5
- 102000006601 Thymidine Kinase Human genes 0.000 claims abstract description 3
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 3
- 241000700605 Viruses Species 0.000 claims description 57
- 230000006798 recombination Effects 0.000 claims description 30
- 238000005215 recombination Methods 0.000 claims description 27
- 229960005486 vaccine Drugs 0.000 claims description 26
- 101150039990 B13R gene Proteins 0.000 claims description 11
- 101150071286 SPI-2 gene Proteins 0.000 claims description 11
- 101100534084 Vaccinia virus (strain Copenhagen) B14R gene Proteins 0.000 claims description 5
- 101100004092 Vaccinia virus (strain Western Reserve) VACWR196 gene Proteins 0.000 claims description 5
- 239000000427 antigen Substances 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 229940023860 canarypox virus HIV vaccine Drugs 0.000 claims 2
- 239000013603 viral vector Substances 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 140
- 206010046865 Vaccinia virus infection Diseases 0.000 description 76
- 239000012634 fragment Substances 0.000 description 76
- 208000007089 vaccinia Diseases 0.000 description 76
- 101710154606 Hemagglutinin Proteins 0.000 description 60
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 60
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 60
- 101710176177 Protein A56 Proteins 0.000 description 60
- 210000004027 cell Anatomy 0.000 description 59
- 241000282472 Canis lupus familiaris Species 0.000 description 55
- 238000012217 deletion Methods 0.000 description 46
- 230000037430 deletion Effects 0.000 description 46
- 238000011081 inoculation Methods 0.000 description 46
- 239000000185 hemagglutinin Substances 0.000 description 42
- 241000712083 Canine morbillivirus Species 0.000 description 41
- 201000005505 Measles Diseases 0.000 description 41
- 241001465754 Metazoa Species 0.000 description 40
- 108020004414 DNA Proteins 0.000 description 35
- 241000283973 Oryctolagus cuniculus Species 0.000 description 35
- 239000013598 vector Substances 0.000 description 32
- 238000000034 method Methods 0.000 description 31
- 108091034117 Oligonucleotide Proteins 0.000 description 30
- 101150039660 HA gene Proteins 0.000 description 29
- 238000010276 construction Methods 0.000 description 27
- 230000003472 neutralizing effect Effects 0.000 description 27
- 108010005774 beta-Galactosidase Proteins 0.000 description 25
- 241000588724 Escherichia coli Species 0.000 description 22
- 210000003501 vero cell Anatomy 0.000 description 22
- 210000002966 serum Anatomy 0.000 description 21
- 230000004044 response Effects 0.000 description 20
- 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 19
- 238000004458 analytical method Methods 0.000 description 19
- 230000001681 protective effect Effects 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 102000005936 beta-Galactosidase Human genes 0.000 description 18
- 230000029087 digestion Effects 0.000 description 17
- 238000001114 immunoprecipitation Methods 0.000 description 17
- 108010017826 DNA Polymerase I Proteins 0.000 description 16
- 102000004594 DNA Polymerase I Human genes 0.000 description 16
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 16
- 238000003780 insertion Methods 0.000 description 16
- 230000037431 insertion Effects 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 230000000405 serological effect Effects 0.000 description 15
- 241000700198 Cavia Species 0.000 description 14
- 230000002238 attenuated effect Effects 0.000 description 14
- 230000003053 immunization Effects 0.000 description 14
- 238000002649 immunization Methods 0.000 description 14
- 208000015181 infectious disease Diseases 0.000 description 14
- 238000012809 post-inoculation Methods 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 238000010790 dilution Methods 0.000 description 13
- 239000012895 dilution Substances 0.000 description 13
- 238000006386 neutralization reaction Methods 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 238000002255 vaccination Methods 0.000 description 11
- 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 10
- 101150060895 I4L gene Proteins 0.000 description 10
- 101150034814 F gene Proteins 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 9
- 230000036039 immunity Effects 0.000 description 9
- 230000036961 partial effect Effects 0.000 description 9
- 238000003556 assay Methods 0.000 description 8
- 230000007910 cell fusion Effects 0.000 description 8
- 239000013553 cell monolayer Substances 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 230000028993 immune response Effects 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 7
- 241000700199 Cavia porcellus Species 0.000 description 7
- 241000282693 Cercopithecidae Species 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 6
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 6
- 108091081024 Start codon Proteins 0.000 description 6
- 230000002458 infectious effect Effects 0.000 description 6
- 206010037742 Rabies Diseases 0.000 description 5
- 230000003141 anti-fusion Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 101710112753 A-type inclusion protein A25 homolog Proteins 0.000 description 4
- 238000012286 ELISA Assay Methods 0.000 description 4
- 102000000505 Ribonucleotide Reductases Human genes 0.000 description 4
- 101150003725 TK gene Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000000120 cytopathologic effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000035931 haemagglutination Effects 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013615 primer Substances 0.000 description 4
- 238000009589 serological test Methods 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000700663 Avipoxvirus Species 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 3
- 241000700662 Fowlpox virus Species 0.000 description 3
- 101150082239 G gene Proteins 0.000 description 3
- 208000004068 Morbillivirus Infections Diseases 0.000 description 3
- 241000282695 Saimiri Species 0.000 description 3
- 108020005038 Terminator Codon Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000036760 body temperature Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 206010014599 encephalitis Diseases 0.000 description 3
- 102000013165 exonuclease Human genes 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 231100000518 lethal Toxicity 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 230000008774 maternal effect Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- 241000271566 Aves Species 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 208000015220 Febrile disease Diseases 0.000 description 2
- 208000000666 Fowlpox Diseases 0.000 description 2
- 206010025327 Lymphopenia Diseases 0.000 description 2
- 101900237959 Measles virus Hemagglutinin glycoprotein Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 101100277251 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HEM12 gene Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229940124590 live attenuated vaccine Drugs 0.000 description 2
- 229940023012 live-attenuated vaccine Drugs 0.000 description 2
- 231100001023 lymphopenia Toxicity 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000012808 pre-inoculation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012770 revaccination Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 238000006677 Appel reaction Methods 0.000 description 1
- 101100129922 Caenorhabditis elegans pig-1 gene Proteins 0.000 description 1
- 241000282421 Canidae Species 0.000 description 1
- 241000701101 Canine adenovirus 1 Species 0.000 description 1
- 241000701114 Canine adenovirus 2 Species 0.000 description 1
- 241001466804 Carnivora Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 101100520057 Drosophila melanogaster Pig1 gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 101710121925 Hemagglutinin glycoprotein Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 208000037309 Hypomyelination of early myelinating structures Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 238000012218 Kunkel's method Methods 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 241001147385 Potos flavus Species 0.000 description 1
- 241000282696 Saimiri sciureus Species 0.000 description 1
- 241000287231 Serinus Species 0.000 description 1
- 241000287219 Serinus canaria Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 241000700622 Vaccinia virus Copenhagen Species 0.000 description 1
- 241000625014 Vir Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- 101150030339 env gene Proteins 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000007499 fusion processing Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 101150061325 mv gene Proteins 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 230000017960 syncytium formation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 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
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
- C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Communicable Diseases (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Pulmonology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
A recombinant poxvirus containing therein DNA from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper, is described. The viral vector may be canary poxvirus or vaccinia virus with the following open reading frames deleted: a thymidine kinase gene, a haemorrhagic gene, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region and a large subunit ribonucleotide reductase gene. The Morbillivirus DNA may be the measles virus glycoproteins haemagglutinin and/or fusion glycoproteins.
Description
MEASLES VIRUS RECOMBINANT POXVIRUS VACCINE
CROSS REFERENCE TO RELATED APPLICATIONS
This patentapplication is a divisional of GB 930914.2 filed 20 November 1991 which was derived from PCT/US91/08703 (W0 92/08789).
FIELD OF THE INVENTION
The present invention relates to a modified poxvirus and to methods of making and using the same. More in particular, the invention relates to recombinant poxvirus, which virus expresses gene products of a
Morbillivirus gene, and to vaccines which provide protective immunity against Morbillivirus infections.
Several publications are referenced in this application by arabic numerals within parentheses. Full citation to these references is found at the end of the specification immediately preceding the claims. These references describe the state-of-the-art to which this invention pertains.
BACKGROUND OF THE INVENTION
Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox
DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Patent No. 4,603,112, the disclosure of which patent is incorporated herein by reference.
First1 the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969;
Maniatis et al., 1982).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA; particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA.
In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Canine distemper virus (CDV) and measles virus (MV) are members of the Morbillivirus subgroup of the family
Paramyxovirus genus (Diallo, 1990; Kingsbury et al., 1978).
The viruses contain a non-segmented single-stranded RNA genome of negative polarity. Canine distemper is a highly infectious febrile disease of dogs and other carnivores.
The mortality rate is high; ranging between 30 and 80 percent. Dogs surviving often have permanent central nervous system damage (Fenner, et al., 1987). Similarly, measles virus causes an acute infectious febrile disease characterized by a generalized macropapular eruption. The disease mainly affects children.
The characteristics of Morbilliviruses have recently been reviewed by Norrby and Oxman (1990) and Diallo (1990). As reported for other Paramyxoviruses (Avery and
Niven, 1979; Merz et al., 1980) two structural proteins are trucial for the induction of a protective immune response.
These are the membrane glycoprotein hemagglutinin (HA), which is responsible for hemagglutination and attachment of the virus to the host cell, and the fusion glycoprotein (F), which causes membrane fusion between the virus and the infected cell or between the infected and adjacent uninfected cells (Graves et al., 1978). The order of genes in the MV genome has been deduced by Richardson et al.
(1985) and Dowling et al. (1986). The nucleotide sequence of the MVHA gene and MVF gene has been determined by
Alkhatib and Briedis (1986) and Richardson et al. (1986), respectively.
CDV and MV are structurally similar and share a close serological relationship. Immunoprecipitation studies have shown that antiserum to MV will precipitate all CDV proteins (P, NP, F, HA and M). By contrast, antiserum to
CDV will precipitate all MV proteins except the HA glycoprotein (Hall et al., 1980; Orvell et al., 1980;
Stephenson, et al., 1979). In light of this close serological relationship, it has previously been demonstrated that vaccination with MV will elicit protection against CDV challenge in dogs (Gillespie et al., 1960; Moura et al., 1961; Warren et al., 1960). Neutralizing antibodies against CDV have been reported in human anti-MV sera (Adams et al., 1957; Imagawa et al., 1960; Karzon, 1955; Karzon, 1962) but neutralizing antibodies against MV have not been found in anti-CDV sera from dogs (Delay et al., 1965;
Karzon, 1962; Roberts, 1965).
MV HA and F genes have been expressed in several viral vectors including vaccinia virus (Drillien et al., 1988; Wild et al., 1991), fowlpox virus (Spehner et al., 1990; Wild et al., 1990), adenovirus (Alkhatib et al., 1990) and baculovirus (Vialard et al., 1990). In these studies, authentic MV proteins were expressed which were functional in hemagglutination (Vialard et al., 1990) hemolysis (Alkhatib et al., 1990; Vialard et al., 1990) or cell fusion (Alkhatib et al., 1990; Vialard et al., 1990; Wild et al., 1991) assays. When inserted into a vaccinia virus vector, the expression of either the HA or the F protein was capable of eliciting a protective immune response in mice against MV encephalitis (Drillien et al., 1988). Similarly, expression of the F protein in a fowlpox virus vector elicited protective immunity against MV encephalitis in mice (Wild et al., 1990). No protection studies were reported with other vectors.
European Patent Application No. 0 314 569 relates to the expression of an MV gene in fowlpox.
Perkus et al. (1990.) recently described the definition of two unique host range genes in vaccinia virus.
These genes encode post range functions which permit vaccinia virus replication on various cell substrates in vitro. The genes encode host range functions for vaccinia virus replication on human cells as well as cells of rabbit and porcine origin. Definition of these genes provides for the development of a vaccinia virus vector, which, while still expressing foreign genes of interest, would be severely restricted in its ability to replicate in defined cells. This would greatly enhance the safety features of vaccinia virus recombinants.
An attenuated vector has been developed by the sequential deletion of six non-essential regions from the
Copenhagen strain of vaccinia virus. These regions are known to encode proteins that may have a role in viral virulence. The regions deleted are the tk gene, the hemorrhagic gene, the A-type inclusion gene, the hemagglutinin gene and the gene encoding the large subunit of the ribonucleotide reductase as well as the C7L through
K1L sequences defined previously (Perkus et al., 1990). The sequences and genomic locations of these genes in the
Copenhagen strain of vaccinia virus have been defined previously (Goebel et al., 1990 a,b). The resulting attenuated vaccinia strain is designated as NYVAC.
The technology of generating vaccinia virus recombinants has recently been extended to other members of the poxvirus family which have a more restricted host range.
The avipoxvirus, fowlpox, has been engineered as a recombinant virus expressing the rabies G gene (Taylor et al., 1988b). This recombinant virus is also described in
PCT Publication No. W089/03429. On inoculation of the recombinant into a number of non-avian species an immune response to rabies is elicited which in mice, cats and dogs is protective against a lethal rabies challenge.
Both canine distemper and measles are currently controlled by the use of live attenuated vaccines (Fenner et al., 1987; Preblud et al., 1988). Immunization is recommended for control of CDV using a live attenuated vaccine at eight weeks of age and again at 12 to 16 weeks of age. Although immunity to CDV is life-long, because of the highly infectious nature of the agent and the severity of the disease, annual revaccination is usually recommended.
One problem with the current policy of continual revaccination is that CDV immune mothers pass neutralizing antibody to offspring in the colostrum. It is difficult to ascertain when these antibody levels will wane such that pups can be vaccinated. This leaves.a window when pups may be susceptible to CDV infection. Use of a recombinant vaccine expressing only the measles virus glycoproteins may provide a means to overcome the inhibitory effects of maternal antibody and allow vaccination of newborns. In fact, it has been demonstrated that CDV-specific antibodies in pups that suckled CDV immune mothers did not prevent the development of MV-specific antibodies when inoculated with a MV vaccine (Baker et al., 1966).
Other limitations of the commonly used modified live CDV vaccines have been previously documented (Tizard, 1990) and are linked to the ability of these vaccine strains to replicate within the vaccinated animals. These deleterious effects are most notable when the CDV vaccine strain is co-inoculated with canine adenovirus 1 and 2 into dogs resulting in immunosuppression, thrombocytopenia, and encephalitis (Bestetti et al., 1978; Hartley, 1974; Phillips et al., 1989). The modified live CDV vaccines have also been shown to induce distemper in other animal species including foxes, Kinkajous, ferrets, and the panda (Bush et al., 1976; Carpenter et al., 1976; Kazacos et al., 1981).
Therefore, the use of a recombinant CDV vaccine candidate would eliminate the continual introduction of modified live
CDV into the environment and potential vaccine-associated and vaccine-induced complications which have arisen with the use of the conventional CDV vaccines.
The use of poxvirus vectors may also provide a means of overcoming the documented inhibitory effect that maternal antibody has on vaccination with presently utilized live attenuated CDV strains in dogs. Pups born to mothers previously immunized at a young age with a poxvirus recombinant may avoid the interference of CDV-specific maternal antibody. Additionally, the ability of both vaccinia virus and canarypox virus vectors harbouring MV HA and F genes to elicit these responses and the lack of serological cross-reactivity between the two poxviruses provides a further advantage in that one vector could be utilized early in the pup's life and the other later, to boost CDV-specific immunity. This would eliminate the release of live attenuated CDV strains into the environment, an event linked to the occurrence of vaccine-induced and vaccine-associated complications (Tizard, 1990).
It can thus be appreciated that provision of a Morbillivirus recombinant poxvirus, and of vaccines which provide protective immunity against Morbillivirus infections, would be a highly desirable advance over the current state of technology.
It is an object of this invention to provide a vaccine which is capable of eliciting
Morbillivirus neutralizing antibodies, hemagglutination-inhibiting antibodies and protective immunity against Morbillivirus infection and a lethal Morbillivirus challenge, for providing cross-protection of dogs against canine distemper, particularly using a measles virus recombinant poxvirus vaccine.
It is also an object of this invention to provide recombinant poxviruses for use as the vaccine according to the present invention, which viruses express gene products of
Morbillivirus, and to provide a method of making such recombinant poxviruses.
It is also an object of this invention to provide for the cloning and expression of
Morbillivirus coding sequences, particularly measles virus coding sequences, in a poxvirus vector, particularly vaccinia virus or canarypox virus vectors, for use as the vaccine according to the present invention.
These and other objects and advantages of the present invention will become more readily apparent after consideration of the following.
According to the present invention there is provided a recombinant poxvirus containing therein DNA.from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper. According to the present invention there is also provided use of a recombinant poxvirus containing therein DNA from Morbillivirus in a nonessential region of the poxvirus genome for the preparation of a medicament for protecting a dog against canine distemper.
Therefore, the present invention relates to a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine including a carrier and a recombinant poxvirus containing, in a nonessential region thereof, DNA from
Morbillivirus, particularly measles virus. Advantageously, the DNA codes for an expresses a measles virus glycoprotein, particularly measles virus hemagglutinin glycoprotein and measles virus fusion glycoprotein. A plurality of measles virus glycoproteins advantageously are co-expressed in the host. The poxvirus used in the vaccine according to the present invention is advantageously a vaccinia virus or an avipox virus, such as canarypox virus.
For use as the vaccine according to the present invention, the present invention provides a recombinant poxvirus containing therein a DNA sequence from
Morbillivirus in a nonessential region of the poxvirus genome. The poxvirus is advantageously a vaccinia virus or an avipox virus, such as canarypox virus. The
Morbillivirus is advantageously measles virus. For use as the vaccine according to the present invention, the recombinant poxvirus expresses gene products of the foreign Morbillivirus gene. In particular, the foreign DNA codes for a measles virus glycoprotein, advantageously measles virus hemagglutinin glycoprotein and measles virus fusion glycoprotein. Advantageously, a plurality of measles virus glycoproteins are co-expressed in the host by the recombinant poxvirus.
A better understanding of the present invention will be had by referring to the accompanying drawings, in which:
Figure 1 schematically shows a method for the construction of plasmid pSPM2LHAVC used to derive recombinant vaccinia virus vP557 expressing the MV hemagglutinin gene;
FIG. 2 schematically shows a method for the construction of plasmid pSPMFVC used to derive recombinant vaccinia virus: vP455 expressing the MV fusion gene;
FIG. 3 schematically shows a method for the construction of plasmid pRW843 used to derive recombinant vaccinia virus vP756 expressing the MV hemagglutinin gene;
FIG. 4 schematically shows a method for the construction of plasmid pRW850 used to derive recombinant vaccinia virus vP800 expressing the MV fusion gene;
FIG. 5 schematically shows a method for the construction of plasmid pRW800 used to derive recombinant canarypox virus vCP40 expressing the MV fusion gene;
FIG. 6 schematically shows a method for the construction of plasmid pRW810 used to derive recombinant canarypox viruses yCP5O expressing the MV hemagglutinin gene and vCP57 co-expressing the MV fusion and hemagglutinin genes;;
FIG. 7 schematically shows a method for the construction of plasmid pRW852 used to derive recombinant canarypox virus vCP85 expressing the MV hemagglutinin gene;
FIG. 8 schematically shows a method for the construction of plasmid pRW853A used to derive recombinant canarypox virus vCP82 co-expressing the MV hemagglutinin and fusion genes;
FIG. 9 schematically shows a method for the construction of plasmid pSD460 for deletion of thymidine kinase gene and generation of recombinant vaccinia virus vP410;
FIG. 10 schematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus vP553;
FIG. 11 schematically shows a method for the construction of plasmid pMP494A for deletion of ATI region and generation of recombinant vaccinia virus vP618;;
FIG. 12 schematically shows a method for the construction of plasmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus vP723;
FIG. 13 schematically shows a method for the construction of plasmid pNPCSK1t for deletion of gene cluster [C7L - KlL)"and generation of recombinant vaccinia virus vP804;
FIG. 14 schematically shows a method for the construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant.vaccinia virus vP866 (NYVAC); and
FIG. 15 schematically shows a method for the construction of plasmid pRW857 used to derive recombinant
NYVAC virus vP913 co-expressing the MV hemagglutinin and fusion genes.
DETAILED DESCRIPTION OF THE INVENTION
A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.
Example 1 - GENERATION OF VACCINIA VIRUS RECOMBINANTS CONTAINING THE MEASLES HEMAGGLUTININ GENE
The rescuing virus used in the production of both recombinants was the Copenhagen strain of vaccinia virus from which the thymidine kinase gene had been deleted. All viruses were grown and titered on VERO cell monolayers.
The early/late vaccinia virus H6 promoter (Rosel et al., 1986; Taylor et al., 1988a,b) was constructed by annealing four overlapping oligonucleotides, H6SYN A-D. The resultant H6 sequence is as follows:
Vaccinia Virus H6 Promoter (SEQ ID NO:1/SEQ ID NO:2):
HindIII 5' AGCTTCTTTATTCTATACTTAAAAAGTGAAAATAAATACAAAGGTTCTTGAGG GTTGT AGAAATAAGATATGAATTTTTCACTTTTATTTATGTTTCCAAGAACTCC CAACA
GTTAAATTGAAAGCGAGAAATAATCATAAATTATTTCATTATCG CGATATCCGTT AAGTT CAATTTAACTTTCG CTCTTTATTAGTATTTAATAAAGTAATAG CG CTATAGG CAA TTCAA
TGTATCGTAC-3'
ACATAGCATGAGCT-5'
XhoI;;
Referring now to Figure 1, the annealed H6SYN oligonucleotides were ligated into pMP2LVC digested with
XhoI/HindIII to yield plasmid pSP131. The plasmid pMP2LVC contains the leftmost 0.4kbp of the vaccinia virus (Copenhagen strain) HindIII K region within pUCl8. The construction of pMP2LVC was performed as follows: a 0.4kbp
HindIII/SalI fragment from the HindIII K region was isolated and blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. This fragment was inserted into pUC18 which had been digested with PvuII. The resulting plasmid was designated pMP2VC.The plasmid pMP2VC was linearized with SspI. Synthetic oligonucleotides
MPSYN52 (SEQ ID NO:3) (5'-ATTATTTTTATAAGCTTGGA
TCCCTCGAGGGTACCCCCGGGGAGCTCGAATTCT-3') and MPSYN53 (SEQ ID
NO:4) (5'
AGAATTCGAGCTCCCCGGGGGTACCCTCGAGGGATCCAAGCTTATAAAAATAAT-3') were annealed and inserted into the leftmost of the two SsDI sites located within the vaccinia virus sequences. The resultant plasmid pMP2LVC contains a multiple cloning region in the intergenic region between the K1L and K2L open reading frames.
Annealed oligonucleotides 3P1 (SEQ ID NO:5) (5' GGGAAG-ATGGAACCAATCGCAGATAG-3') and 3P2 (SEQ ID NO:6) (5' AATTCTATCTG-CGATTGGGGTTCCATCTTCCC-3') containing the extreme 3' sequences of the HA gene and a sticky EcoRI end were ligated to a 1.8kbp XhoI/SmaI fragment from pMH22 containing the remainder of the HA gene and pSP131 digested with XhoI and EcoRI. The resultant plasmid was designated pSPMHA11.
The plasmid pNH22 was derived from a full length cDNA clone of the measles HA gene by creating a XhoI site at the ATG initiation codon (Alkhatib et al., 1986).
A 1.9kbp HindIII/EcoRI fragment from pSPMHA11, containing the measles HA gene, was isolated and blunt-ended with the Klenow fragment of the E. coii DNA polymerase in the presence of 2mM dNTPs. The isolated fragment was inserted into pMP409DVC (Guo et al., 1989) digested with Balli and blunt-ended by. treatment with mung bean nuclease.
Insertion into this vector yielded plasmid pSPMHA41. The
XhoI site between the H6 promoter and the initiation codon of the HA gene was removed by oligonucleotide directed double strand break mutagenesis (Mandecki, 1982) using oligonucleotide HAXHOD (SEQ ID NO:7) (5' ATATCCGTTAAGTTTGTATCGTAATGTCACCACAACGAGACCGGAT-3'). Plasmid pSPM2LHAVC was generated by this procedure. Insertion plasmid pSPM2LHAVC was used in in vitro recombination experiments with vaccinia virus vP458 as the rescue virus to generate recombinant vP557. vP458 contains the E. coli lac
Z gene in the M2L insertion site of vP410. This vaccinia virus recombinant contains the measles HA gene in the M2L locus of the genome, replacing the lac Z gene.
Example 2 - GENERATION OF VACCINIA VIRUS RECOMBINANT S CONTAINING THE MEASLES FUSION GENE
Referring now to Figure 2, annealed oligonucleotides 3PA (SEQ ID NO:8) (5' CCTAAAGCCTGATCTTACGGGAACATCAAAATCCTAT- GTAAGGTCGCTCTGATTTTTATCGGCCGA-3') and 3PB (SEQ ID NO:9) (5' AGCTTCGGCCGATAAAAATCAGAGCGACCTTACATAGGATTTTGATGTTCCCGTAAG- ATCAGGCTTTAGG-3') containing the 3' end of the measles fusion gene, a vaccinia virus early transcription termination signal (Yuen et al., 1987) and Eacti and HindIII ends were ligated to a lkbp SalI/HaeIII fragment from pCRF2 (obtained from C. Richardson, National Research Council of
Canada (Biotechnology Institute), Montreal, Canada H3A lAl) and pUC8 digested with SalI and HindIII.The resulting plasmid pMF3PR14 contains the 3' end of the lkbp fragment of the measles fusion gene.
Annealed oligonucleotides 5PA (SEQ ID NO:10) (5'
GGGATGGGTCTCAAGGTGAACGTCTCTGCCATATTC-3') and 5PB (SEQ ID NO:11) (5'-ATGGCAGAGACGTTCACCTTGAGACCCATCCC-3'), containing a 5' SmaI site and a 3' BstXI site, were ligated to a 820bp
BstXI/SalI fragment from pCRF2 and pUC8 digested with SmaI and SalI. The resultant plasmid pSPMF5Pl6 contains the 5' portion of the measles fusion gene.The 820bp SmaI/SalI fragment from pSPMF5Pl6 and the lkbp SalI/EaqS fragment from pMF3PR14 were ligated into pTPl5 digested with SmaI and East, The plasmid pTPl5 (Guo et al., 1989) contains the vaccinia virus early/late H6 promoter flanked by sequences from the HA locus of the vaccinia virus (Copenhagen strain) genome. The resultant plasmid containing the measles fusion gene juxtaposed 3' to the H6 promoter within the HA insertion plasmid was designated pSPHMF7.
Oligonucleotide directed mutagenesis was performed on pSPHMF7. Initially an in vitro mutagenesis reaction (Mandecki, 1982) was performed to create a precise ATG:ATG linkage of the H6 promoter with the measles fusion gene by removing the SmaI site using the oligonucleotide SPMAD (SEQ
ID NO:12) (5 '-TATCCGTTAAGT-TTGTATGGTAATGGGTCTCAAGGTGAACGTCT- 3'). This resulted in the generation of pSPMF75M20.
Subsequently, the BqlII site at the 5' end of the H6 promoter was removed using oligonucleotide SPBGLD (SEQ ID NO:13) (5 '-AATAAATCACTTTTTATACTAATTCTTTATTCTATACTT- AAAAAGT-3') according to a known procedure (Mandecki, 1982).
The resultant plasmid was designated pSPMFVC. This plasmid was used in in vitro recombination experiments with vaccinia virus vP410 as rescue virus to generate vP455.
Example 3 - IMMUNOPRECIPITATION ANALYSIS
In order to determine that recombinants vP455 and vP557 expressed authentic proteins, immunoprecipitation experiments were performed essentially as described (Taylor et al., 1990). Briefly, VERO cell monolayers were infected at 10 pfu per cell with either parental or recombinant viruses in the presence of 35S-methionine. The fusion protein was specifically precipitated from the infected cell lysate using a rabbit antiserum directed against a carboxy terminal fusion peptide. The hemagglutinin protein was specifically precipitated from the infected cell lysate using a polyclonal monospecific anti-hemagglutinin serum.
With respect to immunoprecipitation using a fusion specific serum, no radiolabelled products were detected in uninfected VERO cells, parentally infected VERO cells, or cells infected with the HA recombinant vP557. In cells infected with the fusion recombinant vP455, the fusion precursor F, with a molecular weight of approximately 60 kd and the two cleavage products F1 and F2 with molecular weights of 44 kd and 23 kd were detected. Similarly, with respect to immunoprecipitation of the glycosylated form of the HA protein with a molecular weight of approximately 7577 kd, no products were detected in uninfected VERO cells, parental infected cells, or VERO cells infected with vP455.
In addition, immunofluorescence studies indicated that both proteins were expressed on the infected cell surface.
Example 4 - CELL FUSION EXPERIMENTS
A characteristic of Morbillivirus cytopathogenicity is the formation of syncytia which arise by fusion of infected cells with surrounding uninfected cells followed by migration of the nuclei toward the center of the syncytium (Norrby et al., 1982). This has been shown to be an important method of viral spread, which for
Paramyxoviruses can occur in the presence of hemagglutinin specific antibody (Merz et al., 1980). This ability has been assigned by analogy with other Paramyxoviruses to the amino terminus of the F1 peptide (Choppin et al., 1981;
Novick et al., 1988; Paterson et al., 1987).
In order to determine that the measles proteins expressed in vaccinia virus were functionally active, VERO cell monolayers were inoculated with parental or recombinant viruses vP455 and vP557, respectively, at 1 pfu per cell.
After 1 h absorption at 370C the inoculum was removed, the overlay medium replaced, and the dishes incubated overnight at 370C. At 18 h post-infection, plates were examined with a microscope and photographed. No cell fusing activity was evident in VERO cells inoculated with parental virus, vP455 or vP557. However, when vP455 and vP557 were co-inoculated, efficient cell fusing activity was observed.
This result has recently been confirmed by Wild et al. (1991) who determined that syncytium formation in a variety of cell lines infected with measles/vaccinia virus recombinants required expression of both fusion and hemagglutinin genes. The result, however, is in contrast to a previous report (Alkhatib, 1990) which described cell fusion in 293 cells infected with high multiplicities of an adenovirus recombinant expressing the measles fusion protein. Similarly, it has been reported (Vialard et al., 1990) that cell fusion was observed in insect cells infected with a baculovirus recombinant expressing the measles fusion protein but only when incubated at pH 5.8. In neither case was the fusion activity enhanced by co-infection with the appropriate recombinant expressing the measles hemagglutinin protein. Variables which may be involved in the fusion process are cell type (Giraudon et al., 1984), pH of medium (Vialard et al., 1990) and level of expression of the fusion protein (Norrby et al., 1982).
EXamD1e 5 - SEROLOGICAL TESTS
The technique for virus neutralizing (VN) antibody testing was previously described in detail (Appel et al., 1973). Testing for CDV-VN antibody titers was made in VERO cells with the adapted Onderstepoort strain of CDV. Testing for MV-VN antibody titers was made in VERO cells with the adapted Edmonston strain of MV. The results of the serological tests are shown in Table 1.
Dogs immunized as described in Example 6 with either the vaccinia parental virus or vP455 expressing the measles fusion protein did not develop neutralizing antibody to MV. Dogs immunized with either vP557 expressing the HA protein or co-inoculated with both recombinants vP455 and vP557 did develop neutralizing antibodies after one inoculation. Levels of antibody were equivalent to those induced by inoculation with the attenuated Edmonston strain of MV.
Table 1
Measles virus neutralizing antibody titers in response to vaccination
Dats past vaccination
Immunization Dog No. 0a 7 14 12b 28 35c
Vacc. 4/1 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
4/2 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 vP455 4/3 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
4/4 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 vP557 4/5 < 1.0 2.2d 2.9 2.9 3.4 3.4
4/6 < 1.0 2.7 2.9 2.9 3.9 3.4 vP445 αvP557 4/7 < 1.0 2.7 3.4 3.2 3.4 3.4
4/8 < 1.0 2.5 2.9 2.9 3.6 2.9
NV 4/14 < 1.0 2.9
4/15 < 1.0 3.2 a) Time of first immunization b) Time of second immunization (first immunization with MV) c) Time of challenge d) Titer expressed as log10 of last antibody dilution showing complete neutralization of infectivity in a microtiter neutralization test as described by Appel et al. (1973).
Example 6 - ANIMAL PROTECTION STUDIES
In order to determine whether expression of the measles virus proteins in dogs inoculated with the recombinants was sufficient to induce a protective immune response against CDV challenge, fourteen 10 week old specific pathogen free beagle dogs were studied. Blood samples were collected at the initiation of the experiment and repeatedly thereafter. Four groups with two dogs in each group were immunized with two injections three weeks apart. The first group received vaccinia virus only. The second group received vaccinia virus with an insert for the
F protein of measles virus (vP455). The third group received vaccinia virus with an insert for the HA antigen of
MV (vP557), and the fourth group received a combination of 2 and 3.Each dog was inoculated with approximately 4 x 108 pfu of vaccinia virus in 1 ml amounts (0.6 ml subcutaneously and 0.4 ml intramuscularly). Two control dogs received 105 50t tissue culture infectious doses (TCID50) of the attenuated Edmonston strain of MV intramuscularly (1 ml amount) and two control dogs received 104 TCID50 of the attenuated Rockborn strain of CDV subcutaneously two weeks before challenge with virulent CDV. Two control dogs remained uninoculated before challenge.
All dogs were challenged by intranasal inoculation of 1 ml of tissue culture fluid containing 104 TCID50 of the
Snyder Hill strain of virulent CDV two weeks after the last inoculation. The clinical reactions of the dogs were monitored by daily observations and recording of body temperature and by biweekly recording of weight gain or losses. Circulating blood lymphocytes were counted before challenge and on days post challenge (dpc) 3, 5, 7 and 10.
Virus isolation from buffy coat cells by co-cultivation with dog lung macrophages (Appel et al., 1967) was attempted on dpc 3, 5, 7 and 10. Blood samples for serological tests were collected before vaccination and in weekly intervals until time of challenge, and on dpc 7, 10 and 20.
The results of challenge are shown in Table 2. Table 2
Effects of immunization on clinical signs after exposure of dogs to virulent CDV
No. of days after inoculation with virulent CDV
Dog Weight Elevated Lympho- Virus
Immunization Number Depression Loss Body Temp. a peniab Isolationc Deaqth
Vacc. 4/1 4-10d 3-10 4,5,7,10 7-10 3-7 10
4/2 4-10d 3-10 4,5,8-10 3-10 3-7 10 vP455 4/3 4-8 7-10 4,5,7-10 5,7 5-7
4/4 4-6 7-10 4-6 7 5-7 vP577 4/5 NDe ND 6,7 10 7
4/6 ND ND ND ND ND vP455 α vP557 4/7 ND ND 7 7 7
4/8 ND 7 6 ND 7
MV 4/14 ND ND ND 7 ND
4/15 ND 7 5 5 ND
CDV-Ro 4/16 ND ND ND ND ND
4/17 ND ND ND ND ND
None 4/18 6,14-17d 7-17 5,7 13-17 10 17
4/19 4-10d 3-10 4,5,7 3,7,10 3-10 10 a) Above 39.5 C b) Less than 2x10 Lymphocytes per mm c) Isolated from buffy coat cells co-cultivated with dog lung macrophages d) Dog became dehydrated and was euthanized e) None detected Non-immunized control dogs and dogs vaccinated with parental vaccinia virus developed clinical signs of severe disease and.were euthanized when dehydration was evident. Both dogs immunized with vP455 showed some signs of infection with CDV including weight loss, elevated body temperature, and lymphopenia although these symptoms were of shorter duration than were seen in control dogs.
Nonetheless, both dogs survived lethal challenge with CDV.
Dogs inoculated with vP557 or co-inoculated with both recombinants showed minimal signs of infection and survived challenge. Dogs inoculated with either attenuated Edmonston strain of MV or the attenuated Rockborn strain of CDV also survived challenge with minimal signs of disease.
Example 7 - ADDITIONAL VACCINIA/NEASLES CONSTRUCTS
Referring now to Figure 3, a second vaccinia virus recombinant containing the measles HA gene within the tk locus was generated (vP756) using insertion plasmid pRW843.
pRW843 was constructed in the following manner. A 1.8kbp
EcoRV/SmaI fragment containing the 3'-most 24bp of the H6 promoter fused in a precise ATG:ATG configuration with the
HA gene lacking the 3'-most 26bp was isolated from pSPM2LHAVC. This fragment was used to replace the 1.8kbp
EcoRV/SmaI fragment of pSPMHA11 to generate pRW803. Plasmid pRW803 contains the entire H6 promoter linked precisely to the entire measles HA gene.
In the confirmation of previous constructs with the measles HA gene it was noted that the sequence for cod on 18 (CCC) was deleted as compared to the published sequence (Alkhatib et al., 1986). The CCC sequence was replaced by oligonucleotide mutagenesis via the Kunkel method (Kunkel, 1985) using oligonucleotide RW117 (SEQ ID NO:14) (5'
GACTATCCTACTT CCCTTGGGATGGGGGTTATCTTTGTA-3').
Pro 18
Single stranded template was derived from plasmid pRW819 which contains the H6/HA cassette from pRW803 in pIBI25 (IBI, New Haven, CT.). The mutagenized plasmid containing the inserted (CCC) to encode for a proline residue at codon 18 was designated pRW820. The sequence between the HindIII and XbaI sites of pRW820 was confirmed by nucleotide sequence analysis. .The HindIII site is situated at the 5 border of the H6 promoter while the XbaI site is located 230bp downstream from the initiation codon of the HA gene.
A 1.6kbp XbaI/EcoRI fragment from pRW803, containing the HA coding sequences downstream from the XbaI and including the termination codon, was used to replace the equivalent fragment of pRW820 resulting in the generation of pRW837.
The mutagenized expression cassette contained within pRW837 was derived by digestion with HindIII and EcoRI, blunt-ended using the Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs, and inserted into the SmaI site of pSD573VCVQ to yield pRW843. The plasmid pRW843 was used in in vitro recombination experiments with vP618 as the rescue virus to yield vP756. Parental virus vP618 is a Copenhagen strain virus from which the thymidine kinase, hemorrhagic and A-type inclusion genes have been deleted. Recombinant vP756 has been shown by immunoprecipitation analysis to correctly express a hemagglutinin glycoprotein of approximately 75kd.
Referring now to Figure 4, a second vaccinia virus recombinant (vP800) harboring the measles fusion gene in the
ATI locus of the genome was generated using insertion plasmid pRW850. To construct pRW850, the following manipulations were performed. The plasmid pSPMF75M20 containing the measles fusion gene linked in a precise
ATG:ATG configuration with the H6 promoter was digested with
NruI and East. The 1.7kbp blunt ended fragment containing the 3'-most 28bp of the H6 promoter and the entire fusion gene was isolated and inserted into pRW823 digested with
NruI and XbaI and blunt-ended. The resultant plasmid pRW841 contains the H6 promoter linked to the measles fusion gene in the pIBI25 plasmid vector (IBI, New Haven, CT.).The
H6/measles fusion expression cassette was derived from pRW841 by digestion with SmaI and the resulting 1.8kbp fragment was inserted into pSD494VC digested with SmaI to yield pRW850. The plasmid pRW850 was used in in vitro recombination experiments with vP618 as the rescue virus to yield vP800. Recombinant vP800 has been shown by immunoprecipitation analysis to express an authentically processed fusion glycoprotein.
ExamPle 8 - ASSESSMENT OF MEASLES NEUTRALIZING ANTIBODY
IN GUINEA PIGS AND RABBITS INOCULATED WITH
vP455
Two rabbits were inoculated intradermally at 5 sites with a total of 1x108 pfu of recombinant vP455 expressing the measles fusion protein. Both rabbits were boosted with an identical inoculation at week 12. Serial bleeds were collected, and at week 14, two weeks after the boost, the rabbits were tested for the presence of serum neutralizing antibodies.
Four guinea pigs were inoculated subcutaneously with 1xl08 pfu each of recombinant vP455. An identical booster inoculation was given at 21 days. Serial bleeds were collected.
The presence of measles virus serum neutralizing antibody was assessed using a microtiter test (Appel et al.
1973) using 10 TCID50 of virus per microtiter well. The results are shown in Table 3.
Table 3
Results of measles vires serum neutralizing antibodies in guinea pigs and rabbits inoculated with vP455
Week Post-Inoculation
0 2 3 4 5 7 14
Animal
Guines Pig
1 N.D.@ N.D. N.D.-.8b 1.3-1.3 1.3-1.5 1.3 N.Tc
2 N.D. N.D. .8-1.0 .8-1.3 1.3-1.5 N.D. N.T.
3 N.D. N.D. N.D.-.8 .8-1.0 1.0-1.3 1.0 N.T.
6 N.D. N.D. .8 -.8 .8-1.5 1.0-1.3 1.0 N.T.
Rabbit
W44 N.D. N.T. N.T. N.T. N.T. N.T. 1.5
W86 N.D. N.T. N.T. N.T. N.T. N.T. 1.5 a) Not detectable b) Results of two assays c) Not tested Example 9 - GENERATION OF MEASLES VIRUS RECOMBINANT
CANARYPOX VIRUS Measles/cssnarypox virus recombinants were developed using a similar strategy to that previously described for fowlpox virus (Taylor et al., 1988a,b).
Plasmids for insertion of the measles F and HA genes into canarypox virus were generated as follows.
Referring now to Figure 5, the 1.8kbp blunt-ended BalII/EaqI fragment from pSPMF75M20 containing the H6 promoted measles F gene was inserted into the blunt-ended
EcoRI site of pRW764.2. Plasmid pRW764.2 contains a 3.4kbp
PvuII fragment from the canarypox genome having a unique
EcoRI site which has been determined to be non-essential for viral replication. The resultant plasmid containing the measles F gene was designated pRW800 and was used in recombination experiments with canarypox as the rescuing virus to generate vCP40.
Referring now to Figure 6, the 1.8kbp EcoRV/SmaI fragment from pSPM2LHA containing the 3'-most 28bp of the H6 promoter fused in a precise ATG:ATG configuration with HA was inserted between the EcoRV and SmaI sites of pSPMHA11.
The resultant plasmid was designated pRW803. A 2kbp
HindIII/EcoRI fragment of pRW803 containing the H6 promoted measles HA gene was blunt-ended and inserted into the bluntended BalII site of plasmid pRW764.5. Plasmid pRW764.5 contains an 800bp PvuII fragment of the canarypox genome having a unique BqlII site which has previously been determined to be non-essential for viral growth. This insertion created plasmid pRW810 which was used in recombination tests to generate vCP50.
Insertion of the measles F and HA sequences individually led to the development of recombinants vCP40 and vCP50, respectively. In order to create a double recombinant, the single F recombinant vCP40 was used as a rescue virus for insertion of the HA gene contained in pRW810. This led to the development of double recombinant vCP57.
Example 10 - IMMUNOPRECIPITATION ANALYSIS
In order to confirm that recombinants vCP40, vCP50 and vCP57 expressed.authentic proteins, immunoprecipitation analysis was performed using mono-specific sera directed against either the HA or F proteins. A correctly processed fusion polypeptide was specifically precipitated from lysates of cells infected with vCP40 and vCP57. The fusion precursor F0 with a molecular weight of approximately 60kd and the two cleavage products F1 and F2 with molecular weights of approximately 44 and 23kd, respectively, were detected. No fusion specific products were detectable in uninfected CEF cells, parentally infected CEF cells or CEF cells infected with the HA recombinant vCP50.Similarly, a glycoprotein of approximately 75kd was specifically precipitated from CEF cells infected with the single HA recombinant vCP50 and double recombinant vCP57. No HA specific products were detected in uninfected cells, parentally infected cells or cells infected with fusion recombinant vCP4 0.
Example 11 - CELL FUSION EXPERIMENTS
In order to determine that the measles virus recombinants were functionally active, cell fusion assays were performed. VERO cell monolayers were infected with 1 pfu per cell of CP parental or recombinant viruses and examined for cytopathic effects at 18 hours post infection.
No cell fusing activity was evident in VERO cells inoculated with parental, vCP40 or vCP50 viruses. However, when VERO cells were inoculated with the double recombinant vCP57 or when cells are co-infected with both vCP40 and vCP50, efficient cell fusing activity is evident.
Example 12 - SEROLOGICAL TESTS
Dogs inoculated as described in Example 13 with the canarypox/HA recombinant vCP50, vaccinia/HA recombinant vP557, the canarypox/HA/F double recombinant vCP57 or coinoculated with vP455 and vP557 developed significant serum neutralizing antibody to measles virus after one inoculation. Neither of the two dogs inoculated with the canarypox/F recombinant vCP40 developed neutralizing antibody after one or two inoculations. The results of the serological tests are shown in Table 4.
In addition, guinea pigs inoculated with the vCP40 recombinant did develop low but reproducible levels of serum neutralizing antibody.
Table 4
Measles virus neutralizing antibody titers (in log10)
Days post vaccination
Immunization Dog No. 0a 7 14 21b 28 35c
Canary pox virus 9/ 1 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 (CPV) 9/ 2 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
VCP50 9/ 3 < 1.0 2.7d 2.9 3.2 4.4 4.1
9/ 4 < 1.0 1.7 2.7 2.7 3.9 3.9
VCP40 9/ 5 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
9/ 6 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
VCP57 9/ 7 < 1.0 2.0 2.7 2.5 3.9 3.6
9/ 8 < 1.0 1.0 2.2 2.0 3.6 3.4
VP455 9/ 9 < 1.0 < 1.0 < 1.0 1.0 1.0 1.0
VP557 9/10 < 1.0 2.9 2.5 3.2 3.4 3.4 vP455 α VP557 9/11 < 1.0 1.3 2.9 2.9 2.9 2.9
MV 9/12 < 1.0 2.5 2.5
Control 9/13 < 1.0
9/14 < 1.0
CDV-Ro 9/15 < 1.0 < 1.0 < 1.0 a) Time of first immunization.
b) Time of second immunization. (First immunization with MV and CDV-Ro).
c) Time of challenge.
d) Assessment of serum neutralization titers assessed in known manner (Appel et al., 1973).
ExamPle 13 - ANIMAL PROTECTION STUDIES
In order to determine whether non-replicating canarypox vectors expressing measles virus proteins would induce a protective immune response against CDV challenge, ten week old specific pathogen free beagle dogs were inoculated with canarypox parental and recombinant viruses.
Two dogs were inoculated simultaneously with two subcutaneous injections of 1x10 8 pfu of each recombinant at three week intervals. For comparison, one dog was inoculated in the same regimen with each of the single vaccinia virus recombinants vP455 and vP557 and a combination of both. One dog was also inoculated intramuscularly with one dose of 105 TCID50 of the attenuated
Edmonston strain of MV. One dog was inoculated subcutaneously with one dose of 104 TCID50 of the attenuated
Rockborn strain of CDV. Dogs were challenged two weeks after the final inoculation via intranasal inoculation with a lethal dose of 104 TCID50 of the virulent Snyder Hill strain of CDV. Clinical reactions of dogs were monitored daily. The results are shown in Table 5.
Table 5
Effects of immunization on clinical signs after exposure of dogs to virulent CDV
No. of days after inoculation with virulent CDV
Elevated Lympho Virus
Immunization Dog No. Depression Wt. Loss Body Temp.a peniab Isolationc
Canary pox 9/ 1 4-10d 3-10 4,5,7,8 5-10 3-10 virus (CPV) 9/ 2 4-10d 7-10 4,5,7,8 3-10 3-10 vCP50 9/ 3 NDe 7-10 5-7 5-7 7
9/ 4 ND 7 6,7 7 ND vCP40 9/ 5 4-10 3-10 4 5-10 5-7
9/ 6 4-6 3-10 4-6 5 5-7 vCP57 9/ 7 ND 7-10 5,6 5 ND
9/ 8 ND 7-10 4-7,10 7-10 5-7 vP455 9/ 9 6-8 7-10 4,5,8,9 5-10 3-10 vP577 9/10 ND 3-10 ND ND ND vP455 α 9/11 ND 3-10 ND 5-7 ND vP577
MV 9/12 ND 7-10 ND 5 5
None 9/13 4-10d 3-10 4-6 5-10 5-10
9/14 4-10d 3-10 4-5 3-10 5-7
CDV-Ro 9/15 ND ND ND ND ND a) Above 39.5 C. b) Less than 2x10 Lymphocytes per mm . c) Isolated form buffy coat cells co-cultivated with dog lung macrophages in Known manner (Appel et al., 1967).
d) Dog became dehydrated and was euthanized. e) None detected.
No adverse reactions to vaccination were noticed in any of the dogs during the course of the experiment. The two dogs immunized with parental canarypox virus and two non-immunized control dogs showed severe disease after challenge with virulent CDV. All four dogs became depressed, showed elevated body temperature, weight loss, lymphopenia and severe dehydration. Dogs immunized with
CDV-Rockborn developed serum neutralizing antibodies against
CDV but not against MV prior to challenge and survived challenge, symptom free. Dogs immunized with attenuated MV developed serum neutralizing antibodies to MV but not CDV prior to challenge, and survived challenge with mild signs of infection.Dogs inoculated with vCP50, vCP57, vP557 or co-inoculated with vP455 and vP557 developed significant serum neutralizing antibody to MV after one inoculation and survived challenge with only minor signs of infection.
Example 14 - ADDITIONAL CANARYPOXJMEASLES CONSTRUCTS
Referring now to Figure 7, to generate a canarypox virus recombinant expressing the MV HA gene the following insertion plasmids were created. A 1.8kbp EcoRV/EcoRI fragment from pRW837 containing the 3'-most 26bp of the H6 promoter linked precisely to the measles HA, was ligated to a 3.2kbp EcoRV/EcoRI fragment from pRW838. The pRW838 derived fragment includes the 5' portion of the H6 promoter and C5 locus flanking arms. Plasmids pRW838 and pRW831 (see below) were derived as follows.
An 880 bp PvuII canarypox genomic fragment was inserted between the PvuII sites of pUC9. the resultant plasmid was designated pRW764.5. The nucleotide sequence of the 880 bp canarypox fragment was determined using the modified T7 enzyme SequenaseTM Kit (United States
Biochemical, Cleveland, OH) according to manufacturer's specifications. Sequence reactions utilized custom synthesized primers (17-18 mers) prepared with the Biosearch 8700 (San Rafael, CA) or Applied Biosystems 3800 (Foster
City, CA). This enabled the definition of the C5 open reading frame.
To specifically delete the C5 open reading frame, pRW764.5 was partially cut with RsaI and the linear product was isolated. The ksaI linear fragment was recut with BallI and the pRW764.5 fragment with a RsaI-BglII deletion from position 156 to position 462 was isolated and used as a vector for the following synthetic oligonucleotides: RW145 (SEQ ID NO:15): (5'-ACTCTCA AAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA-3') RWl4 6 (SEQ ID
NO:16): (5' GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT-3') Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW764.5 RsaI-BalII vector described above. The resulting plasmid is pRW831.
This C5 deletion plasmid was constructed without interruption of other canarypox virus open reading frames.
The C5 coding sequence was replaced with the above annealed oligonucleotides (RW145 and RW146) which include the restriction sites for HindIII, Smal, and EcoRI.
The plasmid pRW838, was derived from pRW831 by the insertion of a SmaI fragment containing the Rabies G gene (Taylor et al., 1988b) juxtaposed 3' to the vaccinia virus
H6 promoter. Ligation of the 1.8 kbp EcoRV/EcoRI fragment from pRW837 with the 3.2 kbp EcoRV/EcoRI fragment from pRW838 led to the construction of plasmid pRW852. Plasmid pRW852 was used in recombination experiments with a canarypox isolate designated ALVAC to yield vCP85. ALVAC is a plaque cloned isolate of canarypox virus (CPV) derived from the Rentschler strain, a highly attenuated strain of
CPV used for vaccination of canaries. Replication of ALVAC and derived recombinants is restricted to avian species.
Immunoprecipitation analysis has confirmed that a protein of approximately 75kd recognized by a rabbit anti-HA serum is expressed in CEF cells infected with recombinant vCP85.
Referring now to Figure 8, to generate a canarypox virus recombinant harboring both the MV HA and F genes the following constructs were engineered. SmaI restriction sites were added to the ends of the H6 promoted measles fusion gene. To accomplish this, pRW823, which is pIBI25 containing the vaccinia virus H6 promoter, was digested downstream of the promoter sequence at the XbaI site. The ends were blunted with the Klenow fragment of the E. coli
DNA polymerase in the presence of 2mM dNTPs. The bluntended DNA was subsequently digested with NruI to liberate a 3.Okbp fragment containing the 5'-most 100bp of the H6 promoter. This fragment was isolated and ligated to a 1.7kbp blunt-ended EaqI/NruI fragment from pSPMF75. The resultant plasmid was designated as pRW841.
The 1.8kbp SmaI fragment derived by digestion of pRW841 was inserted into the C5 deletion vector, pRW83l.
The plasmid pRW851 was linearized at the EcoRI site situated 3' to the fusion gene and was blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. The plasmid:pRW837, containing the measles HA gene juxtaposed 3' to the H6 promoter sequences, was digested with HindIII and EcoRI and blunt-ended with the
Klenow fragment. The resultant 1.8kbp fragment was isolated and inserted into pRW851 that had been linearized with EcoRI and blunt-ended. The resultant plasmid, which contains both genes in a tail to tail configuration, was designated pRW853A and was utilized in in vitro recombination experiments with canarypox (ALVAC) as the rescue virus to generate vCP82 also designated ALVAC-MV.Expression analysis using immunoprecipitation and immunofluorescence confirmed that in cells infected with recombinant vCP82 authentically processed HA and F proteins were expressed.
The recombinant was also functional for cell fusing activity.
Results of seroloqical analysis of sera of rabbits and guinea pigs inoculated with ALVAC-MV (vCP82)
Four guinea pig were inoculated by the subcutaneous route with ALVAC-MV (vCP82). Two animals (026 and 027) each received 1x108 pfu and two animals (028 and 029) each received lox107 pfu. At 28 days, animals were re-inoculated with an identical dose. Two rabbits were inoculated with lox108 pfu of ALVAC-MV (vCP82) by the subcutaneous route. At 28 days, animals were re-inoculated with an identical dose.Serial bleeds of these animals were analyzed for measles virus neutralizing activity using either a microtite,neutralization test described by Appel and Robson (1973) or a plaque reduction neutralization test described by Albrecht et al. (1981). In addition, sera were analyzed for the presence of antibody capable of blocking measles virus induced cell-cell fusion in an anti-fusion assay performed as described in Merz et al. (1980).
The results of analysis for the presence of measles virus serum neutralizing antibody are shown in
Tables 6 and 7. Both guinea pigs (026 and 027) receiving 1x108 pfu of ALVAC-MV sero-converted after a single inoculation and sera showed an antibody rise after the booster inoculation. One animal (029) receiving 1x107 pfu also sero-converted after one inoculation. The fourth animal (028) did not show a detectable response after one inoculation but did achieve equivalent titers after the second inoculation.
Rabbit sera were also analyzed using a plaque reduction neutralization method. The results are shown in
Table 7. Both animals sero-converted after one inoculation.
Sera of rabbit 063 was tested by both the micro-titer neutralization test and the plaque reduction neutralization test. Titers achieved were similar using both methods. It has been reported that a minimal serum neutralizing titer of 1.2 to 1.9 in vaccinated children is-required for protection from disease (Lennon and Black, 1986; Black et al., 1984).
Using this criteria, all animals, except the one guinea-pig which did not sero-convert until the second inoculation showed a protective level of antibody after one inoculation.
Table 6
Serological analysis of sera of guinea pigs inoculated with
ALVAC-MV (vCP82): Analysis performed by microtiter serum neutralization assay.
Animal Days post-inoculation
0 14 21 28' 42 48 56
Guinea pigs 026d - N.T.a 1.25b 1,49 2.45 2.68 2.92 027 - N.T. 1.97 1.49 2.68 2.45 2.21 028e - N.T. - - 1.73 2.45 1.97 029 - N.T. 0.8 1.49 2.45 2.45 2.45 a) Not tested.
b) Titer expressed as log10 of reciprocal of last dilution
showing complete neutralization of cytopathic effect.
c) Animals boosted at 28 days post-inoculation.
d) Animals 026 and 027 received 1x108 pfu.
e) Animals 028 and 029 received 1x107 pfu.
Table 7
Serological analysis of sera of rabbits inoculated with
ALVAC-MV (vCP82)
Animal Days post-inoculation
0 14 21 28b 42 56
Plaque reduction method 063 - l.9a 2.8 1.6 2.2 2.2 064 - 2.2 2.5 2.8 3.1 2.8
Microtiter neutralization method 063 - 1.5' 1.7 1.5 1.7 a) Titer expressed as log10 of reciprocal of last dilution
showing a 50% reduction in plaque number as compared to
pre-inoculation serum.
b) Animals boosted at 28 days post-inoculation.
c) Titer expressed as log10 of reciprocal of last dilution
showing complete neutralization of cytophatic effect.
Previous studies have shown that an inactivated vaccine was associated with poor protective efficacy and an enhanced measles disease on re-exposure to the virus.
Recipients of the inactivated vaccine demonstrated an absence of antibody to the fusion protein and it was proposed that the inactivation process had rendered the protein non-immunogenic (Norrby and Gollmar, 1975; Norrby et al., 1975). In addition, it has been shown for other paramyxoviruses that antibody to the F protein is able to inhibit cell to cell spread of virus in tissue culture while antibody to the hemagglutinin component is not (Merz et al., 1980).
It was therefore significant to demonstrate that animals inoculated with ALVAC-MV (vCP82) were able to induce antibody to the F component which was capable of blocking cell to cell transmission of measles virus. The results of this anti-fusion assay are shown in Table 8. Anti-fusion activity was evident in sera of both guinea-pigs and rabbits inoculated with ALVAC-MV (vCP82). The sera analyzed was taken two or three weeks after the boost inoculation. No anti-fusion activity could be detected in sera of rabbits inoculated with ALVAC parental virus.
Table 8
Analysis of sera of guinea pigs and rabbits inoculated with
ALVAC-MV for anti-fwsion activity
Animal Designation Immunogen Anti-Fusion Titer
Pre-inoc. Post-Vacc.
Guinea-pig 026 ALVAC-MV - 2.4a. b
027 ALVAC-MV - 1.2
Rabbit 063 ALVAC-MV - 1.8' 064 ALVAC-MV - 1.8
Rabbit W121 ALVAC -
W123 ALVAC - a) Guinea pig sera tested at 7 weeks post-vaccination.
b) Titer expressed as log10 of reciprocal of highest
dilution showing complete inhibition of measles virus
induced cell fusing activity.
c) Rabbit sera test at 6 weeks post-vaccination.
In further tests to demonstrate the presence of antibody to both the MV hemagglutinin and MV fusion proteins in sera of animals inoculated with ALVAC-MV, immunoprecipitation experiments were performed. Sera of rabbits inoculated with ALVAC-MV was shown to specifically precipitate both the hemagglutinin and fusion proteins from radiolabelled lysates of Vero cells infected with Edmonston strain MV.
In a similar study, groups of guinea pigs, rabbits and mice were inoculated by the intra muscular route with
ALVAC-MV, and their serological response to measles virus monitored using the hemagglutination-inhibition (HI) test.
The serological response to canarypox virus was monitored by
ELISA assay. In this study, five guinea pigs were inoculated with 5.5 log10 TCID50, thirty mice were inoculated with 4.8 log10 TCIDso, and five rabbits were inoculated with 5.8 log10 TCID50. All animals were re-inoculated at 28 days with an equivalent dose. Animals were bled at regular intervals and their response to measles virus assessed in an
HI assay. The limit of detection in the HI assay corresponds to a log10 titer of 1 and it is considered tnat sero-positive (protected) children have a serum titer in the range of 1.6 to 2.8. The results of analysis are shown in
Tables 9, 10 and 11.
Sera of mice were analyzed in groups of 5 animals (Table 9). All animals showed a primary response to canarypox virus which was boosted after the second inoculation. The mice did not show a response to MV after one inoculation. Three of the six groups showed titers within the protective range at 8 weeks post-inoculation.
Similarly, all guinea-pigs (Table 10) showed a response to canarypox virus after one inoculation which was boosted after the second inoculation. Four of five animals developed anti-HI titers after one inoculation, one of these being in the protective range. One week after the second inoculation, the titers of all animals were in the protective range. These titers were maintained through 8 weeks post-inoculation when the experiment was concluded.
All rabbits (Table 11) inoculated with ALVAC-MV (vCP82) responded serologically to canarypox inoculation. Four of five animals sero-coinverted to measles virus after one inoculation (one in the protective range). Serum titers of all animals were in the protective range one week after the second inoculation.
Table 9
Serological response of mice to inoculation with ALVAC-MV (vCP82) Anti-canarypox response
ELISA TITER Week post-inoculation
Mouse Group 0 2 4 5 6 8 1a -0.009b 0.364 0.193 1.821 1.616 1.123 2 -0.026 0.047 0.240 1.739 1.963 1.986 3 -0.006 0.148 0.641 -1.860 1.861 1.947 4 -0.005 0.130 0.451 1.506 1.937 1.124 5 0.687 0.542
Mean -0.012 0.275 0.413 1.732 1.844 1.395
Anti-measles response
HI TITER Week post-inoculation
Mouse Group 0 2 4 5 6 8 1 < lC - < 1 < 1 < 1 1 1 2 < 1 < 1 < 1 1 1.6 1.6 3 < 1 < 1 1 1 2. 2 2. 2 4 < 1 < 1 < 1 1.6 1 1.8 5 < 1 < 1 < 1 1.3 1.8 1.2
Mean - - 1 1.2 1.5 1.5 a). Groups of five mice were exsanguinated and sera
pooled.
b) Optical density in an ELISA assay on sera at
dilution of 1:800 c) Limit of detection in HI test corresponds to a log10
titer of 1 i.e. 1:10 dilution. Titer expressed as logl0 of reciprocal of highest dilution showing
inhibition of hemagglutination.
Table 10
Serological response of guinea-pigs to inoculation with
ALVAC-MV (vCP82) Anti-canarvox resDonse
ELISA TITER Week post-inoculation
Guinea-pig 0 2 4 5 6 8 1 0.038a 0.045 0.111 .771 1.970 1.856 2 0.010 0.072 0.234 1.768 1.786 1.785 3 -0.011 0.426 0.529 1.567 1.586 1.700 4 0.016 0.045 0.076 1.583 1.696 1.635 5 -0.020 0.012 0.050 1.583 1.859 1.847
Anti-measles resDonse
HI TITER Week post-inoculation
Guinea pig 0 2 4 5 6 8 1 < 1b 1.18 1.90 3.11 3.41 3.11 2 < 1 < 1 1.00 2.20 2.20 2.08 3 < 1 < 1 1.18 2.51 2.68 2.98 4 < 1 < 1 < 1 1.60 1.90 1.90 5 < 1 < 1 1.30 1.90 2.20 2.20 a) Optical density in an ELISA assay on serum at a
1:3200 dilution.
b) Limit of detection in HI test corresponds to a log10 titer of 1 i.e. 1:10 dilution. Titer expressed as
in legend to Table 9.
Table 11
Serological response of rabbits to inoculation with ALVAC-MV (vCP82)
Anti-canarypox response
ELISA TITER Week post-inoculation
Rabbit 0 2 4 5 6 8 la - 0.009a 0.085 0.113 1.953 1.754 1.249 2 -0.002 0.065 0.068 0.717 0.567 0.353 3 -0.003 0.090 0.079 0.921 0.692 0.481 4 -0.005 0.034 0.068 1.558 1.324 1.076 5 -0.003 0.072 0.092 1.785 1.226 0.710
Anti-measles response
HI TITER Week post-inoculation
Rabbit 0 2 4 5 6 8 1 < 1b < 1 1.00 2.81 2.51 2.20 2 < 1 < 1 < 1 2.20 1.90 1.60 3 < 1 < 1 1.30 2.81 2.51 2.38 4 < 1 1.30 1.60 3.11 3.11 2.51 5 < 1 1.00 1.30 2.68 2.38 1.90 a) Optical density in an ELISA assay on sera at a
dilution of 1:1600.
b) Limit of detection in HI test corresponds to a log10
titer of 1 i.e. 1:10 dilution. Titer expressed as
in legend to Table 9.
Results of serological analysis of sera of squirrel monkeys inoculated with ALVAC-MV (vCP82): Influence of Prior exposure to poxvirus on induction of a measles virus specific immune response
Nine squirrel monkeys (Saimiri sciureus) were inoculated with ALVAC-MV (vCP82). All monkeys were naive to measles virus. Seven of the monkeys had prior exposure to vaccinia virus and/or canarypox virus. The previous immunization history is shown in Table 12. All monkeys were inoculated with one dose of 5.8 log10 pfu by the subcutaneous route. Four of the animals (#39, 42, 53 and 58) were re-inoculated with an equivalent dose fifteen weeks after the primary inoculation. Anti-measles antibody was measured in the HI test. The results are shown in Table 12.
After the first inoculation, two of the nine monkeys showed a low response to inoculation with ALVAC-MV. After the second inoculation, the four monkeys re-inoculated all sero-converted with significant antibody titers in the range required for protective immunity. The titers achieved were equivalent whether the monkey had prior exposure to vaccinia virus and ALVAC or no prior poxvirus exposure.
Table 12
Inoculation of squirrel monkeys with ALVAC-MV (vCP82):
Immune response in the face of pre-existing ALVAC immunity.
Monkey # Previous Immunity Anti-Measles HI response
to Poxviruses Primary a Boostb 36 W, ALVAC < 1 N.B.
37 W, ALVAC-RG < 1 N.B.
39 W ALVAC-RG, CP-FeLV 1 2.2.
40 W, CP-FeLV < 1 N.B.
42 None < 1 2.2.
52 ALVAC < 1 N.B.
53 ALVAC-RG, ALVAC-RG < 1 1.6.
56 CP-FeLV < 1 N.B.
58 None 1 2.2.
W: Vaccinia virus, Copenhagen strain
ALVAC-RG: ALVAC recombinant expressing rabies G gene
CP-FeLV: Canarypox recombinant expressing FeLV env gene
NB: Not boosted a) Animals received 5.8 log10 pfu by S.C. route.
b) Animals 39, 42, 52 and 53 were boosted with an
identical dose 15 weeks after the first inoculation.
ExamPle 15 - ATTENUATED VACCINIA VACCINE STRAIN NYVAC
To develop a new vaccinia vaccine strain, the
Copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors. The sequential deletions are detailed below. All designations of vaccinia restriction fragments, open reading frames and nucleotide positions are based on the terminology reported in Goebel et al. (1990a,b).
The deletion loci were also engineered as recipient loci for the insertion of foreign genes.
The regions sequentially deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designations for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified:
(1) thymidine kinase gene (TK; J2R) vP410; (2) hemorrhagic region (; B13R + B14R) vP553;
(3) A type inclusion body region (ATI; A26L) vP618;
(4) hemagglutinin gene (HA; A56R) vP723;
(5) host range gene region (C7L - K1L) vP804; and
(6) large subunit, ribonucleotide reductase (14L) vP866
(NYVAC).
DNA Cloning and Synthesis
Plasmids were constructed, screened and grown by standard procedures (Maniatis et al., 1986; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from GIBCO/BRL, Gaithersburg, MD, New England
Biolabs, Beverly, MA; and Boehringer Mannheim Biochemicals,
Indianapolis, IN. Klenow fragment of . coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New
England Biolabs. The reagents were used as specified by the various suppliers.
Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously described (Guo et al., 1989). DNA amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp
DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk,
CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler.
Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides.
Cells, Virus, and Transfection
The origins and conditions of cultivation of the
Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for Beta-galactosidase activity are as previously described (Panicali et al., 1982;
Perkus et al., 1989).
Construction of Plasmid pSD460 for Deletion of Thymidine
Kinase Gene (J2R)
Referring now to FIG. 9, plasmid pSD406 contains vaccinia HindIII J (pos. 83359 - 88377) cloned into pUC8.
pSD406 was cut with HindIII and PvuII, and the 1.7 kb fragment from the left side of HindIII J cloned into pUC8 cut with HindIII/SmaI, forming pSD447. pSD447 contains the entire gene for J2R (pos. 83855 - 84385). The initiation codon is contained within an NlaIII site and the termination codon is contained within an Ss > I site. Direction of transcription is indicated by an arrow in FIG. 9.
To obtain a left flanking arm, a 0.8 kb
HindIII/EcoRI fragment was isolated from pSD447, then digested with NlaIII and a 0.5 kb HindIII/NlaIII fragment isolated. Annealed synthetic oligonucleotides
MPSYN43/MPSYN44 (SEQ ID NO:17/SEQ ID NO:18)
SmaI
MPSYN43 5' TAATTAACTAGCTACCCGGG 3'
MPSYN44 3' GTACATTAATTGATCGATGGGCCCTTAA 5'
NlaIII a EcoRI were ligated with the 0.5 kb HindIII/NlaIII fragment into pUC18 vector plasmid cut with HindIII/EcoRI, generating plasmid pSD449.
To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with SsDI (partial) within vaccinia sequences and
HindIII at the pUC/vaccinia junction} and a 2.9 kb vector fragment isolated. This vector fragment was ligated with annealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID
NO:19/SEQ ID NO:20)
HindIII SmaI
MPSYN45 5' AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA MPSYN46 3' AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT
NotI SspI
ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5' MPSYN46 generating pSD459.
To combine the left and right flanking arms into one plasmid, a 0.5 kb HindIII/SmaI fragment was isolated from pSD449 and ligated with pSD459 vector plasmid cut with
HindIII/SmaI, generating plasmid pSD460. pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2. 32P labeled probe was synthesized by primer extension using MPSYN45 (SEQ ID
NO:19) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:21) (5'-TTAGTTAATTAGGCGGCCGC-3') as primer. Recombinant virus vP410 was identified by plaque hybridization.
Construction of Plasmid pSD486 for Deletion of Hemorrhagic Region (B13R + B14R) Referring now to FIG. 10, plasmid pSD419 contains vaccinia SalI G (pos. 160,744-173,351) cloned into pUC8.
pSD422 contains the contiguous vaccinia SalI fragment to the right, SalI J (pos. 173,351-182,746) cloned into pUC8. To construct a plasmid deleted for the hemorrhagic region, u,
B13R - B14R (pos. 172,549 - 173,552), pSD419 was used as the source for the left flanking arm and pSD422 was used as the source of the right flanking arm. The direction of transcription for the u region is indicated by an arrow in
FIG. 10.
To remove unwanted sequences from pSD419, sequences to the left of the NcoI site (pos. 172,253) were removed by digestion of pSD419 with NcoI/SmaI followed by blunt ending with Klenow fragment of E. coli polymerase and ligation generating plasmid pSD476. A vaccinia right flanking arm was obtained by digestion of pSD422 with HpaI at the termination codon of B14R and by digestion with NruI 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb HincII vector fragment isolated from pSD476, generating plasmid pSD477. The location of the partial deletion of the vaccinia u region in pSD477 is indicated by a triangle.The remaining B13R coding sequences in pSD477 were removed by digestion with ClaI/HPaI, and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID NO:22/SEQ ID NO:23)
ClaI BamHI HpaI
SD22mer 5' CGATTACTATGAAGGATCCGTT 3'
SD20mer 3' TAATGATACTTCCTAGGCAA 5' generating pSD479. pSD479 contains an initiation codon (underlined) followed by a BamHI site.To place E. coli
Beta-galactosidase in the B13-B14 () deletion locus under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactosidase gene (Shapira et al., 1983) was inserted into the BamHI site of pSD479, generating pSD479BG. pSD479BG was used as donor plasmid for recombination with vaccinia virus vP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque in the presence of chromogenic substrate X-gal. In vP533 the B13R
B14R region is deleted and is replaced by Betagalactosidase.
To remove Beta-galactosidase sequences from vP533, plasmid pSD486, a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized. First the ClaI/HDaI vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:24/SEQ
ID NO::25)
ClaI SacI XhoI HraI SD42mer 5' CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3'
SD40mer 3' TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5' BglII SmaI BamHI generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia junction was destroyed by digestion of pSD478 with EcoRI followed by blunt ending with Klenow fragment of
E. coli polymerase and ligation, generating plasmid pSD478E pSD478E was digested with BamHI and HpaI and ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ ID
NO:26/SEQ ID NO::27)
BamHI EcoRI HpaI
HEMS 5 GATCCGAATTCTAGCT 3'
HEM6 3' GCTTAAGATCGA 5' generating plasmid pSD486. pSD486 was used as donor plasmid for recombination with recombinant vaccinia virus vP533, generating vP553, which was isolated as a clear plaque in the presence of X-gal.
Construction of Plasmid PMP494A for Deletion of ATI Region tA26L) Referring now to FIG. 11, pSD414 contains SalI B cloned into pUC8. To remove unwanted DNA sequences to the left of the A26L region, pSD414 was cut with XbaI within vaccinia sequences (pos. 137,079) and with HindIII at the pUC/vaccinia junction, then blunt ended with Klenow fragment of E. coli polymerase and ligated, resulting in plasmid pSD483. To remove unwanted vaccinia DNA sequences to the right of the A26L region, pSD483 was cut with EcoRI (pos.
140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding region, pSD484 was cut with NdeI (partial) slightly upstream from the A26L ORF (pos. 139,004) and with HpaI (pos. 137,889) slightly downstream from the A26L ORF. The 5.2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:28/SEQ ID NO:29)
NdeI
ATI3 5' TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT
ATI4 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA Bql I 1;;EcoRI HPaI
TATATAAATAGATCTGAATTCGTT 3' ATI3
ATATATTTATCTAGACTTAAGCAA 5' ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region containing the restriction sites Ball, EcoRI and HpaI, as indicated above.
The resulting plasmid was designated pSD485. Since the BalII and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted BqlII and EcoRI sites were removed from plasmid pSD483 (described above) by digestion with BalII (pos. 140,136) and with EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenow fragment of E. coli polymerase and ligation: The resulting plasmid was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcoRV (pos. 139,048) fragment from pSD489 containing the A26L ORF was replaced with the corresponding 0.7 kb polylinkercontaining ClaI/EcoRV fragment from pSD485, generating pSD492. The BalII and EcoRI sites in the polylinker region of pSD492 are unique.
A 3.3 kb Balil cassette containing the E. coli Betagalactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985;
Perkus et al., 1990) was inserted into the BglII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus vP553. Recombinant vaccinia virus, vP581, containing Beta-galactosidase in the
A26L deletion region, was isolated as a blue plaque in the presence of X-gal.
To generate a plasmid for the removal of Betagalactosidase sequences from vaccinia recombinant virus vP581, the polylinker region of plasmid pSD492 was deleted by mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN177 (SEQ ID NO:30) (5'
AAAATGGGCGTGGATTGTTAACTTTATATA-ACTTATTTTTTGAATATAC-3'). In the resulting plasmid, pMP494A, vaccinia DNA encompassing positions [137,889 - 138,937), including the entire A26L ORF is deleted. Recombination between the pMp494A and the Betagalactosidase containing vaccinia recombinant, vP581, resulted in vaccinia" deletion mutant vP618, which was isolated as a clear plaque in the presence of X-gal.
Construction of Plasmid pSD467 for Deletion of Hemaqqlutinin Gene (A56R)
Referring now to FIG. 12, vaccinia SalI G restriction fragment (pos. 160,744-173,351) crosses the
HindIII A/B junction (pos. 162,539). pSD419 contains vaccinia SalI G cloned into pUC8. The direction of transcription for the hemagglutinin (HA) gene is indicated by an arrow in FIG. 12. Vaccinia sequences derived from
HindIII B were removed by digestion of pSD419 with HindIII within vaccinia sequences and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to the right.A56R coding sequences were removed by cutting pSD456 with RsaI (partial; pos. 161,090) upstream from A56R coding sequences, and with EaqI (pos. 162,054) near the end of the gene. The 3.6 kb RsaI/EaaI vector fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:31), MPSY62 (SEQ ID
NO:32), MPSYN60 (SEQ ID NO:33), and MPSYN 61 (SEQ ID NO::34)
RsaI
MPSYN59 5' ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGTAGTTGATAGA
MPSYN62 3' TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCATCCACTATCT 5'
MPSYN59 -ACAAAATACATAATTT' B,el11 MPSYN60 5' T6TAAAAATAAATCACrTTITATACTAAGATCT- MPSYN61 3' TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATATGATTCTAGA Smal PstI EagI MPSYN6O -CCCGGGCTGCAGC 3'
MPSYN61 QGGGCCCGACGTCGCCGG 5' reconstructing the DNA sequences upstream from the A56R ORF and replacing the A56R ORF with a polylinker region as indicated above. The resulting plasmid is pSD466. The vaccinia deletion in pSD466 encompasses positions [161,185162,053]. The site of the deletion in pSD466 is indicated by a triangle in FIG. 12.
A 3.2 kb BqlII/BamHI (partial) cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) was inserted into the BqlII site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination with rescuing virus vP618.
Recombinant vaccinia virus, vP708, containing Betagalactosidase in the A56R deletion, was isolated as a blue plaque in the presence of X-gal.
Beta-galactosidase sequences were deleted from vP708 using donor plasmid pSD467. pSD467 is identical to pSD466, except that EcoRI, SmaI and BamHI sites were removed from the pUC/vaccinia junction by digestion of pSD466 with
EcoRI/BamHI followed by blunt ending with Klenow fragment of
E. coli polymerase and ligation. Recombination between vP708 and pSD467 resulted in recombinant vaccinia deletion mutant, vP723, which was isolated as a clear plaque in the presence of X-gal.
Construction of Plasmid DMPCSK1t for Deletion of OPen
Reading Frames rC7L-KlLl Referring now to FIG. 13, the following vaccinia clones were utilized in the construction of pMPCSK1L.
pSD420 is SalI H cloned into pUC8. pSD435 is KDnI F cloned into pUC18. pSD435 was cut with SphI and religated, forming pSD451. In pSD451, DNA sequences to the left of the S > hI site (pos. 27,416) in HindIII M are removed (Perkus et al., 1990). pSD409 is HindIII M cloned into pUC8.
To provide a substrate for the deletion of the [C7L K1L) gene cluster from vaccinia, E. coli Beta-galactosidase was first inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminate the BqlII site in pSD409, the plasmid was cut with BslII in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique SPhI site (pos. 27,416).M2L coding sequences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide BslII MPSYN82 (SEQ ID NO:35) 5' TTTCTGTATATTTG CACCAATTTAGATCTTACTCAAAA TATGTAACAATA 3'
The resulting plasmid, pMP409D, contains a unique BqlII site inserted into the M2L deletion locus as indicated above. A 3.2 kb BamHI (partial)/BalII cassette containing the E. coli
Beta-galactosidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with BqlII. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination with rescuing vaccinia virus vP723.Recombinant vaccinia virus, vP784, containing Betagalactosidase inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal.
A plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUC8 cut with SmaI, HindIII and blunt ended with Klenow fragment of E. coli polymerase. The left flanking arm consisting of vaccinia HindIII C sequences was obtained by digestion of pSD420 with XbaI (pos. 18,628) followed by blunt ending with Klenow fragment of E. coli polymerase and digestion with BalII (pos. 19,706). The right flanking arm consisting of vaccinia HindIII K sequences was obtained by digestion of pSD451 with BqlII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP581CK is deleted for vaccinia sequences between the BalII site (pos. 19,706) in HindIII C and the BglII site (pos. 29,062) in HindIII K. The site of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 13.
To remove excess DNA at the vaccinia deletion junction, plasmid pMP581CK, was cut at the NcoI sites within vaccinia sequences (pos. 18,811; 19,655), treated with Bal31 exonuclease and subjected to mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:36) 5'
TGTCATTTAACACTA TACTCATATTAATAAAAATAATATTTATT-3'. The resulting plasmid, pMPCSK1A, is deleted for vaccinia sequences positions 18,805-29,108, encotpassing 12 vaccinia open reading frames [C7L - K1L). Recombination between pMPCSKln and the Betagalactosidase containing vaccinia recombinant, vP784, resulted in vaccinia deletion mutant, vP804, which was isolated as a clear plaque in the presence of X-gal.
Construction of Plasmid PSD548 for Deletion of Large Subunit, Ribonucleotide Reductase (I4L) Referring now to FIG. 14, plasmid pSD405 contains vaccinia HindIII I (pos. 63,875-70,367) cloned in pUC8.
pSD405 was digested with EcoRV within vaccinia sequences (pos. 67,933) and with SmaI at the pUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 was used as the source of all the vaccinia restriction fragments used in the construction of pSD548.
The vaccinia I4L gene extends from position 67,37165,059. Direction of transcription for I4L is indicated by an arrow in FIG. 14. To obtain a vector plasmid fragment deleted for a portion of the I4L coding sequences, pSD518 was digested with BamHI (pos. 65,381) and HDaI (pos. 67,001) and blunt ended using Klenow fragment of E. coli polymerase.
This 4.8 kb vector fragment was ligated with a 3.2 kb SmaI cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985, Perkus et al., 1990), resulting in plasmid pSD524KBG. pSD524KBG was used as donor plasmid for recombination with vaccinia virus vP804.
Recombinant vaccinia virus, vP855, containing Betagalactosidase in a partial deletion of the I4L gene, was isolated as a blue plaque in the presence of X-gal.
To delete Beta-galactosidase and the remainder of the I4L ORF from vP855, deletion plasmid pSD548 was constructed. The left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and presented schematically in FIG. 14.
To construct a vector plasmid to accept the left vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518A1/518A2 (SEQ ID NO:37/SEQ ID NO:38)
BamHI RsaI 518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT 518A2 3' GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA BqlII EcoRI
TTGAGAATAAAAAGATCTTAGG 3' 518A1
AACTCTTATTTTTCTAGAATCCTTAA 5 518A2 forming plasmid pSD531. pSD531 was cut with RsaI (partial) and BamHI and a 2.7 kb vector fragment isolated. pSD518 was cut with BallI (pos. 64,459)/ RsaI (pos. 64,994) and a 0.5 kb fragment isolated.The two fragments were ligated together, forming pSD537, which contains the complete vaccinia flanking arm left of the I4L coding sequences.
To construct a vector plasmid to accept the right vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID NO:39/SEQ ID NO:40)
BamHI BalII SmaI 518B1 5' GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAGGGATTT 518B2 3' GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATCCCTAAA RsaI EcoRI
GACGTATGTAGCGTACTAGG 3 518B1
CTGCATACTACGCATGATCCTTAA 5' 518B2 forming plasmid pSD532. pSD532 was cut with RsaI (partial)/EcoRI and a 2.7 kb vector fragment isolated.
pSD518 was cut with RsaI within vaccinia sequences (pos.
67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two fragments were ligated together, forming pSD538, which contains the complete vaccinia flanking arm to the right of I4L coding sequences.
The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BqlII fragment from pSD538 and ligated into pSD537 vector plasmid cut with EcoRI/BglII. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a polylinker region, which is flanked by 0.6 kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right, all in a pUC background. The site of deletion within vaccinia sequences is indicated by a triangle in FIG. 14.
To avoid possible recombination of Beta-galactosidase sequences in the ppC-derived portion of pSD539 with Betagalactosidase sequences in recombinant vaccinia virus vP855, the vaccinia I4L deletion cassette was moved from pSD539 into pRC11, a pUC derivative from which all Betagalactosidase sequences have been removed and replaced with a polylinker region (Colinas et al., 1990). pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment isolated. This fragment was ligated into pRC11 cut with EcoRI/PstI (2.35 kb), forming pSD548. Recombination between pSD548 and the
Beta-galactosidase containing vaccinia recombinant, vP855, resulted in vaccinia deletion mutant vP866, which was isolated as a clear plaque in the presence of X-gal.
DNA from recombinant vaccinia virus vP866 was analyzed by restriction digests followed by electrophoresis on an agarose gel. The restriction patterns were as expected. Polymerase chain reactions (PCR) (Engelke et al., 1988) using vP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected. Recombinant vaccinia virus vP866, containing the six engineered deletions as described above, was designated vaccinia vaccine strain "NYVAC." ExamDle 16 - CONSTRUCTION OF NYVAC-MV RECOMBINANT
EXPRESSING MEASLES FUSION AND
HEMAGGLUTININ GLYCOPROTEINS
cDNA copies of the sequences encoding the HA and F proteins of measles virus MV (Edmonston strain) were inserted into NYVAC to create a double recombinant designated NYVAC-MV (vP913). The recombinant authentically expressed both measles glycoproteins on the surface of infected cells. Immunoprecipitation analysis demonstrated correct processing of both F and HA glycoproteins. The recombinant was also shown to induce syncytia formation.
Cells and Viruses
The rescuing virus used in the production of NYVAC
MV was the modified Copenhagen strain of vaccinia virus designated NYVAC. All viruses were grown and titered on
Vero cell monolayers.
Plasmid Construction
Referring now to Fig. 15 and Taylor et al. (1991), plasmid pSPM2LHA contains the entire measles HA gene linked in a precise ATG to ATG configuration with the vaccinia virus H6 promoter which has been previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989). A 1.8kpb EcoRV/SmaI fragment containing the 3' most 24 bp of the H6 promoter fused in a precise ATG:ATG configuration with the HA gene lacking the 3' most 26 bp was isolated from pSPM2LHA. This fragment was used to replace the 1.8 kbp EcoRV/SmaI fragment of pSPMHA11 (Taylor et al., 1991) to generate pRW803. Plasmid pRW803 contains the entire H6 promoter linked precisely to the entire measles HA gene.
Plasmid pSD513VCVQ was derived from plasmid pSD460 by the addition of polylinker sequences. Plasmid pSD460 was derived to enable deletion of the thymidine kinase gene from vaccinia virus (FIG. 9).
To insert the measles virus F gene into the HA insertion plasmid, manipulations were performed on pSPHMF7.
Plasmid pSPHMF7 (Taylor et al., 1991) contains the--measles F gene juxtaposed 3' to the previously described vaccinia virus H6 promoter. In order to attain a perfect ATG for ATG configuration and remove intervening sequences between the 3' end of the promoter and the ATG of the measles F gene oligonucleotide directed mutagenesis was performed using oligonucleotide SPMAD (SEQ ID NO:41).
SPMAD: 5' -TATCCGTTAAGTTTGTATCGTAATGGGTCTCAAGGTGAACGTCT-3' The resultant plasmid was designated pSPMF75M20.
The plasmid pSPMF75M20 which contains the measles F gene now linked in a precise ATG for ATG configuration with the H6 promoter was digested with NruI and EaqI. The resulting 1.7 kbp blunt ended fragment containing the 3' most 27 bp of the H6 promoter and the entire fusion gene was isolated and inserted into an intermediate plasmid pRW823 which had been digested with NruI and XbaI and blunt ended.
The resultant plasm it pRW841 contains the H6 promoter linked to the measles F gene in the pIBI25 plasmid vector (IBI, New
Haven, CT). The H6/measles F cassette was excised from pRW841 by digestion with SmaI and the resulting 1.8 kb fragment was inserted into pRW843 (containing the measles HA gene). Plasmid pRW843 was first digested with NotI and blunt-ended with Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs. The resulting plasmid, pRW857, therefore contains the measles virus F and HA genes linked in a tail to tail configuration. Both genes are linked to the vaccinia virus H6 promoter.
Development of NYVAC-MV.
Plasmid pRW857 was transfected into NYVAC (vP866) infected Vero cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of in situ plaque hybridization to specific MV
F and HA radiolabeled probes and subjected to 6 sequential rounds of plaque purification until a pure population was achieved. One representative plaque was then amplified and the resulting recombinant was designated NYVAC-MV (vP913).
Immunofluorescence
Indirect immunofluorescence was performed as previously described (Taylor et al., 1990). Mono-specific reagents used were sera generated by inoculation of rabbits with canarypox recombinants expressing either the measles F or HA genes.
Immunoprecipitation
Immunoprecipitation reactions were performed as previously described (Taylor et al., 1990) using a guineapig anti measles serum (Whittaker M.A. Bioproducts,
Walkersville, MD).
Cell Fusion Experiments
Vero cell monolayers in 6Omm dishes were inoculated at a multiplicity.oif 1 pfu per cell with parental or recombinant viruses. After 1 h absorption at 370C the inoculum was removed, the overlay medium replaced and the dishes inoculated overnight at 370C. At 20 h postinfection, dishes were examined.
In order to determine that the expression products of both measles virus F and HA genes were presented on the infected cell surface, indirect immunofluorescence analysis was performed using mono-specific sera generated in rabbits against canarypox recombinants expressing either the measles
F or HA genes. The results indicated that both F and HA gene products were expressed on the infected cell surface, as demonstrated by strong surface fluorescence with both mono-specific sera. No background staining was evident with either sera on cells inoculated with the parental NYVAC strain, nor was there cross-reactive staining when monospecific sera were tested against vaccinia single recombinants expressing either the HA or F gene.
In order to demonstrate that the proteins expressed by NYVAC-MV were immunoreactive with measles virus specific sera and were authentically processed in the infected cell, immunoprecipitation analysis was performed. Vero cell monolayers were inoculated at a multiplicity of 10 pfu/cell of parental or recombinant viruses in the presence of 35Smethionine. Immunoprecipitation analysis revealed a HA glycoprotein of approximately 76 kDa and the cleaved fusion products F1 and F2 with molecular weights of 44 kDa and 23 kDa, respectively. No measles specific products were detected in uninfected Vero cells or Vero cells infected with the parental NYVAC virus.
A characteristic of MV cytopathology is the formation of syncytia which arise by fusion of infected cells with surrounding infected or uninfected cells followed by migration of the nuclei toward the center of the syncytium (Norrby et al., 1982). This has been shown to be an important method of viral spread, which for
Paramyxoviruses, can occur in the presence of HA-specific virus neutralizing antibody (Merz et al., 1980). In order to determine that the MV proteins expressed in vaccinia virus were functionally active, Vero cell monolayers were inoculated with NYVAC and NYVAC-MV and observed for cytopathic effects. Strong cell fusing activity was evident in NYVAC-MV infected Vero cells at approximately 18 hours post infection. No cell fusing activity was evident in cells infected with parental NYVAC.
Results of serological analysis of sera of rabbits inoculated with NYVAC-XV (vP913)
In this study, two rabbits were inoculated with 1x108 pfu of NYVAC-MV (vP913) by the subcutaneous route. At 28 days, animals were boosted with an equivalent dose. Serial bleeds were analyzed for MV neutralizing activity using the plaque reduction method. The results are shown in Table 13.
The results indicate that neither rabbit responded to the initial inoculation of NYVAC-MV. However, the sharply rising response after the second inoculation indicates that the animals were primed. Both animals achieved neutralizing antibody titers in the protective range.
The in vivo analysis of immunogenicity of ALVAC-MV (vCP82) shown in Example 14 indicates that on inoculation of a range of species, the recombinant is able to induce a serological response which is measurable in standard serological tests. The titers achieved are in the range required for protection from disease. Inoculation of NYVAC
MV (vP913) into rabbits similarly induces a level of measles virus neutralizing antibody which would be protective.
Table 13
Anti-measles neutralizing antibody titers (log10) in sera of rabbits inoculated with NYVAC-MV (vP913)
Animal Titer at weeks post-inoculation
WO W2 W4C W5 W6 W7
Rabbita A116 < 1 < 1 < 1 2.8b 2.2 2.2
A117 < 1 < 1 < 1 1.9 1.9 1.9 a) Rabbits received 8.0 log10 pfu of NYVAC-MV (vP913) by
S.C. route.
b) Titer expressed as log10 of reciprocal of last
dilution showing a 50% reduction in plaque number as
compared to pre-inoculation serum.
c) Animals were re-inoculated at 28 days.
REFERENCES 1. Adams, J.M., and D.T. Imagawa, Proc. Soc. Exper.
Biol. Med. 96, 240-244 (1957).
2. Albrecht, P., K. Herrman, and G.R. Burns, J. Virol.
Methods 3, 251-260 (1981).
3. Alkhatib, G., and D. Briedis, Virology 150, 479-490
(1986).
4. Alkhatib, G., C. Richardson, and S-H. Shen, Virology
175, 262-270 (1990).
5. Appel, M.J.G., and O.R. Jones, Proc. Soc. Exp. Biol.
Med. 126, 571-574 (1967).
6. Appel, M.J.G., and D.S. Robson, Am. J. Vet. Res. 34,
1459-1463 (1973).
7. Avery, R.J., and J. Niven, Infect. Immun. 26, 795
801 (1979).
8. Baker, J.A., B.E. Sheffy, D.S. Robson, J. Gilmartin,
Cornell Vet (USA) 56, 588-594 (1966).
9. Bertholet, C., R. Drillien, and R. Wittek, Proc.
Natl. Acad. Sci. USA 82, 2096-2100 (1985).
10. Bestetti, G., R. Fatzer, and R. Frankhauser, Acta
Neuropathol. 43, 69-75 (1978).
11. Black, F.L., L.L. Berman, M. Libel, C.A. Reichelt,
F. de P. Pinheiro, A.T. da Rosa, F. Figuera, and
E.S. Gonzales, Bull, W.H.O. 62, 315-319 (1984).
12. Bush, M., R.J. Montali, D. Brownstein, A.E. James,
Jr., and M.J.G. Appel, J. Am. Vet. Med. Assoc. 169,
959-960 (1976).
13. Carpenter, J.W., M.J.G. Appel, R.C. Erickson, and
M.N. Novilla, J. Am. Vet. Med. Assoc. 169, 961-964
(1976).
14. Choppin, P.W., C.D. Richardson, D.C. Merz, W.W.
Hall, and A. Scheid, J. Infect. Dis. 143, 352-363
(1981).
15. Clewell, D.B., J. Bacteriol. 110, 667-676 (1972).
16. Clewell, D.B., and D.R. Helinski, Proc. Natl. Acad.
Sci. USA 62, 1159-1166 (1969).
17. Colinas, R.J., R.C. Condit, and E. Paoletti, Virus
Research 18, 49-70 (1990).
18. DeLay, P.D., S.S. Stone, D.T. Karzon, S. Katz, and
J. Enders, Am. J. Vet. Res. 26, 1359-1373 (1965).
19. Diallyl, A., Vet. Micro. 23, 155-163 (1990).
20. Dowling, P.C., B.M. Blumberg, J. Menonna, J.E.
Adamus, P. Cook, J.C. Crowley, D. Kolakofsky, and
S.D. Cook, J. Gen. Virol. 67, 1987-1992 (1986).
21. Drillien, R., D. Spehner, A. Kirn, P. Giraudon, R.
Buckland, F. Wild, and J.P. Lecocq, Proc. Natl.
Acad. Sci. USA 85, 1252-1256 (1988).
22. Engelke, D.R., P.A. Hoener, and F.S. Collins, Proc.
Natl. Acad. Sci. USA 85; 544-548 (1988).
23. Fenner, F., P.A. Bachmann, E.P.J. Gibbs, F.A.
Murphy, M.J. Studdert, and D.O. White, In Veterinary
Virology, ed. F. Fenner, (Academic Press, Inc., New
York) pp. 485-503 (1987).
24. Gillespie, J.H., and D.T. Karzon, Proc. Soc. Exp.
Biol Med. 105, 547-551 (1960).
25. Giraudon, P., Ch. Gerald, and T.F. Wild,
Intervirology 21, 110-120 (1984).
26. Goebel, S.J., G.P. Johnson, M.E. Perkus, S.W. Davis,
J.P. Winslow, and E. Paoletti, Virology 179, 247-266 (1990a).
27. Goebel, S.J., G.P. Johnson, M.E. Perkus, S.W. Davis,
J.P. Winslow, and E. Paoletti, Virology 179, 517-563
(1990b).
28. Graves, M.C., S.M. Silver, and P.W. Choppin,
Virology 86, 254-263 (1978).
29. Guo, P., S. Goebel, S. Davis, M.E. Perkus, B.
Languet, P. Desmettre, G. Allen, and E. Paoletti, J.
Virol. 63, 4189-4198 (1989).
30. Guo, P., S. Goebel, S. Davis, M.E. Perkus, J.
Taylor, E. Norton, G. Allen, B. Languet, P.
Desmettre, and E. Paoletti, J. Virol. 64, 2399-2406
(1990).
31. Hall, W.W., R.A. Lamb, and P.W. Choppin, Virology
100, 433-449 (1980).
32. Hartley, W.J., Vet. Path. 11, 301-312 (1974).
33. Imagawa, D.T., P. Goret, and J.M. Adams, Proc. Natl.
Acad. Sci. USA 46, 1119-1123 (1960).
34. Karzon, D.T., Pediatrics 16, 809-818 (1955).
35. Karzon, D.T., Annals of the N.Y. Academy of Sci.
101, 527-539 (1962).
36. Kazacos, KR., H.L. Thacker, H.L. Shivaprasad, and
P.P. Burger, J. Am. Vet. Med. Assoc. 179, 1166-1169 (1981).
37. Kingsbury, D.W., M.A. Bratt, P.W. Choppin, R.P.
Hanson, T. Hosaka, V. ter Meulen, E. Norrby, W.
Plowright, R. Rott, and W.H. Wunner, Intervirology
10, 137-152 (1978).
38. Kunkel, T.A., Proc. Natl. Acad. Sci. USA 82, 488-492
(1985).
39. Lennon, J.L., and F.L. Black, J. Ped. 108, 671-676
(1986).
40. Mandecki, W., Proc. Natl. Acad. Sci. USA 83,
7177-7181 (1982).
41. Mandecki, W., Proc. Natl. Acad. Sci. USA 83,
7177-7181 (1986).
42. Maniatis, T., E.F. Fritsch, and J. Sambrook,
Molecular Cloning, Cold Spring Harbor Laboratory, NY
545 pages (1982).
43. Maniatis, T., E.F. Fritsch, and J. Sambrook,
Molecular Cloning, Cold Spring Harbor Laboratory, NY
545 pages (1986).
44. Merz, D.C., A. Schied, and P. Choppin, J. Exp. Med.
151, 275-288 (1980).
45. Moura, R.A., and J. Warren, J. Bact. 82, 702-705
(1961).
46. Norrby, E., and Y. Gollmar, Infect. Immun. 11, 231
239 (1975).
47. Norrby, E., G. Enders-Ruckle, and V. ter Meulen, J.
Infect. Dis. 132, 262-269 (1975).
48. Norrby, E., S.N. Chen, T. Togashi, H. Shesberadaran,
and K.P. Johnson, Archives of Virology 71, 1-11
(1982).
49. Norrby, E., and M.N. Oxman, In Fields Virology 2nd
Ed., B.N. Fields and D.M. Knipe, eds. (Raven Press,
NY) pp. 1013-1044 (1990).
50. Novick, S.L. and D. Hoekstra, Proc. Natl. Acad. Sci.
USA 85, 7433-7437 (1988).
51. Orvell, C., and E. Norrby, J. Gen. Virol. 50, 231
245 (1980).
52. Panicali, D., and E. Paoletti, Proc. Natl. Acad.
Sci. USA 79, 4927-4931 (1982).
53. Paterson, R.G., and R.A. Lamb, Cell 48, 441-452
(1987).
54. Perkus, M.E., A. Piccini, B.R. Lipinskas, and E.
Paoletti, Science 229, 981-984 (1985).
55. Perkus, M.E., K. Limbach, and E. Paoletti, J. Virol.
63, 3829-3836 (1989).
56. Perkus, M.E., S.J. Goebel, S.W. Davis, G.P Johnson,
K. Limbach, E.K. Norton, and E. Paoletti, Virology
179, 276-286 (1990).
57. Phillips, T.R., J.L. Jensen, M.J. Rubino, W.C. Yang,
and R.D. Schultz, Can. J. Vet. Res. 53, 154-160
(1989).
58. Piccini, A., M.E. Perkus, and E. Paoletti, In
Methods in Enzymology, Vol. 153, eds. Wu, R., and
Grossman, L., (Academic Press) pp. 545-563 (1987).
59. Preblud, S.R., and S.L. Katz, In Vaccines, eds. S.A.
Plotkin and E.A. Mortimer, (W.B. Saunders Co.) pp.
182-222 (1988).
60. Richardson, C.D., A. Berkovich, S. Rozenblatt, and
W. Bellini, J. Virol. 54, 186-193 (1985).
61. Richardson, C., D. Hull, P. Greer, K. Hasel, A.
Berkovich, G. Englund, W. Bellini, B. Rima, and R.
Lazzarini, Virology 155, 508-523 (1986).
62. Roberts, J.A., J. Immunol. 94, 622-628 (1965).
63. Rosel, J.L., P.L. Earl, J.P. Weir, and B. Moss, J.
Virol. 60, 436-449 (1986).
64. Sanger, F., S. Nicklen, and A.R. Coulson, Proc.
Natl. Acad. Sci. USA 74, 5463-5467 (1977).
65. Shapira, S.K., J. Chou, F.V. Richaud, and M.J.
Casadaban, Gene 25, 71-82 (1983).
66. Spehner, D., R. Drillien, and J.P. Lecocq, J. Virol.
64, 527-533 (1990).
67. Stephenson, J.R. and V. ter Meulen, Proc. Nat. Acad.
Sci. USA 76, 6601-6605 (1979).
68. Tabor, S., and C.C. Richardson, Proc. Natl. Acad.
Sci. USA 84, 4767-4771 (1987).
69. Taylor, J., R. Weinberg, Y. Rawaoka, R.G. Webster,
and E. Paoletti, Vaccine 6, 504-508 (1988a).
70. Taylor, J., R. Weinberg, B. Languet, P. Desmettre,
and E. Paoletti, Vaccine 6, 497-503 (1988b).
71. Taylor, J.,.-C. Edbauer, A. Rey-Senelonge, J.F.
Bouquet, E. Norton, S. Goebel, P. Desmettre, and E.
Paoletti, J. Virol. 64, 1441-1450 (1990).
72. Taylor, J., S. Pincus, J. Tartaglia, C. Richardson,
G. Alkhatib, D. Briedis, M. Appel, E. Norton, and E.
Paoletti, J. Virol. 65, 4263-4272 (1991).
73. Tizard, I., J. Am. Vet. Med. Assoc. 196, 1851-1858
(1990).
74. Vialard, J., M. Lalumiere, T. Vernet, D. Briedis, G.
Alkhatib, D. Henning, D. Levin, and C. Richardson,
J. Virol. 64, 37-50 (1990).
75. Warren, J., M.K. Nadel, E. Slater, and S.J. Millian,
Amer. J. Vet. Res. 21, 111-119 (1960).
76. Wild, T.F., E. Malvoisin, and R. Buckland, J. Gen.
Virol. 72, 439-442 (1991).
77. Wild, F., P. Giraudon, D. Spehner, R. Drillien, and
J-P. Lecocq, Vaccine 8, 441-442 (1990).
78. Yuen, L., and B. Moss, Proc. Natl. Acad. Sci. USA
84, 6417-6421 (1987).
Claims (18)
1. A recombinant poxvirus containing therein DNA from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper.
2. A recombinant poxvirus for the use according to claim I wherein the recombinant poxvirus comprises in a nonessential region of its genome exogenous
Morbillivirus DNA coding for an antigen characterised in that the poxvirus is either a modified vaccinia virus having at least the following open reading frames deleted therefrom: a thymidine kinase gene, a haemorrhagic gene region, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region, and a large subunit, ribonucleotide reductase gene, or an ALVAC canarypox virus.
3. A recombinant poxvirus for the use according to claim 1 or 2 wherein the exogenous Morbillivirus DNA is exogenous measles virus DNA.
4. A recombinant poxvirus for the use according to claim 3 wherein the exogenous Morbillivirus DNA codes for a measles virus glycoprotein, a measles virus haemagglutinin glycoprotein, or a measles virus fusion glycoprotein.
5. A recombinant poxvirus for the use according to claim 4 wherein the exogenous Morbillivirus DNA codes for two measles virus glycoproteins, preferably a haemagglutinin glycoprotein and a fusion glycoprotein.
6. A recombinant poxvirus for the use according to any one of claims 3 to 5 wherein the exogenous Morbillivirus DNA is from the Edmonston strain of measles virus.
7. A recombinant poxvirus for the use according to any one of claims 2 to 6 wherein the exogenous Morbillivirus DNA is introduced into the poxvirus by recombination.
8. A recombinant poxvirus for the use according to any one of claims 2 to 7 wherein the exogenous Morbillivirus DNA further includes a promoter for expressing the antigen.
9. A recombinant poxvirus for the use according to claim 8 wherein the promoter is an H6 promoter.
10. A recombinant poxvirus for the use according to any of claims 2 to 9 wherein the poxvirus is a modified vaccinia virus.
11. A recombinant poxvirus for the use according to claim 10 wherein the deleted open reading frames comprise C7L-K1L, J2R, B13R+B14R, A26L, A56R and I4L.
12. A recombinant pox virus for the use according to claim 11 wherein the poxvirus is the NYVAC vaccinia virus.
13. A recombinant poxvirus for the use according to claim 1 wherein the recombinant poxvirus vP913.
14. A recombinant poxvirus for the use according to any one of claims 1 to 8 wherein the poxvirus is the ALVAC canarypox virus.
15. A recombinant poxvirus for the use according to claim 1 wherein the recombinant poxvirus vCP82.
16. A recombinant poxvirus for the use according to any one of the preceding claims admixed with a vaccine carrier.
17. Use of a recombinant poxvirus containing therein DNA from Morbillivirus in a nonessential region of the poxvirus genome for the preparation of a medicament for protecting a dog against canine distemper.
18. Use according to claim 17 wherein the recombinant poxvirus is that defined in any one of claims 2 to 16.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62161490A | 1990-11-20 | 1990-11-20 | |
US77686791A | 1991-10-22 | 1991-10-22 | |
GB9309414A GB2264949B (en) | 1990-11-20 | 1991-11-20 | Measles virus recombinant poxvirus vaccine |
Publications (3)
Publication Number | Publication Date |
---|---|
GB9500214D0 GB9500214D0 (en) | 1995-03-01 |
GB2283021A true GB2283021A (en) | 1995-04-26 |
GB2283021B GB2283021B (en) | 1995-07-05 |
Family
ID=27089007
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9309414A Expired - Lifetime GB2264949B (en) | 1990-11-20 | 1991-11-20 | Measles virus recombinant poxvirus vaccine |
GB9500214A Expired - Lifetime GB2283021B (en) | 1990-11-20 | 1991-11-20 | A recombinant poxviral vaccine for canine distemper |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9309414A Expired - Lifetime GB2264949B (en) | 1990-11-20 | 1991-11-20 | Measles virus recombinant poxvirus vaccine |
Country Status (12)
Country | Link |
---|---|
JP (2) | JP3617668B2 (en) |
AU (1) | AU1253892A (en) |
BE (1) | BE1005908A5 (en) |
CA (1) | CA2096633A1 (en) |
CH (1) | CH683921A5 (en) |
DE (2) | DE4192786T1 (en) |
FR (1) | FR2669346B1 (en) |
GB (2) | GB2264949B (en) |
IE (2) | IE71643B1 (en) |
IT (1) | IT1252687B (en) |
NL (2) | NL195058C (en) |
WO (1) | WO1992008789A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001030851A2 (en) * | 1999-10-28 | 2001-05-03 | Universitätsklinikum Freiburg | Idiotype vaccines |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7767449B1 (en) | 1981-12-24 | 2010-08-03 | Health Research Incorporated | Methods using modified vaccinia virus |
US5338683A (en) | 1981-12-24 | 1994-08-16 | Health Research Incorporated | Vaccinia virus containing DNA sequences encoding herpesvirus glycoproteins |
US5505941A (en) * | 1981-12-24 | 1996-04-09 | Health Research, Inc. | Recombinant avipox virus and method to induce an immune response |
US5833975A (en) * | 1989-03-08 | 1998-11-10 | Virogenetics Corporation | Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence |
US6248333B1 (en) | 1990-04-04 | 2001-06-19 | Health Research Inc. | Isolated nucleic acid sequence of equine herpesvirus type 1 glycoprotein D (EHV-1 gD) |
US5756102A (en) * | 1990-11-20 | 1998-05-26 | Virogenetics Corporation | Poxvirus-canine distemper virus (CDV) recombinants and compositions and methods employing the recombinants |
WO1992022641A1 (en) * | 1991-06-14 | 1992-12-23 | Virogenetics Corporation | Immunodeficiency virus recombinant poxvirus vaccine |
US5989561A (en) * | 1991-03-07 | 1999-11-23 | Virogenetics Corporation | Recombinant poxvirus-calicivirus rabbit hemorrhagic disease virus (RHDV) compositions and uses |
KR100242671B1 (en) * | 1991-03-07 | 2000-03-02 | 고돈 에릭 | Genetically engineered vaccine strain |
EP0575544A4 (en) * | 1991-03-20 | 1997-05-21 | Virogenetics Corp | Malaria recombinant poxvirus vaccine |
US5643578A (en) | 1992-03-23 | 1997-07-01 | University Of Massachusetts Medical Center | Immunization by inoculation of DNA transcription unit |
CN1089370C (en) * | 1994-12-08 | 2002-08-21 | 中国预防医学科学研究院病毒学研究所 | Recombination vaccinia virus for expression of measles virus L4 strain hemagglutinin and fusion protein gene |
WO1997028265A1 (en) * | 1996-02-05 | 1997-08-07 | University Of Massachusetts Medical Center | Measles immunization by dna transcription unit inoculation |
AU750322B2 (en) | 1997-05-09 | 2002-07-18 | Trustees Of The University Of Pennsylvania, The | Methods and compositions for reducing ischemic injury of the heart by administering adenosine receptor agonists and antagonists |
AU2002252199B2 (en) | 2001-03-08 | 2008-01-03 | Emory University | MVA expressing modified HIV envelope, GAG, and POL genes |
AR052743A1 (en) * | 2006-04-11 | 2007-03-28 | Inst Nac De Tecnologia Agropec | PLASMIDIC TRANSFER VECTOR AND RECOMBINANT CANARYPOX VIRUS |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989003429A1 (en) * | 1987-08-28 | 1989-04-20 | Health Research Inc. | Recombinant avipox virus |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2617715B1 (en) * | 1987-07-07 | 1990-08-31 | Transgene Sa | VIRAL VECTOR AND RECOMBINANT DNA ENCODING FOR ONE OR MORE SURFACE PROTEINS (HA AND / OR F) OF A MORBILLIVIRUS, INFECTED CELL CULTURE, PROTEINS OBTAINED, VACCINE AND ANTIBODIES OBTAINED |
FR2632863B2 (en) * | 1987-10-29 | 1990-08-31 | Transgene Sa | RECOMBINANT FOWLPOX VIRUS AND VACCINES DERIVED FROM SUCH VIRUSES |
JPH01218590A (en) * | 1988-02-29 | 1989-08-31 | Toa Nenryo Kogyo Kk | Rinderpest virus vaccine using recombinant vaccinia virus |
-
1991
- 1991-11-13 IE IE960091A patent/IE71643B1/en not_active IP Right Cessation
- 1991-11-13 IE IE396091A patent/IE68404B1/en not_active IP Right Cessation
- 1991-11-19 BE BE9101066A patent/BE1005908A5/en not_active IP Right Cessation
- 1991-11-19 FR FR9114256A patent/FR2669346B1/en not_active Expired - Lifetime
- 1991-11-20 GB GB9309414A patent/GB2264949B/en not_active Expired - Lifetime
- 1991-11-20 CH CH2364/92A patent/CH683921A5/en not_active IP Right Cessation
- 1991-11-20 CA CA002096633A patent/CA2096633A1/en not_active Abandoned
- 1991-11-20 AU AU12538/92A patent/AU1253892A/en not_active Abandoned
- 1991-11-20 WO PCT/US1991/008703 patent/WO1992008789A1/en active Application Filing
- 1991-11-20 DE DE4192786T patent/DE4192786T1/en active Pending
- 1991-11-20 JP JP50520492A patent/JP3617668B2/en not_active Expired - Fee Related
- 1991-11-20 NL NL9120026A patent/NL195058C/en not_active IP Right Cessation
- 1991-11-20 GB GB9500214A patent/GB2283021B/en not_active Expired - Lifetime
- 1991-11-20 IT ITMI913092A patent/IT1252687B/en active IP Right Grant
- 1991-11-20 DE DE4192786A patent/DE4192786B4/en not_active Expired - Lifetime
-
2003
- 2003-07-15 NL NL9900036A patent/NL195095C/en not_active IP Right Cessation
-
2004
- 2004-06-09 JP JP2004170929A patent/JP3824619B2/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989003429A1 (en) * | 1987-08-28 | 1989-04-20 | Health Research Inc. | Recombinant avipox virus |
Non-Patent Citations (1)
Title |
---|
Proc.Natl.Acad.Sci. USA, Vol 85, 1988, pages 1252-1256 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001030851A2 (en) * | 1999-10-28 | 2001-05-03 | Universitätsklinikum Freiburg | Idiotype vaccines |
WO2001030851A3 (en) * | 1999-10-28 | 2001-09-20 | Universitaetsklinikum Freiburg | Idiotype vaccines |
Also Published As
Publication number | Publication date |
---|---|
GB2283021B (en) | 1995-07-05 |
DE4192786T1 (en) | 1994-01-13 |
GB2264949A (en) | 1993-09-15 |
IE68404B1 (en) | 1996-06-12 |
NL195058C (en) | 2003-07-01 |
AU1253892A (en) | 1992-06-11 |
GB9309414D0 (en) | 1993-07-14 |
NL195095C (en) | 2004-01-21 |
NL9120026A (en) | 1993-09-01 |
JPH06502996A (en) | 1994-04-07 |
FR2669346A1 (en) | 1992-05-22 |
BE1005908A5 (en) | 1994-03-08 |
NL9900036A (en) | 2003-10-01 |
ITMI913092A1 (en) | 1993-05-20 |
JP2005040129A (en) | 2005-02-17 |
GB2264949B (en) | 1995-07-05 |
FR2669346B1 (en) | 1995-07-21 |
JP3824619B2 (en) | 2006-09-20 |
GB9500214D0 (en) | 1995-03-01 |
IE71643B1 (en) | 1997-02-26 |
CA2096633A1 (en) | 1992-05-21 |
JP3617668B2 (en) | 2005-02-09 |
ITMI913092A0 (en) | 1991-11-20 |
IE913960A1 (en) | 1992-05-20 |
WO1992008789A1 (en) | 1992-05-29 |
CH683921A5 (en) | 1994-06-15 |
IT1252687B (en) | 1995-06-23 |
DE4192786B4 (en) | 2006-08-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR970011149B1 (en) | Recombinant avipox virus | |
US5364773A (en) | Genetically engineered vaccine strain | |
JP4108742B2 (en) | Poxvirus-canine distemper virus (CDV) recombinants and compositions and methods using said recombinants | |
US7144578B2 (en) | Poxvirus-rabies recombinants and compositions and methods employing the recombinants | |
CA1341234C (en) | Recombinant fowlpox virus | |
Taylor et al. | Nonreplicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins | |
JP4140862B2 (en) | Recombinant poxvirus-rabies composition and combination composition and use | |
JP3602844B2 (en) | Genetically engineered vaccine strain | |
Taylor et al. | Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge | |
NL195058C (en) | Recombinant vaccinia virus and vaccine containing this recombinant vaccinia virus. | |
JP2007082551A (en) | Recombinant poxvirus-calicivirus [rabbit hemorrhagic disease virus (rhdv)] composition and use thereof | |
AU724172B2 (en) | Recombinant poxvirus-feline infectious peritonitis virus, compositions thereof and methods for making and using them | |
US5503834A (en) | Measles virus recombinant poxvirus vaccine | |
US5759841A (en) | Immunological composition of measles virus utilizing recombinant poxvirus | |
EP0623172B1 (en) | Marek's disease virus recombinant vaccinia virus vaccine | |
AU699903B2 (en) | Measles virus recombinant poxvirus vaccine | |
AU720448B2 (en) | Poxvirus-canine distemper virus (CDV) recombinants and compositions and methods employing the recombinants | |
CA2187207C (en) | Poxvirus-canine distemper virus (cdv) recombinants and compositions and methods employing the recombinants | |
AU769221B2 (en) | Recombinant poxvirus-feline infectious peritonitis virus, compositions thereof and methods for making and using them |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PE20 | Patent expired after termination of 20 years |
Expiry date: 20111119 |