WO1997033999A1 - Immunity to trypanosomatids species - Google Patents
Immunity to trypanosomatids species Download PDFInfo
- Publication number
- WO1997033999A1 WO1997033999A1 PCT/EP1997/001289 EP9701289W WO9733999A1 WO 1997033999 A1 WO1997033999 A1 WO 1997033999A1 EP 9701289 W EP9701289 W EP 9701289W WO 9733999 A1 WO9733999 A1 WO 9733999A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- leishmania
- gene
- species
- histones
- histone
- Prior art date
Links
- 230000036039 immunity Effects 0.000 title claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 110
- 108010033040 Histones Proteins 0.000 claims abstract description 57
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 44
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 38
- 208000015181 infectious disease Diseases 0.000 claims abstract description 33
- 102000006947 Histones Human genes 0.000 claims abstract description 27
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 13
- 229960005486 vaccine Drugs 0.000 claims abstract description 9
- 238000002405 diagnostic procedure Methods 0.000 claims abstract description 7
- 239000002671 adjuvant Substances 0.000 claims abstract description 6
- 101710194807 Protective antigen Proteins 0.000 claims abstract description 4
- 108020004511 Recombinant DNA Proteins 0.000 claims abstract description 4
- 241000222722 Leishmania <genus> Species 0.000 claims description 43
- 208000004554 Leishmaniasis Diseases 0.000 claims description 19
- 229920001184 polypeptide Polymers 0.000 claims description 17
- 108020004999 messenger RNA Proteins 0.000 claims description 16
- 241000894007 species Species 0.000 claims description 14
- 108020004414 DNA Proteins 0.000 claims description 13
- 206010047505 Visceral leishmaniasis Diseases 0.000 claims description 13
- 239000002299 complementary DNA Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 201000000626 mucocutaneous leishmaniasis Diseases 0.000 claims description 13
- 230000001681 protective effect Effects 0.000 claims description 13
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 10
- 241000222724 Leishmania amazonensis Species 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- 241000222740 Leishmania braziliensis Species 0.000 claims description 7
- 241000178949 Leishmania chagasi Species 0.000 claims description 7
- 108091034117 Oligonucleotide Proteins 0.000 claims description 7
- 208000030852 Parasitic disease Diseases 0.000 claims description 7
- 230000003834 intracellular effect Effects 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 230000036281 parasite infection Effects 0.000 claims description 7
- 108091033319 polynucleotide Proteins 0.000 claims description 7
- 102000040430 polynucleotide Human genes 0.000 claims description 7
- 239000002157 polynucleotide Substances 0.000 claims description 7
- 241000222696 Leishmania guyanensis Species 0.000 claims description 6
- 241000222695 Leishmania panamensis Species 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 230000036961 partial effect Effects 0.000 claims description 6
- 241000222727 Leishmania donovani Species 0.000 claims description 5
- 241000222697 Leishmania infantum Species 0.000 claims description 5
- 206010057168 Leishmania infections Diseases 0.000 claims description 5
- 241000222704 Leishmania peruviana Species 0.000 claims description 5
- 102000007999 Nuclear Proteins Human genes 0.000 claims description 5
- 108010089610 Nuclear Proteins Proteins 0.000 claims description 5
- 244000020186 Nymphaea lutea Species 0.000 claims description 5
- 241001522283 Viannia Species 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 108020004635 Complementary DNA Proteins 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 239000012678 infectious agent Substances 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- 238000011321 prophylaxis Methods 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000000069 prophylactic effect Effects 0.000 claims description 3
- 238000002560 therapeutic procedure Methods 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000000032 diagnostic agent Substances 0.000 claims description 2
- 229940039227 diagnostic agent Drugs 0.000 claims description 2
- 230000002163 immunogen Effects 0.000 claims description 2
- 240000005528 Arctium lappa Species 0.000 claims 3
- 230000001225 therapeutic effect Effects 0.000 claims 2
- 206010001935 American trypanosomiasis Diseases 0.000 claims 1
- 241000223104 Trypanosoma Species 0.000 claims 1
- 241000223105 Trypanosoma brucei Species 0.000 claims 1
- 241000223109 Trypanosoma cruzi Species 0.000 claims 1
- 244000104547 Ziziphus oenoplia Species 0.000 claims 1
- 235000005505 Ziziphus oenoplia Nutrition 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 210000001124 body fluid Anatomy 0.000 claims 1
- 239000010839 body fluid Substances 0.000 claims 1
- 239000000969 carrier Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000002934 lysing effect Effects 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 244000045947 parasite Species 0.000 abstract description 22
- 108020004707 nucleic acids Proteins 0.000 abstract description 7
- 102000039446 nucleic acids Human genes 0.000 abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 abstract description 7
- 239000008194 pharmaceutical composition Substances 0.000 abstract 1
- 239000000546 pharmaceutical excipient Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 27
- 241000222732 Leishmania major Species 0.000 description 18
- 230000003902 lesion Effects 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 description 11
- 108020001507 fusion proteins Proteins 0.000 description 11
- 102000037865 fusion proteins Human genes 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 239000000284 extract Substances 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 238000003119 immunoblot Methods 0.000 description 8
- 206010011668 Cutaneous leishmaniasis Diseases 0.000 description 7
- 230000004224 protection Effects 0.000 description 7
- 238000011725 BALB/c mouse Methods 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 210000004940 nucleus Anatomy 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241000255129 Phlebotominae Species 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 108090000992 Transferases Proteins 0.000 description 3
- 102000004357 Transferases Human genes 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 108091092330 cytoplasmic RNA Proteins 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- 235000003969 glutathione Nutrition 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101001095863 Enterobacteria phage T4 RNA ligase 1 Proteins 0.000 description 2
- 102000003814 Interleukin-10 Human genes 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 102000013462 Interleukin-12 Human genes 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 210000002683 foot Anatomy 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 244000000056 intracellular parasite Species 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000007937 lozenge Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- HTAGIZQYGRLQQX-AUUYWEPGSA-N Amurine Chemical compound C1C2=CC=3OCOC=3C=C2[C@]23C=C(OC)C(=O)C=C3[C@@H]1N(C)CC2 HTAGIZQYGRLQQX-AUUYWEPGSA-N 0.000 description 1
- 241000870366 Atherion Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000005996 Blood meal Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000020446 Cardiac disease Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010061619 Deformity Diseases 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 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
- 241000237537 Ensis Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 101100336468 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) gem-1 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 206010071368 Psychological trauma Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 101000836384 Rattus norvegicus Serpin H1 Proteins 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- HTAGIZQYGRLQQX-UHFFFAOYSA-N amurine Natural products C1C2=CC=3OCOC=3C=C2C23C=C(OC)C(=O)C=C3C1N(C)CC2 HTAGIZQYGRLQQX-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000002787 antisense oligonuctleotide Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000022602 disease susceptibility Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000000548 hind-foot Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 229940028885 interleukin-4 Drugs 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000012768 mass vaccination Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229940079938 nitrocellulose Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108700020942 nucleic acid binding protein Proteins 0.000 description 1
- 102000044158 nucleic acid binding protein Human genes 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000007691 rabbit blood agar Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 231100000019 skin ulcer Toxicity 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000011806 swiss nude mouse Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- -1 vanadyl ribonucleoside Chemical compound 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
-
- 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
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- 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
Definitions
- the present invention relates to genes and corresponding gene products ⁇ DNAs, RNAs and proteins) for use in vaccines and diagnostics specific for intracellular infectious agents, in particular for parasite infection, more in particular for trypanosomatids infection, in particular for Leishmania species infection, more in particular for cutaneous lesions inducing Leishmania, more in particular for Leishmania ma or.
- Leishmania a member of the trypanosomatid family, is endemic in the tropical regions of America, Africa and the Indian subcontinent, in the sub-tropics of south-west Asia and in the Mediterranean. Infection with different species of the protozoan Leishmania.
- leishmaniasis which is transmitted by sandflies, manifests itself as either self-healing cutaneous lesions, as neurological and cardiac disorders or as fatal visceral infections.
- the disease caused by Leishmania is generally called leishmaniasis.
- Some forms of the disease are anthroponotic
- Visceral leishmaniasis causes large scale epidemics and the number of cases varies greatly between the years. During 1991 there were large epidemics in India and Sudan. The number of cases in India alone may have been of the order of 250.000. It is estimated that visceral leishmaniasis may have killed 75.000 people in 1991.
- the parasites are transmitted to the human host where they are phagocytosed by resident skin macrophages. Within the macrophage lysosome, the parasites differentiate into round, non- flagellated amastigotes where they replicate until the macrophage is lysed. They are then released to infect other macrophages, or are taken up by a sandfly where they di ferentiate back to the pro astigote stage to repeat their life cycle.
- Thl/Th2 balance L.major infection.
- Thl/Th2 balance Detailed discussions on the importance of the Thl/Th2 balance and the cytokine network(s) have been reported in recent reviews (Liew and O'Donnell, 1993; Milon et al . , 1995) .
- the Thl response is associated with the production of the cytokines IL-2 and ⁇ -IFN, parasite killing, healing and protection, while the Th2 response is associated with the production of IL- 4 and/or IL-10, disease progression and susceptibility (Heinzel et al . , 1991;- Scott, 1989) .
- This pattern of lymphokine production has also been observed in human leishmaniasis (Reed and Scott, 1993) .
- IL-12 facilitates the development of a Thl response by stimulating the production of ⁇ -IFN and down-regulating that of IL-4 (Heinzel et al, 1993; Sypek et al, 1993) .
- Leishmania major promastigotes derived antigens such as the surface membranes gp63 and lipophosphoglycan (Russell et al, 1988) , soluble extract (Scott et al, 1987) or gp63 epitopes (Yang et al, 1991) have shown that protection can be achieved at various levels in the susceptible BALB/c mice.
- dp72 and GP46/M2 have been used against Leishmania donovani and Leishmania amazonensis respectively, but only partical protection has been observed (Rachamim and Jaffe, 1993; McMahon- Pratt et al, 1993) . In these trials, only promastigote derived antigens were used.
- vaccines comprising antigens present in different stages of the life cycle of the parasite might increase the percentage of protection. Furthermore, it was found that some proteins are exported as peptides to the macrophage surface and are involved in T-cell mediated immune response towards intracellular parasites. Such parasite antigen (s) involved in inducing protective immunity have not previously been identified. The identification and characterization of such gene products are imperative to allow an understanding of intracellular parasitism as well as the design of diagnostic tests and vaccines.
- genes and their derivatives like mRNA or proteins that may prove to be useful in diagnosis, prophylaxis and treatment of infection by intracellular infectious agents like parasites, in particular trypanosomatids, like Leishmania species.
- the invention pertains to the general finding that intacellular antigens (e.g. histones) that are already involved in the T-cell mediated immune response towards intracellular parasites are a good starting point for the design and development of vaccines, pharmaceuticals and diagnostic tests. In the following this principle will be illustrated by reference to trypanosomatids in general and Leishmania species in particular.
- intacellular antigens e.g. histones
- the invention in its most basic form thus provides for trypanosomatid histones in substantially isolated form for use as a protective antigen against trypanosomatid infection.
- the histones may be isolated from the corresponding parasite species but are preferably produced by means of recombinant DNA techniques .
- the invention also provides genes encoding the protective histones. Common to these genes is their ability to hybridise to: a) at least a distinguishing part of the polynucleotide of the sequence depicted in Fig. 6A; or b) those parts of the sequence of Fig.
- the recombinant histone may be the complete histone encoded by the gene isolated from the parasite species. In a more advanced embodiment only parts of the gene, either one or more, encoding one or more parts of the histone, at least some of which parts are responsible for its protective capacity, may be used.
- Fig. 6A The phrase "at least a distinguishing part of the polynucleotide" is intended to indicate that genes of the invention need not necessarily hybridise to the complete sequence depicted in Fig. 6A.
- the invention in fact provides for a gene family the members of which share a sequence similarity and the fact that they encode a histone, in particular histone HI. These two features will enable the skilled person to define the extent to which the sequence of Fig. 6A should hybridise to the other members of the family.
- One possibility to establish whether a gene that has been isolated belongs to the family of the invention is comparison of its derived amino acid sequence with an amino acid database, such as the Swiss Prot database or the EMBL database. Such a database will give information on the homology of the derived amino acid sequence with known sequences. On the basis hereof the skilled person can establish whether he actually isolated a histone.
- a further tool in establishing whether a histone gene was isolated, is chemical and physical characterisation of the gene product. For example, histones are nuclear proteins and bind DNA.
- nucleotide sequence depicted in Fig. 6A is the sequence designated SW3 and already described previously.
- the invention is intended to encompass both the use of this gene and its derivatives and its family members as defined above.
- the invention relates to derivatives of the genes, which derivatives comprise fragments of the gene, complete or partial cDNAs of the gene, complete or partial mRNAs to the gene, complete or partial proteins encoded by the gene, peptides comprising at least an immunogenic part of the protein, antisense oligo- or polynucleotides, fusion products between at least part of the gene, cDNA, mRNA, protein or peptide and at least part of another gene, cDNA, RNA, protein or peptide, antibodies against the gene, cDNA, mRNA, protein, peptides or fusion products thereof, primers specific for the gene, cDNA or mRNA, wherein each derivative may either be isolated or may be obtained through recombinant DNA techniques.
- nucleic acids genes, cDNA”, mRNA, oligonucleotide, and “polynucleotide” as well as their antisense or complementary counterparts may be referred to as "nucleic acids”.
- Proteins refer to the products obtainable by transcription and translation of the nucleic acids and are generally referred to as "gene products”.
- RNAs for antisense polynucleotides or RNAs
- RNAs for antisense polynucleotides or RNAs
- their activity by their function, by recognizing the gene product or parts thereof (for antibodies)
- helper or cytotoxic T lymphocyte epitopes can be identified by molecu ⁇ lar hybridisation to the gene (for antisense polynucleotides or RNAs) , by their activity, by their function, by recognizing the gene product or parts thereof (for antibodies) , by the immune system as helper or cytotoxic T lymphocyte epitopes.
- the invention further relates to expression vectors harboring the nucleic acids for producing mRNA or gene products.
- expression vectors harboring the nucleic acids for producing mRNA or gene products Again the skilled person will be very well capable of selecting a suitable vector and the necessary expiession signals, such as transcription and translation initiation and termination sequences based on his common knowledge and the information contained in this application.
- the invention provides for probes directed against the nucleic acids, antibodies directed against the gene products, nucleic acid- molecules or polypeptides recogni ⁇ sing the amino acid sequence of the gene products.
- the gene product is a nuclear protein that may be isolated due to the fact that it binds to DNA.
- the invention provides for diagnostic agents comprising said nucleic acids, antibo ⁇ dies or said gene products for use in assaying infection and thus diagnosing Leishmania infections, and the use of said nucleic acids, antibodies or said gene products for prophylactic purpose, e.g. as component in a vaccine, or in therapy.
- this invention relates to intracellular protein, in particular to nucleic acid binding protein, more in particular to nuclear proteins.
- this invention relates to histones, and more in particular to histones HI, in particular to histone HI gene family, isolated by molecular hybridisation or polymerase chain reaction, more in particular to the polypeptide of the SW3 gene.
- the gene products in particular products of the SW3 gene of Leishmania major or SW3 analogs of other Leishmania species, were found to elicit a strong immune response and demonstrate protective capacities.
- mice an immune response in mice was elicited by injecting a recombinant protein derived from the gene.
- a specific protection was obtained when the protein was injected sub-cutaneously and animals were subsequently challenged with live parasites.
- Another example teaches a nuclear polypeptide sequence, histone HI, and its use in immunisation regimens.
- Cross- hybridising mRNAs were detected in other Leishmania species. More particular the SW3 polypeptide was expressed in E.coli as a recombinant protein by a fusion with glutathion S transferase (GST) .
- Mice were immunised with purified GST-SW3 polypeptide. The raised antibodies recognised the injected product. Immunised mice were infected with Leishmania major. Specific recognition was observed and protective effect of the polypeptide was shown in challenge experiments.
- the present invention is described in the above under reference to L.major.
- the invention is also intended to encompass further Leishmania species, such as species of the subgenus Leishmania, comprising the complex L.major, which only comprises L.major species, the complex L.dono ani , comprising for example L.chaqasi, L.donovani, and L. infantum, and the complex L.mexicana, comprising inter alia L.amazonensis and
- L.t ⁇ exicana as well as species of the subgenus Viannia, comprising the complex L.braziliensis, comprising L.braziliensis en L.peruviana and the complex L.guyanensis, comprising the species L.guyanensis and L.panamensis.
- Leishmania major promastigotes (strain LV39- MHRO/SU/59/P or MRHO/IR/75/ER) are cultivated at 26°C in Dulbecco's modified Eagle medium (DMEM; Gibco-BRL) on a solid rabbit blood agar (Louis et al, 1979) , supplemented with 10% fetal calf serum (Seromed) and gentamicin (10 ⁇ g/ml) . Amastigotes were produced in vivo. BALB/c mice were injected subcutaneously in the hind footpad with 2 to 5xl0 7 parasites/ml of stationary phase promastigotes.
- DMEM Dulbecco's modified Eagle medium
- Seromed 10% fetal calf serum
- gentamicin (10 ⁇ g/ml)
- amastigotes were obtained by 5 passing parasites in the back of Swiss nude mice.
- L.major amastigotes were purified from back lesions and extracted according to a described protocol (Glaser et al . , 1990) .
- the nuclear pellet is frozen in liquid nitrogen and stored at -70°C. Isolation of amastigotes nuclei was performed using a similar protocol.
- Histones are soluble in HCl and, among histones, HI is selectively soluble in perchloric acid. Cells were collected, washed twice in lxPBS and lysed in
- NP4 Nuclei were pelleted at 6000 g for 3 min (Kontron) . Nuclei were resuspended in 1.25N HCl for total histone extraction or 5% perchloric acid for histone HI recovery, vortexed for 30 seconds and mixed on a rotating wheel at
- Promastigotes were collected by centrifugation (10 min at 3000 revs/min at 4°C) , washed three times in lxPBS and resuspended in gel sample buffer for SDS-PAGE (Lammli, 1970) . Amastigotes were isolated according to a published method (Glaser et al . , 1990) and are then handled as described for promastigotes. Protein gel electrophoresis was performed as described. Routinely, the proteins from 3xl0 7 cells boiled in 1 x Lammli buffer for 5 min, and loaded in a 4 mm wide slot were separated by 15% SDS-PAGE. Proteins were electrotransferred onto nitro-cellulose (Immunoblots) .
- Immunoblotting from SDS- polyacrylamide gels was carried out as described by Harlow and Lane (1988) and a 1: 1000 dilution of the rabbit ⁇ 415 peptide serum was added to the filter incubated overnight at room temperature.
- Goat anti-rabbit secondary antibody and peroxydase conjugated protein A were used and a chemiluminescence reaction substrate (Amersham) was used to reveal presence of reactive polypeptide (s) .
- S-SW3 (5 ' -cccgtcgacggatgtcctctaattc-3 ' ) and: A-SW3 (5' -agagtcgacctatgatgcgtcttcgggcacgt-3 ' ) in a buffer containing 20mM Tris-HCl (pH 8.3) , lOOmM KCl, 3 mM MgCl 2 , 2.5 mM of each of the dNTPs and one unit of Taq polymerase.
- RNA isolation and analysis Cytoplasmic RNA of different Leishmania species was isolated as described previously (Fasel et al . , 1994) . 15 ⁇ g RNA were fractionated on 0.8% agarose gel and transferred to Genescreen plus membrane (NEN research products) . Radioactive antisense probes were generated by in vitro transcription of SW3 cDNA inserted in p GEM-1/2 vectors containing T7 and Sp6 RNA polymerases promotors . Hybridisation and washing were carried out as described (Fasel et al . , 1994) .
- amino acid sequences were compared to the Swiss Prot sequence data bank and the degree of similarity was assessed by using Multiple Sequence Alignment (http:/www2pasteur. fr/-takaia/MAcours/ multalign.html) .
- SW3 expression of the SW3 in E.coli
- the open reading frame SW3 was amplified by polymerase chain reaction from the construct pCRII-SW3 (Fasel et al . , 1994) using the oligonucleotides : S-SW3 (5 ' -cccgtcgacggatgtcctctaattc-3 ' ) and: A-SW3 (5'-aga gtcgacctatgatgcgtcttcgggcacgt-3" ) in a buffer containing 20mM Tris-HCl (pH 8.3), lOOmM KCl, 3mM MgCl 2 ,2.5mM of each of the dNTPs and one unit of Taq polymerase.
- the nucleotide sequence in bold characters correspond to Sail restriction sites which can be used to insert the PCR product in the Sail site of an expression vector.
- Amplification was performed using the following cylces: 8 min at 98°C, 3 min at 60°C, 2 min at 72°C followed by 33 cycles of one min at 94°C, 1.5 min at 60°C and 2 min at 72°C.
- the fragment was inserted into the Sail site of the vector pGEX-KG (Guan and Dixon, 1991) in phase with the reading frame of the glutathion S- transferase (GST) .
- E.coli (DH5 or) were transformed by electroporation and the orientation of the insert was determined by Haelll restriction enxyme which generates a fragment of different length according to the orientation of the insert. Orientation and sequence of the insert was confirmed by double stranded DNA sequencing.
- the DNA construct named pGEX-KG-415 was further used to obtain expression of a fusion protein GST-SW3.
- An E.coli colony containing the pGEX-KG-415 plasmid was used to inoculate bacterial culture medium (2xTY) supplemented with lOO ⁇ g/ l of ampicilline and grown overnight at 37°C.
- Cells are resuspended in 6 ml of STE containing 5 mg/ml of lysozyme. 10 ⁇ l of DNase I at a concentration of 10 mg/ml are added to the mix and the solution is incubated 15 min on ice. Dithiothreitol and sarkosyl are added to final concentration of 5 mM and 1.5% respectively. Additional lysis is obtained by two sonication of 30 sec (Sonifier 250, Brandson) . The lysate is centrifuged 10 min at lO.OOOxg and Triton X-100 is added to the supernatant at a final concentration of 1.4% before an incubation of 15 tot 30 min on ice.
- Fusion protein is then purified by gentle stirring the mix with agarose-glutation beads (Sigma, Nr.G-4510) in lxPBS for 30 min at room tempera ⁇ ture. Beads are sedimented by centrifuging during 5 min at 1000 xg and washed 4 to 5 times with 1 ml of cold PBS. Finally fusion protein is eluted from the resin by resuspending the beads in a solution of 10 mM glutathion (pH 8.0) (Merck Nr 4090) and incubating them 10 to 30 min at room temperature with constant stirring. The mix is centrifuged 5 min at 500xg and the supernatant is transferred to a new tube.
- agarose-glutation beads Sigma, Nr.G-4510
- Beads are sedimented by centrifuging during 5 min at 1000 xg and washed 4 to 5 times with 1 ml of cold PBS.
- fusion protein is eluted from the resin by resuspending the beads in
- Fusion protein can be quantified by measuring absorbance at 280 n or by comparing intensity of the fusion protein after Coomassie R-250 staining with the staining of defined quantities of molecular weight markers .
- the SW3 gene has a higher expression at the RNA level in the intracellular amastigote stage as compared to the free-living promastigote stage. It encodes a protein with sequence similarity to an histone HI protein and has been described as an histone HI-like protein (Fasel et al . , 1994) but no evidence for it has been provided.
- To characterize the SW3 gene product we generated a rabbit antiserum directed against a peptide ("415") corresponding to the amino-terminus of the deduced amino acid sequence of SW3 (see Materials and methods) and analysed cytoplasmic or nuclear lysates of LV39 strain.
- SW3 polypeptide Biochemical analysis of the SW3 polypeptide confirmed the histone nature of the encoded polypeptide.
- SW3 protein can be purified out of a nuclear fraction to a high degree using the histones (HCl) and histone HI preparative (perchloric acid) method (Fig. 2, lanes b, d and f) . No signal is present when the protein extracts are analysed with pre-immune serum (Fig. 2, lanes a, c 5 and e) .
- GST glutathion-S-transferase
- a product of the expected molecular weight' can be detected after purification on a 0 GST-agarose column (Fig. 3, lane c, indicated by arrow 1) .
- Arrow 2 indicates GST-415 fusion protein containing GST linked to 50 amino acids of SW3.
- the purified recombinant protein mix was injected in susceptible mouse strains (BALB/c) to demonstrate its potential as a 5 protective antigen in parasite challenge experiments. Groups of mice were immunized subcutaneously (at the basis of the tail) either with a mixture of recombinant protein GST-415 and incomplete Freund' s adjuvant (Fig.
- SW3 and SW3 related polypeptides are interesting if it can be used to control other Leishmania species. For this reason, search for cross-hybridising mRNA transcripts of various sizes have been detected in New World Leishmania species such as L.chagasi . L.guvanensis, L.panamensis and L.amazonensis (Fig. 7) . This result provides evidence for presence of related genes and gene products in other Leishmania which could be used to obtain protection.
- Biochemical characterisation of the SW3 gene product Western blot analysis of nuclei (lanes a and b) and of 0. IN HCl (lanes c and d) or 5% perchloric acid (lanes e and f) nuclear extract of stationary phase promastigotes with rabbit antiserum raised against peptide 415 (lanes b, d, and f) or with rabbit preimmune serum (lanes a, c, and f) . Extracts were separated on 12.5% SDS-polyacrylamide gels before immunoblotting.
- mice immunised either IFA (Panel A) , with GST (Panel B) , with the 415 peptide (Panel C) or the GST-415 fusion protein (Panel D) were infected in the right hind foot pad (filled lozenge symbols) with LV39 parasites.
- the left foot pad (open lozenge symbols) is used as an internal control .
- Panel A schematic representation of the SW3 transcript, the SW3 gene and of the antisense SW3 riboprobe.
- Panel B Cytoplasmic RNA iso- ' ated from promastigotes of L.major (lane a) , L.guyanensis (lane b) , L.pana ensis (lane c) , L.chagasi (lane d) and L.amazonensis (lane e) were separated on a 0.8% agarose gel, transferred to a nylon membrane and hybridised with a SW3 anti-sense riboprobe as shown in panel A.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU20272/97A AU733720B2 (en) | 1996-03-12 | 1997-03-12 | Immunity to trypanosomatids species |
JP9532298A JP2000507098A (en) | 1996-03-12 | 1997-03-12 | Immunity to Trypanosomaceae species |
BR9708188-4A BR9708188A (en) | 1996-03-12 | 1997-03-12 | Immunity to trypanosomatidae species. |
IL12614597A IL126145A0 (en) | 1996-03-12 | 1997-03-12 | Immunity to trypanosomatids species |
EP97908233A EP0896623A1 (en) | 1996-03-12 | 1997-03-12 | Immunity to trypanosomatids species |
NZ331889A NZ331889A (en) | 1996-03-12 | 1997-03-12 | Trypanosomatids histones for use as an antigen against trypanosomatic infection, including diagnostic tests, pharmaceutical compositions and vaccines of these antigens |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96200665 | 1996-03-12 | ||
EP96200665.6 | 1996-03-12 | ||
EP96201343.9 | 1996-05-15 | ||
EP96201343 | 1996-05-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997033999A1 true WO1997033999A1 (en) | 1997-09-18 |
Family
ID=26142593
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1997/001289 WO1997033999A1 (en) | 1996-03-12 | 1997-03-12 | Immunity to trypanosomatids species |
Country Status (12)
Country | Link |
---|---|
EP (1) | EP0896623A1 (en) |
JP (1) | JP2000507098A (en) |
CN (1) | CN1216584A (en) |
AU (1) | AU733720B2 (en) |
BR (1) | BR9708188A (en) |
CA (1) | CA2248684A1 (en) |
IL (1) | IL126145A0 (en) |
MA (1) | MA25182A1 (en) |
NZ (1) | NZ331889A (en) |
TN (1) | TNSN97045A1 (en) |
TR (1) | TR199801776T2 (en) |
WO (1) | WO1997033999A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005044301A3 (en) * | 2003-11-05 | 2005-06-30 | C B F Leti S L Unipersonal | Use of specific histones for the treatment of parasitic diseases |
WO2011153602A2 (en) * | 2010-06-08 | 2011-12-15 | Universidade Federal de Viçosa | Recombinant e-ntpdases, use for producing a diagnostic kit for detecting antibodies in various types of leishmaniasis caused by species of the leishmania genus |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2952382B1 (en) * | 2009-11-10 | 2011-11-25 | Univ Victor Segalen Bordeaux 2 | VACCINES AND DIAGNOSES AGAINST AFRICAN ANIMAL TRYPANOSOMOSES |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0309898A2 (en) * | 1987-09-28 | 1989-04-05 | F. Hoffmann-La Roche Ag | Method for the induction of a cytotoxic T cell response |
-
1997
- 1997-03-11 MA MA24516A patent/MA25182A1/en unknown
- 1997-03-11 TN TNTNSN97045A patent/TNSN97045A1/en unknown
- 1997-03-12 NZ NZ331889A patent/NZ331889A/en unknown
- 1997-03-12 CN CN97193835A patent/CN1216584A/en active Pending
- 1997-03-12 WO PCT/EP1997/001289 patent/WO1997033999A1/en not_active Application Discontinuation
- 1997-03-12 CA CA002248684A patent/CA2248684A1/en not_active Abandoned
- 1997-03-12 TR TR1998/01776T patent/TR199801776T2/en unknown
- 1997-03-12 EP EP97908233A patent/EP0896623A1/en not_active Withdrawn
- 1997-03-12 BR BR9708188-4A patent/BR9708188A/en not_active IP Right Cessation
- 1997-03-12 IL IL12614597A patent/IL126145A0/en unknown
- 1997-03-12 AU AU20272/97A patent/AU733720B2/en not_active Ceased
- 1997-03-12 JP JP9532298A patent/JP2000507098A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0309898A2 (en) * | 1987-09-28 | 1989-04-05 | F. Hoffmann-La Roche Ag | Method for the induction of a cytotoxic T cell response |
Non-Patent Citations (13)
Title |
---|
ASLUND ET AL.: "A gene family encoding heterogeneous histone H1 proteins in Trypanosoma cruzi", MOL. BIOCHEM. PARASITOL., vol. 65, no. 2, June 1994 (1994-06-01), pages 317 - 330, XP000574776 * |
BELLI ET AL.: "Molecular karyotyping of the developmentally regulated Leishmania histone H1-like gene sw3", EXPERIENTIA, vol. 51, February 1995 (1995-02-01), pages A25, XP002007560 * |
BENDER ET AL.: "Sequence differences between histones of procyclic Trypanosoma brucei brucei and higher eukaryotes", PARASITOLOGY, vol. 105, no. pt1, August 1992 (1992-08-01), pages 97 - 104, XP000574758 * |
BURRI ET AL.: "Characterization of the histones of Trypanosoma brucei brucei bloodstream forms", ACTA TROP., vol. 58, no. 3-4, December 1994 (1994-12-01), pages 291 - 305, XP000574771 * |
BURRI ET AL.: "Partial amino acid sequence and functional aspects of histone H1 proteins in Trypanosoma brucei brucei", BIOL. CELL., vol. 83, no. 1, 1995, pages 23 - 31, XP000574770 * |
FASEL ET AL.: "Identification of a histone-like gene expressed in Leishmania major", MOL. BIOCHEM. PARASITOL., vol. 62, no. 2, 1994, pages 321 - 324, XP000574083 * |
FORMENTON ET AL.: "The genomic organization of the Leishmania histone-H1 like coding gene SW3", EXPERIENTIA, 28TH ANN. MEET. SWISS SOC. EXP. BIOL., vol. 52, 27 March 1996 (1996-03-27) - 29 March 1996 (1996-03-29), BASEL, pages a63, XP002033274 * |
NOLL ET AL.: "Stage specific & anti-sense transcription in the histone H1 locus of Leishmania major", EXPERIENTIA, 28TH ANN. MEET. SWISS SOC. EXP. BIOL., vol. 52, 27 March 1996 (1996-03-27) - 29 March 1996 (1996-03-29), BASEL, pages A63, XP002033273 * |
NOLL ET AL.: "Stage-specific and antisense transcription in the histone H1-like locus of Laishmania major", EXPERIENTIA, vol. 51, February 1995 (1995-02-01), pages a87, XP002007559 * |
SOTO ET AL.: "Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with Leishmania", IMMUNOL. LETT., vol. 48, no. 3, 1995, pages 209 - 214, XP000574777 * |
SOTO ET AL.: "Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A", EUR. J. BIOCHEM., vol. 205, 1992, pages 211 - 216, XP000574752 * |
SOTO ET AL.: "The Leishmania infantum histone H3 possesses an extremely divergent N-terminal domain", BIOCHIM. BIOPHYS. ACTA, vol. 1219, no. 2, 1994, pages 533 - 535, XP000574774 * |
TORO ET AL.: "H1 histone and histone variants in Trypanosoma cruzi", EXP. CELL. RES., vol. 174, no. 1, 1988, pages 16 - 24, XP000574781 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005044301A3 (en) * | 2003-11-05 | 2005-06-30 | C B F Leti S L Unipersonal | Use of specific histones for the treatment of parasitic diseases |
WO2011153602A2 (en) * | 2010-06-08 | 2011-12-15 | Universidade Federal de Viçosa | Recombinant e-ntpdases, use for producing a diagnostic kit for detecting antibodies in various types of leishmaniasis caused by species of the leishmania genus |
WO2011153602A3 (en) * | 2010-06-08 | 2013-02-07 | Universidade Federal de Viçosa | Recombinant e-ntpdases, use for producing a diagnostic kit for detecting antibodies in various types of leishmaniasis caused by species of the leishmania genus |
Also Published As
Publication number | Publication date |
---|---|
BR9708188A (en) | 2000-01-18 |
IL126145A0 (en) | 1999-05-09 |
CA2248684A1 (en) | 1997-09-18 |
TR199801776T2 (en) | 1998-12-21 |
JP2000507098A (en) | 2000-06-13 |
TNSN97045A1 (en) | 2005-03-15 |
MA25182A1 (en) | 2001-07-02 |
AU733720B2 (en) | 2001-05-24 |
NZ331889A (en) | 2000-12-22 |
EP0896623A1 (en) | 1999-02-17 |
AU2027297A (en) | 1997-10-01 |
CN1216584A (en) | 1999-05-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Duffy et al. | A novel malaria protein, Pfs28, and Pfs25 are genetically linked and synergistic as falciparum malaria transmission-blocking vaccines | |
EP0716697B1 (en) | DIFFERENTIALLY EXPRESSED $i(LEISHMANIA) GENES AND PROTEINS | |
US8343499B2 (en) | Anti-arthropod vector vaccines, methods of selecting and uses thereof | |
Culpepper et al. | Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen | |
WO1997011180A9 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
EP0172865B2 (en) | Dna sequences, recombinant dna and processes for producing antigens of plasmodium falciparum | |
Omara-Opyene et al. | Molecular cloning, characterization and overexpression of two distinct cysteine protease cDNAs from Leishmania donovani chagasi | |
US6613337B1 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
JPH0235085A (en) | Recombination useful as coccidiosis vaccine and natural b-group eimeria tenera immunogen | |
Binger et al. | Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus | |
US5965143A (en) | Immunity to trypanosomatids species | |
AU733720B2 (en) | Immunity to trypanosomatids species | |
US6375955B1 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
US6500437B1 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
US6365165B1 (en) | Leishmania antigens for use in the therapy and diagnosis of Leishmaniasis | |
EP0981624A2 (en) | $i(LEISHMANIA) ANTIGENS FOR USE IN THE THERAPY AND DIAGNOSIS OF LEISHMANIASIS | |
WO1998035045A9 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
US6607731B1 (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
EP1666489B1 (en) | DNA encoding an antigenic protein of eimeria apical membrane antigen 1 and use thereof | |
EP0215059A1 (en) | cDNA CODING FOR PLASMODIUM FALCIPARUM GLYCOPHORIN BINDING PROTEINS WEIGHING 130,000 DALTONS AND 155,000 DALTONS | |
CA2105538C (en) | Differentially expressed leishmania genes and proteins | |
PT1422238E (en) | Leishmania antigens for use in the therapy and diagnosis of leishmaniasis | |
MXPA98002284A (en) | Antigens of leishmania to be used in the therapy and diagnosis of leishmania | |
MXPA99007477A (en) | Leishmania | |
JP2006180872A (en) | Nucleic acid encoding antigenic protein of apical membrane antigen 1 of avian coccidium protozoan, and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 97193835.0 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF |
|
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: 1998/01776 Country of ref document: TR |
|
ENP | Entry into the national phase |
Ref document number: 1997EP 9701289 Country of ref document: KE Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997EP9701289 Country of ref document: KE |
|
ENP | Entry into the national phase |
Ref document number: 2248684 Country of ref document: CA Ref document number: 2248684 Country of ref document: CA Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 331889 Country of ref document: NZ Ref document number: PA/A/1998/007432 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997908233 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: 1997908233 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1997908233 Country of ref document: EP |