WO1996016184A1 - Expression of the bovine parainfluenza virus type 3 hemagglutinin/neuraminidase (hn) glycoprotein in two heterologous systems - Google Patents
Expression of the bovine parainfluenza virus type 3 hemagglutinin/neuraminidase (hn) glycoprotein in two heterologous systems Download PDFInfo
- Publication number
- WO1996016184A1 WO1996016184A1 PCT/US1995/013482 US9513482W WO9616184A1 WO 1996016184 A1 WO1996016184 A1 WO 1996016184A1 US 9513482 W US9513482 W US 9513482W WO 9616184 A1 WO9616184 A1 WO 9616184A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- bhv
- neuraminidase
- glycoprotein
- hemagglutinin
- Prior art date
Links
- 241000712005 Bovine respirovirus 3 Species 0.000 title claims abstract description 58
- 102000005348 Neuraminidase Human genes 0.000 title claims abstract description 32
- 108010006232 Neuraminidase Proteins 0.000 title claims abstract description 32
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 26
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 26
- 101710154606 Hemagglutinin Proteins 0.000 title claims abstract description 20
- 101710093908 Outer capsid protein VP4 Proteins 0.000 title claims abstract description 20
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 title claims abstract description 20
- 101710176177 Protein A56 Proteins 0.000 title claims abstract description 20
- 239000000185 hemagglutinin Substances 0.000 title claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 61
- 241000700605 Viruses Species 0.000 claims abstract description 53
- 229960005486 vaccine Drugs 0.000 claims abstract description 28
- 241000283690 Bos taurus Species 0.000 claims abstract description 15
- 108020004440 Thymidine kinase Proteins 0.000 claims description 9
- 102000006601 Thymidine Kinase Human genes 0.000 claims description 8
- 230000003362 replicative effect Effects 0.000 claims description 5
- 230000006806 disease prevention Effects 0.000 claims description 2
- 241000701083 Bovine alphaherpesvirus 1 Species 0.000 abstract description 61
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 239000002299 complementary DNA Substances 0.000 abstract description 8
- 241000701447 unidentified baculovirus Species 0.000 abstract description 5
- 208000023504 respiratory system disease Diseases 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 50
- 241001465754 Metazoa Species 0.000 description 29
- 238000000034 method Methods 0.000 description 25
- 101150008820 HN gene Proteins 0.000 description 22
- 201000010099 disease Diseases 0.000 description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 17
- 101710133291 Hemagglutinin-neuraminidase Proteins 0.000 description 14
- 239000013598 vector Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 13
- 239000012634 fragment Substances 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 208000015181 infectious disease Diseases 0.000 description 11
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 description 10
- 241000256251 Spodoptera frugiperda Species 0.000 description 10
- 239000013605 shuttle vector Substances 0.000 description 10
- 230000003612 virological effect Effects 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 238000010276 construction Methods 0.000 description 7
- 230000002265 prevention Effects 0.000 description 7
- 208000002289 Bovine Respiratory Disease Complex Diseases 0.000 description 6
- 101150003725 TK gene Proteins 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 238000002255 vaccination Methods 0.000 description 6
- 238000012217 deletion Methods 0.000 description 5
- 239000003085 diluting agent Substances 0.000 description 5
- 230000003472 neutralizing effect Effects 0.000 description 5
- 210000002845 virion Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229960004452 methionine Drugs 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000013599 cloning vector Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000003156 radioimmunoprecipitation Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 229960004854 viral vaccine Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- 101710182846 Polyhedrin Proteins 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 108010006025 bovine growth hormone Proteins 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108010074605 gamma-Globulins Proteins 0.000 description 2
- 229930195712 glutamate Natural products 0.000 description 2
- 102000057593 human F8 Human genes 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000010369 molecular cloning Methods 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
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229940047431 recombinate Drugs 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 244000052613 viral pathogen Species 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- WOVKYSAHUYNSMH-UHFFFAOYSA-N BROMODEOXYURIDINE Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 241000711895 Bovine orthopneumovirus Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 230000007023 DNA restriction-modification system Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 101150054371 UL24 gene Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000000454 anti-cipatory effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000008952 bacterial invasion Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 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
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 101150118163 h gene Proteins 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- -1 such as Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000017960 syncytium formation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000002103 transcriptional effect Effects 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
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 210000001944 turbinate Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 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
-
- 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
- A61K39/12—Viral antigens
- A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- 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
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16641—Use of virus, viral particle or viral elements as a vector
- C12N2710/16643—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
- C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
- C12N2760/18622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention relates to the field of Bovine Parainfluenza Virus Type 3, and vaccines for the treatment thereof.
- Bovine parainfluenza virus-3 belongs to the genus Paramyxo ⁇ irus of the Paramyxo ⁇ iridae. Virions are pleomorphic, enveloped particles which contain a nucleocapsid that incorporates a single stranded RNA genome. The genome serves as a template for transcription of six unique structural proteins, nucleoprotein (NP), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L). The HN and F proteins project as spike-like structures from the virion surface, and are also expressed on the surface of infected cells.
- NP nucleoprotein
- M matrix protein
- F fusion protein
- HN hemagglutinin-neuraminidase protein
- L large protein
- the HN protein is strongly hydrophobic at the amino terminus and this domain is important in anchoring the protein into the virion envelope and host-cell membrane.
- the hemagglutination reaction which is widely used in sero-diagnosis and experimental laboratory studies, is also a manifestation of this type of interaction and is the reason that this glycoprotein is designated as a hemagglutinin.
- the HN protein also cleaves sialic acid residues from the virion surface by virtue of its neuraminidase activity. After attachment to the cell the virus has to deliver the nucleocapsid into the cytoplasm and this is mediated by the second surface glycoprotein, the F protein. This occurs with the fusion of the viral lipoprotein envelope with the lipoprotein surface of the host cell.
- the F glycoprotein is also expressed on the surface of infected cells which allows fusion of contiguous infected and uninfected cells to occur, leading to syncytium formation and an extension of the infection.
- the crucial role which these two proteins have in the infective process, and their presence on the surface of infected cells, makes them obvious targets for the immune system and studies have shown that animals which have recovered from infection have neutralizing antibody to both of these glycoproteins.
- BPIV-3 bovine respiratory disease complex
- BRDC bovine respiratory disease complex
- BHV-1 bovine herpesvirus type-1
- BPIV-3 bovine viral diarrhea virus
- BVDV bovine viral diarrhea virus
- bovine respiratory syncytial virus bovine respiratory syncytial virus
- This invention will provide the user with an effective vaccine for prevention of the BPIV-3 and/or BHV-1 caused components of the BDRC complex. SUMMARY OF THE INVENTION.
- This invention comprises new vaccines to combat respiratory diseases in cattle.
- the vaccines are produced by inserting the cDNA that codes for the bovine parainfluenza virus hemagglutinin/neuraminidase (HN) glycoprotein into bovine herpesvinis type 1 (BHV-1) or into baculovinis, such that the protein is expressed by the respective viruses.
- HN hemagglutinin/neuraminidase
- the invention comprises a replicating nonpathogenic virus, comprised of a gene or gene combination encoding a Hemagglutinin/Neuraminidase (HN) glycoprotein inserted into and expressed in any appropriate locus of bovine herpesvinis type 1 (BHV-1), a virus where the gene or gene combination that encodes the Hemagglutinin/Neuraminidase (HN) glycoprotein is from bovine parainfluenza virus type 3 (BPIV-3), a virus where the gene or gene combination that encodes the Hemagglutinin/Neuraminidase (HN) glycoprotein is inserted into and expressed in the thymidine kinase locus of BHV-1.
- a vaccine for the prevention of diseases caused by bovine parainfluenza virus type-3 consisting of a replicating nonpathogenic virus containing a gene or gene combination that encodes a
- Hemagglutinin/Neuraminidase (HN) glycoprotein inserted into and expressed in any appropriate locus of bovine herpesvinis type 1 (BHV-1), a vaccine where the gene or gene combination that encodes the Hemagglutinin Neuraminidase (HN) glycoprotein is from bovine parainfluenza virus type 3 (BPIV-3), a vaccine, where the gene or gene combination that encodes the Hemagglutinin/Neuraminidase (HN) glycoprotein is inserted into and expressed in the thymidine kinase locus of BHV-1.
- BHV-1 bovine herpesvinis type 1
- BPIV-3 bovine parainfluenza virus type 3
- a vaccine consisting of a gene or gene combination encoding a Hemagglutinin/Neuraminidase (HN) glycoprotein inserted into and expressed in the baculovinis expression system, a vaccine where the gene or gene combination that encodes the Hemagglutinin/Neuraminidase (HN) glycoprotein is from bovine parainfluenza virus type-3 (BPTV-3).
- BPTV-3 bovine parainfluenza virus type-3
- Pharmaceutical compositions, carriers, diluents, adjuvants appropriate for the viral vaccine are also provided or understood by one skilled in the art.
- Figure 2 Construction of a recombinant baculovinis transfer vector for expression of the BPIV-3 HN gene in the baculovinis expression system.
- compositions and Administrations A pharmaceutically effective amount of the vaccine of the present invention can be employed along with a pharmaceutically acceptable carrier, diluent or adjuvant as a vaccine against BHV-1 and BPIV-3 in animals, such as bovine, sheep and goats.
- Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include any physiological buffered medium, i.e., about pH 7.0 to 7.4, containing from about 2.5 to 15% serum which does not contain antibodies to BHV-1, i.e., is seronegative for BHV-1. Serum which does not contain gamma globulin is preferred to serum which contains gamma globulin.
- Examples of serum to be employed in the present invention include fetal calf serum, lamb serum, horse serum, swine serum, and goat serum. Serum protein such as porcine albumin or bovine serum albumin (hereinafter "BSA") in an amount of from about 0.5 to 3.0% can be employed as a substitute for the serum.
- BSA bovine serum albumin
- the virus may be diluted in any of the conventional stabilizing solutions containing phosphate buffer, glutamate, casitone, and sucrose or sorbose, or containing phosphate buffer, lactose, dextran and glutamate.
- the vaccine viruses of the present invention be stored at a titer of at least 10 5 to 10 6 PFU/ml at -70°C to -90°C or in a lyophilized state at 2°C to 7°C.
- the lyophilized virus may be reconstituted for use with sterile distilled water or using an aqueous diluent containing preservatives such as gentamicin and amphotericin B or penicillin and streptomycin.
- the useful dosage to be administered will vary depending upon the age, weight and species of the animal vaccinated and the mode of administration.
- a suitable dosage can be, for example, about 10 ° to 10 PFU/animal, preferably about 10 4 - 5 to 10 5 - 5 PFU.
- the vaccines of the present invention can be administered intranasally, intravaginally or intramuscularly.
- Intranasally is the preferred mode of administration for the BHV-1 component
- intramuscularly is the preferred mode of administration for the baculovinis component.
- This cDNA available from the procedure described above or as obtained from Dr. Hiroshi Shibuta, Tokyo University, is inserted into shuttle vectors for recombination and expression in BHV-1 and baculovinis (BV).
- BV baculovinis
- the HN gene is inserted into the shuttle vector pHAS4DBXex-l (the construction of which is described in this section). The insertion is made such that the HN gene disrupt the BHV-1 thymidine kinase gene.
- the HN gene is inserted into a standard, commercially available shuttle vector (PVL1392, Pharmingen, San Diego, CA) under the control of the polyhedron promoter.
- PVL1392 Pharmingen, San Diego, CA
- tk-negative BHV-1 and polyhedron- negative BV are isolated and screened for the presence of the HN gene by Southern hybridization and for the expression of HN by radioimmunoprecipitation.
- Bovine turbinate (BT) cells are available from the American Type Culture Collection (ATCC, Rockville, MD). The BT cells are grown in Dulbecco's modifed eagle medium (DMEM) supplemented with penicillin, streptomycin, non-essential amino acids and 10% horse serum.
- DMEM Dulbecco's modifed eagle medium
- Thymidine kinase deficient rabbit skin (RabBU) cells were provided by Saul Kit and Malon Kit; however, any other thymidine kinase-deficient cell lines that support the growth of BHV-1 can be used to reproduce this invention.
- the tk deficient cells are grown in DMEM supplemented with penicillin, streptomycin, 10% fetal bovine serum and were periodically passaged in lOOug/ml 5-bromo-2'-deoxyuridine (BDUR, Sigma, St. Louis, MO). Spodoptera frugiperda (SF9) cells were propagated in Grace's Media (complete) supplemented with penicillin, streptomycin, fungizone and 10% fetal bovine serum. Tissue culture media, supplements and sera were all obtained from Gibco BRL (Grand Island, NY). Bovine herpesvinis type 1 (BHV-1) strain "Iowa" can be obtained from the National Animal Disease Center of the USDA (Ames, Iowa). Other BHV-1 strains can be used.
- BHV-1 strain "Iowa" can be obtained from the National Animal Disease Center of the USDA (Ames, Iowa). Other BHV-1 strains can be used.
- BPIV-3 bovine parainfluenzavirus type 3
- Ames, LA National Veterinary Services Laboratory
- Viruses are grown and titered by standard methods that would be well known to those skilled in the art. Initial plaque isolations are carried out under 0.8% agarose, and plaque purifications were accomplished by limiting dilution in 96 well plates.
- Frozen competent cells can be purchased from Gibco/BRL or other suppliers. DNA fragments excised from agarose gels can be purified using Qiaex (Qiagen,
- Plasmid DNAs can be purified using Qiagen columns (Qiagen) or by other methods.
- BHV-1 DNA is purified from infected BT cells on sodium iodide gradients essentially by the method described by Wallboomers and Ter Shegett (J.M. Walboomers, et al., Virology, 74:256-258 (1976)), or for small- scale preparations by the "mini-prep" procedure as described by Engel, et al (J.P. Engel, et al., Virology, 192:112-120 (1993)).
- DNA hybridizations of viral DNA restriction digests are carried out using the digoxygenin/anti-digoxygenin reagents supplied with the Genius Kit (BMB) or by other applicable methods.
- Plasmid pHAS4 is a 2.7kb subfragment of the BHV-1 Hindlll "A" fragment (from strain Cooper) cloned into vector pUC18.
- This plasmid can be assembled according to procedures well known in the art or by following the procedures described in (J.E. Mayfield, et al., J Virol, 47:259-264 (1983)), incorporated by reference herein.
- This Sail fragment contains the entire thymidine kinase (tk) gene, the BHV-1 homolog of the HSV-1 UL24 gene and a small portion of the BHV-1 glycoprotein H gene (L.J.
- a 424bp deletion is made in the tk gene by digesting with Bglll and Xhol and blunt-end ligating the filled-in ends. This manipulation regenerates the Bglll site, and the resulting plasmid is designated pHAS4DBX. Creation of a shuttle vector for expression of foreign genes in the BHV-1 tk locus.
- pHAS4DBXex-l In order to express various different viral antigens in BHV-1, a shuttle vector, pHAS4DBXex-l was created to allow recombination of the foreign genes into the viral thymidine kinase locus.
- the construction of this vector is detailed in FIGURE 1.
- pHAS4 a 2.7kb BHV-1 Sail fragment which contains the entire tk gene and flanking regions on either side.
- a 424bp deletion Is made in the tk gene by digesting with enzymes Bglll and Xhol, filling in the sticky ends and religating the resulting blunt ends (this manipulation regenerated the Bglll restriction site), resulting in plasmid pHAS4DBX.
- this manipulation regenerated the Bglll restriction site
- plasmid pHAS4DBX Into this Bglll restriction site insert an expression cassette consisting of the Human Cytomegalovirus (HCMV) immediate early promoter and the polyadenylation signal from bovine growth hormone, which are separated by a unique Hindlll restriction site for the insertion of foreign genes to be expressed (R.J. Brideau, et al, J Gen Virol, 1 ⁇ 11-A11 (1993)).
- This shuttle vector is called pHAS4DBXex-l.
- the promoter/polyadenylation signal cassette in this vector is inserted in the opposite transcriptional orientation to that of the original tk gene.
- the HN gene was cloned MR2-9tr from strain MR (Y. Sakai, et al, J Virol, 63:3661-8 (1989), H. Shibuta, et al, Virology, 155:688-96 (198 )),.
- This cDNA contains the complete HN mRNA cloned into an Okayama and Berg-type cloning vector (H. Okayama, et al, Molec Cell Biol, 2:161-170 (1982)).
- This HN gene clone can be obtained or constructed according to procedures well known in the art or by following the procedures described in Y. Sakai, et al, Nucleic Acids Res, 15:2927-44 (1987), incorporated by reference herein.
- recombinant BHV-1 unit length viral DNA and plasmids containing the sequences of interest are cotransfected into BT cells by the calcium phosphate method as described by Graham and Van der Eb (R.L. Graham, et al, Virology, 52:456-467 (1973)), and as modified by Lawrence, et. al. (W.C. Lawrence, et al, J Virol, 60:405-14 (1986)).
- the shuttle plasmids Prior to cotransfection, the shuttle plasmids are linearized by digestion with Sse8387I and the resulting linear fragments are purified away from the enzyme using the Qiaex glass matrix (Qiagen) according to manufacturer supplied methods. Tk-negative viruses arising from the cotransfections are selected by two passages on RabBU cells in the presence of lOOug/ml BDUR. Selected BDUR-resistant viruses are then plaque purified three times on BT cells (in the absence of BDUR) by limiting dilution.
- Qiaex glass matrix Qiagen
- the BaculoGold system (Pharmingen, San Diego, CA) can be utilized for the generation of recombinant baculoviruses (BV), using protocols provided by the manufacturer.
- the shuttle plasmid carrying the foreign gene of interest is cotransfected with a defective genome of AcNPV such that only those genomes rescued by the recombination of flanking sequences on the shuttle plasmid will be viable in wild-type cells.
- viral progeny from the transfection need only be plaque purified and screened for expression of the gene of interest. Metabolic labelling of infected cells.
- cells can be infected with BHV-1 or BPIV-3 and SF9 cells infected with recombinant BV at a multiplicity of infection of 5-10.
- the normal media is replaced with media containing 10% cold methionine and lOOuCi/ml S-Methionine (50 mCI/mMol, Amersham, Arlington Hights, IL) at 8 hours post-infection, and the cells allowed to label for another 24 hours.
- the media is replaced with methionine-free Grace's media containing lOOuCi/ml S-methionine at 24 hours post-infection, and the labelling continues for another 24 hours.
- Neuraminidase assays Neuraminidase activity is assayed using the fluorescent substrate 2'-(4-methylumbelliferyl)-a-D-N-Acetylneuraminic acid (Sigma) by the method of Myers, et al (R.W. Meyers, et al, Anal Biochem, 101:166-174 (1980)). The assays are nm at pH 5.1, which was shown to be optimal for the HN protein of BPI3 strain MR by Shibuta, et al (H. Shibuta, et al, Infect Immun, 41:780-788 (1983)).
- 225cm 2 flasks of BT cells (approximately 6xl0 6 cells) are infected at an MOI of 1 with the BHV-1 viruses of interest, and infection is allowed to proceed until 100% CPE is observed (approximately 36 hours).
- Spinner r flasks of SF9 cells (approximately 5x10 cells) are infected with the baculoviruses of interest at an MOI of 1 and the infection is allowed to proceed for 48 hours.
- the various harvested cell pellets are resuspended in 1.0ml cold PBS each, subjected to three freeze/thaw cycles in a dry ice/ethanol bath and then centrifuged at 12000Xg for 15 minutes at 4°C.
- the resulting supernatants are adjusted to lmg ml using the BCA protein assay (Pierce, Rockford, IL). Protein samples (50ug each) are then incubated with 80nmole of substrate in a lOOul total volume of 50mM NaAcetate (pH 5.1) at 37°C for 0-60 minutes. The reactions are stopped by the addition of 3.0ml 250mM glycine (pH 10.4), and the fluorescence intensity can be read on a Perkin Elmer LS50-B luminescence spectrophotometer (excitation 360nm, lO.Onm slit width, emission, 440nm, 2.5nm slit width), or other appropriate instrument.
- the sodium salt of 4-methylumbelliferone (Sigma) can be used as the standard, and under the above conditions results in a linear range between 0.2 nmoles and 10 nmoles.
- Immunological methods For immunoprecipitations, S-methionine labelled infected or mock infected cells are solubilized in RIPA buffer (lOmM Tris-HCl (pH7.5), l ⁇ OmM NaCl, 0.1% SDS, 1% TritonX-100, 1% NaDeoxycholate, and lmg/ml ovalbumin) for 1 hour at room temperature, and then the insoluble fraction is removed by centrifugation at 15Kxg for 10 minutes at RT in a microcentrifuge. The cleared lysates are then preadsorbed with lOOul of protein A Sepharose 4B
- the immune complexes thus adsorbed to the protein A sepharose are washed three times with HO buffer (lOmM Tris-HCl (pH 7.5), lOOmM NaCl, lmM EDTA, 1%NP40, and 0.5% Na deoxycholate), and then resuspended in 50 ul SDS sample buffer with 2-mercapatoethanol (Sigma) and boiled for 5 minutes to dissociate the immune complexes from the matrix.
- the samples are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography.
- the BPIV-3 HN gene is expressed in two heterologous systems; a recombinant herpesvinis and a recombinant baculovinis are described.
- the recombinant proteins are very similar to native HN with respect to antigenicity and electrophoretic mobility, and they retain the neuraminidase activity expected for native HN, as illustrated in the following examples.
- EXAMPLE 1 Introduction of the BPIV-3 HN into BHV-1, and selection of recombinant viruses.
- a map of the BPIV-3 HN cDNA clone and its insertion into the BHV-1 shuttle vector is shown in FIGURE 1.
- For cloning into the BHV-1 vector remove the HN gene from the cDNA cloning vector with Hindlll and BamHI (this fragment includes the 3' poly A tail retained from the cloning of the cDNA, and also includes a small portion of the SV40 polyadenylation signal region present in the vector).
- the Hindlll site is 80bp upstream of the ATG initiation codon, FIGURE 1.
- the linearized BHV-1 shuttle vector containing the BPIV-3 HN gene is allowed to recombine with the wild type BHV-1 tk locus via cotransfection into BT cells of the plasmid and unit length BHV-1 Strain "Iowa” DNA.
- "Iowa” is a highly virulent disease isolate strain of BHV-1.
- plasmid pHAS4DBX which contains a deleted version of the BHV-1 tk gene with no inserted promoters or genes
- BHV-1 Iowa to obtain a tk-deleted control virus without an inserted gene. Tk-negative progeny from these cotransfections are selected and isolated by passage on RabBU cells in the presence of BDUR.
- the DNA from these viruses is isolated for restriction digestion and Southern hybridization using either the BPIV-3 HN gene or pHAS4 as probes (data not shown).
- BDUR resistant transfection progeny two BHV-1 viral clones that hybridized independently with the HN gene, HN16 and HN21 were isolated. This latter virus is referred to in the animal studies (see “utility of the invention", below) as BHVDtk HN.
- BHVDtk HN Several BDUR-resistant transfection progeny that contained the tk-deletion as assessed by a drop in size of the pHAS4 fragment in these clones were also isolated.
- a representative virus was isolated and called IowaDtk (referred to in the animal studies as BHVDtk).
- EXAMPLE 2 Introduction of the BPIV-3 HN into baculoviurs, and selection of recombinant viruses.
- HN gene For expression in the BV system, we cloned the HN gene into the polyhedrin-based transfer vector pVL1392.
- FIGURE 2 shows the insertion of HN into this vector.
- the HN gene is digested with Hindlll, the single stranded protruding end is filled in with Klenow, and then the insert is excised from plasmid vector pSP72 by digestion with BamHI.
- Plasmid pVL1392 is digested with Bglll, and the ends are filled in with Klenow fragment, then the plasmid is digested with BamHI.
- the HN insert is ligated into the digested pVL1392 vector via one blunt end and one sticky end, assuring the proper orientation for expression.
- the HN gene properly cloned into pVL1392, is allowed to recombine into the AcNPV genome via cotransfection with BaculoGold DNA into SF9 cells.
- EXAMPLE 3 Demonstration of expression and biological activity of the HN protein in BHV-1 and Baculovinis.
- Metabolically labelled infected cell lysates were incubated with polyclonal bovine antiserum against BPIV-3 (obtained from NVSL, Ames, LA).
- HN21, HN16 (lanes 3 and 4) as well as a representative viral clone purified from the BV/HN transfection (lane 6, designated baculoHN) expressed large amounts of an approximately 70KD BPIV-3 immunoreactive protein that appeared to comigrate with native BPIV-3 HN (Lane 2).
- This protein was not present in wild-type BHV-1-infected (lane 1) or in mock- infected BT cells (data not shown), nor was it present in SF9 cells infected with an irrelevant recombinant baculovinis (lane 5).
- NA activity was not present in the negative controls, consisting of the BHV-1 IowaDtk virus, and a recombinant baculovinis expressing BHV-1 gill protein.
- This invention is intended to provide the user with an effective vaccine for prevention of BPIV-3 and BHV-1 caused disease.
- BPIV-3 As a pathogen is its association with Bovine Respiratory Disease Complex (BRDC).
- the vaccine of this invention is created with the intention of treating disease, preferably through prevention.
- prevent or prevention applicant means to keep the host from developing symptoms of the disease or to mitigate the effects of the disease, that is to avert the typical diseased state.
- Prevention implies decisive action to stop, impede or delay the onset of disease.
- Prevention can include the following concepts: to hinder, frustrate, to obstruct; to intercept, possibly prohibit, impede or preclude.
- Preclude would suggest the onset of the disease state either does not occur or the disease pathogen is largely ineffectual in causing the disease state.
- Prevent or prevention can indicate the disease state is forestalled, meaning that anticipatory action to prevent or hinder the disease has occurred but the conditions creating the disease have not been eliminated.
- the usefulness of this invention is illustrated by the ability of the vaccine to provide effective protection against BPIV-3 disease.
- Vaccination for this disease includes eliciting strong neutralizing antibody responses to the HN glycoprotein.
- This invention consists of different embodiments that include a recombinant baculovinis and a recombinant bovine herpesvinis. These recombinant viruses express the HN protein from bovine parainfluenza virus- 3, thus conferring a protective effect in the vaccinated animals.
- Animals vaccinated with the HN recombinant viruses exhibited increased levels of serum neutralizing antibodies to BPIV-3, demonstrating the immunogenicity of both embodiments of this invention. Animals also showed little or no signs of clinical disease after challenge.
- the animals vaccinated with the HN recombinant viruses claimed in this invention were also protected from challenge, as shown by the durations of challenge virus shedding. TABLE 2. Virus shedding is illustrated by shading within squares for each day post challenge, and the percentage of virus excretion over both 7 and 14 days is illustrated in the right two columns of the table.
- pHAS4 is a DNA fragment from the BHV-1 genome. The gene and gene fragments encoded in the fragment are noted. pHAS4DBX depicts the 424bp deletion made in the tk gene. The various cassettes inserted into pHAS4DBX are labelled. pHAS4DBXexl was made by the insertion of the CMV-IE promoter and the bovine growth hormone polyadenylation signal into the tk deletion. In pHAS4DBXexl::HN (bottom) the direction of transcription of HN is noted by an arrow.
- Figure 2 Construction of a recombinant baculovinis transfer vector for expression of the BPIV-3 HN gene in the baculovinis expression system.
- TOP pVL1392, a polyhedrin promoter-based transfer vector for the expression of foreign genes.
- BOTTOM Insertion of the HN gene into this vector.
- FIG. 3 Immunoprecipitations of infected-cell lysates showing expression of BPIV-3 HN from BHV-1 and baculovinis.
- the immunoprecipitates were brought down with a polyclonal antiserum (from calf) against BPIV-3.
- Lanes 1-4 were metabolically labelled infected (or mock infected) BT cells, and lanes 5 and 6 were metabolically labelled infected SF-9 cells.
- Lane 1 mock infected cells
- lane 2 BPIV- 3 strain SF4 (the HN protein runs at approximately 70kD)
- lane 3 HN16 (recomb. BHV-1)
- lane 4 HN21 (recomb.
- FIG. 1 HN activity in the BHV-1 and baculovinis recombinants.
- the graph shows enzymatic release of 4-methylumbelliferone (4-MUBF) over time from the 2'-(4-methylubelliferyl)-a-D-N-acetylmeruaminic acid substrate, as catalyzed by freeze-thaw lysates of the various viral preps.
- the X axis is time in minutes.
- the Y axis is nmoles of 4-MUBF released.
- KEY Open squares, HN21 (BHVDtk-HN); closed diamonds, baculoHN; open circles, BHVDtk; closed squares, baculo-gC (a recombinant baculovinis expressing the gC protein of BHV-1, used here as an negative control); closed circles, BPIV-3.
- a virus was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, zip code 20852, USA. That deposit was designated UC VR-59 by the Upjohn Company and given the following number by the depository, ATCC No. VR 2478, it corresponds to the virus described herein as "HN21", or "BHVDtk HN". This deposit was received by the American Type Culture Collection depository on 14 September 1994. TABLES
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Pulmonology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU45005/96A AU706454B2 (en) | 1994-11-18 | 1995-11-08 | Expression of the bovine parainfluenza virus type 3 hemagglutinin/neuraminidase (HN) glycoprotein in two heterologous systems |
JP8516849A JPH10509593A (en) | 1994-11-18 | 1995-11-08 | Expression of hemagglutinin / neuraminidase (HN) glycoprotein of bovine parainfluenza virus type 3 in two heterologous systems |
EP95943566A EP0793728A1 (en) | 1994-11-18 | 1995-11-08 | Expression of the bovine parainfluenza virus type 3 hemagglutinin/neuraminidase (hn) glycoprotein in two heterologous systems |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US34219894A | 1994-11-18 | 1994-11-18 | |
US08/342,198 | 1994-11-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996016184A1 true WO1996016184A1 (en) | 1996-05-30 |
Family
ID=23340791
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/013482 WO1996016184A1 (en) | 1994-11-18 | 1995-11-08 | Expression of the bovine parainfluenza virus type 3 hemagglutinin/neuraminidase (hn) glycoprotein in two heterologous systems |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0793728A1 (en) |
JP (1) | JPH10509593A (en) |
KR (1) | KR970707293A (en) |
AU (1) | AU706454B2 (en) |
CA (1) | CA2204252A1 (en) |
WO (1) | WO1996016184A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002062381A1 (en) * | 2001-02-05 | 2002-08-15 | Hisamitsu Pharmaceutical Co., Inc. | Baculovirus vector vaccine |
US8513006B2 (en) | 2010-09-14 | 2013-08-20 | University of Pittsburgh—of the Commonwealth System of Higher Education | Tetravalent influenza vaccine and use thereof |
US9061041B2 (en) | 2011-04-13 | 2015-06-23 | Merck Sharp & Dohme Corp. | 2′-substituted nucleoside derivatives and methods of use thereof for the treatment of viral diseases |
US9150603B2 (en) | 2011-04-13 | 2015-10-06 | Merck Sharp & Dohme Corp. | 2′-cyano substituted nucleoside derivatives and methods of use thereof useful for the treatment of viral diseases |
US9416154B2 (en) | 2011-07-13 | 2016-08-16 | Merck Sharp & Dohme Corp. | 5′-substituted nucleoside derivatives and methods of use thereof for the treatment of viral diseases |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2161814A (en) * | 1984-07-18 | 1986-01-22 | Grace W R & Co | Synthetic genes for bovine parainfluenza virus |
JPS63283578A (en) * | 1987-05-16 | 1988-11-21 | Hiroshi Shibuta | Recombinant vaccinia virus |
WO1994024296A2 (en) * | 1993-04-19 | 1994-10-27 | University Of Saskatchewan | Recombinant bovine herpesvirus type 1 vaccines |
-
1995
- 1995-11-08 JP JP8516849A patent/JPH10509593A/en active Pending
- 1995-11-08 CA CA002204252A patent/CA2204252A1/en not_active Abandoned
- 1995-11-08 KR KR1019970703328A patent/KR970707293A/en not_active Application Discontinuation
- 1995-11-08 AU AU45005/96A patent/AU706454B2/en not_active Ceased
- 1995-11-08 EP EP95943566A patent/EP0793728A1/en not_active Withdrawn
- 1995-11-08 WO PCT/US1995/013482 patent/WO1996016184A1/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2161814A (en) * | 1984-07-18 | 1986-01-22 | Grace W R & Co | Synthetic genes for bovine parainfluenza virus |
JPS63283578A (en) * | 1987-05-16 | 1988-11-21 | Hiroshi Shibuta | Recombinant vaccinia virus |
WO1994024296A2 (en) * | 1993-04-19 | 1994-10-27 | University Of Saskatchewan | Recombinant bovine herpesvirus type 1 vaccines |
Non-Patent Citations (2)
Title |
---|
K.L.VAN WYKE COELINGH ET AL.: "Expression of biologically active and antigenically authentic parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus", VIROLOGY, vol. 160, 1987, ORLANDO US, pages 465 - 472, XP002000517 * |
PATENT ABSTRACTS OF JAPAN vol. 013, no. 107 (C - 576) 14 March 1989 (1989-03-14) * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002062381A1 (en) * | 2001-02-05 | 2002-08-15 | Hisamitsu Pharmaceutical Co., Inc. | Baculovirus vector vaccine |
US8513006B2 (en) | 2010-09-14 | 2013-08-20 | University of Pittsburgh—of the Commonwealth System of Higher Education | Tetravalent influenza vaccine and use thereof |
US9061041B2 (en) | 2011-04-13 | 2015-06-23 | Merck Sharp & Dohme Corp. | 2′-substituted nucleoside derivatives and methods of use thereof for the treatment of viral diseases |
US9150603B2 (en) | 2011-04-13 | 2015-10-06 | Merck Sharp & Dohme Corp. | 2′-cyano substituted nucleoside derivatives and methods of use thereof useful for the treatment of viral diseases |
US9416154B2 (en) | 2011-07-13 | 2016-08-16 | Merck Sharp & Dohme Corp. | 5′-substituted nucleoside derivatives and methods of use thereof for the treatment of viral diseases |
Also Published As
Publication number | Publication date |
---|---|
KR970707293A (en) | 1997-12-01 |
JPH10509593A (en) | 1998-09-22 |
EP0793728A1 (en) | 1997-09-10 |
AU4500596A (en) | 1996-06-17 |
CA2204252A1 (en) | 1996-05-30 |
AU706454B2 (en) | 1999-06-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Schmaljohn et al. | Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants | |
KR970011149B1 (en) | Recombinant avipox virus | |
Drillien et al. | Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the hemagglutinin or the fusion protein. | |
Taylor et al. | Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens | |
EP0390799B1 (en) | Respiratory syncytial virus: vaccines | |
US5905040A (en) | Parvovirus empty capsids | |
US5187087A (en) | Recominbant herpesvirus of turkeys and live vector vaccines derived thereof | |
Parker et al. | Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV | |
Lüschow et al. | Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene | |
Hunt et al. | Retrovirus-expressed hemagglutinin protects against lethal influenza virus infections | |
Bertagnoli et al. | Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein | |
Osterrieder et al. | Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gB) | |
US5420026A (en) | Self-assembling replication defective hybrid virus particles | |
Riviere et al. | Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants | |
HUT67138A (en) | Attenvated, genetically-engineered pseudorabies virus s-prv-155 and uses thereof | |
Wathen et al. | Immunization of cotton rats with the human respiratory syncytial virus F glycoprotein produced using a baculovirus vector | |
EP2129390A2 (en) | Turkey herpesvirus vectored recombinant containing avian influenza genes | |
EP0334530B1 (en) | A recombinant marek's disease virus and a vaccine | |
JPH08500969A (en) | Recombinant turkey herpesviruses and their use | |
Gogev et al. | Induction of protective immunity to bovine herpesvirus type 1 in cattle by intranasal administration of replication-defective human adenovirus type 5 expressing glycoprotein gC or gD | |
US6544526B1 (en) | Equine herpesvirus glycoproteins | |
Haanes et al. | The bovine parainfluenza virus type-3 (BPIV-3) hemagglutinin/neuraminidase glycoprotein expressed in baculovirus protects calves against experimental BPIV-3 challenge | |
Yokoyama et al. | Recombinant viral vector vaccines for the veterinary use | |
JP2007020574A (en) | Bhv-1 gene-deleted virus vaccine | |
Ray et al. | Expression of the fusion glycoprotein of human parainfluenza type 3 virus in insect cells by a recombinant baculovirus and analysis of its immunogenic property |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA US UZ VN |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2204252 Country of ref document: CA Ref country code: CA Ref document number: 2204252 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1019970703328 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1995943566 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref country code: US Ref document number: 1997 849064 Date of ref document: 19970827 Kind code of ref document: A Format of ref document f/p: F |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1995943566 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1019970703328 Country of ref document: KR |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1019970703328 Country of ref document: KR |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1995943566 Country of ref document: EP |