WO1994010201A1 - Peptides and vaccines derived from nematode tubulin - Google Patents
Peptides and vaccines derived from nematode tubulin Download PDFInfo
- Publication number
- WO1994010201A1 WO1994010201A1 PCT/CA1993/000431 CA9300431W WO9410201A1 WO 1994010201 A1 WO1994010201 A1 WO 1994010201A1 CA 9300431 W CA9300431 W CA 9300431W WO 9410201 A1 WO9410201 A1 WO 9410201A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glu
- tubulin
- peptide
- monoclonal antibody
- gin
- Prior art date
Links
- 108090000704 Tubulin Proteins 0.000 title claims abstract description 94
- 241000244206 Nematoda Species 0.000 title claims abstract description 34
- 229960005486 vaccine Drugs 0.000 title claims abstract description 28
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 61
- 102000004243 Tubulin Human genes 0.000 title description 91
- 102000004196 processed proteins & peptides Human genes 0.000 title description 13
- 208000036815 beta tubulin Diseases 0.000 claims abstract description 16
- 239000012634 fragment Substances 0.000 claims abstract description 16
- 208000030852 Parasitic disease Diseases 0.000 claims abstract description 13
- 239000003096 antiparasitic agent Substances 0.000 claims abstract description 7
- 229940125687 antiparasitic agent Drugs 0.000 claims abstract description 6
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 6
- 239000000032 diagnostic agent Substances 0.000 claims abstract description 5
- 229940039227 diagnostic agent Drugs 0.000 claims abstract description 5
- 229940124452 immunizing agent Drugs 0.000 claims abstract description 5
- 102000000131 Beta tubulin Human genes 0.000 claims abstract 2
- 150000001413 amino acids Chemical group 0.000 claims description 26
- 244000045947 parasite Species 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 16
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 claims description 13
- OGNJZUXUTPQVBR-BQBZGAKWSA-N Glu-Gly-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OGNJZUXUTPQVBR-BQBZGAKWSA-N 0.000 claims description 13
- WBINSDOPZHQPPM-AVGNSLFASA-N Ser-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)O WBINSDOPZHQPPM-AVGNSLFASA-N 0.000 claims description 13
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 claims description 13
- 108010055341 glutamyl-glutamic acid Proteins 0.000 claims description 13
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 claims description 12
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 201000006353 Filariasis Diseases 0.000 claims description 9
- 241000124008 Mammalia Species 0.000 claims description 8
- 230000003053 immunization Effects 0.000 claims description 7
- 230000037396 body weight Effects 0.000 claims description 5
- 231100000433 cytotoxic Toxicity 0.000 claims description 5
- 230000001472 cytotoxic effect Effects 0.000 claims description 5
- 230000036281 parasite infection Effects 0.000 claims description 4
- 241000282465 Canis Species 0.000 claims description 2
- 241000002163 Mesapamea fractilinea Species 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 6
- 102000036639 antigens Human genes 0.000 abstract description 6
- 108091007433 antigens Proteins 0.000 abstract description 6
- 239000000203 mixture Substances 0.000 abstract description 6
- 241000243982 Brugia pahangi Species 0.000 description 37
- 108090000623 proteins and genes Proteins 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 210000004556 brain Anatomy 0.000 description 18
- 230000035899 viability Effects 0.000 description 18
- 102000001708 Protein Isoforms Human genes 0.000 description 16
- 108010029485 Protein Isoforms Proteins 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 16
- OPXLLQIJSORQAM-UHFFFAOYSA-N mebendazole Chemical compound C=1C=C2NC(NC(=O)OC)=NC2=CC=1C(=O)C1=CC=CC=C1 OPXLLQIJSORQAM-UHFFFAOYSA-N 0.000 description 16
- 229960003439 mebendazole Drugs 0.000 description 16
- 239000000499 gel Substances 0.000 description 13
- 239000000020 Nitrocellulose Substances 0.000 description 11
- 229920001220 nitrocellulos Polymers 0.000 description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 11
- 238000001262 western blot Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 10
- 230000009467 reduction Effects 0.000 description 10
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 10
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 9
- 230000004899 motility Effects 0.000 description 9
- 241000243990 Dirofilaria Species 0.000 description 8
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 230000007423 decrease Effects 0.000 description 7
- 241000243988 Dirofilaria immitis Species 0.000 description 6
- 240000007860 Heteropogon contortus Species 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 210000002950 fibroblast Anatomy 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000003071 parasitic effect Effects 0.000 description 6
- 229920002401 polyacrylamide Polymers 0.000 description 6
- 241000244188 Ascaris suum Species 0.000 description 5
- 241000244036 Brugia Species 0.000 description 5
- 102000029749 Microtubule Human genes 0.000 description 5
- 108091022875 Microtubule Proteins 0.000 description 5
- 108020004511 Recombinant DNA Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 150000001556 benzimidazoles Chemical class 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 210000004688 microtubule Anatomy 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 241000244038 Brugia malayi Species 0.000 description 4
- 241000699694 Gerbillinae Species 0.000 description 4
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 3
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 206010014950 Eosinophilia Diseases 0.000 description 3
- 241000224468 Giardia muris Species 0.000 description 3
- 108010051815 Glutamyl endopeptidase Proteins 0.000 description 3
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 3
- 238000000134 MTT assay Methods 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000005063 solubilization Methods 0.000 description 3
- 230000007928 solubilization Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 206010009168 Chyluria Diseases 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- -1 SEQ ID NO : 1 Chemical compound 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 206010065258 Tropical eosinophilia Diseases 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 241000244005 Wuchereria bancrofti Species 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- KQHXBDOEECKORE-UHFFFAOYSA-L beryllium sulfate Chemical compound [Be+2].[O-]S([O-])(=O)=O KQHXBDOEECKORE-UHFFFAOYSA-L 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 208000006036 elephantiasis Diseases 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process 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
- 230000002147 killing effect Effects 0.000 description 2
- 210000004324 lymphatic system Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- IWDCLRJOBJJRNH-UHFFFAOYSA-N p-cresol Chemical compound CC1=CC=C(O)C=C1 IWDCLRJOBJJRNH-UHFFFAOYSA-N 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 210000003200 peritoneal cavity Anatomy 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- ZTOJFFHGPLIVKC-UHFFFAOYSA-N 3-ethyl-2-[(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical group S1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C1=NN=C1SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-UHFFFAOYSA-N 0.000 description 1
- LVSPDZAGCBEQAV-UHFFFAOYSA-N 4-chloronaphthalen-1-ol Chemical compound C1=CC=C2C(O)=CC=C(Cl)C2=C1 LVSPDZAGCBEQAV-UHFFFAOYSA-N 0.000 description 1
- WLHCBQAPPJAULW-UHFFFAOYSA-N 4-methylbenzenethiol Chemical compound CC1=CC=C(S)C=C1 WLHCBQAPPJAULW-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 1
- 208000020154 Acnes Diseases 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 206010003399 Arthropod bite Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000009079 Bronchial Spasm Diseases 0.000 description 1
- 208000014181 Bronchial disease Diseases 0.000 description 1
- 206010006482 Bronchospasm Diseases 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 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 1
- 241000258955 Echinodermata Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000004166 Lanolin Substances 0.000 description 1
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 1
- 206010052315 Lymphatic obstruction Diseases 0.000 description 1
- 108091077621 MAPRE family Proteins 0.000 description 1
- 238000003222 MTT reduction assay Methods 0.000 description 1
- 241000699684 Meriones unguiculatus Species 0.000 description 1
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- RMSTYYWFZAAEQO-NXNBBRKUSA-N N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(N)=O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H]([C@@H](C)CC)C(=O)O.N[C@@H](CC1=CC=C(C=C1)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CO)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(N)=O)C(=O)O.N[C@@H](CC(C)C)C(=O)O.N[C@@H](CC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CC(=O)O)C(=O)O Chemical compound N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(N)=O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H]([C@@H](C)CC)C(=O)O.N[C@@H](CC1=CC=C(C=C1)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CO)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(N)=O)C(=O)O.N[C@@H](CC(C)C)C(=O)O.N[C@@H](CC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.N[C@@H](CC(=O)O)C(=O)O RMSTYYWFZAAEQO-NXNBBRKUSA-N 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 241001044169 Parum Species 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241000224485 Physarum Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000242594 Platyhelminthes Species 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 244000028344 Primula vulgaris Species 0.000 description 1
- 235000016311 Primula vulgaris Nutrition 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010046996 Varicose vein Diseases 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 1
- 239000000921 anthelmintic agent Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000001001 anti-filiarial effect Effects 0.000 description 1
- 230000001946 anti-microtubular Effects 0.000 description 1
- 230000002927 anti-mitotic effect Effects 0.000 description 1
- 230000002141 anti-parasite Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002501 antifilarial agent Substances 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003080 antimitotic agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- SMTOKHQOVJRXLK-UHFFFAOYSA-N butane-1,4-dithiol Chemical compound SCCCCS SMTOKHQOVJRXLK-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000004081 cilia Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000009699 differential effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000010265 fast atom bombardment Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 239000003664 filaricide agent Substances 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 229960001614 levamisole Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- HEHQDWUWJVPREQ-XQJZMFRCSA-N lipid X Chemical compound CCCCCCCCCCC[C@@H](O)CC(=O)N[C@H]1[C@@H](OP(O)(O)=O)O[C@H](CO)[C@@H](O)[C@@H]1OC(=O)C[C@H](O)CCCCCCCCCCC HEHQDWUWJVPREQ-XQJZMFRCSA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 201000003265 lymphadenitis Diseases 0.000 description 1
- 206010025226 lymphangitis Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229940031348 multivalent vaccine Drugs 0.000 description 1
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 229960005030 other vaccine in atc Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 108700006586 polylysine-agarose Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 201000004409 schistosomiasis Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000004706 scrotum Anatomy 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- KEAYESYHFKHZAL-OUBTZVSYSA-N sodium-24 Chemical compound [24Na] KEAYESYHFKHZAL-OUBTZVSYSA-N 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 208000027185 varicose disease Diseases 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/10—Anthelmintics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to a monoclonal antibody which substantially binds to β-tubulin of nematode origin and fragments thereof. There is provided a hybridoma cell line producing the monoclonal antibody of the present invention which has been deposited at the ATCC under the accession number HB 11129. The antibody of the present invention can be used as an anti-parasitic agent and a diagnostic agent for parasitic diseases. The present invention also relates to the use of an antigen, which is recognized by the monoclonal antibody, as an immunizing agent and in vaccine compositions.
Description
PEPTIDES AND VACCINES DERIVED FROM NEMATODE TUBULIN
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to a monoclonal antibody which specifically binds to β-tubulin of nematode origin, which antibody can be used as an anti- parasitic agent and a diagnostic agent for parasitic diseases.
The present invention also relates to the use of immunogenic peptides useful in vaccine compositions for protecting mammals, such as humans and canines, againsr parasites of the Brυgia and D±rofiiaria genuses .
The present invention relates to the use of peptide fragments which provide improved means to protect a mammal against parasites of the Brug±a and D±r-f±la -±a genuses. More specifically, a dog exposed to the peptide fragments of the present invention is protected from heartworm by cytotoxic antibodies induced by the peptide.
2. Brief description of Prior Art
Parasitic diseases such as schistoscmiasis (Bilharziasis ) malaria and filariasis affect large numbers of people and are frequent causes of gastrointestinal, circulatory and other disorders. Parasitic infections often are chronic or recurrent, and it is not surprising that immunologic types of diseases have been described.
Filariasis consists of a group of diseases occurring in tropical and subtropical countries and caused by Filario±dea .
Filariasis involves the lymphatic system, with obstruction leading to chyluria, hydrocoele, and elephantiasis that may involve the scrotum, legs and the arms .
Etiology and Pathogenesis of Filariasis
W chereria bancrofti is found only in humans ; Brugia malay± is often spread to man from animal hosts . The adult filarial worms live in the human lymphatic system. Microfilariae released by gravid females are found in the peripheral blood, usually at night. Infection is spread by many species of mosquitoes. The microfilariae are ingested by the mosquito, undergo development in the insect's thoracic muscles, and, when mature, migrate to its outhparts . When the infected mosquito bites a new host, the microfilariae penetrate the bite puncture and eventually reach the lymphatics, where they develop to the adult stage.
Pathology
Inflammation and fibrosis occurring in the vicinity of the juvenile and adult worms produce pro¬ gressive lymphatic obstruction.
Symptoms and signs
The incubation period may be as short as two months. The "prelatent" period, from the time of infection to the appearance of microfilariae in the blood, is at least eight months. Clinical manifestations depend on the severity of the infection; they may include lymphangitis, lymphadenitis, orchitis, funiculitis, epididymitis, lymph varices, and chyluria. Chills, fever, headache, and malaise may also be present. Elephantiasis and other late severe sequelae occur with long-time residence in endemic areas and repeated reinfection. An aberrant form of filariasis (tropical eosinophilia) is characterized by hypereosinophilia, presence of microfilariae in the tissues but not in the blood, and high titers of antifilarial antibodies (tropical eosinophilia). Clinically, the patient may present with lymphadeno-splenomegly or with cough, bronchospasm, and chest infiltrates.
Diagnosis
Microfilariae may be found in blood or lymph fluid. A number of serologic tests are available, but are not completely reliable. Antigen detection proce¬ dures are being investigated.
Tubulin
Microtubules are proteinaceous organelles that are implicated in a variety of cellular functions including mitosis, intracellular transport, the maintenance of cell shape and the formation of cilia, flagella and sensory organelles. The major structural component of microtubules is tubulin, which is composed of α- and β- subunits, the dimer having a molecular weight of 110 kDa. Both α- and β-tubulins are expressed as heterogeneous but closely related families of multiple isoforms, in different organisms, tissues and even within single cells of the same organism. The heterogeneous population of tubulin isoforms may result from both the differential expression of distinct tubulin genes and post- translational modifications. It has been suggested that the diversity in tubulin isoforms may have implications for specific MT • functions (Lewis and Cowan, J. of Cell Biol., 1988, 106:2023-2033 ) . The precise nature or role of α- and β-tubulin isoforms have not yet been elucidated, although several groups of researchers have demonstrated that many in vivo functions of tubulin are to some extent, isoform specific (Gundersen et al., Cell, 1984, 38:779- 789).
Benzimidazoles, anti-mitotic and anti-fungal agents are widely used in the chemotherapy of parasitic diseases. Several chemicals such as colchicine, vinblastine and benzimidazoles have been shown to bind to tubulin. Benzimidazoles exert toxic effects on nematodes by binding to tubulin and inhibiting polymerization of the heterodimer into microtubules. Benzimidazoles induce paralysis and slow growth in the free-living nematode Caenoxhabditis elegans . These drugs are potent filaricides
for B. pahangi and B. malay . However, the precise benzimidazoles binding site has not been determined.
Monoclonal antibodies have made it possible to recognize different domains of tubulin in different species in order to study the structure, distribution and functions of tubulin. Tang and Prichard (Mol. & Biochem. Parasitology, 1989, 3.2: 145-152) reported the presence of 4 to 5 ?-tubulin isoforms in the tubulin-enriched extracts of adult B. pahangi . In addition, immunogold studies with B . malayi adult and microfilariae using anti- tubulin monoclonal antibodies have revealed the presence of ?-tubulin in the somatic muscle blocks beneath the cuticle, intestinal brush border and intra-uterine microfilariae of the adult worms (Helm et al. , Parasite Immunology, 1989, 11:479-502) .
Several other anti-tubulin monoclonal antibodies raised against parasitic protozoa and nematodes have been isolated but these have been found to cross-react with tubulin from other species. For example, Draber et al (Protoplasma, 1985, 128: 201-207) reported a monoclonal antibody raised against pig brain tubulin which reacted with microtubules from diverse species (mammalian, bird, amphibian, fungi, echinoderm, platyhelminth, slime moulds) but not protozoan tubulin. Similarly, Birkett et al. (FEBS Letters, 1985, 187 : 211-218) generated an anti- /?-tubulin monoclonal antibody against Physarum myxamoebae which reacts with ^-tubulin from various fungi, algae, higher plants, avian, insect and several mammalian sources. In addition, Helm et al. (Parasite Immunology, 1989, 2λ- 479-502) have raised monoclonal antibodies against microfilariae of Brugia species. Contrary to the anti-B. pahangi ?-tubulin monoclonal antibodies of the present invention, their monoclonal antibodies cross- reacted with mammalian tubulin.
All these monoclonal antibodies of the prior art are not specific against tubulin of nematode origin.
It would be highly desirable to have a monoclonal antibody which specifically binds to nematode tubulin and
which could be used as an anti-parasitic agent and as a reliable diagnostic agent for parasitic diseases.
It would be also highly desirable to have a peptide which can be used to immunize mammals against parasites such as Brugia and Dirofilaria .
The desired peptide could be used in vaccine composition to provide an immune protection against these parasites.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided a monoclonal antibody which specifically binds to /?-tubulin of nematode origin and fragments thereof. The monoclonal antibody of the present invention can be used as an anti-parasitic agent and as a diagnostic agent for parasitic diseases.
In accordance with the present invention, there is also provided a hybridoma cell line which produces the monoclonal antibody of the present invention.
The monoclonal antibody of the present invention recognizes the C-terminal of nematode 7-tubulin which corresponds to a peptide of eighteen amino acids.
In accordance with another embodiment of the present invention, there is provided the use of a peptide as an immunizing agent against parasites wherein said peptide has the following amino acid sequence:
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu, SEQ ID NO: 1.
The use of the peptide in accordance with the present invention induces by a host the production of cytotoxic antibodies against parasites such as Brugia and Dirofilaria .
In accordance with the present invention, there is also provided a vaccine for parasite infection comprising a peptide which has the following amino acid sequence:
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin
1 5 10 15
Glu Glu, SEQ ID NO:1.
Finally, in accordance with the present invention, there is provided a method of immunizing mammals against parasites comprising the administration of the vaccine of the present invention. The vaccine of the present invention can be administered in a dosage range of 0.015 μg to 0.15 mg per kg body weight, preferably in a dosage range of 1.5 μg to 15 mg per kg body weight.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the nature of the invention, reference will not be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and wherein:
Fig. 1 is a Western blot analysis of extracts from parasites, pig brain and 3T3 fibroblasts using anti-B. pahangi β tubulin monoclonal antibody in Figure IA and anti-chick brain tubulin monoclonal antibody in Figure IB;
Fig. 2 is a Western blot analysis of the total protein extract of adult B. pahangi using anti-B. pahangi β tubulin monoclonal antibody P3D in Figure 2A or anti-B. pahangi β tubulin monoclonal antibody 1B6 in Figure 2B;
Fig. 3 is a Western blot analysis of products from limited proteolysis of anti-B. pahangi β tubulin monoclonal antibody P3D in Figure 3A and anti-B. pahangi β tubulin monoclonal antibody 1B6 in Figure 3B;
Fig. 4 is a graph of the effects of anti-B. pahangi tubulin monoclonal antibody P3D on the viability of adult female B.phangi in vitro;
Fig. 5 is a graph of the effects of anti-B. pahangi tubulin monoclonal antibody 1B6 on the viability of adult female B .phangi in vitro;
Fig. 6 is a graph of the effects of anti-chick brain tubulin monoclonal antibody 357 on the viability of adult female B.phangi in vitro.
-6A-
DETAILED DESCRIPTION OF THE INVENTION I-Monoclonal Antibody
A first embodiment of the present invention relates to the production and characterization of a monoclonal anti-B. phangi tubulin monoclonal antibody.
The monoclonal antibody of the present invention, denoted P3D, specifically reacts to the C-terminal portion of 3-tubulin from B . pahangi and Dirofilaria and
hence is capable of killing these parasites. The hybridoma P3D producing the monoclonal antibody of the present invention has been deposited at the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland, USA 20852) under accession number HB 11129 on September 18, 1992. This deposit is available to be public upon the grant of a patent to the assignee, McGill University, disclosing same. The deposit is also available as required by Foreign Patent laws in countries wherein counterpart applications are filed.
In total, fifty-four anti-B. pahangi tubulin mono¬ clonal antibodies were obtained after immunization of mice with purified B. pahangi tubulin. Because of their remarkable specificity for tubulin, monoclonal antibodies P3D and 1B6 among others, have been selected for more extensive characterization. Western blot analysis of one-dimensional SDS-PAGE showed that the anti-B. pahangi monoclonal antibodies of the present invention recognized tubulin from a number of filarial nematodes ( B . pahangi , B malayi and D. immitis ) and an intestinal nematode ( H. contor- tus ) . However, the monoclonal antibodies did not cross- react with tubulin from pig brain, 3T3 mouse fibroblast cells or the parasitic protozoan c muris. On the other hand, anti-chick monoclonal antibody 357 reacted with pig brain, 3T3 mouse fibroblast and G. muris tubulins as strongly as it did with filarial and other nematode β- tubulins. The anti-B. pahangi tubulin monoclonal antibodies of the present invention recognize an epitope that is conserved between filarial and intestinal nematode ^-tubulin but not in protozoan and mammalian β- tubulin. Whereas, cross-reactive anti-chick monoclonal antibody 357 recognizes an epitope that is conserved among filarial and intestinal nematodes as well as protozoan and mammalian ^-tubulin. The epitope recognized by monoclonal antibody 357 has been localized to a region of ?-tubulin between amino acid 339-417 in the proteolytic fragments of pig brain tubulin (Serrano et al., Analytical Biochemistry, 1986, 159: 253-259). The anti-B. pahangi tubulin monoclonal antibodies of the
- 8 -
present invention are highly specific to nematode tubulin.
The monoclonal antibodies of the present invention specific for the - or ?-subunit of tubulin allow the subcellular localization and the function of each subunit of tubulin to be studied. Proteins of the size of tubulin are generally built of several structural domains that have distinct functions. In the case of tubulin, such functions include binding of anti-microtubule drugs, GTP or microtubule-associated proteins and the association between monomers, dimers or protofilaments . The nematode-specific anti-tubulin monoclonal antibodies of the present invention may serve to characterize the structure and distribution of B. pahangi tubulin molecule, and to define microtubule stability and functional domains.
The following procedures are used in the prepa¬ ration and the characterization of the monoclonal antibody of the present invention.
Enzyme-linked im unosorbent assay (ELISA)
ELISA was performed in icrotiter plates (Falcon) coated with the* polylysine-purified tubulin or an 18 amino acid peptide corresponding to the extreme C- terminal residues 430-448 of B. pahangi tubulin (Guenette et al., Mol. and Biochem. Parasitology, 1991, .44.: 153- 164) at a concentration of lOμg/ml in phosphate buffer saline (PBS). Plates are incubated with 200 μl of 1% bovine serum albumin (BSA) in PBS. Horseradish peroxidase-labeled anti-mouse IgG or IgM (Bio-Can, Mississauga, Ontario) at dilutions of 1:5000 and 1:20,000, respectively, is added to each well and incubated for 1 hour at 37°C. The substrate is 2,2'- azino-bis( 3-ethylbenzthiazoline-6-sulfonic acid) (Sigma). The plates are read on a Titertek multiskan™ plate (Flow Laboratories, Irvine, Ayrshire, UK) at 414 nm. Normal mouse serum or culture medium used to grow hybridoma cells (Iscoves modified Dulbecco's medium (IMDM) with 20% FCS, 10% NCTC 135 and HT) is used as a negative control.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Samples are run in a Mini Protean II ™ dual slab cell (Bio-Rad, Richmond, CA) using 4% polyacrylamide as stacking and 12% polyacrylamide as separating gels.
Isoelectric focusing and two-dimensional electrophoresis (IEF-2D SDS-PAGE)
IEF gels are prepared and run in tube gels (1.5 x 8cms) containing 9.5M urea (LKB) and 2% (w/v) ampholines (LKB) (1.6% pH 4-6 and 0.4% pH 3.5-10). IEF is conducted at 400 V for a period of 16 hours and then at 800 V for 3 h. Electrophoresis is performed in 4% polyacrylamide stacking and 12% polyacrylamide separating gels, running at 50 V for 30 min and at 150 V for 60 min, in the Mini Protean II ™ slab cell. After 2-dimensional (2D) SDS- PAGE, gels are either stained with silver stain (Bio- Rad), or the proteins are transferred onto nitrocellulose (NC) sheets for Western blot analysis.
Western blotting
After 1 . and 2D SDS-PAGE, tubulin subunits, individual tubulin isoforms and peptides are elec- trophoretically transferred onto nitrocellulose sheets for 2 hours at 4°C. The nitrocellulose sheets are cut into several strips containing an identical pattern of separated proteins. To visualize protein bands, two nitrocellulose strips are stained with amido black. The remaining strips are washed in PBS and incubated for 2 hours at room temperature in 10% newborn calf serum (Gibco) in Tris-buffer saline (140 mM NaCl2/ 50 mM Tris- HC1, pH 7.4, with 0.1% (v/v) Tween 20™ (TBS-T)) to saturate the unoccupied protein binding sites of the nitrocellulose. After washing, the strips are incubated overnight at 4°C with anti-tubulin monoclonal antibodies (MAbs) or IMDM (negative control). The nitrocellulose strips are then washed 6 x 5 min with TBS-T, immersed in peroxidase-conjugated goat anti-mouse IgM or IgG (Bio-
- -
Can) diluted at 1:500 with high salt buffer (1 M NaCl2, 10 mM Tris-HCl, pH 7.4; 0.5% (v/v) Tween 20™ (HSB-T) with 10% NBCS)), and incubated for 2 hours at room temperature. After washing the nitrocellulose strips with TBS-T for 30 min, the bound peroxidase is detected with the substrate 4-chloro-l-naphthol (Sigma) at 3 mg/ml in methanol/PBS, 1:5 (vol/vol), containing 0.075% of 30% hydrogen peroxide.
1- Preparation of antigens
Gerbils ( Meriones unguiculatus ) , 9-10 months old and previously infected intraperitoneally with 400 B . pahangi infective larvae, are obtained from Dr. J. McCall (University of Georgia, USA). The adult B . pahangi (0.7 g) are harvested from the peritoneal cavities of gerbils in warm physiological saline (0.85% NaCl), washed with 0.025M buffer containing 1 mM ethyleneglycol-bis-(^- aminoethyl ether)N,N,N',N' -tetraacetic acid (EGTA), 0.5mM MgSθ and 1 mM guanosine-5 ' -triphosphate (GTP), and are homogenized in 7 ml of 2[N-morpholino)-ethanesulfonic acid (MES) buffer. The homogenate is centrifuged at 100,000 g for 1 hour at 4°C. The supernatant is retained and the pellet discarded. The same procedure is used to prepare tubulin from other filarial ( B . malayi and D. immitis ) and non-filarial nematodes ( A . suum, benzimidazole- susceptible and resistant strains of H. contortus ) .
Tubulin from pig brain is prepared by 2 cycles of polymerization-depolymerization.
Giardia Muris antigen is prepared as a sonicate.
A peptide corresponding to amino acid residues 430-448 of B. pahangi yS-tubulin, synthesized using an Applied Bisystems Peptide Synthesizer™, HPLC purified, sequenced and coupled to the carrier protein, keyhole Limpet He ocyanin (KLH) (The Alberta Peptide Institute), is also used as an antigen in enzyme-linked immunosorbent assay (ELISA).
- 11 -
2- Purification of parasite tubulin
B. pahangi, B. malayi, D. immitis, A. suum and H. contortus tubulins are partially purified using polylysine affinity chromatography (Lacey & Prichard, Mol. & Biochem. Para- sitology, 1986, _1£: 171-181). The elution profile consisted of three distinct peaks. The first protein peak is eluted with MES buffer, the second with 1% aqueous (NH4)2Sθ4- Fractions for each peak are pooled and concentrated separately in centriflo™ (Amicon) at 400 g-
Polylysine-purified proteins are separated on SDS- PAGE, protein bands of the molecular weight corresponding to tubulin are excised, and the protein is electro-eluted (Electroeluter™, Bio-Rad) (Blose et al. , J. of Cell Biol. , 1984, £8.: 847-858). The eluted protein is precipitated three times with 80% acetone at -20°C for 5 hours and then dissolved in 0.125 M Tris-HCl (pH 6.8), 0.1% SDS and 1 mM EDTA, dialysed overnight against this buffer at 4°C and stored at -70°C until used.
Crude supernatant of adult B. pahangi is chro- matographed on a polylysine agarose column. The protein content of each fraction is determined. The elution profile consisted of 3 distinct protein peaks. The protein concentrations in the first and second peaks are very high compared with that in the third peak, but in contrast to this last peak the first two peaks contained little if any tubulin. This is consistent with the previous report by Tang & Prichard (Mol. & Biochem. Parasitology, 1989, .32.: 145-152). Third peak proteins are concentrated and then subjected to SDS-PAGE, respectively. The tubulin band is cut out of the SDS- gels and subjected to electro-elution for further purification.
3- Immunization and preparation of monoclonal anti¬ bodies
Six week old female BALB/c mice (Charles River Canada Inc., St. Constant, Quebec) are injected subcu- taneously at three week intervals with purified eluted B .
pahangi tubulin (100 μg/injection) using equal volumes of complete Freund' s adjuvant for the first injection and incomplete adjuvant for the second injection. The third immunization of 100 μg of tubulin in PBS is administered intraperitoneally (i.p.). At this stage, mice are bled and serum is tested for anti-tubulin antibodies by ELISA and Western blotting. The spleen cells from the mouse giving the highest titer are fused with the myeloma cell line, P3X63.Ag8 (American Type Culture Collection (ATCC), accession number CRL1580, Rockville, MD), as described by Hurrell ("Monoclonal hybridoma antibodies: Techniques and applications", 1982, CRC Press, Boca Raton, Florida, p. 22). Positive cultures as determined by ELISA and Western blotting, are cloned twice by limiting dilution.
Two different isotypes of anti-B. pahangi mono¬ clonal antibodies were obtained. Seven out of fifty-four monoclonal antibodies were polyreactive IgM, recognizing tubulin as well as other high and low molecular weight proteins, whereas the remaining monoclonal antibodies represented two populations of the IgG isotype. Of these, four out of fifty-four reacted with tubulin and other low molecular weight proteins; however, forty-three monoclonal antibodies were specific for tubulin. Monoclonal antibodies P3D and 1B6 specific to nematode tubulin, were chosen for further characterization. These monoclonal antibodies are of IgG isotype. 4- Monoclonal antibodies (MAbs)
Three monoclonal antibodies, all specific for tubulin, are investigated. Anti-chick brain monoclonal antibody 357, which cross-reacts with ?-tubulins from a spectrum of eukaryotic cell types, was purchased from the Radiochemical Centre (Amersham, England) and monoclonal antibodies P3D and 1B6 are raised against the tubulin of adult B . pahangi . All anti-tubulin monoclonal antibodies are of IgG isotype.
5- Specificity of monoclonal antibodies (MAbs) P3D, 1B6 and 357
The specificity of these monoclonal antibodies is investigated by determining their reactivity to proteins from a variety of filarial and non-filarial nematodes, protozoa and mammalian cells using ELISA and Western blot.
In ELISA, the anti-B. pahangi monoclonal antibodies P3D and 1B6 do not react with G. muris tubulin, which is recognized by anti-chick brain tubulin monoclonal antibody 357. Crude and partially purified extracts of adults and microfilariae of B. pahangi , adult B malayi and D. immitis , eggs of H. contortus , adult A. suum, pig brain and 3T3 mouse fibroblast cell tubulins are separated on SDS- PAGE and electrophoretically transferred onto nitrocellulose sheets. The blots are treated with: (1) amido black; (2) monoclonal antibody 1B6; (3) monoclonal antibody P3D; and monoclonal antibody 357. Analysis of amido black stained blots revealed that crude extracts of adults and microfilariae of B. pahangi , adult B. malayi and D. immitis , eggs of susceptible and resistant strains of H. contortus, adult A. suum, pig brain and 3T3 mouse fibroblast cell contained many bands in the tubulin region. Tubulin from the various nematodes and mammalian extracts are separated into two bands designated α and β. Anti-B. pahangi monoclonal antibody P3D recognized specifically β- tubulin from adult and microfilariae of the filarial worms B. pahangi , B malayi and D. immitis (Fig. IA, lane 1-4). It also reacted with equal intensity to tubulin from the intestinal nematode H. contortus (BZ-susceptible and benz- imidazole-resistant strains) (Fig. IA, lane 5-6). Tubulin from A . suum do not show very strong reactivity with this monoclonal antibody (Fig. IA, lane 7), no reactivity to 3T3 mouse fibroblast cells or pig brain tubulins is detected (Fig. IA, lane 8-9). Similar results are obtained using monoclonal antibody 1B6 (not shown). Whereas, cross-reactive anti-chick /?-tubulin monoclonal antibody 357 recognized ^-tubulin from all nematodes and mammalian cells (Fig. IB, lane 1 to "9 ) .
6- Identification of tubulin isoforms
Anti-B. pahangi ?-tubulin monoclonal antibodies P3D and 1B6, and anti-chick /?-tubulin monoclonal antibody 357, are used to characterize ^-tubulin isoforms in B. pahangi tubulin. Monoclonal antibodies P3D (Fig. 2A) and 357 recognized the same isoform pattern, reacting with two /?-tubulin isoforms in the crude as well as partially purified extracts of B. pahangi (not shown). Whereas, monoclonal antibody 1B6 specifically recognized only one ?-tubulin isoform in the extract of .B. pahangi (Fig. 2B). The ?-tubulin isoforms are in the pH range of 5.1-5.3.
Monoclonal antibody 357 probed blots are re-probed with monoclonal antibodies P3D and 1B6 respectively, to demonstrate that the same spots are recognized by this monoclonal antibody. Furthermore, to show the full complement of /0-tubulin isoforms, P3D and 1B6 probed blots are re-probed with monoclonal antibody 357. The results indicated that all these monoclonal antibodies recognized the same isoforms in tubulin-enriched extracts of adult B. pahangi . However, monoclonal antibody 1B6 is specific to one isoform.
7- Limited Proteolysis of tubulin
Limited proteolysis of tubulin in gel slices is performed. Gel pieces corresponding to the tubulin are cut out of the polyacrylamide gels and placed directly into the sample well of a second 15% SDS-polyacrylamide gel. Gel pieces are overlaid with one of the following proteases:α-chymotrypsin from bovine pancreas (Sigma) or s. aureus V8 protease (Boehringer Mannheim). The SDS-PAGE is performed at 50 V until bromophenol blue dye reached the bottom of the stacking gel and then increased to 150 V for the remainder of the electrophoresis. After SDS- PAGE, the digested peptides are either stained with silver stain or transferred onto nitrocellulose sheets, in the same way as described for the Western blot analysis, and reacted either with anti-B. pahangi tubulin
monoclonal antibodies or anti-chick tubulin monoclonal antibody 357.
8- Interaction of anti-tubulin monoclonal antibodies with tubulin proteolytic fragments
Three identical gels are run and the peptide fragments transferred onto nitrocellulose, Three of which are immunostained with anti-B. pahangi tubulin monoclonal antibodies P3D and 1B6 (Fig. 3) and anti-chick brain β- tubulin monoclonal antibody 357 (not shown).
Western blots of peptides digested with chy¬ motrypsin showed that monoclonal antibody P3D reacted with a 21 kDa chymotrypsin fragment (Fig. 3A, lane 2) and a 21 kDa V8 protease ?-tubulin fragment (Fig. 3A, lane 3). In contrast, monoclonal antibody 1B6 reacted with the two chymotrypsin-digested fragments of 42 and 34 kDa (Fig. 3B, lane 2). It reacted strongly with the 42 kDa and weakly with the 34 kDa fragment. However, the same protease (Fig. 3B, lane 3). These results of the limited proteolysis analysis indicate that the antigenic site recognized by monoclonal antibody P3D differs from that recognized by monoclonal antibody 1B6.
Although -monoclonal antibody 357 reacts strongly to intact ?-tubulin from B. pahangi , no interaction was seen with ?-tubulin fragments digested with chymotrypsin or V8 protease (not shown). Protease digestion appears to destroy the reactivity of B. pahangi tubulin towards monoclonal antibody 357.
II-Peptide used as an immunizing agent against parasite
The monoclonal antibody P3D of the present inven¬ tion recognizes the C-terminal of nematode ?-tubulin which corresponds to a peptide of eighteen amino acids .
A second embodiment of the present invention relates to the use of a peptide, recognized by the antibody of the present invention, which consists of the following eighteen amino acid sequence:
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : l , which is located at the C-terminal of nematode ^-tubulin . Furthermore , the present invention relates to a vaccine which comprises a peptide that consists of the following amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : 1 .
The present invention provides a peptide having the amino acid sequence derived from the eighteen amino acids at the C-terminal of ^-tubulin from B. pahangi and Dirofilaria . The peptide can be made using a peptide sequence or using recombinant DNA technology.
A vaccine comprising the peptide of the present invention, a fragment thereof or a larger peptide which comprises the amino acid sequence of the peptide of the present invention is effective in conferring protection against parasite infection. Such vaccines can be prepared by one having ordinary skill in the art.
It has been discovered that monoclonal antibodies which specifically react to the C-terminal portion of β- tubulin from B. pahangi or Dirofilaria are capable of killing these parasites. •
Accordingly, using a vaccine that comprises peptide with the epitope of the C-terminal of B. pahangi or Dirofilaria >3-tubulin will elicit cytotoxic antibodies in vaccinated mammals that can kill these parasites and therefore protect the mammal against the parasite.
The present invention relates to vaccines which comprise a peptide which consist of the eighteen amino acid residues from the C terminus of B. pahangi or Dirofilaria ?-tubulin or fragment thereof and to vaccines which comprise a peptide that have portions which are the eighteen amino acid sequence.
The amino acids at the carboxy terminus of Brugia and Dirofilaria ?-tubulin are:
DEEGDLQEGESEYIEQEE or
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : 1 , or aspartate-glutamate-glutamate-glycine-aspartate-leucine- glutamine-glutamate-glycine-glutamate-serine-glutamate- tyrosine-isoleucine-glutamate-glutamine-glutamate- glutamate.
Production of the peptide of the present invention, fragment thereof or larger peptides which include this sequence can be accomplished using standard peptide synthesis or recombinant DNA techniques both well known to those having ordinary skill in the art. Peptide synthesis is the preferred method of making polypeptides which comprise about 50 amino acids or less. For larger molecules, production in host cells using recombinant DNA technology is preferred.
Smaller peptides according to the present invention can be synthesized, for example, by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, California) as described in detail below.
For larger molecules, production in host cells using recombinant DNA is preferred. There are several different methods available to one having ordinary skill in the art who wishes to use recombinant DNA technology to produce proteins. Typically, genes encoding desired polypeptides are inserted in expression vectors which are then used to transform or transfect suitable host cells. The inserted gene is then expressed in the host cell and the desired polypeptide is produced.
Methods and materials for preparing genes and recombinant vectors, transforming or transfecting host cells using the same, replicating the vectors in host cells and expressing biologically active foreign peptides and proteins are described in Principles of Gene Manipulation, by Old and Primrose, 2nd edition, 1981 and Sambrook et al., Molecular Cloning, 2nd Edition, Cold
Spring Harbor Laboratory Press, NY (1989), the disclosure of both is incorporated herein by reference.
European Patent application 322,237 published on June 28, 1989, U.S. Patent 4,735,801 (Stocker, April 5, 1988) and U.S. Patent 4,837,151 (Stocker, June 6, 1989) describe attenuated microorganisms useful in vaccine and which microorganisms have non-reverting mutation in two discrete genes in their aromatic biosynthetic pathway. These microorganisms can usefully form the basis of an oral live vaccine and can be genetically engineered so as to express antigens from other pathogens. These references are all incorporated herein by reference.
Example I in vitro assay of B. p-h-rιg± inhibition Measurement of the in vitro activity of anti-B. pahangi tubulin monoclonal antibodies P3D, 1B6 and anti- chick brain tubulin monoclonal antibody 357 against female B. pahangi or anti-chick tubulin monoclonal anti¬ bodies can independently cause any damage to the intact adult worms. Mebendazole (MBZ) is used to determine whether the presence of MBZ drug alone or in synergy with monoclonal antibodies has any differential effect.
Inhibitors
Anti-B. pahangi /?-tubulin monoclonal antibodies P3D, 1B6 (in culture medium)), anti-chick ^-tubulin monoclonal antibody 357 (in ascites fluid) and mebendazole (MBZ) (in DMSO), a benzimidazole anthelmintic drug, are used as inhibitors in the in vitro assays. Anti-B. pahangi anti- chick brain monoclonal antibody 357 is in ascites fluid and is diluted to 1:1000 concentration with culture medium IMDM/FCS.
Culture in -*±txo
Parasitic nematodes are isolated from their mammalian host. B. pahangi are isolated from peritoneal cavities of gerbils, as described earlier in a sterile hood of Iscove's Modified Dulbecco's Medium/NCTC-135
- -
supplemented with 20% fetal calf serum ( IMDM/FCS) . Following isolation, B. pahangi are washed five times with sterile IMDM/FCS medium, for surface sterilization. Three wells in 24-well plates (Nunc) are set up for each test monoclonal antibody, drug and for the control cultures. To each well was added 2 ml of the appropriate test medium containing pure monoclonal antibody P3D, 1B6, 357 alone or monoclonal antibody and MBZ and two adult worms. The plates are incubated at 37°C in a humidified incubator in the presence of 95% air and 5% Cθ2- Worm activity is observed every two hours, and motility is assessed subjectively by observation with a naked eye. Experiment is terminated after 48 hours. During the 48 hour incubation the culture medium is not changed. Control medium contained an identical volume of the IMDM/FCS without monoclonal antibodies or drug.
Optimization of MTT reduction assay
Female live B. pahangi worm is placed in 0.5 ml of IMDM containing 0.5 mg/ml [3-(4, 5-dimethyl(thiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] (Sigma) (MTT) and incubated at 37°C for various time intervals ranging from 0-90 min (MTT-reduction) . Female worms that had previously been heat-killed are also incubated with MTT for selected time intervals over this range. For each time point three replicate worms are used. At the end of the MTT incubations worms are removed and carefully transferred to a separate well of a microtiter plate containing 200μl of DMSO and allowed to stand at room temperature for 1 hour (formazan solubilization) , with occasional gentle agitation to evenly disperse the color. The absorbance of the resulting formazan solution is then determined at 550 n , using an ELISA reader and compared with a DMSO blank.
Quantification of B. pahangi viability
A three-step colorimetric assay based on MTT is used to assess viability of parasitic nematodes. MTT is dissolved in PBS at a concentration of 5.0 mg/ml and sub-
sequently diluted to 0.5 mg/ml with PBS. Worms are incu¬ bated for 30 min at 37°C (MTT reduction). After incu¬ bation, worms are transferred to 96 well plates con¬ taining 200μl of DMSO. The plates are allowed to stand for 1 hour at room temperature (formazan solubilization) . The absorbance is determined at 550 nm in the presence and absence of worm and compared with a DMSO blank. Worms are killed for control purposes by heating in PBS at 100°C for 10 min.
Previous studies have demonstrated the utility of MTT-formazan colorimetry in proliferation and cytotoxicity assays in anti-cancer chemotherapy. Sub¬ sequently it has been demonstrated that the application of this assay was successful to determine filarial viability and for in vitro anti-filarial drug screening. MTT is pale-yellow in solution but when incubated with living cells is reduced by active mitochondria to yield a dark blue crystalline deposit (formazan) within cells, which once solubilized can be quantified colorimetrically.
In accordance with the present invention, MTT assays are performed to determine the effects of anti- tubulin monoclonal antibodies on the viability of parasitic nematodes. Viable control female B. pahangi showed rates of formazan formation that are maximal and linear during the first 30 min of the incubation with MTT. By one hour rate of formazan formation had begun to decline and plateaued between 60-90 min. Heat-killed worms shows only background levels of formazan formation.
Worms treated with anti-B. pahangi monoclonal antibody P3D alone and in synergy with MBZ show a detectable decrease in motility 12 hours post-treatment. The other anti-B. pahangi monoclonal antibody 1B6 alone and in synergy with MBZ, also exhibit an apparent decline in the motility of worms, however, no mortalities are observed using these monoclonal antibodies during the experiment. No noticeable reduction is observed in the motility of the worms treated with MBZ alone or anti- chick brain monoclonal antibody 357 alone or in synergy
- -
with MBZ or the control worms, during the period the worms are in culture. From these observations, it is suggested that the reduction in the worm motility is caused mainly by the anti-B. pahangi monoclonal antibody alone, since MBZ alone do not have any effect on the motility of the worms.
Analysis of MTT assays demonstrates that mono¬ clonal antibody P3D treated B. pahangi shows significant decline in their ability to reduce MTT to formazan (Fig. 4). This monoclonal antibody alone caused a highly significant 80% reduction in worm viability, compared with untreated live worms, 48 hours post-treatment. MBZ in synergy with monoclonal antibody P3D caused significant 70% reduction in the viability of worms. The high reduction in the viability of worms seems to be due to the presence of monoclonal antibody P3D and not MBZ. As MBZ alone induced a minimal decrease (10%) in the viability of worms. Exposure to monoclonal antibody 1B6 resulted in 40% decrease in the ability of worms to reduce MTT (Fig. 5). Monoclonal antibodies P3D and 1B6 had the same respective effects on the viability of males as for females . Anti-chick β-tubulin monoclonal antibody 357 did not show- any significant effect on the viability of worms (Fig. 6). The properties of anti-B. pahangi and anti-chick brain monoclonal antibodies appear qualitatively similar. Differences in their inhibitory effects on the motility and viability of B. pahangi may depend on their different binding affinities.
Control untreated live female worms show a linear rate of formazan production and gave an absorbance reading of 1.1 at 550 nm. In contrast, heat killed worms show no ability to reduce MTT (Fig. 4 to 6). After DMSO solubilization for 1 hour the absorbance of the resulting formazan solution is determined in the presence or absence of the female worms. This is done to determine if the presence of worm had any effect on the absorbance values. In the presence of worm, there is a slight increase in the absorbance values obtained. Inhibition of MTT reduction does not always occur uniformly along
the entire length of treated worms and areas retaining viability are observed. Thus by close observation of the worms during MTT reduction it is sometimes possible to determine sites of selective damage.
Conclusion
The results of Example I demonstrate an apparent decline in the motility, when the worms are cultured with the anti-tubulin monoclonal antibodies P3D and 1B6 of the present invention. However, no noticeable reduction in the motility is observed, when the worms are treated with anti-chick monoclonal antibody 357, MBZ or IMDM/FCS culture medium without antibodies. The viability of the worms was assessed by MTT assay. The anti-B. pahangi , monoclonal antibodies P3D and 1B6 of the present invention, significantly reduced the viability of parasitic nematodes. No reduction in viability was observed when adult B. pahangi were exposed to anti-chick monoclonal antibody 357 and/or MBZ.
Example II Anti-parasitic Antibody composition
An antibody composition to be administered to a gerbil as an anti-parasitic agent in dosage varying from lmg/0.5ml to 10mg/0.5ml in a pharmaceutical carrier suitable for intraperitoneally administration.
The carrier for such administration is an IMDM culture media.
Example III Production of the eighteen Amino Acid peptide
The peptide consists of the amino acid sequence: sp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu, SEQ ID NO: 1.
To prepare the peptide for use in a vaccine, the peptide is synthesized by solid phase methodology on an Applied
Biosystems Inc. (ABI) 430A peptide synthesizer using ABI's Small Scale Rapid Cycles (SSRC) on a 0.1 mmole scale or other similar synthesizer. SSRC utilizes abbreviated single couple cycles with standard Boc chemistry. The t-Boc-L-amino acids used (1 mmole) are supplied by ABI with standard side-chain protecting groups. The completed peptide is removed from the supporting PAM (phenylacetamidomethyl) resin, concur¬ rently with the side-chain protecting groups, by a standard HF procedure using appropriate cation scavengers (10% v/v amisole, p-cresol plus p-thiocresol, 1,4- butanedithiol plus anisole or DMS plus anisole) depending on the amino acid sequence of the peptide.
The crude peptide, after HF cleavage, is purified by preparative reverse phase chromatography on a Phenomenex C-18 Column (250 x 22.5 mm) using water acetonitrile gradients, each phase containing 0.1% TFA. The pure fractions (as determined by analytical HPLC) are pooled, acetonitrile evaporated and the aqueous solution lyophilized. The peptide is analyzed by fast atom bombardment mass spectrocopy and resulting (M+H)+ is compared with the anticipated (M+H)+.
Example IV Vaccine Comprising Eighteen Amino Acid Peptide
The peptide can be prepared in vaccine dose form by well-known procedures. The vaccine can be administered sublingually, intramuscularly, subcuta- neously or intranasally. For parenteral administration, such as intramuscular injection, the immunogen may be combined with a suitable carrier, for example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunomodulating agents such as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium
- 24 -
alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Co., Inc., Rahway, NJ) .
The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (AI2O3 basis). On a per dose basis, the concentration of the immunogen can range from about 0.015 μg to about 1.5 mg per kilogram per patient body weight for an animal or human patient. A preferable dosage range in humans is about 0.1 - 1 ml, preferably about 0.1 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.1 ml containing immunogen in admixture with 0.5% aluminum hydroxide.
The vaccine of the present invention may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments such as antibiotics.
While the invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modification and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows from the scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: McGill University
845 Sherbrooke Street West Montreal, Quebec, CANADA H3A 1B1
(ii) TITLE OF INVENTION: Peptides and Vaccines Derived From Nematode Tubulin
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MERCHANT & GOULD
(B) STREET: 3100 Norwest Center
(C) CITY: Minneapolis
(D) STATE: MN
(E) COUNTRY: USA
(F) ZIP: 55402
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/967,829
(B) FILING DATE: 28-OCT-1992
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Woessner, Warren D.
(B) REGISTRATION NUMBER: 30,440
(C) REFERENCE/DOCKET NUMBER: 10022.3-WO01
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-332-5300
(B) TELEFAX: 612-332-9081
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu
Claims
1. A monoclonal antibody which substantially binds to β-tubulin of nematode origin and fragments thereof .
2. A hybridoma cell line producing the monoclonal antibody of claim 1 and deposited at the ATCC under accession number HB 11129.
3 . The use of the antibody of claim 1 as an anti- parasitic agent .
4. The use of the antibody of claim 1 as a diagnostic agent for parasitic diseases .
5 . The use of claim 3 directed against filariasis- causing parasites .
6. The use of claim 4 , wherein the parasitic disease is filariasis .
7 . The use of a peptide as an immunizing agent against parasites , wherein said peptide has the amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu, SEQ ID NO : 1 , or a fragment thereof .
8. The use of a peptide as an immunizing agent against parasites , wherein said peptide has at least the amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : 1 .
9. The use of a peptide to induce production by a host of cytotoxic antibodies against parasites, which peptide has the amino acid sequence: -28-
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : 1 , or a fragment thereof .
10. The use of a peptide to induce production by a host of cytotoxic antibodies against parasites , which peptide has at least the amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu, SEQ ID N0 : 1 .
11. A vaccine for parasite infection comprising a peptide which has the amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu, SEQ ID NO : 1 , or a fragment thereof in association with a pharmaceutical carrier .
12. A vaccine for parasite infection comprising a peptide which has at least the amino acid sequence :
Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
Glu Glu , SEQ ID NO : l .
13. A method of immunizing mammals against parasites comprising the administration of a vaccine according to claim 11 or 12.
14. The method of immunizing of claim 13, wherein the parasite is a nematode.
15. The method of immunizing of claim 14, wherein the nematode is a heartworm and the mammal is a canine.
16. The method according to claim 13, 14 or 15, wherein said vaccine is administered in a dosage range of 0.015 μg to 0.15 mg per kg body weight. -29-
17. A method according to claim 16, wherein said vaccine is administered in a dosage range of 1.5 μg to 0.15 mg per kg body weight.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP93922866A EP0674659A1 (en) | 1992-10-28 | 1993-10-22 | Peptides and vaccines derived from nematode tubulin |
| JP6510491A JPH08503935A (en) | 1992-10-28 | 1993-10-22 | Nematode tubulin-derived peptides and vaccines |
| AU52821/93A AU5282193A (en) | 1992-10-28 | 1993-10-22 | Peptides and vaccines derived from nematode tubulin |
| KR1019950701712A KR950704359A (en) | 1992-10-28 | 1993-11-22 | Peptides and Vaccines Derived from the Nematode Tubulin |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US96782992A | 1992-10-28 | 1992-10-28 | |
| US07/967,829 | 1992-10-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994010201A1 true WO1994010201A1 (en) | 1994-05-11 |
Family
ID=25513395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CA1993/000431 WO1994010201A1 (en) | 1992-10-28 | 1993-10-22 | Peptides and vaccines derived from nematode tubulin |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0674659A1 (en) |
| JP (1) | JPH08503935A (en) |
| KR (1) | KR950704359A (en) |
| AU (1) | AU5282193A (en) |
| CA (1) | CA2147756A1 (en) |
| WO (1) | WO1994010201A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7203902B2 (en) | 2003-07-24 | 2007-04-10 | Hewlett-Packard Development Company, L.P. | Method and apparatus for document composition |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1983001739A1 (en) * | 1981-11-17 | 1983-05-26 | Brigham & Womens Hospital | Monoclonal antibodies against brugia malayi |
| GB2152510A (en) * | 1984-01-10 | 1985-08-07 | Kenneth K Lew | Method for the production of helminths vaccines |
| WO1992003549A1 (en) * | 1990-08-16 | 1992-03-05 | Euro-Diagnostics B.V. | Specific dna sequences of a nematode which can be used for the diagnosis of infection with the nematode |
-
1993
- 1993-10-22 WO PCT/CA1993/000431 patent/WO1994010201A1/en not_active Application Discontinuation
- 1993-10-22 AU AU52821/93A patent/AU5282193A/en not_active Abandoned
- 1993-10-22 EP EP93922866A patent/EP0674659A1/en not_active Withdrawn
- 1993-10-22 JP JP6510491A patent/JPH08503935A/en active Pending
- 1993-10-22 CA CA002147756A patent/CA2147756A1/en not_active Abandoned
- 1993-11-22 KR KR1019950701712A patent/KR950704359A/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1983001739A1 (en) * | 1981-11-17 | 1983-05-26 | Brigham & Womens Hospital | Monoclonal antibodies against brugia malayi |
| GB2152510A (en) * | 1984-01-10 | 1985-08-07 | Kenneth K Lew | Method for the production of helminths vaccines |
| WO1992003549A1 (en) * | 1990-08-16 | 1992-03-05 | Euro-Diagnostics B.V. | Specific dna sequences of a nematode which can be used for the diagnosis of infection with the nematode |
Non-Patent Citations (5)
| Title |
|---|
| BUGHIO ET AL.: "Characterization and biological activities of monoclonal antibodies specific to nematode tubulin", AM. J. TROP. MED. HYG., vol. 45, no. 3SUP, September 1991 (1991-09-01), pages 186 * |
| BUGHIO ET AL.: "Identification of tubulin isoforms in different tissues of Ascaris suum using antitubulin monoclonal antibodies", INTERNATIONAL JOURNAL OF PARASITOLOGY, vol. 21, no. 8, 1991, pages 913 - 918 * |
| FORTITH ANNUAL MEETING OF THE AMERICAN SOCIETY OF TROPICAL MEDICINE AND HYGIENE , 1-5 /12 / 1991, BOSTON, USA * |
| GUENETTE ET AL.: "Characterization of a beta-tubulin gene and beta-tubulin gene products of Brugia pahangi", MOL. BIOCHEM. PARASITOL., vol. 44, no. 2, 1991, pages 153 - 164 * |
| HELM ET AL.: "Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi", PARASITE IMMUNOLOGY, vol. 11, no. 5, September 1989 (1989-09-01), pages 479 - 502 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7203902B2 (en) | 2003-07-24 | 2007-04-10 | Hewlett-Packard Development Company, L.P. | Method and apparatus for document composition |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0674659A1 (en) | 1995-10-04 |
| JPH08503935A (en) | 1996-04-30 |
| KR950704359A (en) | 1995-11-20 |
| AU5282193A (en) | 1994-05-24 |
| CA2147756A1 (en) | 1994-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1341166C (en) | Antigenic proteins and vaccines containing them for prevention of coccidiosis | |
| Newton | Progress on vaccination against Haemonchus contortus | |
| WO1997036612A1 (en) | Anti-cryptosporidium parvum preparations | |
| US6015882A (en) | Vaccines, antibodies, proteins, glycoproteins, DNAs and RNAs for prophylaxis and treatment of Cryptosporidium parvum infections | |
| KR100244828B1 (en) | Coccidiosis vaccines | |
| KR100553646B1 (en) | Malaria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen | |
| KR20010011330A (en) | Crude extract from Viscum album coloratum, protein and lectins isolated therefrom | |
| AU759316B2 (en) | Chimeric gene encoding the antigenic determinants of four proteins of L. infantum | |
| EP0846176A1 (en) | Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccine | |
| AU2017261603B2 (en) | Tick toxin compositions | |
| AU663862B2 (en) | Nematode vaccine | |
| WO1994010201A1 (en) | Peptides and vaccines derived from nematode tubulin | |
| FR2863499A1 (en) | Vaccine for treating or preventing infection by Echinococcus, useful especially for control of hydatid cysts and alveolar echinococcosis, comprises a specific parasite protein or its fragments | |
| US20180311327A1 (en) | Aquaporin 2 protects cattle from ticks and tick-borne parasites | |
| JP3582663B2 (en) | Gene clone expressing chicken leukocytozone immunogenic protein and transgenic vaccine against chicken leukocytosonosis | |
| EP0283829A1 (en) | Plasmodium falciparum merozoite antigen peptides | |
| WO1996032132A1 (en) | Peptides of nematode tubulin and methods of use | |
| US6962704B2 (en) | Chimeric gene formed by the dna sequences that encode the antigenic determinants of four proteins of l. infantum and protein encoded by said gene, and pharmaceutical composition useful for preventing and/or treating leishmaniosis in animals or humans | |
| Sawangjaroen et al. | Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera | |
| CA2256124C (en) | Chimeric gene formed of the dna sequences that encode the antigenic determinants of four proteins of l. infantum, and protein encoded by said gene, and pharmaceutical composition useful for preventing and/or treating leishmaniosis in animals or humans | |
| NZ224597A (en) | Taenia ovis antigens and vaccines | |
| FR2850384A1 (en) | New antigen from intracellular pathogens, useful in vaccines against bacterial and viral infections, comprises sequences from extracellular domains of type III membrane proteins, also derived antibodies | |
| GB2219590A (en) | Antigenic polypeptides of taena ovis | |
| CA2253834A1 (en) | B. burgdorferi polypeptides expressed in vivo | |
| WO2006009862A2 (en) | Amebiasis subunit vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU MG MN MW NL NO NZ PL PT RO RU SD SE SK UA VN |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| WWE | Wipo information: entry into national phase |
Ref document number: 2147756 Country of ref document: CA |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 1993922866 Country of ref document: EP |
|
| REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
| WWP | Wipo information: published in national office |
Ref document number: 1993922866 Country of ref document: EP |
|
| WWW | Wipo information: withdrawn in national office |
Ref document number: 1993922866 Country of ref document: EP |