WO2011035422A1 - Method of preparing plant-derived vlps - Google Patents
Method of preparing plant-derived vlps Download PDFInfo
- Publication number
- WO2011035422A1 WO2011035422A1 PCT/CA2010/001488 CA2010001488W WO2011035422A1 WO 2011035422 A1 WO2011035422 A1 WO 2011035422A1 CA 2010001488 W CA2010001488 W CA 2010001488W WO 2011035422 A1 WO2011035422 A1 WO 2011035422A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- plant
- vlps
- fraction
- protein
- derived
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 144
- 210000001938 protoplast Anatomy 0.000 claims abstract description 67
- 239000000203 mixture Substances 0.000 claims abstract description 60
- 102000004190 Enzymes Human genes 0.000 claims abstract description 59
- 108090000790 Enzymes Proteins 0.000 claims abstract description 59
- 210000002421 cell wall Anatomy 0.000 claims abstract description 51
- 230000000593 degrading effect Effects 0.000 claims abstract description 16
- 101710154606 Hemagglutinin Proteins 0.000 claims description 112
- 101710093908 Outer capsid protein VP4 Proteins 0.000 claims description 112
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 claims description 112
- 101710176177 Protein A56 Proteins 0.000 claims description 112
- 239000000185 hemagglutinin Substances 0.000 claims description 111
- 108090000623 proteins and genes Proteins 0.000 claims description 97
- 102000004169 proteins and genes Human genes 0.000 claims description 95
- 229940088598 enzyme Drugs 0.000 claims description 58
- 108010059820 Polygalacturonase Proteins 0.000 claims description 40
- 206010022000 influenza Diseases 0.000 claims description 39
- 210000004027 cell Anatomy 0.000 claims description 36
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 34
- 108010059892 Cellulase Proteins 0.000 claims description 29
- 239000000284 extract Substances 0.000 claims description 28
- 229940106157 cellulase Drugs 0.000 claims description 27
- 150000007523 nucleic acids Chemical group 0.000 claims description 26
- 108090000565 Capsid Proteins Proteins 0.000 claims description 24
- 238000005119 centrifugation Methods 0.000 claims description 23
- 230000003612 virological effect Effects 0.000 claims description 20
- 108020004707 nucleic acids Proteins 0.000 claims description 19
- 102000039446 nucleic acids Human genes 0.000 claims description 19
- 238000001914 filtration Methods 0.000 claims description 16
- 108010006232 Neuraminidase Proteins 0.000 claims description 15
- 102000005348 Neuraminidase Human genes 0.000 claims description 15
- 102100023321 Ceruloplasmin Human genes 0.000 claims description 13
- 108010087302 Viral Structural Proteins Proteins 0.000 claims description 12
- 238000011118 depth filtration Methods 0.000 claims description 12
- 108010003533 Viral Envelope Proteins Proteins 0.000 claims description 11
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 claims description 10
- 108091005804 Peptidases Proteins 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 10
- 210000004779 membrane envelope Anatomy 0.000 claims description 10
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 9
- 102000004882 Lipase Human genes 0.000 claims description 9
- 108090001060 Lipase Proteins 0.000 claims description 9
- 239000004367 Lipase Substances 0.000 claims description 9
- 239000003729 cation exchange resin Substances 0.000 claims description 9
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- 235000019421 lipase Nutrition 0.000 claims description 9
- 230000001052 transient effect Effects 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 230000001413 cellular effect Effects 0.000 claims description 7
- 101710085938 Matrix protein Proteins 0.000 claims description 3
- 101710127721 Membrane protein Proteins 0.000 claims description 3
- 239000002198 insoluble material Substances 0.000 claims description 3
- 241000700605 Viruses Species 0.000 abstract description 23
- 239000002245 particle Substances 0.000 abstract description 22
- 241000196324 Embryophyta Species 0.000 description 216
- 235000018102 proteins Nutrition 0.000 description 86
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 62
- 230000029087 digestion Effects 0.000 description 60
- 239000000872 buffer Substances 0.000 description 46
- 239000000306 component Substances 0.000 description 35
- 108090000765 processed proteins & peptides Proteins 0.000 description 33
- 239000011780 sodium chloride Substances 0.000 description 31
- 230000000694 effects Effects 0.000 description 30
- 238000000605 extraction Methods 0.000 description 30
- 229920001184 polypeptide Polymers 0.000 description 30
- 102000004196 processed proteins & peptides Human genes 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 30
- 230000014509 gene expression Effects 0.000 description 29
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 25
- 229930195725 Mannitol Natural products 0.000 description 25
- 239000000594 mannitol Substances 0.000 description 25
- 235000010355 mannitol Nutrition 0.000 description 25
- 150000002632 lipids Chemical class 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- 229960005486 vaccine Drugs 0.000 description 24
- 238000004519 manufacturing process Methods 0.000 description 22
- 230000006862 enzymatic digestion Effects 0.000 description 21
- 238000001764 infiltration Methods 0.000 description 21
- 230000035931 haemagglutination Effects 0.000 description 20
- 238000002360 preparation method Methods 0.000 description 17
- 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 16
- 241000207746 Nicotiana benthamiana Species 0.000 description 16
- 239000000427 antigen Substances 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 230000003834 intracellular effect Effects 0.000 description 15
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 14
- 238000001542 size-exclusion chromatography Methods 0.000 description 14
- 101150073246 AGL1 gene Proteins 0.000 description 13
- 241000631130 Chrysophyllum argenteum Species 0.000 description 11
- 230000008595 infiltration Effects 0.000 description 11
- 210000000170 cell membrane Anatomy 0.000 description 10
- 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 10
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 9
- 238000005352 clarification Methods 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 108010002430 hemicellulase Proteins 0.000 description 9
- 230000028993 immune response Effects 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 241000223261 Trichoderma viride Species 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 108020001507 fusion proteins Proteins 0.000 description 8
- 102000037865 fusion proteins Human genes 0.000 description 8
- 238000003306 harvesting Methods 0.000 description 8
- 229940059442 hemicellulase Drugs 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 7
- 108090000051 Plastocyanin Proteins 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 238000000265 homogenisation Methods 0.000 description 7
- 229960003971 influenza vaccine Drugs 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 7
- 229940001584 sodium metabisulfite Drugs 0.000 description 7
- 235000010262 sodium metabisulphite Nutrition 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 108010084185 Cellulases Proteins 0.000 description 6
- 102000005575 Cellulases Human genes 0.000 description 6
- 241000208125 Nicotiana Species 0.000 description 6
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 6
- 108010064851 Plant Proteins Proteins 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 210000001723 extracellular space Anatomy 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 230000003067 hemagglutinative effect Effects 0.000 description 6
- 239000000419 plant extract Substances 0.000 description 6
- 235000021118 plant-derived protein Nutrition 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000013638 trimer Substances 0.000 description 6
- -1 Ca2+ Chemical class 0.000 description 5
- 241000219823 Medicago Species 0.000 description 5
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 5
- 241001494479 Pecora Species 0.000 description 5
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 5
- 241000710145 Tomato bushy stunt virus Species 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 5
- 230000004186 co-expression Effects 0.000 description 5
- 239000000356 contaminant Substances 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 230000036961 partial effect Effects 0.000 description 5
- 229940068065 phytosterols Drugs 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 241000712461 unidentified influenza virus Species 0.000 description 5
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 4
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 4
- 241000272517 Anseriformes Species 0.000 description 4
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 4
- 238000009010 Bradford assay Methods 0.000 description 4
- 241000723655 Cowpea mosaic virus Species 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 4
- 239000000232 Lipid Bilayer Substances 0.000 description 4
- 108010006519 Molecular Chaperones Proteins 0.000 description 4
- 102000005431 Molecular Chaperones Human genes 0.000 description 4
- 108010067390 Viral Proteins Proteins 0.000 description 4
- 238000005341 cation exchange Methods 0.000 description 4
- 125000002091 cationic group Chemical group 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 238000011067 equilibration Methods 0.000 description 4
- 239000006167 equilibration buffer Substances 0.000 description 4
- 230000030279 gene silencing Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000012160 loading buffer Substances 0.000 description 4
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 4
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 4
- 238000004161 plant tissue culture Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 235000020138 yakult Nutrition 0.000 description 4
- 108020005345 3' Untranslated Regions Proteins 0.000 description 3
- 241000228245 Aspergillus niger Species 0.000 description 3
- 241000271566 Aves Species 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000905450 Grapevine leafroll-associated virus 2 Species 0.000 description 3
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 3
- 101800000653 Helper component proteinase Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 108010085220 Multiprotein Complexes Proteins 0.000 description 3
- 102000007474 Multiprotein Complexes Human genes 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 3
- 101150084101 RNA2 gene Proteins 0.000 description 3
- 101100353432 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP2 gene Proteins 0.000 description 3
- 229930182558 Sterol Natural products 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000011436 enzymatic extraction method Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 230000008105 immune reaction Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000010297 mechanical methods and process Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108020003519 protein disulfide isomerase Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000001932 seasonal effect Effects 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 125000005629 sialic acid group Chemical group 0.000 description 3
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 3
- 229950005143 sitosterol Drugs 0.000 description 3
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 3
- 150000003432 sterols Chemical class 0.000 description 3
- 235000003702 sterols Nutrition 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 210000002845 virion Anatomy 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 229920001221 xylan Polymers 0.000 description 3
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 2
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 2
- SGNBVLSWZMBQTH-QGOUJLTDSA-N (3s,8s,9s,10r,13r,14s,17r)-17-[(2r)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCC(C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-QGOUJLTDSA-N 0.000 description 2
- CPQUIAPJXYFMHN-UHFFFAOYSA-N 24-methylcholesterol Natural products C1CC2=CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(C)C(C)C)C1(C)CC2 CPQUIAPJXYFMHN-UHFFFAOYSA-N 0.000 description 2
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 241000700198 Cavia Species 0.000 description 2
- 108010022172 Chitinases Proteins 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 2
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101710156496 Endoglucanase A Proteins 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 2
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 2
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 2
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 2
- 101001016865 Homo sapiens Heat shock protein HSP 90-alpha Proteins 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 229940127470 Lipase Inhibitors Drugs 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 101710175974 Pathogenesis-related protein 2 Proteins 0.000 description 2
- 241000286209 Phasianidae Species 0.000 description 2
- 241000725643 Respiratory syncytial virus Species 0.000 description 2
- 241000952054 Rhizopus sp. Species 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000034303 cell budding Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 150000002339 glycosphingolipids Chemical class 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000001814 pectin Substances 0.000 description 2
- 235000010987 pectin Nutrition 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 108010004568 plant pathogenesis-related proteins Proteins 0.000 description 2
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 2
- 230000003334 potential effect Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 230000012846 protein folding Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000015500 sitosterol Nutrition 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 2
- 235000016831 stigmasterol Nutrition 0.000 description 2
- 229940032091 stigmasterol Drugs 0.000 description 2
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OSELKOCHBMDKEJ-UHFFFAOYSA-N (10R)-3c-Hydroxy-10r.13c-dimethyl-17c-((R)-1-methyl-4-isopropyl-hexen-(4c)-yl)-(8cH.9tH.14tH)-Delta5-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(=CC)C(C)C)C1(C)CC2 OSELKOCHBMDKEJ-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- MCWVPSBQQXUCTB-UHFFFAOYSA-N (24Z)-5alpha-Stigmasta-7,24(28)-dien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(=CC)C(C)C)CCC33)C)C3=CCC21 MCWVPSBQQXUCTB-UHFFFAOYSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- 101710154868 60 kDa heat shock protein, mitochondrial Proteins 0.000 description 1
- WRDABNWSWOHGMS-UHFFFAOYSA-N AEBSF hydrochloride Chemical compound Cl.NCCC1=CC=C(S(F)(=O)=O)C=C1 WRDABNWSWOHGMS-UHFFFAOYSA-N 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228257 Aspergillus sp. Species 0.000 description 1
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 1
- VGGGPCQERPFHOB-UHFFFAOYSA-N Bestatin Natural products CC(C)CC(C(O)=O)NC(=O)C(O)C(N)CC1=CC=CC=C1 VGGGPCQERPFHOB-UHFFFAOYSA-N 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 241001429249 Blueberry scorch virus Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108700016947 Bos taurus structural-GP Proteins 0.000 description 1
- OILXMJHPFNGGTO-NRHJOKMGSA-N Brassicasterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@H]([C@@H](/C=C/[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 OILXMJHPFNGGTO-NRHJOKMGSA-N 0.000 description 1
- 101100184662 Caenorhabditis elegans mogs-1 gene Proteins 0.000 description 1
- 102100021868 Calnexin Human genes 0.000 description 1
- 108010056891 Calnexin Proteins 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101100507655 Canis lupus familiaris HSPA1 gene Proteins 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- 101710119774 Chitodextrinase Proteins 0.000 description 1
- 241000710177 Citrus tristeza virus Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241000724252 Cucumber mosaic virus Species 0.000 description 1
- 102000001493 Cyclophilins Human genes 0.000 description 1
- 108010068682 Cyclophilins Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- YAHZABJORDUQGO-NQXXGFSBSA-N D-ribulose 1,5-bisphosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)C(=O)COP(O)(O)=O YAHZABJORDUQGO-NQXXGFSBSA-N 0.000 description 1
- 101100001914 Danio rerio apmap gene Proteins 0.000 description 1
- MCWVPSBQQXUCTB-AMOSEXRZSA-N Delta7-Avenasterol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@@H]4C[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C MCWVPSBQQXUCTB-AMOSEXRZSA-N 0.000 description 1
- LTLYEAJONXGNFG-DCAQKATOSA-N E64 Chemical compound NC(=N)NCCCCNC(=O)[C@H](CC(C)C)NC(=O)[C@H]1O[C@@H]1C(O)=O LTLYEAJONXGNFG-DCAQKATOSA-N 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 description 1
- 101710156500 Endoglucanase D Proteins 0.000 description 1
- 101710198178 Endopolygalacturonase A Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920002449 FKM Polymers 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000218627 Garlic common latent virus Species 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241001492240 Grapevine virus A Species 0.000 description 1
- 241001492237 Grapevine virus B Species 0.000 description 1
- 102100023737 GrpE protein homolog 1, mitochondrial Human genes 0.000 description 1
- 102000004447 HSP40 Heat-Shock Proteins Human genes 0.000 description 1
- 108010042283 HSP40 Heat-Shock Proteins Proteins 0.000 description 1
- 241000237369 Helix pomatia Species 0.000 description 1
- 241000232846 Heracleum latent virus Species 0.000 description 1
- 241000282375 Herpestidae Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101710121996 Hexon protein p72 Proteins 0.000 description 1
- 101000829489 Homo sapiens GrpE protein homolog 1, mitochondrial Proteins 0.000 description 1
- 101001014213 Homo sapiens Morphogenetic neuropeptide Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000222342 Irpex Species 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241000282333 Martes foina Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- RXSVYGIGWRDVQC-UHFFFAOYSA-N N-[6-[[(cyclohexylideneamino)oxy-oxomethyl]amino]hexyl]carbamic acid (cyclohexylideneamino) ester Chemical compound C1CCCCC1=NOC(=O)NCCCCCCNC(=O)ON=C1CCCCC1 RXSVYGIGWRDVQC-UHFFFAOYSA-N 0.000 description 1
- 241000772415 Neovison vison Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282373 Panthera pardus Species 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 108010029182 Pectin lyase Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241000283216 Phocidae Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000710181 Potato virus M Species 0.000 description 1
- 241000710179 Potato virus S Species 0.000 description 1
- 241000709992 Potato virus X Species 0.000 description 1
- 241000723762 Potato virus Y Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710147478 Probable endo-beta-1,4-glucanase D Proteins 0.000 description 1
- 101710191534 Probable endopolygalacturonase A Proteins 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102000018679 Tacrolimus Binding Proteins Human genes 0.000 description 1
- 108010027179 Tacrolimus Binding Proteins Proteins 0.000 description 1
- 241000708500 Tomato crinkle virus Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- OILXMJHPFNGGTO-ZRUUVFCLSA-N UNPD197407 Natural products C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)C=C[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZRUUVFCLSA-N 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- MCWVPSBQQXUCTB-OQTIOYDCSA-N avenasterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CC/C(=C/C)C(C)C)CC[C@H]33)C)C3=CC[C@H]21 MCWVPSBQQXUCTB-OQTIOYDCSA-N 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 229940076810 beta sitosterol Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 235000004420 brassicasterol Nutrition 0.000 description 1
- OILXMJHPFNGGTO-ZAUYPBDWSA-N brassicasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZAUYPBDWSA-N 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- HFNQLYDPNAZRCH-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O.OC(O)=O HFNQLYDPNAZRCH-UHFFFAOYSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000009295 crossflow filtration Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- OQMZNAMGEHIHNN-CIFIHVIMSA-N delta7-stigmasterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)CC[C@H]33)C)C3=CC=C21 OQMZNAMGEHIHNN-CIFIHVIMSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 108010081495 driselase Proteins 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 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
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 229920001973 fluoroelastomer Polymers 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 239000001056 green pigment Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 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
- 239000002054 inoculum Substances 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 210000003093 intracellular space Anatomy 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 235000015250 liver sausages Nutrition 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 102000035123 post-translationally modified proteins Human genes 0.000 description 1
- 108091005626 post-translationally modified proteins Proteins 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 239000011546 protein dye Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- XYSQXZCMOLNHOI-UHFFFAOYSA-N s-[2-[[4-(acetylsulfamoyl)phenyl]carbamoyl]phenyl] 5-pyridin-1-ium-1-ylpentanethioate;bromide Chemical compound [Br-].C1=CC(S(=O)(=O)NC(=O)C)=CC=C1NC(=O)C1=CC=CC=C1SC(=O)CCCC[N+]1=CC=CC=C1 XYSQXZCMOLNHOI-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 201000010740 swine influenza Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229950009811 ubenimex Drugs 0.000 description 1
- 108020005087 unfolded proteins Proteins 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 108010082737 zymolyase Proteins 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
- C12N7/02—Recovery or purification
-
- 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
- A61K2039/517—Plant cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16123—Virus like particles [VLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
Definitions
- the present invention relates to methods of preparing plant-derived virus-like particles (VLPs).
- Virus-like particles may be employed to prepare influenza vaccines.
- VLPs mimic the structure of the viral capsid, but lack a genome, and thus cannot replicate or provide a means for a secondary infection.
- VLPs offer an improved alternative to isolated (soluble) recombinant antigens for stimulating a strong immune response.
- VLPs are assembled upon expression of specific viral proteins and present an external surface resembling that of their cognate virus but, unlike true viral particle, do not incorporate genetic material.
- the presentation of antigens in a particulate and multivalent structure similar to that of the native virus achieves an enhanced stimulation of the immune response with balanced humoral and cellular components.
- enveloped VLPs present the surface antigens in their natural membrane-bound state (Grgacic and Anderson, 2006, Methods 40, 60- 65).
- Influenza VLPs with their nanoparticle organization, have been shown to be better vaccine candidates compared to recombinant hemagglutinin (HA) (i.e. monomeric HA, or HA organized into rosettes; assembly of 3-8 trimers of HA), and they are able to activate both humoral and cellular immune response. (Bright, R.A., et. al.,. 2007, Vaccine 25, 3871-3878).
- HA hemagglutinin
- influenza vaccines are composed of viral particle or virus antigens obtained from egg-grown virions.
- the production of egg-derived vaccines relies on the culture of live viruses in embryonated hen eggs.
- Split-influenza vaccines are obtained after chemical inactivation and disruption of purified virions with a detergent.
- Recombinant influenza antigens are an effective alternative to virus-derived antigens as pandemic vaccine products.
- Recombinant antigens can be produced from information on the genetic makeup of a new strain once this information is made available, and allows a rapid initiation of the production process.
- Influenza VLPs have been obtained in cultured mammalian cells from the co-expression of all 10 influenza proteins (Mena et al., 1996, J. Virol. 70, 5016-5024).
- Several viral proteins are dispensable for the production of VLPs, and influenza VLPs in vaccine development programs have been produced from the co-expression of the 2 major antigenic envelope proteins (HA and NA) with Ml or from the co-expression of HA and Ml only (Kang et al., 2009, Virus Res. 143, 140-146). Chen et al. (2007, J. Virol.
- HA alone is capable of driving VLP formation and budding and Ml co-expression could be omitted in their system.
- HA was found to bind to sialylated glycoproteins on the surface of the mammalian cells producing the VLPs, a viral sialidase was co-expressed to allow the release of VLPs from the producing cell after budding.
- VLP production system for example, one that relies on the expression of only one or a few viral proteins without requiring expression of non-structural viral proteins is desirable to accelerate the development of vaccines.
- Production of viral antigens, including VLPs, in plant systems provides an advantage for production, in that they may be grown in a greenhouse or field, and don't require aseptic tissue culture methods and handling.
- PCT Publication WO 2006/1 19516 discloses expression of full length and truncated human-codon optimized H5 HA of Influenza A/Vietnam/1194/2004 in plants. The truncated construct lacks the membrane anchoring domain. The highest
- influenza HA VLPs that comprise a lipid envelope
- WO 2009/009876 and WO 2009/076778 to D'Aoust et al.; both of which are incorporated herein by reference.
- a lipid layer or membrane to be retained by the virus.
- the composition of the lipid may vary with the system (e.g. a plant-produced enveloped virus would include plant lipids or phytosterols in the envelope), and may contribute to an improved immune response.
- HBV surface antigen HBV surface antigen
- NVCP viral capsid protein
- Non-enveloped VLPs have also been obtained from the expression of HBV core antigen (HBcAg; Huang et al., 2009,
- VLPs may be desirable to separate the VLPs from some, or all of the proteins, carbohydrates, etc. present in the plant or plant matter before the VLP is used in vaccine formulation.
- centrifugation process to provide an interstitial fluid extract comprising the protein of interest is described in PCT Publication WO 00/09725 (to Turpen et al.). This approach is suitable for small proteins (of 50 kDa or smaller) that pass through pores under vacuum and centrifugation, but is not suitable for larger superstructure proteins or protein complexes such as a VLP.
- McCormick et al 1999 (Proc Natl Acad Sci USA 96:703-708) discloses use of a rice amylase signal peptide fused to a single-chain Fv (scFv) epitope to target the expressed protein to the extracellular compartment, followed by vacuum infiltration of leaf and stem tissue for recovery of the scFv polypeptides.
- scFv single-chain Fv
- Moehnke et al., 2008 Biotechnol Lett 30: 1259-1264
- PCT Publication WO 2003/025124 discloses expression of scFv immunoglobulins in plants, targeting to the apoplastic space using murine signal sequences.
- VLPs Given the complexity of VLPs and the plant tissue in which they may be produced, methods of preparing VLPs that are substantially free of, or easily separated from plant proteins, yet retain the structural and immunogenic characteristics of the enveloped virus are desired.
- the present invention relates to methods of preparing plant-derived virus-like particles (VLPs). More specifically, the present invention is directed to methods of preparing VLPs comprising influenza antigens.
- the present invention provides a method (A) of preparing plant derived VLPs comprising obtaining a plant or plant matter comprising plant-derived VLPs localized within the apoplast; producing a protoplast and an apoplast fraction, the apoplast fraction comprising plant-derived VLPs; and recovering the apoplast fraction.
- the method may further comprise a step of purifying the plant derived VLPs from the apoplast fraction.
- the plant-derived VLP may be a chimeric plant-derived VLP.
- the plant derived VLP may be selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins.
- the plant derived VLPs may comprise influenza hemagglutinin.
- the apoplast and protoplast fractions may be produced by treatment of the plant or plant matter by an enzyme composition.
- the enzyme composition may comprise one or more than one pectinase, one or more than one cellulase, or one or more than one pectinase and one or more than one cellulase.
- the enzyme composition does not include a lipase or protease, or the composition does not include an added lipase or protease.
- Plant or plant matter may be obtained by growing, harvesting or growing and harvesting the plant.
- the plant matter may comprise some or all of the plant, one ore more than one plant cell, leaves, stems, roots or cultured plant cells.
- the present invention provides a method of preparing plant derived VLPs as described above (Method A), wherein a nucleic acid encoding the VLP selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins is introduced into the plant in a transient manner. Alternatively, the nucleic acid is stably integrated within a genome of the plant.
- the present invention provides a method of preparing plant derived VLPs as described above (Method A) further comprising a step of purifying the plant derived VLPs from the apoplast fraction.
- the step of purifying may comprise filtering the apoplast fraction using depth filtration to produce a clarified extract, followed by chromatography of the clarified extract using a cation exchange resin.
- proteins obtained from the apoplast are more homogenous, as the intermediate forms of post-translationally modified proteins, or proteins comprising other types of processing that occurs in various intracellular compartments are not co-extracted.
- a higher degree of homogeneity of a recombinant protein typically results in a higher quality of a preparation comprising the protein, and may result in a product with beneficial properties including higher potency, longer half-life, or better immunogenic capacity.
- blood proteins containing high-mannose glycosylation are eliminated in blood circulation more rapidly than proteins comprising complex glycosylation.
- a glycosylated protein produce in the apoplastic fraction exhibits more complex-type glycosylation. Therefore, an apoplast-derived protein prepared using the methods described herein, involving cell-wall digestion, exhibit, for example, a better half life in circulation.
- the present invention also provides for a method (B) of preparing plant-derived VLPs comprising a plant-derived lipid envelope, the method comprising, obtaining a plant, or plant matter comprising VLPs localized within the apoplast; treating the plant or plant matter with an enzyme composition to produce a protoplast fraction, and one or more than one apoplastic protein composition; separating the one or more than one apoplastic protein complex from the protoplast fraction, wherein the one or more than one apoplastic protein complexes comprise the VLPs.
- the enzyme composition may comprise one or more than one pectinase, one or more than one cellulose, or one or more than one pectinase and one or more than one cellulase.
- the enzyme composition does not include a lipase or protease, or the composition does not include an added lipase or protease.
- the plant-derived VLP may be a chimeric plant-derived VLP.
- the plant derived VLP may be selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins.
- the plant derived VLPs may comprise influenza hemagglutinin.
- the present invention provides a method of preparing plant derived VLPs as described above (Method B), wherein a nucleic acid encoding the VLP selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins is introduced into the plant in a transient manner. Alternatively, the nucleic acid is stably integrated within a genome of the plant.
- the present invention provides a method of preparing plant derived VLPs as described above (Method B) further comprising a step of purifying the plant derived VLPs from the apoplast fraction.
- the step of purifying may comprise filtering the apoplast fraction using depth filtration to produced a clarified extract, followed by chromatography of the clarified extract using a cation exchange resin.
- the plant derived VLPs may include VLPs comprising one or more influenza HA polypeptides.
- the influenza HA polypeptide may also be a chimeric HA polypeptide.
- the plant- derived VLPs may further comprise hemagglutinating activity.
- Plant or plant matter may be obtained by growing, harvesting or growing and harvesting the plant.
- the plant matter may comprise some or all of the plant, or one or more than one plant cell, leaves, stems, roots or cultured cells.
- the present invention also provides a method (C) of preparing plant derived VLPs, comprising obtaining a plant or plant matter comprising plant-derived VLPs, digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction, and filtering the digested fraction to produced a filtered fraction and recovering the plant-derived VLPs from the filtered fraction.
- the enzyme composition may comprise one or more than one pectinase, one or more than one cellulose, or one or more than one pectinase and one or more than one cellulase.
- the enzyme composition does not include a lipase or protease, or the composition does not include an added lipase or protease.
- the plant-derived VLP may be a chimeric plant-derived VLP.
- the plant derived VLP may be selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins.
- the plant derived VLPs may comprise influenza hemagglutinin.
- the present invention provides a method of preparing plant derived VLPs as described above (Method C), wherein a nucleic acid encoding the VLP selected from the group of viral envelope proteins, viral structural proteins, viral capsid proteins, and viral coat proteins is introduced into the plant in a transient manner. Alternatively, the nucleic acid is stably integrated within a genome of the plant.
- the present invention provides a method of preparing plant derived VLPs as described above (Method C) further comprising a step of separating the VLPs in the filtered fraction from the cellular debris and insoluble materials.
- the step of separating may be performed by centrifugation, by depth filtration, or bother centrifugation and depth filtration to produce a clarified fraction.
- the plant derived VLPs may be further purified by chromatography, for example, the clarified extract may be purified using a cation exchange resin.
- the plant derived VLPs may include VLPs comprising one or more influenza HA polypeptides.
- the influenza HA polypeptide may also be a chimeric HA polypeptide.
- the plant- derived VLPs may further comprise hemagglutinating activity.
- Plant or plant matter may be obtained by growing, harvesting or growing and harvesting the plant.
- the plant matter may comprise some or all of the plant, or one or more than one plant cell, leaves, stems, roots or cultured cells.
- plant-made VLPs comprising plant derived lipids, may induce a stronger immune reaction than VLPs made in other manufacturing systems and that the immune reaction induced by these plant-made VLPs is stronger when compared to the immune reaction induced by live or attenuated whole virus vaccines.
- composition of a protein extract obtained from a host cell is complex and typically comprises intercellular and intracellular components along with a protein or suprastructure of interest that is to be isolated.
- Preparation of an apoplastic fraction, followed by a step to segregate the intracellular proteins and components is advantageous since the protein or suprastructure of interest can be enriched and increase efficiency within a manufacturing process. Having a simpler process, comprising fewer efficient steps, may result in significant yield increases, and cost reduction.
- the process of digesting the cell wall using cell wall degrading enzymes increases VLP protein yield even if protoplasts do not remain intact during the extraction procedure.
- the step of cell wall digestion may loosen the polymeric components of the cells wall and assist in release of the VLPs otherwise associated within the cell wall. This protocol may also minimize contamination of the VLPs within intracellular components.
- the methods described herein result in less disruption, and contamination of a plant- derived VLP extract when compared to methods for preparing plant-derived VLPs involving homogenization, blending or grinding.
- the methods described herein provide an apoplast fraction of the plant tissue and that may maintain the integrity of protoplasts and their
- the method as described herein is effective in purifying VLPs even if the protoplasts, or a portion of the protoplasts, lose their integrity and are no longer intact.
- VLPs provide a higher yield of VLPs when compared to methods of VLP extraction involving standard tissue disruption techniques, for example, homogenization, blending or grinding.
- the greater yield may be due to, in part, a reduction of the shearing forces that disrupt the structural integrity of the VLPs and/or the lipid envelope.
- Preparation of VLPs from an apoplastic fraction may be advantageous, as apoplastic fractions are significantly reduced, or free of, cytoplasmic proteins. Therefore, VLP separation from other proteins and matter, including HA monomers, trimers or fragments of HA, in the apoplastic fraction is easily carried out.
- increased yields of VLPs may also be obtained using the methods described herein, even if the protoplast preparation, or a portion of the protoplast preparation, is not intact.
- the VLPs of the present invention are also characterized as exhibiting a greater hemagglutinating activity than those obtained using standard tissue disruption techniques. This improved hemagglutinating activity may result from a greater yield of intact VLPs (fewer HA monomers or trimers free in solution), a greater yield of intact VLPs with intact lipid envelopes, or a combination thereof.
- Vaccines made using VLPs provide the advantage, when compared to vaccines made of whole viruses, that they are non-infectious. Therefore, biological containment is not an issue and it is not required for production.
- Plant-made VLPs provide a further advantage by allowing the expression system to be grown in a greenhouse or field, thus being significantly more economical and suitable for scale-up.
- VLPs may be produced in the absence of neuraminidase (NA), and there is no need to co-express NA, or to treat the producing cells or extract with sialidase
- NA neuraminidase
- Figure 1 shows a schematic representation of CPMVHT-based expression cassette (construct 685) for the expression of H5 A/Indonesia/5/05 hemagglutinin.
- Figure 2 shows A) the nucleic acid sequence (SEQ ID NO. 1) of a portion of construct for expressing H5/Indo (construct number 685) from Pad (upstream of the 35S promoter) to Ascl (immediately downstream of the NOS terminator). Coding sequence of H5 from
- Figure 2B shows the amino acid sequence (SEQ ID NO. 2) of H5 A/Indonesia/5/05 hemagglutinin encoded by construct number 685.
- Figure 3 shows characterization of hemagglutinin (HA)-containing structures by size exclusion chromatography (SEC). Following centrifugation of the digested plant extract, the pellet was resuspended and fractionated by SEC.
- Figure 3A shows the total soluble protein content per fraction (solid triangles; % of maximum, left-side Y-axis; determined using the Bradford method). The hemagglutinating activity of the collected fractions (solid bars; right-side Y axis) is also shown.
- FIG. 3B shows SDS-PAGE analysis of SEC eluted fractions. Fractions were precipitated by acetone and re-suspended in 1/40 volume of reducing sample loading buffer prior to analysis. Gel was stained with 0.1% Coomassie R-250 solution. Purified VLPs were run as a control. The band corresponding to the HA0 monomer is indicated by an arrow. MW - Molecular weight standards (kDa); C - Purified VLPs (control); lanes 7 through 10 and 14 through 16 correspond to fractions number eluted from SEC analysis, shown in Figure 3 A.
- Figure 4 shows a comparison of protein profiles obtained after enzymatic digestion and by mechanical homogenization using a ComitrolTM homogenizer.
- Samples were treated in denaturing sample loading buffer and proteins were separated by SDS-PAGE analysis of elution fractions. Gels were stained with 0.1% Coomassie R-250 solution.
- MW - Molecular weight standards (kDa); lane 1— 25 ⁇ enzyme mixture; lane 2 - 25 ⁇ enzymatic digestion of plant tissue and lane 3 - 5 ⁇ extract obtained with the Comitrol homogenizer.
- Figure 5 shows the nucleic acid sequence (SEQ ID NO: 9) of an HA expression cassette comprising alfalfa plastocyanin promoter and 5' UTR, hemagglutinin coding sequence of H5 from A/Indonesia/5/2005 (Construct # 660), alfalfa plastocyanin 3' UTR and terminator sequences.
- Figure 6 shows the capture of HA-VLP on cationic exchange resin directly form separation of HA-VLP in the apoplastic fraction.
- Samples were treated in non-reducing, denaturing sample loading buffer and proteins were separated by SDS-PAGE. Gels were stained with 0.1% Coomassie R-250 solution.
- Lane 1 Apoplastic fraction after centrifugation, Lane 2-3: Apoplastic fraction after successive microfiltration;
- Lane 4 Load of the cationic exchange;
- Lane 5 Flow through fraction of the cationic exchange.
- Lane 7 Molecular weight standards (kDa).
- FIG. 7 shows the Nanoparticle Tracking analysis (NTA) profile of H5/Indo VLP (A) and Hl/Cal VLP (B) after clarification without addition of NaCl to digestion buffer and of Hl/Cal VLP (C) with this addition.
- NTA experiments were carried out with NanoSight LM20 (NanoSight, Amesbury, UK). The instrument is equipped with a blue laser (405 nm), a sample chamber and a Viton fluoroelastomer o-ring. Videos were recorded at room temperature and analysed using the NTA 2.0 software. The samples were recorded for 60 sec. The shutter and gain were manually chosen so that optimal particle resolution was obtained.
- Figure 8 shows a Western blot of extract of H3/Brisbane VLP generated by enzymatic digestion using different buffers.
- Lane 1 Pure recombinant HA standard (5 ⁇ g, from Immune Technology Corp. IT-003-0042p)
- Lane 2 to 5 contain 7 ⁇ of centrifuged enzymatic extract performed in the following buffers: Lane 2) 600mM Mannitol + 125mM citrate+ 75mM NaP0 4 + 25mM EDTA + 0.04% bisulfite pH6.2, Lane 3) 600mM Mannitol + 125mM citrate+ 75mM NaP0 4 + 50mM EDTA + 0.04% bisulfite pH6.2, Lane 4) 200mM Mannitol + 125mM citrate+ 75mM NaP0 4 + 25mM EDTA + 0.03% bisulfite pH6.2, Lane 5) 200mM Mannitol + 125mM citrate+ 75mM NaP0 4 + 50mM EDTA +
- the present invention relates to methods of preparing plant-derived virus-like particles (VLPs). More specifically, the present invention is directed to methods of preparing VLPs comprising influenza hemagglutinin (HA).
- VLPs plant-derived virus-like particles
- HA influenza hemagglutinin
- the present invention provides a method for obtaining a protein, or protein suprastructure of interest.
- the protein of interest may be present in the apoplast or extracellular compartment, corresponding to the plant cell portion excluding the protoplast/spheroplast compartment.
- the method involves removing, digesting or both digesting and removing the cellulosic plant cell wall that surrounds plant cells. By digesting the cell wall the polymeric components of the cell wall are loosened, and the protein or protein suprastructure of interest may be more readily released.
- the protein or protein suprastructure of interest is enriched since the protoplast/spheroplast compartment that contains a majorly host-cell proteins and
- protein suprastructures are structures comprised of two or more polypeptides; the polypeptides may be the same, or different; if different, they may be present in a ratio of about 1 : 1 to about 10: 1 or greater.
- the protein suprastructure may further comprise one or more lipids, phospholipids, nucleic acids, membranes or the like. The two or more
- polypeptides may be connected by a covalent bond, a disulfide bridge, charge interaction, hydrophobic attraction, van der waals forces, hydrogen bonds or the like.
- An example of a protein suprastructure is a virus like particle (VLP), which may be enveloped, or non-enveloped, for example, a viral envelope protein, a viral structural protein, a viral capsid protein, or a viral coat protein.
- VLP virus like particle
- the present invention also provides a method of preparing plant-derived virus like particles (VLPs).
- the method involves obtaining a plant or plant matter comprising plant- derived VLPs localized within the apoplast; producing a protoplast/spheroplast fraction, and an apoplast fraction from the plant matter, the apoplast fraction comprising plant-derived VLPs, and recovering the apoplast fraction.
- the plant derived VLPs may be purified from the apoplast fraction.
- the present invention also provides a method of preparing VLPs comprising a plant- derived lipid envelope.
- the method includes obtaining a plant, or plant matter comprising VLPs, treating the plant or plant matter with an enzyme composition to produce one or more than one apoplastic protein complex and a protoplast/spheroplast fraction, and separating the one or more than one apoplastic protein complex from the protoplast fraction.
- the one or more than one apoplastic protein complex comprises the VLPs comprising a plant derived lipid envelope.
- the present invention also provides a method of preparing plant derived VLPs, comprising obtaining a plant or plant matter that comprise the plant-derived VLPs, digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction, and filtering the digested fraction to produced a filtered fraction and recovering the plant-derived VLPs from the filtered fraction.
- integrity of the protoplasts may not be required.
- a protoplast is a plant cell that has had its cell wall completely or partially removed.
- a spheroplast may have partial removal of the cell wall.
- a protoplast, a spheroplast, or both a protoplast and spheroplast may be used as described herein, and the terms as used herein are interchangeable.
- the cell wall may be disrupted and removed mechanically (e.g. via homogenization, blending), the cell wall may be fully or partially digested enzymatically, or the cell wall may be removed using a combination of mechanical and enzymatic methods, for example homogenization followed by treatment with enzymes for digestion of the cell wall.
- Protoplasts may also be obtained from cultured plant cells, for example liquid cultured plant cells, or solid cultured plant cells.
- Standard reference works setting forth the general principles of plant tissue culture, cultured plant cells, and production of protoplasts, spheroplasts and the like include:
- Enzymes useful for digesting or degrading plant cell walls for release or protoplasts or spheroplasts are known to one of skill in the art and may include cellulase (EC 3.2.1.4), pectinase (EC 3.2.1.15), xylanase (EC 3.2.1.8), chitinases (EC 3.2.1.14), hemicellulase, or a combination thereof.
- Non- limiting examples of suitable enzymes includes a multi-component enzyme mixture comprising cellulase, hemicellulase, and pectinase, for example MACEROZYMETM (containing approximately: Cellulase: O.lU/mg, Hemicellulase: 0.25U/mg, and Pectinase:
- Alternate names, and types of cellulases include endo-l,4-P-D-glucanase; ⁇ -1 ,4- glucanase; P-l ,4-endoglucan hydrolase; cellulase A; cellulosin AP; endoglucanase D; alkali cellulase; cellulase A 3; celludextrinase; 9.5 cellulase; avicelase; pancellase SS and 1 ,4-(1 ,3; 1,4)- ⁇ -D-glucan 4-glucanohydrolase.
- pectinases include pectin depolymerase; pectinase; endopolygalacturonase; pectolase; pectin hydrolase; pectin polygalacturonase; endo-polygalacturonase; poly-a-l,4-galacturonide glycanohydrolase; endogalacturonase; endo-D-galacturonase and poly(l ,4-a-D-galacturonide) glycanohydrolase.
- Alternate names, and types of xylanases include hemicellulase, endo-(l- ⁇ 4)-P-xylan 4- xylanohydrolase; endo- 1 ,4-xylanase; xylanase; p-l,4-xylanase; endo-l,4-xylanase; endo-p-1,4- xylanase; endo-l ,4-P-D-xylanase; 1 ,4- -xylan xylanohydrolase; ⁇ -xylanase; P-l,4-xylan xylanohydrolase; endo-l,4-p-xylanase; ⁇ -D-xylanase.
- chitinases include chitodextrinase; l,4- -poly-N-acetylglucosaminidase; poly-P-glucosaminidase; ⁇ -1,4- poly-N-acetyl glucosamidinase; poly[l,4-(N-acetyl-p-D-glucosaminide)] glycanohydrolase.
- Choice of a particular enzyme or combination of enzymes, and concentration and reaction conditions may depend on the type of plant tissue used from which the protoplast and apoplast fraction comprising the VLPs is obtained.
- a mixture of cellulase, hemicellulase and pectinase, for example, a pectinase MACEROZYMETM or Multifect may be used in a concentration ranging from 0.01% to2.5% (v/v), for example 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,- 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5% (v/v), or any amount therebetween.
- MACEROZYMETM or Multifect may be used alone, or in combination with other enzymes, e.g cellulase, pectinase, hemicellulase, or a combination thereof.
- Cellulase may be used in a concentration ranging from 0.1% to 5%, for example 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75. 3.0. 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0% (w/v) or any amount therebetween.
- the enzyme solution (alternately referred to as a cell wall degrading composition, digesting solution) will generally comprise a buffer or buffer system, an osmoticum, and one or more than one salts, divalent cations or other additives.
- the buffer or buffer system is selected to maintain a pH in the range suitable for enzyme activity and the stability of the protein(s), or VLP, to purify, for example, within the range of about pH 5.0 to about 8.0, or any value therebetween.
- the selected pH used may vary depending upon the VLP to be recovered, for example the pH may be 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or any pH therebetween.
- buffers or buffer systems include, but are not limited to, MES, phosphate, citrate and the like.
- One or more buffers or buffer systems may be combined in an enzyme solution (digesting solution); the one or more buffers may be present at a concentration from 0 mM to about 200 mM, or any amount therebetween, for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 mM or any amount therebetween.
- an osmoticum component can be added if desired. The osmoticum and its concentration are selected to raise the osmotic strength of the enzyme solution.
- osmoticum examples include mannitol, sorbitol or other sugar alcohols, polyethylene glycol (PEG) of varying polymer lengths, and the like. Concentration ranges of osmoticum may vary depending on the plant species, the type of osmoticum used, and the type of plant tissue selected (species or organ of origin e.g. leaf or stem) - generally the range is from 0M to about 0.8 M, for example 0.05, 0.1, 0.15, 0.2, 0.25, 0.3.
- osmoticum 0.35, 0.4, 0.5, 0.6, 0.7, or 0.75 M, or any amount therebetween, for example, 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 nM mannitol, or any amount therebetween.
- concentration of osmoticum may also be expressed as a percentage (w/v).
- the osmoticum can also be omitted during digestion.
- Temperature may be controlled if desired during the digestion process. Useful temperature range should be between 4°C and 40 °C or any temperature therebetween, for example from about 4°C to about 15°C, or any amount therebetween, or from about 4°C to about 22°C, or any temperature therebetween. Depending to the temperature chosen, the other digestion experimental parameters may be adjusted to maintain optimal extraction conditions.
- Cations, salts or both may be added to improve plasma membrane stability, for example divalent cations, such as Ca 2+ , or Mg 2+ , at 0.5-50mM, or any amount therebetween, salts, for example CaCl 2 , NaCl, CuS0 4 , N0 3 , and the like, from about 0 to about 750 mM, or any amount therebetween, for example 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700 or 750 mM.
- divalent cations such as Ca 2+ , or Mg 2+
- salts for example CaCl 2 , NaCl, CuS0 4 , N0 3 , and the like, from about 0 to about 750 mM, or any amount therebetween, for example 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700 or 750 mM.
- additives may also be added including a chelator for example, but not limited to, EDTA, EGTA, from about 0 to about 200 mM, or any amount therebetween, for example 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200 mM, or any amount therebetween, a reducing agent to prevent oxidation such as, but not limited to, sodium bisulfite or ascorbic acid, at 0.005-0.4% or any amount therebetween, for example 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0,25, 0.3, 0.35, 0.4%, or any amount therebetween, specific enzyme inhibitors (see below), and if desired, an inhibitor of foliar senescence, for example, cycloheximide, kinetin, or one or more polyamines.
- a chelator for example, but not limited to, EDTA, EGTA, from about 0 to about 200 mM
- the digestion solution may also comprise one or more of mannitol from about 0 to about 600 mM, NaCl from about 0 to about 500 mM, EDTA from about 0 to about 50 mM, cellulose from about 1% to about 2% v/v, pectinase from about 0 to about 1% v/v, sodium metabisulfite from about 0.03 to about 0.04%, citrate from about 0 to about 125 mM or NaP0 4 from about 0 to 75 mM.
- the plant matter may be treated to enhance access of the enzymes or enzyme composition to the plant cell wall.
- the epidermis of the leaf may be removed or 'peeled' before treatment with an enzyme composition.
- the plant matter may be cut into small pieces (manually, or with a shredding or cutting device such as an Urschel slicer); the cut up plant matter may be further infiltrated with an enzyme composition under a partial vacuum (Nishimura and Beevers 1978, Plant Physiol 62:40-43; Newell et al., 1998, J. Exp Botany 49:817-827).
- Mechanical perturbation of the plant matter may also be applied to the plant tissues (Giridhar et al., 1989. Protoplasma 151 :151-157) before or during treatment with an enzyme composition.
- cultured plant cells either liquid or solid cultures, may be used to prepare protoplasts or spheroplasts.
- an enzyme composition that lacks, or that has inactivated lipases or proteases.
- one or more protease, or lipase inhibitors may be included in the enzyme composition.
- lipase inhibitors include RHC80267 (SigmaAldrich); examples of protease inhibitors include E-64, Na 2 EDTA, Pepstatin, aprotinin, PMSF, Pefabloc, Leupeptin, bestatin and the like.
- any suitable method of mixing or agitating the plant matter in the enzyme composition may be used.
- the plant matter may be gently swirled or shaken in a tray or pan or via a rotary shaker, tumbled in a rotating or oscillating drum.
- Precaution should be taken in order to minimize the protoplast (and/or spheroplast) damage until they are removed form the digestion soup.
- the digestion vessel should be selected accordingly.
- an enzyme composition comprising 1.5% cellulase (Onozuka R-10) and 0.375% MACEROZYMETM in 500 mM mannitol, 10 m CaCl 2 and 5 mM MES (pH 5.6) may be used for protoplast (or spheroplast) production from some Nicotiana tissues.
- the concentration of mannitol may also be varied from about 0 to about 500mM, or any amount therebetween.
- One of skill in the art provided with the information disclosed herein, will be able to determine a suitable enzyme composition for the age and strain of the Nicotiana sp, or for another species used for production of VLPs.
- a protoplast fraction comprising protoplasts and/or spheroplasts
- an "apoplast fraction” are obtained.
- a "digested fraction” may be obtained.
- integrity of the protoplast fraction may not be required to produce high yields of protein as described herein, therefore, an apoplast fraction or a digested fraction may be used for the extraction of proteins, for example, but not limited to, VLPs, viral envelope proteins, viral structural proteins, viral capsid proteins, viral coat proteins.
- apoplast fraction it is meant a fraction that is obtained following enzymatic digestion, or partial enzymatic digestion, using cell wall degrading enzymes of the plant matter in the presence of an osmoticum and/or other ingredients that may be used to assist in maintaining integrity of the protoplast.
- the apoplast fraction may comprise some components arising from disrupted protoplasts (or spheroplasts).
- the apoplast fraction may comprise from about 0 to about 50% (v/v) or any amount therebetween, of the components from the protoplast fraction, or 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% (v/v) or any amount
- a digested fraction it is meant the fraction that remains following enzymatic digestion, or partial enzymatic digestion, using cell wall degrading enzymes of the plant matter, however, integrity of the protoplast is not required, and the digested fraction may comprise intact, disrupted, or both intact and disrupted protoplasts.
- the composition comprising the cell wall degrading enzymes used to produce the digested fraction may comprise an osmoticum, or the osmoticum may be present at a reduced amount when compared to the amount present in standard procedures used to obtain protoplasts, or the osmoticum may be absent from the composition.
- the digested fraction comprises the apoplast fraction and the
- the protoplast/spheroplast fraction may or may not be intact.
- the digested fraction contains intracellular components and extracellular components. Intracellular components may be found in the form of protoplasts/spheroplasts if an osmoticum is used to maintain the protoplast/spheroplast intact. If no osmoticum is used in the digestion solution, then the protoplasts/spheroplasts may be disrupted and the intracellular and
- the VLPs may be separated from components of the digested fraction using any suitable technique.
- the step of cell wall digestion may loosen the polymeric components of the cells wall and assist in release VLPs, otherwise trapped within the cell wall. This protocol also minimizes contamination of the VLPs, with the intracellular components.
- the VLPs may be separated from cellular debris following enzymatic digestion using low speed centrifugation followed by filtration, depth filtration, sedimentation, precipitation for example , but not limited to ammonium sulfate precipitation, or a combination thereof to obtain a separated fraction comprising the proteins or suprastructure proteins of interest.
- the protoplast/spheroplast fraction, or fraction comprising protoplasts may be separated from the apoplast fraction using any suitable technique, for example but not limited to, centrifugation, filtration, depth filtration, sedimentation,
- the protoplast (and spheroplast) fraction, or fraction comprising protoplasts may be separated from the apoplast fraction using any suitable technique, for example but not limited to, centrifugation, filtration, depth filtration, sedimentation, precipitation, or a combination thereof to obtain a separated fraction.
- the separated fraction may be for example a supernatant (if centrifuged, sedimented, or precipitated), or a filtrate (if filtered), and is enriched for VLPs.
- the separated fraction may be further processed to isolate, purify, concentrate or a combination thereof, the VLPs by, for example, additional centrifugation steps, precipitation, chromatographic steps (e.g. size exclusion, ion exchange chromatography), tangential flow filtration, or a combination thereof.
- additional centrifugation steps, precipitation, chromatographic steps e.g. size exclusion, ion exchange chromatography
- tangential flow filtration tangential flow filtration
- the presence of purified VLPs may be confirmed by, for example, native or SDS-PAGE, Western analysis using an appropriate detection antibody, capillary electrophoresis, or any other method as would be evident to one of skill in the art.
- the apoplast is the portion of the plant cell outside the plasma membrane, and includes the cell wall and intercellular spaces of the plant. While it is preferred that the integrity of the protoplasts (and/or spheroplasts) be maintained during digestion and further processing, it is not required that the protoplasts remain intact in order to enrich for VLPs. [0077] During synthesis, VLPs are excreted outside of the plasma membrane.
- VLPs are of an average size of about 20 nm to 1 ⁇ , or any amount therebetween, for example 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150 160, 170, 180, 190, or 200 nm, or any amount therebetween, for example 100 nm, and may include a lipid membrane. Due to their size, once synthesized, VLPs may remain trapped between the plasma membrane and cell wall and may be inaccessible for isolation or further purification using standard mechanical methods used to obtain plant proteins.
- VLPs In order to maximize yields, minimize contamination of the VLP fraction with cellular proteins, maintain the integrity of the VLPs and, in some embodiments, the associated lipid envelope or membrane, methods of disrupting the cell wall to release the VLPs that minimize mechanical damage to the protoplast (and/or spheroplasts) may be useful, such as the enzymatic methods described herein. However, it is not required that the integrity of all of the protoplasts be retained during the procedure.
- a VLP produced in a plant according to some aspects of the invention may be complexed with plant-derived lipids.
- the VLP may comprise an HA0 precursor form, or the HA1 or HA2 domains retained together by disulphide bridges form.
- the plant-derived lipids may be in the form of a lipid bilayer, and may further comprise an envelope surrounding the VLP.
- the plant derived lipids may comprise lipid components of the plasma membrane of the plant where the VLP is produced, including, but not limited to, phosphatidylcholine (PC),
- phosphatidylethanolamine PE
- glycosphingolipids PEG
- phytosterols PEG
- a plant-derived lipid may alternately be referred to as a 'plant lipid'.
- phytosterols are known in the art, and include, for example, stigmasterol, sitosterol, 24-methylcholesterol and cholesterol (Mongrand et al., 2004, J. Biol Chem 279:36277-86).
- VLPs Correct folding of viral structural proteins such as HA, and formation of trimers of HA is desired for assembly of VLPs.
- VLPs and in particular VLPs comprising a plant derived lipid envelope, may provide for a superior immune response when administered to a subject, relative to administration of the structural protein monomer.
- polypeptide expression may be targeted to any intracellular or extracellular space, organelle or tissue of a plant.
- the nucleic acid encoding the polypeptide may be linked to a nucleic acid sequence encoding a signal peptide.
- a signal peptide may alternately be referred to as a transit peptide or signal sequence.
- Signal peptides or peptide sequences for directing localization of an expressed polypeptide to the apoplast include, but are not limited to, a rice amylase signal peptide (McCormick 1999, Proc Natl Acad Sci USA 96:703-708), protein disulfide isomerase signal peptide (PDI) having the amino acid sequence:
- an expressed polypeptide may accumulate in specific intercellular or extracellular space (such as the apoplast), organelle or tissue, for example when the polypeptide is expressed and secreted in the absence of a signal peptide or transit peptide.
- virus like particle refers to structures that self-assemble and comprise viral surface proteins, for example an influenza HA protein, or a chimeric influenza HA protein.
- VLPs and chimeric VLPs are generally
- VLPs and chimeric VLPs may be produced in suitable host cells including plant host cells, and if desired further purified.
- influenza VLPs and chimeric influenza VLPs are exemplified herein, the methods described herein may be used for any plant-derived VLPs that localize in, or are secreted to, the apoplast.
- chimeric protein or “chimeric polypeptide”
- chimeric polypeptide it is meant a protein or polypeptide that comprises amino acid sequences from two or more than two sources, for example but not limited to, two or more influenza types or subtypes, that are fused as a single polypeptide.
- the chimeric protein or polypeptide may include a signal peptide that is the same (i.e. native) as, or heterologous with, the remainder of the polypeptide or protein.
- the chimeric protein or chimeric polypeptide may be produced as a transcript from a chimeric nucleotide sequence, and remain intact, or if required, the chimeric protein or chimeric polypeptide may be cleaved following synthesis.
- a chimeric protein or a chimeric polypeptide may also include a protein or polypeptide comprising subunits that are associated via disulphide bridges (i.e. a multimeric protein).
- a chimeric polypeptide comprising amino acid sequences from two or more than two sources may be processed into subunits, and the subunits associated via disulphide bridges to produce a chimeric protein or chimeric polypeptide.
- the polypeptide may be influenza hemagglutinin (HA), and each of the two or more than two amino acid sequences that make up the polypeptide may be obtained from different HA's to produce a chimeric HA, or chimeric influenza HA.
- a chimeric HA may also include a amino acid sequence comprising heterologous signal peptide (a chimeric HA pre-protein) that is cleaved after synthesis. Examples of HA proteins that may be used in the invention described herein may be found in WO 2009/009876; WO 2009/076778; WO 2010/003225 (which are incorporated herein by reference).
- a nucleic acid encoding a chimeric polypeptide may be described as a "chimeric nucleic acid", or a "chimeric nucleotide sequence”.
- a virus-like particle comprised of chimeric HA may be described as a "chimeric VLP”.
- Chimeric VLPs are further described in PCT Application No. PCT/CA2010/000983 filed June 25, 2010, and.U.S. Provisional
- VLPs can be obtained from expression of native or chimeric HA.
- the HA of the VLPs prepared according to a method provided by the present invention include known sequences and variant HA sequences that may be developed or identified.
- VLPs produced as described herein do not comprise neuraminidase (NA) or other components for example Ml (M protein), M2, NS and the like.
- NA and Ml may be co-expressed with HA should VLPs comprising HA and NA be desired.
- lipid refers to a fat-soluble (lipophilic), naturally-occurring molecules.
- a chimeric VLP produced in a plant according to some aspects of the invention may be complexed with plant-derived lipids.
- the plant-derived lipids may be in the form of a lipid bilayer, and may further comprise an envelope surrounding the VLP.
- the plant derived lipids may comprise lipid components of the plasma membrane of the plant where the VLP is produced, including phospholipids, tri-, di- and monoglycerides, as well as fat-soluble sterol or metabolites comprising sterols. Examples include phosphatidylcholine (PC),
- phosphatidylethanolamine PE
- phosphatidylinositol phosphatidyl serine
- glycosphingolipids phytosterols or a combination thereof.
- a plant-derived lipid may alternately be referred to as a 'plant lipid'.
- phytosterols include campesterol, stigmasterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol, daunosterol, sitosterol, 24- methylcholesterol, cholesterol or beta-sitosterol (Mongrand et al., 2004, J. Biol Chem 279:36277-86).
- the lipid composition of the plasma membrane of a cell may vary with the culture or growth conditions of the cell or organism, or species, from which the cell is obtained.
- Cell membranes generally comprise lipid bilayers, as well as proteins for various functions. Localized concentrations of particular lipids may be found in the lipid bilayer, referred to as 'lipid rafts'. These lipid raft microdomains may be enriched in sphingolipids and sterols. Without wishing to be bound by theory, lipid rafts may have significant roles in endo and exocytosis, entry or egress of viruses or other infectious agents, inter-cell signal transduction, interaction with other structural components of the cell or organism, such as intracellular and extracellular matrices.
- VLPs comprising a lipid envelope has been previously described in WO 2009/009876; WO 2009/076778, and WO 2010/003225 (which are incorporated herein by reference).
- hemagglutinin or "HA” as used herein refers to a structural glycoprotein of influenza viral particles.
- the HA of the present invention may be obtained from any subtype.
- the HA may be of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, or H16, or of influenza types B or C.
- the recombinant HA of the present invention may also comprise an amino acid sequence based on the sequence of any hemagglutinin.
- the structure of influenza hemagglutinin is well-studied and demonstrates a high degree of conservation in secondary, tertiary and quaternary structure. This structural conservation is observed even though the amino acid sequence may vary (see, for example, Skehel and Wiley, 2000 Ann Rev Biochem 69:531-69; Vaccaro et al 2005; which is incorporated herein by reference).
- the present invention also pertains to methods of preparing, isolating, or both preparing and isolating VLPs, including influenza VLPs of viruses which infect humans, or host animals, for example primates, horses, pigs, birds, sheep, avian water fowl, migratory birds, quail, duck, geese, poultry, chicken, camel, canine, dogs, feline, cats, tiger, leopard, civet, mink, stone marten, ferrets, house pets, livestock, mice, rats, seal, whale and the like.
- influenza viruses may infect more than one host animal.
- Amino acid variation is tolerated in hemagglutinins of influenza viruses. This variation provides for new strains that are continually being identified. Infectivity between the new strains may vary. However, formation of hemagglutinin trimers, which subsequently form VLPs is maintained.
- the present invention also includes methods of preparing any plant-derived VLPs, regardless of the HA subtype or sequence, or chimeric HA comprising the VLP, or species of origin.
- Correct folding of the hemagglutinins may be important for stability of the protein, formation of multimers, formation of VLPs and function of the HA (ability to hemagglutinate), among other characteristics of influenza hemagglutinins.
- Folding of a protein may be influenced by one or more factors, including, but not limited to, the sequence of the protein, the relative abundance of the protein, the degree of intracellular crowding, the availability of cofactors that may bind or be transiently associated with the folded, partially folded or unfolded protein, the presence of one or more chaperone proteins, or the like.
- Heat shock proteins or stress proteins are examples of chaperone proteins, which may participate in various cellular processes including protein synthesis, intracellular trafficking, prevention of misfolding, prevention of protein aggregation, assembly and disassembly of protein complexes, protein folding, and protein disaggregation.
- chaperone proteins include, but are not limited to, Hsp60, Hsp65, Hsp 70, Hsp90, Hsp 100, Hsp20-30, Hsp 10, HsplOO-200, HsplOO, Hsp90, Lon, TF55, FKBPs, cyclophilins, ClpP, GrpE, ubiquitin, calnexin, and protein disulfide isomerases (see, for example, Macario, A.J.L., Cold Spring Harbor Laboratory Res. 25:59-70. 1995; Parsell, D.A. & Lindquist, S. Ann. Rev. Genet. 27:437-496 (1993); U.S. Patent No. 5,232,833).
- Chaperone proteins for example but not limited to Hsp40 and Hsp70 may be used to ensure folding of a chimeric HA (PCT Application No.
- VLPs may be assessed for structure, size potency or activity by, for example, hemagglutination assay, electron microscopy, light scattering, size exclusion chromatography, HPLC, Western blot analysis, or electrophoresis. These and other methods for assessing size, concentration, activity and composition of VLPs are known in the art.
- a preparation comprising VLPs may be obtained by the methods described herein, and insoluble material removed by centrifugation. Precipitation with PEG may also be of benefit.
- the recovered protein may be quantified using conventional methods (for example, Bradford Assay, BCA), and the extract passed through a size exclusion column, using for example SEPHACRYLTM, SEPHADEXTM, or similar medium, and the fractions collected. Blue Dextran 2000 or a suitable protein, may be used as a calibration standard.
- the extract may also be passed through a cation exchange column and active fractions collected. Following chromatography, fractions may be further analyzed by protein
- a hemagglutination assay may be used to assess the hemagglutinating activity of the VLP-containing fractions, using methods well-known in the art. Without wishing to be bound by theory, the capacity of HA to bind to RBC from different animals is driven by the affinity of HA for sialic acids a2,3 or 2,3 and the presence of these sialic acids on the surface of RBC.
- Equine and avian HA from influenza viruses agglutinate erythrocytes from all several species, including turkeys, chickens, ducks, guinea pigs, humans, sheep, horses and cows; whereas human HAs will bind to erythrocytes of turkey, chickens, ducks, guinea pigs, humans and sheep (Ito T. et al, 1997, Virology, 227:493-499; Medeiros R et al, 2001. Virology 289:74-85).
- a hemagglutination inhibition (HI, or HAI) assay may also be used to demonstrate the efficacy of antibodies induced by a vaccine, or vaccine composition comprising chimeric HA or chimeric VLP can inhibit the agglutination of red blood cells (RBC) by recombinant HA.
- HI hemagglutination inhibition
- Hemagglutination inhibitory antibody titers of serum samples may be evaluated by microtiter HAI (Aymard et al 1973). Erythrocytes from any of several species may be used - e.g. horse, turkey, chicken or the like. This assay gives indirect information on assembly of the HA trimer on the surface of VLP, confirming the proper presentation of antigenic sites on HAs.
- Cross-reactivity HAI titres may also be used to demonstrate the efficacy of an immune response to other strains of virus related to the vaccine subtype.
- serum from a subject immunized with a vaccine composition comprising a chimeric hemagglutinin comprising an HDC of a first influenza type or subtype may be used in an HAI assay with a second strain of whole virus or virus particles , and the HAI titer determined.
- transformation it is meant the interspecific transfer of genetic information (nucleotide sequence) that is manifested genotypically, phenotypically or both.
- the interspecific transfer of genetic information from a chimeric construct to a host may be heritable (i.e.
- Plant matter any material derived from a plant.
- Plant matter may comprise an entire plant, tissue, cells, or any fraction thereof.
- plant matter may comprise intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or a combination thereof.
- plant matter may comprise plants, plant cells, tissue, a liquid extract, or a combination thereof, from plant leaves, stems, fruit, roots or a combination thereof.
- Plant matter may comprise a plant or portion thereof which has not been subjected to any processing steps.
- a portion of a plant may comprise plant matter. Plants or plant matter may be harvested or obtained by any method, for example, the whole plant may be used, or the leaves or other tissues specifically removed for use in the described methods.
- Transgenic plants expressing and secreting VLPs may also be used as a starting material for processing as described herein.
- constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, infiltration, and the like.
- Ti plasmids Ri plasmids
- plant virus vectors direct DNA transformation, micro-injection, electroporation, infiltration, and the like.
- Transient expression methods may be used to express the constructs of the present invention (see Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348; which is incorporated herein by reference).
- a vacuum-based transient expression method as described in PCT Publications WO 00/063400, WO 00/037663 (incorporated herein by reference) may be used.
- These methods may include, for example, but are not limited to, a method of Agro-inoculation or Agro-infiltration, however, other transient methods may also be used as noted above. With either Agro-inoculation or Agro-infiltration, a mixture of
- Agrobacterium infect and transfer t-DNA copies into the cells.
- the t-DNA is episomally transcribed and the mRNA translated, leading to the production of the protein of interest in infected cells, however, the passage of t-DNA inside the nucleus is transient.
- influenza VLPs prepared by methods of the present invention may be used in conjunction with an existing influenza vaccine, to supplement the vaccine, render it more efficacious, or to reduce the administration dosages necessary.
- the vaccine may be directed against one or more than one influenza virus.
- suitable vaccines include, but are not limited to, those commercially available from Sanofi-Pasteur, ID Biomedical, Merial, Sinovac, Chiron, Roche, Medlmmune, GlaxoSmithKline, Novartis, Sanofi-Aventis, Serono, Shire Pharmaceuticals and the like.
- the VLPs of the present invention may be admixed with a suitable adjuvant as would be known to one of skill in the art.
- the VLP may be used in a vaccine composition comprising an effective dose of the VLP for the treatment of a target organism, as defined above.
- the VLP produced according to the present invention may be co-expressed with other protein components or reconstituted with other VLPs or influenza protein components, for example, neuraminidase (NA), Ml, and M2, . It can also be co-expressed or reconstituted with other VLP made of vaccinal proteins such as malaria antigens, HIV antigens, respiratory syncytial virus (RSV) antigens, and the like.
- NA neuraminidase
- Ml neuraminidase
- M2 neuraminidase
- vaccinal proteins such as malaria antigens, HIV antigens, respiratory syncytial virus (RSV) antigens, and the like.
- CPMV-H77 expression cassettes included the 35S promoter to control the expression of an mRNA comprising a coding sequence of interest flanked, in 5' by nucleotides 1-512 from the Cowpea mosaic virus (CPMV) RNA2 with mutated ATG at positions 1 15 and 161 and in 3', by nucleotides 3330-3481 from the CPMV RNA2 (corresponding to the 3' UTR) followed by the NOS terminator.
- Plasmid pBD-C5-l LC Plasmid pBD-C5-l LC, (Sainsbury et al. 2008; Plant
- the primers for the second amplification were Mut-ATG161.c (SEQ ID NO: 5) and LC-C5-1.1 lOr (SEQ ID NO: 6).
- the two fragments were then mixed and used as template for a third amplification using pBinPlus.2613c (SEQ ID NO: 3) and LC-C5-1.1 lOr (SEQ ID NO: 6) as primers.
- the resulting fragment was digested with Pad and Apal and cloned into pBD-C5-lLC digested with the same enzyme.
- the expression cassette generated was named 828.
- Construct 660 comprises an alfalfa plastocyanin promoter and 5' UTR, hemagglutinin coding sequence of H5 from
- Post-transcriptional gene silencing may be involved in limiting expression of transgenes in plants, and co-expression of a suppressor of silencing from the potato virus Y (HcPro) may be used to counteract the specific degradation of transgene mRNAs (Brigneti et al., 1998).
- Alternate suppressors of silencing are well known in the art and may be used as described herein (Chiba et al., 2006, Virology 346:7-14; which is incorporated herein by reference), for example but not limited to, TEV-pl/HC-Pro (Tobacco etch virus-pl/HC-Pro), BYV -p21, pi 9 of Tomato bushy stunt virus (TBSV pi 9), capsid protein of Tomato crinkle virus (TCV -CP), 2b of Cucumber mosaic virus; CMV-2b), p25 of Potato virus X (PVX-p25), pi 1 of Potato virus M (PVM-pl 1), pi 1 of Potato virus S (PVS-pl 1), pi 6 of Blueberry scorch virus, (BScV -pi 6), p23 of Citrus tristeza virus (CTV-p23), p24 of Grapevine leafroll-associated virus-2, (GLRaV-2 p24), plO of Grapevine virus A, (G
- a suppressor of silencing for example, but not limited to, HcPro, TEV -pl/HC-Pro, BYV-p21, TBSV pi 9, TCV-CP, CMV-2b, PVX-p25, PVM-pl 1, PVS-pl 1 , BScV-pl6, CTV-p23, GLRaV-2 p24, GBV-pl4, HLV-plO, GCLV-pl6 or GVA-plO, may be co-expressed along with the nucleic acid sequence encoding the protein of interest to further ensure high levels of protein production within a plant.
- pi 9 The construction of pi 9 is described in described in WO 2010/0003225 (which is incorporated herein by reference). Briefly, the coding sequence of pi 9 protein of tomato bushy stunt virus (TBSV) was linked to the alfalfa plastocyanin expression cassette by the PCR-based ligation method presented in Darveau et al. (Methods in Neuroscience 26: 77-85(1995)). In a first round of PCR, a segment of the plastocyanin promoter was amplified using primers Plasto-443c:
- the plasmids were used to transform Agrobacteium tumefaciens (AGL1 ; ATCC, Manassas, VA 20108, USA) by electroporation (Mattanovich et al., 1989). The integrity of all A. tumefaciens strains were confirmed by restriction mapping.
- the A. tumefaciens strain comprising R472 is termed "AGL1/R472 ".
- HcPro construct 35HcPro was prepared as described in Hamilton et al. (2002). All clones were sequenced to confirm the integrity of the constructs. The plasmids were used to transform Agrobacteium tumefaciens (AGL1 ; ATCC, Manassas, VA 20108, USA) by electroporation (Mattanovich et al., 1989). The integrity of all A. tumefaciens strains were confirmed by restriction mapping.
- Nicotiana benthamiana plants were grown from seeds in flats filled with a commercial peat moss substrate. The plants were allowed to grow in the greenhouse under a 16/8 photoperiod and a temperature regime of 25°C day/20°C night. Three weeks after seeding, individual plantlets were picked out, transplanted in pots and left to grow in the greenhouse for three additional weeks under the same environmental conditions. After six weeks, plants have an average weight of 80 g and 30 cm in height.
- Agrobacterium strain AGL1 was transfected (electroporation) with constructs as identified below, using the methods described by D'Aoust et al 2008 (Plant Biotechnology Journal 6:930-940). Transfected Agrobacterium were grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 ⁇ acetosyringone, 50 ⁇ g/ml kanamycin and 25 ⁇ g/ml of carbenicillin pH5.6 to an OD 6 oo between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgCl 2 and 10 mM MES pH 5.6).
- MES 2-(N-morpholino)ethanesulfonic acid
- Leaf tissue was collected from the Nicotiana benthamiana plants and cut into ⁇ 1 cm pieces. The leaf pieces were soaked in 500 mM mannitol for 30 minutes at room temperature (RT). The mannitol solution was then removed and changed with the enzyme mix (mixture of cellulases from Trichoderma viride (Onozuka R-10; 3% v/v) and a mixture of pectinases from Rhizopus sp. (MACEROZYMETM; 0.75% v/v; both from Yakult Pharmaceuticals) in
- protoplasting solution 500 mM mannitol, lOmM CaCl 2 and 5 mM MES/KOH (pH 5.6)). The ratio used was 20 g of leaf pieces per 100 mL solution. This preparation was spread evenly into a shallow vessel (—1 1x18 cm) and incubated for 16 hours on a rotary shaker at 40 rpm and 26°C.
- VLP extraction may be performed as follows: plants were
- Leaf tissue was collected from the N. benthamiana plants at day 6 post-infiltration and cut into ⁇ 1 cm 2 pieces.
- Multifect Pectinase FE, Multifect CX CG and Multifect CX B (Genencor) were added to 1.0% each (v/v) in a 600 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer using a ratio of 1 :2.5 (w/v) fresh biomass; digestion buffer. The biomass was digested for 15h at room temperature in a orbital shaker. [00120] Following incubation, leaf debris was removed by filtration (nylon filter of 250 or 400 ⁇ mesh).
- Protoplasts in suspension were collected by centrifugation at 200xg (15 min), followed by centrifugation of the supernatant at 5000xg (15 min) to further clarify the supernatant. Alternately, a single centrifugation step at 5000 xg for 15 minutes may be employed. Seventy mL of the supernatant was then centrifuged at 70,000xg for 30 minutes. The resulting pellet was resuspended in 1.7mL of PBS and analyzed immediately or frozen.
- a hemagglutination assay for H5 was based on a method described by Nayak and Reichl (2004). Briefly, serial double dilutions of the test samples (100 ⁇ _) were made in V- bottomed 96-well microtiter plates containing 100 PBS, leaving 100 of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, NY) were added to each well, and plates were incubated for 2h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as hemagglutination activity. In parallel, a recombinant HA5 standard (A/Vietnam/ 1203/2004 H5N1) (Protein Science Corporation, Meriden, CT) was diluted in PBS and run as a control on each plate.
- HA5 standard was prepared with purified virus-like particles which were disrupted by treatment with 1% Triton X-100 followed by mechanical agitation in a Tissue LyserTM (Qiagen) for 1 min.
- Tissue LyserTM Qiagen
- U-bottom 96-well microtiter plates were coated with 10 ⁇ g/mL of capture antibody (Immune Technology Corporation, #IT-003-005I) in 50 raM carbonate- bicarbonate coating buffer (pH 9.6) for 16-18 hours at 4°C. All washes were performed with 0.01 M PBS (phosphate-buffered saline), pH 7.4 containing 0.1% Tween-20.
- HA5 standard was diluted in a mock extract (prepared from leaf tissue infiltrated with AGL1/R472 alone) to generate a standard curve from 500 to 50 ng/mL. Samples to quantify were treated in 1% Triton X-100 prior to loading the microplate. Plates were further incubated for 1 hour at 37°C. After washing, sheep polyclonal antibody raised against HA5 (CBER/FDA) diluted 1 : 1000 was added and the plates were incubated for 1 hour at 37°C.
- CBER/FDA sheep polyclonal antibody raised against HA5
- Example 1 Enzymatic extraction of plant tissue releases high quantities of HA having an elevated relative activity.
- HA obtained from the present enzymatic extraction method were compared with that of HA obtained from common mechanical extraction methods.
- N. benthamiana plants were infiltrated with AGL1/685 and the leaves were harvested after a five to six-day incubation period.
- Leaf homogenates were prepared as follows : Two grams of leaves were homogenized with a Polytron homogenizer; 4g of leaves were ground with a mortar and a pestle; and 25kg of leaves were homogenized with a COMITROLTM processor (Urschel Laboratories) in an extraction buffer (50 mM Tris, 150 mM NaCl pH 8.0, ratio of 1 :1 w/v).
- Enzymatic extraction was carried as follow.
- Table 3 Comparative quantities and relative activities of HA obtained from mechanical and enzymatic extraction of plant leaves. All data have been adjusted to take into account the differences in volume of liquid added for each extraction method. The ComitrolTM extraction method was chosen as the standard value for the purpose of the present comparative analysis.
- a combination of differential centrifugation and size exclusion chromatography (SEC) was used to demonstrate that the HA obtained by the enzymatic extraction method described herein were organized as VLPs.
- N. benthamiana plants were agroinfiltrated with AGL1/685 as described in Example 1. Leaves were collected from the plants 6 days post- infiltration, cut into ⁇ 1 cm 2 pieces and then digested, coarse-filtered and centrifuged as described in Example 1.
- the clarified samples were then centrifuged at 70,000xg to allow for segregation of VLPs.
- the centrifugation pellet, containing the VLPs was gently resuspended in 1/50 volume of Phosphate buffered saline (PBS; 0.1M sodium phosphate, 0.15 NaCl pH 7.2) before being loaded on a SEC column.
- PBS Phosphate buffered saline
- equilibration/elution buffer The eluate was collected in fractions of 1.7 mL, and the protein content of each fraction was evaluated by mixing 10 of the eluate fraction with 200 of diluted Bio-Rad protein dye reagent (Bio-Rad, Hercules, CA). Each separation was preceded by a calibration with Blue Dextran 2000 (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). Comparison of the elution profiles of both Blue Dextran 2000 and host proteins was performed for each separation to ensure uniformity of the separations.
- test samples 100 ⁇ were made in V-bottomed 96-well microtiter plates containing 100 PBS, leaving 100 ⁇ , of diluted sample per well.
- a 0.25% turkey red blood cells suspension Bio Link inc., Syracuse, NY
- Figure 3 A shows that the hemagglutination activity is concentrated in the fractions corresponding to the void volume of the column, confirming that the hemagglutination activity originates from a high molecular weight structural organization.
- SDS-PAGE analysis revealed that those same void volume fractions (fractions 7-10) also present the highest HA content, with a band corresponding to the HA0 monomer being detectable at approximately 75 kDa.
- Example 3 Enzymatic digestion of plant tissue releases HA-VLPs with fewer contaminants
- N. benthamiana plants were agroinfiltrated with AGL1/685 as described in Example 1. Leaves were collected on day 6 post-infiltration, cut into ⁇ 1 cm pieces, digested, coarse- filtered and centrifuged as described in Example 1.
- Figure 4 shows a SDS-PAGE analysis of the resulting solution obtained following the controlled enzymatic digestion of leaves tissue as described previously, with lane 1 showing the enzyme mixture used and lane 2 showing the resulting solution following the enzymatic digestion.
- the protein content of a crude extract from ComitrolTM is provided on lane 3 for comparison.
- the biomass:buffer ratio for the extract presented in lane 2 was 1 :5 (w/v) while it was 1 : 1 (w/v) for that in lane 3.
- Each of lanes 2 and 3 therefore contain proteins derived from an equivalent quantity of starting material.
- a mechanical plant extract contained a protein concentration of approximately 3.5-4 mg/ml, while the enzymatic plant extract obtained according to the present method presented a protein concentration of approximately 1 mg/ml.
- the major contaminant present in lane 3 was found to be RubisCo (Ribulose-1,5- bisphosphate carboxylase oxygenase), which is made of two types of protein subunits: a large- chain (L, about 55 kDa) and a small-chain (S, about 13 kDa). A total of eight large-chain dimers and eight small-chains usually assemble with each other into a RubisCo 540 kDa larger complex. While this plant protein contaminant is found in large amount in plant extracts originating from mechanical extraction method (see arrow in Figure 4), it is virtually absent in plant extracts obtained by the enzymatic digestion method described herein. Therefore, the present method allows for the elimination of this major plant protein contaminant, amongst others, at an early stage of the process.
- RubisCo Rostase
- Example 4 Enzymatic digestion of plant tissue releases HA-VLP in conditions where it can be directly captured on a cation exchange resin.
- N. benthamiana plants were agroinfiltrated with AGL 1/685 as described in Example 1. Leaves were collected on day 6 post-infiltration, cut into ⁇ 1 cm pieces and digested for 15h at room temperature in an orbital shaker.
- the digestion buffer contained 1.0% (v/v) Multifect Pectinase FE, 1.0% (v/v) Multifect CX CG orand 1.0% (v/v) Multifect CX B (all from Genencor), each in a solution of 600 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer using a biomass : digestion buffer ratio of 1 : 2.5 (w/v).
- the apoplastic fraction was filtered through a 400 ⁇ nylon filter to remove coarse undigested vegetal tissue ( ⁇ 5% of starting biomass). The filtered extract was then centrifuged at room temperature for 15 min at 5000xg to remove protoplasts and intracellular contaminants (proteins, DNA, membranes, vesicles, pigments, etc). Next, the supernatant was depth-filtered (for clarification) using a 0.65 ⁇ glass fiber filter
- N. benthamiana plants were agroinfiltrated with Agrobacterium AGL1 strains carrying a construct expressing a hemagglutinin of interest (Hl/Cal WT, B/Flo, H5/Indo or Hl/Cal XI 79 A) as described in Example 1. Leaves were collected on day 6 post-infiltration, cut
- Example 4 Then, ⁇ 1 cm pieces were digested according to Example 4, except where noted below. Filtration, centrifugation and clarification were performed as described in Example 4.
- NaCl was added to digestion buffer to evaluate its potential effect on the HA-VLP recovery rate.
- the suspected advantages were the potential prevention of non-specific association of HA with plant cells or with particle in suspension that are removed during clarification and potential effect on achievement and/or maintenance and/or improvement of colloidal stability of the HA-VLP.
- Table 5 Effect of the addition of NaCl to the digestion step on the HA-VLP recovery yield (as measured by hemagglutination activity unit) after the clarification step.
- N. benthamiana plants were agroinfiltrated with Agrobacteriwn AGL1 strains carrying a construct expressing a hemagglutinin of interest (H5/Indo) as described in Example 1. Leaves were collected on day 6 post-infiltration, cut into ⁇ 1 cm pieces, and digested as described in Example 4, with addition of either 500 mM NaCl or 500 mM NaCl and 25 mM EDTA to the digestion buffer. Filtration, centrifugation and clarification were performed as described in Example 4.
- N. benthamiana plants were agroinfiltrated with Agrobacterium AGL1 strains carrying a construct expressing a hemagglutinin of interest (H5/Indo) as described in Example 1. Leaves were collected on day 6 post-infiltration, cut into ⁇ 1 cm pieces and digested according to Example 4, with modification of digestion buffer to include 0%, 0.25%, 0.5%, 0.75% or 1% v/v Multifect Pectinase FE, Multifect CX- CG cellulase and Multifect CX B cellulose as noted in Tables 7-9. Filtration, centrifugation and clarification were as described in Example 4.
- pectinase has been demonstrated to be nonessential in the digestion buffer. Similar levels of H5/Indo or Hl/Cal WT VLP can be extracted with the present method either in the presence or absence of pectinase. Furthermore, it has been found that reducing the concentration of cellulase when compared to previous examples had no significant impact on the quality of extraction (Table 9).
- Controlling the pH during the digestion can be critical for the extraction of some VLPs. Taking into account that the depolymerisation of the cell wall occurring during the digestion step can release acid sugars that could acidify the solution (i.e. from pH 6 to 5) in the presence of appropriate buffers, and that some VLPs (such as those comprising H3/Bris and B/Flo HA) have already demonstrated a strong sensitivity to mildly acidic conditions, impact of such a potential acidification on the yield of VLP produced was investigated.
- N. benthamiana plants were agroinfiltrated with Agrobacterium AGL1 strains carrying a construct expressing a hemagglutinin of interest (B/Flo, H5/Indo, H3/Bris) as described in Example 1. Leaves were collected on day 6 post-infiltration, cut into ⁇ 1 cm 2 pieces and digested according to Example 4, with modification of digestion conditions to include 500 mM NaCl; 25 or 50 mM EDTA; 0.03 or 0.04 % sodium bisulfite; 0, 100, 200 or 600 mM mannitol, 75, 125 or 150 mM citrate; and/or 75 mM NaP0 ; with the pH of the digestion buffer adjusted as set out in Tables 10-14. Filtration, centrifugation and clarification were as described in Example 4.
- Table 10 Effect of the digestion buffer composition on the extraction yield of B/Flo VLPs.
- All buffers contained 600 mM mannitol, sodium metabisulfite 0.04%
- Tablell Effect of the initial pH of the digestion buffer on the extraction yield of H5/Indo VLPs.
- All digestion buffers contained 600 mM mannitol, sodium metabisulfite 0.04%,125 mM Citrate +
- Table 12 Effect of various digestion buffer components on the extraction yield of B/Flo VLPs.
- Buffer composition was further modified to improve the extraction yield of H3/Brisbane VLPs (Table 13)
- Table 13 Effect of the concentrations of mannitol and sodium bisulfite in the digestion solution on the extraction yield of H3/Bris VLPs.
- mannitol concentration could be reduced to 200 mM without significantly affecting VLPs extraction yield. Further reduction of mannitol concentrations to 100 mM, and even the total omission of mannitol from the digestion solution, did not significantly affect the level of HA-VLP obtained (Table 14). Table 14: Released of H5/Indo VLP from digestion of biomass performed in buffers with different concentration of mannitol.
- Trial 1 mannitol
- Trial 2 lOOmM mannitol
- Example 9 Suitability of enzymatic digestion to a broad variety of HA-VLPs
- the enzymatic digestion method for plant biomass described herein has the potential to be applied to extracting of a broad variety of HA-VLPs. Adding to the extraction of HA-VLPs comprising H5/Indo, Hl/Cal WT VLP, H3/Bris and B/Flo shown in previous examples, the method described herein was also shown to be suitable for the extraction of HA- VLPs from seasonal Hl/Bris and Hl/NC, as shown in Table 15.
- Table 15 Release of seasonal Hl/Bris and Hl/NC VLP from digestion of agroinfiltrated N. benthamiana leaves, (concentration in HA measured by hemagglutination activity, dil :
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Pulmonology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Water Supply & Treatment (AREA)
- Analytical Chemistry (AREA)
Abstract
Description
Claims
Priority Applications (22)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
MX2012003373A MX2012003373A (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps. |
NO10818190A NO2480658T3 (en) | 2009-09-22 | 2010-09-21 | |
IN2637DEN2012 IN2012DN02637A (en) | 2009-09-22 | 2010-09-21 | |
CN2010800423330A CN102549148A (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
US13/497,757 US11826419B2 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived VLPs |
SI201031558T SI2480658T1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
RU2012115661/10A RU2579903C2 (en) | 2009-09-22 | 2010-09-21 | Method of production of virus-like particles of plants |
AU2010300033A AU2010300033B2 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived VLPS |
NZ598481A NZ598481A (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
JP2012530059A JP5554414B2 (en) | 2009-09-22 | 2010-09-21 | Method for preparing plant-derived VLPs |
KR1020127009357A KR101773431B1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
DK10818190.0T DK2480658T3 (en) | 2009-09-22 | 2010-09-21 | PROCEDURE FOR PREPARING PLANT DERIVATIVE VLPs |
ES10818190.0T ES2642631T3 (en) | 2009-09-22 | 2010-09-21 | Method of preparation of plant-derived VLP |
PL10818190T PL2480658T3 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
CA2772962A CA2772962C (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
EP10818190.0A EP2480658B1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
SG2012014254A SG178918A1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
BR112012006415A BR112012006415B1 (en) | 2009-09-22 | 2010-09-21 | method for preparing plant-derived vlps |
IL218393A IL218393A0 (en) | 2009-09-22 | 2012-02-29 | Method of preparign plant-derived vlps |
ZA2012/02836A ZA201202836B (en) | 2009-09-22 | 2012-04-18 | Method of preparing plant-derived vlps |
HRP20171564TT HRP20171564T1 (en) | 2009-09-22 | 2017-10-16 | Method of preparing plant-derived vlps |
IL270661A IL270661B (en) | 2009-09-22 | 2019-11-14 | Method of preparing plant-derived vlps |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US24478609P | 2009-09-22 | 2009-09-22 | |
US61/244,786 | 2009-09-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011035422A1 true WO2011035422A1 (en) | 2011-03-31 |
Family
ID=43795254
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2010/001488 WO2011035422A1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived vlps |
PCT/CA2010/001489 WO2011035423A1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived proteins |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2010/001489 WO2011035423A1 (en) | 2009-09-22 | 2010-09-21 | Method of preparing plant-derived proteins |
Country Status (27)
Country | Link |
---|---|
US (2) | US11833200B2 (en) |
EP (3) | EP3354657B1 (en) |
JP (2) | JP5554414B2 (en) |
KR (4) | KR102115226B1 (en) |
CN (4) | CN107129973A (en) |
AR (2) | AR078433A1 (en) |
AU (2) | AU2010300033B2 (en) |
BR (2) | BR112012006414B1 (en) |
CA (2) | CA2772964C (en) |
DK (3) | DK2480560T3 (en) |
ES (3) | ES2662778T3 (en) |
HR (3) | HRP20220478T1 (en) |
HU (3) | HUE036776T2 (en) |
IL (4) | IL218393A0 (en) |
IN (2) | IN2012DN02637A (en) |
MX (2) | MX2012003373A (en) |
MY (2) | MY164704A (en) |
NO (2) | NO2480658T3 (en) |
NZ (2) | NZ598508A (en) |
PL (3) | PL2480658T3 (en) |
PT (3) | PT3354657T (en) |
RU (2) | RU2567012C2 (en) |
SG (2) | SG178947A1 (en) |
SI (3) | SI2480560T1 (en) |
TR (1) | TR201803560T4 (en) |
WO (2) | WO2011035422A1 (en) |
ZA (2) | ZA201202836B (en) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012083445A1 (en) | 2010-12-22 | 2012-06-28 | Medicago Inc. | Virus like particle production in plants |
WO2012171104A1 (en) | 2011-06-13 | 2012-12-20 | Medicago Inc. | Rabies virus like particle production in plants |
WO2013044390A1 (en) | 2011-09-30 | 2013-04-04 | Medicago Inc. | Increasing virus-like particle yield in plants |
WO2013044203A3 (en) * | 2011-09-23 | 2013-05-23 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Novel influenza hemagglutinin protein-based vaccines |
JP2013198481A (en) * | 2012-02-22 | 2013-10-03 | Mitsubishi Shoji Foodtech Co Ltd | Method for softening vegetable food material, softening preparation, softened vegetable food material, and food using the food material |
WO2014036645A1 (en) | 2012-09-05 | 2014-03-13 | Medicago Inc. | Picornavirus-like particle production in plants |
US20150086589A1 (en) * | 2012-03-22 | 2015-03-26 | Fraunhofer Usa Inc. | Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof |
EP2798059A4 (en) * | 2011-12-21 | 2015-07-29 | Apse Llc | Processes using vlps with capsids resistant to hydrolases |
KR20150134423A (en) * | 2013-03-28 | 2015-12-01 | 메디카고 인코포레이티드 | Influenza virus-like particle production in plants |
WO2016004536A1 (en) | 2014-07-11 | 2016-01-14 | Medicago Inc. | Modifying protein production in plants |
US9452210B2 (en) | 2007-07-13 | 2016-09-27 | Medicago Inc. | Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant |
US9458470B2 (en) | 2007-11-27 | 2016-10-04 | Medicago Inc. | Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin |
US9815873B2 (en) | 2011-03-23 | 2017-11-14 | Medicago Inc. | Method for recovering plant-derived proteins |
CN109310061A (en) * | 2016-03-31 | 2019-02-05 | 日本烟草产业株式会社 | The method that substance is imported into plant |
WO2019104439A1 (en) * | 2017-11-30 | 2019-06-06 | Medicago Inc. | Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins |
WO2020010428A1 (en) * | 2018-07-13 | 2020-01-16 | Medicago Inc. | Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins |
US10744199B2 (en) | 2013-10-11 | 2020-08-18 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Epstein-Barr virus vaccines |
US11191727B2 (en) | 2014-12-31 | 2021-12-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Multivalent nanoparticle-based vaccines |
US11338033B2 (en) | 2016-09-02 | 2022-05-24 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof |
US11390878B2 (en) | 2011-09-30 | 2022-07-19 | Medicago Inc. | Increasing protein yield in plants |
CN114934007A (en) * | 2022-07-01 | 2022-08-23 | 广东省农业科学院作物研究所 | Preparation method of sweet potato protoplast |
US11441150B2 (en) | 2014-01-10 | 2022-09-13 | Medicago Inc. | CPMV enhancer elements |
WO2022260849A1 (en) | 2021-06-09 | 2022-12-15 | Nant Holdings Ip, Llc | Methods and systems for producing a protein of interest in a plant |
US11826419B2 (en) | 2009-09-22 | 2023-11-28 | Medicago Inc. | Method of preparing plant-derived VLPs |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010148511A1 (en) | 2009-06-24 | 2010-12-29 | Medicago, Inc. | Chimeric influenza virus-like particles comprising hemagglutinin |
US9617297B2 (en) * | 2012-10-11 | 2017-04-11 | The Regents Of The University Of California | Apoplast wash fluid recovery for improved recombinant endoglucanase extraction in tabacco leaves |
JP6358257B2 (en) | 2013-09-06 | 2018-07-18 | 三菱ケミカル株式会社 | Protein production method using plants |
CN109112093B (en) * | 2018-09-05 | 2021-11-09 | 上海交通大学 | Method for efficient separation and instantaneous transformation of artemisia apiacea protoplast |
JP2022529503A (en) * | 2019-04-25 | 2022-06-22 | フラッグシップ パイオニアリング イノベーションズ シックス,エルエルシー | Compositions and Methods for Plant Messenger Packs |
CN110483616B (en) * | 2019-07-31 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Method for separating apoplast effector protein secreted by pathogenic bacteria from plant tissue infected by pathogenic bacteria |
CN111060696B (en) * | 2019-12-27 | 2023-09-08 | 湖南农业大学 | Method for reducing false positive rate of plant small molecule signal peptide |
CN111718394A (en) * | 2020-06-28 | 2020-09-29 | 广西壮族自治区农业科学院 | Method for extracting sugarcane tissue denatured protein based on BPP method |
CN111826394A (en) * | 2020-08-07 | 2020-10-27 | 浙江华缔药业集团医药开发有限公司 | Construction and application of plant transient expression vector |
Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4945050A (en) | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5036006A (en) | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5100792A (en) | 1984-11-13 | 1992-03-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues |
US5232833A (en) | 1988-09-14 | 1993-08-03 | Stressgen Biotechnologies Corporation | Accumulation of heat shock proteins for evaluating biological damage due to chronic exposure of an organism to sublethal levels of pollutants |
US5625136A (en) | 1991-10-04 | 1997-04-29 | Ciba-Geigy Corporation | Synthetic DNA sequence having enhanced insecticidal activity in maize |
WO2000009725A2 (en) | 1998-08-11 | 2000-02-24 | Biosource Technologies, Inc. | Method for recovering proteins from the interstitial fluid of plant tissues |
WO2000037663A2 (en) | 1998-12-23 | 2000-06-29 | The Samuel Roberts Noble Foundation, Inc. | Plant transformation process |
WO2000063400A2 (en) | 1999-04-21 | 2000-10-26 | The Samuel Roberts Noble Foundation | Plant transformation process |
US6403865B1 (en) | 1990-08-24 | 2002-06-11 | Syngenta Investment Corp. | Method of producing transgenic maize using direct transformation of commercially important genotypes |
WO2003025124A2 (en) | 2001-09-14 | 2003-03-27 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Immunoglobulin having particular framework scaffold and methods of making and using |
WO2004098533A2 (en) * | 2003-05-05 | 2004-11-18 | Boyce Thompson Institute For Plant Research | Vectors and cells for preparing immunoprotective compositions derived from transgenic plants |
WO2006119516A2 (en) | 2005-04-29 | 2006-11-09 | University Of Cape Town | Expression of viral proteins in plants |
WO2009009876A1 (en) | 2007-07-13 | 2009-01-22 | Medicago Inc. | Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant |
WO2009076778A1 (en) | 2007-11-27 | 2009-06-25 | Medicago Inc. | Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin |
WO2010003325A1 (en) | 2008-07-10 | 2010-01-14 | 华为技术有限公司 | A method, apparatus and system for replacing advertisements |
WO2010003225A1 (en) | 2008-07-11 | 2010-01-14 | Medicago, Inc. | Influenza virus-like particles (vlps) comprising hemagglutinin |
Family Cites Families (61)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0203177A4 (en) | 1984-11-29 | 1987-04-28 | Scripps Clinic Res | Polypeptides and antibodies related to deglycosylated viral glycoproteins. |
JPS61265086A (en) * | 1985-05-21 | 1986-11-22 | Mitsui Toatsu Chem Inc | Method of cultivating protoplast |
EP0509656B1 (en) | 1991-03-28 | 1996-09-18 | Rooperol (Na) Nv | Compositions of phytosterols and phytosterolins as immunmodulators |
US6326470B1 (en) | 1997-04-15 | 2001-12-04 | The Penn State Research Foundation | Enhancement of accessibility of cellulose by expansins |
US5762939A (en) | 1993-09-13 | 1998-06-09 | Mg-Pmc, Llc | Method for producing influenza hemagglutinin multivalent vaccines using baculovirus |
GB9414118D0 (en) | 1994-07-13 | 1994-08-31 | Axis Genetics Ltd | Modified plant viruses as vectors of heterologous peptides |
US6020169A (en) | 1995-07-20 | 2000-02-01 | Washington State University Research Foundation | Production of secreted foreign polypeptides in plant cell culture |
US5773695A (en) * | 1996-01-26 | 1998-06-30 | North Carolina State University | Plant nuclear scaffold attachment region and method for increasing gene expression in transgenic cells |
US6042832A (en) | 1996-08-28 | 2000-03-28 | Thomas Jefferson University | Polypeptides fused with alfalfa mosaic virus or ilarvirus capsid proteins |
US20010006950A1 (en) | 1998-02-11 | 2001-07-05 | Juha Punnonen | Genetic vaccine vector engineering |
US6489537B1 (en) | 1998-08-07 | 2002-12-03 | The Trustees Of The University Of Pennsylvania | Phytochelatin synthases and uses therefor |
US6287570B1 (en) | 1998-11-23 | 2001-09-11 | Patricia L. Foley | Vaccine against swine influenza virus |
FR2791358B1 (en) | 1999-03-22 | 2003-05-16 | Meristem Therapeutics | CHEMICAL EXPRESSION PROMOTERS, EXPRESSION CASSETTES, PLASMIDS, VECTORS, PLANTS AND TRANSGENIC SEEDS CONTAINING THEM AND METHODS OF OBTAINING THEM |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
WO2002012454A1 (en) * | 2000-08-08 | 2002-02-14 | The Kitasato Institute | Virus-like micrograins and process for producing the same |
DK1362109T3 (en) | 2001-01-18 | 2009-07-27 | Vlaams Interuniv Inst Biotech | Recombinant oligomeric protein complexes with enhanced immunogenic potential |
ES2806412T3 (en) | 2002-02-13 | 2021-02-17 | Wisconsin Alumni Res Found | Signal for packaging of influenza virus vectors |
AU2003213065A1 (en) | 2002-02-14 | 2003-09-04 | Novavax, Inc. | Novel insect cell line |
AU2003219402B2 (en) | 2002-03-19 | 2008-05-08 | Stichting Dienst Landbouwkundig Onderzoek | GnTIII (UDP-n-acethylglucosamine:beta-D mannoside beta (1,4)-N-acethylglucosaminy ltransferase III) expression in plants |
KR20060029214A (en) | 2003-05-05 | 2006-04-05 | 다우 아그로사이언시즈 엘엘씨 | Stable immunoprophylactic and therapeutic compositions derived from transgenic plant cells and methods for production |
WO2005018539A2 (en) | 2003-06-16 | 2005-03-03 | Medimmune Vaccines, Inc. | Influenza hemagglutinin and neuraminidase variants |
US8592197B2 (en) | 2003-07-11 | 2013-11-26 | Novavax, Inc. | Functional influenza virus-like particles (VLPs) |
CN1560227A (en) * | 2004-02-25 | 2005-01-05 | 福建师范大学 | Preparation and regenerating technology for protoplasm of rainy red ball alga |
CN101065145B (en) | 2004-08-13 | 2010-12-15 | 印度科学工业研究所 | A chimeric G protein based rabies vaccine |
US9155483B2 (en) | 2004-12-03 | 2015-10-13 | The Invention Science Fund I, Llc | Vision modification with reflected image |
NZ594810A (en) * | 2005-03-15 | 2012-12-21 | Verenium Corp | Cellulases, nucleic acids encoding them and methods for making and using them |
CN100410378C (en) | 2005-05-09 | 2008-08-13 | 中国农业科学院生物技术研究所 | Coding hemaagglutinin gene of poultry influenza virus, plant expressing carrier and application thereof |
CN101203612B (en) * | 2005-06-24 | 2013-03-20 | 拜尔作物科学公司 | Methods for altering the reactivity of plant cell walls |
CN101227920A (en) | 2005-07-19 | 2008-07-23 | 陶氏环球技术公司 | Recombinant flu vaccines |
US7871626B2 (en) | 2005-08-04 | 2011-01-18 | St. Jude Children's Research Hospital | Modified influenza virus for monitoring and improving vaccine efficiency |
CA2625406C (en) | 2005-10-18 | 2016-08-09 | Novavax, Inc. | Functional influenza virus like particles (vlps) |
ES2514316T3 (en) | 2005-11-22 | 2014-10-28 | Novartis Vaccines And Diagnostics, Inc. | Norovirus and Sapovirus virus-like particles (VLPs) |
EP1984405A4 (en) | 2006-02-13 | 2010-06-30 | Fraunhofer Usa Inc | Influenza antigens, vaccine compositions, and related methods |
JP2009535306A (en) | 2006-04-21 | 2009-10-01 | ダウ・アグロサイエンス・エル・エル・シー | Vaccines against bird flu and methods of use |
US8778353B2 (en) | 2006-05-01 | 2014-07-15 | Technovax, Inc. | Influenza virus-like particle (VLP) compositions |
CA2657849A1 (en) | 2006-05-18 | 2008-05-08 | Pharmexa Inc. | Inducing immune responses to influenza virus using polypeptide and nucleic acid compositions |
EP2029755A1 (en) | 2006-05-22 | 2009-03-04 | Plant Bioscience Limited | Bipartite system, method and composition for the constitutive and inducible expression of high levels of foreign proteins in plants |
US20070286873A1 (en) | 2006-05-23 | 2007-12-13 | Williams John V | Recombinant Influenza H5 Hemagluttinin Protein And Nucleic Acid Coding Therefor |
US8523925B2 (en) | 2007-01-17 | 2013-09-03 | Lerner Medical Devices, Inc. | Fiber optic phototherapy device |
US7730950B2 (en) | 2007-01-19 | 2010-06-08 | Halliburton Energy Services, Inc. | Methods for treating intervals of a subterranean formation having variable permeability |
HUE031698T2 (en) | 2007-06-15 | 2017-07-28 | Medicago Inc | Modifying glycoprotein production in plants |
KR100964462B1 (en) | 2007-07-10 | 2010-06-16 | 성균관대학교산학협력단 | An avian influenza virus vaccine and a method for preparing same |
RU2358981C2 (en) * | 2007-08-07 | 2009-06-20 | Центр "Биоинженерия" Российской Академии Наук | Universal avian influenza virus vaccine |
CA2696764A1 (en) | 2007-08-20 | 2009-02-26 | Fraunhofer Usa, Inc. | Prophylactic and therapeutic influenza vaccines, antigens, compositions, and methods |
CN101422128B (en) * | 2007-10-29 | 2011-06-08 | 中国水产科学研究院黄海水产研究所 | Separation and regeneration method of gulfweed protoplast |
CN101909454A (en) * | 2007-12-28 | 2010-12-08 | 荷兰联合利华有限公司 | Process for recovering aroma from tea |
US8799801B2 (en) | 2008-01-16 | 2014-08-05 | Qualcomm Incorporated | Interactive ticker |
CN102089432A (en) | 2008-07-08 | 2011-06-08 | 麦迪卡格公司 | Soluble recombinant influenza antigens |
CN102165062B (en) | 2008-07-18 | 2017-09-08 | 麦迪卡格公司 | New influenza virus immunizing epitope |
WO2010025285A1 (en) | 2008-08-27 | 2010-03-04 | Arizona Board Of Regents For And On Behalf Of Arizona State University | A dna replicon system for high-level rapid production of vaccines and monoclonal antibody therapeutics in plants |
WO2010042551A2 (en) * | 2008-10-06 | 2010-04-15 | Genvault Corporation | Methods for providing cellular lysates from cell wall-containing samples |
WO2010077712A1 (en) | 2008-12-09 | 2010-07-08 | Novavax, Inc. | Bovine respiratory syncytial virus virus-like particle (vlps) |
EP2391644B1 (en) | 2009-01-30 | 2016-04-13 | System of Systems Analytics, Inc. | Conformationally dynamic peptides |
EP3682895A1 (en) | 2009-06-10 | 2020-07-22 | NoNO Inc. | Co-administration of an agent linked to a tat-internalization peptide with a mast cell degranulation inhibitor |
PL3431076T3 (en) | 2009-06-10 | 2022-01-31 | Arbutus Biopharma Corporation | Improved lipid formulation |
WO2010148511A1 (en) | 2009-06-24 | 2010-12-29 | Medicago, Inc. | Chimeric influenza virus-like particles comprising hemagglutinin |
SG178947A1 (en) * | 2009-09-22 | 2012-04-27 | Medicago Inc | Method of preparing plant-derived proteins |
AU2011325827B2 (en) | 2010-11-04 | 2016-08-04 | Medicago Inc. | Plant expression system |
DK2635257T3 (en) | 2010-11-05 | 2017-09-04 | Novavax Inc | Rabies virus-like glycoprotein particles (VLP) |
TWI526539B (en) | 2010-12-22 | 2016-03-21 | 苜蓿股份有限公司 | Method of producing virus-like particles (vlps) in plants and vlp produced by such method |
TWI620816B (en) | 2011-03-23 | 2018-04-11 | 苜蓿股份有限公司 | Method of recovering plant-derived proteins |
-
2010
- 2010-09-21 SG SG2012014718A patent/SG178947A1/en unknown
- 2010-09-21 PL PL10818190T patent/PL2480658T3/en unknown
- 2010-09-21 SI SI201031656T patent/SI2480560T1/en unknown
- 2010-09-21 CN CN201710164586.9A patent/CN107129973A/en active Pending
- 2010-09-21 KR KR1020197012547A patent/KR102115226B1/en active IP Right Grant
- 2010-09-21 MX MX2012003373A patent/MX2012003373A/en active IP Right Grant
- 2010-09-21 PT PT181576299T patent/PT3354657T/en unknown
- 2010-09-21 IN IN2637DEN2012 patent/IN2012DN02637A/en unknown
- 2010-09-21 CN CN2010800423364A patent/CN102549008A/en active Pending
- 2010-09-21 HU HUE10818190A patent/HUE036776T2/en unknown
- 2010-09-21 EP EP18157629.9A patent/EP3354657B1/en active Active
- 2010-09-21 ES ES10818191.8T patent/ES2662778T3/en active Active
- 2010-09-21 BR BR112012006414-2A patent/BR112012006414B1/en active IP Right Grant
- 2010-09-21 AU AU2010300033A patent/AU2010300033B2/en active Active
- 2010-09-21 BR BR112012006415A patent/BR112012006415B1/en active IP Right Grant
- 2010-09-21 PT PT108181900T patent/PT2480658T/en unknown
- 2010-09-21 JP JP2012530059A patent/JP5554414B2/en active Active
- 2010-09-21 CA CA2772964A patent/CA2772964C/en active Active
- 2010-09-21 JP JP2012530060A patent/JP5551780B2/en active Active
- 2010-09-21 EP EP10818190.0A patent/EP2480658B1/en active Active
- 2010-09-21 DK DK10818191.8T patent/DK2480560T3/en active
- 2010-09-21 HU HUE10818191A patent/HUE038557T2/en unknown
- 2010-09-21 RU RU2012115996/10A patent/RU2567012C2/en active
- 2010-09-21 HU HUE18157629A patent/HUE058165T2/en unknown
- 2010-09-21 NZ NZ598508A patent/NZ598508A/en unknown
- 2010-09-21 KR KR1020187005775A patent/KR20180026562A/en not_active Application Discontinuation
- 2010-09-21 SI SI201031558T patent/SI2480658T1/en unknown
- 2010-09-21 NO NO10818190A patent/NO2480658T3/no unknown
- 2010-09-21 KR KR1020127010044A patent/KR20120093223A/en not_active Application Discontinuation
- 2010-09-21 KR KR1020127009357A patent/KR101773431B1/en active IP Right Grant
- 2010-09-21 CN CN2010800423330A patent/CN102549148A/en active Pending
- 2010-09-21 PL PL18157629T patent/PL3354657T3/en unknown
- 2010-09-21 DK DK18157629.9T patent/DK3354657T3/en active
- 2010-09-21 ES ES10818190.0T patent/ES2642631T3/en active Active
- 2010-09-21 SI SI201032108T patent/SI3354657T1/en unknown
- 2010-09-21 MY MYPI2012001251A patent/MY164704A/en unknown
- 2010-09-21 CA CA2772962A patent/CA2772962C/en active Active
- 2010-09-21 NZ NZ598481A patent/NZ598481A/en unknown
- 2010-09-21 DK DK10818190.0T patent/DK2480658T3/en active
- 2010-09-21 WO PCT/CA2010/001488 patent/WO2011035422A1/en active Application Filing
- 2010-09-21 SG SG2012014254A patent/SG178918A1/en unknown
- 2010-09-21 IN IN2591DEN2012 patent/IN2012DN02591A/en unknown
- 2010-09-21 EP EP10818191.8A patent/EP2480560B1/en active Active
- 2010-09-21 PL PL10818191T patent/PL2480560T3/en unknown
- 2010-09-21 PT PT108181918T patent/PT2480560T/en unknown
- 2010-09-21 US US13/497,767 patent/US11833200B2/en active Active
- 2010-09-21 ES ES18157629T patent/ES2911037T3/en active Active
- 2010-09-21 RU RU2012115661/10A patent/RU2579903C2/en active
- 2010-09-21 WO PCT/CA2010/001489 patent/WO2011035423A1/en active Application Filing
- 2010-09-21 NO NO10818191A patent/NO2480560T3/no unknown
- 2010-09-21 MY MYPI2012001248A patent/MY158475A/en unknown
- 2010-09-21 CN CN201710005957.9A patent/CN107090021A/en active Pending
- 2010-09-21 MX MX2012003372A patent/MX2012003372A/en active IP Right Grant
- 2010-09-21 TR TR2018/03560T patent/TR201803560T4/en unknown
- 2010-09-21 AU AU2010300034A patent/AU2010300034B2/en active Active
- 2010-09-21 HR HRP20220478TT patent/HRP20220478T1/en unknown
- 2010-09-21 US US13/497,757 patent/US11826419B2/en active Active
- 2010-09-22 AR ARP100103460A patent/AR078433A1/en unknown
- 2010-09-22 AR ARP100103458A patent/AR078432A1/en unknown
-
2012
- 2012-02-29 IL IL218393A patent/IL218393A0/en unknown
- 2012-03-01 IL IL218422A patent/IL218422B/en active IP Right Grant
- 2012-04-18 ZA ZA2012/02836A patent/ZA201202836B/en unknown
- 2012-04-18 ZA ZA2012/02835A patent/ZA201202835B/en unknown
-
2017
- 2017-10-16 HR HRP20171564TT patent/HRP20171564T1/en unknown
-
2018
- 2018-01-31 IL IL257261A patent/IL257261B/en active IP Right Grant
- 2018-03-27 HR HRP20180509TT patent/HRP20180509T1/en unknown
-
2019
- 2019-11-14 IL IL270661A patent/IL270661B/en unknown
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5036006A (en) | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5100792A (en) | 1984-11-13 | 1992-03-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues |
US4945050A (en) | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5232833A (en) | 1988-09-14 | 1993-08-03 | Stressgen Biotechnologies Corporation | Accumulation of heat shock proteins for evaluating biological damage due to chronic exposure of an organism to sublethal levels of pollutants |
US6403865B1 (en) | 1990-08-24 | 2002-06-11 | Syngenta Investment Corp. | Method of producing transgenic maize using direct transformation of commercially important genotypes |
US5625136A (en) | 1991-10-04 | 1997-04-29 | Ciba-Geigy Corporation | Synthetic DNA sequence having enhanced insecticidal activity in maize |
WO2000009725A2 (en) | 1998-08-11 | 2000-02-24 | Biosource Technologies, Inc. | Method for recovering proteins from the interstitial fluid of plant tissues |
WO2000037663A2 (en) | 1998-12-23 | 2000-06-29 | The Samuel Roberts Noble Foundation, Inc. | Plant transformation process |
WO2000063400A2 (en) | 1999-04-21 | 2000-10-26 | The Samuel Roberts Noble Foundation | Plant transformation process |
WO2003025124A2 (en) | 2001-09-14 | 2003-03-27 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Immunoglobulin having particular framework scaffold and methods of making and using |
WO2004098533A2 (en) * | 2003-05-05 | 2004-11-18 | Boyce Thompson Institute For Plant Research | Vectors and cells for preparing immunoprotective compositions derived from transgenic plants |
WO2006119516A2 (en) | 2005-04-29 | 2006-11-09 | University Of Cape Town | Expression of viral proteins in plants |
WO2009009876A1 (en) | 2007-07-13 | 2009-01-22 | Medicago Inc. | Influenza virus-like particles (vlps) comprising hemagglutinin produced within a plant |
WO2009076778A1 (en) | 2007-11-27 | 2009-06-25 | Medicago Inc. | Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin |
WO2010003325A1 (en) | 2008-07-10 | 2010-01-14 | 华为技术有限公司 | A method, apparatus and system for replacing advertisements |
WO2010003225A1 (en) | 2008-07-11 | 2010-01-14 | Medicago, Inc. | Influenza virus-like particles (vlps) comprising hemagglutinin |
Non-Patent Citations (51)
Title |
---|
AUSUBEL ET AL.: "Current Protocols In Molecular Biology", 1998, JOHN WILEY & SONS |
BILANG ET AL., GENE, vol. 100, 1991, pages 247 - 250 |
CHIBA ET AL., VIROLOGY, vol. 346, 2006, pages 7 - 14 |
D'AOUST ET AL., PLANT BIOTECHNOL. J., vol. 6, 2008, pages 930 - 940 |
D'AOUST ET AL., PLANT BIOTECHNOLOGY JOURNAL, vol. 6, 2008, pages 930 - 940 |
D'AOUST, M.-A. ET AL.: "Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice", PLANT BIOTECHNOL. J., vol. 6, December 2008 (2008-12-01), pages 930 - 940, XP002598510 * |
D'AOUST, M.-A. ET AL.: "The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza", PLANT BIOTECHNOL. J., vol. 8, June 2010 (2010-06-01), pages 607 - 619, XP002598511 * |
DARVEAU ET AL., METHODS IN NEUROSCIENCE, vol. 26, 1995, pages 77 - 85 |
DAVEY MR ET AL., BIOTECHNOLOGY ADVANCES, vol. 23, 2005, pages 131 - 171 |
DEBLOCK ET AL., PLANT PHYSIOLOGY, vol. 91, 1989, pages 694 - 701 |
DT. DENNIS, DH TURPIN, DD LEFEBRVE, DB LAYZELL: "Plant Metabolism, 2nd ed.", 1997, ADDISON-WESLEY, LANGMANS LTD., article MIKI; IYER: "Fundamentals of Gene Transfer in Plants", pages: 561 - 579 |
FISCHER ET AL., J IMM METH, vol. 226, 1999, pages 1 - 10 |
GEIERSON; COREY: "Plant Molecular Biology, 2nd ed.", 1988 |
GIRIDHAR ET AL., PROTOPLASMA, vol. 151, 1989, pages 151 - 157 |
GRECO, VACCINE, vol. 25, 2007, pages 8228 - 8240 |
GUERCHE ET AL., PLANT SCIENCE, vol. 52, 1987, pages 111 - 116 |
HORSCH ET AL., SCIENCE, vol. 227, 1985, pages 1229 - 1231 |
HOWELL ET AL., SCIENCE, vol. 208, 1980, pages 1265 |
HUANG ET AL., BIOTECHNOL. BIOENG., vol. 103, 2009, pages 706 - 714 |
HUANG ET AL., VACCINE, vol. 23, 2005, pages 1851 - 1858 |
HUANG, Z. ET AL.: "Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses", VACCINE, vol. 23, March 2005 (2005-03-01), pages 1851 - 1858, XP027652245 * |
ITO T. ET AL., VIROLOGY, vol. 227, 1997, pages 493 - 499 |
KAUFMAN ET AL.: "Handbook Of Molecular And Cellular Methods In Biology And Medicine", 1995, CRC PRESS |
KLEIN ET AL., NATURE, vol. 327, 1987, pages 70 - 73 |
LIU; LOMONOSSOFF, J. VIROL METH, vol. 105, 2002, pages 343 - 348 |
LIU; LOMONOSSOFF, JOURNAL OF VIROLOGICAL METHODS, vol. 105, 2002, pages 343 - 348 |
MACARIO, A.J.L., COLD SPRING HARBOR LABORATORY RES., vol. 25, 1995, pages 59 - 70 |
MASON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 11745 - 11749 |
MASON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 5335 - 5340 |
MCCORMICK ET AL., PROC NATL ACAD SCI USA, vol. 96, 1999, pages 703 - 708 |
MCPHERSON: "Directed Mutagenesis: A Practical Approach", 1991, IRL PRESS |
MEDEIROS R ET AL., VIROLOGY, vol. 289, 2001, pages 74 - 85 |
MK RAZDAN: "Introduction to Plant Tissue Culture, 2nd ed.", 2003, SCIENCE PUBLISHERS |
MOEHNKE ET AL., BIOTECHNOL LETT, vol. 30, 2008, pages 1259 - 1264 |
MONGRAND ET AL., J. BIOL CHEM, vol. 279, 2004, pages 36277 - 86 |
NEUHAUSE ET AL., THEOR. APPL GENET., vol. 75, 1987, pages 30 - 36 |
NEWELL ET AL., J. EXP BOTANY, vol. 49, 1998, pages 817 - 827 |
NISHIMURA; BEEVERS, PLANT PHYSIOL, vol. 62, 1978, pages 40 - 43 |
PARSELL, D.A.; LINDQUIST, S., ANN. REV. GENET., vol. 27, 1993, pages 437 - 496 |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 2nd ed.", 1989, COLD SPRING HARBOR LABORATORY PRESS |
SANTI ET AL., VACCINE, vol. 26, 2008, pages 1846 - 1854 |
SCHEID ET AL., MOL. GEN. GENET., vol. 228, 1991, pages 104 - 112 |
SCHULER AND ZIELINSKI: "Methods in Plant Molecular Biology", 1989, ACADEMIC PRESS INC. |
SKEHEL; WILEY, ANN REV BIOCHEM, vol. 69, 2000, pages 531 - 69 |
SMITH ET AL., VACCINE, vol. 21, 2003, pages 4011 - 4021 |
SQUIRES ET AL., NUCLEIC ACIDS RESEARCH, vol. 36, 2008, pages D497 - D503 |
TACKET ET AL., J. INFECT. DIS., vol. 182, 2000, pages 302 - 305 |
VARSANI ET AL., ARCH. VIROL., vol. 148, 2003, pages 1771 - 1786 |
VOINNET ET AL., THE PLANT JOURNAL, vol. 33, 2003, pages 949 - 956 |
WEISSBACH AND WEISSBACH: "Methods for Plant Molecular Biology", 1988, ACADEMIC PRESS INC. |
WEISSBACH; WEISSBACH: "Methods for Plant Molecular Biology", vol. VIII, 1988, ACADEMY PRESS, pages: 421 - 463 |
Cited By (57)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9492528B2 (en) | 2007-07-13 | 2016-11-15 | Medicago Inc. | Influenza virus-like particles (VLPS) comprising hemagglutinin |
US9452210B2 (en) | 2007-07-13 | 2016-09-27 | Medicago Inc. | Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant |
US10190132B2 (en) | 2007-11-27 | 2019-01-29 | Medicago Inc. | Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin |
US9458470B2 (en) | 2007-11-27 | 2016-10-04 | Medicago Inc. | Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin |
US11434497B2 (en) | 2007-11-27 | 2022-09-06 | Medicago Inc. | Recombinant influenza virus-like particles (VLPS) produced in transgenic plants |
US11833200B2 (en) | 2009-09-22 | 2023-12-05 | Medicago Inc. | Method of preparing plant-derived proteins |
US11826419B2 (en) | 2009-09-22 | 2023-11-28 | Medicago Inc. | Method of preparing plant-derived VLPs |
WO2012083445A1 (en) | 2010-12-22 | 2012-06-28 | Medicago Inc. | Virus like particle production in plants |
US9815873B2 (en) | 2011-03-23 | 2017-11-14 | Medicago Inc. | Method for recovering plant-derived proteins |
EP2718428A4 (en) * | 2011-06-13 | 2015-07-01 | Medicago Inc | Rabies virus like particle production in plants |
AU2012269684B2 (en) * | 2011-06-13 | 2017-06-22 | Medicago Inc. | Rabies virus like particle production in plants |
WO2012171104A1 (en) | 2011-06-13 | 2012-12-20 | Medicago Inc. | Rabies virus like particle production in plants |
CN103957891B (en) * | 2011-09-23 | 2017-01-11 | 美利坚合众国由健康及人类服务部部长代表 | Novel influenza hemagglutinin protein-based vaccines |
US20140302079A1 (en) * | 2011-09-23 | 2014-10-09 | The United States Of America As Represented By The Secretary, Department Of Health & Human Services | Novel influenza hemagglutinin protein-based vaccines |
US10137190B2 (en) | 2011-09-23 | 2018-11-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Nucleic acid molecules encoding ferritin-hemagglutinin fusion proteins |
US9441019B2 (en) * | 2011-09-23 | 2016-09-13 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Influenza hemagglutinin protein-based vaccines |
CN103957891A (en) * | 2011-09-23 | 2014-07-30 | 美利坚合众国由健康及人类服务部部长代表 | Novel influenza hemagglutinin protein-based vaccines |
WO2013044203A3 (en) * | 2011-09-23 | 2013-05-23 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Novel influenza hemagglutinin protein-based vaccines |
US11390878B2 (en) | 2011-09-30 | 2022-07-19 | Medicago Inc. | Increasing protein yield in plants |
US11155581B2 (en) | 2011-09-30 | 2021-10-26 | Medicago Inc. | Increasing virus-like particle yield in plants |
WO2013044390A1 (en) | 2011-09-30 | 2013-04-04 | Medicago Inc. | Increasing virus-like particle yield in plants |
EP3626733A1 (en) | 2011-09-30 | 2020-03-25 | Medicago Inc. | Increasing virus-like particle yield in plants |
EP2798059A4 (en) * | 2011-12-21 | 2015-07-29 | Apse Llc | Processes using vlps with capsids resistant to hydrolases |
JP2013198481A (en) * | 2012-02-22 | 2013-10-03 | Mitsubishi Shoji Foodtech Co Ltd | Method for softening vegetable food material, softening preparation, softened vegetable food material, and food using the food material |
US9364530B2 (en) * | 2012-03-22 | 2016-06-14 | Fraunhofer Usa, Inc. | Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof |
US20150086589A1 (en) * | 2012-03-22 | 2015-03-26 | Fraunhofer Usa Inc. | Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof |
WO2014036645A1 (en) | 2012-09-05 | 2014-03-13 | Medicago Inc. | Picornavirus-like particle production in plants |
RU2705555C2 (en) * | 2013-03-28 | 2019-11-07 | Медикаго Инк. | Obtaining virus-like particles of influenza virus in plants |
US10358652B2 (en) | 2013-03-28 | 2019-07-23 | Medicago Inc. | Influenza virus-like particle production in plants |
AU2014245779B2 (en) * | 2013-03-28 | 2020-02-06 | Aramis Biotechnologies Inc. | Influenza virus-like particle production in plants |
EP3626827A1 (en) * | 2013-03-28 | 2020-03-25 | Medicago Inc. | Influenza virus-like particle production in plants |
IL241692B1 (en) * | 2013-03-28 | 2023-08-01 | Medicago Inc | Influenza virus-like particle production in plants |
KR20150134423A (en) * | 2013-03-28 | 2015-12-01 | 메디카고 인코포레이티드 | Influenza virus-like particle production in plants |
KR102199018B1 (en) * | 2013-03-28 | 2021-01-07 | 메디카고 인코포레이티드 | Influenza virus-like particle production in plants |
RU2742607C1 (en) * | 2013-03-28 | 2021-02-09 | Медикаго Инк. | Obtaining virus-like particles of influenza virus in plants |
US11085049B2 (en) | 2013-03-28 | 2021-08-10 | Medicago Inc. | Influenza virus-like particle production in plants |
EP2978848A4 (en) * | 2013-03-28 | 2016-11-16 | Medicago Inc | Influenza virus-like particle production in plants |
US12005115B2 (en) | 2013-10-11 | 2024-06-11 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Epstein-barr virus vaccines |
US10744199B2 (en) | 2013-10-11 | 2020-08-18 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Epstein-Barr virus vaccines |
US11884929B2 (en) | 2014-01-10 | 2024-01-30 | Medicago Inc. | CPMV enhancer elements |
US11441150B2 (en) | 2014-01-10 | 2022-09-13 | Medicago Inc. | CPMV enhancer elements |
WO2016004536A1 (en) | 2014-07-11 | 2016-01-14 | Medicago Inc. | Modifying protein production in plants |
US11191727B2 (en) | 2014-12-31 | 2021-12-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Multivalent nanoparticle-based vaccines |
US11938221B2 (en) | 2014-12-31 | 2024-03-26 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Multivalent nanoparticle-based vaccines |
CN109310061A (en) * | 2016-03-31 | 2019-02-05 | 日本烟草产业株式会社 | The method that substance is imported into plant |
US11338033B2 (en) | 2016-09-02 | 2022-05-24 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof |
US11793871B2 (en) | 2016-09-02 | 2023-10-24 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof |
US11602558B2 (en) | 2017-11-30 | 2023-03-14 | Medicago Inc. | Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins |
US20200330583A1 (en) * | 2017-11-30 | 2020-10-22 | Medicago Inc. | Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins |
CN111727255A (en) * | 2017-11-30 | 2020-09-29 | 麦迪卡格公司 | Modified norovirus VP1 protein and VLP comprising modified norovirus VP1 protein |
WO2019104439A1 (en) * | 2017-11-30 | 2019-06-06 | Medicago Inc. | Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins |
US12076388B2 (en) | 2017-11-30 | 2024-09-03 | Aramis Biotechnologies Inc. | Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins |
US11759512B2 (en) | 2018-07-13 | 2023-09-19 | Medicago Inc. | Modified norovirus VP1 proteins and VLPs comprising modified norovirus VP1 proteins |
WO2020010428A1 (en) * | 2018-07-13 | 2020-01-16 | Medicago Inc. | Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins |
WO2022260849A1 (en) | 2021-06-09 | 2022-12-15 | Nant Holdings Ip, Llc | Methods and systems for producing a protein of interest in a plant |
CN114934007B (en) * | 2022-07-01 | 2023-11-24 | 广东省农业科学院作物研究所 | Preparation method of sweet potato protoplast |
CN114934007A (en) * | 2022-07-01 | 2022-08-23 | 广东省农业科学院作物研究所 | Preparation method of sweet potato protoplast |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11826419B2 (en) | Method of preparing plant-derived VLPs | |
AU2017202473B2 (en) | Method of recovering plant-derived proteins | |
US9546375B2 (en) | Influenza virus immunizing epitope |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201080042333.0 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10818190 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010300033 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 218393 Country of ref document: IL |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2772962 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12012500566 Country of ref document: PH |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012530059 Country of ref document: JP Ref document number: MX/A/2012/003373 Country of ref document: MX |
|
ENP | Entry into the national phase |
Ref document number: 2010300033 Country of ref document: AU Date of ref document: 20100921 Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13497757 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2637/DELNP/2012 Country of ref document: IN |
|
ENP | Entry into the national phase |
Ref document number: 20127009357 Country of ref document: KR Kind code of ref document: A |
|
REEP | Request for entry into the european phase |
Ref document number: 2010818190 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010818190 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012115661 Country of ref document: RU |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112012006415 Country of ref document: BR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1201001223 Country of ref document: TH |
|
ENP | Entry into the national phase |
Ref document number: 112012006415 Country of ref document: BR Kind code of ref document: A2 Effective date: 20120321 |