EP1003851A2 - Rsv epitope und antikörper dagegen zur verwendung in dagnose und therapie - Google Patents
Rsv epitope und antikörper dagegen zur verwendung in dagnose und therapieInfo
- Publication number
- EP1003851A2 EP1003851A2 EP98939703A EP98939703A EP1003851A2 EP 1003851 A2 EP1003851 A2 EP 1003851A2 EP 98939703 A EP98939703 A EP 98939703A EP 98939703 A EP98939703 A EP 98939703A EP 1003851 A2 EP1003851 A2 EP 1003851A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- protein
- rsv
- nucleotide sequence
- immunogenic agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 24
- 230000000241 respiratory effect Effects 0.000 title abstract 3
- 238000003745 diagnosis Methods 0.000 title description 3
- 238000002560 therapeutic procedure Methods 0.000 title description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 99
- 101710120037 Toxin CcdB Proteins 0.000 claims abstract description 13
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 5
- 230000008878 coupling Effects 0.000 claims description 35
- 238000010168 coupling process Methods 0.000 claims description 35
- 238000005859 coupling reaction Methods 0.000 claims description 35
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 30
- 235000018102 proteins Nutrition 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 210000004027 cell Anatomy 0.000 claims description 23
- 102000014914 Carrier Proteins Human genes 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 18
- 230000002163 immunogen Effects 0.000 claims description 18
- 108010078791 Carrier Proteins Proteins 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 13
- 125000003729 nucleotide group Chemical group 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 10
- 210000004899 c-terminal region Anatomy 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 8
- 230000004927 fusion Effects 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 241000194017 Streptococcus Species 0.000 claims description 5
- 108091008324 binding proteins Proteins 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 4
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 4
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 4
- 108010079246 OMPA outer membrane proteins Proteins 0.000 claims description 4
- 108020003175 receptors Proteins 0.000 claims description 4
- 238000009007 Diagnostic Kit Methods 0.000 claims description 3
- 241000588748 Klebsiella Species 0.000 claims description 3
- 108020004511 Recombinant DNA Proteins 0.000 claims description 3
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 102000009016 Cholera Toxin Human genes 0.000 claims description 2
- 108010049048 Cholera Toxin Proteins 0.000 claims description 2
- 108091006027 G proteins Proteins 0.000 claims description 2
- 102000030782 GTP binding Human genes 0.000 claims description 2
- 108091000058 GTP-Binding Proteins 0.000 claims description 2
- 241000238631 Hexapoda Species 0.000 claims description 2
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 108010071390 Serum Albumin Proteins 0.000 claims description 2
- 102000007562 Serum Albumin Human genes 0.000 claims description 2
- 241000191940 Staphylococcus Species 0.000 claims description 2
- 238000009109 curative therapy Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 claims description 2
- 238000003780 insertion Methods 0.000 claims description 2
- 230000037431 insertion Effects 0.000 claims description 2
- 229940039696 lactobacillus Drugs 0.000 claims description 2
- 210000004962 mammalian cell Anatomy 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 230000003449 preventive effect Effects 0.000 claims description 2
- 241001515965 unidentified phage Species 0.000 claims description 2
- 239000013603 viral vector Substances 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 description 52
- 102000004196 processed proteins & peptides Human genes 0.000 description 37
- 210000004072 lung Anatomy 0.000 description 31
- 241000725643 Respiratory syncytial virus Species 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 210000002966 serum Anatomy 0.000 description 18
- 230000001681 protective effect Effects 0.000 description 16
- 229940024606 amino acid Drugs 0.000 description 15
- 150000001413 amino acids Chemical class 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 230000009257 reactivity Effects 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 108020001507 fusion proteins Proteins 0.000 description 10
- 102000037865 fusion proteins Human genes 0.000 description 10
- 230000003053 immunization Effects 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- BHATUINFZWUDIX-UHFFFAOYSA-N Zwittergent 3-14 Chemical compound CCCCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O BHATUINFZWUDIX-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000005847 immunogenicity Effects 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 238000010511 deprotection reaction Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000000069 prophylactic effect Effects 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- -1 hexafluorophosphate Chemical compound 0.000 description 5
- 230000002685 pulmonary effect Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 4
- 102000007079 Peptide Fragments Human genes 0.000 description 4
- 108010033276 Peptide Fragments Proteins 0.000 description 4
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 4
- 235000011613 Pinus brutia Nutrition 0.000 description 4
- 241000018646 Pinus brutia Species 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 3
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108010088716 attachment protein G Proteins 0.000 description 3
- 238000005251 capillar electrophoresis Methods 0.000 description 3
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- PLVPPLCLBIEYEA-WAYWQWQTSA-N (z)-3-(1h-indol-3-yl)prop-2-enoic acid Chemical compound C1=CC=C2C(\C=C/C(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-WAYWQWQTSA-N 0.000 description 2
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dithiothreitol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000011579 SCID mouse model Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- PLVPPLCLBIEYEA-UHFFFAOYSA-N indoleacrylic acid Natural products C1=CC=C2C(C=CC(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-UHFFFAOYSA-N 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012429 reaction media Substances 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 150000003573 thiols Chemical group 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 239000001974 tryptic soy broth Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- VVQIIIAZJXTLRE-QMMMGPOBSA-N (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](N)C(O)=O VVQIIIAZJXTLRE-QMMMGPOBSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 108010077480 Albumin Receptors Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 206010006448 Bronchiolitis Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101710160621 Fusion glycoprotein F0 Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101710121996 Hexon protein p72 Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-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
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 229940124679 RSV vaccine Drugs 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 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
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 150000001412 amines Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 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
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 150000001510 aspartic acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 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 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- AQEFLFZSWDEAIP-UHFFFAOYSA-N di-tert-butyl ether Chemical compound CC(C)(C)OC(C)(C)C AQEFLFZSWDEAIP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000002307 glutamic acids Chemical class 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- DDMCDMDOHABRHD-UHFFFAOYSA-N methyl 2-hydroxybutanoate Chemical compound CCC(O)C(=O)OC DDMCDMDOHABRHD-UHFFFAOYSA-N 0.000 description 1
- 238000002941 microtiter virus yield reduction assay Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- SHDMMLFAFLZUEV-UHFFFAOYSA-N n-methyl-1,1-diphenylmethanamine Chemical compound C=1C=CC=CC=1C(NC)C1=CC=CC=C1 SHDMMLFAFLZUEV-UHFFFAOYSA-N 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
- C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to respiratory syncytial virus, and. more particularly to the identification of new epitopes and to the corresponding antibodies, useful in particular in the field of treatment, prophylaxis and diagnosis of the conditions caused by this virus.
- Respiratory syncytial virus is one of the most common etiological agents encountered in infants and the elderly. Bronchiolitis is often severe in children and requires hospitalization. Currently, there are no means of prevention against RSV disease, the first RSV infection does not prevent against the next. Treatment of severe cases with antibiotic therapy (Ribavirin) and / or combined with immunotherapy (human immunoglobulins) cannot reduce the worsening of the disease. However, this type of treatment is still very expensive. Recent clinical trials with ORAVAX HNK20 monoclonal antibodies (directed against RSV F protein) have not shown efficacy compared to placebo against RSV infection in children.
- RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a non-segmented RNA genome, of negative polarity, coding for 10 specific proteins.
- Application WO 87/04185 proposed using RSV structural proteins for a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a protein of 9.5 Kd, or the major capsid protein (protein N).
- RSV protein G can be useful in the preparation of products intended for the treatment and / or prevention of disorders caused by RSV, subgroup A or B.
- fragments of the RSV protein G containing specific epitopes, have particularly advantageous properties.
- New peptide fragments of the RSV protein G can thus be prepared, in particular for the following applications: (i) said peptide fragment coupled or fused, by chemical methods or by genetic engineering, to a carrier, constitutes an effective vaccine against RSV infection, regardless of the mode of administration;
- the same peptide fragments can be used to generate polyclonal and monoclonal antibodies which are very effective in the prophylactic or therapeutic treatments of the host infected with RSV;
- these peptide fragments and the monoclonal antibodies can be used as reagents in a diagnostic kit allowing assert and identify infection in the host infected with RSV- ⁇ or RSV-B.
- the subject of the invention is therefore polyclonal or monoclonal antibodies directed against an epitope of the G protein of RSV corresponding to a sequence chosen from one of the peptide sequences included respectively between the amino acid residues 150-159, 176-189 , 194-207 and 155-176 of the total sequence of protein G of RSV A or B, or of sequences having at least 80%, and preferably at least 98% of homology.
- These antibodies will recognize peptides carried by the sequence between amino acid residues 130 and 230 of protein G of RSV, subgroup A or subgroup B.
- G2Na This region corresponding to amino acids 130-230 of protein G of RSV A is hereinafter designated G2Na.
- G2Na was produced in a bacterium such as E. coli, therefore not glycosylated. It confers, in particular when it is coupled to a carrier such as an OmpA of gram negative bacteria, for example of Klebsiella (protein p40, described in WO 96/14415) or a protein derived from streptococcus (such as the protein binding to serum albumin human, hereinafter called BB, described in WO 96/14416), an immune protection against RSV infections.
- BB serum albumin human
- a recombinant protein BBG2al a derivative of BBG2Na where only four residues have been modified on G2Na: the residues Asn (aal91), Lys (aal92), Gly (195) and Thr (aal98) have been replaced by the residues Ser , Asn, Lys and Pro respectively, confers VRS-A or VRS-B cross-protection in BALB / c mice.
- BBG2a2 where the residues Asn (aal 57), Asn (aal60), ⁇ sn (aal61) and Phe (aal63) have been substituted by the residues Lys, Lys, ⁇ sp and Tyr respectively;
- BBG2a3 contains the eight modified residues of BBG2a l and BBG2a2.
- Particularly advantageous peptides according to the invention present in particular one of the sequences chosen from the sequences ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5, ID No.
- such a peptide can, in addition, comprise at least one cysteine residue in the N-terminal or C-terminal position.
- G5a (aal44-159), G1a (aal64-176), G4a (aal72-187) and G9a (aal90-204).
- the invention therefore also relates to antibodies, monoclonal or polyclonal, directed against a peptide having at least one of the sequences ID No. 1 to ID No. 22.
- Peptides having one or more units corresponding to epitopes 150-159, 176-189, 194-207 and 155-176 of the GRS protein G sequence will be very useful for the various embodiments of the invention.
- Peptides according to the invention, coupled to a carrier protein are useful as immunogens.
- the carrier protein is advantageously chosen from the OmpA of gram negative bacteria and their fragments, the TT protein (tetanus toxoid), the human serum albumin binding protein of Streptococcus and its fragments and the B subunit of cholera toxin. (CTB); preferably, the carrier protein is an OmpA of a bacterium of the genus Klebsiella.
- the peptide is conjugated to the carrier protein by a binding protein; this binding protein can in particular be chosen from a receptor for serum mammalian albumin and the receptors present on the surface of mucosal cells.
- the coupling is preferably a covalent coupling, which can be carried out chemically or by recombinant DNA techniques.
- the invention therefore relates to a nucleotide sequence coding for a peptide or an immunogenic agent as defined above. It may in particular be a hybrid DNA molecule produced by insertion or fusion into the DNA molecule coding for the carrier protein, DNA coding for a peptide according to one of claims 4 or 5 or l 'one of their fragments, fused with a promoter; it can also be an RNA molecule.
- the peptides, antibodies, immunogens and nucleotide sequences according to the invention can be used as a medicament, and more particularly for the preparation of a composition intended for the preventive or curative treatment of conditions caused by RSV, group A or B.
- monoclonal antibodies specifically recognizing the peptides G5a and G l ia and G l ⁇ Ca were generated. Passive transfer of monoclonal antibodies 5C2 (anti-G5a) and 18D 1 (anti-G l ⁇ Ca) to naive mice on the one hand prevents infection with RSV-A. And on the other hand, the same 5C2 monoclonal rapidly eliminates a chronic RSV-A infection in immunocompromised mice.
- the subject of the invention is therefore also a pharmaceutical composition, characterized in that it contains at least one mono or polyclonal antibody, a peptide or an epitope according to the invention, an agent immunogen, or a nucleotide sequence as defined above, and pharmacologically acceptable excipients.
- the monoclonal antibodies are preferably humanized and produced by the recombinant route. According to another aspect of the invention, they are obtained by the phage library method.
- the peptides, immunogens, antibodies and nucleotide sequences according to the invention can, according to an embodiment of the invention, enter into the composition of a diagnostic kit.
- the immunogens can be prepared by recombinant DNA technology, by the introduction of a nucleotide sequence according to the invention in a host cell.
- This nucleotide sequence can be a fusion gene which is introduced via a DNA vector which comes from a plasmid, a bacteriophage, a virus and / or a cosmid.
- This fusion gene can, in one embodiment of the preparation process, be integrated into the genome of the host cell.
- the vector may be a viral vector, known to those skilled in the art.
- the host cell can be a prokaryote, in particular chosen from the group comprising: E. coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus.
- the host cell can also be a yeast, a mammalian cell, a cell of plant origin or an insect cell.
- the fusion protein is expressed: secreted, localized in the cytoplasm, or else exposed to the membrane of the host cells.
- Figures 1 and 2 Principle of cloning the genes G2a l, G2a2 and G2a3 in vectors.
- Figure 3 A-SDS-P ⁇ GE 20% Gel, staining with Comassie Blue.
- M Standards of molecular sizes, tracks 1 and 2 proteins BBG2a l (theoretical mass 38.7 Kd) purified by affinity on HSA-Sepharose.
- Figure 5 A- Immunogenicity of peptides G7a and G8a.
- Figure 6 Immunogenicity of BBG2al to RSV A ( Figure 6A) and RSV B (6B) and protective efficacy against RSV A (6C) and RSV B (6D).
- Figure 7 Curative effect of monoclonal antibodies 18D 1 and 5C2.
- Figure 8 A- Prophylactic efficacy of the 18D 1 monoclonal antibody.
- Figure 10 Determination of the recognition zone of the 5B7 monoclonal antibody by the Pepscan B method.
- BHA Bromo hydrosuccimidyl acid
- CE Capillary Electrophoresis
- FZCE Free Zone Capillary Electrophoresis
- HBTU 2- (l H-Benzotriazole- l-yl) - l, l, 3,3-tetramethyluronium hexafluorophosphate
- the peptide G5a is a peptide of 16 amino acids which corresponds to the fragment of the protein G (144-159) of the VRS-A. It is obtained by chemical synthesis on solid phase from the C side to the N-terminal side.
- the peptides CysG5a and G5aCys correspond respectively to this peptide with an additional Cysteine on the N or C-terminal side which is intended for unequivocal coupling on a carrier protein.
- the peptides were synthesized using an automatic solid phase peptide synthesizer from the C side to the N-terminal side (FMOC chemistry on the scale of 0.1, 0.25 or 1.0 mmol).
- the synthesis of the CysG5a peptide is carried out from a Proline preloaded on a resin of HMP type, which allows after cleavage to obtain a free acid function on the C-terminal side or a resin of Rink amide MHBA type, which allows after cleavage to obtain an amide function on the C-terminal side. That of the G5aCys peptide begins with a Cysteine preloaded on one or the other of the resins.
- the reactive functions of the side chains of the amino acids used are protected by groups compatible with chemistry
- a coupling cycle takes place as follows: deprotection of the N-terminal amino function of the first amino acid using piperidine, activation of the acid function of the second amino acid to be coupled using HBTU / HMP and coupling.
- the peptide ' is cleaved from the resin and the side chains are deprotected by reaction with a water / TFA mixture.
- the peptide is precipitated with ether cooled beforehand to -40 ° C and the mixture is centrifuged. The pellet is washed three times with ether and then dried with nitrogen. The pellet is taken up with water containing 0.1% TFA.
- Example 2 Coupling of peptides G5a, G7a, G8a, G9a, Gl la and Gl l ⁇ Ca on a carrier protein (P40, BB, TT, KLH).
- the peptide Gl l ⁇ Ca (Seq ID No. 14) is a peptide of 13 amino acids derived from protein G of the RSV. It corresponds to the sequence 164-176 of this protein. During the synthesis, the Cys residue at position 173 was replaced by a Ser residue in order to keep only one Cys residue in position 176 and avoid the formation of a disulfurc bridge 1 -2 which does not exist in natural protein G (apparently 1 -4 / 2-3).
- This peptide was coupled using glutaraldehyde (homobifunctional reagent, coupling on the amino and thiol functions) or unequivocally using BH ⁇ (heterobi onctional reagent, coupling on the thiol function of Cysteine in position C- terminal).
- the solution is then dialyzed using a 0.1 M phosphate buffer, pH 7 + 0.1% Zwittergent 3-14, overnight at + 4 ° C. with stirring and the solution obtained is stored frozen. • Coupling to the P40 protein using a homobifunctional reagent (glutaraldehyde).
- conjugates are thawed, sterile filtered (0.22 ⁇ m), aliquoted and stored at + 4 ° C to avoid problems of precipitation.
- the conjugates are characterized by assaying the proteins by the Lowry method, electrophoresis of the SDS-PAGE type (development with Coomassie blue) and by assaying the amino acids after acid hydrolysis in the gas phase, derivation by PITC and analysis by HPLC.
- Example 3 Cloning of G2al, G2a2 gene in expression vector pvaBB308 and production of BBG2al and BBG2a2 fusion proteins in E. coli
- the principle of cloning of G2al gene (Seq ID No. 15), G2a2 (Seq ID No. 16 ) and G2a3 (Seq ID No. 17) in the vectors is explained in Figures 1 and 2.
- G2al The gene coding for the protein G2al (Seq ID No. 15) is constructed by site-directed mutagenesis using the plasmid pRIT28G2Na as starting material. For this, two PCR reactions (Polymerase chain reaction) are carried out with the pairs of oligonucleotides RIT29 / TH 137 (PCR 1) on the one hand and RIT30 / TH 136 (PCR 2) on the other hand under the following conditions:
- the fragments obtained, respectively 262 bp and 208 bp for reactions 1 and 2 are fixed on magnetic beads.
- 25 ⁇ l of DYNAL® M-280 magnetic beads coupled to streptavidin are rinsed twice beforehand with TE buffer (10 mM Tris; EDTA ImM, pH 7.5) and then incubated for 20 minutes at 37 ° C with 90 ⁇ l of amplification reactions 1 and 2.
- the fragments are denatured by incubating the magnetic beads with 50 ⁇ l of 0.15 M NaOH for 10 minutes at room temperature. The two supernatants are recovered, precipitated with absolute ethanol and resuspended in 50 ⁇ l of I ⁇ O.
- the amplified fragment of 509 bp is digested with the restriction enzymes Psû and HindIII.
- the generated fragment of 169 bp is cloned in the vector pRIT28 digested with the same enzymes.
- the plasmid obtained pRIT28G2al down is sequenced with the Dye Deoxy Terminator chemistry according to the protocol described by Applied Biosystem (Perkin Elmer).
- the plasmid pRIT28G2al is obtained by cloning the PstI / Hindlll fragment from pRIT28G2aldown (fragment downstream of the Pst 1 site) into the vector pRIT28G2Na.
- the G2al gene is then cloned into the expression vector pvaBB308 at the EcoRI / HindIII restriction sites generating the vector pvaBBG2al.
- oligonucleotide sequence list is shown below: RIT27: 5 '- GCTTCCGGCTCGTATGTTGTGTG - 3'
- TH 136 5'- CCG ⁇ GA ⁇ AAACCG ⁇ CGACC ⁇ ACCG ⁇ CC - 3 ' ⁇ -1137: 5' - TTTIT ⁇ CTTCGGTITGTTGJCTCGGG - 3 'RIT29: RIT27 biotinylated in 5' RIT30: RIT28 biotinylated in 5 '
- oligonucleotides used in this construction are the following: RIT29 / TNG 193 (PCR 1) on the one hand and TNG 192 / RIT30 (PCR 2) on the other go.
- PCR 1 RIT29 / TNG 193
- TNG 192 / RIT30 PCR 2
- sequences of the oligonucleotides are described below:
- TNG 192 5'- CCGCCGAAAAAACCGAAAGACGAT - 3 '
- TNG 193 5 '- CGAAATGGTAATCGTCTTTCGG - 3'
- the three insert fragments G2al, G2a2 and G2a3 were cloned into different expression vectors in E. coli, in particular in our examples the vectors pvaBB308 where BB is the gene coding for the albumin receptor.
- the fusion proteins obtained BBG2al, BBG2a2 and BBG2a3 can be easily purified by affinity on an HSA-Sepharose column (Human serum Albumin).
- the parameters controlled during fermentation are: pH, agitation, temperature, oxygenation rate, feeding of combined sources (glycerol or glucose).
- the pH is regulated at 7.0.
- the temperature is set at 37 ° C.
- a 30 g fraction of wet biomass is resuspended in 70 ml of TST solution (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.05% Tween 20 and 0.5 mM EDTA).
- the cells are disintegrated by sonication (Vibracell 72401, Sonics & Materials). After centrifugation of the cell lysate, the supernatant is filtered (1.2 ⁇ m) and diluted in 500 ml of TST.
- the fusion proteins thus obtained in soluble form are purified on an affinity column: HSA-Sepharose (human serum albumin) according to the protocol described by (Stahl et al, J. Immunol. Methods, 1989; 124: 43-52).
- the insoluble lysate after centrifugation, is washed once with a buffer (50 mM Tris-HCl pH 8.5; 5 mM MgCl 2 ). After washing, the pellet is dissolved in 30 ml of 7 M guanidine hydrochloride, 25 mM Tris-HCl (pH 8.5), 10 mM Dithiotreitol (DTT), followed by incubation at 37 ° C for 2 hours. The solubilized proteins are added to a renaturation buffer (25 mM Tris-HCl (pH 8.5); 150 mM NaCl and 0.05% Tween 20).
- the concentration of guanidine hydrochloride is adjusted to the final concentration of 0.5 M in the renaturation buffer before the addition of the solubilized fusion proteins.
- the mixture is incubated at room temperature, with moderate shaking, for 16 hours. After centrifugation, the soluble products in the supernatant are purified on an HSA-Sepharose column.
- the purified fusion proteins are analyzed on SDS-PAGE gel (12%), on the MINI PROTEAN II SYSTEM device (BIORADS). Proteins are visualized with Coomassie brilliant blue R250.
- the analysis of the recombinant proteins by Immunoblot with antibodies specific for RSV shows that the proteins are antigenic (see example of SDS gel and BBG2al immunoblot in FIG. 3).
- Example 4 Immunogenicity and protective efficacy of peptides G5a and G9a coupled to P40.
- mice Groups of 7 mice were immunized twice ip with 20 ⁇ g of P40-G9aCys, P40-CysG5a, or P40-G5aCys. Control mice were immunized with PBS. Alhygrogel (20% v / v) was used as an adjuvant for all immunizations. The mice were taken from the retro-orbital sinus 2 weeks after the last immunization to confirm their seroconvcrsion against VRS-V, challenged a week later with 10 5 TCID 50 VRS- ⁇ by the in route, and sacrificed 5 days post-challenge . The lungs were removed and the titer of virus in the lungs determined.
- the immunogenicity of the G5a peptide is dependent on the orientation of the coupling to P40. After coupling by the C-terminal part of the peptide, low to moderate anti-RSV-A antibody titers were induced in the serum. On the other hand, after coupling by the N-terminal part, G5a did not induce such antibodies.
- the G9a peptide, coupled in C-terminal to P40 was weakly immunogenic in terms of induction of anti-RSV-A antibodies.
- P40-G9acys developed a high anti-RSV-A antibody titer.
- mice out of 7 immunized with P40-G5acys were protected without evidence of virus in the lungs, the last one had the virus only at the limit of detection of the test.
- mice immunized with P40-cysG5a had virus titers in the lungs as high as control mice, immunized with PBS.
- the peptides G5a and G9a contain protective epitopes against an RSV-A infection of the lungs.
- the orientation of the coupling of G5a to a carrier protein is crucial for its protective efficacy.
- Example 5 Immunogenicity and protective efficacy of peptides G7a and G8a.
- mice Groups of 3 to 4 mice were immunized twice ip with 20 ⁇ g of G7a, G8a, BB-G7a, or BB-G8a.
- Control mice were immunized with PBS.
- Alhygrogel (20% v / v) was used as an adjuvant for all immunizations.
- the mice were removed from the retro-orbital sinus 2 weeks after the last immunization to confirm their seroconversion vis-à-vis VRS-A, challenged a week later with 10 TCID 50 VRS-A by the inhalation route, and sacrificed for 5 days. post-challenge.
- the lungs were removed and the nasal passages washed. The virus titers in the lungs and nasal passages were determined. Results - Humoral immune responses.
- the peptides G7a and G8a are both immunogenic vis-à-vis the RSV- ⁇ coupled or not coupled to 1313. If we consider the titers of serum antibodies, the unlinked peptides seem to be a little more immunogenic than the coupled peptides.
- the G7a and G8a peptides contain protective epitopes against the lungs.
- the peptides are effective whether or not coupled to BB.
- Example 6 Immunogenicity and protective efficacy of the BBG2al fusion protein.
- mice were immunized 2 and 3 times, respectively, by ip route with 20 ⁇ g of protein 2 weeks apart .
- Control mice were immunized with PBS.
- Alhygrogel (20% v / v) was used as an adjuvant for all immunizations.
- the mice were removed from the retro-orbital sinus 2 weeks after the last immunization to confirm their seroconversion with RSV-A, challenged a week later with 10 5 TCID 50 VRS- ⁇ by in route, and sacrifices 5 days post-challenge. The lungs were removed and the titer of virus in the lungs determined.
- BBG2al is capable of inducing anti-RSV- ⁇ and B antibodies.
- the anti-RSV- ⁇ antibody titer is much higher than the anti-RSV-B antibody titer. Nevertheless, antibody titers against the two strains of RSV are high.
- mice challenged with VRS- ⁇ were protected without evidence of virus in the lungs (2 mice / 3) or only at the detection limit (1 mouse / 3).
- the mice challenged with VRS-B were protected, either without evidence of virus in the lungs (1 mouse / 3) or with virus only at the detection limit (1 mouse / 3) or just above this detection limit (1 mouse / 3).
- the BBG2al fusion protein is very immunogenic vis-à-vis VRS-A and vis-à-vis VRS-B.
- BBG2al is capable of inducing responses which protect the lungs against a challenge with the 2 subgroups of RSV.
- EXAMPLE 7 Obtaining the Antibodies 18D1, 5C2 and 5B7
- Immunization peptide (i) G l ⁇ Ca coupled to KLI I (Kcyhole Lempct Haemocyanin), (ii) G2 ⁇ Ca and (iii) BBG2Na.
- mice were immunized at OJ with 50 ⁇ g of CF ⁇ antigen (complete Freund Adjuvant) in ip, on D 14 with 10 ⁇ g of each antigen in IFA (Incomplete Freund Adjuvant) in ip, then on D38 in iv with 10 ⁇ g of each peptide without adjuvant.
- the spleens are removed and fused with the myeloma cells SP2-O at D42 in a 1/1 ratio.
- the hybridomas positive against each antigen are kept. These hybridomas were injected into mice to obtain ascites and then the different antibodies obtained were selected on different peptides in order to determine the specificity of the antibodies obtained.
- the 18D 1, 5C2 monoclonal antibodies selected by their specificity against the peptides G l ⁇ Ca, G5a, specifically recognize VRS- ⁇ .
- the monoclonal antibody 5B7 obtained from BBG2Na and recognizing the peptide G 1 recognizes it VRS-A.
- Example 8 Curative effect of the 18D 1 and 5C2 monoclonal antibodies on chronic RSV-A infection obtained in SCID mice.
- mice 10 5 TCID 50 of VRS-A, under 50 ⁇ l. Twenty-six days later, the mice received 50 ⁇ l of an 18D 1 antibody or 5C2 antibody preparation with an anti-RSV-A ELISA titre of 10 4, in and at the rate of 7 mice per group. . Control mice received anti-BB serum with an anti-BB ELISA titer of 10. The mice were sacrificed 5 days later and their lungs were removed for virus titration. Results.
- Fig. 7 demonstrate that the 18D 1 and 5C2 monoclonal antibodies are capable of eliminating a chronic infection with VRS-A in SCID mice and this in a sterilizing manner. No traces of virus were detected in the lungs at the time of the sacrifice. The results obtained in the lungs of the mice treated with 5C2 correspond to the average of the detection limits of the test, a higher limit due to the lack of sample availability, and not to the presence of virus.
- the monoclonal antibodies 5C2 and 18D 1 could be used as a therapeutic treatment in the context of pulmonary infections with RSV-A.
- Example 9 Prophylactic effect of the 18D 1 monoclonal antibody on RSV-A infection in mice.
- VRS-A received by intraperitoneal injection 200 ⁇ l of an 18D 1 antibody preparation adjusted for the anti-VRS-A ELISA titre of 10 ⁇ .
- a group of ip transfer control mice received in parallel 200 ⁇ l of an anti-P40 serum preparation (irrelevant serum) adjusted to the anti-P40 ELISA titre of 10%. All the mice are infected the following day by the in route with 50 ⁇ l of a viral suspension containing 10 ⁇ TCID50 of VRS-A. They are sacrificed 5 days later for assay of virus in the lungs. Results.
- the antibody 18 1 is capable after ip transfer of inducing protection in the lungs of naive mice during a challenge with VRS- ⁇ . All the mice are protected after injection of 200 ⁇ l of 18D 1 as 10 5 . Three out of 7 mice are protected without evidence of virus in the lungs. The others show traces of virus only at the detection limit of the trial (3 mice / 7), or just above this limit (1 mouse / 7). Control mice have titers between log i Q 3.70 and 4.45 per gram of lungs.
- the 18D 1 antibody is capable of preventing a RSV-V pulmonary infection in B ⁇ LB / c mice. It demonstrates significant prophylactic efficacy.
- Example 10 Prophylactic effect of 5C2 monoclonal antibody on RSV-A infection in mice.
- Naive mice seronegative vis-à-vis VRS-A receive by transfer in, 50 ⁇ l of a preparation of 5C2 adjusted to the ELISA anti-VRS-A title of 10 4 .
- Control mice receive in parallel anti-BB serum adjusted to the anti-BB ELISA titer of 10 4 . All mice are infected the next day by the in route with
- mice treated with 5C2 at 10 4 were protected at the pulmonary level. Only 1 mouse out of 7 shows traces of virus. Control mice have titers between log i Q 3.70 and 4.70 per gram of lungs.
- the 5C2 antibody is capable of preventing an RSV- ⁇ pulmonary infection in BALB / c mice. It demonstrates significant prophylactic efficacy.
- Example 11 Pepscan: example of the multiple synthesis of 94 octapeptides covering the sequence of aa 130-230 of G2Na and overlapping with an amino acid.
- peptides are synthesized on a solid support at the end of 96 "pins" (8 x 12) complementary to an ELISA microtiter plate in which the screening of monoclonal or polyclonal antibodies (sera) will be carried out directly.
- control peptide # 1 PLAQGGGG A2 (l): control peptide # 2: GL ⁇ QGGGG
- the synthesis corresponds to several cycles of deprotection, washing and coupling until the desired peptide sequences are obtained. At the end of the synthesis, the peptides are N-acetylated, before the step of deprotection of the side chains.
- the amino acids used for synthesis on pins are protected by an Fmoc group (9-Fluorenylmethoxycarbonyl) and the following side chain protecting groups: t-Butyl ether (tBu) for Serine, Threonine and Tyrosine, t-Butyl ester (OtBu) for Aspartic and Glutamic Acids, t-Butoxycarbonyl (Boc) for Lysine, Histidine and Tryptophan, 2,2,5,7,8-pcntamethylchroman-6-sulfonyl (Pmc) for Arginine, Trityle (Trt) for Cysteine, Asparagine and Glutamine.
- the activation of the acid function is carried out with Diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) dissolved in DMF.
- DIC Diisopropylcarbodiimide
- HOBt 1-hydroxybenzotriazole
- Fmoc-amino acids undergo an activation step before they can be coupled.
- the duration of a coupling step is 2 hours for a concentration of 100 mM with 1.5 equivalent of HOBt and 1.2 equivalent of DIC.
- Software is used to calculate the quantities of reagents required for the coupling step.
- the weighings are carried out in tubes classified in alphabetical order of the letter code of amino acids. The tubes are filled outside the balance on a sheet of paper-linen, then weighed until a mass close to that indicated on the weighing sheet is obtained. The mass must not be less than 0.2 mg nor more than 0.9 mg compared to the theoretical quantity.
- Coupling step The pins are gently introduced into the wells. The box is closed carefully and left in a hood for the duration of the coupling for 2 hours for an amino acid concentration of 100 mM. A 2 hour coupling allows 3 couplings per day.
- Acetylation of terminal amines the pine heads are incubated in the wells of a plate containing 150 ⁇ l of the following reactive mixture: DMF / acetic anhydride / trietylamine 50/5/1 (v / v / v). The block is enclosed in a box for 90 min. at room temperature. The block is then washed with 200 ml of MeOH for 15 min. then dried for 15 min.
- the protective groups of the side chains are eliminated by immersing the pins in 200 ml of a TFA / anisole / Ethanedithiol 190/5/5 ml mixture for 2 h 30 at room temperature. After this deprotection step, the block of pins is removed from the acid solution. The box is rinsed once with MeOH, then filled with MeOH to completely immerse the block of pins in it for 10 min. The block is then struck on tissue paper and then immersed in 200 ml of a MeOH / water / acetic acid mixture (100/100/1 ml) for 1 hour and struck again on tissue paper. The block is placed under vacuum in a desiccator overnight.
- Example 12 ELISA The plate carrying the pins is saturated for 1 hour at 37 ° C. in saturation buffer (PBS, Tween 0, 1%, gelatin 1%), washed for 10 min in PBS and incubated overnight at 4 ° C. with shaking. with the diluted serum to be analyzed. The plate is then washed 4 times 10 min in PBS and incubated for one hour at room temperature with a secondary antibody labeled with peroxidase, diluted to 1/5000 e. After 4 washes in PBS, the TMB substrate is added. The reaction is stopped by adding sulfuric acid.
- saturation buffer PBS, Tween 0, 1%, gelatin 1%
- FIGS. 9 ⁇ and 9B show a reactivity of the anti-BBG2Na mouse serum against 4 regions of the G2Na molecule (the residues in bold lines represent the amino acids playing an important role in ⁇ c / Ag recognition): - region 1 located between residues 150 and 159 whose sequence is QRQNKPPNKP. This region is included in the peptide G5a (144-159) and corresponds to the reactivity zone of the 5C2 monoclonal antibody;
- Region 2 located between residues 176 and 189 whose sequence is CSNNPTCWAICKRI. It is a region located at the level of the peptide G l ⁇ Ca (174-187) and corresponding to the reactivity of the monoclonal antibodies 18D 1 and 5D3;
- G l la the recognition zone of the monoclonal antibody 5B7 (obtained after immunization of BALB / c mice by the vaccine candidate BBG2Na) whose pepsean B is shown below in Figure 10.
- This monoclonal antibody recognizes the sequence FEVFNFVP (165-172).
- Table I shows that the anti-BBG2Na serum indeed exhibits "anti-G4a, G5a cys, G9a cys and Gl1a" activities.
- Table I Anti-G4a, G5aCys, G9aCys and Gl l ⁇ Ca Titers Expressed in Logio of the Anti-BBG2Na Serum Ref. BE-02.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pulmonology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Immunology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9709079A FR2766192B1 (fr) | 1997-07-17 | 1997-07-17 | Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie |
FR9709079 | 1997-07-17 | ||
PCT/FR1998/001570 WO1999003987A2 (fr) | 1997-07-17 | 1998-07-17 | Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1003851A2 true EP1003851A2 (de) | 2000-05-31 |
Family
ID=9509314
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98939703A Withdrawn EP1003851A2 (de) | 1997-07-17 | 1998-07-17 | Rsv epitope und antikörper dagegen zur verwendung in dagnose und therapie |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1003851A2 (de) |
JP (1) | JP2001510039A (de) |
CN (1) | CN1264425A (de) |
AU (1) | AU756110B2 (de) |
BR (1) | BR9810907A (de) |
CA (1) | CA2296736A1 (de) |
FR (1) | FR2766192B1 (de) |
WO (1) | WO1999003987A2 (de) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2790959B1 (fr) * | 1999-03-15 | 2003-06-27 | Pf Medicament | Utilisation de fractions membranaires bacteriennes a effet adjuvant, leurs procedes de preparation et composition pharmaceutique les contenant |
GB9920000D0 (en) * | 1999-08-25 | 1999-10-27 | Imp Cancer Res Tech | Polypeptides |
FR2798857B1 (fr) * | 1999-09-23 | 2003-06-06 | Pf Medicament | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale |
FR2805163A1 (fr) * | 2000-02-21 | 2001-08-24 | Pf Medicament | Utilisation d'un detergent de type zwittergent pour la preparation d'une composition pharmaceutique destinee a etre administree par voie nasale |
EP1174506A1 (de) * | 2000-06-28 | 2002-01-23 | Stichting Dienst Landbouwkundig Onderzoek | C-terminales Erns-Peptide und Analoge davon |
FR2819810B1 (fr) * | 2001-01-23 | 2004-05-28 | Pf Medicament | Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin |
FR2827605B1 (fr) | 2001-07-20 | 2004-07-16 | Pf Medicament | Nouveaux peptides derives de la proteine g du vrs et leur utilisation dans un vaccin |
SI1472286T1 (sl) * | 2002-02-05 | 2007-08-31 | Geymonat Spa | Postopek proizvodnje rekombinantnega placentnega rastnega faktorja |
CN100528896C (zh) * | 2004-02-03 | 2009-08-19 | 中国人民解放军军事医学科学院毒物药物研究所 | 用于预防、诊断和治疗呼吸道合胞病毒感染的嵌合抗原及其抗体 |
EP1768993A4 (de) * | 2004-06-16 | 2008-07-23 | Univ Johns Hopkins | Cysteinreiche region des humanen respiratorischen synzytial-virus und verwendungsverfahren dafür |
CN101130765B (zh) * | 2006-08-21 | 2011-04-06 | 北京阿斯可来生物工程有限公司 | 呼吸道合胞病毒检测试剂盒 |
CN101808663B (zh) * | 2007-10-25 | 2015-09-30 | 特雷利斯生物科学股份有限公司 | 抗rsv g蛋白抗体 |
EP2461825B1 (de) * | 2009-08-04 | 2017-05-31 | The Government of The United States of America, as represented by the Secretary, Department of Health and Human Services, | Anti-rsv-immunogene und immunisierungsverfahren damit |
CN103163302B (zh) * | 2011-12-10 | 2015-06-03 | 河北菲尼斯生物技术有限公司 | 一种采用定向交叉偶联技术制备的短肽抗体试剂盒 |
CN106432439A (zh) * | 2016-11-28 | 2017-02-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106397547A (zh) * | 2016-11-28 | 2017-02-15 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106518988A (zh) * | 2016-11-28 | 2017-03-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106749555A (zh) * | 2016-11-28 | 2017-05-31 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106432437A (zh) * | 2016-11-28 | 2017-02-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106432438A (zh) * | 2016-11-28 | 2017-02-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106432436A (zh) * | 2016-11-28 | 2017-02-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106432435A (zh) * | 2016-11-28 | 2017-02-22 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN106749556A (zh) * | 2016-11-28 | 2017-05-31 | 烟台偌帝生物工程有限公司 | 一种牛呼吸道胞合体病毒抗原蛋白 |
CN112409481B (zh) * | 2020-12-01 | 2024-03-26 | 福州捷赫生物科技有限公司 | 抗p40蛋白单克隆抗体、细胞系及其制备方法和应用 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5223254A (en) * | 1987-09-29 | 1993-06-29 | Praxis Biologics, Inc. | Respiratory syncytial virus: vaccines |
SE9101433D0 (sv) * | 1991-05-13 | 1991-05-13 | Marianne Hansson | Recombinant dna sequence and its use |
FR2718452B1 (fr) * | 1994-04-06 | 1996-06-28 | Pf Medicament | Elément d'immunogène, agent immunogène, composition pharmaceutique et procédé de préparation. |
DE69509103T2 (de) * | 1994-08-25 | 1999-09-30 | Pepscan Systems Bv | Antigene peptide abgeleitet vom g-protein des rsv (respiratorisches synzytialvirus) für die typen- und subtypen spezifische diagnose einer rsv-infektionen |
FR2726576B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Production de peptides analogues de peptides hydrophobes, peptide recombinant, sequence d'adn correspondante |
FR2726472B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Proteine porteuse a effet adjuvant, complexe immunogene la contenant, leur procede de preparation, sequence nucleotidique et vaccin |
FR2726471B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Procede pour ameliorer l'immunogenicite d'un compose immunogene ou d'un haptene et application a la preparation de vaccins |
FR2726577B1 (fr) * | 1994-11-07 | 1997-01-31 | Pf Medicament | Procede d'obtention d'un peptide derive du virus respiratoire syncitial, polypeptide et bacterie l'exprimant, et leurs applications a titre de medicament |
AUPO026596A0 (en) * | 1996-06-05 | 1996-06-27 | Biomolecular Research Institute Limited | Viral peptide |
-
1997
- 1997-07-17 FR FR9709079A patent/FR2766192B1/fr not_active Expired - Fee Related
-
1998
- 1998-07-17 CN CN98807304A patent/CN1264425A/zh active Pending
- 1998-07-17 AU AU88123/98A patent/AU756110B2/en not_active Ceased
- 1998-07-17 JP JP2000503193A patent/JP2001510039A/ja active Pending
- 1998-07-17 EP EP98939703A patent/EP1003851A2/de not_active Withdrawn
- 1998-07-17 BR BR9810907-3A patent/BR9810907A/pt not_active Application Discontinuation
- 1998-07-17 CA CA002296736A patent/CA2296736A1/fr not_active Abandoned
- 1998-07-17 WO PCT/FR1998/001570 patent/WO1999003987A2/fr not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9903987A2 * |
Also Published As
Publication number | Publication date |
---|---|
FR2766192A1 (fr) | 1999-01-22 |
WO1999003987A2 (fr) | 1999-01-28 |
AU756110B2 (en) | 2003-01-02 |
BR9810907A (pt) | 2000-08-01 |
CA2296736A1 (fr) | 1999-01-28 |
WO1999003987A3 (fr) | 1999-04-08 |
CN1264425A (zh) | 2000-08-23 |
AU8812398A (en) | 1999-02-10 |
JP2001510039A (ja) | 2001-07-31 |
FR2766192B1 (fr) | 2001-07-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1003851A2 (de) | Rsv epitope und antikörper dagegen zur verwendung in dagnose und therapie | |
EP0754231B1 (de) | Peptidfragment des g-proteins des respiratorischen syncytialvirus, immunogene verbindung und pharmazeutische zusammensetzung, die es enthalten, und herstellungsverfahren | |
EP0791064B1 (de) | Verfahren zur verbesserung der immunogenität einer immunogenen zusammensetzung oder eines haptens und verwendung zur herstellung von impfstoffen | |
EP0712414A1 (de) | Rekombinanter vektor mit gensequenz von lipoproteine zur nukleotidefrequenzen expression | |
JPH03502687A (ja) | レスピラトリイ・シンシチアル・ウイルス:ワクチンおよび診断法 | |
BE1000811A4 (fr) | Anticorps monoclonaux, peptides et compositions les contenant, destinees au diagnostic et au traitement des infections par le virus hiv. | |
NO313917B1 (no) | Antigen-presenterende kapsid med fusert MS2-kappeprotein | |
ES2270420T3 (es) | Genes receptores de la transferrina de haemophilus. | |
FR2827605A1 (fr) | Nouveaux peptides derives de la proteine g du vrs et leur utilisation dans un vaccin | |
EP0791063B1 (de) | Trägerprotein mit adjuvant aktivität, diese enthaltende immunogene komplexe, ihre herstellung, nukleotidsequenz und impfstoff | |
JP2002511847A (ja) | Porphyromonas gingivalisに関連した歯周病の診断および治療のための合成ペプチド構築物 | |
KR20090092764A (ko) | 신규한 리노바이러스 중화 면역원 및 이의 백신 용도로의 이용 | |
JPH03128399A (ja) | エイズ用複合免疫原 | |
WO2003102170A1 (fr) | Souches de bordetella rendues deficientes par attenuation genetique | |
CA2385404A1 (fr) | Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale | |
EP0953050A1 (de) | Mittel zum diagnostik, zur vorbeugung und zur behandlung von ansteckungenoder infektionen durch mucotrophe virusen | |
FR2650954A1 (fr) | Composition resultant de la reunion d'un epitope b de la glycoproteine d'enveloppe d'un retrovirus du type iv et d'un epitope t issu d'une proteine distincte codee par ce retrovirus et leur application a la production d'anticorps protecteurs contre le sida | |
FR2532850A1 (fr) | Conjugues immunogenes entre un haptene et une molecule porteuse derivee d'une toxine, les vaccins les composant et procede pour leur obtention | |
JP2002518033A (ja) | 農場動物の成長促進のための合成ソマトスタチン免疫原 | |
MXPA00000587A (en) | Epitopes of syndrical respiratory virus and antibodies that understand them, useful in diagnosis and tera | |
FR2699538A1 (fr) | Sous-unité d'une capsule protéique CS31A modifiée par au moins un peptide hétérologue, capsule protéique CS31A et microorganismes portant ces sous-unités; procédés d'obtention et utilisation de ceux-ci. | |
WO1989006971A1 (en) | Conserved rotavirus gene segments and use in immunization and neutralization |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20000131 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20050201 |