US20090274717A1 - Genes and proteins, and their use - Google Patents
Genes and proteins, and their use Download PDFInfo
- Publication number
- US20090274717A1 US20090274717A1 US11/892,024 US89202407A US2009274717A1 US 20090274717 A1 US20090274717 A1 US 20090274717A1 US 89202407 A US89202407 A US 89202407A US 2009274717 A1 US2009274717 A1 US 2009274717A1
- Authority
- US
- United States
- Prior art keywords
- gene
- identified
- pho3
- amino acid
- homologues
- 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.)
- Abandoned
Links
- 108090000623 proteins and genes Proteins 0.000 title abstract description 130
- 102000004169 proteins and genes Human genes 0.000 title description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 39
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 6
- 230000028993 immune response Effects 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 3
- 229940037003 alum Drugs 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims 7
- 230000028996 humoral immune response Effects 0.000 claims 1
- 230000021633 leukocyte mediated immunity Effects 0.000 claims 1
- 241000193990 Streptococcus sp. 'group B' Species 0.000 abstract description 36
- 239000012634 fragment Substances 0.000 abstract description 14
- 229960005486 vaccine Drugs 0.000 abstract description 14
- 208000015181 infectious disease Diseases 0.000 abstract description 10
- 238000002360 preparation method Methods 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000013612 plasmid Substances 0.000 description 42
- 101150009573 phoA gene Proteins 0.000 description 30
- 241000193996 Streptococcus pyogenes Species 0.000 description 26
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 19
- 241000194032 Enterococcus faecalis Species 0.000 description 16
- 239000002773 nucleotide Substances 0.000 description 16
- 125000003729 nucleotide group Chemical group 0.000 description 16
- 241000588724 Escherichia coli Species 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 229940032049 enterococcus faecalis Drugs 0.000 description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 description 8
- 241000193998 Streptococcus pneumoniae Species 0.000 description 8
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 7
- 241000194019 Streptococcus mutans Species 0.000 description 7
- 101150081160 dltB gene Proteins 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 229940023064 escherichia coli Drugs 0.000 description 5
- 101150015333 kup gene Proteins 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 108700020474 Penicillin-Binding Proteins Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000013615 primer Substances 0.000 description 4
- 241000589969 Borreliella burgdorferi Species 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 108090000860 Endopeptidase Clp Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000606768 Haemophilus influenzae Species 0.000 description 3
- 244000199866 Lactobacillus casei Species 0.000 description 3
- 206010040047 Sepsis Diseases 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 229940047650 haemophilus influenzae Drugs 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 206010053555 Arthritis bacterial Diseases 0.000 description 2
- 101100455969 Bacillus subtilis (strain 168) mdxK gene Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000193764 Brevibacillus brevis Species 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 208000035357 Focal Infection Diseases 0.000 description 2
- 101100077927 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) mrdA gene Proteins 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- 208000004575 Infectious Arthritis Diseases 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- 241000186840 Lactobacillus fermentum Species 0.000 description 2
- 241000186604 Lactobacillus reuteri Species 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 101710084556 Low affinity potassium transport system protein kup Proteins 0.000 description 2
- 101710183187 Major outer membrane lipoprotein Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010061308 Neonatal infection Diseases 0.000 description 2
- 108010053775 Nisin Proteins 0.000 description 2
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010031252 Osteomyelitis Diseases 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 101100295833 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) oprI gene Proteins 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 101100054091 Schizosaccharomyces pombe (strain 972 / ATCC 24843) par2 gene Proteins 0.000 description 2
- 241000607762 Shigella flexneri Species 0.000 description 2
- 241000191965 Staphylococcus carnosus Species 0.000 description 2
- 241000264435 Streptococcus dysgalactiae subsp. equisimilis Species 0.000 description 2
- 244000057717 Streptococcus lactis Species 0.000 description 2
- 241000194020 Streptococcus thermophilus Species 0.000 description 2
- 241000607477 Yersinia pseudotuberculosis Species 0.000 description 2
- 206010000269 abscess Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 102000004432 dTDP-4-dehydrorhamnose reductase Human genes 0.000 description 2
- 108010083506 dTDP-4-dehydrorhamnose reductase Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 108010060371 endo-N-acetylmuramidase Proteins 0.000 description 2
- 101150110946 gatC gene Proteins 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 101150109648 lppC gene Proteins 0.000 description 2
- 101150003248 malK gene Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 208000004396 mastitis Diseases 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000004309 nisin Substances 0.000 description 2
- 235000010297 nisin Nutrition 0.000 description 2
- 101150003900 oprI gene Proteins 0.000 description 2
- 101150055083 ospC gene Proteins 0.000 description 2
- 101150053585 perM gene Proteins 0.000 description 2
- 230000004260 plant-type cell wall biogenesis Effects 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 101150117748 rfbC gene Proteins 0.000 description 2
- 101150016581 rmlC gene Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 201000001223 septic arthritis Diseases 0.000 description 2
- 101150044856 spaR gene Proteins 0.000 description 2
- 229960000268 spectinomycin Drugs 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 208000019206 urinary tract infection Diseases 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 101150086824 yutD gene Proteins 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 1
- FKJSFKCZZIXQIP-UHFFFAOYSA-N 2-bromo-1-(4-bromophenyl)ethanone Chemical compound BrCC(=O)C1=CC=C(Br)C=C1 FKJSFKCZZIXQIP-UHFFFAOYSA-N 0.000 description 1
- 101710088758 23kDa protein Proteins 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 102000005416 ATP-Binding Cassette Transporters Human genes 0.000 description 1
- 108010006533 ATP-Binding Cassette Transporters Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000893512 Aquifex aeolicus Species 0.000 description 1
- 101100289923 Bacillus subtilis (strain 168) lytG gene Proteins 0.000 description 1
- 101100268003 Bacillus subtilis (strain 168) yutD gene Proteins 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101710166119 Basic membrane protein Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 125000000030 D-alanine group Chemical class [H]N([H])[C@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 244000024708 Dioscorea cayenensis Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101100321238 Escherichia coli (strain K12) yubE gene Proteins 0.000 description 1
- 101100053968 Escherichia coli (strain K12) znuC gene Proteins 0.000 description 1
- 101100173396 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) fbpC1 gene Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- 241001625930 Luria Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241001123650 Schwanniomyces occidentalis Species 0.000 description 1
- 102100027287 Serpin H1 Human genes 0.000 description 1
- 108050008290 Serpin H1 Proteins 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 241001464947 Streptococcus milleri Species 0.000 description 1
- 241000194024 Streptococcus salivarius Species 0.000 description 1
- 241000194021 Streptococcus suis Species 0.000 description 1
- 108010023191 Streptomycin 3''-adenylyltransferase Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108010006886 Vitrogen Proteins 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000001055 blue pigment Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108060002399 dTDP-4-dehydrorhamnose 3,5-epimerase Proteins 0.000 description 1
- ZOSQFDVXNQFKBY-ZORVDHEWSA-N dTDP-rhamnose Chemical compound OC1C(O)C(O)C(C)OC1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)C[C@H](N2C(NC(=O)C(C)=C2)=O)O1 ZOSQFDVXNQFKBY-ZORVDHEWSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 101150020570 fbpC gene Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 101150005487 ftsI gene Proteins 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- 229940012969 lactobacillus fermentum Drugs 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 101150080142 lpoA gene Proteins 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 101150064202 mrdA gene Proteins 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 101150005569 pbpA gene Proteins 0.000 description 1
- 101150060462 pbpB gene Proteins 0.000 description 1
- 101150028051 pbpC gene Proteins 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 101150016899 potA gene Proteins 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 101150017775 sgcC gene Proteins 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 229940098232 yersinia enterocolitica Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
-
- 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
-
- 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
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- 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
-
- 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
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates to the identification of bacterial genes and proteins, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.
- GBS Group B Streptococcus
- Streptococcus agalactiae is the causative agent of various conditions.
- GBS causes:
- This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.
- This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis.
- GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.
- GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
- GBS infections can be treated with antibiotics.
- immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.
- the present invention is based on the identification of a series of genes in GBS, and also related organisms, the products of which may be localised on the outer surface of the organism and therefore may be used as a target for immuno-therapy.
- a peptide is encoded by an operon including any of the genes identified herein as pho1-13, pho3-21, pho2-15, pho3-18, pho3-22, pho3-3, pho3-17, pho2-2, pho1-5, pho3-1, pho3-23, pho3-50, pho1-14, pho2-10, pho3-14, pho3-24 and pho3-29, obtainable from Group B Streptococcus , or a homologue or functional fragment thereof.
- Such a peptide is suitable for therapeutic use, e.g. when isolated.
- a functional fragment of the peptide is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide.
- a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.
- a gene fragment may be used to encode the active peptide.
- the gene fragment may have utility in gene therapy, targeting the wild-type gene in vivo to exert a therapeutic effect.
- a peptide according to the present invention may comprise any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35, or a functional fragment thereof.
- peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS.
- therapeutically-effective is intended to include the prophylactic effect of vaccines.
- a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection.
- the vaccine may be administered to females prior to or during pregnancy to protect mother and neonate against infection by GBS.
- the peptides or genes may be used for screening potential antimicrobial drugs or for the detection of virulence.
- a further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.
- the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention.
- the peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.
- the present invention is described with reference to Group B Streptococcal strain M732.
- GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732.
- Organisms likely to contain the peptides include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. milleri , Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.
- the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.
- sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank.
- Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.
- Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms.
- suitable carriers or adjuvants e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms.
- suitable carriers or adjuvants e.g. alum
- a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.
- a partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3 (Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess a constitutive spectinomycin adenyltransferase antibiotic resistance marker, which confers a high level of spectinomycin resistance and is therefore easily selected. Furthermore, these vectors contain a truncated (leaderless) Escherichia coli phoA gene for alkaline phosphatase.
- the three vectors differ only with respect to the reading frame in which the leaderless phoA gene exists, as compared to an upstream in-frame BamHI restriction enzyme site. Because this truncated E. coli phoA gene lacks the appropriate leader sequence for export of this enzyme across the bacterial membrane, extracellular alkaline phosphatase activity is absent when these plasmids are propagated in an E. coli phoA mutant (e.g. strain DH5 ⁇ ).
- the chromogenic alkaline phosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate)
- the chromogenic XP substrate is cleaved to yield a blue pigment and the corresponding bacterial colonies can be identified by their blue colour.
- Plasmid DNA was digested to completion with BamHI and dephosphorylated using shrimp alkaline phosphatase.
- GBS genomic DNA was partially digested with Sau3AI, size fractionated on a sucrose gradient and fragments ⁇ 1 kb in size were ligated into the prepared pFW-phoA vectors.
- E. coli strain DH5 ⁇ was chosen as the cloning host since it lacks a functional phoA gene.
- Recombinant plasmids were selected on Luria agar containing 100 ⁇ g/ml of spectinomycin and 40 ⁇ g/ml of the chromogenic XP substrate.
- oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an ‘outward’ direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5′ and 3′ termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5′ end the presence of an AUG start codon (or accepted equivalent) preceded by a Shine-Dalgarno consensus sequence, and for the 3′ end, the presence of a translation termination (Stop) codon.
- AUG start codon or accepted equivalent
- primers were designed for amplification of full-length product. Primers used included restriction enzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end) to allow subsequent cloning of the product into the Lactococcal expression system used.
- PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (In Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I.
- the vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21).
- This vector harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10 ⁇ His tags, flanked on the 5-most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a Stop (termination) codon at the 3′ end of the His tags.
- the gene of interest was inserted so that a 10 ⁇ His tag was in the 3′ position relative to the coding region.
- L. lactis strain NZ9000-Kuipers, O. P. et al. (1998) supra
- a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture.
- the cells were harvested and lysed by bead beating.
- the resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.
- the recombinant protein obtained was then used to immunize New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.
- Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5 ml PBS. 50 ⁇ l aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4° C. overnight to allow for absorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3% BSA in PBS for 1 hr at 37° C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50 ⁇ l of these dilutions were added to the wells of the plate, in duplicate.
- the plate was covered and incubated for 1 hr at 37° C. The plate was washed, then 50 ⁇ l anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37° C. for an hour, the plate was washed again. 50 ⁇ l substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30 min before the absorbance was read at 405 nm.
- PNPP substrate
- GBS M732 was grown up in THB until mid-log phase was reached—approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2 ⁇ 10 7 bacteria per ml in pre-immune or test sera. 50 ⁇ l of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.
- a first clone contained a gene sequence identified herein as SEQ ID NO. 1, with an amino acid sequence identified as SEQ ID NO. 2, and classified as pho1-13.
- Homologues to the GBS pho1-13 gene product can be identified in Streptococcus pyogenes, S. pneumoniae, S. salivarius, Escherichia coli, Yersinia enterocolitica, Aquifex aeolicus, Helicobacter pylori and Haemophilus influenzae .
- the S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In all other cases, the above homologues can be identified as ATP-dependent Clp protease proteolytic subunits.
- Clp proteases results in the hydrolysis of proteins to small peptides in the presence of ATP and magnesium (Giffard, P. M. et al. 1993. J. Gen. Microbiol. 139:913-920). Furthermore, the ClpP component of Clp proteases has been shown to be induced as part of the heat shock response (Kroh, H. E. and L. D. Simon. 1990. J. Bacteriol. 172:6026-6034) and it is probable that this subunit or the complete proteolytic domain would associated with the bacterial surface.
- a second clone was selected containing a plasmid designated pho1-14.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 3 and 4, respectively.
- Homologues to the GBS pho1-14 gene product can be identified in Streptococcus pyogenes, Enterococcus faecalis and Streptococcus pneumoniae . These homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Additionally, two possible homologues were also identified from Shigella flexneri (SpaR) and Yersinia pseudotuberculosis (YscT). These latter two homologues are related proteins, believed to be anchored in the bacterial membrane (Bergman, T. et al. 1994. J. Bacteriol. 176:2619-2626). In S.
- the product of the spaR gene has been shown to be important for invasion of epithelial cells (Sasakawa, C. et al. 1993. J. Bacteriol. 175:2334-2346). Furthermore, the product of the spaR gene is also required for surface presentation of invasion plasmid antigens.
- the analogous protein in Y. pseudotuberculosis is a component of the Yop secretion system and is also important for virulence in this organism.
- a third clone was selected containing a plasmid designated pho1-5.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 5 and 6.
- Homologues to the GBS pho1-5 gene product can only be identified in streptococcus pyogenes and Staphylococcus carnosus (sceA).
- S. pyogenes homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Furthermore, little information is available on the function of the sceA gene product from S. carnosus .
- the sceA gene product shows some sequence similarity to the aggregation promoting protein from Lactobacillus gasseri . Based on analysis of the sceA gene product, this molecule contains a well-conserved signal sequence and is apparently secreted or associated with the bacterial cell surface.
- a further clone was selected containing a plasmid designated pho3-3.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 7 and 8.
- Homologues to the GBS pho3-3 gene product can be identified in Streptococcus mutans (rmiC), (cpsM) S. pneumoniae and S. pyogenes .
- the S. pyogenes homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene product.
- the homologue can be identified as dTDP-4-keto-6-deoxy glucose-3,5-epimerase.
- the above homologues can be identified as dTDP-4-keto-L-rhamnose reductase (rmlC).
- rmlC dTDP-4-keto-L-rhamnose reductase
- mutants the gene encoding this enzyme, rmlC, is part of the rml locus.
- the rml locus consists of three genes which exhibit significant similarity to enzymes involved in the biosynthesis of dTDP-rhamnose, the immediate precursor of the rhamnose component in the S. mutans polysaccharide capsule (Tsukioka, Y. et al. 1997. J. Bacteriol. 179:1126-1134).
- An analogous locus has also been identified in S. pneumoniae (Coffey, T. J. et al. 1998. Mol. Micobiol. 17:73-83).
- Streptococci characteristically possess rhamnose in their cell wall associated polysaccharides (Schleifer, K. H. and R. Kilper-Bälz. 1987. Syst. Appl. Microbiol. 10:1-19), and it is highly probable that dTDP-4-keto-L-rhamnose reductase would be associated with the outer surface in Streptococci.
- a further clone was selected containing a plasmid designated pho2-10.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide sequence is shown as SEQ ID NO. 9. From this, upstream and downstream coding regions were identified, and the deduced amino acid sequences shown as SEQ ID NOS. 10 and 11.
- Homologues to the GBS pho2-10 gene product can be identified in Streptococcus pyogenes, Enterococcus faecalis, Debaryomyces occidentalis (hatI) and Escherichia coli (trkD).
- the S. pyogenes and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- the yeast D. occidentalis the hak1 gene is a homologue of the trkD gene from E. coli (Banuelos, M. A. et al. 1995. EMBO J. 14:3021-3027).
- the coli is part of the kup potassium uptake system.
- the specific homolog identified here is the kup system potassium uptake protein.
- the kup system is a constitutive potassium uptake system in E. coli .
- the kup system potassium uptake protein contains a highly hydrophobic N-terminus that is predicted to span the membrane at least 12 times. Kup is not homologous to other known membrane protein sequences. There is no indication of ATP binding, and it is proposed that the system is driven by a chemiosmotic gradient (Schleyer, M. & E. P. Bakker, 1993. J. Bacteriol. 175:6925-6931).
- a further clone was selected containing a plasmid designated pho2-15.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 12 and 13.
- Homologues to the GBS pho2-15 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis and Escherichia coli (gatC and SgcC).
- the S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- the gatC and sgcC gene products can be identified as being the IIC component of phosphoenolypyruvate-dependent sugar phosphotransferase systems (PTS), a major carbohydrate active-transport system.
- PTS phosphoenolypyruvate-dependent sugar phosphotransferase systems
- the IIC component is typically involved in binding of extracellular carbohydrates and forms a complex with the IID component to constitute a membrane channel (Nobelmann, B. and J. W. Lengeler. 1995. Biochim. Biophys. Acta 1262:69-72).
- a further clone was selected containing a plasmid designated pho2-2.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 14 and 15, respectively.
- Homologues to the GBS pho2-2 gene product can be identified in Enterococcus faecalis, Escherichia coli (malK and afuC), Bacillus subtilis (glnO), Haemophilus influenzae (yebM and potA), Streptococcus pyogenes, Streptococcus pneumoniae and Salmonella typhimurium (malK).
- the E. faecalis, S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- homologues represented ATP-binding transport proteins that are part of ABC type transporters. Many of the components of ABC type transporters are membrane or cell surface associated, as these systems are involved in the transport of macromolecules from the extracellular environment to the intracellular compartment.
- a further clone was selected containing a plasmid designated pho3-14.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 16 and 17.
- a further clone was selected containing a plasmid designated pho3-17.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 18 and 19.
- GBS Pho3-17 gene product Homologues to the GBS Pho3-17 gene product can be identified in Streptococcus mutans and Lactococcus lactis , with similarity being shown to N-acetyl muramidase. Similarity is also seen with an unidentified gene, yubE from Bacillus subtilis.
- N-acetylmuramidase is an autolysin that is involved in cell division. Using this limited information along with the fact that pho3-17 complemented the leaderless phoA gene, it can be concluded that the pho3-17 product would most probably be located extracellularly.
- a further clone was selected containing a plasmid designated pho3-18.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 20 and 21.
- Homologues to the GBS pho3-18 gene product can be identified in Streptococcus pyogenes and Streptococcus pneumoniae . These homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Using these S. pyogenes and S. pneumoniae homologues to search the public databases showed some similarity to outer surface and membrane spanning proteins. Since the ORF3-18 product complemented the leaderless phoA gene, it can be concluded that this protein (or part thereof) would most probably be located extracellularly.
- a further clone was selected containing a plasmid designated pho3-1.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 22 and 23.
- Homologues to the GBS pho3-1 gene product can be identified in Streptococcus pyogenes, streptococcus pneumoniae, Bacillus subtilis (yutD) and Enterococcus faecalis .
- the S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- B. subtilis the function of the yutD gene product is unknown. It can be noted however, that the yutD gene is located on the B. subtilis chromosome in a region containing genes involved in cell wall synthesis. The fact that this DNA sequence complemented the leaderless phoA gene suggests that this gene product is extracellularly located.
- a further clone was selected containing a plasmid designated pho3-21.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 24 and 25.
- Homologues to the GBS pho3-21 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus fermentum (bspA) and Lactobacillus reuteri (cnb).
- the S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- the bspA gene product has been identified as being a basic cell surface-located protein that has some sequence similarity to family III of the bacterial solute-binding proteins (Turner, M. S. et al. 1997. J. Bacteriol. 179:3310-3316).
- L. fermentum the bspA gene product has been identified as being a basic cell surface-located protein that has some sequence similarity to family III of the bacterial solute-binding proteins (Turner, M. S. et al. 1997. J. Bacteriol. 179
- the cnb gene product has been identified as a collagen binding protein that has some sequence similarity to the solute-binding component of bacterial ABC transporters (Roos, S. et al. 1996. FEMS Microbiol. Lett. 144:33-38).
- a further clone was selected containing a plasmid designated pho3-22.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 26 and 27.
- Homologues to the GBS pho3-22 gene product can be identified in Enterococcus faecalis, Streptococcus equisimilis (lppC), Pseudomonas fluorescens (oprI) and Streptococcus thermophilus (orf142).
- the E. faecalis homolog was identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- the lppC gene product has been identified as being a lipoprotein that is homologous to the E(P4) outer membrane protein from Haemophilus influenzae (Gase, K. et al. 1997. Med. Microbiol. Immunol.
- the P. fluorescens oprI gene encodes a major outer membrane lipoprotein (Cornelis, P. et al. 1989. Mol. Microbiol. 3:421-428).
- the orf142 product has been putatively identified as a cell surface exposed lipoprotein that may act as a receptor for the bacteriophages TP-J34 and Sfi21 (Neve, H. et al. 1998. Virology 241:61-72).
- the ORF3-22 product showed good similarity to the above homologues, particularly at the N-terminus. This is most likely the region required for complementation of the leaderless phoA gene, and therefore serves as a leader sequence.
- a further clone was selected containing a plasmid designated pho3-23.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the genes are shown as SEQ ID NOS. 28 and 29.
- Homologues to the GBS pho3-23 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis and Streptococcus mutans (perM).
- the S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- S. mutans the perM gene product has been presumptively identified as a permease, but no other information is available as to the function of this protein.
- the pho3-23 coding region complements the leaderless phoA gene, it can be concluded that the pho3-17 gene product would most probably be located extracellularly.
- a further clone was selected containing a plasmid designated pho3-24.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 30 and 31.
- Homologues to the GBS pho3-24 gene product can be identified in Streptococcus mutans (dltB), Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Lactobacillus casei (dltB) and Bacillus subtilis (dltB).
- the S. pneumoniae, S. pyogenes and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
- S. mutans L. casei and B.
- subtilis the dltB gene product has been identified as being a basic membrane protein that is involved in the transport of activated D-alanine through the cell membrane.
- the dltB gene product is involved in the biosynthesis of D-alanyl-lipoteichoic acid (Heaton, M. P. and F. C. Neuhaus. 1992. J. Bacteriol. 174:4707-4717).
- the dltB gene product is believed to contain at least 9 membrane spanning domains, indicating that the protein or portions thereof are exposed to the outside of the cell.
- a further clone was selected containing a plasmid designated pho3-29.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 32 and 33.
- Homologues to the GBS pho3-29 gene product can be identified in Borrelia burgdorferi (p23 or ospC), Bacillus brevis (owp) and Pseudomonas aeruginosa (oprI). Although these homologues are not related to each other, they all represent major outer surface proteins.
- the ospC gene product has been identified as being a 23-kDa protein that is the immunodominant antigen on the surface of this bacterium (Padula, S. J. et al. 1993. Infect. Immun. 61:5097-5105).
- a further clone was selected containing a plasmid designated pho3-50.
- This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- the nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 34 and 35.
- Homologues to the GBS pho3-50 gene product can be identified in a variety of Streptococci (penA, pbp2B, pbpB2), Borrelia burgdorferi (pbp2), Enterococcus faecalis (pbpC), Staphylococcus aureus (pbpA), Mycobacterium laprae (pbpB) and Helicobacter pylori (pbp2).
- the above homologues can be identified as penicillin binding proteins (PBPs). Genes encoding penicillin binding proteins are often located in a cluster of genes associated with cell wall synthesis (Pucci, M. J. et al. 1997. J. Bacteriol. 179:5632-5635).
- PBPs are typically integrated into the cell wall of a bacterium with some or all of the protein being exposed on the outer bacterial surface.
Abstract
Description
- This invention relates to the identification of bacterial genes and proteins, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.
- Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is the causative agent of various conditions. In particular, GBS causes:
- This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.
- This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis.
- These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis.
- GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.
- GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
- GBS infections can be treated with antibiotics. However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.
- The present invention is based on the identification of a series of genes in GBS, and also related organisms, the products of which may be localised on the outer surface of the organism and therefore may be used as a target for immuno-therapy.
- According to one aspect of the invention, a peptide is encoded by an operon including any of the genes identified herein as pho1-13, pho3-21, pho2-15, pho3-18, pho3-22, pho3-3, pho3-17, pho2-2, pho1-5, pho3-1, pho3-23, pho3-50, pho1-14, pho2-10, pho3-14, pho3-24 and pho3-29, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.
- The term “functional fragments” is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.
- A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targeting the wild-type gene in vivo to exert a therapeutic effect.
- A peptide according to the present invention may comprise any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35, or a functional fragment thereof.
- Because of the extracellular or cell surface location, the peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS. The term “therapeutically-effective” is intended to include the prophylactic effect of vaccines. For example, a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection. The vaccine may be administered to females prior to or during pregnancy to protect mother and neonate against infection by GBS.
- According to another aspect of the invention, the peptides or genes may be used for screening potential antimicrobial drugs or for the detection of virulence.
- A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.
- Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.
- The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732. Organisms likely to contain the peptides include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.
- Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.
- Having characterised a gene according to the invention, it is possible to use the gene sequence to establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank.
- Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.
- The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms. The preparation of vaccine formulations will be apparent to the skilled person.
- More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.
- The products of the present invention were identified as follows:
- A partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3 (Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess a constitutive spectinomycin adenyltransferase antibiotic resistance marker, which confers a high level of spectinomycin resistance and is therefore easily selected. Furthermore, these vectors contain a truncated (leaderless) Escherichia coli phoA gene for alkaline phosphatase. The three vectors differ only with respect to the reading frame in which the leaderless phoA gene exists, as compared to an upstream in-frame BamHI restriction enzyme site. Because this truncated E. coli phoA gene lacks the appropriate leader sequence for export of this enzyme across the bacterial membrane, extracellular alkaline phosphatase activity is absent when these plasmids are propagated in an E. coli phoA mutant (e.g. strain DH5α). The chromogenic alkaline phosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate), does not enter intact bacterial cells and therefore only exported or surface associated alkaline phosphatase activity can be detected. When exported or surface associated alkaline phosphatase activity is present, the chromogenic XP substrate is cleaved to yield a blue pigment and the corresponding bacterial colonies can be identified by their blue colour.
- Plasmid DNA was digested to completion with BamHI and dephosphorylated using shrimp alkaline phosphatase. GBS genomic DNA was partially digested with Sau3AI, size fractionated on a sucrose gradient and fragments <1 kb in size were ligated into the prepared pFW-phoA vectors. E. coli strain DH5α was chosen as the cloning host since it lacks a functional phoA gene. Recombinant plasmids were selected on Luria agar containing 100 μg/ml of spectinomycin and 40 μg/ml of the chromogenic XP substrate. E. coli transformants harbouring plasmids containing GBS insert DNA that complements the export signal sequence of the leaderless phoA gene were identified by the blue colour of the colonies. Approximately 30000 different recombinant plasmids containing GBS insert DNA were screened in this manner and 83 recombinant plasmids, which complemented the leaderless phoA, were chosen for further study.
- From these experiments, several clones were selected each containing a plasmid containing a gene (or part thereof), which complemented the leaderless phoA.
- Having identified the gene in each clone it is then possible to obtain the full-length gene sequence, as follows.
- Using the identified and sequenced gene fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an ‘outward’ direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5′ and 3′ termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5′ end the presence of an AUG start codon (or accepted equivalent) preceded by a Shine-Dalgarno consensus sequence, and for the 3′ end, the presence of a translation termination (Stop) codon.
- Upon identification of the full-length gene, primers were designed for amplification of full-length product. Primers used included restriction enzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end) to allow subsequent cloning of the product into the Lactococcal expression system used.
- PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (In Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I.
- The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10× His tags, flanked on the 5-most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a Stop (termination) codon at the 3′ end of the His tags.
- The gene of interest was inserted so that a 10× His tag was in the 3′ position relative to the coding region. Following transformation of the recombinant plasmid into L. lactis (strain NZ9000-Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.
- To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti-His antibodies.
- The recombinant protein obtained was then used to immunize New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.
- Using the sera obtained from the animal studies, immunosorption studies were carried out.
- Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4° C. overnight to allow for absorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3% BSA in PBS for 1 hr at 37° C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50 μl of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37° C. The plate was washed, then 50 μl anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30 min before the absorbance was read at 405 nm.
- Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits.
- GBS M732 was grown up in THB until mid-log phase was reached—approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2×107 bacteria per ml in pre-immune or test sera. 50 μl of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.
- The following Examples illustrate the invention.
- A first clone contained a gene sequence identified herein as SEQ ID NO. 1, with an amino acid sequence identified as SEQ ID NO. 2, and classified as pho1-13.
- A comparison of the amino acid sequence of pho1-13 was performed.
- Homologues to the GBS pho1-13 gene product can be identified in Streptococcus pyogenes, S. pneumoniae, S. salivarius, Escherichia coli, Yersinia enterocolitica, Aquifex aeolicus, Helicobacter pylori and Haemophilus influenzae. The S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In all other cases, the above homologues can be identified as ATP-dependent Clp protease proteolytic subunits. The catalytic activity of Clp proteases results in the hydrolysis of proteins to small peptides in the presence of ATP and magnesium (Giffard, P. M. et al. 1993. J. Gen. Microbiol. 139:913-920). Furthermore, the ClpP component of Clp proteases has been shown to be induced as part of the heat shock response (Kroh, H. E. and L. D. Simon. 1990. J. Bacteriol. 172:6026-6034) and it is probable that this subunit or the complete proteolytic domain would associated with the bacterial surface.
- Immunisation studies, carried out as described above, yielded the following results.
-
No animals surviving at time (hrs) Treatment No animals 24 48 PBS 10 7 0 Pre-immunised 37 13 0 Immunised 38 17 9 - A second clone was selected containing a plasmid designated pho1-14. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 3 and 4, respectively.
- A comparison of the amino acid sequence of pho1-14 was performed.
- Homologues to the GBS pho1-14 gene product can be identified in Streptococcus pyogenes, Enterococcus faecalis and Streptococcus pneumoniae. These homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Additionally, two possible homologues were also identified from Shigella flexneri (SpaR) and Yersinia pseudotuberculosis (YscT). These latter two homologues are related proteins, believed to be anchored in the bacterial membrane (Bergman, T. et al. 1994. J. Bacteriol. 176:2619-2626). In S. flexneri, the product of the spaR gene has been shown to be important for invasion of epithelial cells (Sasakawa, C. et al. 1993. J. Bacteriol. 175:2334-2346). Furthermore, the product of the spaR gene is also required for surface presentation of invasion plasmid antigens. The analogous protein in Y. pseudotuberculosis is a component of the Yop secretion system and is also important for virulence in this organism.
- A third clone was selected containing a plasmid designated pho1-5. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 5 and 6.
- A comparison of the amino acid sequence of pho1-5 was performed.
- Homologues to the GBS pho1-5 gene product can only be identified in streptococcus pyogenes and Staphylococcus carnosus (sceA). The S. pyogenes homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Furthermore, little information is available on the function of the sceA gene product from S. carnosus. The sceA gene product shows some sequence similarity to the aggregation promoting protein from Lactobacillus gasseri. Based on analysis of the sceA gene product, this molecule contains a well-conserved signal sequence and is apparently secreted or associated with the bacterial cell surface.
- A further clone was selected containing a plasmid designated pho3-3. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences are shown as SEQ ID NOS. 7 and 8.
- A comparison of the amino acid sequence of pho3-3 was performed.
- Homologues to the GBS pho3-3 gene product can be identified in Streptococcus mutans (rmiC), (cpsM) S. pneumoniae and S. pyogenes. The S. pyogenes homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene product. In S. pneumoniae, the homologue can be identified as dTDP-4-keto-6-deoxy glucose-3,5-epimerase. In the other two cases, the above homologues can be identified as dTDP-4-keto-L-rhamnose reductase (rmlC). In S. mutants, the gene encoding this enzyme, rmlC, is part of the rml locus. The rml locus consists of three genes which exhibit significant similarity to enzymes involved in the biosynthesis of dTDP-rhamnose, the immediate precursor of the rhamnose component in the S. mutans polysaccharide capsule (Tsukioka, Y. et al. 1997. J. Bacteriol. 179:1126-1134). An analogous locus has also been identified in S. pneumoniae (Coffey, T. J. et al. 1998. Mol. Micobiol. 17:73-83). Almost all Streptococci characteristically possess rhamnose in their cell wall associated polysaccharides (Schleifer, K. H. and R. Kilper-Bälz. 1987. Syst. Appl. Microbiol. 10:1-19), and it is highly probable that dTDP-4-keto-L-rhamnose reductase would be associated with the outer surface in Streptococci.
- A further clone was selected containing a plasmid designated pho2-10. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA.
- The nucleotide sequence is shown as SEQ ID NO. 9. From this, upstream and downstream coding regions were identified, and the deduced amino acid sequences shown as SEQ ID NOS. 10 and 11.
- A comparison of the amino acid sequences of pho2-10 was performed.
- Homologues to the GBS pho2-10 gene product can be identified in Streptococcus pyogenes, Enterococcus faecalis, Debaryomyces occidentalis (hatI) and Escherichia coli (trkD). The S. pyogenes and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In the yeast D. occidentalis, the hak1 gene is a homologue of the trkD gene from E. coli (Banuelos, M. A. et al. 1995. EMBO J. 14:3021-3027). The trkd gene of E. coli is part of the kup potassium uptake system. The specific homolog identified here is the kup system potassium uptake protein. The kup system is a constitutive potassium uptake system in E. coli. The kup system potassium uptake protein contains a highly hydrophobic N-terminus that is predicted to span the membrane at least 12 times. Kup is not homologous to other known membrane protein sequences. There is no indication of ATP binding, and it is proposed that the system is driven by a chemiosmotic gradient (Schleyer, M. & E. P. Bakker, 1993. J. Bacteriol. 175:6925-6931).
- A further clone was selected containing a plasmid designated pho2-15. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 12 and 13.
- A comparison of the amino acid sequence of pho2-15 was performed.
- Homologues to the GBS pho2-15 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis and Escherichia coli (gatC and SgcC). The S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In E. coli, the gatC and sgcC gene products can be identified as being the IIC component of phosphoenolypyruvate-dependent sugar phosphotransferase systems (PTS), a major carbohydrate active-transport system. In PTS systems, the IIC component is typically involved in binding of extracellular carbohydrates and forms a complex with the IID component to constitute a membrane channel (Nobelmann, B. and J. W. Lengeler. 1995. Biochim. Biophys. Acta 1262:69-72).
- A further clone was selected containing a plasmid designated pho2-2. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 14 and 15, respectively.
- A comparison of the amino acid sequence of pho2-2 was performed.
- Homologues to the GBS pho2-2 gene product can be identified in Enterococcus faecalis, Escherichia coli (malK and afuC), Bacillus subtilis (glnO), Haemophilus influenzae (yebM and potA), Streptococcus pyogenes, Streptococcus pneumoniae and Salmonella typhimurium (malK). The E. faecalis, S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In all other cases, homologues represented ATP-binding transport proteins that are part of ABC type transporters. Many of the components of ABC type transporters are membrane or cell surface associated, as these systems are involved in the transport of macromolecules from the extracellular environment to the intracellular compartment.
- A further clone was selected containing a plasmid designated pho3-14. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 16 and 17.
- A comparison of the amino acid sequence of pho3-14 was performed and no homologues could be identified in any of the public databases. One homologue to the GBS pho3-14 gene product can be identified in Streptococcus pyogenes, but this homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene product. Using this S. pyogenes homologue to search the public databases yielded no further information. Since the pho3-14 product complemented the leaderless phoA gene, it can be concluded that this protein (or part thereof) would most probably be located extracellularly.
- A further clone was selected containing a plasmid designated pho3-17. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 18 and 19.
- A comparison of the amino acid sequence of pho3-17 was performed.
- Homologues to the GBS Pho3-17 gene product can be identified in Streptococcus mutans and Lactococcus lactis, with similarity being shown to N-acetyl muramidase. Similarity is also seen with an unidentified gene, yubE from Bacillus subtilis.
- N-acetylmuramidase is an autolysin that is involved in cell division. Using this limited information along with the fact that pho3-17 complemented the leaderless phoA gene, it can be concluded that the pho3-17 product would most probably be located extracellularly.
- A further clone was selected containing a plasmid designated pho3-18. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 20 and 21.
- A comparison of the amino acid sequence of pho3-18 was performed.
- Homologues to the GBS pho3-18 gene product can be identified in Streptococcus pyogenes and Streptococcus pneumoniae. These homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Using these S. pyogenes and S. pneumoniae homologues to search the public databases showed some similarity to outer surface and membrane spanning proteins. Since the ORF3-18 product complemented the leaderless phoA gene, it can be concluded that this protein (or part thereof) would most probably be located extracellularly.
- A further clone was selected containing a plasmid designated pho3-1. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 22 and 23.
- A comparison of the amino acid sequence of pho3-1 was performed.
- Homologues to the GBS pho3-1 gene product can be identified in Streptococcus pyogenes, streptococcus pneumoniae, Bacillus subtilis (yutD) and Enterococcus faecalis. The S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In B. subtilis, the function of the yutD gene product is unknown. It can be noted however, that the yutD gene is located on the B. subtilis chromosome in a region containing genes involved in cell wall synthesis. The fact that this DNA sequence complemented the leaderless phoA gene suggests that this gene product is extracellularly located.
- A further clone was selected containing a plasmid designated pho3-21. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 24 and 25.
- A comparison of the amino acid sequence of pho3-21 was performed.
- Homologues to the GBS pho3-21 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus fermentum (bspA) and Lactobacillus reuteri (cnb). The S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In L. fermentum, the bspA gene product has been identified as being a basic cell surface-located protein that has some sequence similarity to family III of the bacterial solute-binding proteins (Turner, M. S. et al. 1997. J. Bacteriol. 179:3310-3316). In L. reuteri, the cnb gene product has been identified as a collagen binding protein that has some sequence similarity to the solute-binding component of bacterial ABC transporters (Roos, S. et al. 1996. FEMS Microbiol. Lett. 144:33-38).
- A further clone was selected containing a plasmid designated pho3-22. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 26 and 27.
- A comparison of the amino acid sequence of pho3-22 was performed.
- Homologues to the GBS pho3-22 gene product can be identified in Enterococcus faecalis, Streptococcus equisimilis (lppC), Pseudomonas fluorescens (oprI) and Streptococcus thermophilus (orf142). The E. faecalis homolog was identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In S. equisimilis, the lppC gene product has been identified as being a lipoprotein that is homologous to the E(P4) outer membrane protein from Haemophilus influenzae (Gase, K. et al. 1997. Med. Microbiol. Immunol. 186:63-73). Likewise, the P. fluorescens oprI gene encodes a major outer membrane lipoprotein (Cornelis, P. et al. 1989. Mol. Microbiol. 3:421-428). In S. thermophilus, the orf142 product has been putatively identified as a cell surface exposed lipoprotein that may act as a receptor for the bacteriophages TP-J34 and Sfi21 (Neve, H. et al. 1998. Virology 241:61-72). The ORF3-22 product showed good similarity to the above homologues, particularly at the N-terminus. This is most likely the region required for complementation of the leaderless phoA gene, and therefore serves as a leader sequence.
- A further clone was selected containing a plasmid designated pho3-23. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the genes are shown as SEQ ID NOS. 28 and 29.
- A comparison of the amino acid sequence of pho3-23 was performed.
- Homologues to the GBS pho3-23 gene product can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis and Streptococcus mutans (perM). The S. pyogenes, S. pneumoniae and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In S. mutans, the perM gene product has been presumptively identified as a permease, but no other information is available as to the function of this protein. Considering that the pho3-23 coding region complements the leaderless phoA gene, it can be concluded that the pho3-17 gene product would most probably be located extracellularly.
- A further clone was selected containing a plasmid designated pho3-24. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 30 and 31.
- A comparison of the amino acid sequence of pho3-24 was performed.
- Homologues to the GBS pho3-24 gene product can be identified in Streptococcus mutans (dltB), Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Lactobacillus casei (dltB) and Bacillus subtilis (dltB). The S. pneumoniae, S. pyogenes and E. faecalis homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In S. mutans, L. casei and B. subtilis, the dltB gene product has been identified as being a basic membrane protein that is involved in the transport of activated D-alanine through the cell membrane. The dltB gene product is involved in the biosynthesis of D-alanyl-lipoteichoic acid (Heaton, M. P. and F. C. Neuhaus. 1992. J. Bacteriol. 174:4707-4717). In L. casei and B. subtilis, the dltB gene product is believed to contain at least 9 membrane spanning domains, indicating that the protein or portions thereof are exposed to the outside of the cell.
- A further clone was selected containing a plasmid designated pho3-29. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 32 and 33.
- A comparison of the amino acid sequence of pho3-29 was performed.
- Homologues to the GBS pho3-29 gene product can be identified in Borrelia burgdorferi (p23 or ospC), Bacillus brevis (owp) and Pseudomonas aeruginosa (oprI). Although these homologues are not related to each other, they all represent major outer surface proteins. In B. burgdorferi, the ospC gene product has been identified as being a 23-kDa protein that is the immunodominant antigen on the surface of this bacterium (Padula, S. J. et al. 1993. Infect. Immun. 61:5097-5105). The owp gene product from B. brevis is one of two major cell wall proteins involved in the surface layer lattice (Tsuboi, A. 1988. J. Bacteriol. 170:935-945). Finally, the oprI gene from P. aeruginosa encodes a major outer membrane lipoprotein precursor (Saint-Onge, A. et al. 1992. J. Gen. Microbiol. 138:733-741).
- A further clone was selected containing a plasmid designated pho3-50. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 34 and 35.
- A comparison of the amino acid sequence of pho3-50 was performed.
- Homologues to the GBS pho3-50 gene product can be identified in a variety of Streptococci (penA, pbp2B, pbpB2), Borrelia burgdorferi (pbp2), Enterococcus faecalis(pbpC), Staphylococcus aureus (pbpA), Mycobacterium laprae(pbpB) and Helicobacter pylori (pbp2). In all cases, the above homologues can be identified as penicillin binding proteins (PBPs). Genes encoding penicillin binding proteins are often located in a cluster of genes associated with cell wall synthesis (Pucci, M. J. et al. 1997. J. Bacteriol. 179:5632-5635). Furthermore, PBPs are typically integrated into the cell wall of a bacterium with some or all of the protein being exposed on the outer bacterial surface.
Claims (11)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/892,024 US20090274717A1 (en) | 1998-12-22 | 2007-08-17 | Genes and proteins, and their use |
Applications Claiming Priority (38)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9828352.6 | 1998-12-22 | ||
GBGB9828353.4A GB9828353D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containig it |
GBGB9828357.5A GB9828357D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828345.0 | 1998-12-22 | ||
GBGB9828349.2A GB9828349D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828356.7 | 1998-12-22 | ||
GBGB9828354.2 | 1998-12-22 | ||
GBGB9828349.2 | 1998-12-22 | ||
GBGB9828353.4 | 1998-12-22 | ||
GBGB9828352.6A GB9828352D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828350.0A GB9828350D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828350.0 | 1998-12-22 | ||
GBGB9828354.2A GB9828354D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828356.7A GB9828356D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828359.1A GB9828359D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828345.0A GB9828345D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828359.1 | 1998-12-22 | ||
GBGB9828355.9A GB9828355D0 (en) | 1998-12-22 | 1998-12-22 | Protein and compositions containing it |
GBGB9828355.9 | 1998-12-22 | ||
GBGB9828357.5 | 1998-12-22 | ||
GBGB9900086.1A GB9900086D0 (en) | 1999-01-04 | 1999-01-04 | Protein and compositions containing it |
GBGB9900085.3 | 1999-01-04 | ||
GBGB9900086.1 | 1999-01-04 | ||
GBGB9900083.8A GB9900083D0 (en) | 1999-01-04 | 1999-01-04 | Protein and compositions containing it |
GBGB9900083.8 | 1999-01-04 | ||
GBGB9900085.3A GB9900085D0 (en) | 1999-01-04 | 1999-01-04 | Protein and compositions containing it |
GBGB9900082.0 | 1999-01-04 | ||
GBGB9900084.6A GB9900084D0 (en) | 1999-01-04 | 1999-01-04 | Protein and compositions containing it |
GBGB9900082.0A GB9900082D0 (en) | 1999-01-04 | 1999-01-04 | Protein and compositions containing it |
GBGB9900084.6 | 1999-01-04 | ||
GBGB9901916.8A GB9901916D0 (en) | 1999-01-28 | 1999-01-28 | Protein and compositions containing it |
GBGB9901922.6A GB9901922D0 (en) | 1999-01-28 | 1999-01-28 | Protein and compositions containing it |
GBGB9901922.6 | 1999-01-28 | ||
GBGB9901916.8 | 1999-01-28 | ||
PCT/GB1999/004377 WO2000037646A2 (en) | 1998-12-22 | 1999-12-22 | Group b streptococcus proteins, and their use |
US09/868,352 US6812021B1 (en) | 1998-12-22 | 1999-12-22 | Genes and proteins and their use |
US10/799,072 US7419672B2 (en) | 1998-12-22 | 2004-03-12 | Genes and proteins, and their use |
US11/892,024 US20090274717A1 (en) | 1998-12-22 | 2007-08-17 | Genes and proteins, and their use |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/799,072 Division US7419672B2 (en) | 1998-12-22 | 2004-03-12 | Genes and proteins, and their use |
Publications (1)
Publication Number | Publication Date |
---|---|
US20090274717A1 true US20090274717A1 (en) | 2009-11-05 |
Family
ID=27585888
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/868,352 Expired - Fee Related US6812021B1 (en) | 1998-12-22 | 1999-12-22 | Genes and proteins and their use |
US10/799,072 Expired - Fee Related US7419672B2 (en) | 1998-12-22 | 2004-03-12 | Genes and proteins, and their use |
US11/892,024 Abandoned US20090274717A1 (en) | 1998-12-22 | 2007-08-17 | Genes and proteins, and their use |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/868,352 Expired - Fee Related US6812021B1 (en) | 1998-12-22 | 1999-12-22 | Genes and proteins and their use |
US10/799,072 Expired - Fee Related US7419672B2 (en) | 1998-12-22 | 2004-03-12 | Genes and proteins, and their use |
Country Status (23)
Country | Link |
---|---|
US (3) | US6812021B1 (en) |
EP (3) | EP2105504A3 (en) |
JP (1) | JP2002533083A (en) |
KR (3) | KR100790653B1 (en) |
CN (1) | CN101066447A (en) |
AP (1) | AP1502A (en) |
AT (1) | ATE353367T1 (en) |
AU (1) | AU776735B2 (en) |
CA (1) | CA2354135A1 (en) |
CY (1) | CY1106559T1 (en) |
CZ (1) | CZ20012172A3 (en) |
DK (1) | DK1141308T3 (en) |
EA (1) | EA004444B1 (en) |
ES (1) | ES2281977T3 (en) |
HK (1) | HK1039353A1 (en) |
HU (1) | HUP0104825A3 (en) |
NO (1) | NO20013102L (en) |
NZ (3) | NZ526879A (en) |
OA (1) | OA11734A (en) |
PL (1) | PL201887B1 (en) |
PT (1) | PT1141308E (en) |
SI (1) | SI1141308T1 (en) |
WO (1) | WO2000037646A2 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PL201887B1 (en) * | 1998-12-22 | 2009-05-29 | Microscience Ltd | Genes and proteins, and their use |
CN1733800A (en) * | 1998-12-22 | 2006-02-15 | 微科学有限公司 | Outer surface proteins, their genes, and their use |
GB0105922D0 (en) * | 2001-03-09 | 2001-04-25 | Microscience Ltd | Genes and proteins, and their use |
US6890539B2 (en) | 1998-12-22 | 2005-05-10 | Microscience, Ltd. | Genes and proteins, and their use |
EP1360324A2 (en) | 2000-08-31 | 2003-11-12 | Mayo Foundation For Medical Education And Research | Detection of varicella-zoster virus |
GB0021977D0 (en) | 2000-09-07 | 2000-10-25 | Pyrosequencing Ab | Method of sequencing DNA |
US7691571B2 (en) | 2001-01-31 | 2010-04-06 | Mayo Foundation For Medical Education And Research | Detection of bordetella |
US7074598B2 (en) | 2002-09-25 | 2006-07-11 | Mayo Foundation For Medical Education And Research | Detection of vancomycin-resistant enterococcus spp. |
US7365176B2 (en) | 2002-09-26 | 2008-04-29 | Mayo Foundation For Medical Education And Research | Detection of Epstein-Barr virus |
AU2003298770A1 (en) * | 2002-12-02 | 2004-06-23 | Biosynexus Inc | Wall teichoic acid as a target for anti-staphylococcal therapies and vaccines |
US7427475B2 (en) | 2003-11-18 | 2008-09-23 | Mayo Foundation For Medical Education And Research | Detection of group B streptococcus |
WO2008152448A2 (en) * | 2006-12-21 | 2008-12-18 | Emergent Product Development Uk Limited | Streptococcus proteins, and their use in vaccination |
US10114982B2 (en) | 2012-05-18 | 2018-10-30 | Tata Consultancy Services Limited | Method and system for determining and implementing a viable containment design of a data center |
CN106822885B (en) * | 2017-02-16 | 2020-06-30 | 清华大学 | Streptococcus pneumoniae vaccine |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5328996A (en) * | 1989-03-29 | 1994-07-12 | University Of Florida Research Foundation, Inc. | Bacterial plasmin receptors as fibrinolytic agents |
US5807978A (en) * | 1995-06-07 | 1998-09-15 | Kokolus; William J. | Immunogenic peptides of prostate specific antigen |
US6015889A (en) * | 1993-03-19 | 2000-01-18 | Gunnar Lindahl | Protein rib, a cell surface protein that confers immunity to many strains of the group B streptococcus: process for purification of the protein, reagent kit and pharmaceutical composition |
US6248329B1 (en) * | 1998-06-01 | 2001-06-19 | Ramaswamy Chandrashekar | Parasitic helminth cuticlin nucleic acid molecules and uses thereof |
US20030104000A1 (en) * | 1998-12-22 | 2003-06-05 | Hughes Martin John Glenton | Genes and proteins, and their use |
US6605709B1 (en) * | 1999-04-09 | 2003-08-12 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Proteus mirabilis for diagnostics and therapeutics |
US6699703B1 (en) * | 1997-07-02 | 2004-03-02 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
US20040044191A1 (en) * | 1997-07-08 | 2004-03-04 | Fischer Carrie L. | 123 human secreted proteins |
US6812021B1 (en) * | 1998-12-22 | 2004-11-02 | Microscience Limited | Genes and proteins and their use |
US20060104990A1 (en) * | 1998-12-22 | 2006-05-18 | Hughes Martin J G | Outer surface proteins, their genes, and their use |
US7098023B1 (en) * | 1997-07-02 | 2006-08-29 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
US20070053936A1 (en) * | 1997-08-14 | 2007-03-08 | Doucette-Stamm Lynn A | Nucleic acid and amino acid sequences relating to Staphylococcus epidermidis for diagnostics and therapeutics |
Family Cites Families (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0672123A1 (en) | 1992-01-08 | 1995-09-20 | The Rockefeller University | Multifunctional surface protein of streptococci |
HU217582B (en) * | 1993-03-19 | 2000-02-28 | Gunnar Lindahl | Protein rib, a cell surface protein that confers immunity to strains of the group b streptococcus; process for purification of the protein; reagent kit, and pharmaceutical composition |
CA2269663A1 (en) | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
EP2280072B1 (en) | 1998-02-20 | 2015-03-25 | ID Biomedical Corporation of Quebec | Group B streptococcus antigens |
CA2333578C (en) | 1998-07-14 | 2010-11-16 | American Cyanamid Company | Adjuvant and vaccine compositions containing monophosphoryl lipid a |
JP2002531054A (en) | 1998-07-27 | 2002-09-24 | マイクロビアル テクニクス リミティッド | Nucleic acids and proteins from group B streptococci |
EP1124978A4 (en) | 1998-10-30 | 2003-11-12 | Janssen Pharmaceutica Nv | An unusual retrotransposon from the yeast candida albicans |
AU1818200A (en) | 1998-11-05 | 2000-05-22 | Daniel Carucci | Chromosome 2 sequence of the human malaria parasite (plasmodium falciparum) and proteins of said chromosome useful in anti-malarial vaccines and diagnostic reagents |
DE60028195T2 (en) | 1999-04-09 | 2007-03-15 | Heska Corp., Fort Collins | PROTEINS AND NUCLEIC ACID MOLECULES OF THE HEAD, NERVENKORDS, DEEP PEEL AND MALPIGHI FLOW AND THEIR USES |
GB9921125D0 (en) | 1999-09-07 | 1999-11-10 | Microbial Technics Limited | Proteins |
AU1478301A (en) | 1999-11-09 | 2001-06-06 | Glaxo Group Limited | Staphylococcus epidermidis nucleic acids and proteins |
CA2404988A1 (en) | 2000-04-11 | 2001-10-18 | Institut Pasteur | Listeria monocytogenes genome, polypeptides and uses |
US6974667B2 (en) | 2000-06-14 | 2005-12-13 | Gene Logic, Inc. | Gene expression profiles in liver cancer |
CA2881568C (en) | 2000-10-27 | 2019-09-24 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from streptococcus groups a & b |
AU2002306849A1 (en) | 2001-03-21 | 2002-10-08 | Elitra Pharmaceuticals, Inc. | Identification of essential genes in microorganisms |
GB0107658D0 (en) | 2001-03-27 | 2001-05-16 | Chiron Spa | Streptococcus pneumoniae |
GB0107661D0 (en) | 2001-03-27 | 2001-05-16 | Chiron Spa | Staphylococcus aureus |
EP2258842A1 (en) | 2001-04-16 | 2010-12-08 | Wyeth Holdings Corporation | Streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof |
WO2004030608A2 (en) | 2001-06-05 | 2004-04-15 | The Regents Of The University Of Michigan | Nanoemulsion vaccines |
EP1409013B1 (en) | 2001-07-26 | 2009-11-18 | Novartis Vaccines and Diagnostics S.r.l. | Vaccines comprising aluminium adjuvants and histidine |
GB0210128D0 (en) | 2002-05-02 | 2002-06-12 | Chiron Spa | Nucleic acids and proteins from streptococcus groups A & B |
WO2004041157A2 (en) | 2002-09-13 | 2004-05-21 | Chiron Corporation | Group b streptococcus vaccine |
CA2522986A1 (en) | 2003-05-07 | 2004-11-18 | Intercell Ag | Streptococcus agalactiae antigens i + ii |
EP1663303B1 (en) | 2003-09-15 | 2011-05-04 | Novartis Vaccines and Diagnostics, Inc. | Immunogenic compositions for streptococcus agalactiae |
CA2582137A1 (en) | 2004-10-05 | 2007-02-15 | Wyeth | Probe arrays for detecting multiple strains of different species |
US20090104218A1 (en) | 2004-12-22 | 2009-04-23 | J. Craig Venter Institute | Group B Streptococcus |
-
1999
- 1999-12-22 PL PL349320A patent/PL201887B1/en not_active IP Right Cessation
- 1999-12-22 NZ NZ526879A patent/NZ526879A/en unknown
- 1999-12-22 EP EP09159100A patent/EP2105504A3/en not_active Withdrawn
- 1999-12-22 NZ NZ535122A patent/NZ535122A/en unknown
- 1999-12-22 NZ NZ512297A patent/NZ512297A/en unknown
- 1999-12-22 KR KR1020017007911A patent/KR100790653B1/en not_active IP Right Cessation
- 1999-12-22 CA CA002354135A patent/CA2354135A1/en not_active Abandoned
- 1999-12-22 PT PT99962422T patent/PT1141308E/en unknown
- 1999-12-22 SI SI9930959T patent/SI1141308T1/en unknown
- 1999-12-22 US US09/868,352 patent/US6812021B1/en not_active Expired - Fee Related
- 1999-12-22 WO PCT/GB1999/004377 patent/WO2000037646A2/en active IP Right Grant
- 1999-12-22 AT AT99962422T patent/ATE353367T1/en not_active IP Right Cessation
- 1999-12-22 CN CNA2007101018407A patent/CN101066447A/en active Pending
- 1999-12-22 AU AU18780/00A patent/AU776735B2/en not_active Ceased
- 1999-12-22 HU HU0104825A patent/HUP0104825A3/en unknown
- 1999-12-22 EP EP06019847A patent/EP1757696A3/en not_active Withdrawn
- 1999-12-22 AP APAP/P/2001/002189A patent/AP1502A/en active
- 1999-12-22 EA EA200100700A patent/EA004444B1/en not_active IP Right Cessation
- 1999-12-22 EP EP99962422A patent/EP1141308B1/en not_active Expired - Lifetime
- 1999-12-22 JP JP2000589700A patent/JP2002533083A/en active Pending
- 1999-12-22 OA OA1200100163A patent/OA11734A/en unknown
- 1999-12-22 DK DK99962422T patent/DK1141308T3/en active
- 1999-12-22 ES ES99962422T patent/ES2281977T3/en not_active Expired - Lifetime
- 1999-12-22 CZ CZ20012172A patent/CZ20012172A3/en unknown
-
2001
- 2001-06-21 NO NO20013102A patent/NO20013102L/en not_active Application Discontinuation
- 2001-12-20 HK HK01108928A patent/HK1039353A1/en unknown
-
2004
- 2004-03-12 US US10/799,072 patent/US7419672B2/en not_active Expired - Fee Related
-
2007
- 2007-05-04 CY CY20071100589T patent/CY1106559T1/en unknown
- 2007-08-17 US US11/892,024 patent/US20090274717A1/en not_active Abandoned
- 2007-09-28 KR KR1020070098153A patent/KR20070101197A/en not_active Application Discontinuation
-
2008
- 2008-05-09 KR KR1020080043336A patent/KR20080050550A/en not_active Application Discontinuation
Patent Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5328996A (en) * | 1989-03-29 | 1994-07-12 | University Of Florida Research Foundation, Inc. | Bacterial plasmin receptors as fibrinolytic agents |
US6015889A (en) * | 1993-03-19 | 2000-01-18 | Gunnar Lindahl | Protein rib, a cell surface protein that confers immunity to many strains of the group B streptococcus: process for purification of the protein, reagent kit and pharmaceutical composition |
US5807978A (en) * | 1995-06-07 | 1998-09-15 | Kokolus; William J. | Immunogenic peptides of prostate specific antigen |
US7098023B1 (en) * | 1997-07-02 | 2006-08-29 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
US6699703B1 (en) * | 1997-07-02 | 2004-03-02 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
US6800744B1 (en) * | 1997-07-02 | 2004-10-05 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
US7196164B2 (en) * | 1997-07-08 | 2007-03-27 | Human Genome Sciences, Inc. | Secreted protein HHTLF25 |
US20040044191A1 (en) * | 1997-07-08 | 2004-03-04 | Fischer Carrie L. | 123 human secreted proteins |
US20070053936A1 (en) * | 1997-08-14 | 2007-03-08 | Doucette-Stamm Lynn A | Nucleic acid and amino acid sequences relating to Staphylococcus epidermidis for diagnostics and therapeutics |
US6248329B1 (en) * | 1998-06-01 | 2001-06-19 | Ramaswamy Chandrashekar | Parasitic helminth cuticlin nucleic acid molecules and uses thereof |
US20030104000A1 (en) * | 1998-12-22 | 2003-06-05 | Hughes Martin John Glenton | Genes and proteins, and their use |
US20050131210A1 (en) * | 1998-12-22 | 2005-06-16 | Glenton Hughes Martin J. | Genes and proteins, and their use |
US20060104990A1 (en) * | 1998-12-22 | 2006-05-18 | Hughes Martin J G | Outer surface proteins, their genes, and their use |
US6890539B2 (en) * | 1998-12-22 | 2005-05-10 | Microscience, Ltd. | Genes and proteins, and their use |
US6812021B1 (en) * | 1998-12-22 | 2004-11-02 | Microscience Limited | Genes and proteins and their use |
US7217415B1 (en) * | 1998-12-22 | 2007-05-15 | Emergent Product Development Uk Limited | NADP-dependent glyceraldehyde-3-phosphate dehydrogenase for therapeutic use |
US6605709B1 (en) * | 1999-04-09 | 2003-08-12 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Proteus mirabilis for diagnostics and therapeutics |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20090274717A1 (en) | Genes and proteins, and their use | |
KR101684292B1 (en) | Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use | |
US20040265933A1 (en) | Proteins | |
US20080226641A1 (en) | Outer surface proteins, their genes, and their use | |
US6890539B2 (en) | Genes and proteins, and their use | |
AU2004201404B2 (en) | Genes and proteins, and their use | |
NZ543923A (en) | Pho3-18 for a theraputic use, particulary in bacterial infection. | |
WO2002072623A1 (en) | Genes and proteins, and their use | |
ZA200104818B (en) | Genes and proteins, and their use. | |
AU2007200004A1 (en) | Genes and proteins, and their use | |
PL195869B1 (en) | Outer surface proteins, their genes, and their use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: MICROSCIENCE LIMITED, UNITED KINGDOM Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HUGHES, MARTIN JOHN GLENTON;SANTANGELO, JOSEPH DAVID;LANE, JONATHAN DOUGLAS;AND OTHERS;REEL/FRAME:023073/0964;SIGNING DATES FROM 20010626 TO 20010712 Owner name: EMERGENT PRODUCT DEVELOPMENT UK LIMITED, UNITED KI Free format text: CHANGE OF NAME;ASSIGNOR:EMERGENT EUROPE LIMITED;REEL/FRAME:023074/0044 Effective date: 20060601 Owner name: EMERGENT EUROPE LIMITED, UNITED KINGDOM Free format text: CHANGE OF NAME;ASSIGNOR:MICROSCIENCE LIMITED;REEL/FRAME:023074/0016 Effective date: 20050722 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE |