CN118725052A - Respiratory syncytial virus F protein with stable pre-fusion conformation - Google Patents
Respiratory syncytial virus F protein with stable pre-fusion conformation Download PDFInfo
- Publication number
- CN118725052A CN118725052A CN202410376573.8A CN202410376573A CN118725052A CN 118725052 A CN118725052 A CN 118725052A CN 202410376573 A CN202410376573 A CN 202410376573A CN 118725052 A CN118725052 A CN 118725052A
- Authority
- CN
- China
- Prior art keywords
- protein
- amino acid
- rsv
- acid residue
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000725643 Respiratory syncytial virus Species 0.000 title claims abstract description 614
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 title claims abstract description 429
- 230000004927 fusion Effects 0.000 title claims abstract description 230
- 230000035772 mutation Effects 0.000 claims abstract description 366
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 246
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 238
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 61
- 239000000203 mixture Substances 0.000 claims abstract description 59
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 57
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 57
- 239000013598 vector Substances 0.000 claims abstract description 51
- 229960005486 vaccine Drugs 0.000 claims abstract description 25
- 230000000087 stabilizing effect Effects 0.000 claims abstract description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 629
- 235000018102 proteins Nutrition 0.000 claims description 227
- 150000001413 amino acids Chemical class 0.000 claims description 150
- 210000004027 cell Anatomy 0.000 claims description 62
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 39
- 239000002245 particle Substances 0.000 claims description 36
- 108020004705 Codon Proteins 0.000 claims description 22
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 22
- 235000001014 amino acid Nutrition 0.000 claims description 21
- 229940024606 amino acid Drugs 0.000 claims description 21
- 230000027455 binding Effects 0.000 claims description 20
- 238000005829 trimerization reaction Methods 0.000 claims description 20
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 19
- 108010033276 Peptide Fragments Proteins 0.000 claims description 15
- 102000007079 Peptide Fragments Human genes 0.000 claims description 15
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 claims description 14
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 14
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 14
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 13
- 239000002671 adjuvant Substances 0.000 claims description 12
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 12
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 11
- 230000028993 immune response Effects 0.000 claims description 11
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims description 10
- 230000002163 immunogen Effects 0.000 claims description 9
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims description 8
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 7
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims description 7
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 230000003612 virological effect Effects 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 6
- 108010075254 C-Peptide Proteins 0.000 claims description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 5
- 108020001507 fusion proteins Proteins 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 5
- 210000005260 human cell Anatomy 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims description 4
- 210000004962 mammalian cell Anatomy 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 101100107610 Arabidopsis thaliana ABCF4 gene Proteins 0.000 claims description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 3
- 101100068078 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GCN4 gene Proteins 0.000 claims description 3
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims description 3
- 230000003308 immunostimulating effect Effects 0.000 claims description 3
- 239000002502 liposome Substances 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 241000699802 Cricetulus griseus Species 0.000 claims description 2
- 206010016654 Fibrosis Diseases 0.000 claims description 2
- 241000238631 Hexapoda Species 0.000 claims description 2
- 241000124008 Mammalia Species 0.000 claims description 2
- 241000144300 Peromyscus gossypinus Species 0.000 claims description 2
- 241000288906 Primates Species 0.000 claims description 2
- 241000283984 Rodentia Species 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 239000003443 antiviral agent Substances 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 230000004761 fibrosis Effects 0.000 claims description 2
- 229960001438 immunostimulant agent Drugs 0.000 claims description 2
- 239000003022 immunostimulating agent Substances 0.000 claims description 2
- 210000001672 ovary Anatomy 0.000 claims description 2
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 claims description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims 1
- 108090000565 Capsid Proteins Proteins 0.000 claims 1
- 102100023321 Ceruloplasmin Human genes 0.000 claims 1
- 241000702421 Dependoparvovirus Species 0.000 claims 1
- 102000008857 Ferritin Human genes 0.000 claims 1
- 108050000784 Ferritin Proteins 0.000 claims 1
- 241000233866 Fungi Species 0.000 claims 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims 1
- 241000712079 Measles morbillivirus Species 0.000 claims 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims 1
- 235000004279 alanine Nutrition 0.000 claims 1
- 229920006317 cationic polymer Polymers 0.000 claims 1
- 239000003814 drug Substances 0.000 claims 1
- 239000002086 nanomaterial Substances 0.000 claims 1
- 241000701161 unidentified adenovirus Species 0.000 claims 1
- 241001529453 unidentified herpesvirus Species 0.000 claims 1
- 230000003472 neutralizing effect Effects 0.000 abstract description 26
- 241000700605 Viruses Species 0.000 abstract description 8
- 239000000427 antigen Substances 0.000 abstract description 7
- 108091007433 antigens Proteins 0.000 abstract description 5
- 102000036639 antigens Human genes 0.000 abstract description 5
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 230000009545 invasion Effects 0.000 abstract description 2
- 210000002966 serum Anatomy 0.000 description 50
- 239000000243 solution Substances 0.000 description 50
- 238000004458 analytical method Methods 0.000 description 42
- 241000699670 Mus sp. Species 0.000 description 39
- 230000003053 immunization Effects 0.000 description 31
- 238000002649 immunization Methods 0.000 description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 28
- 238000002965 ELISA Methods 0.000 description 25
- 238000006243 chemical reaction Methods 0.000 description 21
- 239000000523 sample Substances 0.000 description 19
- 229920001213 Polysorbate 20 Polymers 0.000 description 18
- 230000000903 blocking effect Effects 0.000 description 18
- 238000011156 evaluation Methods 0.000 description 18
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 18
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 18
- 239000013638 trimer Substances 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 238000000746 purification Methods 0.000 description 13
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 12
- 239000011248 coating agent Substances 0.000 description 12
- 238000000576 coating method Methods 0.000 description 12
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 12
- 229920001184 polypeptide Polymers 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 230000001086 cytosolic effect Effects 0.000 description 11
- 238000003860 storage Methods 0.000 description 11
- 238000003776 cleavage reaction Methods 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 230000007017 scission Effects 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 9
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 8
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000001493 electron microscopy Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 229910052802 copper Inorganic materials 0.000 description 7
- 239000010949 copper Substances 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000010369 molecular cloning Methods 0.000 description 6
- 239000012089 stop solution Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 102220634571 AP-2 complex subunit alpha-1_E92V_mutation Human genes 0.000 description 5
- 241000711895 Bovine orthopneumovirus Species 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 102000004961 Furin Human genes 0.000 description 4
- 108090001126 Furin Proteins 0.000 description 4
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 4
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000002808 molecular sieve Substances 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 3
- 108010073385 Fibrin Proteins 0.000 description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 206010061598 Immunodeficiency Diseases 0.000 description 3
- 102400001093 PAK-2p27 Human genes 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000010224 classification analysis Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 229950003499 fibrin Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 239000008174 sterile solution Substances 0.000 description 3
- 229940031626 subunit vaccine Drugs 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 238000011537 Coomassie blue staining Methods 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 102000030782 GTP binding Human genes 0.000 description 2
- 108091000058 GTP-Binding Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000711920 Human orthopneumovirus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010007127 Pulmonary Surfactant-Associated Protein D Proteins 0.000 description 2
- 102100027845 Pulmonary surfactant-associated protein D Human genes 0.000 description 2
- 229940124679 RSV vaccine Drugs 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 230000009137 competitive binding Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000013480 data collection Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- -1 e.g. Chemical group 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012527 feed solution Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 238000012835 hanging drop method Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 2
- 229940126582 mRNA vaccine Drugs 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000004845 protein aggregation Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 230000012743 protein tagging Effects 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 102220511128 APC membrane recruitment protein 1_V56I_mutation Human genes 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 206010006448 Bronchiolitis Diseases 0.000 description 1
- 241001491790 Bupalus piniaria Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102220499772 Carbonic anhydrase 2_N67L_mutation Human genes 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 101710160621 Fusion glycoprotein F0 Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- 206010024971 Lower respiratory tract infections Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 241000200174 Noctiluca Species 0.000 description 1
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102220470717 Transcription factor IIIB 90 kDa subunit_S38A_mutation Human genes 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- NTGGOTYRTOXKMQ-UHFFFAOYSA-K aluminum;potassium;phosphate Chemical compound [Al+3].[K+].[O-]P([O-])([O-])=O NTGGOTYRTOXKMQ-UHFFFAOYSA-K 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 102220346476 c.199A>T Human genes 0.000 description 1
- 102220411436 c.247C>T Human genes 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940001442 combination vaccine Drugs 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 244000309457 enveloped RNA virus Species 0.000 description 1
- 239000012537 formulation buffer Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 208000030500 lower respiratory tract disease Diseases 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000008384 membrane barrier Effects 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 229940023146 nucleic acid vaccine Drugs 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 102200024822 rs121908823 Human genes 0.000 description 1
- 102220028408 rs386352357 Human genes 0.000 description 1
- 102220272830 rs80357350 Human genes 0.000 description 1
- 102220082272 rs863223860 Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000002676 xenobiotic agent Substances 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
- A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
- C07K14/135—Respiratory syncytial virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Pulmonology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to recombinant Respiratory Syncytial Virus (RSV) F protein with stable pre-fusion conformation, a coding nucleic acid molecule thereof, a vector and a composition containing the same, and application of the protein, the nucleic acid molecule, the vector and the composition in preparing vaccines and compositions for preventing and treating. By introducing a series of mutations in RSV F, a RSV F protein is obtained with a stable pre-fusion conformation, the mutation sites not involving the introduction of any unnatural disulfide bond and not being in an important epitopeAny mutation is introduced nearby and on the exposed surface of other critical epitopes of RSV F, so that the immunogenicity and critical conformation of RSV F can be minimized while stabilizing the pre-fusion conformation of RSV F, thereby ensuring that the designed antigen can stably induce the generation of neutralizing antibodies to the critical epitopes for preventing virus invasion.
Description
Technical Field
The present invention relates to the medical field, more precisely to recombinant Respiratory Syncytial Virus (RSV) F protein or corresponding coding nucleic acid molecules with stable pre-fusion conformation and their use, e.g. in vaccine immunization of infants, elderly and immunocompromised persons, which can cause severe respiratory diseases after infection with RSV.
Background
Respiratory Syncytial Virus (RSV) is an enveloped RNA virus of the genus pneumovirus of the family paramyxoviridae, whose genome is a non-segmented single-stranded negative-strand RNA. RSV viruses are currently considered to have only one serotype, which can be divided into A, B subtypes. About 6400 thousands of RSV infection cases worldwide, with 160,000 infected deaths, are counted by the world's health organization. RSV infection produces severe clinical symptoms in premature infants, the elderly and immunocompromised individuals. Almost all children are reported to be infected with RSV before age two. RSV is the major pathogen causing severe lower respiratory disease in children under 2 years of age and its infection is a serious threat to children, especially infants less than 6 months. Also, RSV infection can cause lower respiratory tract infections in the elderly, immunocompromised, and severely underlying disease populations, resulting in mortality in these populations comparable to influenza virus infection.
The RNA genome of RSV encodes 11 viral proteins, of which the adhesion protein G and fusion protein F located on the surface of the envelope are the two major antigens that elicit protective immunity and neutralizing antibodies in the body. RSV G protein functions to adhere to host cells during viral infection of the host cells. The G protein is the least conserved protein among RSV encoding proteins, and its sequence varies widely among different RSV strains. In order to infect host cells, RSV, like other enveloped viruses (e.g., influenza and HIV), requires the break through of the cell membrane barrier by mediating fusion of the viral membrane with the host cell membrane by its encoded proteins. The main function of the RSV surface-conserved fusion protein F (RSV F protein) is to mediate fusion of the virus with the host cell membrane during viral infection, and plays a key role in not being replaced by other viral proteins during viral invasion into host cells. The different subtypes of RSV surface F proteins have a high degree of homology. RSV F induces the production of high titer neutralizing antibodies in the body.
Similar to other envelope virus mediated membrane fusion surface glycoproteins (e.g., influenza HA protein, coronavirus S protein, etc.), RSV F is post-translationally treated with a series of proteases and folded to form a metastable trimer having a "pre-fusion" conformation, as shown in figure 1 of the present specification for pre-and post-fusion conformations and epitopes. During viral entry into the cell, the F protein in its metastable pre-fusion conformation undergoes refolding and conformational changes, transforming into a more stable "post-fusion" conformation. The process of converting RSV F protein from pre-fusion conformation to post-fusion conformation is irreversible. A series of structural studies showed that there was a large structural difference between pre-and post-fusion trimer conformations for RSV-F (McLellan JS et al.science 340 (6136): 1113-7 (2013) and McLellan JS et al.science 342 (6158): 592-8 (2013)). Whereas a series of studies on the immune-related properties of the F protein showed that RSV F proteins with pre-and post-fusion conformations differ significantly in immunogenicity (Calder, LJ et al virology 271,122-131 (2000)), where pre-fusion RSV F has more critical neutralizing antibody binding epitopes, such as the neutralizing antibody binding epitope located near the top of the trimer as shown in FIG. 2Epitope V. While only a portion of antibodies directed against the post-fusion conformation of RSV-F are capable of cross-reacting with the native conformation of the pre-fusion spike on the viral surface. As shown in figure 2 of the specification, the important neutralizing antibody binds to an epitopeEpitope V is present only in RSV F, which has a pre-fusion conformation. Recombinant RSV F protein studies have shown that its pre-fusion conformation is very unstable, with the vast majority of recombinant RSV F proteins in the post-fusion conformation.
There is no vaccine currently on the market against RSV infection. The related vaccine development experiments have been performed by the company xenobiotics using RSV inactivated virus. But failed due to severe infection enhancement phenomena caused by the relevant candidate vaccine. RSV F protein is an important subunit vaccine candidate antigen, and immunization based on RSV F protein does not cause related infection enhancement phenomenon, but vaccine candidates based on RSV F protein have not been breakthrough developed at present due to the problems of low stability, insufficient purity, poor reproducibility, low efficacy, and the like. Current research on RSV vaccines has focused on developing RSV F protein or nucleic acid vaccines with stable pre-fusion conformational forms (see, e.g., WO2010/1149745, WO2010/1149743, WO2009/1079796, WO 2012/158613), and no RSV F protein vaccine currently approved for use in humans (particularly infants).
Disclosure of Invention
Unless otherwise indicated, reference herein to RSV refers specifically to respiratory syncytial virus that infects humans. RSV viruses that infect other hosts will be prefixed with a prefix before RSV, e.g., bovine RSV refers specifically to bovine-infected RSV viruses. The amino acids described in the present application may be any of the twenty naturally occurring (or "standard" amino acids) or variants thereof, such as D-amino acids (D-enantiomers of amino acids with chiral centers), or any variants that do not naturally occur in proteins, such as norleucine. Standard amino acids can be divided into groups according to their nature, depending on the charged, hydrophilic or hydrophobic, size and functional groups of the amino acid. The nature of the amino acids in the different groups is important for protein structure and protein-protein interactions. Some amino acids have specific properties, such as cysteine, which can form covalent disulfide bonds (or disulfide bonds) with other cysteine residues; charged amino acids such as arginine, lysine, aspartic acid, and glutamic acid, and positively charged amino acid residues can form a salt bond between them to stabilize the protein structure; proline induces turning of the protein backbone; hydrophobic amino acids such as leucine, isoleucine, methionine and the like form a hydrophobic core stabilizing protein structure within the protein structure, which may cause protein aggregation, such as exposure to the outer surface of the protein structure; and glycine, which is more flexible than other amino acids, allows increased flexibility of the peptide chain in the polypeptide chain. Amino acids in a polypeptide chain are collectively referred to as amino acid residues, e.g., leucine in a polypeptide chain is referred to as leucine residues. The nucleotide sequences referred to throughout the present application are in the 5 'to 3' direction and the amino acid sequences are from N-terminal to C-terminal as is conventional in the art.
Fusion protein F of Respiratory Syncytial Virus (RSV) mediates fusion of viral membrane with host cell membrane during viral infection of host cells, a critical protein necessary for infection. RSV F mRNA is translated into a precursor protein designated F0. F0 polypeptide chain contains 574 amino acid residues (SEQ ID NO: 1) and has a signal peptide sequence containing 26 amino acid residues at the N-terminus (amino acid residues 1-26 in SEQ ID NO: 1) which is removed by a signal peptidase in the endoplasmic reticulum. F0 is cleaved by cellular proteases, particularly furin, at two sites (between amino acid residues 109/110 and 136/137) (SEQ ID NO: 1) as shown in FIG. 3 of the specification, resulting in three peptide stretches, including F1 (amino acid residues 137-574 in SEQ ID NO: 1), F2 (amino acid residues 27-109 in SEQ ID NO: 1) and P27 (amino acid residues 110-136 in SEQ ID NO: 1). The folding of the two peptide fragments of F1 and F2 results in two domains or subunits, designated as the F1 domain and the F2 domain. The F1 domain (amino acid residues 137-574) comprises at its N-terminus a hydrophobic fusion peptide (amino acid residues 137-153 in SEQ ID NO: 1), and at its C-terminus a Transmembrane (TM) (amino acid residues 530-550 in SEQ ID NO: 1) and a cytoplasmic domain (amino acid residues 551-574 in SEQ ID NO: 1). The peptide stretch (amino acid residues 154 to 207, hra) of F1 immediately adjacent to its N-terminal hydrophobic fusion peptide and the peptide stretch (amino acid residues 486 to 513, hrb) immediately adjacent to its C-terminal transmembrane region contain a heptad repeat. Heptad repeats are markers that can form coiled-coil structures. As shown in FIG. 1 of the specification, it is known that in the pre-fusion conformation of RSV F, F1 and F2 are spatially interleaved together to form a heterodimer F1-F2, and F1 is covalently linked to F2 (amino acid residues 27-109 of SEQ ID NO: 1) via two disulfide bonds (C69-C212, C37-C439). Three F1-F2 heterodimers assemble into a homotrimer on the virus surface, the trimer with pre-fusion conformation (F1-F2) 3 is overall ellipsoidal in shape, and when the trimer (F1-F2) 3 is transformed into post-fusion conformation, it is overall long-cone-shaped. The HRB in F1 forms a segment of alpha helix structure which is located at one end of the ellipsoidal near-viral membrane in the trimer with pre-fusion conformation (F1-F2) 3 and loosely aggregates with the HRB alpha helices in the other two F1 molecules. During the transition of the trimer (F1-F2) 3 structure from pre-fusion to post-fusion, the structure of the F2 peptide is not changed much, but refolding and rearrangement occurs in most regions of the F1 peptide. In the post-fusion conformation of trimers (F1-F2) 3, the HRA region containing the heptad repeat sequence is folded 180 degree inverted to form a long alpha helix and stacked with HRB, which is also 180 degree inverted, to form a 6 helix bundle structure containing 6 stable alpha helices. In the pre-fusion structure of trimer (F1-F2) 3, the C-terminus of F2 is not too far from the N-terminus of F1. however, in the post-fusion structure of trimer (F1-F2) 3, the C-terminus of F2 is far from the N-terminus of F1, so that although cleavage site mutation studies of the protease between F1 and F2 indicate that uncleaved F0 can form a trimer having a pre-fusion conformation, F0 must be converted to F1 and F2 after cleavage before the conversion from the pre-fusion conformation to the post-fusion conformation can be properly completed.
RSV is known to have two subtypes a and B. RSV F is highly sequence-conserved among different RSV subtype strains. As shown in fig. 4 and 5 of the specification, the F protein sequences derived from different strains in the RSV a subtype and the F protein sequences derived from different strains in the RSV B subtype are compared, the amino acid sequences of the F proteins in different strains of the same subtype are about 98% identical, and the F protein sequences derived from different RSV subtypes are compared, and as shown in fig. 6 of the specification, the amino acid sequences of the F proteins between the two different strains of the two subtypes are about 93% identical. Further, as shown in fig. 7, it was found that the sequence of RSV F was about 80% identical to that of bovine RSV F by comparison of the sequence of the virus strain A2 RSV F with that of bovine RSV F. The amino acid sequence of a representative strain F protein in the RSV subtype B is shown in SEQ ID NO: 2.
Amino acid residue positions used throughout the present application refer to the RSV F protein sequence (SEQ ID NO: 1) from the A2 strain. Thus, the phrase as used herein, such as "amino acid residue (x) of RSV F protein" refers to a polypeptide that hybridizes to the sequence of SEQ ID NO:1, and the amino acid residue corresponding to the amino acid residue at (x) position in the protein F of the strain A2 of RSV. In the numbering system used throughout the present application, 1 refers to the N-terminal amino acid residue of the precursor protein F0 (SEQ ID NO: 1). When using RSV strains other than the A2 strain, the amino acid residue position of the F protein is referred to the A2 strain F protein numbering of SEQ ID NO. 1. The sequence of the F protein of another RSV strain may be numbered by alignment with the F protein of SEQ ID NO. 1 and inserted into the gap as desired. Sequence alignment can be performed using art-recognized methods, such as by software CLUSTALW or manually performing related multiple sequence alignments, typical results of which are shown in figures 4,5,6 and 7 of the specification. Reference may also be made directly to the RSV F protein sequence from the RSV subtype B (SEQ ID NO: 2).
One potential method of developing RSV vaccines is based on purified RSV F protein subunit vaccines. However, for this approach, the ideal RSV subunit vaccine is to use the purified RSV F protein in a stable pre-fusion conformation, and its conformation is stable for long periods of time under certain storage conditions, and this form of RSV F protein can be produced in large quantities. Furthermore, because RSV F is a transmembrane protein, its C-terminal peptide stretch comprises a Transmembrane (TM) (amino acid residues 530-550) and a cytoplasmic region (amino acid residues 551-574) (SEQ NO: 1), which is currently very challenging in industry for both expression and purification of membrane proteins. The transmembrane region of RSV F (amino acid residues 530-550) functions to anchor the F protein to the viral envelope on the one hand and to aid in the trimerization of the F protein on the other hand. The Transmembrane (TM) and cytoplasmic regions (amino acid residues 551-574) of RSV F (amino acid residues 530-550) do not constitute important neutralizing antibody binding epitopes and can therefore be removed or substituted. For vaccines based on RSV F protein, to meet the industrial mass production and purification requirements, the Transmembrane (TM) and cytoplasmic regions of the RSV F protein are deleted to produce the soluble secreted F protein (sF). The soluble F protein without the transmembrane region is less stable than the full-length protein and is easily refolded to a post-fusion final state.
To stabilize sF, the Transmembrane (TM) and cytoplasmic domains (amino acid residues 530 to 574) of RSV F can also be replaced by well-known trimerization domains. Common trimerization domains include the T4 bacteriophage fibrin trimerization domain, also known as "Foldon" (Letarov et al Biochemistry Moscow 64:817-823 (1993); S-Guthe et al J.mol. Biol.337:905-915 (2004)); the modified Foldon domain (SEQ ID NO: 3); GCN4 and its modified form GCN4II (SEQ ID NO: 4) have been reported to form stable triple helical coiled coil structures; other naturally or artificially designed trimerization domains and multimerization domains, such as the mammalian lung surfactant protein D neck trimerization domain disclosed in WO 2005037852: VASLRQQVEALQGQ (SEQ ID NO: 5), U.S. Pat. No. 3, 9630004 discloses I53-50A & B. In particular embodiments, the selection of substituted peptide fragments may involve 10-15 amino acid residues comprising the Transmembrane (TM) and cytoplasmic domains of RSV F and adjacent thereto, such as peptide fragments comprising amino acid residues 495-575, or peptide fragments comprising amino acid residues 515-575, etc., or peptide fragments comprising amino acid residues 529-575. Multiple GS-linked sequences, such as GSGS, may be added between the trimerization domain and truncated RSV F.
HRB alpha helices (486-513) immediately adjacent to the F1 transmembrane region are loosely packed together in the pre-fusion conformation of RSV F, and another strategy to stabilize sF is to use trimerization domains to replace HRB, transmembrane region, and cytoplasmic region simultaneously. In particular embodiments, the selection of substituted peptide segments may involve 10-15 amino acid residues comprising the Transmembrane (TM) and cytoplasmic domains of RSV F and adjacent thereto, such as peptide segments comprising amino acid residues 486-575, or peptide segments comprising amino acid residues 495-575, etc. Multiple GS-linked sequences, such as GSGS, may be added between the trimerization domain and truncated RSV F.
Further, as noted above, the proteolytic cleavage of F0 to F1 and F2 is also important for the transition from the pre-fusion conformation to the post-fusion conformation of RSV F. Thus, altering the proteolytic cleavage site between amino acid residues 109/110 and 136/137 by mutation is also one way to prevent the transition of RSV F from the pre-fusion to post-fusion conformation. In practice, mutations may be made at the relevant sites, such as the cleavage site into a connecting peptide stretch containing the amino acid residue GS. A connecting peptide containing the amino acid residue GS may be used instead of the peptide containing the cleavage site of the protease, for example GGSGSGSGS.
None of the above strategies, alone or in combination, ensures that either RSV F trimers or sF trimers with stable pre-fusion conformations are obtained. In order to obtain a stable pre-fusion conformation and produce a soluble F protein with high expression and high stability, further modification and stabilization of RSV F are required. Some of the strategies currently in use include: 1. mutations in RSV F introduce unnatural disulfide bonds that utilize covalent bonds of disulfide bonds to bind molecules in RSV F trimers; 2. introducing a mutation of an amino acid residue capable of preventing a conformational change in the segment of the RSV F conformational change; 3. amino acid residue mutations were introduced in RSV F to enhance hydrophobic interactions with pre-fusion conformational F proteins. Some of these strategies have been shown to work well for stabilizing the pre-fusion conformation of RSV F, such as the combined mutations at amino acid residues 155, 190, 207 and 290 in Ds-CaV1 (US 2022323568). However, these strategies to prevent conformational changes in RSV F have corresponding disadvantages, such as the introduction of unnatural disulfide bonds that can lead to aggregation, conformational changes, and loss of some of the native conformation of RSV F proteins; as a further example, the previous series of mutations (WO 2014174018) all involve the use of an epitope of interestNearby mutations, although helping to epitopeStable in a particular conformation but may affect epitopesNearby immunogenicity; also, substitution of protease cleavage sites near the fusion peptide, such as with a linking sequence such as GGSGSGSGS, allows for the appearance of unnatural epitopes near the fusion peptide.
Current mRNA vaccines and other vaccine technology that use vectors to deliver nucleic acid molecules (e.g., vaccines delivered using viral vectors) are becoming mature. mRNA vaccines and other vaccine forms that use vectors to deliver nucleic acid molecules do not involve industrial protein production and purification. For vaccine approaches based on these forms, it is desirable that the vaccine form contain nucleic acid material encoding RSV F protein with a stable pre-fusion conformation, which is stable for long periods of time under certain storage conditions and can be produced in sufficient amounts. However, vaccine methods based on these forms may employ nucleic acid materials encoding RSV F proteins having a stable pre-fusion conformation and containing both Transmembrane (TM) and cytoplasmic regions, as well as nucleic acid materials having a stable pre-fusion conformation but not containing both Transmembrane (TM) and cytoplasmic regions sF.
In the above-described RSV vaccine forms, a number of strategies have been tried in order to stabilize RSV F in a pre-fusion conformation, including, for example: mutation of the F0 furin cleavage site, or substitution of p27 and furin cleavage site with a linking peptide, such a strategy that F0 is not treated as F1 and F2, in some cases (e.g., the furin cleavage site is mutated) the p27 peptide is still retained; 2. introducing additional disulfide bonds between F1 or F2, or F1 and F2; 3. introducing mutations at F1 or F2, or at some amino acid residue positions at F1 and F2, to stabilize the RSV F pre-fusion conformation; fusion of the rsv-F ectodomain with the trimerisation domain (as disclosed in WO2010149743, WO2010149745, WO2009079796, WO 2012158613). However, each of these designs produces stable pre-fusion RSV-F proteins that have certain drawbacks and have not produced a well-behaved RSV vaccine.
The present invention now employs different strategies to design RSV F proteins with stable pre-fusion conformations, i.e., RSV F polypeptides with stable pre-fusion conformations. In the studies of the present invention, a series of mutations were introduced and/or combined in RSV F to obtain the RSV F protein with stable pre-fusion conformation. RSV F proteins designed according to the invention are in a pre-fusion conformation comprising (displaying) at least one pre-fusion conformation F protein-specific epitope. The pre-fusion conformation F protein-specific epitope is an epitope that is not present in the post-fusion conformation. The mutation site designed in the invention does not involve the introduction of any unnatural disulfide bond and is not an important epitopeAny mutation is introduced nearby and on the exposed surface of other critical epitopes of RSV F, so that the immunogenicity and critical conformation of RSV F can be minimized while stabilizing the pre-fusion conformation of RSV F, thereby ensuring that the designed antigen can stably induce the generation of neutralizing antibodies to the critical epitopes for preventing virus invasion. In addition, RSV F containing the mutation site designed in the present invention has significantly higher expression level in mammalian cells, such as HEK293, than wild-type RSV F and other RSV F in the reported designs, thereby being beneficial for industrial production. Furthermore, the RSV F proteins containing the engineered mutation sites of the invention have significantly higher melting point (Tm) temperatures than wild-type RSV F and other RSV F in reported designs.
The recombinant proteins of the invention comprise mutations in at least one of the F1 and/or F2 domains that stabilize the pre-fusion conformation of RSV F, as compared to the RSV Fl and/or F2 domains in the wild-type RSV F protein.
In certain embodiments, RSV F proteins designed according to the invention with stable pre-fusion conformations comprise a mutation of amino acid residue 55 and/or a mutation of amino acid residue 144 and/or a mutation of amino acid residue 355 and/or a mutation of amino acid residue 400 and/or a mutation of amino acid residue 459 and/or a mutation of amino acid residue 481 and/or a mutation of amino acid residue 55. In certain embodiments, RSV F proteins designed according to the invention having stable pre-fusion conformations comprise both a mutation of amino acid residue 400 and a mutation of amino acid residue 481;
in certain embodiments, RSV F proteins designed according to the invention having stable pre-fusion conformations further comprise a mutation of amino acid residue 144 and/or a mutation of amino acid residue 355 and/or a mutation of amino acid residue 459 and/or a mutation of amino acid residue 55.
In certain embodiments, RSV F proteins designed according to the invention having stable pre-fusion conformations comprise both mutations at amino acid residue 144, amino acid residue 355, amino acid residue 400, amino acid residue 459, and amino acid residue 481.
In certain embodiments, RSV F proteins designed according to the invention having stable pre-fusion conformations comprise a mutation of amino acid residue 144, a mutation of amino acid residue 355, a mutation of amino acid residue 400, a mutation of amino acid residue 459, a mutation of amino acid residue 481, and an amino acid residue 55.
In certain embodiments, amino acid residue 481 is mutated to a hydrophilic amino acid residue to reduce possible protein aggregation at high concentrations.
In certain embodiments, mutations to amino acid residue 144, amino acid residue 400 are made to increase the local hydrogen bonding network to stabilize the pre-fusion conformation.
In certain embodiments, mutations at amino acid residue 55, amino acid residue 459, amino acid residue 355 are made to fine tune the local van der Waals interactions to stabilize the pre-fusion conformation.
In certain embodiments, RSV F proteins having stable pre-fusion conformations according to the invention include mutations in the amino acid residue threonine at position 400 and in the amino acid residue leucine at position 481.
In certain embodiments, the RSV F protein having a stable pre-fusion conformation according to the invention further comprises a mutation of the valine residue at amino acid residue 144 and/or a mutation of the alanine residue at amino acid residue 355 and/or a mutation of the valine residue at amino acid residue 459 and/or a mutation of the serine residue at amino acid residue 55.
In certain embodiments, RSV F proteins having stable pre-fusion conformations according to the invention comprise a mutation of the amino acid residue at position 144 to a glutamic acid residue or a glutamine residue and/or at position 355 to a proline residue and/or at position 400 to an aspartic acid residue and/or at position 459 to a leucine residue or a methionine residue and/or at position 481 to an asparagine residue or a serine residue or a threonine residue or a glutamine residue and/or at position 55 to a valine residue or a threonine residue.
The protein may be mutated by routine molecular biology procedures by a skilled artisan. Mutations according to the invention preferably result in increased expression levels and/or increased stability and/or increased conformation specific to the pre-fusion RSV F protein compared to RSV F proteins not comprising these mutations.
In certain embodiments, the pre-fusion RSV F protein is soluble, i.e., in the sF form.
In certain embodiments, the pre-fusion RSV F protein further comprises a heterotrimeric domain linked to the truncated F1 domain. According to the present invention, it was shown that by ligating the heterotrimeric domain to the C-terminal amino acid residue of the truncated F1 domain in combination with the aforementioned stabilizing mutations, the expression level of recombinant RSV F protein as well as the stability of the pre-fusion conformation can be significantly improved. RSV F protein with stable pre-fusion conformation binds to specific antibody D25 with high efficiency. The heavy chain sequence and the light chain sequence of the neutralizing antibody D25 are shown as SEQ ID NO: 6. 7 are shown separately. Neutralizing antibody D25 recognizes a conformational epitope on RSV F rather than a linear polypeptide epitope. Previous studies showed that neutralizing antibody D25 binds only an epitope that is possessed by the pre-fusion conformational RSV F protein
(McLellan JS et al.science 340 (6136): 1113-7 (2013)). Epitope of antigenNear the top of the pre-fusion RSV F, distal to its transmembrane region, contains the alpha helix formed by peptide segments 196-212 and peptide segments 61-70. As shown in FIG. 8 of the specification, the alpha helices formed by peptide segments 196-212 and peptide segments 61-70 in pre-fusion RSV F are adjacent to each other, and the CDR3 of the D25 heavy chain is inserted into the specific structural groove between peptide segments 196-212alpha helices and peptide segments 61-70. As shown in FIG. 8 of the specification, the major structural changes of peptide fragments 196-212alpha and peptide fragments 61-70 are distant from each other in the post-RSV F-fusion conformation, and the major binding site of D25 is absent in the post-RSV F-fusion conformation. Thus, neutralizing antibody D25 can only bind to RSV F protein having a pre-fusion conformation and can be used to detect whether RSV F is in a pre-fusion conformation, specific binding to D25 indicates that RSV F retains the pre-fusion conformation.
In further embodiments, the RSV F protein having a stable pre-fusion conformation further comprises one or more additional mutations selected from the group consisting of:
(a) Mutation of amino acid residue 56;
(b) Mutation of amino acid residue 67;
(c) Amino acid residue 79;
(d) Mutation of amino acid residue 81;
(e) Mutation of amino acid residue 82;
(f) Mutation of amino acid residue 83;
(g) Mutation of amino acid residue 92;
(h) Mutation of 96 amino acid residues;
(i) Mutation of amino acid residue 146;
(j) Mutation of 147 th amino acid residue;
(k) Mutation of amino acid residue at position 155;
(l) Amino acid residue 167;
(m) mutation of amino acid residue 181;
(n) mutation of amino acid residue 185;
(o) mutation of amino acid residue 189;
(p) mutation of amino acid residue 190;
(q) mutation of amino acid residue 225;
(r) mutation of amino acid residue at position 230;
(s) mutation of amino acid residue 254;
(t) mutation of amino acid residue 279;
(u) mutation of amino acid residue 287;
(v) 292 amino acid residue;
(w) mutation of amino acid residue 296;
(x) Mutation of amino acid residue 405;
(y) mutation of amino acid residue 487.
In a preferred embodiment, the one or more additional mutations are selected from the group consisting of:
(a) Mutation of amino acid residue 56 from wild-type RSV F protein to isoleucine residue (I);
(b) Mutation of amino acid residue 67 from wild-type RSV F protein to leucine residue (L) or tyrosine residue (W);
(c) Mutation of amino acid residue 79 from wild-type RSV F protein to phenylalanine residue (F) or methionine residue (M);
(d) Mutation of amino acid residue 81 from wild-type RSV F protein to glutamic acid residue (E); (e) Mutation of amino acid residue 82 from wild-type RSV F protein to leucine residue (L); (f) Mutation of amino acid residue 83 from wild-type RSV F protein to methionine residue (M) or phenylalanine residue (F);
(g) Mutation of amino acid residue 92 from wild-type RSV F protein to serine residue (S) or alanine residue (a);
(h) Mutation of amino acid residue 96 from wild-type RSV F protein to methionine residue (M); (i) Mutation of amino acid residue 146 from wild-type RSV F protein to aspartic acid residue (D); (j) Mutating 147 th amino acid residue from wild type RSV F protein into proline residue (P); (k) Mutation of amino acid residue 155 from wild-type RSV F protein to alanine residue (a) or isoleucine residue (T) or valine residue (V);
(l) Mutation of amino acid residue 167 from wild-type RSV F protein to methionine residue (M) or phenylalanine residue (F);
(m) mutation of amino acid residue 181 from wild-type RSV F protein to valine residue (V);
(n) mutation of amino acid residue 185 from wild-type RSV F protein to methionine residue (M) or aspartic acid residue (D) or glutamic acid residue (E);
(o) mutation of amino acid residue 189 from wild-type RSV F protein to valine residue (V);
(p) mutation of amino acid residue 190 from wild-type RSV F protein to methionine residue (M);
(q) mutation of amino acid residue 225 from wild-type RSV F protein to glutamic acid residue (E); (r) mutation of amino acid residue 230 from wild-type RSV F protein to phenylalanine residue (F); (s) mutation of amino acid residue 254 from wild-type RSV F protein to aspartic acid residue (D) or threonine residue (T);
(t) mutation of amino acid residue 279 from wild-type RSV F protein to histidine residue (H) or phenylalanine residue (F);
(u) mutation of amino acid residue 287 from wild-type RSV F protein to threonine residue (T) or alanine residue (a);
(v) Mutation of amino acid residue 292 from wild-type RSV F protein to leucine residue (L);
(w) mutation of amino acid residue 296 from wild-type RSV F protein to leucine residue (L);
(x) Mutation of amino acid residue 405 from wild-type RSV F protein to methionine residue (M); (y) mutation of amino acid residue 487 from wild-type RSV F protein to leucine residue (L).
Note again that for the position of the amino acid residue reference is made to SEQ ID NO:1. the skilled artisan will be able to determine the corresponding amino acid residues in the F protein of other RSV strains, e.g., SEQ ID NO:2.
In certain embodiments, the RSV F protein comprises at least three mutations.
In certain embodiments, the RSV F protein comprises at least four mutations.
In certain embodiments, the RSV F protein comprises at least five mutations.
In certain embodiments, the RSV F protein comprises at least six mutations.
In certain embodiments, the RSV F protein comprises at least seven mutations.
In certain embodiments, the RSV F protein comprises at least eight mutations.
In certain embodiments, the RSV F protein comprises at least nine mutations.
In certain embodiments, the mutant residues comprised in RSV F are all located remotely from the epitopeAnd the primer is not exposed on the surface of RSV F, so that the influence on important epitope on the surface of RSV F before fusion is greatly reduced.
In certain embodiments, the heterotrimeric domain comprises one of the amino acid sequences from:
(a) T4 phage fibrosis trimerization domain (SEQ ID NO: 3):
SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL；
(b) GCN4 leucine zipper trimerization domain: (SEQ ID NO: 4): RMKNLEDKVEELLSKNYHLENEVARLKKLVGER;
(c) Mammalian lung surfactant protein D neck trimerization domain: VASLRQQVEALQGQ.
As described above, in certain embodiments, the proteins of the invention comprise a truncated F1 domain. As used herein, a "truncated" F1 domain refers to an F1 domain that is not a full-length F1 domain, i.e., wherein one or more amino acid residues have been deleted at the N-terminus or C-terminus. According to the invention, at least the transmembrane domain and cytoplasmic tail have been deleted to allow expression of the soluble extracellular domain of RSV F protein.
In certain other embodiments, the F1 domain is truncated after amino acid residue 495 of the RSV F protein (see SEQ ID NO: 1), i.e., the C-terminal portion of the F1 domain from amino acid residue 496 (see SEQ ID NO: 1) has been deleted. In certain other embodiments, the F1 domain is truncated after amino acid residue 513 of the RSV F protein. In certain embodiments, the F1 domain is truncated after any one of the amino acid residues 486-528 in the peptide stretch.
In certain embodiments, the trimerization domain is linked to amino acid residue 495 of the RSV F1 domain. In certain embodiments, the trimerization domain comprises SEQ ID NO:3,4 or 5 and is attached to the 495 amino acid residues of the RSV F1 domain. In certain embodiments, the trimerization domain comprises SEQ ID NO:3,4 or 5 and is attached to the RSV F1 domain after amino acid residue 513.
In certain embodiments, RSV F proteins having stable pre-fusion conformations according to the invention use one, two or three GS linker sequences to replace the p27 peptide fragment (amino acid residues 106-136 of SEQ ID NO: 1) contained.
In certain embodiments, the F1 domain and/or F2 domain is from an RSV a strain.
In certain embodiments, the F1 domain and/or F2 domain is from the RSV A2 strain of SEQ ID NO. 1.
In certain embodiments, the F1 domain and/or F2 domain is from an RSV B strain.
In certain embodiments, the F1 domain and/or F2 domain is from the RSV B strain of SEQ ID NO. 2.
In certain embodiments, the F1 domain and/or F2 domain are from the same RSV strain. In certain embodiments, the pre-fusion RSV F protein is a chimeric protein, i.e., comprises F1 and F2 domains from different RSV strains.
In certain embodiments, the expression level of the pre-fusion RSV F protein of the invention is increased as compared to the wild-type RSV F protein ectodomain without mutation (i.e., without transmembrane and cytoplasmic regions). In certain embodiments, the expression level is increased by at least 5-fold as compared to the wild-type RSV F protein extracellular domain without mutation. In certain embodiments, the expression level is increased by more than 10-fold (tables 1, 2).
In certain embodiments, the pre-fusion RSV F protein expression levels of the invention are increased by at least 2-fold compared to other constructs SCTM (WO 2014174018; krarup et al, nature Communications,2015, 6:8143) that have been reported. In certain embodiments, the expression level is increased by at least 6-fold compared to the other construct Ds-CaV1 (US 2022323568) that has been reported.
RSV F proteins according to the invention having stable pre-fusion conformations are not easily changed to post-fusion conformations during e.g. purification, freeze-thawing cycles and/or storage, and are highly stable and not easily degraded.
In certain embodiments, the pre-fusion RSV F proteins according to the invention have no distinct degradation bands as compared to the RSV F protein without mutation, as determined by SDS-PAGE gel electrophoresis after 7 days of storage at 37 ℃.
In certain embodiments, the pre-fusion RSV F proteins according to the invention retain stability after storage at 4 ℃ as compared to RSV F proteins without mutations. In certain embodiments, the protein remains stable for at least 30 days at 4 ℃. By "storage stable" is meant that the protein still exhibits at least one specific epitope for an antibody specific for the pre-fusion RSV F conformation (e.g., D25) for at least 30 days after storage of the protein in solution (e.g., PBS buffer) at 4 ℃, e.g., as determined using the methods described in the examples.
In certain embodiments, the pre-fusion RSV F proteins according to the invention retain stability after storage at 37 ℃ as compared to RSV F proteins without mutations. In certain embodiments, the protein is stable for at least 14 days at 37 ℃. By "storage stable" is meant that the protein still exhibits at least one specific epitope for an antibody (e.g., D25) specific for the presplitting conformation of RSV F after storage of the protein in solution (e.g., PBS buffer) at 37 ℃ for at least 14 days, e.g., as determined using the methods described in the examples.
In certain embodiments, the pre-fusion RSV F proteins according to the invention have increased stability when heated compared to RSV F proteins without the mutation. In certain embodiments, the pre-fusion RSV F protein is thermostable at a temperature of 55 ℃ for at least 30 minutes. By "thermostable" is meant that the protein still exhibits at least one RSV F presusion conformation-specific epitope after having been subjected to an elevated temperature (i.e., a temperature of 55 ℃ or above) for at least 30 minutes, e.g., as determined using the methods described in the examples.
In certain embodiments, the protein does not significantly precipitate after undergoing 1 to 6 freeze-thaw cycles in a suitable formulation buffer and retains at least one RSV F prefusion conformation-specific epitope.
In certain embodiments, the pre-fusion RSV F protein according to the invention is in a pre-fusion conformation in a negative electron microscope or cryoelectron microscope data analysis as compared to the RSV F protein without the mutation.
In certain embodiments, the RSV F protein of the invention having a stable pre-fusion conformation consists of an amino acid sequence selected from SEQ ID NO. 8-93. It is noted, however, that in addition to the mutant amino acid residues mentioned, other amino acid residues in the sequence may be replaced by corresponding amino acid residues in the F protein sequences from other RSV strains. The skilled person can know how to substitute by sequence alignment.
In certain embodiments, the encoded proteins according to the invention further comprise a signal sequence or signal peptide corresponding to amino acid residues 1-26 of SEQ ID NO.1 or SEQ ID NO. 2. Such short peptides (typically 5-30 amino acids in length) are present at the nitrogen terminus of most secreted novel synthetic protein polypeptides and function to aid in the localization and secretion of the protein polypeptides. In certain embodiments, RSV F proteins according to the invention may not comprise a leader sequence. In certain embodiments, the leader sequence in the RSV F proteins according to the invention may be replaced by a leader sequence of another protein, such as amino acid sequence MGWSCIILFLVATATGVHS (antibody secretion signal peptide), or amino acid sequence MKTLILAVALVYCVTVNC (CYPRIDNIA NOCTILUCA luciferase secretion signal peptide), or amino acid sequence MMRPIVLVLLFATSALA (pine moth silk protein (Pine Moth Fibroin) light chain secretion signal peptide).
In certain embodiments, the RSV F protein according to the invention comprises a HIS tag. The HIS tag or poly-histidine tag consists of at least five consecutive histidine (H) residues, such as HHHHHH, typically located at the N or C terminus of the protein for purification purposes.
In certain embodiments, RSV F proteins according to the invention comprise 1 x or more repeat strep ii tags. The 1 x strep ii tag has the sequence: WSHPQFEK, usually located at the N or C terminus of the protein, is used for purification purposes.
In certain embodiments, RSV F proteins according to the invention comprise 1x or more repeat FLAG tags. The 1×flag tag has the sequence: dykdddk, typically located at the N or C terminus of a protein, is used for purification purposes.
In certain embodiments, a protease cleavage site, such as an HRV 3C cleavage site (amino acid sequence: LEVLFQGP), may also be added between the RSV F protein according to the present invention and the HIS-tag, FLAG-tag, or STREP II-tag.
In certain embodiments, the RSV F proteins according to the invention can efficiently induce antibody production against pre-fusion RSV F proteins in laboratory animals, and the antibodies produced can compete well with neutralizing antibody D25 for binding to pre-fusion RSV F.
The invention further provides nucleic acid molecules encoding the RSV F proteins described according to the invention.
In a preferred embodiment, the nucleic acid molecule encoding a protein according to the invention may be codon optimized for expression in mammalian cells, preferably primate cells, most preferably human cells. Methods of codon optimisation are known and have been described previously in other documents (e.g. WO 9609378). A sequence is considered codon optimized if at least one non-preferred codon is replaced with a more preferred codon compared to the wild-type sequence. In this context, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, while a more preferred codon is a codon that is used more frequently in an organism than the non-preferred codon. The codon usage frequency of a particular organism can be found in its codon usage frequency table, e.g.http:// www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, most or all of the non-preferred codons are replaced by more preferred codons. Preferably, the codons most commonly used in an organism are used in the codon optimized sequence. Substitution with the preferred codon generally results in higher expression.
The skilled artisan will appreciate that many different polynucleotides and nucleic acid molecules may encode the same protein due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can make nucleotide substitutions using conventional techniques that do not affect the protein sequence encoded by the nucleic acid molecule. Thus, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The RNA nucleotide sequence encoding the protein may or may not include introns.
The invention also provides vectors comprising the nucleic acid molecules as described above. In certain embodiments, the nucleic acid molecules obtained according to the invention are part of a vector. Such vectors can be engineered by methods well known to those skilled in the art, such as replication in prokaryotic and/or eukaryotic cells. The vector used may be any vector suitable for cloning DNA and which can be used for transcription of the nucleic acid of interest. Suitable vectors according to the invention include, but are not limited to, cytomegalovirus vectors, such as pCMV and the like. In addition, many vectors can be used for transformation of eukaryotic cells and integration of all or part of the genome of such cells to produce stable host cells comprising the desired nucleic acid in their genome. Host cells comprising nucleic acid molecules encoding pre-fusion RSV F proteins are therefore also part of the invention.
The pre-fusion RSV F protein can be expressed recombinantly by DNA technology in different host cells, such as Chinese Hamster Ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, per.c6 cells, or yeast, fungal, insect cells, etc., or transgenic animals or plants. In certain embodiments, the cells are derived from multicellular organisms, and in certain embodiments, the cells are derived from vertebrates or invertebrates. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In general, the production of recombinant proteins (e.g., pre-fusion RSV F proteins of the invention), expression in a host cell involves introducing a heterologous nucleic acid molecule encoding the protein into the host cell in an expressible form, culturing the cell under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in the cell.
Nucleic acid molecules encoding the proteins in an expressible form may be in the form of expression cassettes and generally require sequences capable of assisting in the transcriptional or translational expression of the nucleic acid, such as enhancers, promoters, polyadenylation signals, and the like. Those skilled in the art know that a variety of regulatory elements can be used to help achieve efficient expression of genes in host cells. Promoters may be constitutive or regulated, and may be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or artificial designs. Suitable promoters for obtaining high expression in eukaryotic cells are numerous, such as the CMV promoter (US 5,385,839). Polyadenylation signals, such as bovine growth hormone polyA signal (US 5,122,458), which are added after the gene of interest, stabilize the target protein mRNA molecule. The expression vector may be selected from several widely used commercially available expression vectors, such as pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg vectors of BD Sciences, pCMV-Script vector of Stratagene, etc., for recombinant expression of the protein of interest.
Cell culture media are available in large quantities from commercial sources and suitable media can be optimally selected for the host cell to increase expression of the protein of interest. Suitable media may or may not contain serum. Suitable conditions and means for culturing cells are known. The cell culture means may be of any type, including adherent culture, e.g. attached to the surface of a culture vessel or microcarriers, and suspension cell culture, e.g. suspension growth in a specific medium.
The invention further provides compositions comprising RSV F protein and/or nucleic acid molecules and/or vectors having a stable pre-fusion conformation, as described above. Thus, the present invention provides compositions comprising pre-fusion RSV F proteins that exhibit stable pre-fusion conformations of RSV F proteins that exhibit epitopes that are present in the pre-fusion conformation, but not in the post-fusion conformation of RSV F proteins. The invention also provides compositions comprising nucleic acid molecules and/or vectors encoding such pre-fusion RSV F proteins. The invention further provides immunogenic compositions comprising pre-fusion RSV F protein and/or nucleic acid molecules and/or vectors, as described above. The invention also provides a pre-fusion RSV F protein, nucleic acid molecule and/or vector according to the invention for inducing an immune response against the RSV F protein in a subject. Further provided are pre-fusion RSV F proteins and/or nucleic acid molecules and/or vectors according to the invention for inducing an immune response against RSV F proteins in a subject. Also provided are methods of inducing an immune response against RSV F protein in a subject, comprising administering to the subject a pre-fusion RSV F protein and/or nucleic acid molecule and/or vector according to the invention.
The pre-fusion RSV F proteins, nucleic acid molecules or vectors according to the invention can be used to prevent and/or treat RSV infection. In certain embodiments, the prophylaxis and/or treatment may be directed to a patient population susceptible to RSV. Such patient populations include, but are not limited to, for example, elderly (e.g., >50 years), pediatric and infant (e.g., <5 years, <1 year), pregnant women, hospitalized patients, and patients who have been treated with antiviral compounds but have an inadequate antiviral response.
The pre-fusion RSV F proteins, nucleic acid molecules and/or vectors according to the invention can be used, for example, for the treatment and/or prophylaxis of diseases or conditions caused by RSV alone or in combination with other prophylaxis and/or treatment methods, such as (existing or future) vaccines, antiviral drugs and/or monoclonal antibodies.
The invention also provides methods of preventing and/or treating RSV infection in a subject using RSV F proteins, nucleic acid molecules and/or vectors according to the invention having a stable pre-fusion conformation. In a particular embodiment, a method for preventing and/or treating RSV infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion RSV F protein, nucleic acid molecule and/or vector, as described above. Prevention includes inhibiting or reducing the spread of RSV, or inhibiting or reducing the development or progression of one or more symptoms associated with RSV infection. As used herein, improvement may refer to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable manifestation following infection.
For administration to a subject (e.g., a human), the invention can employ a pharmaceutical composition comprising a pre-fusion RSV F protein, nucleic acid molecule, and/or vector as described herein, and a pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable" means that the carrier or excipient does not have any unwanted or deleterious effects on the subject to which it is administered at the dosage and concentration employed. Such pharmaceutically acceptable carriers and excipients are well known in the art.
RSV F protein or nucleic acid molecules having a stable pre-fusion conformation are formulated and administered as sterile solutions, and may also be formulated and administered as lyophilized formulations. Sterile solutions are prepared by sterile filtration or other methods known per se in the art and filled into pharmaceutical dosage containers. Lyophilized formulations are prepared by lyophilizing a solution of RSV F protein having a stable pre-fusion conformation and loading into a pharmaceutical dosage container. The pH of the solution is typically in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. The RSV F protein is typically in solution with a suitable pharmaceutically acceptable buffer, and the composition may further comprise a salt, and optionally a stabilizing agent, such as albumin, may be present. In certain embodiments, a detergent is added. Although lyophilized formulations may also be used, it is preferred to formulate and administer a sterile solution.
In some embodiments, RSV F proteins having a stable pre-fusion conformation can be formulated as injectable formulations.
In certain embodiments, the compositions according to the present invention further comprise one or more adjuvants. Adjuvants are known in the art to further increase the immune response to the antigenic determinants of the application. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the RSV F proteins of the invention. Examples of suitable adjuvants include aluminum salts, such as aluminum hydroxide and/or aluminum phosphate; an oil emulsion composition (or oil-in-water composition) comprising a squalene-water emulsion, such as MF59 (see, e.g., WO 9014837); saponin formulations, such as QS21 and immunostimulatory complexes (ISCOMS) (see, e.g., US 5,057,540;WO 9003184;WO 9611711;WO 2004/004762; wo 2005/002620); bacterial or microbial derivatives, such as monophosphoryl lipid a (MPL), 3-O-deacylated MPL (3 dMPL), cpG-motifs containing oligonucleotides, ADP-ribosylated bacterial toxins or mutants thereof, such as e.coli heat labile enterotoxin LT, cholera toxin CT, etc.; eukaryotic proteins such as interferons may stimulate an immune response. In certain embodiments, the compositions of the present invention comprise aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or a combination thereof, at a concentration of 0.05 to 5 milligrams, for example 0.075 to 1.0 milligrams, per dose of aluminum content.
RSV F protein having a stable pre-fusion conformation can also be administered in combination or conjugation with nanoparticles, e.g., polymers, liposomes, viral particles, virus-like particles. The pre-fusion F protein may be bound to, entrapped in, or conjugated to the nanoparticle with or without an adjuvant. Encapsulation within liposomes is described, for example, in US 4,235,877. Conjugation to macromolecules is disclosed, for example, in US 4,372,945 or US 4,474,757.
In other embodiments, the composition does not comprise an adjuvant.
In certain embodiments, the invention provides methods and uses for preparing a vaccine against Respiratory Syncytial Virus (RSV), comprising providing a composition according to the invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to an agent or composition containing an active ingredient that is effective to induce a degree of immunity against a pathogen or disease in a subject, thereby reducing (until completely absent) the severity of the associated disease, as evidenced by a reduction in symptoms, duration, or other manifestations associated with the pathogen infection or disease. In the present invention, the vaccine comprises an effective amount of RSV F protein having a stable pre-fusion conformation and/or a nucleic acid molecule encoding RSV F protein having a stable pre-fusion conformation and/or a vector comprising said nucleic acid molecule, and administration of the vaccine according to the present invention will result in an immune response against RSV F protein. Immunization of a population with a vaccine developed in accordance with the compositions of the present invention provides a method of preventing severe lower respiratory tract disease resulting in hospitalization, and a method of reducing the frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. The vaccine pharmaceutical compositions according to the present invention generally comprise a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain other active ingredients. In certain embodiments, it may be a combination vaccine, which also comprises other components that induce an immune response.
The composition may be administered to a subject, such as a human subject. The total dose of RSV F protein having a stable pre-fusion conformation in a composition for single administration may be, for example, from about 0.01 μg to about 10mg, such as 0.1-1mg, such as 10 μg to 100 μg. The recommended dose will be determined experimentally and the relevant procedure will be routine to those skilled in the art.
Administration of the compositions according to the invention may be carried out using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal or mucosal administration, such as nasal spray, inhalation, and the like. The skilled person is aware of the various possible ways of administering a composition, such as a vaccine, to induce an immune response to an antigen in the vaccine.
The subject used herein is preferably a mammal, such as a rodent, e.g., a mouse, a cotton mouse, or a non-human primate, or a human.
The proteins, nucleic acid molecules, vectors and/or compositions may also be administered as a prime or as boost in a homologous or heterologous prime-boost regimen. If booster vaccinations are administered, typically, such booster vaccinations will be administered to the same subject between one week and one year after the first administration of the composition to the subject (in this case referred to as "primary vaccination"). In certain embodiments, the administration includes priming and at least one booster administration.
Furthermore, the proteins, nucleic acid molecules, vectors and/or compositions of the invention may be used as diagnostic tools for testing the immune status of an individual, for example by determining whether antibodies capable of binding to the proteins of the invention are present in the serum of the individual. Accordingly, the present invention also relates to an in vitro diagnostic method for detecting the presence or absence of RSV infection in a patient, said method comprising the steps of: a) Mixing a biological sample obtained from the patient with a protein according to the invention; b) Detecting the presence of the antibody-protein complex.
Furthermore, the proteins, nucleic acid molecules, vectors and/or compositions of the invention may be used to prepare compositions for preventing and/or treating RSV infection, for example, by administering to an individual who has been determined to be infected with RSV, to inhibit, delay, prevent progression of infection, to restore the individual from an infected state, or to prevent further worsening of infection.
The invention will be further explained herein in the following examples. The examples are not intended to limit the invention in any way, nor are they intended to be exhaustive of the invention, but are merely illustrative of the invention.
Drawings
FIG. 1 is a comparison of pre-fusion and post-fusion conformations of RSV F;
FIG. 2 is a comparison of neutralizing antibody binding sites before and after fusion of RSV F;
FIG. 3 is a schematic representation of the post-translational treatment of RSV F0 protein by proteases to produce F1, F2 and p 27;
FIG. 4 shows an alignment of F protein sequences from different strains in the RSV A subtype;
FIG. 5 shows a sequence alignment of F proteins from different strains in the RSV subtype B;
FIG. 6 is an alignment of F protein sequences derived from different RSV subtypes;
FIG. 7 is a sequence alignment of strain A2 RSV F and bovine RSV F;
FIG. 8 is an epitope The inclusion of peptide fragments 196-212 and peptide fragments 61-70 are compared in position in pre-fusion RSV F and post-fusion RSV F proteins;
FIG. 9 is a graph showing the results of molecular sieve purification of five-point combined mutant constructs, L481N, T400D, V144E, A355P, V459L, wherein 8.43mL elution peak corresponds to RSV F trimer;
FIG. 10 shows the results of polyacrylamide gel electrophoresis analysis of constructs containing single site mutations;
FIG. 11 shows the results of polyacrylamide gel electrophoresis analysis of constructs containing multiple site mutations;
FIG. 12 shows the results of polyacrylamide gel electrophoresis analysis of constructs containing multiple site mutations after 7 days of 37 degrees of placement;
FIG. 13 is a graph showing the negative staining results and 2-dimensional classification analysis of a pre-fusion RSV F construct containing L481N, T400D, V144E, A355P, V459L, S55V six-locus combined mutation (6M); wherein A is the negative staining result of the construct, B is the 2D class average map of the conformation before fusion (31034 particles), C is the 2D class average map of the conformation after fusion (2950 particles), D is the 2D projection of the conformation before fusion, E is the 2D projection of the conformation after fusion;
FIG. 14 contains a graph of the negative results and 2-dimensional classification analysis of RSV F construct before fusion of the seven-site combined mutation (6M+7190F) of L481N, T400D, V144E, A355P, V459L, S55V, I79F; wherein A is the negative staining result of the construct, B is the 2D class average map of the conformation before fusion (30929 particles), C is the 2D class average map of the conformation after fusion (3398 particles), D is the 2D projection of the conformation before fusion, E is the 2D projection of the conformation after fusion;
FIG. 15 is a graph of a pre-fusion RSV F construct negative stain and 2-dimensional classification analysis comprising L481N, T400D, V144E, A355P, V459L, S55V, S190M seven-locus combined mutation (6M+S190M), wherein A is the construct negative stain, B is the pre-fusion conformational 2D class average map (101797 particles), C is the post-fusion conformational 2D class average map (7247 particles), D is the pre-fusion conformational 2D projection, E is the post-fusion conformational 2D projection;
FIG. 16 is a graph showing the structure analysis and comparison of a pre-fusion RSV F construct containing S55V, L481N, T400D, V144E, A355P, V459L six-locus combined mutation (6M);
FIG. 17 is an assessment of the titer of antibodies induced specifically by RSV F protein 5M in mice having a stable pre-fusion conformation;
figure 18 is an analysis of the ability of RSV F protein to induce neutralizing antibodies in mice with stable pre-fusion conformations.
Detailed Description
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in molecular cloning edited by SammBroker (Sambrook), french (Fritsch), and Meniere et al: laboratory Manual (MOLECULAR CLONING: ALABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel et al, (1987)); the enzyme methods series (METHODS IN ENZYMOLOGY) (academic publishing Co): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Hemsl (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. Fu Lei Xieni (R.I. Freshney) editions (1987)).
In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1 preparation of RSV F protein having a stable pre-fusion conformation
1. Molecular cloning design
The RSV F protein amino acid sequence (UniProtKB/Swiss-Prot: P03420.1) was obtained from the NCBI database and was taken from the region 1-574 of the amino acid sequence of the RSV F protein in the present study (abbreviated as RSV F WT) as shown in SEQ ID NO: 1. A total of 86 mutants were prepared by mutating RSV F WT, specifically as follows:
The protein obtained by mutating the 144 th amino acid residue V of SEQ ID NO.1 into E is named as V144E, and the amino acid sequence of the protein is shown as SEQ ID NO. 8.
The protein obtained by mutating 355 th amino acid residue A of SEQ ID NO.1 into P is named A355P, and the amino acid sequence is shown as SEQ ID NO. 9.
The protein obtained by mutating the 400 th amino acid residue T of SEQ ID NO.1 into D is named as T400D, and the amino acid sequence of the protein is shown as SEQ ID NO. 10.
The protein obtained by mutating the 459 th amino acid residue V of SEQ ID NO.1 into L is named as V459L, and the amino acid sequence of the protein is shown as SEQ ID NO. 11.
The protein obtained by mutating the 481 th amino acid residue L of SEQ ID NO.1 into N is named L481N, and the amino acid sequence is shown as SEQ ID NO. 12.
The protein obtained by mutating the 55 th amino acid residue S of SEQ ID NO. 1 into V is named S55V, and the amino acid sequence is shown as SEQ ID NO. 13.
The protein obtained by mutating the 67 th amino acid residue N of SEQ ID NO. 1 into L is named N67L, and the amino acid sequence is shown as SEQ ID NO. 14.
The protein obtained by mutating the 79 th amino acid residue I of SEQ ID NO. 1 into F is named I79F, and the amino acid sequence of the protein is shown as SEQ ID NO. 15.
The protein obtained by mutating the 145 th amino acid residue G of SEQ ID NO.1 into Y is named G145Y, and the amino acid sequence of the protein is shown as SEQ ID NO. 16.
The protein obtained by mutating 147 th amino acid residue A of SEQ ID NO.1 into P is named A147P, and the amino acid sequence is shown as SEQ ID NO. 17.
The protein obtained by mutating 167 th amino acid residue I of SEQ ID NO.1 into M is named I167M, and the amino acid sequence is shown as SEQ ID NO. 18.
The protein obtained by mutating the 185 th amino acid residue V of SEQ ID NO.1 into M is named V185M, and the amino acid sequence of the protein is shown as SEQ ID NO. 19.
The protein obtained by mutating the 185 th amino acid residue V of SEQ ID NO.1 into E is named V185E, and the amino acid sequence of the protein is shown as SEQ ID NO. 20.
The protein obtained by mutating the 189 th amino acid residue T of SEQ ID NO.1 into V is named as T189V, and the amino acid sequence of the protein is shown as SEQ ID NO. 21.
The protein obtained by mutating the 190 th amino acid residue S of SEQ ID NO.1 into M is named S190M, and the amino acid sequence is shown as SEQ ID NO. 22.
The protein obtained by mutating the 230 th amino acid residue L of SEQ ID NO.1 into F is named L230F, and the amino acid sequence of the protein is shown as SEQ ID NO. 23.
The protein obtained by mutating the 279 th amino acid residue Q of SEQ ID NO.1 into H is named as Q279H, and the amino acid sequence of the protein is shown as SEQ ID NO. 24.
The protein obtained by mutating the 279 th amino acid residue Q of SEQ ID NO.1 into F is named as Q279F, and the amino acid sequence of the protein is shown as SEQ ID NO. 25.
The protein obtained by mutating the 287 th amino acid residue S of SEQ ID NO.1 into T is named S287T, and the amino acid sequence of the protein is shown as SEQ ID NO. 26.
The protein obtained by mutating 292 th amino acid residue I of SEQ ID NO.1 into L is named I292L, and the amino acid sequence of the protein is shown as SEQ ID NO. 27.
The protein obtained by mutating the 296 th amino acid residue V of SEQ ID NO.1 into L is named V296L, and the amino acid sequence of the protein is shown as SEQ ID NO. 28.
The protein obtained by mutating the 144 th amino acid residue V of SEQ ID NO.1 into E and mutating the 400 th amino acid residue T into D is named as V144E+T400D, and the amino acid sequence is shown as SEQ ID NO. 29.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 481 th amino acid residue L is mutated into N, the mutated protein is named as V144E+L481N, and the amino acid sequence is shown as SEQ ID NO. 30.
The 400 th amino acid residue T of SEQ ID NO. 1 is mutated into D and the 481 th amino acid residue L is mutated into N, the mutated protein is named L481N+T400D, and the amino acid sequence is shown as SEQ ID NO. 31.
The 254 th amino acid residue N of SEQ ID NO. 1 is mutated into D and the 92 th amino acid residue E is mutated into S, the mutated protein is named as N254D+E92S, and the amino acid sequence is shown as SEQ ID NO. 32.
The 254 th amino acid residue N of SEQ ID NO. 1 is mutated into D and the 92 th amino acid residue E is mutated into A, the mutated protein is named as N254D+E92A, and the amino acid sequence is shown as SEQ ID NO. 33.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N, the mutated protein is named as V144E+T400D+L481N, and the amino acid sequence is shown as SEQ ID NO. 34.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 459 th amino acid residue V is mutated into L, the mutated protein is named as V144E+T400D+V459L, and the amino acid sequence is shown in SEQ ID NO. 35.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P, the mutated protein is named as V144E+A355P+T400D+L481N, and the amino acid sequence is shown in SEQ ID NO. 36.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L, the mutated protein is named as V144E+A355P+T400D+V459L+L481N or 5M, the amino acid sequence is shown in SEQ ID NO. 37, and the nucleic acid sequence is shown in SEQ ID NO:94 from 1 to 1539.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 55 th amino acid residue S is mutated into V, the mutated protein is named as 5M+S55V or 6M, the amino acid sequence is shown as SEQ ID NO. 38, and the nucleic acid sequence is shown as SEQ ID NO:95 from 1 to 1539.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 39, wherein the mutation of the 144 th amino acid residue V to E and the 400 th amino acid residue T to D and the 481 th amino acid residue L to N and the 355 th amino acid residue A to P and the 459 th amino acid residue V to L and the 67 th amino acid residue N to L in SEQ ID NO.1 is carried out.
The protein after mutation is named as 5M+Il79F, and the amino acid sequence is shown in SEQ ID NO. 40, wherein the mutation is carried out from 144 th amino acid residue V to E and 400 th amino acid residue T to D and 481 th amino acid residue L to N and 355 th amino acid residue A to P and 459 th amino acid residue V to L and 79 th amino acid residue I to F in SEQ ID NO. 1.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 41, wherein the mutation of the 144 th amino acid residue V to E and the mutation of the 400 th amino acid residue T to D and the mutation of the 481 th amino acid residue L to N and the mutation of the 355 th amino acid residue A to P and the mutation of the 459 th amino acid residue V to L and the mutation of the 147 th amino acid residue A to P in SEQ ID NO. 1 is named as 5M+A147P.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 42, wherein the mutation of the 144 th amino acid residue V to E and the 400 th amino acid residue T to D and the 481 th amino acid residue L to N and the 355 th amino acid residue A to P and the 459 th amino acid residue V to L and the 167 th amino acid residue I to M in SEQ ID NO. 1 is carried out.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 43, wherein the mutation of the 144 th amino acid residue V to E and the 400 th amino acid residue T to D and the 481 th amino acid residue L to N and the 355 th amino acid residue A to P and the 459 th amino acid residue V to L and the 185 th amino acid residue V to M in SEQ ID NO.1 is carried out.
The amino acid sequence of the mutated protein is shown as SEQ ID NO 44, wherein the mutation from 144 th amino acid residue V to E and 400 th amino acid residue T to D and 481 th amino acid residue L to N and 355 th amino acid residue A to P and 459 th amino acid residue V to L and 190 th amino acid residue V to M in SEQ ID NO 1 is carried out.
The 144 th amino acid residue V of SEQ ID NO.1 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 279 th amino acid residue Q is mutated into H, and the mutated protein is named as 5M+Q2799H, and the amino acid sequence is shown as SEQ ID NO. 45.
The 144 th amino acid residue V of SEQ ID NO.1 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 287 th amino acid residue S is mutated into T, and the mutated protein is named as 5M+S287T, and the amino acid sequence is shown as SEQ ID NO. 46.
The 144 th amino acid residue V of SEQ ID NO.1 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 292 nd amino acid residue I is mutated into L, and the mutated protein is named as 5M+I292L, and the amino acid sequence is shown as SEQ ID NO. 47.
The 144 th amino acid residue V of SEQ ID NO.1 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 296 th amino acid residue V is mutated into L, and the mutated protein is named as 5M+V296L, and the amino acid sequence is shown as SEQ ID NO. 48.
The protein obtained by mutating the 38 th amino acid residue S of SEQ ID NO.1 into the A mutation is named as S38A, and the amino acid sequence is shown as SEQ ID NO. 49.
The protein obtained by mutating the 49 th amino acid residue R of SEQ ID NO.1 into V mutation is named as R49V, and the amino acid sequence is shown as SEQ ID NO. 50.
The protein obtained by mutating the 56 th amino acid residue V of SEQ ID NO.1 into I mutation is named V56I, and the amino acid sequence of the protein is shown as SEQ ID NO. 51.
The protein obtained by mutating the 67 th amino acid residue N of SEQ ID NO.1 into Y mutation is named as N67Y, and the amino acid sequence of the protein is shown as SEQ ID NO. 52.
The protein obtained by mutating the 79 th amino acid residue I of SEQ ID NO.1 into W mutation is named I79W, and the amino acid sequence of the protein is shown as SEQ ID NO. 53.
The protein obtained by mutating the 81 st amino acid residue Q of SEQ ID NO.1 into E mutation is named as Q81E, and the amino acid sequence of the protein is shown as SEQ ID NO. 54.
The protein obtained by mutating the 82 nd amino acid residue E of SEQ ID NO.1 into L mutation is named as E82L, and the amino acid sequence of the protein is shown as SEQ ID NO. 55.
The protein obtained by mutating the 83 rd amino acid residue L of SEQ ID NO. 1 into F mutation is named L83F, and the amino acid sequence of the protein is shown as SEQ ID NO. 56.
The protein obtained by mutating the 83 rd amino acid residue L of SEQ ID NO. 1 into M mutation is named L83M, and the amino acid sequence of the protein is shown as SEQ NO. 57.
The protein obtained by mutating the 86 th amino acid residue Y of SEQ ID NO. 1 into W mutation is named as Y86W, and the amino acid sequence is shown as SEQ NO. 58.
The protein obtained by mutating the 92 th amino acid residue E of SEQ ID NO. 1 into S mutation is named as E92S, and the amino acid sequence of the protein is shown as SEQ ID NO. 59.
The protein obtained by mutating the 90 th amino acid residue V of SEQ ID NO. 1 into L mutation is named V90L, and the amino acid sequence is shown as SEQ NO. 60.
The protein obtained by mutating the 90 th amino acid residue V of SEQ ID NO. 1 into M mutation is named as V90M, and the amino acid sequence of the protein is shown as SEQ ID NO. 61.
The protein obtained by mutating the 96 th amino acid residue L of SEQ ID NO. 1 into M mutation is named L96M, and the amino acid sequence of the protein is shown as SEQ NO. 62.
The protein obtained by mutating the 139 th amino acid residue G of SEQ ID NO.1 into the A mutation is named as G139A, and the amino acid sequence is shown as SEQ NO. 63.
The protein obtained by mutating the 144 th amino acid residue V of SEQ ID NO.1 into Q mutation is named V144Q, and the amino acid sequence is shown as SEQ NO. 64.
The protein obtained by mutating the 146 th amino acid residue S of SEQ ID NO.1 into D mutation is named S146D, and the amino acid sequence is shown as SEQ NO. 65.
The protein obtained by mutating the 155 th amino acid residue S of SEQ ID NO.1 into the A mutation is named S155A, and the amino acid sequence is shown as SEQ NO. 66.
The protein obtained by mutating the 155 th amino acid residue S of SEQ ID NO.1 into V mutation is named S155V, and the amino acid sequence is shown as SEQ NO. 67.
The protein obtained by mutating 167 th amino acid residue I of SEQ ID NO.1 into F mutation is named as I167F, and the amino acid sequence is shown as SEQ NO. 68.
The protein obtained by mutating the 181 th amino acid residue L of SEQ ID NO.1 into M mutation is named L181M, and the amino acid sequence is shown as SEQ NO. 69.
The protein obtained by mutating 199 th amino acid residue I of SEQ ID NO.1 into L mutation is named I199L, and the amino acid sequence is shown as SEQ ID NO. 70.
The protein obtained by mutating the 203 th amino acid residue L of SEQ ID NO.1 into F mutation is named L203F, and the amino acid sequence is shown as SEQ NO. 71.
The protein obtained by mutating the 219 th amino acid residue T of SEQ ID NO.1 into A mutation is named as T219A, and the amino acid sequence is shown as SEQ NO. 72.
The protein obtained by mutating the 221 st amino acid residue T of SEQ ID NO.1 into L mutation is named I221L, and the amino acid sequence of the protein is shown as SEQ ID NO. 73.
The protein obtained by mutating the 225 th amino acid residue Q of SEQ ID NO.1 into E mutation is named as Q225E, and the amino acid sequence of the protein is shown as SEQ ID NO. 74.
The protein obtained by mutating the 227 th amino acid residue N of SEQ ID NO.1 into L mutation is named as N227L, and the amino acid sequence of the protein is shown as SEQ NO. 75.
The protein obtained by mutating the 250 th amino acid residue Y of SEQ ID NO.1 into E mutation is named as Y250E, and the amino acid sequence of the protein is shown as SEQ NO. 76.
The protein obtained by mutating the 254 th amino acid residue N of SEQ ID NO.1 into D mutation is named as N254D, and the amino acid sequence is shown as SEQ NO. 77.
The protein obtained by mutating the 254 th amino acid residue N of SEQ ID NO.1 into T mutation is named as N254T, and the amino acid sequence is shown as SEQ NO. 78.
The protein obtained by mutating the 281 th amino acid residue V of SEQ ID NO.1 into M mutation is named V281M, and the amino acid sequence of the protein is shown as SEQ NO. 79.
The protein obtained by mutating the 291 th amino acid residue I of SEQ ID NO.1 into Y mutation is named I291Y, and the amino acid sequence of the protein is shown as SEQ ID NO. 80.
The protein obtained by mutating 345 th amino acid residue N of SEQ ID NO.1 into M mutation is named N345M, and the amino acid sequence is shown as SEQ NO. 81.
The protein obtained by mutating the 405 th amino acid residue S of SEQ ID NO.1 into M mutation is named as S405M, and the amino acid sequence is shown as SEQ NO. 82.
The protein obtained by mutating the 459 th amino acid residue V of SEQ ID NO. 1 into M is named as V459M, and the amino acid sequence of the protein is shown as SEQ NO. 83.
The protein obtained by mutating the 487 th amino acid residue E of SEQ ID NO. 1 into L is named as E487L, and the amino acid sequence of the protein is shown as SEQ NO. 84.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 79 th amino acid residue I is mutated into F, the mutated protein is named 6M+Il79F, and the amino acid sequence is shown as SEQ ID NO: 85.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 147 th amino acid residue A is mutated into P, the mutated protein is named 6M+A147P, and the amino acid sequence is shown as SEQ ID NO: 86.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 185 th amino acid residue V is mutated into M, the mutated protein is named 6M+V185M, and the amino acid sequence is shown as SEQ ID NO 87.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and 144 th amino acid residue V is mutated into E and 400 th amino acid residue T is mutated into D and 481 th amino acid residue L is mutated into N and 355 th amino acid residue A is mutated into P and 459 th amino acid residue V is mutated into L and 190 th amino acid residue S is mutated into M, the mutated protein is named 6M+S190M, and the amino acid sequence is shown as SEQ ID NO: 88.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 254 th amino acid residue N is mutated into D, the mutated protein is named 6M+N254D, and the amino acid sequence is shown as SEQ ID NO: 89.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 279 th amino acid residue Q is mutated into H, the mutated protein is named 6M+Q279H, and the amino acid sequence is shown as SEQ ID NO: 90.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 287 th amino acid residue S is mutated into T, the mutated protein is named 6M+S2870T, and the amino acid sequence is shown as SEQ ID NO: 91.
Setting SEQ ID NO:1, wherein the 55 th amino acid residue S is mutated into V and the 144 th amino acid residue V is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 296 th amino acid residue V is mutated into L, the mutated protein is named 6M+V296L, and the amino acid sequence is shown as SEQ ID NO 92.
The 55 th amino acid residue S of SEQ ID NO. 1 is mutated into V and 92 th amino acid residue E is mutated into S and 144 th amino acid residue V is mutated into E and 400 th amino acid residue T is mutated into D and 481 th amino acid residue L is mutated into N and 355 th amino acid residue A is mutated into P and 459 th amino acid residue V is mutated into L and 254 th amino acid residue N is mutated into D, and the mutated protein is named 6M+E92S+N254D, and the amino acid sequence is shown as SEQ ID NO. 93.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 55 th amino acid residue S is mutated into V, and the F1 domain is truncated after the amino acid residue 486 of the RSV F protein, and the mutated protein is named as 5M+S55V (1-486) or 6M (1-486), and the amino acid sequence is shown as SEQ ID NO. 115.
The 144 th amino acid residue V of SEQ ID NO. 1 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 55 th amino acid residue S is mutated into V, and the F1 domain is truncated after the amino acid residue 525 of the RSV F protein, the mutated protein is named as 5M+S55V (1-525) or 6M (1-525), and the amino acid sequence is shown as SEQ ID NO. 116.
The protein obtained by mutating the 55 th amino acid residue S of SEQ ID NO. 1 into V and the 144 th amino acid residue V into E and the 400 th amino acid residue T into D and the 481 th amino acid residue L into N and the 355 th amino acid residue A into P and the 459 th amino acid residue V into L and the 79 th amino acid residue I into F, and mutating the 106 th to 136 th amino acids into Glycine Serine (GS) connecting sequence is named 6M+I79F+1GS, and the amino acid sequence is shown as SEQ ID NO. 117.
The protein obtained by mutating the 55 th amino acid residue S of SEQ ID NO.1 into V and the 144 th amino acid residue V into E and the 400 th amino acid residue T into D and the 481 th amino acid residue L into N and the 355 th amino acid residue A into P and the 459 th amino acid residue V into L and the 79 th amino acid residue I into F, and mutating the 106 th to 136 th amino acids into a double glycine serine (GSGS) connecting sequence is named 6M+I79F+2GS, and the amino acid sequence is shown as SEQ ID NO. 118.
The protein obtained by mutating the 55 th amino acid residue S of SEQ ID NO. 1 into V and the 144 th amino acid residue V into E and the 400 th amino acid residue T into D and the 481 th amino acid residue L into N and the 355 th amino acid residue A into P and the 459 th amino acid residue V into L and the 79 th amino acid residue I into F, and mutating the 106 th to 136 th amino acids into triple glycine serine (GSGSGS) connecting sequence is named 6M+I79F+3GS, and the amino acid sequence is shown as SEQ ID NO. 119.
2. Construction of protein expression clones
The nucleotide sequence encoding one RSV F protein (F WT and the 86 mutants mentioned above) was ligated with the nucleotide sequence encoding the T4 fibrin trimerization domain (SEQ ID NO: 3) and the nucleotide sequence encoding the FLAG tag (DYKDDDDK) peptide fragment, respectively, by means of the nucleotide sequence encoding GSGS amino acids to construct fusion expressed genes as shown in SEQ ID NO:94 and SEQ ID NO: 95. The genes were synthesized by the polymerase chain reaction with KOD-Plus-Neo polymerase onto the multicloning site of eukaryotic expression vector pCMV. 1. Mu.L of the vector carrying the gene of interest was added to 100. Mu.L of DH 5. Alpha. Competent cells (E.coli). The samples were incubated on ice for 20 minutes, heat-shocked at 42℃for 30 seconds, and on ice for 5 minutes. The E.coli after the reaction was then added to LB medium and plated on agarose medium plates containing kanamycin resistance for cultivation. After the colony grows out, a monoclonal colony is picked, a plasmid DNA miniquantity kit of Axygen is used for miniextracting plasmids, and an endotoxin-free large-extraction plasmid kit of the root of the day is used for removing endotoxin-free large-extraction plasmids for subsequent protein eukaryotic expression.
3. Expression and purification of proteins
HEK293 suspension cells in the logarithmic growth phase were prepared and cultured on a cell shaker at 125rpm,37℃with 8% CO 2 to a density of 2-3X 10 6 cells/mL with a viable cell fraction >98%. 30mL of cells were placed in a new cell culture flask as a transfection system. The process is as follows: at transient transfection, 2. Mu.g of plasmid and 6. Mu.g of PEI MAX per ml of cells were added to the medium to prepare PEI solution and plasmid solution, respectively, and allowed to stand for 5min. The PEI solution is slowly added into the plasmid solution to prepare transfection complex, and the transfection complex is gently shaken and mixed uniformly, and then is stood and incubated for 20-30min. The resulting transfection complex was slowly added to a cell culture flask and placed in a carbon dioxide shaker (set at 120 rpm/min) at 37 ℃. After HEK293F cells were transfected and cultured for a further 24 hours, serum-free and protein-free feed solution SMS293-SUPI (35 mL/L culture system) was added and culture was continued for a further 48 hours. Cultured cells were collected, centrifuged at 1000g for 10min, and the supernatant was collected. The supernatant was centrifuged at 3500g for 20min, the supernatant was collected, filtered through a 0.45 μm filter membrane, and the filtrate was collected. The filtrate was purified by anti-Flag beads affinity chromatography. The filtrate was passed through beads 3 times in a gravity flow column, and then the protein impurities were washed thoroughly with buffer, the target protein was eluted with ph=2, 0.1M Glycine solution and neutralized with 1M Tris solution ph=8.5 at a volume ratio of 10:1. The proteins collected after elution were further subjected to molecular sieve purification using a Superdex200 column in 20mM HEPES pH7.5, 150mM NaCl buffer. SDS-PAGE identification (boiling the sample in water for 5min, so that disulfide bonds between heavy chain and light chain of the antibody are opened, see second edition of molecular cloning Experimental guidelines) is carried out on the purified protein, the concentration is measured by adopting a Coomassie brilliant blue staining method, and the protein is split into 1.5mL tubes and stored at-80 ℃ for standby. The expression levels of recombinant RSV F protein in HEK293F cells are shown in tables 1-2. The results of molecular sieve purification of a typical recombinant RSV F protein are shown in fig. 9.
Table 1 contains the expression levels of the constructs for single site mutations (HEK 293F cell expression is an example)
Table 2 shows the expression levels of the constructs containing multiple site mutations (30 mL 293F cell expression is an example)
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L. The 6M table refers to the mutation at six points L481N, T400D, V144E, A355P, V459L, S V. In the table, 6M (1-486) refers to the RSV F protein containing six point mutations of amino acid residues 1-486 and L481N, T, 400D, V, E, A, 355P, V, 459, L, S V.
In the table 6M (1-525) refers to the RSV F protein containing six point mutations of amino acid residues 1-525 and L481N, T400D, V144E, A355P, V459L, S V.
The SCTM in the table refers to the joint mutation of the three sites N67I, S215P and E487Q.
Ds-CaV1 in the table refers to the joint mutation at four sites of S155C, S190F, V207L and S290C.
EXAMPLE 2 polyacrylamide gel electrophoresis analysis
Polyacrylamide gel electrophoresis (SDS-PAGE) experiments were used to analyze the molecular mass of the RSV F protein and 86 mutants described above in the present invention. The upper layer was 5% concentrated (3.4 mL of water, 830. Mu.L of 30% acrylamide, 630. Mu.L of 1M Tris (pH 6.8), 50. Mu.L of 10% SDS, 50. Mu.L of 10% ammonium persulfate and 5. Mu.L of TEMED) were added. The lower layer was 12% split gum (3.3 mL of water, 4mL of 30% acrylamide, 2.5mL of 1M Tris (pH 8.8), 100. Mu.L of 10% SDS, 100. Mu.L of 10% ammonium persulfate and 10. Mu.L of TEMED). Samples were electrophoresed at 160V for 40 min. After electrophoresis, SDS-PAGE gels were stained overnight with 0.25% Coomassie blue and destained with destaining buffer (50 mL methanol, 100mL acetic acid and 850mL double distilled water). The analysis results of the polyacrylamide gel electrophoresis of the typical recombinant RSV F protein are shown in FIGS. 10 and 11.
EXAMPLE 3 determination of recombinant RSV F protein concentration
Coomassie blue staining solution was used to detect the concentration of RSV F protein and 86 mutants described above in the present invention. After mixing Bovine Serum Albumin (BSA) at concentrations of 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 400. Mu.g/mL with Coomassie Brilliant blue staining solution at a ratio of 1:10, the OD595 signal was detected and a linear regression of the concentration signals was fitted to a standard curve. After mixing the sample with coomassie blue staining solution in a ratio of 1:10, detecting an OD595 signal, and calculating the protein concentration of the sample according to a standard curve.
EXAMPLE 4 thermal stability analysis of RSV F protein having stable Pre-fusion conformation
The thermostability of pre-fusion RSV F protein was determined using Uncle high-throughput multiparameter protein stability analyzer. 9 mu L of each of the RSV F protein and the mutant was added to the sample cell and sealed, and the temperature was set to a range of 25℃to 95℃and raised to 0.5℃per minute. The pre-fusion RSV F protein thermostability was characterized by detection of Tm (protein dissolution temperature) values using the SYPRO (DSF method) procedure. The results of the thermal stability analysis of a typical recombinant RSV F protein are set forth in table 3.
Table 3 melting temperature Tm of constructs comprising multiple site-directed combined mutations
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L.
The SCTM in the table refers to the joint mutation of the three sites N67I, S P and E487Q.
Ds-CaV1 in the table refers to the combined mutation at four sites S155C, S, 190F, V L and S290C.
EXAMPLE 5 Pre-fusion RSV F protein epitope profiling
Neutralizing antibody D25 recognizes a conformational epitope on RSV F rather than a linear polypeptide epitope. Previous studies showed that neutralizing antibody D25 binds only an epitope that is possessed by the pre-fusion conformational RSV F protein(McLellan JS et al science 340 (6136): 1113-7 (2013)). Epitope of antigenNear the top of the pre-fusion RSV F, distal to its transmembrane region, contains the alpha helix formed by peptide segments 196-212 and peptide segments 61-70. As shown in FIG. 8 of the specification, the alpha helices formed by peptide segments 196-212 and peptide segments 61-70 in pre-fusion RSV F are adjacent to each other, and the CDR3 of the D25 heavy chain is inserted into the specific structural groove between peptide segments 196-212alpha helices and peptide segments 61-70. As shown in FIG. 8 of the specification, the major structural changes of peptide fragments 196-212alpha and peptide fragments 61-70 are distant from each other in the post-RSV F-fusion conformation, and the major binding site of D25 is absent in the post-RSV F-fusion conformation. Thus, neutralizing antibody D25 can only bind to RSV F protein having a pre-fusion conformation and can be used to detect whether RSV F is in a pre-fusion conformation, specific binding to D25 indicates that RSV F retains the pre-fusion conformation. We determined the amount of D25 bound to the same mass construct D25 (OD 450 value) using D25 as the detection antibody by ELISA to determine the epitope signature presented by each construct and the relative amount of pre-fusion conformation of each construct by comparison to RSV F wt.
The RSV Fwt protein and 85 mutants were diluted with PBS having a pH of 7.2-7.4 to prepare a coating solution with a final concentration of 0.25. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. A monoclonal antibody D25 specific to human RSV F protein recognizing different epitopes is taken (the VH and VL sequences of the D25 antibody are shown as SEQ ID NO:6 and SEQ ID NO:7 respectively). The B3T solution was diluted 4-fold from the initial concentration of 0.5. Mu.g/mL, 6 gradients total. The blocked ELISA plate was taken, 100. Mu.L of diluted antibody sample was added to each well, and the mixture was allowed to react in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PBS7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled anti-human IgG reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. The key epitope analysis results of the recombinant RSV F protein are shown in tables 4-5.
Table 4 contains the relative values of the pre-fusion conformational content of constructs comprising single site mutations (1 for the pre-fusion conformational content in WT)
Table 5 shows relative values of pre-fusion conformational content of constructs comprising multiple site mutations (1 for pre-fusion conformational content in WT)
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L. The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
In the table, 6M (1-486) refers to the RSV F protein containing six point mutations of amino acid residues 1-486 and L481N, T, 400D, V, E, A, 355P, V, 459, L, S V.
In the table 6M (1-525) refers to the RSV F protein containing six point mutations of amino acid residues 1-525 and L481N, T400D, V144E, A355P, V459L, S V.
The SCTM in the table refers to the joint mutation of the three sites N67I, S215P and E487Q.
EXAMPLE 6 analysis of RSV F protein polypeptide stability by polyacrylamide gel electrophoresis
Pre-fusion RSV F protein in the selection invention: 5m comprising a V144e+a355p+v459l+l481n+t400d combined mutation, seq ID NO:37, respectively; 355P +. V459 355P+V459L+L481 6M, SEQ ID NO:38, a step of carrying out the process; RSV F comprising the combination mutation 6m+il79f, SEQ ID NO:85; RSV F comprising the combination mutation 6m+a147p, SEQ ID NO:86; RSV F comprising the combination mutation 6m+v185m, SEQ ID NO:87, a base; contains 6M+S190M,SEQ ID NO:88; RSV F comprising the combination mutation 6m+n254d, SEQ ID NO:89; RSV F comprising the combination mutation 6m+q279h, SEQ ID NO:90; RSV F comprising the combination mutation 6m+v296l, SEQ ID NO:92. the selected RSV F protein was allowed to stand at 37 degrees for 7 days and then used to analyze whether the protein polypeptide was susceptible to degradation using polyacrylamide gel electrophoresis (SDS-PAGE) experiments. The upper layer was 5% concentrated (3.4 mL of water, 830. Mu.L of 30% acrylamide, 630. Mu.L of 1M Tris (pH 6.8), 50. Mu.L of 10% SDS, 50. Mu.L of 10% ammonium persulfate and 5. Mu.L of TEMED) were added. The lower layer was 12% split gum (3.3 mL of water, 4mL of 30% acrylamide, 2.5mL of 1M Tris (pH 8.8), 100. Mu.L of 10% SDS, 100. Mu.L of 10% ammonium persulfate and 10. Mu.L of TEMED). Samples were electrophoresed at 160V for 40 min. After electrophoresis, SDS-PAGE gels were stained overnight with 0.25% Coomassie blue and destained with destaining buffer (50 mL methanol, 100mL acetic acid and 850mL double distilled water). The results of polyacrylamide gel electrophoresis analysis of recombinant RSV F-protein after a typical 37 degree 7 day shelf life are shown in FIG. 12. L+L481N+T L+L481N+T400D combination the mutated construct had no visible degradation bands.
EXAMPLE 7 RSV F protein conformational analysis with stable pre-fusion conformation
7-1 Negative electron microscope analysis
RSV F protein was diluted with PBS pH 7.2-7.4 to a final concentration of 0.02mg/mL. The diluted protein was attached to a copper mesh grid by the hanging drop method and stained with 0.2% uranium acetate. RSV F protein with stable pre-fusion conformation was observed using BioTwin120, 120KV TECNAI SPRIRIT TEM D1319,1319 (FEI) electron microscopy at a magnification of 52000X and negative electron microscopy data was collected for each sample examined. The data collected are projections of RSV F protein particles in one direction. 10000-20000 protein particles on the negative-dyeing photo are selected by Relion, and the classification of the 2-dimensional image is carried out, and the particles similar to the uniform class are averaged to increase the signal-to-noise ratio of the image. The averaged image is compared with a 2-dimensional projection of the pre-and post-fusion conformations of known RSV F, protein particles with pre-fusion conformations are determined and the number in each class is counted while calculating the percentage of a certain number of particles. Representative negative electron microscopy of RSV F-protein particles with stable pre-fusion conformations are shown in FIGS. 13-15. Statistics of the number percent of different RSV F protein particles with stable pre-fusion conformations are presented in table 6. All constructs containing five combination mutations of L481N, T400D, V144E, a355P, V459L had a predominant number of particles of the pre-fusion conformational RSV F protein.
7-2 Cryoelectron microscope structural analysis
The high-resolution structure of the RSV F protein 6M (comprising six site-directed mutations of S55V, V144E, A355P, T400D, V459L and L481N, SEQ ID NO: 38) designed by the invention is further determined by a freezing electron microscope, and the pre-fusion conformation is accurately analyzed. The 6M sample was diluted with PBS at pH 7.2-7.4 to a final concentration of 0.2mg/mL. The 3. Mu.L diluted protein sample was attached to a copper mesh (Quantifoil, 1.2/1.3 μm,400 mesh) with a porous carbon support membrane, the copper mesh was clamped at its edges with forceps, and then placed in a Vitrobot cryoelectron microscope sample machine. In the sample preparation machine, most of the sample solution was sucked away with filter paper in a 100% humidity environment, and the copper mesh on the tweezers was quickly immersed in liquid ethane for freezing while leaving only a thin layer of solution on the copper mesh. The frozen copper net is loaded into Titan Krois kv electron microscope for data collection of the frozen electron microscope, the magnification used for data collection is 22000 times, and the electron dose of each photographing is 50 electrons/square angstrom. The collected data is processed to generate 2200 frozen electron micrographs.
The 456300 m particles were co-picked using Relion automated particle selection procedure. As shown in figure 16 of the specification, the selected particles exhibit distinct pre-fusion conformational features upon 2-dimensional and 3-dimensional classification. Further 3-dimensional structure modifications resulted in a 6m 3.5 angstrom resolution structure that was fully compatible with the reported pre-fusion RSV F structure, as compared to the specific structure shown in figure 16 of the specification.
Table 6 percent of conformational protein particles prior to fusion by negative electron microscopy of constructs containing site mutations
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L.
The SCTM in the table refers to the joint mutation of the three sites N67I, S P and E487Q.
The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
EXAMPLE 8 storage stability analysis of RSV F protein with stable Pre-fusion conformation at different temperatures
The engineered pre-fusion RSV F proteins were placed on ice after at least 30 days at 4 degrees, at least 14 days at 37 degrees, and at least 30 minutes at 55 degrees, respectively, and the pre-fusion conformational ratios in the different temperature treated RSV F WT proteins and mutants were then determined using the pre-fusion RSV F protein epitope profiling method described in example 5 and compared to the non-temperature treated proteins. The results of the conformational stability analysis of RSV F protein with stable pre-fusion conformations are presented in tables 7-9.
Table 7 ratio of conformational content of constructs containing site mutations after 30 days of 4 degree placement compared to protein fusion without 4 degree placement
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L.
The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
In the table, 6M (1-486) refers to the RSV F protein containing six point mutations of amino acid residues 1-486 and L481N, T, 400D, V, E, A, 355P, V, 459, L, S V.
In the table 6M (1-525) refers to the RSV F protein containing six point mutations of amino acid residues 1-525 and L481N, T400D, V144E, A355P, V459L, S V.
The SCTM in the table refers to the joint mutation of the three sites N67I, S215P and E487Q.
Table 8 ratio of conformational content of constructs containing site mutations after 14 days of 37℃heat treatment compared to the non-heat treated protein fusion
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L. The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
The SCTM in the table refers to the joint mutation of the three sites N67I, S P and E487Q.
Table 9 shows the ratio of the conformational content of the construct comprising the site mutation after 30 minutes of heat treatment at 55℃compared to the pre-fusion conformational content of the non-heat treated protein
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L. The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
The SCTM in the table refers to the joint mutation of the three sites N67I, S215P and E487Q.
EXAMPLE 9 analysis of freeze-thaw stability of RSV F protein with stable pre-fusion conformation
0.5Mg/mL of RSV F protein having a stable pre-fusion conformation was snap frozen with liquid nitrogen, allowed to thaw naturally until frozen, and after thawing 13000g of the sample was centrifuged for 3 min, and then collected for concentration determination as described above in example 3, and the above procedure was repeated until the desired number of freeze-thaw cycles was reached. The freeze-thaw stability results are shown in table 10.
TABLE 10 concentration change after 6 repeated freeze thawing of pre-fusion RSV F constructs
In the table 5M refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V459L. The 6M point in the table indicates the combined mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
Ds-CaV1 in the table refers to the joint mutation at four sites of S155C, S190F, V207L and S290C.
The SCTM in the table refers to the joint mutation of the three sites N67I, S215P and E487Q.
EXAMPLE 10 analysis of the ability of RSV F protein with stable Pre-fusion conformation to induce antibody response in mice 10-1 mouse immunization experiments
4-6 Week old C57BL/6 females were randomly assigned to groups of 3 mice each. Immunization process: primary immunization was performed on trial day 1 and booster immunization was performed on trial day 15. The immunization modes are as follows: intramuscular injection of the right hind limb. A single mouse was immunized 100. Mu.L of the immunized material. Experimental group: each 100. Mu.L of the immunomer was emulsified from 50. Mu.L of pre-fusion RSV F-protein solution (5. Mu.g protein content high dose group, 1. Mu.g low dose group) and 50. Mu.L of aluminium gel adjuvant. The pre-fusion RSV F protein used was 5m containing the V144e+a355p+v459l+l481n+t400d combination mutation, respectively, seq ID NO:37, respectively; 355P +. V459 355P+V459L+L481 6M, SEQ ID NO:38, a step of carrying out the process; RSV F comprising the combination mutation 6m+il79f, SEQ ID NO:85; RSV F comprising the combination mutation 6m+a147p, SEQ ID NO:86; RSV F comprising the combination mutation 6m+v185m, SEQ ID NO:87, a base; contains 6M+S190M,SEQ ID NO:88; RSV F comprising the combination mutation 6m+n254d, SEQ ID NO:89; RSV F comprising the combination mutation 6m+q279h, SEQ ID NO:90; RSV F comprising the combination mutation 6m+v296l, SEQ ID NO:92.PBS control group: the immunization was 100. Mu.L PBS buffer. After 21 days of immunization, serum from the mice was collected, and serum samples were inactivated at 56℃for 30 minutes and stored at-20℃for further use.
10-2 Evaluation of Pre-fusion RSV F protein specific antibody titres in serum
Modified pre-fusion RSV F protein (SCTM with combined mutations at three sites N67I, S215P and E487Q, whose pre-fusion conformation has been confirmed by high resolution cryo-electron microscopy and other data) was diluted with PBS pH 7.2-7.4 to prepare a coating solution at a final concentration of 0.25. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. The serum to be detected is subjected to 4-time gradient dilution, 6 gradients are totally diluted, and the initial dilution ratio is 1:10. The ELISA plate coated with pre-fusion RSV F protein was used, 100. Mu.L of diluted serum sample was added to each well, and the mixture was allowed to react in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled anti-mouse IgG reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. The F protein-specific antibody titer was calculated before serum fusion. The results of evaluation of antibody titers specific for the pre-fusion conformational RSV F protein in mouse serum are shown in tables 10-16 and fig. 17.
Table 10: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 5M immunized mice
Table 11: evaluation of Pre-fusion RSV F protein-specific antibody titres in serum of 6M+I79F immunized mice
Table 12: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 6M+S190M immunized mice
Table 13: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 6M+V185M immunized mice
Table 14: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 6M+N254D immunized mice
Table 15: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 6M+Q279H immunized mice
Table 16: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of 6M+V296L immunized mice
Example 11 analysis of ability of RSV F protein with stable Pre-fusion conformation to induce neutralizing antibodies in mice
The engineered pre-fusion RSV F protein (SCTM with combined mutations at three sites N67I, S215P and E487Q, whose pre-fusion conformation has been confirmed by high resolution structure and other data) was diluted with PBS pH 7.2-7.4 to prepare a coating solution at a final concentration of 0.1. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. The serum to be detected is subjected to 4-time gradient dilution, 6 gradients are totally diluted, and the initial dilution ratio is 1:10. The humanized RSV F protein-specific monoclonal antibody D25 recognizing different epitopes was biotinylated and then diluted with PBS having a pH of 7.2-7.4 to a final concentration of 0.1. Mu.g/mL. The ELISA plate coated with pre-fusion RSV F protein was used, 100. Mu.L of diluted serum sample was added to each well and reacted with biotinylated D25 mixture having a final concentration of 0.1. Mu.g/mL or biotinylated D25 alone having a final concentration of 0.1. Mu.g/mL, and the mixture was placed in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled strepitavidine reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. When biotinylated D25 wells with a final concentration of 0.1 μg/mL alone were added as non-neutralizing antibodies in mice, signals from wells mixed with D25 from serum samples diluted at different fold ratios were compared to signals from wells added alone to D25 wells to obtain a ratio of stable pre-fusion conformational RSV F protein production in mice inducing neutralizing antibodies. The ability of the conformational proteins with stable pre-fusion Prefusion to induce neutralizing antibodies in mice was analyzed as shown in tables 17-23 and figure 18.
TABLE 17 analysis of RSV F Capacity of mice serum antibodies after 5M immunization to compete with D25 binding prior to fusion
TABLE 18 analysis of RSV F Capacity of mice serum antibodies after 6M+I79F immunization to compete with D25 for binding to fusion
TABLE 19 analysis of RSV F Capacity of mice serum antibodies after 6M+V185M immunization to compete with D25 binding to fusion
TABLE 20 analysis of RSV F Capacity of mice serum antibodies after 6M+S190M immunization to compete with D25 for binding to fusion
TABLE 21 analysis of RSV F Capacity of mice serum antibodies after 6M+N254D immunization before competitive binding to D25
TABLE 22 analysis of RSV F Capacity of mice serum antibodies after 6M+Q279H immunization before competitive binding to D25
TABLE 23 analysis of RSV F Capacity of 6M+V296L mice serum antibodies after immunization against D25 binding competition fusion
EXAMPLE 12 construction of RSV F with stable Pre-fusion conformation and preparation of proteins thereof with reference to the type B RSV F sequence SEQ ID NO 2
12-1 Molecular cloning design
The amino acid sequence of the RSV subtype B18537 strain F protein (UniProtKB/Swiss-Prot: P13843) was obtained from the NCBI database, and the region 1-574 of this amino acid sequence was the amino acid sequence of the RSV F protein in the present study (abbreviated as subtype B RSV F WT) as shown in SEQ ID NO: 2. Mutations were performed on subtype B RSV F WT, producing a total of 17 mutants, specifically as follows:
The 144 th amino acid residue V of SEQ ID NO. 2 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L, the mutated protein is named as B-V144E+A355P+T400D+V459L+L481 or B-5M, the amino acid sequence is shown in SEQ ID NO. 96, and the nucleic acid sequence is shown in SEQ ID NO:97 from 1 to 1539.
The 144 th amino acid residue V of SEQ ID NO. 2 is mutated into E and the 400 th amino acid residue T is mutated into D and the 481 th amino acid residue L is mutated into N and the 355 th amino acid residue A is mutated into P and the 459 th amino acid residue V is mutated into L and the 55 th amino acid residue S is mutated into V, the mutated protein is named as B-5M+S55V or B-6M, the amino acid sequence is shown as SEQ ID NO. 98, and the nucleic acid sequence is shown as SEQ ID NO:99 from 1 to 1539.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 100, wherein the mutation of the 144 th amino acid residue V to E and the mutation of the 400 th amino acid residue T to D and the mutation of the 481 th amino acid residue L to N and the mutation of the 355 th amino acid residue A to P and the mutation of the 459 th amino acid residue V to L and the mutation of the 79 th amino acid residue I to F in SEQ ID NO. 2 is named as B-5M+I79F.
The amino acid sequence of the mutated protein is shown as SEQ ID NO. 101, wherein the mutation of the 144 th amino acid residue V to E and the 400 th amino acid residue T to D and the 481 th amino acid residue L to N and the 355 th amino acid residue A to P and the mutation of the 459 th amino acid residue V to L and the 147 th amino acid residue A to P in SEQ ID NO. 2 is named as B-5M+A147P.
The amino acid sequence of the mutated protein is shown as SEQ ID NO 102, wherein the mutation of the 144 th amino acid residue V to E and the mutation of the 400 th amino acid residue T to D and the mutation of the 481 th amino acid residue L to N and the mutation of the 355 th amino acid residue A to P and the mutation of the 459 th amino acid residue V to L and the mutation of the 185 th amino acid residue V to M in SEQ ID NO 2 is named as B-5M+V185M.
The amino acid sequence of the mutated protein is shown as SEQ ID NO 103, wherein the mutation of the 144 th amino acid residue V to E and the 400 th amino acid residue T to D and the 481 th amino acid residue L to N and the 355 th amino acid residue A to P and the 459 th amino acid residue V to L and the 190 th amino acid residue V to M in SEQ ID NO 2 is carried out.
The 144 th amino acid residue V of SEQ ID NO. 2 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 279 th amino acid residue Q is mutated into H, the mutated protein is named as B-5M+Q279H, and the amino acid sequence is shown as SEQ ID NO. 104.
The 144 th amino acid residue V of SEQ ID NO. 2 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 254 th amino acid residue N is mutated into D, and the mutated protein is named as B-5M+N254D, and the amino acid sequence is shown as SEQ ID NO. 105.
The 144 th amino acid residue V of SEQ ID NO. 2 is mutated into E, the 400 th amino acid residue T is mutated into D, the 481 th amino acid residue L is mutated into N, the 355 th amino acid residue A is mutated into P, the 459 th amino acid residue V is mutated into L, the 296 th amino acid residue V is mutated into L, and the mutated protein is named as B-5M+V296L, and the amino acid sequence is shown as SEQ ID NO. 106.
Setting SEQ ID NO:2 from amino acid residue 55 to amino acid residue 144 to amino acid residue 55 to amino acid residue 400 to amino acid residue D to amino acid residue 481 to amino acid residue 355 to amino acid residue P to amino acid residue 459 to amino acid residue 79 to amino acid residue I to amino acid residue F, the mutated protein being designated B-6M+Il79F, the amino acid sequence of which is shown in SEQ ID NO 107.
Setting SEQ ID NO:2 from amino acid residue 55 to amino acid residue 144 to amino acid residue 55 to amino acid residue 400 to amino acid residue D to amino acid residue 481 to amino acid residue 355 to amino acid residue 45 to amino acid residue 147 to amino acid residue P, the mutated protein is named B-6M+A147P, and the amino acid sequence is shown in SEQ ID NO. 108.
The amino acid sequence of the protein after mutation is named as B-6M+V185M, and the amino acid sequence is shown as SEQ ID NO 109, wherein the 55 th amino acid residue S of SEQ ID NO 2 is mutated to V and the 144 th amino acid residue V is mutated to E and the 400 th amino acid residue T is mutated to D and the 481 th amino acid residue L is mutated to N and the 355 th amino acid residue A is mutated to P and the 459 th amino acid residue V is mutated to L and the 185 th amino acid residue V is mutated to M.
Setting SEQ ID NO:2 from amino acid residue 55 to amino acid residue 144 to amino acid residue 55 to amino acid residue 400 to amino acid residue D to amino acid residue 481 to amino acid residue 355 to amino acid residue P to amino acid residue 459 to amino acid residue 190, the mutated protein being designated B-6M+S190M, the amino acid sequence of which is shown in SEQ ID NO 110.
The amino acid sequence of the protein after mutation is named as B-6M+N254D, and the amino acid sequence is shown as SEQ ID NO 111, wherein the 55 th amino acid residue S of SEQ ID NO 2 is mutated to V and the 144 th amino acid residue V is mutated to E and the 400 th amino acid residue T is mutated to D and the 481 th amino acid residue L is mutated to N and the 355 th amino acid residue A is mutated to P and the 459 th amino acid residue V is mutated to L and the 254 th amino acid residue N is mutated to D.
The 55 th amino acid residue S of SEQ ID NO. 2 is mutated into V and 144 th amino acid residue V is mutated into E and 400 th amino acid residue T is mutated into D and 481 th amino acid residue L is mutated into N and 355 th amino acid residue A is mutated into P and 459 th amino acid residue V is mutated into L and 279 th amino acid residue Q is mutated into H, and the mutated protein is named as B-6M+Q279H, and the amino acid sequence is shown as SEQ ID NO. 112.
Setting SEQ ID NO:2 from amino acid residue 55 to amino acid residue 144 to amino acid residue 400 to amino acid residue D to amino acid residue 481 to amino acid residue N to amino acid residue 355 to amino acid residue P to amino acid residue 459 to amino acid residue 296 to amino acid residue V to amino acid residue L, and the mutated protein is named B-6M+V296L, the amino acid sequence of which is shown in SEQ ID NO 113.
The 55 th amino acid residue S of SEQ ID NO. 2 is mutated into V and 144 th amino acid residue V is mutated into E and 400 th amino acid residue T is mutated into D and 481 th amino acid residue L is mutated into P and 459 th amino acid residue V is mutated into L and 254 th amino acid residue N is mutated into D and 92 th amino acid residue E is mutated into S, and the mutated protein is named as B-6M+N254D+E92S, and the amino acid sequence is shown as SEQ ID NO. 114.
12-2 Construction of protein expression clones
The nucleotide sequence encoding one type B RSV F protein (17 type B F protein mutants described above) was ligated with the nucleotide sequence encoding the T4 fibrin trimerization domain (SEQ ID NO: 3) and FLAG tag (DYKDDDK) peptide fragment, respectively, by means of the nucleotide sequence encoding GSGS amino acids to construct fusion expression genes as shown in SEQ ID NO:96 and SEQ ID NO: 98. The genes were synthesized by the polymerase chain reaction with KOD-Plus-Neo polymerase onto the multicloning site of eukaryotic expression vector pCMV. 1. Mu.L of the vector carrying the gene of interest was added to 100. Mu.L of DH 5. Alpha. Competent cells (E.coli). The samples were incubated on ice for 20 minutes, heat-shocked at 42℃for 30 seconds, and on ice for 5 minutes. The E.coli after the reaction was then added to LB medium and plated on agarose medium plates containing kanamycin resistance for cultivation. After the colony grows out, a monoclonal colony is picked, a plasmid DNA miniquantity kit of Axygen is used for miniextracting plasmids, and an endotoxin-free large-extraction plasmid kit of the root of the day is used for removing endotoxin-free large-extraction plasmids for subsequent protein eukaryotic expression.
12-3 Expression and purification of proteins
HEK293 suspension cells in the logarithmic growth phase were prepared and cultured on a cell shaker at 125rpm,37℃with 8% CO 2 to a density of 2-3X 10 6 cells/mL with a viable cell fraction >98%. 30mL of cells were placed in a new cell culture flask as a transfection system. The process is as follows: at transient transfection, 2. Mu.g of plasmid and 6. Mu.g of PEI MAX per ml of cells were added to the medium to prepare PEI solution and plasmid solution, respectively, and allowed to stand for 5min. The PEI solution is slowly added into the plasmid solution to prepare transfection complex, and the transfection complex is gently shaken and mixed uniformly, and then is stood and incubated for 20-30min. The resulting transfection complex was slowly added to a cell culture flask and placed in a carbon dioxide shaker (set at 120 rpm/min) at 37 ℃. After HEK293F cells were transfected and cultured for a further 24 hours, serum-free and protein-free feed solution SMS293-SUPI (35 mL/L culture system) was added and culture was continued for a further 48 hours. Cultured cells were collected, centrifuged at 1000g for 10min, and the supernatant was collected. The supernatant was centrifuged at 3500g for 20min, the supernatant was collected, filtered through a 0.45 μm filter membrane, and the filtrate was collected. The filtrate was purified by anti-Flag beads affinity chromatography. The filtrate was passed through beads 3 times in a gravity flow column, and then the protein impurities were washed thoroughly with buffer, the target protein was eluted with ph=3, 0.1M Glycine solution and neutralized with 1M Tris solution at ph=8.5 in a volume ratio of 10:1. The proteins collected after elution were further subjected to molecular sieve purification using a Superdex200 column in 20mM HEPES pH7.5, 150mM NaCl buffer. SDS-PAGE identification (boiling the sample in water for 5min, so that disulfide bonds between heavy chain and light chain of the antibody are opened, see second edition of molecular cloning Experimental guidelines) is carried out on the purified protein, the concentration is measured by adopting a Coomassie brilliant blue staining method, and the protein is split into 1.5mL tubes and stored at-80 ℃ for standby. Recombinant RSV F protein was expressed at levels comparable to constructs corresponding to subtype a in HEK293F cells (table 24).
Table 24 contains the expression levels of the RSV F protein B strain constructs containing multiple site mutations (exemplified by 30mL 293F cell expression)
B-5M in the table refers to the B-type RSV F protein containing five point-site combined mutations of L481N, T400D, V144E, A355P, V459L.
B-6M in the table refers to six point mutations of the B type RSV F protein containing L481N, T400D, V144E, A355P, V459L, S V.
EXAMPLE 13 subtype Pre-fusion conformational RSV F construct stability, pre-fusion conformational feature epitope analysis and pre-fusion conformational analysis
13-1 Pre-fusion RSV F protein epitope profiling
The 17 subtype B F protein mutants were diluted with PBS having a pH of 7.2-7.4 to prepare a coating solution with a final concentration of 0.25. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. A monoclonal antibody D25 specific to human RSV F protein recognizing different epitopes is taken (the VH and VL sequences of the D25 antibody are shown in SEQ ID NO. 6-7 respectively). The B3T solution was diluted 4-fold from the initial concentration of 0.5. Mu.g/mL, 6 gradients total. The blocked ELISA plate was taken, 100. Mu.L of diluted antibody sample was added to each well, and the mixture was allowed to react in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PBS7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled anti-human IgG reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. The results of the critical epitope analysis of the recombinant subtype B RSV F protein showed consistent properties with the corresponding subtype a construct (table 25).
Table 25 contains relative values of pre-fusion conformational content of multiple site mutated B-type RSV F construct (1 in WT pre-fusion conformational content)
B-5M in the table refers to the B-type RSV F protein containing five point-site combined mutations of L481N, T400D, V144E, A355P, V459L.
B-6M in the table refers to six point mutations of the B type RSV F protein containing L481N, T400D, V144E, A355P, V459L, S V.
13-2B subtype pre-fusion conformation RSV F thermal stability analysis
The thermostability of RSV F protein before B subtype fusion was determined using Uncle high throughput multiparameter protein stability analyzer. 9 mu L of each subtype B RSV F mutant was added into a sample cell for sealing, the temperature change range was set to 25-95 ℃ and the temperature was raised to 0.5 ℃ per minute. The protocol was set up to characterize the thermostability of pre-fusion RSV F protein of subtype B using the SYPRO (DSF method) assay Tm (protein dissolution temperature) values. The results of the thermal stability analysis of the F protein of a typical recombinant subtype B RSV show that it has consistent properties with the corresponding subtype A construct.
13-3B subtype pre-fusion conformational RSV F conformation analysis
The pre-fusion conformational RSV F-protein of subtype B was diluted with PBS having a pH of 7.2-7.4 to a final concentration of 0.02mg/mL. The diluted protein was attached to a copper mesh grid by the hanging drop method and stained with 0.2% uranium acetate. Subtype B was observed with a BioTwin120KV TECNAI SPRIRIT TEM D1319 (FEI) electron microscope at magnification 52000X for a stable pre-fusion conformation of RSV F protein and negative electron microscopy data was collected for each sample examined. The data collected are projections of subtype B pre-fusion conformational RSV F protein particles in one direction. 10000-20000 protein particles on the negative-dyeing photo are selected by Relion, and the classification of the 2-dimensional image is carried out, and the particles similar to the uniform class are averaged to increase the signal-to-noise ratio of the image. The averaged image is compared with a 2-dimensional projection of the pre-and post-fusion conformations of known RSV F, protein particles with pre-fusion conformations are determined and the number in each class is counted while calculating the percentage of a certain number of particles. All construct subtype B presusion conformational RSV F protein particles containing five combined mutations L481N, T400D, V144E, a355P, V459L are in predominant numbers (table 26).
Table 26 percent of presusion conformational protein particles for negative electron microscopy analysis of B-type RSV F-construct containing site mutations
B-5M in the table refers to the joint mutation at five positions L481N, T400D, V144E, A355P, V L. B-6M in the table refers to the joint mutation at six points L481N, T400D, V144E, A355P, V459L, S V.
EXAMPLE 14 analysis of the ability of subtype B to induce antibody responses in mice with a stable pre-fusion conformational RSV F protein
14-1 Mouse immunization experiment
4-6 Week old C57BL/6 females were randomly assigned to groups of 3 mice each. Immunization process: primary immunization was performed on trial day 1 and booster immunization was performed on trial day 15. The immunization modes are as follows: intramuscular injection of the right hind limb. A single mouse was immunized 100. Mu.L of the immunized material. Experimental group: each 100. Mu.L of the immunomer was emulsified with 50. Mu. L B subtype pre-fusion RSV F protein solution (5. Mu.g protein content high dose group, 1. Mu.g low dose group) and 50. Mu.L of aluminium gel adjuvant. The used subtype B pre-fusion RSV F protein is B-5M containing the combined mutation of V144E+A355P+V459L+L481N+T400D, respectively, SEQ ID NO:96; B-6M comprising the S55V+V144E+A355P+V459L+L481N+T400D combination mutation, SEQ ID NO:98; RSV F comprising the combination mutation B-5m+il79f, SEQ ID NO:100; RSV F containing the combination mutation B-5m+s190m, SEQ ID NO:103; RSV F comprising the combination mutation B-6m+il79f, SEQ ID NO:107; contains B-6M+S190M,SEQ ID NO:110; RSV F, SEQ ID NO 111 containing the combination mutation B-6m+n254 d. PBS control group: the immunization was 100. Mu.L PBS buffer. After 21 days of immunization, serum from the mice was collected, and serum samples were inactivated at 56℃for 30 minutes and stored at-20℃for further use.
Evaluation of antibody titer specific for pre-fusion RSV F protein in 14-2 serum
Modified pre-fusion RSV F protein (SCTM with combined mutations at three sites N67I, S215P and E487Q, whose pre-fusion conformation has been confirmed by high resolution cryo-electron microscopy and other data) was diluted with PBS pH 7.2-7.4 to prepare a coating solution at a final concentration of 0.25. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. The serum to be detected is subjected to 4-time gradient dilution, 6 gradients are totally diluted, and the initial dilution ratio is 1:10. The ELISA plate coated with pre-fusion RSV F protein was used, 100. Mu.L of diluted serum sample was added to each well, and the mixture was allowed to react in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled anti-mouse IgG reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. The serum subtype B pre-fusion F protein specific antibody titers were calculated. The results of the evaluation of antibody titers of pre-fusion conformational RSV F protein specific against subtype B in mouse serum showed that it was similar to the ability of the corresponding subtype a construct to induce specific antibodies (tables 27-33).
Table 27: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of B-strain 5M immunized mice
Table 28: evaluation of Pre-fusion RSV F protein specific antibody titres in serum of B-strain 6M immunized mice
Table 29: evaluation of antibody titer specific for pre-fusion RSV F protein in serum of B-strain type 5M+I79F immunized mice
Table 30: evaluation of titer of Pre-fusion RSV F protein specific antibody in serum of B-strain type 5M+S190M immunized mice
Table 31: evaluation of Pre-fusion RSV F protein-specific antibody titres in serum of B-strain type 6M+I79F immunized mice
Table 32: evaluation of Pre-fusion RSV F protein-specific antibody titre in serum of B-strain type 6M+S190M immunized mice
Table 33: evaluation of Pre-fusion RSV F protein-specific antibody titres in serum of B-strain type 6M+N254D immunized mice
EXAMPLE 15 analysis of the ability of subtype B to induce neutralizing antibodies in mice with a stable pre-fusion conformational RSV F protein
The engineered pre-fusion RSV F protein (SCTM with combined mutations at three sites N67I, S P and E487Q, whose pre-fusion conformation has been confirmed by high resolution structure and other data) was diluted with PBS pH 7.2-7.4 to prepare a coating solution at a final concentration of 0.1. Mu.g/mL. 100. Mu.L of coating solution was added to each well of the 96-well ELISA plate, and the plate was coated at 4℃for 16 hours. Then, 280. Mu.L of blocking solution B3T (PBS buffer solution containing 3% bovine serum albumin and 0.5% Tween20 and having a pH of 7.2-7.4) was added to each well, blocking was performed at 37℃for 1 hour, and the blocking solution was discarded. The serum to be detected is subjected to 4-time gradient dilution, 6 gradients are totally diluted, and the initial dilution ratio is 1:10. The humanized RSV F protein-specific monoclonal antibody D25 recognizing different epitopes was biotinylated and then diluted with PBS having a pH of 7.2-7.4 to a final concentration of 0.1. Mu.g/mL. The ELISA plate coated with pre-fusion RSV F protein was used, 100. Mu.L of diluted serum sample was added to each well and reacted with biotinylated D25 mixture having a final concentration of 0.1. Mu.g/mL or biotinylated D25 alone having a final concentration of 0.1. Mu.g/mL, and the mixture was placed in a 37℃incubator for 60 minutes. The ELISA plate was washed 3 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of horseradish peroxidase (HRP) -labeled strepitavidine reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 45 minutes. After completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PBS pH7.4, 150mM NaCl,0.1%Tween20), and 100. Mu.L of TMB developer was added to each well to react at room temperature for 2min30s. After completion of the color reaction step, 50. Mu.L of a stop solution (1M sulfuric acid) was added to each well of the reacted microplate, and the OD450 value of each well was measured on a microplate reader. When biotinylated D25 wells with a final concentration of 0.1 μg/mL alone were added as non-neutralizing antibodies in mice, signals from wells mixed with D25 from serum samples diluted at different fold ratios were compared to signals from wells added alone to D25 wells to obtain a subtype B with a stable pre-fusion conformation of RSV F protein to induce neutralizing antibodies in mice. Subtype B has the ability to stabilize the pre-fusion Prefusion conformational proteins to induce neutralizing antibodies in mice consistent with the corresponding subtype A constructs (tables 34-40).
TABLE 34 analysis of RSV F Capacity of mice serum antibodies after B-strain 5M immunization to compete with D25 binding to fusion
TABLE 35 analysis of RSV F Capacity of mice serum antibodies after B-strain 6M immunization to compete with D25 binding to fusion
TABLE 36 analysis of ability of mouse serum antibodies to compete with D25 for binding to Pre-fusion RSV F following B-strain 5M+I79F immunization
TABLE 37 analysis of ability of mouse serum antibodies to compete with D25 for binding to Pre-fusion RSV F after B-strain 5M-S190M immunization
TABLE 38 analysis of ability of mouse serum antibodies to compete with D25 for binding to Pre-fusion RSV F following B-strain 6M+I79F immunization
TABLE 39 analysis of ability of mouse serum antibodies to compete with D25 for binding to Pre-fusion RSV F after B-type 6M+S190M immunization
TABLE 40 analysis of ability of mouse serum antibodies to compete with D25 for binding to Pre-fusion RSV F after B-strain 6M+N254D immunization
Claims (36)
1. A recombinant Respiratory Syncytial Virus (RSV) F protein having a stable pre-fusion conformation, comprising a protein that introduces at least 1 amino acid residue mutation in the F1 and/or F2 domain of a wild-type RSV F protein, and wherein the respiratory syncytial virus F protein having a stable pre-fusion conformation does not introduce non-native disulfide bonds.
2. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 1, wherein the presusion RSV F protein is capable of specifically binding to a specific antibody recognizing the presusion conformation of RSV F.
3. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 2, wherein the specific antibody recognizing the presusion conformation of RSV F is monoclonal antibody D25.
4. A Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-3, characterized in that it comprises 1 or more amino acid mutations as follows:
(1) Mutation of amino acid residue 55 from a wild-type RSV F protein;
(2) Mutation of amino acid residue 56 from wild-type RSV F protein;
(3) Mutation of amino acid residue 67 from a wild-type RSV F protein;
(4) Mutation of amino acid residue 79 from a wild-type RSV F protein;
(5) Mutation of amino acid residue 81 from wild-type RSV F protein;
(6) Mutation of amino acid residue 82 from a wild-type RSV F protein;
(7) Mutation of amino acid residue 83 from a wild-type RSV F protein;
(8) Mutation of amino acid residue 92 from wild-type RSV F protein;
(9) Mutation of amino acid residue 96 from wild-type RSV F protein;
(10) Mutation of amino acid residue 139 from a wild-type RSV F protein;
(11) Mutation of amino acid residue 144 from wild-type RSV F protein;
(12) Mutation of amino acid residue 145 from a wild-type RSV F protein;
(13) Mutation of amino acid residue 146 from a wild-type RSV F protein;
(14) Mutation of amino acid residue 147 from wild-type RSV F protein;
(15) Mutation of amino acid residue 155 from wild-type RSV F protein;
(16) Mutation of amino acid residue 167 from wild-type RSV F protein;
(17) Mutation of amino acid residue 181 from a wild-type RSV F protein;
(18) Mutation of amino acid residue 185 from wild-type RSV F protein;
(19) Mutation of amino acid residue 189 from the wild-type RSV F protein;
(20) Mutation of amino acid residue 190 from wild-type RSV F protein;
(21) Mutation of amino acid residue 225 from wild-type RSV F protein;
(22) Mutation of amino acid residue 227 from a wild-type RSV F protein;
(23) Mutation of amino acid residue 230 from a wild-type RSV F protein;
(24) Mutation of amino acid residue 254 from wild-type RSV F protein;
(25) Mutation of amino acid residue 279 from the wild-type RSV F protein;
(26) Mutation of amino acid residue 287 from a wild-type RSV F protein;
(27) Mutation of amino acid residue 292 from a wild-type RSV F protein;
(28) Mutation of amino acid residue 296 from wild-type RSV F protein;
(29) Mutation of amino acid residue 355 from the wild-type RSV F protein;
(30) Mutation of amino acid residue 400 from wild-type RSV F protein;
(31) Mutation of amino acid residue 405 from a wild-type RSV F protein;
(32) Mutations at amino acid residue 459 from the wild-type RSV F protein;
(33) Mutation of amino acid residue 481 from a wild-type RSV F protein;
and/or, (34) a mutation of amino acid residue 487 from a wild-type RSV F protein.
5. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 4, wherein the 1 or more amino acid mutations comprise:
(1) Mutation of amino acid residue 55 from wild-type RSV F protein to valine residue or threonine residue;
(2) Mutation of amino acid residue 56 from wild-type RSV F protein to isoleucine residue;
(3) Mutation of amino acid residue 67 from wild-type RSV F protein to leucine residue or tyrosine residue;
(4) Mutation of amino acid residue 79 from wild-type RSV F protein to phenylalanine residue or methionine residue;
(5) Mutation of amino acid residue 81 from wild-type RSV F protein to glutamic acid residue;
(6) Mutation of amino acid residue 82 to leucine residue from a wild-type RSV F protein;
(7) Mutation of amino acid residue 83 from wild-type RSV F protein to methionine residue or phenylalanine residue;
(8) Mutation of amino acid residue 92 from wild-type RSV F protein to serine residue or alanine residue;
(9) Mutation of amino acid residue 96 from wild-type RSV F protein to methionine residue;
(10) Mutation of amino acid residue 139 from wild-type RSV F protein to alanine residue;
(11) Mutation of amino acid residue 144 from wild-type RSV F protein to glutamic acid residue or glutamine residue;
(12) Mutation of amino acid residue 145 from wild-type RSV F protein to tyrosine residue;
(13) Mutation of amino acid residue 146 from wild-type RSV F protein to aspartic acid residue;
(14) Mutation of amino acid residue 147 to proline residue from wild-type RSV F protein;
(15) Mutation of amino acid residue 155 from wild-type RSV F protein to an alanine residue or an isoleucine residue or a valine residue;
(16) Mutation of amino acid residue 167 from wild-type RSV F protein to methionine residue or phenylalanine residue;
(17) Mutation of amino acid residue 181 from wild-type RSV F protein to valine residue;
(18) Mutation of amino acid residue 185 from wild type RSV F protein to methionine residue or aspartic acid residue or glutamic acid residue;
(19) Mutating 189 th amino acid residue from wild type RSV F protein to valine residue;
(20) Mutation of amino acid residue 190 from wild-type RSV F protein to methionine residue;
(21) Mutation of amino acid residue 225 to glutamic acid residue from wild-type RSV F protein;
(22) Mutation of amino acid residue 227 from wild-type RSV F protein to leucine residue;
(23) Mutation of amino acid residue 230 from wild-type RSV F protein to phenylalanine residue;
(24) Mutating the 254 th amino acid residue from wild type RSV F protein to an aspartic acid residue or a threonine residue;
(25) Mutation of amino acid residue 279 from wild-type RSV F protein to a histidine residue or a phenylalanine residue;
(26) Mutation of amino acid residue 287 from the wild-type RSV F protein to a threonine residue or an alanine residue;
(27) Mutation of amino acid residue 292 from wild-type RSV F protein to leucine residue;
(28) Mutation of amino acid residue 296 from wild-type RSV F protein to leucine residue;
(29) Mutation of amino acid residue 355 from the wild-type RSV F protein to a proline residue;
(30) Mutation of amino acid residue 400 from wild-type RSV F protein to aspartic acid residue or asparagine residue;
(31) Mutation of amino acid residue 405 from wild-type RSV F protein to methionine residue;
(32) Mutation of amino acid residue 459 from wild-type RSV F protein to leucine residue or methionine residue;
(33) Mutation of amino acid residue 481 from wild type RSV F protein to an asparagine residue or a serine residue or a threonine residue or a glutamine residue;
And/or, (34) mutation of amino acid residue 487 from wild-type RSV F protein to leucine residue.
6. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 4, wherein the RSV F protein comprises 1 or more mutations selected from the group consisting of:
(a) Mutation of amino acid residue 144 from wild-type RSV F protein;
(b) Mutation of amino acid residue 355 from the wild-type RSV F protein;
(c) Mutation of amino acid residue 400 from wild-type RSV F protein;
(d) Mutations at amino acid residue 459 from the wild-type RSV F protein;
(e) Mutation of amino acid residue 481 from a wild-type RSV F protein;
And/or, (F) a mutation from amino acid residue 55 of the wild-type RSV F protein.
7. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 4, wherein the RSV F protein comprises the following mutations:
(a) Mutation of amino acid residue 144 from wild-type RSV F protein;
(b) Mutation of amino acid residue 355 from the wild-type RSV F protein;
(c) Mutation of amino acid residue 400 from wild-type RSV F protein;
(d) Mutations at amino acid residue 459 from the wild-type RSV F protein;
And (e) mutation of amino acid residue 481 from a wild-type RSV F protein.
8. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 7, wherein the RSV F protein further comprises:
(f) Mutation of amino acid residue 55 from wild-type RSV F protein.
9. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 4, wherein the RSV F protein comprises 1 or more mutations as follows:
(a) Mutation of amino acid residue 144 from wild-type RSV F protein to glutamic acid residue or glutamine residue;
(b) Mutation of amino acid residue 355 from the wild-type RSV F protein to a proline residue;
(c) Mutation of amino acid residue 400 from wild-type RSV F protein to aspartic acid residue or asparagine residue;
(d) Mutation of amino acid residue 459 from wild-type RSV F protein to leucine residue or methionine residue;
(e) Mutation of amino acid residue 481 from wild type RSV F protein to an asparagine residue or a serine residue or a threonine residue or a glutamine residue;
and/or, (F) mutation of amino acid residue 55 from wild-type RSV F protein to a valine residue or a threonine residue.
10. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 4, wherein the RSV F protein comprises the following mutations:
(a) Mutation of amino acid residue 144 from wild-type RSV F protein to glutamic acid residue or glutamine residue;
(b) Mutation of amino acid residue 355 from the wild-type RSV F protein to a proline residue;
(c) Mutation of amino acid residue 400 from wild-type RSV F protein to aspartic acid residue or asparagine residue;
(d) Mutation of amino acid residue 459 from wild-type RSV F protein to leucine residue or methionine residue;
And (e) mutation of amino acid residue 481 from wild type RSV F protein to an asparagine residue or a serine residue or a threonine residue or a glutamine residue.
11. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to claim 10, wherein the RSV F protein further comprises:
(f) Amino acid residue 55 from wild-type RSV F protein is mutated to a valine residue or a threonine residue.
12. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to any one of claims 6-11, further comprising one or more mutations as follows:
Mutation of amino acid residue 79 from wild-type RSV F protein to phenylalanine residue or methionine residue;
mutation of amino acid residue 92 from wild-type RSV F protein to serine residue or alanine residue;
mutation of amino acid residue 147 to proline residue from wild-type RSV F protein;
Mutation of amino acid residue 167 from wild-type RSV F protein to methionine residue or phenylalanine residue;
Mutation of amino acid residue 185 from wild type RSV F protein to methionine residue or aspartic acid residue or glutamic acid residue;
Mutation of amino acid residue 190 from wild-type RSV F protein to methionine residue;
mutating the 254 th amino acid residue from wild type RSV F protein to an aspartic acid residue or a threonine residue;
mutation of amino acid residue 279 from wild-type RSV F protein to a histidine residue or a phenylalanine residue;
mutation of amino acid residue 287 from the wild-type RSV F protein to a threonine residue or an alanine residue;
Mutation of amino acid residue 292 from wild-type RSV F protein to leucine residue;
Amino acid residue 296 from wild-type RSV F protein was mutated to a leucine residue.
13. The Respiratory Syncytial Virus (RSV) F protein of claim 12, wherein the mutation is to mutate amino acid residue 254 of the wild-type RSV F protein to aspartic acid and/or to mutate amino acid residue 92 of the wild-type RSV F protein to alanine or serine.
14. The presusion conformation Respiratory Syncytial Virus (RSV) F protein according to any one of claims 1-13, wherein the F1 domain and/or F2 domain is from an RSV subtype a strain or an RSV subtype B strain.
15. The Respiratory Syncytial Virus (RSV) F protein of any one of claims 1-14, wherein the RSV F protein comprises a heterotrimeric domain linked to the C-terminus of the F1 domain, preferably the heterotrimeric domain is selected from one or more of the following:
t4 phage fibrosis trimerization domain, amino acid sequence: SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL;
The amino acid sequence of the GCN4 leucine zipper trimerization domain is as follows: RMKNLEDKVEELLSKNYHLENEVARLKKLVGER;
Other peptide fragments with heterotrimeric function, preferably have the amino acid sequence VASLRQQVEALQGQ.
16. The presusion conformation respiratory syncytial virus F (RSV) protein according to claim 15, wherein the RSV F protein comprises any one of the following heteromultimerization domains linked C-terminally to the F1 domain:
ferritin proteins;
Viral capsid proteins;
An artificially designed multimeric domain;
other proteins or nucleic acids that can be assembled into multiple polymeric states.
17. The presusion conformational Respiratory Syncytial Virus (RSV) F protein according to claim 16, wherein the artificially designed heteromultimerization domain is I53-50a & b, or a DNA nanomaterial capable of self-assembling to form nanoparticles.
18. The Respiratory Syncytial Virus (RSV) F protein of any one of claims 16 or 17, wherein the trimerization domain or multimerization domain is linked to any amino acid residue between peptide segments 486-530 of the RSV F protein, preferably the linkage is via a peptide segment, preferably the peptide segment is GSGSGS.
19. The Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-18, wherein the amino acid residues 106-136 of the RSV F protein are substituted with a connecting peptide, preferably the connecting peptide is a connecting peptide comprising amino acid residue GS, more preferably GS, GSGS, GSGSGS or GGSGSGSGS.
20. Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-19, which is a fusion protein further comprising a heterologous protein, preferably comprising a leader peptide, a tag protein and/or other epitope proteins.
21. A recombinant nucleic acid molecule comprising a nucleic acid molecule encoding the Respiratory Syncytial Virus (RSV) F protein of any one of claims 1-20 in a pre-fusion conformation.
22. The nucleic acid molecule of claim 21, wherein the nucleic acid molecule codons are optimized for expression in a host cell; preferably, the nucleic acid molecule codon optimized for expression in mammalian cells; more preferably, the nucleic acid molecule is codon optimized for expression in primate cells; most preferably, the nucleic acid molecule is codon optimized for expression in a human cell.
23. A vector comprising the nucleic acid molecule of claim 21 or 22.
24. The vector of claim 23, wherein the vector is any one of an adenovirus vector, an adeno-associated virus vector, a measles virus vector, a lentiviral vector, a herpes virus vector, a liposome, or a cationic polymer vector.
25. A virus-like particle comprising a Respiratory Syncytial Virus (RSV) F protein according to the pre-fusion conformation of any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, and/or a vector according to claim 23 or 24.
26. An immunogenic composition comprising Respiratory Syncytial Virus (RSV) F protein according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, a vector according to claim 23 or 24, and/or a virus-like particle according to claim 25, preferably further comprising an immunostimulant, a pharmaceutically acceptable carrier, an adjuvant and/or other components.
27. A composition comprising the Respiratory Syncytial Virus (RSV) F protein according to the pre-fusion conformation of any one of claims 1-20, the nucleic acid molecule according to claim 21 or 22, the vector according to claim 23 or 24, the virus-like particle according to claim 25, and/or the immunogenic composition of claim 26, preferably further comprising a vaccine, an antiviral drug, a monoclonal antibody, a pharmaceutically acceptable carrier, an adjuvant and/or other components.
28. An isolated host cell comprising a Respiratory Syncytial Virus (RSV) F protein according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, and/or a vector according to claim 23 or 24, preferably the host cell is a eukaryotic or prokaryotic cell, more preferably the cell is selected from Chinese Hamster Ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, per.c6 cells, or yeast, fungi, insect cells.
29. A method of preparing a Respiratory Syncytial Virus (RSV) F protein having a stable pre-fusion conformation, obtained from the host cell of claim 28.
30. Use of a Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, a vector according to claim 23 or 24, a virus-like particle according to claim 25, an immunogenic composition according to claim 26, and/or a composition according to claim 27 for the manufacture of a medicament for inducing an immune response and/or boosting against an RSV F protein.
31. The Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-20, the nucleic acid molecule of claim 21 or 22, the vector of claim 23 or 24, the virus-like particle of claim 25, the immunogenic composition of claim 26, and/or the use of the composition of claim 27 for inducing an immune response and/or boosting against the RSV F protein.
32. The use of a Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, a vector according to claim 23 or 24, a virus-like particle according to claim 25, an immunogenic composition according to claim 26, and/or a composition according to claim 27 for the preparation of a vaccine against RSV.
33. Use of a Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, a vector according to claim 23 or 24, a virus-like particle according to claim 25, an immunogenic composition according to claim 26, and/or a composition according to claim 27 for the preparation of a composition for the prevention and/or treatment of an RSV infection.
34. The Respiratory Syncytial Virus (RSV) F protein in a pre-fusion conformation according to any one of claims 1-20, the nucleic acid molecule of claim 21 or 22, the vector of claim 23 or 24, the virus-like particle of claim 25, the immunogenic composition of claim 26, and/or the use of the composition of claim 27 for the prevention and/or treatment of an RSV infection.
35. A method of preventing and/or treating an RSV infection in a subject, characterized in that a respiratory syncytial virus F protein in a pre-fusion conformation according to any one of claims 1-20, a nucleic acid molecule according to claim 21 or 22, a vector according to claim 23 or 24, a virus-like particle according to claim 25, an immunogenic composition according to claim 26, and/or a composition according to claim 27 is administered to a subject, preferably the subject is a mammal, such as a rodent, e.g. a mouse, a cotton mouse or a non-human primate, or a human.
36. A method for stabilizing a presusion conformation of a respiratory syncytial virus protein, comprising preparing a presusion conformation of a respiratory syncytial virus F (RSV) protein according to any one of claims 1-20.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310341930 | 2023-03-31 | ||
| CN2023103419302 | 2023-03-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118725052A true CN118725052A (en) | 2024-10-01 |
Family
ID=92855228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410376573.8A Pending CN118725052A (en) | 2023-03-31 | 2024-03-29 | Respiratory syncytial virus F protein with stable pre-fusion conformation |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN118725052A (en) |
| WO (1) | WO2024199469A1 (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA035909B1 (en) * | 2015-07-07 | 2020-08-31 | Янссен Вэксинс Энд Превеншн Б.В. | Stabilized soluble pre-fusion rsv f polypeptides |
| RU2723039C2 (en) * | 2015-12-23 | 2020-06-08 | Пфайзер Инк. | Protein f rsv mutants |
| CA3015570A1 (en) * | 2016-03-29 | 2017-10-05 | Peter Kwong | Substitutions-modified prefusion rsv f proteins and their use |
| CN116063551A (en) * | 2016-04-05 | 2023-05-05 | 扬森疫苗与预防公司 | Stabilized soluble pre-fusion RSV F proteins |
| CA3043790A1 (en) * | 2016-12-16 | 2018-06-21 | Institute For Research In Biomedicine | Novel recombinant prefusion rsv f proteins and uses thereof |
-
2024
- 2024-03-29 CN CN202410376573.8A patent/CN118725052A/en active Pending
- 2024-03-29 WO PCT/CN2024/084908 patent/WO2024199469A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024199469A1 (en) | 2024-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020200303B9 (en) | Novel multivalent nanoparticle-based vaccines | |
| TWI663175B (en) | Stabilized soluble pre-fusion rsv f polypeptides | |
| JP6679475B2 (en) | Stabilized soluble pre-fusion RSV F polypeptide | |
| AU2016289496B2 (en) | Stabilized soluble pre-fusion RSV F polypeptides | |
| AU2021232702B2 (en) | Stabilized pre-fusion RSV F proteins | |
| AU2021269783A1 (en) | Stabilized coronavirus spike protein fusion proteins | |
| WO2017207477A1 (en) | Stabilized pre-fusion rsv f proteins | |
| WO2024199469A1 (en) | Respiratory syncytial virus f protein having stable pre-fusion conformation | |
| WO2022229817A1 (en) | Vaccines comprising virus-like particles displaying sars-cov-2 antigens and methods of use | |
| OA19273A (en) | Stabilized pre-fusion RSV F proteins. | |
| OA17539A (en) | Stabilized soluble prefusion RSV F polypeptides. | |
| NZ752808A (en) | Stabilized soluble prefusion rsv f polypeptides | |
| NZ713371B2 (en) | Stabilized soluble prefusion rsv f polypeptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication |