EP1751178A2 - Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein - Google Patents
Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike proteinInfo
- Publication number
- EP1751178A2 EP1751178A2 EP05750800A EP05750800A EP1751178A2 EP 1751178 A2 EP1751178 A2 EP 1751178A2 EP 05750800 A EP05750800 A EP 05750800A EP 05750800 A EP05750800 A EP 05750800A EP 1751178 A2 EP1751178 A2 EP 1751178A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- spike
- polypeptide
- nucleic acid
- seq
- antibodies
- 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.)
- Withdrawn
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 150
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 140
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 140
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 82
- 241000315672 SARS coronavirus Species 0.000 title claims abstract description 75
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 74
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 74
- 238000000034 method Methods 0.000 title claims abstract description 72
- 239000000203 mixture Substances 0.000 title claims abstract description 50
- 230000002163 immunogen Effects 0.000 title claims abstract description 26
- 230000014509 gene expression Effects 0.000 title claims description 42
- 108010061994 Coronavirus Spike Glycoprotein Proteins 0.000 title description 2
- 229940096437 Protein S Drugs 0.000 claims abstract description 46
- 101710198474 Spike protein Proteins 0.000 claims abstract description 45
- 229960005486 vaccine Drugs 0.000 claims abstract description 27
- 239000002773 nucleotide Substances 0.000 claims abstract description 19
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims description 74
- 108090000623 proteins and genes Proteins 0.000 claims description 68
- 102000004169 proteins and genes Human genes 0.000 claims description 57
- 239000000427 antigen Substances 0.000 claims description 45
- 108091007433 antigens Proteins 0.000 claims description 45
- 102000036639 antigens Human genes 0.000 claims description 45
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 29
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 28
- 229940037003 alum Drugs 0.000 claims description 26
- 238000001262 western blot Methods 0.000 claims description 24
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 22
- 239000002671 adjuvant Substances 0.000 claims description 18
- 230000004044 response Effects 0.000 claims description 17
- 230000003472 neutralizing effect Effects 0.000 claims description 16
- 238000003018 immunoassay Methods 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- 238000003556 assay Methods 0.000 claims description 12
- 238000000338 in vitro Methods 0.000 claims description 12
- 230000001965 increasing effect Effects 0.000 claims description 12
- 238000006467 substitution reaction Methods 0.000 claims description 12
- 108020004705 Codon Proteins 0.000 claims description 11
- 108020004414 DNA Proteins 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 10
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 9
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 9
- 238000002965 ELISA Methods 0.000 claims description 7
- 239000013060 biological fluid Substances 0.000 claims description 7
- 230000002708 enhancing effect Effects 0.000 claims description 7
- 238000007912 intraperitoneal administration Methods 0.000 claims description 7
- 108700010070 Codon Usage Proteins 0.000 claims description 6
- 241000699802 Cricetulus griseus Species 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 6
- 238000009396 hybridization Methods 0.000 claims description 6
- 238000001114 immunoprecipitation Methods 0.000 claims description 6
- 238000001727 in vivo Methods 0.000 claims description 6
- 108091035707 Consensus sequence Proteins 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 5
- 238000002405 diagnostic procedure Methods 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 230000001900 immune effect Effects 0.000 claims description 4
- 230000035772 mutation Effects 0.000 claims description 4
- 210000003705 ribosome Anatomy 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 238000009007 Diagnostic Kit Methods 0.000 claims description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 claims description 3
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 3
- 239000012620 biological material Substances 0.000 claims description 3
- 238000010166 immunofluorescence Methods 0.000 claims description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 108091033319 polynucleotide Proteins 0.000 claims 4
- 102000040430 polynucleotide Human genes 0.000 claims 4
- 239000002157 polynucleotide Substances 0.000 claims 4
- 230000005867 T cell response Effects 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 description 34
- 210000002966 serum Anatomy 0.000 description 22
- 239000013604 expression vector Substances 0.000 description 14
- 239000011324 bead Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 230000002550 fecal effect Effects 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- 241000700605 Viruses Species 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000002649 immunization Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 241000711573 Coronaviridae Species 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000006386 neutralization reaction Methods 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000012139 lysis buffer Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 230000001681 protective effect Effects 0.000 description 6
- 102100031673 Corneodesmosin Human genes 0.000 description 5
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 5
- 108010020195 FLAG peptide Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 108010008125 Tenascin Proteins 0.000 description 5
- 102100038126 Tenascin Human genes 0.000 description 5
- 108010031318 Vitronectin Proteins 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- 241000699800 Cricetinae Species 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 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 4
- 230000008901 benefit Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229940023146 nucleic acid vaccine Drugs 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 108091023045 Untranslated Region Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 2
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000282472 Canis lupus familiaris 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
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 101100151951 Homo sapiens SARS1 gene Proteins 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102100031156 Prohibitin-2 Human genes 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 229940024545 aluminum hydroxide Drugs 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 2
- -1 for example Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000009851 immunogenic response Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 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 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 239000006147 Glasgow's Minimal Essential Medium Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101710114810 Glycoprotein Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 101000582320 Homo sapiens Neurogenic differentiation factor 6 Proteins 0.000 description 1
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010022004 Influenza like illness Diseases 0.000 description 1
- 108700001097 Insect Genes Proteins 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-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
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 241000699673 Mesocricetus auratus Species 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 102100030589 Neurogenic differentiation factor 6 Human genes 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000605862 Porphyromonas gingivalis Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 229940124680 SARS vaccine Drugs 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229940024546 aluminum hydroxide gel Drugs 0.000 description 1
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 208000030303 breathing problems Diseases 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229960001714 calcium phosphate Drugs 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000016379 mucosal immune response Effects 0.000 description 1
- 125000001446 muramyl group Chemical group N[C@@H](C=O)[C@@H](O[C@@H](C(=O)*)C)[C@H](O)[C@H](O)CO 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000011824 nuclear material Substances 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 229950009506 penicillinase Drugs 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010517 secondary reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000005026 transcription initiation 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
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—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
- A61K2039/53—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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Definitions
- the invention is directed to purified and isolated nucleic acids, polypeptides, purified and isolated polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, and the use of such nucleic acids and polypeptides in diagnostic methods, kits, immunogenic compositions, vaccines, or antiviral therapy.
- SARS severe acute respiratory syndrome
- SARS coronavirus
- coronavirus a diverse group of large, enveloped viruses that cause respiratory and enteric disease in humans and animals.
- SARS CoV was isolated from FRhK-4 and Vero E6 cells that were inoculated with clinical specimens from patients, and macaques inoculated with this virus developed symptoms similar to those observed in human cases of SARS. To date, over 30 different SARS CoV have been isolated and sequenced.
- SARS CoV contains an RNA genome of about 30 kB (Accession No. AY310120), and shares many characteristic features of coronaviruses. Nucleotides 1-72 contain a predicted RNA leader sequence preceding an untranslated region (UTR) spanning 192 nucleotides. Two overlapping open reading frames, which encompass approximately two-thirds of the genome (nucleotides 265-21485) are down stream of the UTR, and encode proteinases as well as the proteins required for replication and transcription (for a review see Stadler et al., 2004).
- UTR untranslated region
- the remaining 3' part of the genome encodes four structural proteins that are arranged in the same order in all CoV: Spike, Envelope, Membrane glycoprotein, and Nucleocapsid protein.
- the structural protein region of the SARS CoV genome also encodes additional non-structural proteins known as 'accessory genes'. Although the overall organization of the SARS CoV genome is similar to other coronaviruses, the amino acid conservation of the encoded proteins is usually low.
- the Spike protein forms large surface projections that are characteristic of coronaviruses.
- Spike is heavily glycosylated and has 1 ,255 amino acids, containing an amino-terminal bulbous head adjacent to a stem, a single transmembrane region, and a short cytoplasmic tail (See Stadler et al.).
- ⁇ -interferon has been reported to interfere with the replication of the SARS virus in vitro, no licensed drug or vaccine is available.
- large-scale screening of existing antivirals or big chemical libraries for potential replication inhibitors has not been very successful. It is also virtually impossible to confirm a SARS diagnosis in the primary care setting, as the sensitivity and specificity of available tests varies with time from onset of contact or symptoms (See Rainer et al.).
- the invention encompasses a purified nucleic acid molecule comprising the DNA sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 6.
- the invention also encompasses nucleic acid molecules complementary to these sequences, such as fully complementary sequences.
- the invention includes double-stranded nucleic acid molecules comprising the DNA sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 6 and purified nucleic acid molecules encoding the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7.
- Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention. These molecules can be used to detect both single- stranded and double-stranded RNA and DNA encompassed by the invention.
- a double- stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of the nucleic acid molecule.
- the invention further encompasses purified nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NOS: 1-3 & 6.
- In vitro mutagenesis includes numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
- the nucleic acid molecules of the invention which include DNA and RNA, are referred to herein as “Spike nucleic acids” or “Spike DNA”, and the amino acids encoded by these molecules are referred to herein as “Spike polypeptides” or “Spike protein.”
- the invention also encompasses purified nucleic acid molecules degenerate from SEQ ID NOS: 1-3 & 6 as a result of the genetic code, purified nucleic acid molecules, which are allelic variants of Spike nucleic acids or a species homolog of Spike nucleic acids.
- the invention encompasses purified nucleic acids that show increased expression of Spike protein as compared to SEQ ID NO: 1.
- the invention also encompasses purified nucleic acids that show increased expression of Spike protein as compared to SEQ ID NO: 1 , wherein at least one negative cis-acting signal has been substituted without changing the sequence of the encoded protein.
- Negative cis-acting signals as encompassed by the invention include, but are not limited to, AU-rich RNA instability motifs, repeating sequences, secondary stretches, splice donor and acceptor sites, and internal poly(A) sites.
- the invention also encompasses purified nucleic acid molecules that show increased expression of Spike protein as compared to SEQ ID NO: 1 , wherein expression is increased through the addition of expression enhancing sequences.
- Expression enhancing sequences include, but are not limited to, Kozak consensus sequence upstream of the starting ATG, as well as additional stop codons. [018] A skilled artisan will know the suitable placement of the Kozak consensus sequence based on the prior art. [019] The invention also encompasses purified nucleic acid molecules that show increased expression of Spike protein as compared to SEQ ID NO: 1 , wherein codon usage has been optimized to the bias of Cricetulus griseus.
- the invention also encompasses purified nucleic acid molecules that show increased expression of Spike protein as compared to SEQ ID NO: 1 , wherein the portion of the purified nucleic acid molecule encoding Spike protein comprises at least about a 10 percent increase in the percentage GC-content as compared to SEQ ID NO: 1, and wherein regions of very high (>80%) or very low ( ⁇ 30%) GC content have been avoided where possible.
- the invention also encompasses purified nucleic acid molecules that show increased expression of Spike protein as compared to SEQ ID NO: 1 , wherein the substitution of at least one negative cis-acting signal and wherein the at least one additional expression enhancing sequence does not include the following: internal TATA-boxes, chi- sites, and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures; and splice donor and acceptor sites.
- the invention also encompasses purified polypeptides encoded by these nucleic acid molecules, including glycosylated and non-glycosylated forms of the purified polypeptide.
- the invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors.
- Purified polyclonal or monoclonal antibodies that bind to Spike polypeptides are encompassed by the invention, as are neutralizing antibodies.
- the invention further encompasses methods for the production of Spike polypeptides, including culturing a host cell under conditions for promoting expression, and recovering the polypeptide from the culture medium. Especially, the expression of Spike polypeptides in animal cells is encompassed by the invention.
- the invention also encompasses labeled Spike polypeptides. Preferably, the labeled polypeptides are in purified form.
- the unlabeled or labeled polypeptide is capable of being immunologically recognized by human body fluid containing antibodies to Spike polypeptide.
- the polypeptides can be labeled, for example, with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
- an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
- this invention provides a method for detecting infection by SARS CoV.
- the method comprises providing a composition comprising a biological material suspected of being infected with SARS CoV, and assaying for the presence of Spike polypeptide of SARS CoV.
- the polypeptides are typically assayed by electrophoresis or by immunoassay with antibodies that are immunologically reactive with the Spike polypeptides of the invention.
- This invention also provides an in vitro diagnostic method for the detection of the presence or absence of antibodies, which bind to an antigen comprising the Spike polypeptides of the invention.
- the method comprises contacting the antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an antigen-antibody complex, and then detecting the formation of the complex.
- the detecting step can further comprise measuring the formation of the antigen-antibody complex.
- the formation of the antigen-antibody complex is preferably measured by immunoassay based on Western blot technique, ELISA (enzyme linked immunosorbent assay), FACS, indirect immunofluorescent assay, or immunoprecipitation assay.
- the invention also encompasses a diagnostic kit for the detection of the presence or absence of antibodies, which bind to the Spike polypeptide of the invention, contains antigen comprising the Spike polypeptide, and means for detecting the formation of immune complex between the antigen and antibodies.
- the antigens and the means are present in an amount sufficient to perform the detection.
- This invention also provides an immunogenic composition comprising a Spike polypeptide of the invention or a mixture thereof in an amount sufficient to induce an immunogenic or protective response in vivo, in association with a pharmaceutically acceptable carrier therefor.
- the immunogenic composition may contain an Alum adjuvant.
- a vaccine composition of the invention comprises a neutralizing amount of the Spike polypeptide and a pharmaceutically acceptable carrier therefor.
- the polypeptides of this invention are thus useful as a portion of a diagnostic composition for detecting the presence of antibodies to antigenic proteins associated with SARS CoV.
- the Spike polypeptides can be used to raise antibodies for detecting the presence of antigenic proteins associated with SARS CoV.
- the polypeptides of the invention can also be employed to raise neutralizing antibodies that either inactivate the virus, reduce the viability of the virus in vivo, or inhibit or prevent viral replication.
- the ability to elicit virus-neutralizing antibodies is especially important when the polypeptides of the invention are used in immunizing or vaccinating compositions to activate the B-cell arm of the immune response or induce a cytotoxic T lymphocyte response (CTL) in the recipient host.
- CTL cytotoxic T lymphocyte response
- the present invention also pertains to vaccine compositions for immunizing humans and mammals against SARS CoV, comprising an immunogenic composition as described above in combination with one or more pharmaceutically compatible excipients (such as, for example, saline buffer), and optionally in combination with at least one adjuvant such as aluminum hydroxide or a compound belonging to the muramyl peptide family.
- This invention also encompasses a method for detecting the presence or absence of SARS CoV comprising: (1 ) contacting a sample suspected of containing viral genetic material of SARS CoV with at least one nucleotide probe, and (2) detecting hybridization between the nucleotide probe and the viral genetic material in the sample, wherein said nucleotide probe is complementary to the full-length sequence of the purified Spike nucleic acids of the invention.
- Lane 1 represents cells transfected with pcDNA-Spike-Pasteur
- lane 2 represents cells transfected with pcDNA-HKU-PRC
- lane 3 represents cells transfected with SFV-Spike- Pasteur-modif
- lane 4 is empty
- lane 5 represents purified Spike from transfected BHK cells.
- Figure 2 shows the sequence in standard single letter abbreviations of the SARS CoV Spike protein with the Flag peptide sequence used for RNA and protein vaccination (SEQ ID NO: 5). The sequence corresponding to the SARS CoV Spike protein is shaded, while the sequence including the Flag peptide is underlined. The protein sequence was expressed in the Semliki (SFV) Forest Virus vector.
- SFV Semliki
- Figure 3 is an SDS-PAGE of pulse-chase labeled SFV-Spike infected BHK cells following immunoprecipitation with M2 (Flag) antibody. Cells were harvested at the indicated time points after chase.
- the "*” denotes high-mannose N-glycan EndoH-sensitive Spike
- the "O” represents complex N-glycan EndoH-resistant Spike
- the "#” represents high-mannose N-glycan EndoH-sensitive deglycosylated Spike.
- Figure 4 shows the plasma membrane expression of Spike in SFV Spike infected BHK cells.
- FIG. 5 is a Western Blot analysis showing that the SARS CoV protein binds sACE2 receptor.
- M2-beads coated with Spike (lanes 1 and 4) or with BAP as a control (lanes 3 and 6) were incubated with sACE2 and run on an SDS-gel prior to Western blot with anti-ACE2 antibody or with Mab M2 as a control. While both Spike and BAP proteins are present in the reaction (lanes 4 and 6), only Spike binds to ACE2 (lanes 1 and
- CTRL preimmune sera from control
- VACC Spike vaccinated
- Lane 7 represents human SARS patient serum
- lane 8 represents a commercial serum from a rabbit immunized with Spike protein at a 1/50 fold dilution.
- Figure 8 shows that mice immunized with the recombinant immunopurified SARS CoV Spike protein produce antibodies against SARS CoV.
- Figures 9(A), 9(B), and 9(C) show the nucleic acid sequence of Spike- Pasteur (SEQ ID NO: 1). Each of the Spe I sites are underlined, and the nucleic residues replaced to form Spike-Pasteur-modif are shaded.
- Figures 10(A), 10(B), and 10(C) show the nucleic acid sequence of Spike- Pasteur-modif (SEQ ID NO: 2). The mutations eliminating the Spe I sites from Spike- Pasteur are shaded.
- Figures 11 (A), 11 (B), 11 (C), 11 (D), 11 (E), and 11 (F) show the nucleic acid sequence of Spike-HKU-PRC (SEQ ID NO: 3), as well as its complementary strand.
- the shaded nucleic acid sequence encodes Spike polypeptide.
- Figures 11(A), 11(B), 11(C), 11 (D), 11 (E), and 11 (F) also show the amino acid sequence of Spike fused to the Flag peptide (SEQ ID NO: 4). Stop codons are labeled with asterisks.
- Figures 12(A) and 12(B) show the optimized nucleic acid sequence (SEQ ID NO: 6) that encodes the SARS CoV Spike polypeptide within Spike-HKU-PRC.
- SEQ ID NO: 6 differs from SEQ ID NO: 3 in that it does not contain sequence that encodes the Flag peptide or upstream or downstream sequences.
- Figure 13 describes the sequence of the SARS CoV Spike polypeptide (SEQ ID NO: 7) encoded by Spike-HKU-PRC.
- Figure 14 describes a plasmid of the invention, labeled 040078pPCR-Script, which contains sequence encoding Spike-HKU-PRC.
- the synthetic gene 040078 was assembled from synthetic oligonucleotides. The fragment was cloned into pPCR-Script Amp (Stratagene, LaJolla, CA, USA) using Kpnl and Sacl restriction sites.
- Figure 15 describes a plasmid of the invention, labeled 040086pcDNA3.1 (+), also called 040078pcDNA3.1(+), which contains sequence encoding Spike-HKU-PRC cloned into pcDNA3.1(lnvitrogen) using the BamHI restriction site.
- Figure 16 describes the purity of Spike protein used for vaccination, and shows an SDS-PAGE gel colored with silver stain.
- FIG. 17 shows an enhanced serum antibody response in animals immunized with TriSpike+ Alum. Sera from vaccinated mice were analyzed for reactivity with TriSpike.
- A A high-titer neutralizing SARS patient serum, a rabbit serum against S1 , and M2 monoclonal antibody against the FLAG peptide were used as controls.
- FIG. 18 shows the induction of a mucosal immune response in TriSpike + Alum vaccinated mice.
- Fecal and nasal lavage samples from immunized mice (TriSpike or TriSpike + Alum) were collected and analysed for reactivity with TriSpike (A-B). M2 monoclonal antibody against the FLAG peptide was used as a control.
- A Fecal samples from vaccinated mice were collected at day 44 and used at 1/500 dilution for Western Blot analysis.
- Immune complexes were detected with HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody.
- B describes the same experiment as (A), except that Western Blot analysis was performed with pooled nasal lavage samples from vaccinated mice collected at day 65. Nasal lavage samples were used at 1/25 dilution for Western Blot analysis.
- C Fecal samples from vaccinated mice were collected and analyzed for neutralizing activity against SARS CoV infection on FRhk4 cells in vitro. Weak neutralizing activity was detected after the third immunization only. Nasal lavage samples from immunized mice were analysed but no observable level of neutralizing activity obtained in vitro.
- FIG 19 shows the immunogenicity of TriSpike in Golden Syrian hamster.
- Sera from hamsters vaccinated with indicated concentrations of TriSpike+Alum and control hamsters were analyzed for reactivity with TriSpike and neutralization.
- Sera, diluted 1/100, from hamster immunized subcutaneously with 2, 10, or 20 ⁇ g of TriSpike (on day 0, 21 and 42) were reacted with live BHK-21 cells expressing TriSpike at the plasma membrane.
- Immune complexes were identified using FITC-conjugated goat anti-hamster IgG polyclonal antibody.
- Results are expressed as MFI (mean fluorescence intensity) values.
- MFI mean fluorescence intensity
- the MFI value reached the maximum after the second immunization (post-dose 2) and remained stable after the third immunization (post-dose 3).
- B Neutralizing activity was obtained in a SARS CoV microneutralization assay (100TCID50/well final) on FRhk4 cells.
- Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to SEQ ID NOS: 2, 3 & 6 herein under conditions of moderate or severe stringency, and which encode Spike polypeptides. As used ereiri, conditions of moderate stringency, as known to those having ordinary skill in the art, and as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol.
- Spike polypeptides or “Spike proteins.” As used herein, these terms refer to a genus of polypeptides that further encompasses proteins having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7, as well as those proteins and polypeptides having a high degree of similarity (at least 90% homology) with such amino acid sequences and which proteins and polypeptides are immunoreactive.
- Spike polypeptides and “Spike proteins” refer to those proteins encoded by nucleic acid molecules which hybridize under conditions of high stringency to the nucleic acid strand complementary to the coding sequences of SEQ ID NO: 3 or SEQ ID NO: 6.
- substantially purified refers to a mixture that contains Spike polypeptides and is essentially free of association with other proteins or polypeptides, but for the presence of known proteins that can be removed using a specific antibody, and which substantially purified Spike polypeptides can be used as antigens.
- a Spike polypeptide "variant” as referred to herein means a polypeptide substantially homologous to native Spike polypeptides, but which has an amino acid sequence different from that of native Spike polypeptides because of one or more deletions, insertions, or substitutions.
- the variant amino acid sequence preferably is at least 80% identical to a native Spike polypeptide amino acid sequence, most preferably at least 90% identical.
- the percent identity can be determined, for example by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981).
- the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.
- Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn.
- Naturally occurring Spike polypeptide variants are also encompassed by the invention.
- examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the Spike polypeptides.
- Variations attributable to proteolysis include, for example, differences in the termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the Spike polypeptides.
- Variations attributable to frameshifting include, for example, differences in the termini upon expression in different types of host cells due to different amino acids.
- the invention provides isolated and purified, or homogeneous, Spike polypeptides, both recombinant and non-recombinant.
- Variants and derivatives of native Spike polypeptides that can be used as antigens can be obtained by mutations of nucleotide sequences coding for native Spike polypeptides. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
- oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion, or insertion.
- Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymoi. 154:367, 1987); and U.S.
- Spike polypeptides can be utilized to prepare antibodies that specifically bind to Spike polypeptides.
- the term "antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2 and Fab fragments, as well as any recombinantly produced binding partners.
- Antibodies are defined to be specifically binding if they bind Spike polypeptides with a K a of greater than or equal to about 10 7 M "1 . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al., Ann.
- the invention further encompasses isolated fragments and oligonucleotides derived from the nucleotide sequence of SEQ ID NOS: 2-3 & 6. The invention also encompasses polypeptides encoded by these fragments and oligonucleotides.
- a DNA sequence can vary from that shown in SEQ ID NOS: 2-3 & 6 and still encode a Spike polypeptide having the amino acid sequence of SEQ ID NO: 7.
- Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.
- the invention thus provides equivalent isolated DNA sequences, encoding Spike polypeptides, selected from: (a) nucleic acid molecules comprising SEQ ID NOS: 2-3 & 6; (b) DNA capable of hybridization to SEQ ID NOS: 3 or 6 under conditions of high stringency; (c) nucleic acid molecules comprising fragments of SEQ ID NOS: 2-3 & 6; and (d) nucleic acid molecules which are degenerate as a result of the genetic code to a DNA defined in (a), (b), or (c) and which encode Spike polypeptides and fragments of Spike polypeptides.
- Spike polypeptides encoded by such nucleic acid equivalent sequences are encompassed by the invention.
- Spike polypeptides encoded by DNA equivalent to SEQ ID NOS: 3 or 6 include, but are not limited to, Spike polypeptide fragments and Spike polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described above.
- Recombinant expression vectors containing a nucleic acid sequence encoding Spike polypeptides can be prepared using well known methods.
- the expression vectors include a Spike DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
- regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
- Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the Spike DNA sequence.
- a promoter nucleotide sequence is operably linked to a Spike DNA sequence if the promoter nucleotide sequence controls the transcription of the Spike DNA sequence.
- the ability to replicate in the desired host cells usually conferred by an origin of replication, and a selection gene by which transformants are identified can additionally be incorporated into the expression vector.
- Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
- a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
- useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids. Commercially available vectors include those that are specifically designed for the expression of proteins.
- plasmids comprising optimized Spike genes of SARS
- CoV include the following: pPCR-Script-040078 deposited at C.N.C.M. on June 8, 2004 under the number 1-3221 ; pcDNA-Spike-HKUPRC-040086 deposited at C.N.C.M. on June 8, 2004 under the number I-3222; and pcSFV-HKUPRC-040091 deposited at C.N.C.M. on June 8, 2004 under the number I-3223.
- Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
- Suitable host cells for expression of Spike polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems can also be employed to produce Spike polypeptides using RNAs derived from DNA constructs disclosed herein. [078] It will be understood that the present invention is intended to encompass the previously described proteins in isolated or purified form, whether obtained using the techniques described herein or other methods.
- the Spike polypeptides are substantially free of human tissue and human tissue components, nucleic acids, extraneous proteins and lipids, and adventitious microorganisms, such as bacteria and viruses. It will also be understood that the invention encompasses equivalent proteins having substantially the same biological and immunogenic properties. Thus, this invention is intended to cover serotypic variants of the proteins of the invention. [079] Depending on the use to be made of the Spike polypeptides of the invention, it may be desirable to label them. Examples of suitable labels are radioactive labels, enzymatic labels, fluorescent labels, chemiluminescent labels, and chromophores.
- the methods for labeling proteins and glycoproteins of the invention do not differ in essence from those widely used for labeling immunoglobulin.
- the need to label may be avoided by using labeled antibody to the antigen of the invention or anti-immunoglobulin to the antibodies to the antigen as an indirect marker.
- the Spike polypeptides of the invention can be used to produce polyclonal and monoclonal antibodies reactive therewith.
- a protein or polypeptide of the invention can be used to immunize an animal host by techniques known in the art. Such techniques usually involve inoculation, but they may involve other modes of administration. A sufficient amount of the protein or the polypeptide is administered to create an immunogenic response in the animal host.
- Any host that produces antibodies to the antigen of the invention can be used. Once the animal has been immunized and sufficient time has passed for it to begin producing antibodies to the antigen, polyclonal antibodies can be recovered.
- the general method comprises removing blood from the animal and separating the serum from the blood.
- the serum which contains antibodies to the antigen, can be used as an antiserum to the antigen.
- the antibodies can be recovered from the serum.
- Affinity purification is a preferred technique for recovering purified polyclonal antibodies to the antigen, from the serum.
- Monoclonal antibodies to the antigens of the invention can also be prepared.
- One method for producing monoclonal antibodies reactive with the antigens comprises the steps of immunizing a host with the antigen; recovering antibody producing cells from the spleen of the host; fusing the antibody producing cells with myeloma cells deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase to form hybridomas; selecting at least one of the hybridomas by growth in a medium comprising hypoxanthine, aminopterin, and thymidine; identifying at least one of the hybridomas that produces an antibody to the antigen, culturing the identified hybridoma to produce antibody in a recoverable quantity; and recovering the antibodies produced by the cultured hybridoma.
- polyclonal or monoclonal antibodies can be used in a variety of applications. Among these is the neutralization of corresponding proteins. They can also be used to detect viral antigens in biological preparations or in purifying corresponding proteins, glycoproteins, or mixtures thereof, for example when used in an affinity chromatographic columns.
- the Spike polypeptides can be used as antigens to identify antibodies to SARS CoV in materials and to determine the concentration of the antibodies in those materials. Thus, the antigens can be used for qualitative or quantitative determination of the virus in a material. Such materials include human tissue and human cells, as well as biological fluids, such as human body fluids, including human sera.
- the antigens of the present invention provide an assay that is convenient, rapid, sensitive, and specific.
- the antigens of the invention can be employed for the detection of SARS CoV by means of immunoassays that are well known for use in detecting or quantifying humoral components in fluids.
- immunoassays that are well known for use in detecting or quantifying humoral components in fluids.
- antigen-antibody interactions can be directly observed or determined by secondary reactions, such as precipitation or agglutination.
- immunoelectrophoresis techniques can also be employed.
- the classic combination of electrophoresis in agar followed by reaction with anti- serum can be utilized, as well as two-dimensional electrophoresis, rocket electrophoresis, and immunolabeling of polyacrylamide gel patterns (Western Blot or immunoblot).
- Other immunoassays in which the antigens of the present invention can be employed include, but are not limited to, radioimmunoassay, competitive immunoprecipitation assay, enzyme immunoassay, and immunofluorescence assay. It will be understood that turbidimetric, colorimetric, and nephelometric techniques can be employed. An immunoassay based on Western Blot technique is preferred.
- Immunoassays can be carried out by immobilizing one of the immunoreagents, either an antigen of the invention or an antibody of the invention to the antigen, on a carrier surface while retaining immunoreactivity of the reagent.
- the reciprocal immunoreagent can be unlabeled or labeled in such a manner that immunoreactivity is also retained.
- enzyme immunoassays such as enzyme linked immunosorbent assay (ELISA) and competitive inhibition enzyme immunoassay (CIEIA).
- ELISA enzyme linked immunosorbent assay
- CIEIA competitive inhibition enzyme immunoassay
- the support is usually a glass or plastic material. Plastic materials molded in the form of plates, tubes, beads, or disks are preferred.
- Suitable plastic materials are polystyrene and polyvinyl chloride. If the immunoreagent does not readily bind to the solid support, a carrier material can be interposed between the reagent and the support. Examples of suitable carrier materials are proteins, such as bovine serum albumin, or chemical reagents, such as gluteraldehyde or urea. Coating of the solid phase can be carried out using conventional techniques.
- the invention provides immunogenic Spike polypeptides, and more particularly, protective polypeptides for use in the preparation of vaccine compositions against SARS CoV. These polypeptides can thus be employed as viral vaccines by administering the polypeptides to a mammal susceptible to SARS CoV infection. Conventional modes of administration can be employed.
- administration can be carried out by oral, respiratory, or parenteral routes.
- Intradermal, subcutaneous, and intramuscular routes of administration are preferred when the vaccine is administered parenteral ly.
- Various methods for achieving adjuvant effect for the vaccine include the use of agents, such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25% solution.
- agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25% solution.
- Another suitable adjuvant compound comprises DDA (dimethyldioctadecyl-ammonium bromide), as well as immune modulating substances, such as lymphokines (e.g., IFN-gamma, IL-1, IL-2, and IL-12) or IFN-gamma inducer compounds, such as poly l:C.
- DDA dimethyldioctadecyl-ammonium bromide
- immune modulating substances such as lymphokines (e.g., IFN-gamma, IL-1, IL-2, and IL-12) or IFN-gamma inducer compounds, such as poly l:C.
- lymphokines e.g., IFN-gamma, IL-1, IL-2, and IL-12
- IFN-gamma inducer compounds such as poly l:C.
- the vaccine composition according to the present invention is advantageously prepared as an injectable form (either as liquid solution or suspension). However, solid forms suitable for solution in or
- the vaccine compositions of the invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
- the quantity to be administered depends on the subject to be treated including, e.g., the capacity of the individual's immune system to induce an immune response.
- the dosage of the vaccine will depend on the route of administration and will vary according to the age of the patient to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.
- the major purpose of the immune response in a SARS CoV infected mammal is to inactivate the free SARS CoV and to eliminate SARS CoV infected cells that have the potential to release infectious virus.
- the B-cell arm of the immune response has the major responsibility for inactivating free SARS CoV virus.
- the principal manner in which this is achieved is by neutralization of infectivity.
- Another major mechanism for destruction of the SARS CoV infected cells is provided by cytotoxic T lymphocytes (CTL) that recognize viral Spike antigens expressed in combination with class I histocompatibility antigens at the cell surface.
- CTLs recognize Spike polypeptides processed within cells from a Spike protein that is produced, for example, by the infected cell or that is internalized by a phagocytic cell.
- this invention can be employed to stimulate a B-cell response to Spike polypeptides, as well as immunity mediated by a CTL response following viral infection.
- the CTL response can play an important role in mediating recovery from primary SARS CoV infection and in accelerating recovery during subsequent infections.
- the ability of the Spike polypeptides and vaccines of the invention to induce protective levels of neutralizing antibody in a host can be enhanced by emulsification with an adjuvant, incorporating in a liposome, coupling to a suitable carrier, or by combinations of these techniques.
- the Spike polypeptides of the invention can be administered with a conventional adjuvant, such as aluminum phosphate and aluminum hydroxide gel, in an amount sufficient to potentiate humoral or cell-mediated immune response in the host.
- the Spike polypeptides can be bound to lipid membranes or incorporated in lipid membranes to form liposomes.
- the use of nonpyrogenic lipids free of nucleic acids and other extraneous matter can be employed for this purpose.
- the immunization schedule will depend upon several factors, such as the susceptibility of the host to infection and the age of the host.
- a single dose of the vaccine of the invention can be administered to the host or a primary course of immunization can be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as needed following the primary course.
- the Spike proteins, polypeptides, and vaccines of the invention can be administered to the host in an amount sufficient to prevent or inhibit SARS CoV infection or replication in vivo.
- the amount administered should be at least sufficient to protect the host against substantial immunosuppression, even though SARS CoV infection may not be entirely prevented.
- An immunogenic response can be obtained by administering the Spike proteins or glycoproteins of the invention to the host in an amount of about 10 to about 500 micrograms antigen per kilogram of body weight, preferably about 50 to about 100 micrograms antigen per kilogram of body weight.
- the proteins and vaccines of the invention can be administered together with a physiologically acceptable carrier.
- a diluent such as water or a saline solution, can be employed.
- Another aspect of the invention provides a method of RNA and/or DNA vaccination.
- the method also includes administering any combination of the nucleic acids encoding Spike polypeptides, the proteins and polypeptides perse, with or without carrier molecules, to an individual.
- the individual is an animal, and is preferably a mammal. More preferably, the mammal is selected from the group consisting of a human, a mouse, a rat, a rabbit, a sheep, a dog, a cat, a bovine, a pig, and a horse. In an especially preferred embodiment, the mammal is a human.
- the methods of treating include administering immunogenic compositions comprising Spike polypeptides, but compositions comprising nucleic acids encoding Spike polypeptides as well.
- nucleic acid based technology allows the administration of nucleic acids encoding Spike polypeptides, naked or encapsulated, directly to tissues and cells without the need for production of encoded proteins prior to administration.
- the technology is based on the ability of these nucleic acids to be taken up by cells of the recipient organism and expressed to produce an immunogenic determinant to which the recipient's immune system responds.
- the expressed antigens are displayed on the surface of cells that have taken up and expressed the nucleic acids, but expression and export of the encoded antigens into the circulatory system of the recipient individual is also within the scope of the present invention.
- nucleic acid vaccine technology includes, but is not limited to, delivery of naked DNA and RNA and delivery of expression vectors encoding Spike polypeptides. Although the technology is termed "vaccine", it is equally applicable to immunogenic compositions that do not result in a protective response. Such non-protection inducing compositions and methods are encompassed within the present invention.
- nucleic acids encoding Spike polypeptides and carrier molecules as naked nucleic acid
- the present invention also encompasses delivery of nucleic acids as part of larger or more complex compositions. Included among these delivery systems are viruses, virus-like particles, or bacteria containing the nucleic acid encoding Spike polypeptides. Also, complexes of the invention's nucleic acids and carrier molecules with cell permeabilizing compounds, such as liposomes, are included within the scope of the invention.
- Protein based SARS vaccine can induce a neutralizing and protective antibody-dependent immune response after a single or double injection of Spike protein.
- Protein based vaccines present considerable safety advantages over vector-expressed (i.e., plasmid, MVA, Adeno) or whole inactivated virus vaccine.
- plasmid plasmid, MVA, Adeno
- whole inactivated virus vaccine i.e., whole inactivated virus vaccine.
- a kit capable of diagnosing a SARS CoV infection contains the antibodies of this invention, which are capable of binding to SARS CoV Spike polypeptide.
- This kit in another embodiment, contains the polypeptides of this invention, which are capable of detecting the presence or absence of antibodies, which bind to the Spike polypeptide.
- This kit in yet another embodiment, contains the nucleic acid molecules of this invention, which are capable of hybridizing to viral RNA or analogous DNA sequences to indicate the presence of a SARS CoV infection.
- Different diagnostic techniques can be used which include, but are not limited to: (I) Southern blot procedures to identify cellular DNA which may or may not be digested with restriction enzymes; (2) Northern blot techniques to identify RNA extracted from cells; and (3) dot blot techniques, i.e., direct filtration of the sample through an ad hoc membrane, such as nitrocellulose or nylon, without previous separation on agarose gel; (4) immunoassay based on Western blot technique; (5) ELISA (enzyme linked immunosorbent assay); (6) FACS; (7) indirect immunofluorescent assay; or (8) immunoprecipitation assay.
- Suitable material for dot blot technique could be obtained from body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by centrifugation.
- body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by centrifugation.
- Spike-Pasteur was subsequently expressed in the SFV viral expression vector, which effectively allowed for expression in transfected BHK cells.
- the yield of SFV viral particles was low, because of the presence of 2 Spe I sites in the Spike gene. Spe I is usually used to linearize the plasmid at the end of the SFV coding sequence. Because Spe I could not be used, PSFV-Spike-Pasteur was linearized with Sph I, resulting in additional 3' RNA sequences of > 2000 bases of vector RNA.
- RNA transfection from Sph I linearized pSFV-Spike-Pasteur usually yields SFV titer of 2X10 7 IP/ml.
- RNA transfection from Spe I linearized pSFV-Spike-Pasteur-modif usually yields SFV titer of 1- 2X10 9 IP/ml.
- EXAMPLE 2 The Spike-Pasteur sequence was further subjected to a bioinformatics analysis.
- the cDNA for Spike-Pasteur contains numerous cis-acting sites which may negatively influence expression.
- 32 of the identified 33 negative cis-acting signals were eliminated from Spike-Pasteur, and additional signals to stimulate gene expression were added, producing Spike-HKU-PRC (SEQ ID NO: 3).
- Spike-HKU-PRC was cloned into pSC, pcDNA, and pSFV vectors.
- Additional expression enhancing sequences added to Spike-HKU-PRC included a Kozak consensus sequence introduced upstream of the starting ATG to increase translation initiation, and two stop codons added to ensure efficient termination.
- the GC-content of Spike-HKU-PRC was increased from 38% to 49%, while avoiding regions of very high (>80%) or very low ( ⁇ 30%) GC content.
- codon usage was adapted to the bias of Cricetulus griseus to increase translation efficiency. Table 2 shows the codon usage of Cricetulus griseus, with the frequency of each codon given as number per thousand codons.
- Spike protein expression of pcDNA-Spike-Pasteur was compared to pcDNA- Spike-HKU-PRC by transfection of plasmids into 293T cells using the calcium-phosphate method. No Spike protein was observed in pcDNA-Spike-Pasteur transfected cells. In contrast, high levels of Spike protein was detected in pcDNA-Spike-HKU-PRC transfected 293T cells.
- Figure 1. The migration and oligomerization pattern of the Spike protein is consistent with previously obtained results, revealing that this plasmid allows for expression of a full length, natively conformed SARS CoV protein.
- Spike protein was tagged with a C-terminal Flag peptide, as shown in Figure 2.
- the Spike protein was expressed as a full-length protein, including the C-terminal and transmembrane domains as well as a C-terminal Flag tag, in the SFV vector system previously described (See Staropoli et al., Lozach et al., and Chanel et al., all of which are herein incorporated by reference.)
- Spike protein was produced alternatively from cells transfected with SFV- Spike-RNA or cells infected with SFV particles coding for SFV-Spike-RNA.
- RNA was transcribed in vitro according to a standard published procedure.
- the Spike protein was produced in BHK cells and purified under native conditions by immunoaffinity using the anti-FLAG M2 antibody. M2-bound Spike protein » was eluted under native conditions with Flag peptide. Peptide and residual detergent were eliminated by dialysis.
- Mice were immunized intramuscularly with SFV Spike RNA, followed by intraperitoneal (IP) injection of Spike protein at day 14 and at day 35.
- IP intraperitoneal
- EXAMPLE 6 [0117] The following reagents and methods can be used in practicing this invention. [0118] Production of SARS CoV Spike subunit vaccine [0119] 1/ Preparation of SFV expression vector RNA [0120] Note: Spike protein can be produced, for example, from cells transfected with SFV-Spike-RNA or cells infected with SFV particles coding for SFV-Spike-RNA. Here an electroporaton procedure is detailed. [0121] Prepare 1.2 X 10 7 cell/ml suspension for electroporation under STERILE conditions 1. Preparation of medium without Serum, mix the following ingredients and filter/sterilize it : i. Hepes 5% 10 mL ii.
- Electroporation should be done for both sample and untransfected control cells. 1. Prepare two 75 ml flasks with one containing 20 ml GMEM. Label properly. 2. Using sterile P1000 filter tips, transfer 800 ⁇ l of cell suspension (in PBS with no Ca +2 or Mg +2 ) into tube containing RNA, mix twice with pipette up & down. 3.
- Procedure is according to the FLAGIPT-1 instruction manual. 2. elution with 3X FLAG peptide. 3. Prepare working 3X FLAG peptide by adding 3ul of 5ug/ul 3X FLAG peptide with 100ul of 1X wash buffer. 4. Add 100ul working 3X FLAG elution solution to the resin. 5. Incubate the mixture for 1 h at 4°C with gentle rotation. 6. Centrifuge the resin for 10 seconds at 13000 rpm. 7. Keep the supernatant and repeat steps 4 - 6 three more times. Concentration and purification of protein by Amicon filter unit
- EXAMPLE 7 The candidate vaccine preparation, trimeric S-protein (TriSpike, the same protein described as Spike-HKU-PRC), was demonstrated to be >90% pure.
- TriSpike the same protein described as Spike-HKU-PRC
- a sample of TriSpike purified for vaccination studies of mice and hamsters was denatured in SDS/DTT buffer (50mM DDT) to dissociate the trimeric protein completely into monomers. After separation by 4-12% SDS-PAGE, the gel was subjected to Silver stain (Current Protocols in Immunology Chapter 8, 9.1 - 9.10)) to reveal all of the proteins contained in the sample.
- Figure 16 shows that only monomeric S-protein can be detected in its complex glycosylated and high-mannose forms. The degree of purity is > 90%.
- EXAMPLE 8 An enhanced serum IgG response was obtained in animals immunized with TriSpike in Alum adjuvant. Previous studies on the mucosal and systemic response to recombinant HagB from Porphyromonas gingivalis indicated that a higher serum IgG and mucosal IgA response from HagB + alum was induced compared to HagB without adjuvant immunization in Balb/c mice (Vaccine, 2003, 21 , 4459-4471). TriSpike candidate vaccine was analyzed to determine whether it could induce not only serum IgG, but also mucosal IgA with neutralizing ability for SARS CoV.
- TriSpike preparation in PBS was compared with TriSpike preparation in Alum adjuvant for their capacity to induce SARS CoV specific serum IgG.
- Two groups of mice were immunized by the intraperitoneal route: group A represents mice which received 3 doses of 20 ⁇ g of TriSpike protein alone and group B represents mice which received 3 doses of 20 ⁇ g of TriSpike pre-mixed with 1mg of Alum adjuvant.
- Western blot analysis indicated a stronger antibody response from mice immunized with TriSpike + alum as compared with mice immunized with TriSpike alone (Figure 17).
- the TriSpike + Alum group also showed a higher neutralization titer ( Figure 17).
- TriSpike + Alum adjuvant induced a strong neutralizing and long lasting serum IgG response.
- TriSpike in Alum adjuvant induced an enhanced mucosal IgG and IgA response.
- SARS CoV can be detected in the upper and lower respiratory tract of humans and infected laboratory animals. In addition to the respiratory tract, SARS CoV can be detected in intestinal tissue of fatal cases (AJG, 2005, 100, 169-176).
- fecal and nasal lavage samples from mice immunized with TriSpike +/- Alum adjuvant by the intraperitoneal route. Fecal samples were prepared as described previously (PNAS, 2004, 101 , 13584-13589).
- fecal pellets (-100 mg) were collected on indicated days. Fecal extracts were prepared by adding 0.5 ml of PBS containing 0.02% Na-azide for 30 min at 40°C with gentle rotation and cleared by centrifugation (13,000 rpm). Generally, 0.2 ml of clear supernatant could be obtained from one tube of fecal pellet suspension.
- Western blot analysis (Figure 18) showed the presence of mucosal IgG and IgA response from fecal sample only in mice immunized with TriSpike + alum but not in mice immunized with TriSpike alone.
- mice were performed with twice the volume of ketamine/xylazine solution injected intraperitonically into na ⁇ ve or immunized mice.
- the thoracic cavity was opened and 25G needle was inserted with 0.5 ml PBS/aprotinin injected into the tracheal lumen cephalic to the obstruction.
- About 0.5 ml of nasal wash sample could be collected from each mouse.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US86064104A | 2004-06-04 | 2004-06-04 | |
US57834804P | 2004-06-10 | 2004-06-10 | |
PCT/EP2005/006512 WO2005118813A2 (en) | 2004-06-04 | 2005-06-03 | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1751178A2 true EP1751178A2 (en) | 2007-02-14 |
Family
ID=35463454
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP05750800A Withdrawn EP1751178A2 (en) | 2004-06-04 | 2005-06-03 | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1751178A2 (en) |
CA (1) | CA2568838A1 (en) |
WO (1) | WO2005118813A2 (en) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070003577A1 (en) * | 2005-06-28 | 2007-01-04 | Kam Yiu W | Purified trimeric S protein as vaccine against severe acute respiratory syndrome virus infections |
GB0711858D0 (en) * | 2007-06-19 | 2007-07-25 | Glaxosmithkline Biolog Sa | Vaccine |
EP3058370A4 (en) * | 2013-10-14 | 2017-08-30 | The University of North Carolina at Chapel Hill | Methods and compositions for coronavirus diagnostics and therapeutics |
MD3718565T2 (en) | 2015-10-22 | 2022-09-30 | Modernatx Inc | Respiratory virus vaccines |
BR102021003012A2 (en) | 2020-02-19 | 2021-11-30 | Univ Berlin Charite | Methods for diagnosing sars-cov-2 infection, kit and uses |
EP3715847A1 (en) * | 2020-02-20 | 2020-09-30 | Euroimmun Medizinische Labordiagnostika AG | A method and reagents for the diagnosis of sars-cov-2 |
WO2021184391A1 (en) * | 2020-03-20 | 2021-09-23 | 广州市康润生物科技有限公司 | Method for earlier screening of novel coronavirus |
WO2021186029A1 (en) * | 2020-03-20 | 2021-09-23 | Deutsches Krebsforschungszentrum | Sars spike peptides and uses thereof |
JP2023519837A (en) * | 2020-03-30 | 2023-05-15 | ヴァリエーション バイオテクノロジーズ インコーポレイテッド | Vaccine composition for treating coronavirus |
WO2021223647A1 (en) * | 2020-05-06 | 2021-11-11 | The University Of Hong Kong | Compositions and methods for making and using vaccines against covid-19 |
DE20175031T1 (en) | 2020-05-15 | 2021-09-09 | Euroimmun Medizinische Labordiagnostika Ag | Method for determining the effectiveness of a SARS-CoV-2 vaccine |
WO2021244601A1 (en) * | 2020-06-04 | 2021-12-09 | 山东博安生物技术股份有限公司 | Neutralizing antibody of sars-cov-2 virus and application thereof |
WO2021249010A1 (en) * | 2020-06-10 | 2021-12-16 | Sichuan Clover Biopharmaceuticals, Inc. | Coronavirus diagnostic compositions, methods, and uses thereof |
WO2021254476A1 (en) * | 2020-06-19 | 2021-12-23 | 南京金斯瑞生物科技有限公司 | Magnetic microparticle chemiluminescence reagent kit for detecting sars-cov-2 virus neutralising antibodies and application therefor |
DE102020125915B3 (en) | 2020-10-02 | 2022-03-03 | Institut für Molekulare Diagnostik und Bioanalytik | IMMUNDIAGNOSTIC MEANS AND METHODS FOR DETECTION AND DIFFERENTIATION OF CORONAVIRUS INFECTIONS |
AR125373A1 (en) * | 2021-04-19 | 2023-07-12 | Bharat Serums & Vaccines Ltd | POLYCLONAL ANTIBODIES AGAINST SARS-CoV-2 AND ITS APPLICATIONS |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004110483A1 (en) * | 2003-05-31 | 2004-12-23 | Yiyou Chen | Method and composition of a novel vaccine design for the prevention and treatment of sars |
WO2005012360A2 (en) * | 2003-07-22 | 2005-02-10 | Crucell Holland B.V. | Binding molecules against sars-coronavirus and uses thereof |
-
2005
- 2005-06-03 CA CA002568838A patent/CA2568838A1/en not_active Abandoned
- 2005-06-03 EP EP05750800A patent/EP1751178A2/en not_active Withdrawn
- 2005-06-03 WO PCT/EP2005/006512 patent/WO2005118813A2/en active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2005118813A3 * |
Also Published As
Publication number | Publication date |
---|---|
WO2005118813A2 (en) | 2005-12-15 |
CA2568838A1 (en) | 2005-12-15 |
WO2005118813A3 (en) | 2006-03-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1751178A2 (en) | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein | |
US11759516B2 (en) | Nucleic acid vaccine against the SARS-CoV-2 coronavirus | |
US6210675B1 (en) | PT-NANB hepatitis polypeptides | |
Saito et al. | Plasmid DNA-based immunization for hepatitis C virus structural proteins: immune responses in mice | |
JPH06501845A (en) | Expression of human CMV glycoprotein-H using a baculovirus-insect cell expression system | |
CN112094327A (en) | Truncation body based on novel coronavirus RBD-SD1 protein and application thereof | |
JPH08504096A (en) | Recombinant proteins of hepatitis E Pakistan strains and their use in diagnostics and vaccines | |
US20070190065A1 (en) | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with SARS corona virus spike protein | |
US6060457A (en) | DNA plasmid vaccine for immunization of animals against BVDV | |
US20070270361A1 (en) | Sars Nucleic Acids, Proteins, Vaccines, and Uses Thereof | |
JP3889808B2 (en) | Recombinant protein of Pakistani strain of hepatitis E and its diagnostic method and use in seed pods | |
US6673601B1 (en) | Chimeric lyssavirus nucleic acids and polypeptides | |
US20090087832A1 (en) | Nucleic acids and new polypeptides associated with and/or overlapping with hepatitis C virus core gene products | |
CN116240222A (en) | Codon-optimized bovine viral diarrhea virus type 1E2 protein gene and application thereof | |
CN114315984A (en) | N protein epitope mutation marker for preparing PRRSV gene II type epitope deletion vaccine strain and application thereof | |
JP4390293B2 (en) | Pakistani strain recombinant proteins of hepatitis E and their use in diagnostic methods and vaccines | |
US6254869B1 (en) | Cryptopain vaccines, antibodies, proteins, peptides, DNA and RNA for prophylaxis, treatment and diagnosis and for detection of cryptosporidium species | |
KR20230008707A (en) | Vaccine composition for treatment of coronavirus | |
US20040234542A1 (en) | Recombinant orf2 proteins of the swine hepatitis e virus and their use as a vaccine and as a diagnostic reagent for medical and veterinary applications | |
US20050069865A1 (en) | Synthetic hcv envelope proteins and their use for vaccination | |
CN111732667B (en) | Peste des petits ruminants virus genetic engineering subunit vaccine | |
MXPA01010481A (en) | Chimeric lyssavirus nucleic acids and polypeptides. | |
US7166287B1 (en) | Viral agent | |
CN115894713A (en) | Heterotrimeric fusion proteins, compositions and uses thereof | |
ES2201289T3 (en) | EXPRESSION OF ANTIGENS OF HGV AND ITS USE. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20061218 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR |
|
17Q | First examination report despatched |
Effective date: 20070614 |
|
DAX | Request for extension of the european patent (deleted) | ||
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KIEN, FRANCOIS Inventor name: MANUGUERRA, JEAN-CLAUDE Inventor name: ALTMEYER, RALF Inventor name: KAM, YIU, WING Inventor name: STAROPOLI, ISABELLE Inventor name: SIU, YU, LAM Inventor name: TSE, KONG, SAN Inventor name: CHAN, CHEMAN Inventor name: NAL-ROGIER, BEATRICE |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1102820 Country of ref document: HK |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: SIU, YU, LAM Inventor name: ALTMEYER, RALF Inventor name: CHAN, CHEMAN Inventor name: KAM, YIU, WING Inventor name: TSE, KONG, SAN Inventor name: MANUGUERRA, JEAN-CLAUDE Inventor name: STAROPOLI, ISABELLE Inventor name: NAL-ROGIER, BEATRICE Inventor name: KIEN, FRANCOIS |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: MANUGUERRA, JEAN-CLAUDE Inventor name: STAROPOLI, ISABELLE Inventor name: ALTMEYER, RALF Inventor name: KIEN, FRANCOIS Inventor name: NAL-ROGIER, BEATRICE Inventor name: CHAN, CHEMAN Inventor name: TSE, KONG, SAN Inventor name: KAM, YIU, WING Inventor name: SIU, YU, LAM |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20081210 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1102820 Country of ref document: HK |