EP1194561A2 - Donor strand complemented pilin and adhesin broad-based vaccines - Google Patents
Donor strand complemented pilin and adhesin broad-based vaccinesInfo
- Publication number
- EP1194561A2 EP1194561A2 EP00947305A EP00947305A EP1194561A2 EP 1194561 A2 EP1194561 A2 EP 1194561A2 EP 00947305 A EP00947305 A EP 00947305A EP 00947305 A EP00947305 A EP 00947305A EP 1194561 A2 EP1194561 A2 EP 1194561A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- pilus
- strand
- fimh
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108010000916 Fimbriae Proteins Proteins 0.000 title claims abstract description 79
- 229960005486 vaccine Drugs 0.000 title claims abstract description 51
- 108010037896 heparin-binding hemagglutinin Proteins 0.000 title claims description 64
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 135
- 229920001184 polypeptide Polymers 0.000 claims abstract description 134
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 131
- 238000000034 method Methods 0.000 claims abstract description 64
- 230000000295 complement effect Effects 0.000 claims abstract description 37
- 230000001580 bacterial effect Effects 0.000 claims abstract description 33
- 241000588724 Escherichia coli Species 0.000 claims abstract description 31
- 201000010099 disease Diseases 0.000 claims abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 128
- 108010006519 Molecular Chaperones Proteins 0.000 claims description 115
- 102000004169 proteins and genes Human genes 0.000 claims description 105
- 230000002163 immunogen Effects 0.000 claims description 42
- 101100043709 Mus musculus Stradb gene Proteins 0.000 claims description 39
- 101100396916 Drosophila funebris PapD gene Proteins 0.000 claims description 35
- 239000013598 vector Substances 0.000 claims description 29
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 22
- 241000894006 Bacteria Species 0.000 claims description 20
- 108091033319 polynucleotide Proteins 0.000 claims description 15
- 102000040430 polynucleotide Human genes 0.000 claims description 15
- 239000002157 polynucleotide Substances 0.000 claims description 15
- 241000588921 Enterobacteriaceae Species 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 7
- 108091026890 Coding region Proteins 0.000 claims description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 6
- 208000019206 urinary tract infection Diseases 0.000 claims description 5
- 241000124008 Mammalia Species 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 208000026723 Urinary tract disease Diseases 0.000 claims 1
- 208000012931 Urologic disease Diseases 0.000 claims 1
- 208000014001 urinary system disease Diseases 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 6
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 4
- 241000305071 Enterobacterales Species 0.000 abstract 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 99
- 210000004027 cell Anatomy 0.000 description 53
- 235000001014 amino acid Nutrition 0.000 description 31
- 102000005431 Molecular Chaperones Human genes 0.000 description 30
- 150000001413 amino acids Chemical class 0.000 description 29
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 26
- 230000027455 binding Effects 0.000 description 26
- 239000012634 fragment Substances 0.000 description 19
- 210000001322 periplasm Anatomy 0.000 description 19
- 230000003993 interaction Effects 0.000 description 17
- 230000000875 corresponding effect Effects 0.000 description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- 210000004899 c-terminal region Anatomy 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 14
- 230000002209 hydrophobic effect Effects 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 14
- 239000004202 carbamide Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 12
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 11
- 230000002441 reversible effect Effects 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 239000002671 adjuvant Substances 0.000 description 8
- 239000013078 crystal Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 230000008436 biogenesis Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 6
- 239000000853 adhesive Substances 0.000 description 6
- 230000001070 adhesive effect Effects 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 239000003623 enhancer Substances 0.000 description 6
- 239000000835 fiber Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 241001442654 Percnon planissimum Species 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 201000003146 cystitis Diseases 0.000 description 5
- 125000001165 hydrophobic group Chemical group 0.000 description 5
- 238000010348 incorporation Methods 0.000 description 5
- 210000003463 organelle Anatomy 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 208000035143 Bacterial infection Diseases 0.000 description 4
- -1 FimC Proteins 0.000 description 4
- 108090001090 Lectins Proteins 0.000 description 4
- 102000004856 Lectins Human genes 0.000 description 4
- 206010037596 Pyelonephritis Diseases 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 101150015947 fimH gene Proteins 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000002523 lectin Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000002028 premature Effects 0.000 description 4
- 235000004252 protein component Nutrition 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 3
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 108010038196 saccharide-binding proteins Proteins 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 101150084750 1 gene Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000938885 Escherichia coli NU14 Species 0.000 description 2
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 2
- 101100109397 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) arg-8 gene Proteins 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 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 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229960000723 ampicillin Drugs 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
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000007248 cellular mechanism Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000002983 circular dichroism Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 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 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000006384 oligomerization reaction Methods 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 101100238293 Arabidopsis thaliana MOR1 gene Proteins 0.000 description 1
- 206010056396 Asymptomatic bacteriuria Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101100512078 Caenorhabditis elegans lys-1 gene Proteins 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101100110385 Escherichia coli (strain K12) atpB gene Proteins 0.000 description 1
- 101100218161 Escherichia coli (strain K12) atpG gene Proteins 0.000 description 1
- 101100242758 Escherichia coli papD gene Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000009112 Mannose-Binding Lectin Human genes 0.000 description 1
- 108010087870 Mannose-Binding Lectin Proteins 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 101100082836 Pediococcus acidilactici pedC gene Proteins 0.000 description 1
- 101100029151 Pediococcus acidilactici pedD gene Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 206010037597 Pyelonephritis acute Diseases 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 201000005661 acute cystitis Diseases 0.000 description 1
- 201000001555 acute pyelonephritis Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- GZCGUPFRVQAUEE-KVTDHHQDSA-N aldehydo-D-mannose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-KVTDHHQDSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000033847 bacterial urinary tract infection Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 210000003443 bladder cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 102000022324 chaperone binding proteins Human genes 0.000 description 1
- 108091012160 chaperone binding proteins Proteins 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000002447 crystallographic data Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 101150056310 gem1 gene Proteins 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 238000003875 gradient-accelerated spectroscopy Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 229920000140 heteropolymer Polymers 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000000302 molecular modelling Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 101150061305 papC gene Proteins 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229940021993 prophylactic vaccine Drugs 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000030788 protein refolding Effects 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to immunogenic polypeptides and complexes useful in preventing and treating bacterial diseases and to methods of preparing such polypeptides and complexes.
- Newly formed protein chains can quickly fold in vitro to form the native conformation, a process often requiring no outside assistance and wherein the steric information for the three dimensional structure of the protein resides in the amino acid sequence.
- ATP-dependent proteins called “chaperones,” may aid folding of some proteins in vivo.
- chaperones are not requiring ATP for their functioning. These include the Pap-D-like periplasmic chaperones found in bacteria. As used herein, and unless expressly stated otherwise, the term “chaperone” means exclusively this latter class of periplasmic chaperones having the further distinguishing characteristics as described below.
- this latter class of chaperones is responsible for mediating the synthesis of large scale oligomeric structures, for example, the construction of pili, the adhesive fibers expressed in most bacteria of the Enterobacteriaceae family (e.g., Escherichia coli).
- Pili are heteropolymeric structures that are composed of several different structural proteins required for pilus assembly. Pili, also called fimbriae or fibrillae, facilitate the adhesive qualities of bacteria that often lead to colonization and infection of various tissues of the host animal, especially on mucosal surfaces. Such adhesion is facilitated by the presence in the pilus of a protein called an "adhesin,” of which FimH is an example.
- Type 1 pili-carrying bacteria recognize and bind to D-mannose in glycolipids and glycoproteins of bladder epithelial cells. Proteins forming the pili have been considered good candidates for vaccines.
- P pili are adhesive organelles encoded by eleven genes in the pap (pilus associated with pyelonephritis) gene cluster found on the chromosome of uropathogenic strains of E. coli. The biogenesis of P pili and Type 1 pili occurs via the highly conserved chaperone/usher pathway. (Thanassi et al, Curr. Op. Microbiol. 1 ,223 (1998); Hung et al, EMBO J. 15, 3792 (1994)).
- Type 1 pili are composite fibers consisting of a short thin tip fibrillum joined to a thicker, rigid pilus rod.
- the pilus fiber is an ordered array of homologous pilins (FimA, FimF, FimH, and FimG) with the FimH adhesin at its tip. FimH and FimG have been purified as a complex and comprise the bulk of the tip fibrillum, which may also contain FimF.
- the rod is comprised of FimA monomers arranged in a right-handed helical cylinder (Brinton (1965)) Genes important in type 1 pilus biogenesis (fimA-fimH) are organized into the fim operon (Orndorff and Falkow, J. Bacteriol. 159, 736 (1 984)) More specifically, FimH mediates binding to mannose-oligosaccharides present on mucosal surfaces. Thus, FimH mediates adherence to mannosylated receptors on the bladder epithelium and is critical to the ability of uropathogenic Escherichia coli to cause cystitis. (See: Mulvery et al.
- PapD-like superfamily of periplasmic chaperones directs the assembly of over 30 diverse adhesive surface organelles that mediate the attachment of many different pathogenic bacteria to host tissues, a critical early step in the development of disease.
- PapD the prototypical chaperone, is necessary for the assembly of P pili (Lindberg et al, J. Bacterioi. 171 , 6052 (1989)) whereas its homologue, called FimC, directs the assembly of type 1 pili (Jones et al, Proc. Natl. Acad. Sci. USA 90, 8397 (1993)).
- E. coli is the most common pathogen of the urinary tract, accounting for greater than 85% of cases of asymptomatic bacteriuria, acute cystitis and acute pyelonephritis, as well as greater than 60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis infections. Because of the high incidence, continued persistence, and significant expense associated with E. coli urinary tract infections, there is a need for a prophylactic vaccine to reduce susceptibility to this disease.
- type 1 pili are believed to be important in initiating colonization of the bladder and inducing cystitis, whereas P pili are thought to play a role in ascending infections and the ensuing pyelonephritis.
- pili mediate microbial attachment to the surfaces of cells, often the essential first step in the development of a disease, by binding to receptors present in host tissues.
- pilus-based vaccines have been the fact that the major immunodominant components of pilus fibers are often highly variable antigenically and therefore afford protection against only a limited number of bacterial strains.
- pilus associated adhesins such as FimH
- FimH is relatively conserved proteins among different species and strains of bacteria.
- FimH is relatively conserved not only among uropathogenic strains of E. coli, but also among a wide range of gram-negative bacteria.
- many members of the family Enterobacteriaceae produce FimH and vaccines incorporating the FimH antigen should exhibit a broad spectrum of protection.
- adhesin based vaccines of any kind has been the fact that adhesins are often only a minor component of the pilus, cannot be produced in large quantities, and therefore will tend not to elicit a particularly strong immunogenic effect.
- recombinant technology has succeeded in producing adhesin proteins in pure form, these are often rapidly proteolytically degraded when the corresponding chaperone is absent.
- adhesins are readily stabilized by the presence of periplasmic chaperone molecules (the latter also being important in proper synthesis of adhesins).
- Gram negative bacteria of which Escherichia coli is an example, have a characteristic cell surface arrangement. They possess an inner plasma membrane, surrounded by a peptidoglycan cell wall, which in turn is surrounded by an outer membrane, the latter being highly permeable to many substances. Between the cell wall and the outer membrane lies the periplasmic space. Proteins destined for secretion or assembly across the outer membrane often must fold within the periplasmic space prior to their secretion and/or assembly. Chaperones are often to be found within this periplasmic space.
- pilus, pili, fimbrium, fimbriae, fibrillum and fibrilla are be used interchangeably, with incidental use of the singular or plural form of any of these terms in no way limiting the breadth of the disclosed invention.
- a "periplasmic chaperone” is defined herein as a protein localized in the periplasm of bacteria that is capable of forming complexes with a variety of proteins, especially pilus-proteins, including adhesins, especially FimH (where the corresponding chaperone is FimC) via recognition of a common binding epitope (or epitopes).
- Such chaperones are characterized by their similarity in properties to PapD, especially by their possession of an immunoglobulin-like fold for binding to pilus-proteins, such as adhesins. Chaperones serve as templates upon which proteins exported from the bacterial cell into the periplasm fold into their native conformations, especially where such proteins are intended to form oligomeric structures such as pili.
- association of the chaperone-binding protein with the chaperone also serves to protect the binding proteins from degradation by proteases localized within the periplasm, increases their solubility in aqueous solution, and leads to their sequentially correct incorporation into an assembling pilus.
- PapD maintains target structures in native-like conformations [Lecher et al, (1989) EMBO J. 8, 2703-2709; Kuehn et al,
- Such periplasmic chaperones have an effector function, specifically targeting the subunits to outer membrane assembly sites for their incorporation into pili and are characterized in part by the presence of an immunoglobulin-like fold.
- Two of the genes in the pap operon - papD and papC - encode the chaperone and usher, respectively.
- Six genes encode structural pilus subunits, PapA, PapH, PapK, PapE, PapF, and PapG, which assemble into a heteropolymeric surface fiber with an adhesive tip (PapG). (Hultgren et al. Cell 73,887 (1993).
- the ability of PapG to bind to galabiose receptors in the human kidney is a critical event in the pathogenesis of pyelonephritis.
- the pilus consists of two major sub-assemblies, a thick, rigid rod made up of repeating PapA subunits arranged in a right-handed helical cylinder and a thin, flexible tip fiber (the tip fibrillum) extending from the distal end of the rod and composed primarily of repeating PapE subunits arranged in an open-helical configuration.
- PapK is thought to link the pilus rod to the base of the tip fibrillum and regulates its length: its incorporation terminates its growth and nucleates the formation of the pilus rod.
- PapF is thought to join the PapG adhesin to the distal end of the flexible tip fibrillum.
- FimC uses its immunoglobulin-like domains to recognize and bind to pilus subunit proteins, such as the adhesin FimH.
- Type 1 pilus biogenesis proceeds via a highly conserved pathway that is involved in the assembly of over 30 adhesive organelles assembled by the adhesin-usher pathway in gram-negative bacteria.
- the assembly machinery is comprised of two specialized classes of proteins, a periplasmic chaperone and an outer membrane usher. [Thanassi et al, Curr. Opin. Microbiol.
- the periplasmic chaperone FimC forms periplasmic complexes with each of the pilus subunits prior to their incorporation into the pilus.
- chaperones recognize a highly conserved motif present in the C-terminal portions of all subunits assembled by all PapD-like chaperones. [Hung et al., EMBO J.
- FimC The chaperone activity of FimC has been demonstrated and FimC has been shown to bind FimH, the adhesin of type 1 pili, to form periplasmic preassembly complexes.
- a FimH-FimC complex has been isolated using mannose-Sepharose chromatography of periplasm and then eluting with D- mannose.
- FimH is folded in the FimH-FimC complex in such a way that the mannose binding domain is in a native state and accessible for substrate binding.
- amino acid sequence of FimC is known, [see Jones et al (1993)]
- PapD the prototypical periplasmic chaperone
- periplasmic chaperone The crystal structure of PapD, the prototypical periplasmic chaperone, has been solved (Holmgren and Branden, Nature 342,248 (1989) and refined to 2.0 A resolution, revealing a molecule with two immunoglobulin-like domains oriented in an L shape to form a cleft at their surface. All 30 + members of the periplasmic chaperone superfamily have a conserved hydrophobic core that maintains the overall features of the two domains.
- PapD binds to and caps interactive surfaces on pilus subunits and prevents their premature aggregation in the periplasm.
- the G1 ⁇ -strand of PapD forms a ⁇ -zipper interaction with the highly conserved COOH-terminal motif of pilus subunits.
- the COOH- terminal motif also comprises at least part of a primary surface for subunit- subunit assembly interactions, indicating that the direct capping of a primary assembly surface is part of the molecular basis by which periplasmic chaperones prevent the premature oligomerization of pilus subunits.
- the ⁇ -zipper interaction has been proposed to facilitate the folding of the subunit into a native-like conformation via a template-mediated mechanism. (Soto et al, EMBO J. 17: 6155 (1998).
- adhesins While the utility of adhesins as vaccines has been demonstrated, large scale production of adhesins and other pilus-derived proteins has been complicated by the requirement of a chaperone that must be co-expressed with the adhesin in order for it to properly fold and result in a stable structure. It would therefore be highly advantageous if adhesins could be produced in pure form without the need of co-expressing the chaperone, and without the need for the chaperone, or any other protein, at all, thereby permitting large scale production of pure adhesins, or any other pilus subunits, for use, inter alia, as vaccines.
- the present invention relates to immunogenic complexes and polypeptides, comprising a pilus protein component or portion, and a donor strand component, or portion, wherein the pilus protein and donor strand may or may not be covalently bonded together.
- adhesins such as FimH or a pilin such as PapK
- a portion of another protein acting as a donor complement especially where the latter is a chaperone, such as FimC or PapD, or a portion of another subunit, such as FimG or FimF.
- the present invention provides a polypeptide comprising a pilin protein, such as FimH, attached to a donor strand, such as a strand composed of the first 13 residues of FimG, separated by a short intervening sequence, such as a tetrapeptide, to form a single chain immunogenic polypeptide.
- a pilin protein such as FimH
- a donor strand such as a strand composed of the first 13 residues of FimG
- a short intervening sequence such as a tetrapeptide
- Other specific embodiments include a similar structure employing PapK with a sequence of PapD attached to the C-terminus of PapK via a short amino acid linking sequence to form a single chain polypeptides.
- the invention also encompasses such structures wherein the pilin and donor strand segments are non-covalently linked to each other so long as the correct conformation of the pilin is maintained.
- polypeptides, or complexes thereof, of the present invention comprise a pilus subunit and a donor strand derived from the N-terminal extension of another pilus subunit.
- Such methodology will thereby facilitate the production of such pilus-based (pilin-based and adhesin- based) vaccines as a single polypeptide chain, thus reducing time, cost and complexity of production and facilitate large scale vaccine production.
- Figure 1 shows a comparison of the amino acid sequences of the various Fim proteins that compose the pili of bacteria such as E. coli.
- the sequences of FimH, FimA, FimF and FimG are all aligned to show corresponding sequences and domains.
- the end of the mannose-binding lectin domain and the start of the pilin domain in FimH are indicated by vertical arrowheads above the sequences.
- the type 1 pilin subunits (FimA, FimF and FimG) are aligned with the pilin domain of FimH using clustal W [see: Thompson et al, Nucleic Acids Res. 22, 4673 (1994)] and manually adjusted to minimize gaps in secondary structure elements. Gaps in the alignment are indicated by dots.
- Sequence numbering for FimH starts at position 22 in the pre-protein.
- Pilus subunits including FimH are expressed in the cytoplasm as pre-proteins with an amino terminal signal sequence that is cleaved during transport across the inner membrane.
- the first residue in FimH that is visible in our maps corresponds to Phe 22 in the gene-derived sequence, which is the expected start of the FimH chain.
- FimH residues will be denoted with an "H” and FimC residues with a "C” after the residue number. Residues involved in chaperone binding are indicated by an open circle above the residue.
- Residues in the carbohydrate binding pocket are boxed, with a large box marking the NH 2 -terminal extensions in the pilin subunits.
- the conserved ⁇ -zipper motif found in all pilin subunits corresponds to the F ⁇ -strand. Limits and nomenclature for secondary structure elements are shown below the sequence.
- Figure 2 shows the sequence of FimC. Residues involved in subunit binding are indicated by an open circle above the residue. Residues that are identical or conserved in all periplasmic chaperones are set against a darker background. Limits and nomenclature for secondary structure elements are shown below the residues.
- Figure 3 shows ⁇ -sheet topology diagrams of the mannose binding domain (Fig. 3A) and the pilin domain (Fig. 3B) of FimH.
- the F strand is at the C-terminal end of the pilin domain and thus would appear at the C-terminus of the FimH molecule.
- Each strand depicted is readily correlated with the corresponding sequences of Figure 1 wherein the strand designation appears below the arrow indicating the residues making up that strand.
- Figure 4 shows a molscript (P.J. Kraulis, J. Appl. Cryst., 24, 946 (1991 ) ribbon diagram of the FimC-FimH complex, with FimH vertically and FimC oriented horizontally, as depicted by the arrows (with the G1 strand indicated).
- the upper right shows a ball-and-stick representation of the CJHEGA molecule bound to the lectin domain of FimH and indicates the position of the carbohydrate-binding site at the tip of the domain.
- Figure 5 shows a sequence alignment of P-pilus subunits (PapA, PapK, PapE, and PapF).
- the secondary structural elements of PapK are indicated above the aligned sequences, with ⁇ -strands and helices (including both ⁇ and 3 10 ) indicated.
- Residue numbers of PapK are indicated above the PapK sequence.
- Residues involved in contact with domains 1 and 2 of PapD are boxed.
- Residues strictly conserved among pilins are shown as the vertical quartets of G, C and Y residues (using standard one letter code) under residue numbers 7, 14, 54, 144 and 1 56.
- FIG. 6 shows the sequence and secondary structure definition of
- Residue numbers are indicated above the sequence, while secondary structural elements are indicated below it. Residues that contact PapK are boxed.
- Figure 7 shows the topology of PapK with the nomenclature for secondary structures as described in Jones, Curr. Op. Struct. Biol. 3, 846 (1993).
- the ⁇ -strands are indicated as arrows, while helices are shown as cylinders.
- the inserting ⁇ -strand of PapD is indicated in the figure as G1 .
- Figure 8 shows the structure of the PapD-PapK complex and definition of secondary structure notation.
- the molecular surface of PapK was calculated and displayed using GRASP [Nichols et al, Proteins: Struct. F ⁇ nct. Genet. 1 1 , 281 (1991 )]
- the structure of PapD is shown as a ribbon.
- the insertion of the G1 ⁇ -strand of the PapD into a groove on the surface of PapK is shown.
- Figure 9 shows the general arrangement for one embodiment of the present invention wherein a FimH has been linked to residues 1 -13 of FimG using a tetrapeptide (DNKQ - using standard one letter amino acid code) linker to form a donor strand complemented FimH (dscFimH).
- DNKQ tetrapeptide
- dscFimH donor strand complemented FimH
- Figure 10 shows the results of an experiment in which C3H/HeJ mice were immunized with pilus-protein plus CFA/IFA.
- IM intramuscularly
- cfu colony forming units
- Figure 1 1 shows the results of an experiment in which C3H/HeJ mice were immunized with pilus-proteins plus MF59 with 4 and 16 week boosts IM (intramuscularly) with the indicated immunogens with endpoint titers shown at the left.
- Figure 12 shows the results of an experiment in which C3H/HeJ mice were immunized with the indicated pilus-protein plus MF 59.
- IM intramuscularly
- 7x10 7 cfu colony forming units
- the present invention is directed to polypeptides, especially immunogenic polypeptides, formed from a pilus-protein, such as, for example, an adhesin, and either a chaperone fragment or a pilin fragment, as well as dimeric complexes thereof, wherein the components of said complexes may or may not be covalently bound to each other.
- a pilus-protein such as, for example, an adhesin, and either a chaperone fragment or a pilin fragment
- dimeric complexes thereof such as, for example, an adhesin, and either a chaperone fragment or a pilin fragment, as well as dimeric complexes thereof, wherein the components of said complexes may or may not be covalently bound to each other.
- Such polypeptides, and complexes thereof have the advantage of being synthesized in pure form and in a fully functional state without the need of accessory proteins, although the latter may be used to form specific complexes within the invention.
- the term "pilus-protein” means any protein or polypeptide present in a pilus, especially a type 1 pilus or a P pilus. This term encompasses all subunits of a pilus but excludes chaperones because, while they are integral in formation of a pilus, they are not incorporated into this organelle and thus are not subunits of it.
- the term “adhesin” means a protein, especially a subunit of a pilus, with specific receptor binding properties, for example, FimH that has a lectin-binding domain for specifically binding mannose-bearing sites on cell surfaces, especially the surfaces of mucosal cells, most especially bladder cell of a mammal.
- the chaperone binds to and forms stable complexes with individual pilus subunits.
- the chaperones consist of two immunoglobulin-like (Ig-like) domains oriented toward each other to form L-shaped molecules. [Holmgren and Branden, Nature 342, 248 (1 989); Pellucchia et al, Nature Struct. Biol. 5, 885 (1998)]
- the FimH adhesin has both a pilin domain and a receptor-binding domain.
- the PapK pilin and the pilin domain of FimH have Ig-like folds but lack the seventh C-terminal ⁇ -strand (strand G of Figure 3) present in canonical Ig-folds.
- the absence of this strand produces a deep groove along the surface of the pilin domain and exposes the hydrophobic core, thereby accounting for the instability of pilins when expressed without the chaperone (see examples hereinbelow).
- the G1 strand of the chaperone completes an atypical Ig fold of the subunit by occupying the groove and runnig parallel to the subunit C-terminal F strand.
- this "donor strand complementation" interaction simultaneously stabilizes pilus subunits and caps their interactive surfaces, preventing their premature oligomerization in the periplasm.
- the highly conserved N-terminal extension of one subunit displaces the chaperone G1 strand from a neighboring subunit by a process referred to herein as "donor strand exchange.”
- the N-terminal strand then inserts anti-parallel to the F-strand of the neighboring subunit to afford a mature pilus comprising an array of perfectly canonical Ig domains, each of which contributes a strand to the fold of its neighboring subunit.
- the contribution of a chaperone, such as FimC or PapD, to the overall structure of a pilin, such as in the FimC-FimH complex, or in the PapD-PapK complex was determined by solving the structure of such complexes by X-ray diffraction [see: Choudhury et al, X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic E.
- the crystal structure revealed the presence of a pocket in the otherwise flat surface of the lectin domain. This pocket is large enough to accommodate a single mannose unit and is located at the tip of the domain, distal to the connection with the pilin domain.
- the bottom of the pocket is defined by the N-terminus of the FimH molecule and is lined with typical carbohydrate binding side chains from Asn, Gin, and Asp residues in 3 loop regions.
- FimH was folded into 2 domains of the all-beta class. These domains are aligned end to end so that the FimH molecule spans a length of over 100 A.
- the amino terminal domain of the adhesin, FimH contains the mannose binding domain, used to bind to the surface of a mucosal cell, and the C-terminal end forms the pilin domain, which is used to anchor the adhesin to the pilus.
- the NH 2 -terminal mannose binding domain comprises residues 1 H-156H while the C-terminal pilin domain, which is used to anchor the adhesin to the pilus, comprises residues 160H-279H (see Figure
- a short extended linker (residues 1 57H-1 59H) connects the two domains.
- FimC has 2 immunoglobulin-like domains oriented at about 90° to each other and with a deep cleft between the two domains.
- the pilin domain of FimH binds in the cleft of the chaperone but, with the seventh fold missing (see Figure 3), the hydrophobic core is exposed and the terminal portions of the pilin domain lie next to each other instead of at opposite ends of the pilin domain.
- the pilin domain of FimH has the same topology as the NH 2 - terminal domain of a number of chaperones but with the critical difference that the seventh strand of the fold is missing.
- a similar situation would occur in other pilus-proteins, such as FimG and FimF.
- the G1 strand (i.e., the seventh strand) of the chaperone is used to complement the pilin domain by being inserted between the second half of the A strand and the F strand of the pilin.
- the C-terminal, or F, strand of FimH (Figs. 1 and 3) forms a parallel beta pleated strand interaction with the G1 ⁇ -strand of FimC (Fig.
- the mechanism by which this process occurs, wherein the chaperone binds to the adhesin, here FimH, to direct proper folding of the adhesin is referred to herein as "donor strand complementation" and the strand that complements the fold or structure of the adhesin , such as the G1 ⁇ -strand of the chaperone FimC, is referred to as the "donor complement” segment or the "donor strand.” More specifically, the G1 ⁇ -zipper strand of periplasmic chaperones contains a conserved motif of solvent exposed hydrophobic residues at positions 103, 105, and 107 in FimC (see Figure 2). In the FimC-
- the pilin domain is an incomplete protein domain to which the chaperone donates its G1 strand so as to complete it.
- pilus-proteins, or pilins, as a class are missing necessary steric information needed for stability as well as for folding into the native 3 dimensional conformation and so require the presence of the chaperone, FimC in the case of FimH, in order to provide the necessary complementary, and compensating, structure.
- the G1 strand of the chaperone simply inserts itself into the groove of FimH (the F and A2 line groove) so that engineering the same sequence at the C-terminal end of the pilin domain of FimH results in a FimH having the same structure (i.e., same overall shape) as if the chaperone were present.
- the N-terminal sequence of FimG may be employed since it is better for forming a completely canonical fold.
- a donor strand can be engineered onto PapK by modeling the appropriate strand from the chaperone PapD or a strand from another suitable pilus subunit structure.
- the sequences missing from the structure of the putative-pilus protein immunogen are supplemented with appropriate donor strand sequences to take the place of the missing chaperone sequence.
- appropriate donor strand sequences are provided.
- rules providing guidance in facilitating the determination of appropriate sequences to use for donor complementing a wide variety of bacterial pilus proteins, especially where these are to be used in the development of compositions for preventing and/or treating bacterial diseases.
- FimA, FimF, and FimG all have a highly conserved extension of about 10-20 amino acids at the N-terminus compared to the FimH pilin domain.
- the PapK amino terminal extension is disordered and thus the first ⁇ -strand begins after the first cysteine, just as in the FimH pilin domain.
- an amino terminal extension of one subunit is used as a donor strand to provide the missing seventh strand in the neighboring subunit. Use of such neighboring subunits produces a complete canonical fold whereas the chaperone itself would complete an atypical fold.
- crystallographic procedures likewise show that PapK has the same overall variable-region Ig-like fold as the amino terminal domain of PapD, with 2 ⁇ -sheets coming together in a ⁇ -sandwich.
- the Ig fold of PapK is incomplete in that it lacks the COOH-terminal seventh strand, G, which in canonical Ig folds forms an antiparallel B-sheet interaction with strand F and contributes to the hydrophobic core of the protein.
- the first 8 amino acid residues of PapK are disordered and the Ig fold of
- PapK begins with a short ⁇ -strand, A1 (see Figure 7), that makes typical anti- parallel hydrogen bonds with the COOH-terminal residues of strand B.
- This short ⁇ -sheet arrangement is interrupted by the insertion of the 3 10 helical turn that results in strand A switching sides in the ⁇ -sandwich in order to make antiparallel B-strand interactions with the G1 strand of the chaperone.
- a and B are connected by a short ⁇ -helix and it is strand B that forms the edge of one of the two ⁇ -sheets in the ⁇ -sandwich running antiparallel to strand E.
- strand B Following strand B, the structure crosses over to the other side of the ⁇ - sandwich through a short 310 helix to form strand C1 , which then runs antiparallel to strand F.
- the total buried surface area in the PapD-PapK complex is 3434A 2 and there are two distinct sites on PapK that interact with two corresponding sites on PapD.
- Site K1 of PapK (see Figure 7) interacts with domain 1 of PapD (site D1 of Figure 6) and K1 contains a deep groove that runs the length of the subunit. The edges of this groove consist of strands A and F and its base is formed by the hydrophobic core of PapK. This groove is the result of the missing G ⁇ -strand in the Ig fold of PapK.
- Residues 101 to 1 12 (in site D1 ) of the G1 ⁇ -strand in the Ig fold of PapD insert into the K1 groove and make a ⁇ -zipper interaction with strand F of PapK ( Figure 7) on one side of the groove.
- Residues 101 to 105 also make a ⁇ -zipper interaction with strand A2 on the other side of the groove. Insertion of the G1 ⁇ -strand also results in the formation of a continuous 5-stranded ⁇ -sheet which includes strands C1 , F1 , and G1 of PapD and the F and C1 strands of PapK.
- Alternating hydrophobic residues in the G1 ⁇ -strand of PapD interact with the hydrophobic base of the groove.
- donor strand complementation by the G1 ⁇ -strand of PapD shields the hydrophobic core of the pilin from exposure to the aqueous milieu of the periplasm.
- site K2 of PapK interacts with a site on the COOH-terminal domain (domain 2) of PapD.
- domain 2 of PapD strand F of PapK forms one side of the groove into which the G1 ⁇ - strand of the chaperone inserts and is likely to assume the same structural role in pilins.
- alternating hydrophobic side chains in the N-terminal extension can replace the hydrophobic side chains donated to the pilin core by the G1 strand of the chaperone, via the donor strand exchange mechanism of the present invention.
- the net result is that every subunit completes the immunoglobulin-like fold of its neighboring subunit.
- the NH 2 -terminal portion of pilins corresponding to the disordered NH 2 - terminus of PapK, forms an assembly surface on the pilin.
- the 8 NH 2 -terminal residues are disordered in the Pap-D-PapK complex and protrude away from the main body of the structure, where they are free to interact with the groove of the preceding subunit located at the usher.
- the NH 2 -terminus of an incoming subunit inserts into the groove of the preceding subunit, displacing the G1 ⁇ -strand of the chaperone (which process is facilitated by the usher).
- the present invention is thus directed to pilus-proteins, including adhesins, such as FimH, or non-adhesins such as FimG and FimA, in which the missing fragment normally supplied by a chaperone, such as FimC, or a pilin via the donor strand exchange mechanism during pilus assembly, has been added to its amino acid sequence derived from that chaperone or pilin.
- adhesins such as FimH
- non-adhesins such as FimG and FimA
- this result is accomplished by engineering a G1 ⁇ -strand of FimC (SEQ ID NO: 3 and 4), or an N-terminal extension strand of
- FimG SEQ ID NO: 5 and 6
- FimF SEQ ID NO: 7 and 8
- other similar sequence of related pilins and in either a forward or reverse (i.e., inverted) sequence orientation onto the COOH-terminus of a pilus-protein, such as an adhesin, thereby removing the requirement of a chaperone and/or donor strand complementation.
- a pilus-protein such as an adhesin
- the addition of the C-terminal ⁇ -strand to the adhesin completes the immunoglobulin fold and thus removes the need for any type of accessory protein, such as a chaperone.
- the chaperone not only contributes to the stability of the pilin but also prevents other pilins in the periplasm from binding to the groove of the chaperone-bound subunit.
- PapG is the adhesin.
- the donor sequence can be engineered in a forward or reverse (i.e., inverted) amino acid sequence orientation with respect to the pilus-protein sequence being "complemented.”
- this donor strand must fold around and loop back onto the pilus strand in an anti-parallel orientation, such donor complemented structures can be engineered with the donor strand in a forward or reverse orientation.
- Whether enginered in a forward or reverse direction depends on the source of the sequence used to prepare the strand, for example, whether the donor strand is a match from the G1 strand of the corresponding chaperone or is modeled on the N-terminal strand of a neighboring subunit, such as where the N-terminal sequence of FimG is used to complement FimH. If engineered in a forward orientation, the N-terminal residue of the donor strand is bonded to the C-terminal residue of the pilus protein (e.g., FimH), while in the reverse orientation, the C-terminal residue of the donated strand is bonded to the C-terminal residue of the adhesin.
- the C-terminal residue of the donor strand is bonded to the C-terminal residue of the pilus protein (e.g., FimH)
- the C-terminal residue of the donated strand is bonded to the C-terminal residue of the adhesin.
- linker sequence (perhaps as short as 4 residues or a structure of similar length and conformation) between the pilus-protein sequence and the donor strand sequence in order to afford sufficient flexibility for appropriate conformational folding necessary to attain the proper 3 dimensional structure (and such was done in carrying out embodiments of the present invention).
- linker sequence perhaps as short as 4 residues or a structure of similar length and conformation
- Such engineering is readily accomplished either by direct chemical synthesis of the desired adhesin or by using a polynucleotide sequence in which the order of the codons encoding the donated strand sequence have the desired orientation.
- Such donated strand sequence may, of course, be derived from the G 1 strand of FimC or PapD, or the N-terminal extension strand of a pilus subunit, such as FimG. It should be mentioned that because the chaperone is not incorporated into the assembled pilus, such chaperones are not "pilus-proteins.”
- the ability to produce a fully formed and functional pilin protein without the need to provide for the chaperone, either by addition or as a co-expressed protein, is highly advantageous in that it permits large scale production of a single polypeptide chain, thereby reducing the time, cost and complexity of such production.
- the present invention is directed to modified pilus-polypeptides, i.e., polypeptides or proteins derived from pili, especially type 1 pili and P pili, and forming subunits thereof, including adhesins such as FimH, and pilins such as PapK, comprising a pilus-protein portion and a donor complement portion all within the same amino acid sequence.
- modified pilus-polypeptides i.e., polypeptides or proteins derived from pili, especially type 1 pili and P pili, and forming subunits thereof, including adhesins such as FimH, and pilins such as PapK, comprising a pilus-protein portion and a donor complement portion all within the same amino acid sequence.
- a donor complement portion and an adhesin or pilin portion is merely for convenience and in fact only a single polypeptide is contemplated herein, although such donor complement fragment may certainly be chemically attached to the adhesin portion by some chemical linkage other than a conventional peptide bond.
- modified proteins include FimH and PapK following donor strand complementation.
- donor strand complemented FimH would be denoted dscFimH (where dsc stands for "donor strand complemented").
- the donor strand need not be covalently attached at all but can merely be bonded non-covalently as part of a complex the overall shape of which is the native shape of the adhesin or pilin protein, such as FimH.
- the problem of producing pure pilins or adhesins, free of a chaperone complex, and available for use as a vaccine, is solved by the present invention by providing any desired pilus protein (a term that does not include chaperones) from any bacterial structure assembled by the chaperone-usher pathway (see Hung and Hultgren, J. Struct. Biol. 124, 201 - 220 (1998) for a review of this pathway) whose fold is complemented by a peptide, or even a non-peptide moiety, to form one structure or complex that is readily prepared in pure form and without the need for accessory proteins, such as chaperones.
- any desired pilus protein a term that does not include chaperones
- the present invention relates to an immunogenic complex comprising a pilus protein component and a donor strand component, wherein said pilus protein component may or may not be covalently bound to the donor strand component.
- the pilus and donor components may be held together by non-covalent interactions, including, but not limited to, electrostatic interactions, hydrophobic interactions, including van der Waals forces and general entropic forces, London forces, and the like.
- Such pilus protein component may or may not be an adhesin. Where it is an adhesin, especially preferred adhesins are FimH and PapG.
- pilus proteins are those selected from the group consisting of FimF and FimA (both from type 1 pili) and PapK, PapA, PapF, PapE, and PapH (all from type P pili).
- Donor strand components comprise an amino acid, or other polymeric strand, that is able to substitute for the missing strand of the pilus protein. Such substitution is determined by resort to the rules disclosed herein for utilizing the disclosed strands from PapD, FimC and FimG to replace missing strands of PapK and
- the present invention also relates to polypeptides, especially immunogenic polypeptides, comprising a pilus protein portion and a donor complement portion, both of which may be part of the same protein complex.
- the donor complement portion may be a bacterial pilin or adhesin, a portion thereof, or may be derived from a bacterial pilin or adhesin.
- the pilus-protein portion of such a novel polypeptide will commonly be formed from the amino acid sequence of a native pilus-protein molecule, such as that making up the normal pilus from a bacterial cell (including all of the pilus proteins, including adhesins, just mentioned for the immunogenic complexes of the invention).
- Such bacterial cells may be any bacterial cells that have pilus structures, preferably from the enterobacteriaceae family, most especially Escherichia coli.
- the missing strand of the pilus protein can be compensated for using any appropriate donor strands, regardless of the source, so long as said donor strands will provide a functional donor complement (wherein the term "functional" means that the donor complementary strand, when attached to the C-terminus of the adhesin sequence, will permit the adhesin to assume its native three dimensional conformation without the need for the entire chaperone, or other separate conformation-directing molecule, to be present).
- any sequence may be used if it can be modeled to fit into the groove.
- adhesin sequence provides the pilus-protein portion of the polypeptides of the present invention
- said adhesin will most preferably be the FimH protein found in bacterial cells such as E. coli.
- the amino acid sequence of FimH might conceivably vary slightly from one strain to another so that all such sequences are contemplated by the polypeptides of the invention.
- the FimH sequence of SEQ ID NO: 1 forms the adhesin portion, segment or fragment of the polypeptides of the invention when the pilus-protein is an adhesin.
- portion refers to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
- residues such as amino acid residues
- fragment refers to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
- the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.
- the donor complement portion is derived from a chaperone, especially a periplasmic bacterial chaperone
- said chaperone is preferably FimC or PapD, and most preferably where the chaperone or pilin is derived from a bacterial cell of the family enterobacteriaceae, especially E. coli, and most preferably where the donor complement is the G1 ⁇ -strand of FimC or PapD (see Figure 2 and Figure 6, respectively, for location of G1 sequence) or an amino terminal extension sequence from a pilin such as FimG, especially where said N-terminal sequence comprises at least.
- the donor complement, or donor strand, portion of the polypeptides of the invention, or immunogenic complexes of the invention may contain any amino acid, or other polymeric, sequence that provides appropriate compensation for the missing strand of the pilis protein.
- the appropriate strand to use as the donor complement is readily determined using the teaching of the present disclosure. Thus, to find a useful donor strand to complement a selected pilus protein, it is necessary to model the selected pilus protein with the appropriate chaperone and thereby determine the location and extent of the complementary sequence to use in forming the appropriate donor strand. Of course, the amino acid sequence of the relevant strands must be known.
- sequences provided for these proteins in the Sequence Listing can vary from species to species and from strain to strain within a species so that the sequences provided herein represent only one particular polypeptide.
- SEQ ID NO: 19, for PapG is for PapGII, which specifically binds sugars on human epithelial cells.
- the latter sequence also shows the signal sequence for secretion (which comprises the first 20 residues at the N-terminal end).
- the present invention is equally operable with any proteins, and not just similar proteins, and regardless of the organism, so long as a desired conformation of the protein, preferably but not necessarily the native one, is achieved by donor strand complementationas disclosed herein.
- the mechanism of the present invention is directed to proteins that require a strand donated by another protein to achieve a given conformation, such as the native conformation, and is not necessarily restricted to use with the bacterial proteins used herein to describe the present invention.
- the mechanism described herein may be a general one.
- an appropriately selected donor strand is engineered onto the pilus protein to be complemented, either by synthesizing said pilus protein with the donor strand as part of the amino acid sequence, or by expressing a gene encoding the complemented structure (the so-called "dsc-pilus protein"), or by forming some other attachment, with the only requirement being that such dsc-pilus protein have the native three dimensional shape characteristic of the pilus protein as it occurs within the pilus, a structure stabilized in vivo by the presence of the chaperone.
- dsc-pilus protein a gene encoding the complemented structure
- the amino terminal extension of a pilin, especially FimG is used as the donor strand.
- such a sequence normally comprises a sequence of about 6 or more amino acids, preferably about 6 to 20 amino acids, most preferably about 8 to 18 amino acids, especially 8 to 1 7 amino acids, where said sequence is identical to, or derived from, any of the N-terminal extensions of pilus subunits assembled by PapD-like chaperones.
- sequences could have amino acid substitutions at one or more positions to enhance stability, solubility, or other properties.
- the present invention encompasses any such sequences, especially those with two or more alternating hydrophobic residues, that completes the fold of and thus stabilizes the pilus subunit.
- any sequence that accepts the donor strand complement can be complemented by the strands of the invention herein and any strand that completes the groove in the recipient protein to achieve the natural conformation can be used.
- the usefulness of such sequences will be most advantageous where one or more of the amino acids of the native G1 sequence have been replaced by amino acids of the same character (hydrophobicity, acidity, basicity, etc.). Consequently, the donor strands useful within the present invention do not have to be derived from a natural source but may be wholly synthetic in nature and character so long as they complement the recipient protein to produce the desired overall conformation, regardless of the use to which the donor strand complemented protein is to be put (so that it need not be for use as a vaccine but may have some other utility).
- the first 13 amino acids of FimG are linked to the C-terminal end of FimH (SEQ ID NO: 1 ) with the two sequences separated by a tetrapeptide linker composed of the sequence DNKQ (Asp-Asn-Lys-Gln) with the three segments
- pilus protein - linker -- donor strand linked together by conventional peptide bonds The production of such a sequence (using FimH as pilus protein to form dscFimH) is provided in Example 1 below.
- novel polypeptides of the invention also include polypeptides having sequence homology, possibly less than 20% sequence identity, with the sequences of pilus proteins such as FimH, FimA, FimG, FimF, PapG, PapK, PapA, PapE, and PapF with the appropriate donor strand attached thereto to form the corresponding dsc-proteins with an optional linker sequence (for example, the N-terminal extension of the appropriate subunit fused to the corresponding protein to be complemented).
- PapE and PapK have less than 20% sequence identity).
- the methods of the present invention are successful with any proteins assembled by the chaperone-usher pathway (i.e, assembly of a type 1 pilus).
- selected amino acids as a means of increasing antigenic ability, or to increase or decrease the extent of other properties, is considered well within the skill of those in the art. Because the presence of FimC is not required to prepare the polypeptides of the invention, selected segments of the FimH portion that may be required for interaction and/or binding with FimC and serve no other purpose may advantageously be either eliminated or replaced with other amino acids, even those of different character. It is therefore deemed well within the skill of those in the art to make appropriate substitutions that may increase solubility, or any other desirable property, without sacrificing antigenicity.
- the term “percent identity” or “percent identical,” when referring to a sequence means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”).
- the Percent Identity is then determined according to the following formula:
- C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
- the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.
- the present invention is not limited to singly engineered proteins but includes also engineered protein complexes.
- a non-limiting example of such a complex would be a complex formed between FimH and donor complemented FimG.
- FimH i.e., a native FimH without the donor strand added and thus having an exposed tail groove
- FimG i.e., a native FimH without the donor strand added and thus having an exposed tail groove
- the amino terminal extension of the latter pilus-protein would then insert into the exposed tail groove of FimH but would be prepared by the methods disclosed herein having a G1 ⁇ -strand of FimC, or other suitable donor complement strand, such as the N-terminal extension of FimF, attached to the FimG C-terminus (with the donor strand in its forward or reverse sequence orientation).
- FimG-H complex such as a dimer, especially a heterodimer, useful in an immunogenic composition, such as a vaccine.
- Donor complemented FimA may combine with FimH in the same way that FimG does and forms a similar complex of an adhesin and a donor- complemented pilus-protein.
- An especially preferred embodiment of the present invention comprises a polypeptide having an amino acid sequence comprising the amino acid sequence of FimH (or some variant thereof) as the pilus-protein portion and the sequence of the donor complement, such as the G1 ⁇ -strand of FimC or the amino terminal extension of FimG, especially where the G1 sequence of FimC (but not the amino terminal extension sequence of FimG) is inverted and attached through an amino acid linker to the C-terminal of FimH.
- Such linker may be composed of different amino acids, especially sequences capable or readily forming a loop structure so as to cause the donor strand to loop back toward the pilus protein and form an anti-parallel structure in place of the missing strand.
- SEQ ID NO: 10 Such a preferred embodiment is presented as SEQ ID NO: 10 wherein the linker is alternating serine/glycines and the donor strand is from FimC (SEQ ID NO:3).
- SEQ ID NO: 3 A similar preferred embodiment with FimH as the pilus- protein and the N-terminal extension of FimG separated by a DNKQ linker sequence is shown in SEQ ID NO: 1 1 (prepared in Example 1 below).
- An embodiment with PapG (SEQ ID NO: 19), DNKQ linker, and donor strand from PapF SEQ ID NO: 21 .
- Donor strand sequences according to the present invention include the following:
- any sequence that stabilizes the pilin or adhesin can be utilized advantageously as the donor strand or donor complement.
- the donor strand complemented FimH (dscFimH) produced according to this particular embodiment was expressed in the absence of the chaperone in the periplasm and exhibited the properties of native FimH.
- the donor strand binds to the pilus-protein structure in an anti-parallel arrangement so that it will commonly be preferred to attach the donor strand from a chaperone at the C-terminus, for example, of an adhesin, in a reverse orientation so that when it bends back it permits an anti-parallel orientation or in a forward direction in the case of an N-terminal sequence of a pilus subunit as donor strand, again to facilitate the desired anti-parallel orientation.
- any such donor strand regardless of source, should be permitted to bend back in order to form the appropriate strand complementation activity, the donor strand and the C- terminus of the pilus protein portion of a polypeptide of the present invention will usually be separated by an appropriate loop, or other suitable amino acid sequence.
- a looped linking structure could comprise a sequence of up to 20 amino acids, especially 1 to 10 residues, for example, about 4 or 5 residues, for example, the sequence of residues that occurs in the F1 -G1 loop of the FimC chaperone, or especially useful would be the B1 -C1 loop of PapD.
- the donor strand will find its way to the appropriate complementation strand with no further guidance required, so long as sufficient conformational flexibility is provided to the engineered adhesin as a whole.
- polypeptides of the present invention may be in isolated or purified form.
- polypeptides or polynucleotides
- isolated in the context of the present invention with respect to polypeptides means that the material is removed from its original environment (e.g. , the cells used to recombinantly produce the polypeptides disclosed herein). Such peptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
- the recombinant and/or immunogenic polypeptides, disclosed in accordance with the present invention may also be in "purified” form.
- the term “purified” does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art.
- polypeptides from individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
- claimed polypeptides having a purity of preferably 0.001 %, or at least 0.01 % or 0.1 %, and even desirably 1 % by weight or greater is expressly contemplated.
- expression product means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
- polypeptides of the present invention may also be present in the form of a composition.
- Such composition where used for pharmaceutical purposes, will commonly have the polypeptide of the present invention suspended in a pharmacologically acceptable diluent or excipient.
- the present invention is also directed to polynucleotides capable of coding for the polypeptides of the invention, especially polynucleotides encoding the amino acid sequence of SEQ ID NO: 1 1 .
- Such polynucleotides would therefore contain at least one coding region for the polypeptides of the present invention, which would thus be an expression product thereof.
- coding region refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
- the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
- nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
- DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
- expression product means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
- primer means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3'OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
- promoter means a region of DNA involved in binding of RNA polymerase to initiate transcription.
- reference to a DNA sequence includes both single stranded and double stranded DNA.
- specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.
- the present invention is also directed to antibodies specific for, and antisera generated in response to, polypeptides of the invention.
- Such antibodies may be either polyclonal or monoclonal and may be generated, where monoclonal, from a cell, especially a hybridoma cell, by standard methods in the art.
- the present invention also relates to cells, and cell lines, genetically engineered to produce such antibodies after being transfected, or otherwise transformed, so that their genomes contain, within the main chromosome or as part of a plasmid or other vector, a polynucleotide encoding the genes for an antibody specific for a polypeptide of the invention, especially where said engineered cell is a cell capable of forming and secreting a fully formed antibody, such technology being known in the art.
- the present invention also relates to vectors, such as plasmids, comprising the polynucleotides of the invention, said polynucleotides encoding polypeptides disclosed herein, and wherein such vectors are useful for transforming cells and permitting said transformed cells to express the polypeptides of the invention.
- the present invention also relates to cells transformed by such vectors and thereby expressing, with or without subsequent secretion thereof, of the polypeptides of the invention.
- the present invention is also directed to vaccines and vaccine compositions comprising the polypeptides disclosed herein.
- a vaccine would comprise a composition containing an immunogenically effective amount of a polypeptide of the invention.
- a preferred embodiment of the invention is a vaccine composition comprising the polypeptide whose sequences are shown as SEQ ID NO: 10 and 1 1 .
- an immunogenic composition for a vaccine or to produce antibodies for use as a diagnostic or as a passive vaccine
- a bacterial polypeptide of the invention comprising a bacterial polypeptide of the invention.
- proteins and fragments naturally or recombinantly produced, as well as functional analogs from bacteria that produce type 1 or type P pili are contemplated.
- E. coli is contemplated as the source.
- such an immunogenic composition may be utilized to produce antibodies to diagnose urinary tract infections, or to produce vaccines for prophylaxis and/or treatment of such infections as well as booster vaccines to maintain a high titer of antibodies against the immunogen(s) of the immunogenic composition.
- the present invention is directed to an immunogenic composition
- a purified dsc-pilus protein polypeptide or immunogenic complex thereof comprises a native adhesin, preferably FimH, and a donor complement, such as one derived from a periplasmic chaperone, preferably FimC, most preferably the G 1 strand of FimC, or an amino terminal extension of a pilin, preferably FimG, most preferably no more than the first 17 N-terminal residues of FimG, especially the first 13 residues thereof, with the dsc-pilus-protein maintained in the complex in an immunogenic form capable of inducing an immune response when appropriately introduced into a human or other mammalian species.
- a preferred embodiment is one that includes the N-terminal extension of another subunit.
- the dsc-polypeptides and complexes of the present invention are primarily intended for use as vaccines.
- vaccines are prepared as injectables, in the form of aqueous solutions or suspensions.
- Vaccines in an oil base are also well known such as for inhaling.
- Solid forms which are dissolved or suspended prior to use may also be formulated.
- Pharmaceutical carriers are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used.
- Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.
- Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Such vaccines can also be formulated as suppositories or for oral administration, using methods known in the art.
- the amount of vaccine sufficient to confer immunity to pathogenic bacteria is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered will be determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of 50 to 500 ⁇ g purified protein may be given.
- polypeptides of the present invention can be used as immunogens to stimulate the production of antibodies for use in passive immunotherapy, for use as diagnostic reagents, and for use as reagents in other processes such as affinity chromatography.
- the present invention also provides for a recombinant production or synthesis of the proteins and polypeptides of the invention without the need for any chaperone being present or the need to co-express any chaperone during production of the replacement adhesin for use as a vaccine (or as an immunogen to produce antibodies for diagnostic or therapeutic purposes).
- Recombinant polypeptides of the invention are readily produced by methods of genetic engineering already well known in the art (e.g., Example 1 , below) or by direct synthesis by well known, even automated, methods. Therefore, a compendium of procedures for preparing the polypeptides and complexes of the invention need not be recited herein.
- the ability to engineer a recombinant adhesin-donor complement protein, a dsc-pilus polypeptide, without the requirement of co-producing a chaperone, or adding an exogenous chaperone, or fragments thereof, to the expression medium, to enable the completion of the adhesin or pilin native structure and conformation readily permits large scale synthesis of immunogenic polypeptides for use as vaccines.
- the polynucleotides encoding the polypeptides of the invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
- the marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al.. Cell, 37:767 (1984)).
- the vector may be, for example, a cloning vector or an expression vector.
- the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides which encode such polypeptides.
- the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- Vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
- any other vector may be used as long as it is replicable and viable in the host.
- the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
- the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
- the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
- promoter for example, LTR or SV40 promoter, the E. coli. lac or t ⁇ o, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression.
- the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in coli.
- the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.
- bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
- fungal cells such as yeast
- insect cells such as Drosophi/a S2 and Spodoptera Sf9
- animal cells such as CHO, COS or Bowes melanoma
- adenoviruses plant cells, etc.
- the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
- the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
- the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
- a promoter operably linked to the sequence.
- Bacterial pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
- Eukaryotic pWLNEO, pSV2CAT, pOG44, pXT1 , pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
- any other plasmid or vector may be used as long as they are replicable and viable in the host.
- Promoter regions can be selected from any desired gene using CAT
- Two appropriate vectors are pKK232-8 and pCM7.
- Particular named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda P R , P L and TRP.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
- Host cells containing the above-described constructs can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
- constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
- the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
- Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.
- Cell-free translation systems can also be employed to produce such proteins using
- RNAs derived from the DNA constructs of the present invention are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wu et al. Methods in Gene
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to
- a cytomegalovirus early promoter enhancer the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
- promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
- PGK 3-phosphoglycerate kinase
- the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
- the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
- Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
- the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
- Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
- useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
- cloning vector pBR322 ATCC 37017
- Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wl, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
- the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
- Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
- Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, a french press, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
- mammalian cell culture systems can also be employed to express recombinant protein.
- mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23: 175 (1981 ), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
- Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
- the polypeptides can be recovered and/or purified from recombinant cell cultures by well-known protein recovery and purification methods. Such methodology may include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. In this respect, chaperones may be used in such a refolding procedure. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- polypeptides that are useful as immunogens in the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Particularly preferred immunogens are FimH- ⁇ -strand polypeptides or mannose-binding fragments thereof since FimH is highly conserved among many bacterial species.
- polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can also be used as an immunogen to produce antibodies thereto.
- These antibodies can be, for example, polyclonal or monoclonal antibodies.
- the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
- Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides.
- any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-
- buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein.
- the DNA sequence encoding the first 13 amino acids of FimG (see Figure 1 ), referred to herein as the donor strand, was provided to FimH in cis, by fusing it directly to the 3'-end of the FimH coding sequence, with the resulting DNA sequence encoding the donor strand complemented FimH protein (called dscFimH).
- dscFimH FimH protein
- a hairpin loop region in PapD consisting of Asp-Asn-Lys-Gln was inserted upstream of the donor strand to form a hinge region in the expressed protein and thereby allow the donor strand to fold back and form an anti-parallel arrangement with FimH (with the arrangement shown in Figure 9).
- dsc-FimH To construct the dsc-FimH, the following two oligonucleotides were annealed together and ligated into the Clal and BamHI sites of pUC1 8-FimH to create pUC18-dscFimH (here, with DNKQ as linker - the latter are the 4 amino acids used for the loop or hinge region, as described above, using standard 1 letter code for amino acids) where the top (coding strand) is:
- pUC18-dscFimH was sequenced followed by subcloning into the EcoR1 and BamHI sites of pTrc99A (Amann et al, Gene 69, 301 (1 988). to create pTrc-dscFimH.
- the fimH was then subcloned from pUC18-FimH into pTrc99A using the EcoR1 and BamHI sites to create pTrc-FimH.
- FimH, FimH + FimC, and dscFimH were expressed separately.
- the plasmids encoding the FimH (pTrc-FimH), dscFimH (pTrc-dscFimH), and FimC (pJP4) were expressed in C600 (see Jones et al, Proc. Natl. Acad. Sci. USA, 90, 8397 (1 993)). Overnight cultures were diluted 1 : 100 into Luria broth and grown to an OD 600 of 0.6 followed by induction with 0.5 mM IPTG for 1 hour. Periplasms were prepared by a known procedure (see Slonim et al., EMBO J. 1 1 , 4747 (1992).
- FimC and FimH or dscFimH in periplasmic extracts were monitored by immunoblotting using anti-FimCH antibodies.
- mannose-sepharose chromatography (Jones et al, 1993, above) was used to determine the ability of FimH or dscFimH to bind its receptor. FimH was degraded when expressed alone but was stabilized by co-expression of the chaperone. In contrast to FimH, dscFimH was stable in the periplasm in the absence of FimC. FimH bound to mannose-sepharose beads when it was co- expressed with FimC and thus eluted as a FimCH complex.
- FimH Since FimH is degraded in the absence of FimC, no full length FimH eluted from the mannose-sepharose beads. In contrast, dscFimH bound to, and specifically eluted from, the mannose beads when expressed alone. When FimC was co- expressed with dscFimH it did not form a complex with dscFimH and thus did not co-elute with dscFimH from the mannose-sepharose beads.
- fimH and dscFimH were subcloned from pUC1 8-FimH and pUC18-dscFimH into pBad18-Kn (see Guzman et al, J. Bacterioi , ⁇ ll ', 41 21 (1 995). using the EcoR1 and Xba1 sites to create pBad-FimH and pBad- dscFimH which were transformed into ORN 103/pETS10.
- ORN103 does not produce type 1 pili and pETSI O encodes a fimH type 1 gene cluster.
- the strains were diluted 1 : 100 into Luria broth and grown to an OD 600 of 0.8 followed by induction with 0.1 mM IPTG and 0.02% arabinose for one hour.
- the cells were harvested and hemagglutination assays were performed by methods known in the art (see: Hultgren et al, Mol. Microbiol. 4, 131 1 (1990).
- the added donor strand occupied the groove and completed the Ig fold of the FimH pilin domain, thus shielding the surface that would normally interact first with the chaperone and then with another subunit in the pilus.
- dscFimH did not bind FimC nor assemble into the pilus.
- FimH and dscFimH were purified and their denaturation curves were obtained.
- FimH was isolated from a FimCH complex by incubating the complex in 3M urea and subjecting it to cation exchange chromatography which yielded 2 peaks, one of which contained pure FimH.
- the FimCH complex was purified from the periplasm of C600/pHJ9205/pHJ20 (see: Jones et al (1 993) and Slonim et al (1 992, above). The periplasmic extracts were dialyzed against 20 mM MES pH 5.4, injected onto a Source 1 5S column (Pharmacia), and FimCH eluted with 65 mM NaCl.
- the eluate was injected onto a Butyl4FF column in 0.55M (NH 4 ) 2 SO 4 /20 mM MES pH 5.4 and FimCH eluted at 0.3 M (NH 4 ) 2 S0 4 .
- the FimCH complex was brought to 3M urea to separate the two subunits. Pure FimH in 3M urea was collected from the flow through of a Source 1 5S column. FimH retained its native structure in 3M urea as determined by circular dichroism (CD) and its ability to bind mannose.
- CD circular dichroism
- DscFimH was also purified from the periplasm of C600/pTrc-dscFimH (see Slonim et al (1992)).
- the periplasm was dialyzed against 20 mM Tris-CI pH 8.8 and dscFimH was collected from the flow through of a Source 1 5Q column. This flow through was injected onto a Butyl4FF column in 0.9M (NH 4 ) 2 SO 4 /20 mM Tris-CL pH 8.8 and dscFimCH eluted at 0.4 M (NH 4 ) 2 S0 4 .
- the eluate was then loaded onto a Source 1 5S column in 20 mM MES pH 4.7 and dscFimH eluted at 55 mM NaCl.
- FimH and dscFimH had similar denaturation curves with denaturation complete only at concentrations above 8.5 M urea ( + 4mM DTT) as determined by tyrosine fluorescence spectroscopy emission maxima (350 nm).
- 22.5 ⁇ g of FimH or dscFimH in 20 mM MES pH 6.5 + 4 mM DTT was incubated with the appropriate urea concentration. Fluorescence was measured using an excitation wavelength of 290 nM with emission at 350 nm on an AlphaScan PTI fluorometer. FimH did not begin to denature until a concentration of 6.5 M urea was reached, with the midpoint of the denaturation curve occurring at approximately 7.5 M urea.
- An in vitro folding assay was used to demonstrate that the missing steric information in the amino acid sequence of pilus subunit proteins can be provided in cis.
- This assay was based on attempted refolding of urea- denatured FimH (obtained from a FimCH complex as already described) and dscFimH as determined by examination of the CD spectra after rapid dilution of the denatured proteins. Spectra were measured from 1 50 ⁇ g of protein in 20 mM MES pH 6.5 using a 0.02 cm cell in a JASCO J71 5 spectropolarimeter. Denatured proteins were diluted to 0.45 M urea.
- FimH 3 M urea
- 9 M urea + 4 mM DTT
- the CD spectra of FimH and dscFimH became characteristic of non-native proteins.
- Light scattering of the denatured dscFimH indicated the presence of large aggregates.
- rapid dilution of dscFimH led to the refolding of the protein into its native ⁇ -sheet structure.
- the refolded dscFimH bound mannose and was monodisperse ( as shown by light scattering) indicating that it had refolded into its native structure.
- FimC was unable to bind to denatured FimH after its rapid dilution.
- FimC was present in the diluent, FimH formed a complex with FimC and folded into its native mannose-binding ⁇ -sheet structure.
- dscFimH folded independently whereas FimH folded in the presence of, but not in the absence of, FimC.
- FimC was capable of binding to native FimH separated from the FimCH complex by 3 M urea, confirming that the chaperone can indeed bind to folded subunits.
- FimH based vaccines have been shown to protect mice and monkeys from experimental bladder infections (see: Langermann et al, Science 276, 607 (1997); Langermann et al, J. Infect. Dis. in press). However, the production of such vaccines required co-expression of FimC and the purification of a FimCH complex.
- donor strand complementation now permits production of dscFimH vaccines.
- vaccines are advantageous in that they comprise a single subunit and are expected to be more stable due to the anti-parallel donor strand.
- vaccination with dscFimH has been found to produce anti-FimH titers that are comparable to those achieved with the FimCH complex.
- the methods taught by the present invention readily facilitate the production of adhesin-based vaccines generally.
- This experiment evaluated dosages of the dsc immunogen (dose down) at 1 5 ⁇ g, 3 ⁇ g, and 0.6 ⁇ g. CFA/IFA was used as an adjuvant. Here, 30, 6 and 1 .2 ⁇ g FimCH was used for comparison. It was expected that the ratio between the dsc and FimCH would be similar since the amount of FimH at each dose of FimCH complex (1 : 1 ratio of FimH to FimC) is equivalent to the amount present in each dose of dscFimH. Endpoint titers using anti- FimH T3 and Anti-FimdscFimH as detecting antigens were used to illustrate this correlation, evaluate immune responses and demonstrate comparability.
- the dsc antigen was compared to control (CFA/IFA - complete Freund's adjuvant/incomplete Freund's adjuvant) and naive (no adjuvant or antigen) as well as to high dose FimCH (CFA/IFA).
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14358299P | 1999-07-13 | 1999-07-13 | |
US143582P | 1999-07-13 | ||
US14435999P | 1999-07-16 | 1999-07-16 | |
US144359P | 1999-07-16 | ||
US18444200P | 2000-02-23 | 2000-02-23 | |
US184442P | 2000-02-23 | ||
PCT/US2000/019066 WO2001004148A2 (en) | 1999-07-13 | 2000-07-13 | Donor strand complemented pilin and adhesin broad-based vaccines |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1194561A2 true EP1194561A2 (en) | 2002-04-10 |
Family
ID=27385951
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00947305A Withdrawn EP1194561A2 (en) | 1999-07-13 | 2000-07-13 | Donor strand complemented pilin and adhesin broad-based vaccines |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1194561A2 (en) |
JP (1) | JP2003504083A (en) |
AU (1) | AU6094000A (en) |
CA (1) | CA2373534A1 (en) |
HK (1) | HK1045713A1 (en) |
WO (1) | WO2001004148A2 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004502447A (en) * | 2000-07-07 | 2004-01-29 | メディミューン,インコーポレイテッド | FimH adhesin proteins and methods of use |
WO2002015928A1 (en) * | 2000-08-18 | 2002-02-28 | Medimmune, Inc. | METHOD OF ADMINISTERING FimH PROTEIN AS A VACCINE FOR URINARY TRACT INFECTIONS |
WO2002059156A2 (en) | 2000-12-22 | 2002-08-01 | Medimmune, Inc. | Therapeutic compounds structurally-linked to bacterial polypeptides |
EP1855718B1 (en) * | 2005-01-11 | 2016-10-12 | The United States of America as represented by The Secretary of The Navy | Donor strand complemented adhesins as immunogen against Escherichia coli. |
WO2007117339A2 (en) | 2006-01-11 | 2007-10-18 | The United States Of America As Respresented By The Secretary Of The Navy | Adhesin-enterotoxin chimera based immunogenic composition against entertoxigenic escherichia coli |
WO2012028697A1 (en) | 2010-09-01 | 2012-03-08 | Eth Zürich, Institute Of Molecular Biology And Biophysics | Affinity purification system based on donor strand complementation |
US9017698B2 (en) | 2013-09-25 | 2015-04-28 | Sequoia Sciences, Inc. | Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections |
US9149521B2 (en) | 2013-09-25 | 2015-10-06 | Sequoia Sciences, Inc. | Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections |
US9504743B2 (en) | 2013-09-25 | 2016-11-29 | Sequoia Sciences, Inc | Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections |
US9149522B2 (en) | 2013-09-25 | 2015-10-06 | Sequoia Sciences, Inc. | Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections |
WO2018218360A1 (en) * | 2017-05-31 | 2018-12-06 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | Vaccine against necrotic enteritis in poultry |
KR20220092572A (en) * | 2019-11-01 | 2022-07-01 | 화이자 인코포레이티드 | Escherichia coli composition and method therefor |
US20240115688A1 (en) * | 2020-12-02 | 2024-04-11 | Glaxosmithkline Biologicals Sa | Novel Antigens |
WO2023223796A1 (en) * | 2022-05-16 | 2023-11-23 | 株式会社アデノプリベント | Antibody binding to colibactin-producing escherichia coli bacterium |
-
2000
- 2000-07-13 WO PCT/US2000/019066 patent/WO2001004148A2/en not_active Application Discontinuation
- 2000-07-13 CA CA002373534A patent/CA2373534A1/en not_active Abandoned
- 2000-07-13 EP EP00947305A patent/EP1194561A2/en not_active Withdrawn
- 2000-07-13 AU AU60940/00A patent/AU6094000A/en not_active Abandoned
- 2000-07-13 JP JP2001509757A patent/JP2003504083A/en not_active Withdrawn
-
2002
- 2002-09-25 HK HK02106980.7A patent/HK1045713A1/en unknown
Non-Patent Citations (2)
Title |
---|
LANGERMANN ET AL: "Prevention of Mucosal Escherichia coli Infection by FimH-Adhesin-Based Systemic Vaccination", SCIENCE, vol. 276, 25 April 1997 (1997-04-25), pages 607 - 611 * |
SOTO ET AL: "Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.", EMBO J., vol. 17, no. 21, 1998, pages 6155 - 6167 * |
Also Published As
Publication number | Publication date |
---|---|
WO2001004148A2 (en) | 2001-01-18 |
HK1045713A1 (en) | 2002-12-06 |
WO2001004148A9 (en) | 2001-07-26 |
CA2373534A1 (en) | 2001-01-18 |
JP2003504083A (en) | 2003-02-04 |
AU6094000A (en) | 2001-01-30 |
WO2001004148A3 (en) | 2001-06-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7262024B2 (en) | Streptococcus antigens | |
EP2261358B1 (en) | Novel streptococcus antigen | |
FI113009B (en) | Procedure for Preparing a Vaccine against the Neisseria meningitidis Bacteria and Neisseria meningitidis Strain | |
US7635482B2 (en) | Streptococcus antigens | |
KR100771148B1 (en) | Group B streptococcus antigens | |
WO2001004148A2 (en) | Donor strand complemented pilin and adhesin broad-based vaccines | |
US10279026B2 (en) | Antigens and antigen combinations | |
US20190290748A1 (en) | Antigens and antigen combinations | |
CA2931685C (en) | Immunogenic compositions and vaccines derived from bacterial surface receptor proteins | |
CA2325055A1 (en) | Vaccines containing recombinant pilin against neisseria gonorrhoeae or neisseria meningitidis | |
CA2784450C (en) | Polypeptides of pseudomonas aeruginosa | |
WO2001005978A1 (en) | FimH ADHESIN-BASED VACCINES | |
EP1343895B1 (en) | Streptococcus pyogenes antigens and corresponding dna fragments | |
EP1444349B1 (en) | Polypeptides of moraxella (branhamella) catarrhalis | |
AU2002325109B2 (en) | Moraxella (branhamella) catarrhalis polypeptides and corresponding DNA fragments | |
EP1790659B1 (en) | Polypeptides of pseudomonas aeruginosa | |
EP1950302B1 (en) | Streptococcus antigens | |
AU2007207883A1 (en) | Streptococcus antigens | |
CA2379129A1 (en) | Fima pilus-based vaccines | |
MXPA00008111A (en) | Group b streptococcus antigens |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20020111 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: MEDIMMUNE, INC. Owner name: WASHINGTON UNIVERSITY |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: WASHINGTON UNIVERSITY Owner name: MEDIMMUNE, INC. |
|
17Q | First examination report despatched |
Effective date: 20040416 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20050531 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1045713 Country of ref document: HK |