WO2000006736A2 - Nucleic acids and proteins from group b streptococcus - Google Patents
Nucleic acids and proteins from group b streptococcus Download PDFInfo
- Publication number
- WO2000006736A2 WO2000006736A2 PCT/GB1999/002444 GB9902444W WO0006736A2 WO 2000006736 A2 WO2000006736 A2 WO 2000006736A2 GB 9902444 W GB9902444 W GB 9902444W WO 0006736 A2 WO0006736 A2 WO 0006736A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- group
- dna
- streptococcus
- proteins
- protein
- Prior art date
Links
- 241000193990 Streptococcus sp. 'group B' Species 0.000 title claims abstract description 95
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 27
- 102000004169 proteins and genes Human genes 0.000 title claims description 91
- 108020004707 nucleic acids Proteins 0.000 title claims description 21
- 102000039446 nucleic acids Human genes 0.000 title claims description 21
- 108091005461 Nucleic proteins Proteins 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 39
- 108091007433 antigens Proteins 0.000 claims abstract description 32
- 102000036639 antigens Human genes 0.000 claims abstract description 31
- 238000012216 screening Methods 0.000 claims abstract description 23
- 229960005486 vaccine Drugs 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 174
- 108020004414 DNA Proteins 0.000 claims description 105
- 239000012634 fragment Substances 0.000 claims description 58
- 239000013598 vector Substances 0.000 claims description 52
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 44
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 36
- 239000000427 antigen Substances 0.000 claims description 28
- 208000015181 infectious disease Diseases 0.000 claims description 27
- 229920001184 polypeptide Polymers 0.000 claims description 25
- 238000001514 detection method Methods 0.000 claims description 21
- 241000194035 Lactococcus lactis Species 0.000 claims description 19
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 17
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 15
- 230000014509 gene expression Effects 0.000 claims description 15
- 101710163270 Nuclease Proteins 0.000 claims description 13
- 241000192125 Firmicutes Species 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 230000002163 immunogen Effects 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000013519 translation Methods 0.000 claims description 9
- 230000028327 secretion Effects 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims description 3
- 239000003623 enhancer Substances 0.000 claims description 3
- 230000000415 inactivating effect Effects 0.000 claims description 3
- 238000011321 prophylaxis Methods 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 241001505901 Streptococcus sp. 'group A' Species 0.000 claims description 2
- 230000000845 anti-microbial effect Effects 0.000 claims description 2
- 239000004599 antimicrobial Substances 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 239000013615 primer Substances 0.000 description 92
- 230000004083 survival effect Effects 0.000 description 65
- 241000699670 Mus sp. Species 0.000 description 55
- 108010041986 DNA Vaccines Proteins 0.000 description 33
- 229940021995 DNA vaccine Drugs 0.000 description 33
- 230000002441 reversible effect Effects 0.000 description 32
- 238000002105 Southern blotting Methods 0.000 description 27
- 239000000523 sample Substances 0.000 description 21
- 238000010367 cloning Methods 0.000 description 20
- 238000002474 experimental method Methods 0.000 description 20
- 208000024891 symptom Diseases 0.000 description 20
- 101150100619 nuc gene Proteins 0.000 description 17
- 238000002649 immunization Methods 0.000 description 16
- 150000004676 glycans Chemical class 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 15
- 229920001282 polysaccharide Polymers 0.000 description 15
- 239000005017 polysaccharide Substances 0.000 description 15
- 241000193985 Streptococcus agalactiae Species 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 11
- 239000003298 DNA probe Substances 0.000 description 11
- 238000007619 statistical method Methods 0.000 description 11
- 230000036039 immunity Effects 0.000 description 10
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 239000002054 inoculum Substances 0.000 description 9
- 230000034994 death Effects 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 108020003215 DNA Probes Proteins 0.000 description 7
- 239000003155 DNA primer Substances 0.000 description 7
- 108010052285 Membrane Proteins Proteins 0.000 description 7
- 102000018697 Membrane Proteins Human genes 0.000 description 7
- 230000001681 protective effect Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 101000865057 Thermococcus litoralis DNA polymerase Proteins 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 230000002035 prolonged effect Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 230000036961 partial effect Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- 101710117545 C protein Proteins 0.000 description 3
- 208000003643 Callosities Diseases 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 239000006161 blood agar Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 231100000518 lethal Toxicity 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 210000003705 ribosome Anatomy 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 238000013207 serial dilution Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108010060123 Conjugate Vaccines Proteins 0.000 description 2
- 238000011238 DNA vaccination Methods 0.000 description 2
- 206010011906 Death Diseases 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 208000035752 Live birth Diseases 0.000 description 2
- 201000009906 Meningitis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 2
- 208000006816 Neonatal Sepsis Diseases 0.000 description 2
- 206010061308 Neonatal infection Diseases 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 102100024952 Protein CBFA2T1 Human genes 0.000 description 2
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 206010043376 Tetanus Diseases 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 2
- 229940031670 conjugate vaccine Drugs 0.000 description 2
- 230000001268 conjugating effect Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000012215 gene cloning Methods 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000003019 stabilising effect Effects 0.000 description 2
- 229960000814 tetanus toxoid Drugs 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 108010087967 type I signal peptidase Proteins 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000193401 Clostridium acetobutylicum Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000701867 Enterobacteria phage T7 Species 0.000 description 1
- 101100277543 Escherichia coli (strain K12) deoR gene Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101100349541 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) nucS2 gene Proteins 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 101150036121 MARCKS gene Proteins 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical compound CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 206010035039 Piloerection Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 108090000233 Signal peptidase II Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 108010015795 Streptogramin B Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- -1 adhesion molecules Proteins 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 101150040225 bca gene Proteins 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229940041028 lincosamides Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000005371 pilomotor reflex Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- YGXCETJZBDTKRY-DZCVGBHJSA-N pristinamycin IA Chemical compound N([C@@H]1C(=O)N[C@@H](C(N2CCC[C@H]2C(=O)N(C)[C@@H](CC=2C=CC(=CC=2)N(C)C)C(=O)N2CCC(=O)C[C@H]2C(=O)N[C@H](C(=O)O[C@@H]1C)C=1C=CC=CC=1)=O)CC)C(=O)C1=NC=CC=C1O YGXCETJZBDTKRY-DZCVGBHJSA-N 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 229920006298 saran Polymers 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 150000004992 toluidines Chemical class 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
-
- 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
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
Definitions
- the present invention relates to proteins derived from Streptococcus agalactiae, nucleic acid molecules encoding such proteins, and the use of the proteins as antigens and/or immunogens and in detection/diagnosis. It also relates to a method for the rapid screening of bacterial genomes to isolate and characterise bacterial cell envelope associated or secreted proteins.
- the Group B Streptococcus (GBS) (Streptococcus agalactiae) is an encapsulated bacterium which emerged in the 1970s as a major pathogen of humans causing sepsis and meningitis in neonates as well as adults.
- GBS Group B Streptococcus
- the incidence of early onset neonatal infection during the first 5 days of life varies from 0.7 to 3.7 per 1000 live births and causes mortality in about 20% of cases. Between 25-50% of neonates surviving early onset infections frequently suffer neurological sequalae. Late onset neonatal infections occur from 6 days to three months of age at a rate of about 0.5 - 1.0 per 1000 live births.
- GBS colonisation of the maternal genetic tract by GBS at the time of birth and the risk of neonatal sepsis.
- the rectum may act as a reservoir for GBS.
- Susceptibility in the neonate is correlated with the a low concentration or absence of IgG antibodies to the capsular polysaccharides found on GBS causing human disease.
- Type VIII GBS is the major cause of neonatal sepsis in Japan.
- a possible means of prevention involves intra or postpartum administration of antibiotics to the mother but there are concerns that this might lead to the emergence of resistant organisms and in some cases allergic reactions.
- Vaccination of the adolescent females to induce long lasting maternally derived immunity is one of the most promising approaches to prevent GBS infections in neonates.
- the capsular polysaccharide antigens of these organisms have attracted most attention as with regard to vaccine development. Studies in healthy adult volunteers have shown that serotype la, II and III polysaccharides are non-toxic and immunogenic in approximately 65%, 95% and 70% of non-immune adults respectively.
- capsule antigens as vaccines.
- the response rates vary according to pre-immunisation status and the polysaccharide antigen and not all vaccinees produce adequate levels of IgG antibody as indicated in vaccination studies with GBS polysaccharides in human volunteers.
- Rib A protein which is found on most serotype III strains but rarely on serotypes la, lb or II confers immunity to challenge with Rib expressing GBS in animal models (Stalhammar-Carlemalm et al, Journal of Experimental Medicine 177:1593-1603 (1993)).
- Another surface protein of interest as a component of a vaccine is the alpha antigen of the C proteins which protected vaccinated mice against lethal infection with strains expressing alpha protein. The amount of antigen expressed by GBS strains varies markedly.
- This invention seeks to overcome the problem of vaccination against GBS by using a novel screening method specifically designed to identify those Group B Streptococcus genes encoding bacterial cell surface associated or secreted proteins (antigens).
- the proteins expressed by these genes may be immunogenic, and therefore may be useful in the prevention and treatment of Group B Streptococcus infection.
- immunogenic means that these proteins will elicit a protective immune response within a subject.
- the present invention provides a Group B Streptococcus protein, having a sequence selected from those shown in figure 1 , or fragments or derivatives thereof.
- proteins and polypeptides included within this group may be cell surface receptors, adhesion molecules, transport proteins, membrane structural proteins, and/or signalling molecules.
- Alterations in the amino acid sequence of a protein can occur which do not affect the function of a protein. These include amino acid deletions, insertions and substitutions and can result from alternative splicing and/or the presence of multiple translation start sites and stop sites. Polymorphisms may arise as a result of the infidelity of the translation process. Thus changes in amino acid sequence may be tolerated which do not affect the proteins function.
- the present invention includes derivatives or variants of the proteins, polypeptides, and peptides of the present invention which show at least 50%> identity to the proteins, polypeptides and peptides described herein.
- the degree of sequence identity is at least 60%> and preferably it is above 75%o. More preferably still is it above 80%, 90% or even 95%.
- identity can be used to describe the similarity between two polypeptide sequences.
- a software package well known in the art for carrying out this procedure is the CLUSTAL program. It compares the amino acid sequences of two polypeptides and finds the optimal alignment by inserting spaces in either sequence as appropriate.
- amino acid identity or similarity identity plus conservation of amino acid type
- BLASTx a software package such as BLASTx. This program aligns the largest stretch of similar sequence and assigns a value to the fit. For any one pattern comparison several regions of similarity may be found, each having a different score.
- two polypeptides of different lengths may be compared over the entire length of the longer fragment. Alternatively small regions may be compared. Normally sequences of the same length are compared for a useful comparison to be made.
- Manipulation of the DNA encoding the protein is a particularly powerful technique for both modifying proteins and for generating large quantities of protein for purification purposes. This may involve the use of PCR techniques to amplify a desired nucleic acid sequence.
- sequence data provided herein can be used to design primers for use in PCR so that a desired sequence can be targeted and then amplified to a high degree.
- primers will be at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases primers of at least thirty or at least thirty-five nucleotides in length may be used.
- chemical synthesis may be used. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.
- the present invention provides , a nucleic acid molecule comprising or consisting of a sequence which is: (i) any of the DNA sequences set out in figure 1 herein or their RNA equivalents; (ii) a sequence which is complementary to any of the sequences of (i); (iii) a sequence which codes for the same protein or polypeptide, as those sequences of (i) or (ii);
- identity can also be used to describe the similarity between two individual
- the 'bestfit' program (Smith and Waterman, Advances in applied Mathematics, 482-489 (1981)) is one example of a type of computer software used to find the best segment of similarity between two nucleic acid sequences, whilst the GAP program enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
- RNA equivalent' when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA 'U' replaces 'T' in the genetic code.
- the nucleic acid molecule may be in isolated or recombinant form.
- the nucleic acid molecule may be in an isolated or recombinant form.
- DNA constructs can readily be generated using methods well known in the art. These techniques are disclosed, for example in J. Sambrook et al, Molecular Cloning 2 nd Edition, Cold Spring Harbour Laboratory Press (1989). Modifications of DNA constructs and the proteins expressed such as the addition of promoters, enhancers, signal sequences, leader sequences, translation start and stop signals and DNA stability controlling regions, or the addition of fusion partners may then be facilitated.
- the DNA construct will be inserted into a vector which may be of phage or plasmid origin. Expression of the protein is achieved by the transformation or transfection of the vector into a host cell which may be of eukaryotic or prokaryotic origin.
- Such vectors and suitable host cells form yet further aspects of the present invention.
- the Group B Streptococcus proteins (antigens) described herein can additionally be used to raise antibodies, or to generate affibodies. These can be used to detect Group B Streptococcus.
- the present invention provides, an antibody, affibody, or a derivative thereof which binds to any one or more of the proteins, polypeptides, peptides, fragments or derivatives thereof, as described herein.
- Antibodies within the scope of the present invention may be monoclonal or polyclonal.
- Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a protein as described herein, or a homologue, derivative or fragment thereof, is injected into the animal.
- a suitable animal host e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey
- an adjuvant may be administered together with the protein.
- Well- known adjuvants include Freund 's adjuvant (complete and incomplete) and aluminium hydroxide.
- the antibodies can then be purified by virtue of their binding to a protein as described herein.
- Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 (1975)) or subsequent variations upon this technique can be used.
- the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein.
- the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
- Antibody fragments include, for example, Fab, F(ab') 2 and Fv fragments.
- Fab fragments include, for example, Fab, F(ab') 2 and Fv fragments.
- Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V n and Vi regions, which contributes to the stability of the molecule.
- Other synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.
- Synthetic constructs include chimaeric molecules.
- humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention.
- a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PNAS, 81, 6851-6855 (1984) and by Takeda et al in Nature. 314, 452-454 (1985).
- Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding.
- the moiety may be a label (e.g. a fluorescent or radioactive label).
- it may be a pharmaceutically active agent.
- Affibodies are proteins which are found to bind to target proteins with a low dissociation constant. They are selected from phage display libraries expressing a segment of the target protein of interest (Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Department of Biochemistry and Biotechology, Royal Institute of Technology (KTH), Sweden).
- the invention provides an immunogenic composition comprising one or more proteins, polypeptides, peptides, fragments or derivatives thereof, or nucleotide sequences described herein.
- a composition of this sort may be useful in the treatment or prevention of Group B Streptococcus infection in subject.
- the immunogenic composition is a vaccine.
- the invention provides:
- an immunogenic composition as described herein in the preparation of a medicament for the treatment or prophylaxis of Group B Streptococcus infection.
- the medicament is a vaccine.
- a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one antibody, affibody, or a derivative thereof, as described herein.
- a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one protein, polypeptide, peptide, fragments or derivatives as described herein.
- a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one nucleic acid molecule as described herein.
- a kit for the detection of Group B Streptococcus comprising at least one antibody, affibody, or derivatives thereof, described herein.
- kits for the detection of Group B Streptococcus comprising at least one Group B Streptococcus protein, polypeptide, peptide, fragment or derivative thereof, as described herein.
- kits for the detection of Group B Streptococcus comprising at least one nucleic acid of the invention.
- novel proteins described herein are identified and isolated using a novel screening method which specifically identifies those Group B Streptococcus genes encoding bacterial cell envelope associated or secreted proteins.
- Staphylococcal nuclease is a naturally secreted heat-stable, monomeric enzyme which has been efficiently expressed and secreted in a range of Gram positive bacteria (Shortle., Gene 22:181-189 (1983), Kovacevic et al, J. Bacteriol. 162:521-528 (1985), Miller et al, J. Bacteriol. 169:3508-3514 (1987), Liebl et al, J. Bacteriol. 174:1854-1861(1992), Le Loir et al, J. Bacteriol. 176:5135-5139 (1994), Poquet et al, 1998 [supra]).
- the screening vector contains the pAM ⁇ l replicon which functions in a broad host range of Gram-positive bacteria in addition to the ColEl replicon that promotes replication in Escherichia coli and certain other Gram negative bacteria.
- Unique cloning sites present in the vector can be used to generate transcriptional and translational fusions between cloned genomic DNA fragments and the open reading frame of the truncated nuc gene devoid of its own signal secretion leader.
- the nuc gene makes an ideal reporter gene because the secretion of nuclease can readily be detected using a simple and sensitive plate test: Recombinant colonies secreting the nuclease develop a pink halo whereas control colonies remain white
- a direct screen to identify and isolate DNA encoding bacterial cell envelope associated or secreted proteins (antigens). in pathogenic bacteria has been developed by the present inventors which utilises a vector-system (pTREPl expression vector) in
- Lactococcus lactis that specifically detects DNA sequences which are adjacent to, and associated with DNA encoding surface proteins from Group B Streptococcus.
- the screening vector also incorporates the nuc gene encoding the Staphylococcal nuclease as a reporter gene.
- nuc gene encoding the mature nuclease protein (minus its signal peptide sequence) is cloned into the pTREPl expression vector in L. lactis. In this form, the nuc-encoded nuclease cannot be secreted even when expressed intracellularly.
- the reporter vector is then randomly combined with appropriately digested genomic DNA from Group B Streptococcus, cloned into L. lactis and used as a screening system for sequences permitting the export of nuclease. In this way gene/partial gene sequences encoding exported proteins from Group B Streptococcus are isolated. Once a partial gene sequence is obtained, full length sequences encoding exported proteins can readily be obtained using techniques well known in the art.
- the pTREPl -nuc vectors differ from the pFUN vector described by Poquet et al. (1998) [supra], which was used to identify L. lactis exported proteins by screening directly for Nuc activity directly in L. lactis.
- the pFUN vector does not contain a promoter upstream of the nuc open reading frame the cloned genomic DNA fragment must also provide the signals for transcription in addition to those elements required for translation initiation and secretion of Nuc. This limitation may prevent the isolation of genes that are distant from a promoter for example genes which are within polycistronic operons. Additionally there can be no guarantee that promoters derived from other species of bacteria will be recognised and functional in L. lactis.
- promoters may be under stringent regulation in the natural host but not in L. lactis.
- the presence of the PI promoter in the pTREPl -nuc series of vectors ensures that promoterless DNA fragments (or DNA fragments containing promoter sequences not active in L. lactis) may still be transcribed.
- promoterless DNA fragments or DNA fragments containing promoter sequences not active in L. lactis
- genes missed in other screening methods may be identified.
- the present invention provides a method of screening for DNA encoding bacterial cell wall associated or surface antigens in gram positive bacteria comprising the steps of:
- the reporter vector is one of the pTREPl -nuc vectors shown in figure 4.
- the present invention provides a vector as shown in figure 4 for use in screening for DNA encoding exported or surface antigens in gram positive bacteria.
- gram positive bacteria which may be screened include Group B Streptococcus, Streptococcus pneumoniae, Staphylcoccus aureus or pathogenic Group A Streptococci.
- the present invention also provides a method of determining whether a protein or polypeptide as described herein represents a potential anti-microbial target which comprises inactivating said protein and determining whether Group B Streptococcus is still viable.
- a suitable method for inactivating the protein is to effect selected gene knockouts, ie prevent expression of the protein and determine whether this results in a lethal change. Suitable methods for carrying out such gene knockouts are described in Li et al , P.N.A.S., 94: 13251-13256 (1997) and Kolkman et al
- the present invention provides the use of an agent capable of antagonising, inhibiting or otherwise interfering with the function or expression of a protein or polypeptide of the invention in the manufacture of a medicament for use in the treatment or prophylaxis of Group B Streptococcus infection.
- Fig 1 Shows a number of full length nucleotide sequences encoding antigenic Group B Streptococcus proteins.
- B Shows the corresponding amino acid sequences.
- Fig 2 Shows a number of oligonucleotide primers used in the screening process
- nucSl primer designed to amplify a mature form of the nuc A gene
- nucS2- primer designed to amplify a mature form of the nuc A gene
- nucS3 primer designed to amplify a mature form of the nuc A gene
- nucR primer designed to amplify a mature form of the nuc A gene
- nucseq primer designed to sequence DNA cloned into the pTREP-Nuc vector pTREPF nucleic acid sequence containing recognition site for ECORV. Used for cloning fragments into pTREX7.
- pTREPR nucleic acid sequence containing recognition site for BAMH1. Used for cloning fragments into pTREX7.
- PUCF forward sequencing primer enables direct sequencing of cloned DNA fragments.
- VR example of gene specific primer used to obtain further antigen DNA sequence by the method of DNA walking VI example of gene specific primer used to obtain further antigen DNA sequence by the method of DNA walking.
- Fig 3 (i) Schematic presentation of the nucleotide sequence of the unique gene cloning site immediately upstream of the mature nuc gene in pTREPl-nwcl, pTREPl -nuc2 and pTREPl -nuc3.
- Each of the pTREP- ⁇ uc vectors contain an EcoRV (a Smal site in pTREPl -nuc2) cleavage site which allows cloning of genomic DNA fragments in 3 different frames with respect to the mature nuc gene.
- Fig 4 Shows the results of various DNA vaccine trials
- Fig 5 Shows the results of a second group of DNA vaccine trials
- Figs 6-11 Show various Southern Blot analyses of different Group B streptococcus strains.
- All putative surface proteins are analysed for leader/signal peptide sequences.
- Bacterial signal peptide sequences share a common design. They are characterised by a short positively charged N-terminus (N region) immediately preceding a stretch of hydrophobic residues (central portion-h region) followed by a more polar C-terminal portion which contains the cleavage site (c-region).
- Computer software is used to perform hydropathy profiling of putative proteins (Marcks, Nuc. Acid. Res., 16:1829-1836 (1988)) which is used to identify the distinctive hydrophobic portion (h-region) typical of leader peptide sequences.
- the presence/absence of a potential ribosomal binding site is also noted.
- Putative S. agalactiae surface proteins are also be assessed for their novelty. Some of the identified proteins may or may not possess a typical leader peptide sequence and may not show homology with any DNA/protein sequences in the database. Indeed these proteins may indicate the primary advantage of our screening method, i.e. isolating atypical surface-related proteins, which would have been missed in all previously described screening protocols.
- the pTREPl plasmid is a high-copy number (40-80 per cell) theta-replicating gram positive plasmid, which is a derivative of the pTREX plasmid which is itself a derivative of the the previously published pIL253 plasmid.
- pIL253 incorporates the broad Gram-positive host range replicon of pAM ⁇ l (Simon and Chopin, 1988) and is non-mobilisable by the L lactis sex-factor.
- pIL253 also lacks the tra function which is necessary for transfer or efficient mobilisation by conjugative parent plasmids exemplified by pIL501.
- the Enterococcal pAM ⁇ l replicon has previously been transferred to various species including Streptococcus, Lactobacillus and Bacillus species as well as Clostridium acetobutylicum, (LeBlanc et al, Proceedings of the National Academy of Science USA 75:3484-3487 (1978)) indicating the potential broad host range utility.
- the pTREPl plasmid represents a constitutive transcription vector.
- the pTREX vector was constructed as follows. An artificial DNA fragment containing a putative RNA stabilising sequence, a translation initiation region (TIR), a multiple cloning site for insertion of the target genes and a transcription terminator was created by annealing 2 complementary oligonucleotides and extending with Tfl DNA polymerase. The sense and anti-sense oligonucleotides contained the recognition sites for Nhel and BamHI at their 5' ends respectively to facilitate cloning. This fragment was cloned between the Xbal and BamHI sites in pUC19NT7, a derivative of pUC19 which contains the T7 expression cassette from pLETl (Wells et al, J. Appl. Bacteriol.
- pUCLEX The complete expression cassette of pUCLEX was then removed by cutting with Hindlll and blunting followed by cutting with EcoRI before cloning into EcoRI and Sad (blunted) sites of pIL253 to generate the vector pTREX (Wells and Schofield, In Current advances in metabolism, genetics and applications-NATO ASI Series. H 98:37-62. (1996)).
- RNA stabilising sequence and TIR are derived from the Escherichia coli T7 bacteriophage sequence and modified at one nucleotide position to enhance the complementarity of the Shine Dalgarno (SD) motif to the ribosomal 16s RNA of Lactococcus lactis (Schofield et al. pers. corns. University of Cambridge Dept. Pathology.).
- a Lactococcus lactis MG1363 chromosomal DNA fragment exhibiting promoter activity which was subsequently designated P7 was cloned between the EcoRI and Bglll sites present in the expression cassette, creating pTREX7. This active promoter region had been previously isolated using the promoter probe vector pSB292 (Waterfield et al, Gene 165:9-15 (1995)). The promoter fragment was amplified by
- the pTREPl vector was then constructed as follows. An artificial DNA fragment which included a transcription terminator, the forward pUC sequencing primer, a promoter multiple cloning site region and a universal translation stop sequence was created by annealing two overlapping partially complementary synthetic oligonucleotides together and extending with sequenase according to manufacturers instructions.
- the sense and anti-sense (pTREPF and pTREP ) oligonucleotides contained the recognition sites for EcoRV and BamHI at their 5' ends respectively to facilitate cloning into pTREX7.
- the transcription terminator was that of the Bacillus penicillinase gene, which has been shown to be effective in Lactococcus (Jos et al, Applied and Environmental Microbiology 50:540-542 (1985)). This was considered necessary as expression of target genes in the pTREX vectors was observed to be leaky and is thought to be the result of cryptic promoter activity in the origin region (Schofield et al. pers. corns. University of Cambridge Dept. Pathology.).
- the forward pUC primer sequencing was included to enable direct sequencing of cloned DNA fragments.
- the translation stop sequence which encodes a stop codon in 3 different frames was included to prevent translational fusions between vector genes and cloned DNA fragments.
- the pTREX7 vector was first digested with EcoRI and blunted using the 5' - 3' polymerase activity of T4 DNA polymerase (NEB) according to manufacturer's instructions.
- the EcoRI digested and blunt ended pTREX7 vector was then digested with Bgl II thus removing the P7 promoter.
- the artificial DNA fragment derived from the annealed synthetic oligonucleotides was then digested with EcoRV and Bam HI and cloned into the EcoRI(blunted)-Bgl II digested pTREX7 vector to generate pTREP.
- a Lactococcus lactis MG1363 chromosomal promoter designated PI was then cloned between the EcoRI and Bglll sites present in the pTREP expression cassette forming pTREPl.
- This promoter was also isolated using the promoter probe vector pSB292 and characterised by Waterfield et al, (1995) [supra].
- the PI promoter fragment was originally amplified by PCR using vent DNA polymerase according to manufacturers instructions and cloned into the pTREX as an EcoRI-Bglll DNA fragment.
- the EcoRI-Bglll PI promoter containing fragment was removed from pTREXl by restriction enzyme digestion and used for cloning into pTREP (Schofield et al pers. corns. University of Cambridge, Dept. Pathology.).
- the nucleotide sequence of the S. aureus nuc gene (EMBL database accession number V01281) was used to design synthetic oligonucleotide primers for PCR amplification.
- the primers were designed to amplify the mature form of the nuc gene designated nuc A which is generated by proteolytic cleavage of the N-terminal 19 to 21 amino acids of the secreted propeptide designated Snase B (Shortle, 1983 [supra]).
- Three sense primers «wcSl, nucS2 and nucS3, shown in figure 3
- Bglll and BamHI were incorporated at the 5' ends of the sense and anti-sense primers respectively to facilitate cloning into BamHI and Bglll cut pTREPl.
- the sequences of all the primers are given in figure 3.
- Three nuc gene DNA fragments encoding the mature form of the nuclease gene (NucA) were amplified by PCR using each of the sense primers combined with the anti-sense primer.
- the nuc gene fragments were amplified by PCR using S. aureus genomic DNA template, Vent DNA Polymerase (NEB) and the conditions recommended by the manufacturer.
- the purified nuc gene fragments described in section b were digested with Bgl II and BamHI using standard conditions and li gated to BamHI and Bglll cut and dephosphorylated pTREPl to generate the pTREPl -nucl, pTREPl -nuc2 and pTREPl -nuc3 series of reporter vectors. These vectors are described in figure 4.
- General molecular biology techniques were carried out using the reagents and buffers supplied by the manufacturer or using standard techniques (Sambrook and Maniatis, Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbour (1989)).
- the expression cassette comprises a transcription terminator, lactococcal promoter PI, unique cloning sites (Bglll, EcoRV or Smal) followed by the mature form of the nuc gene and a second transcription terminator.
- a transcription terminator lactococcal promoter PI
- unique cloning sites Bglll, EcoRV or Smal
- sequences required for translation and secretion of the nuc gene were deliberately excluded in this construction.
- Such elements can only be provided by appropriately digested foreign DNA fragments (representing the target bacterium) which can be cloned into the unique restriction sites present immediately upstream of the nuc gene.
- Genomic DNA isolated from and Group B Streptococcus (S. agalactiae) was digested with the restriction enzyme Tru9I.
- This enzyme which recognises the sequence 5'- TTAA -3' was used because it cuts A/T rich genomes efficiently and can generate random genomic DNA fragments within the preferred size range (usually averaging 0.5 - 1.0 kb).
- This size range was preferred because there is an increased probability that the PI promoter can be utilised to transcribe a novel gene sequence.
- the PI promoter may not be necessary in all cases as it is possible that many Streptococcal promoters are recognised in L. lactis.
- DNA fragments of different size ranges were purified from partial Tru9I digests of and S.
- Tru9I digested DNA was dissolved in a solution (usually between 10-20 ⁇ l in total) supplemented with T4 DNA ligase buffer (New England Biolabs; NEB) (IX) and 33 ⁇ M of each of the required dNTPs, in this case dATP and dTTP.
- Klenow enzyme was added (1 unit Klenow enzyme (NEB) per ⁇ g of DNA) and the reaction incubated at 25 °C for 15 minutes. The reaction was stoped by incubating the mix at 75 °C for 20 minutes. EcoRV or Smal digested pTREP- ⁇ wc plasmid DNA was then added (usually between 200-400 ng). The mix was then supplemented with 400 units of T4 DNA ligase (NEB) and T4 DNA ligase buffer (IX) and incubated overnight at 16°C. The ligation mix was precipiated directly in 100%) Ethanol and 1/10 volume of 3M sodium acetate (pH 5.2) and used to transform L. lactis MG1363 (Gasson, J.
- the gene cloning site of the pTREP- «uc vectors also contains a Bglll site which can be used to clone for example Sau3AI digested genomic DNA fragments.
- pcDNA3.1 The commercially available pcDNA3.1+ plasmid (Invitrogen), referred to as pcDNA3.1 henceforth, was used as a vector in all DNA immunisation experiments involving gene targets derived using the LEEP system.
- pcDNA3.1 is designed for high-level stable and transient expression in mammalian cells and has been used widely and successfully as a host vector to test candidate genes from a variety of pathogens in DNA vaccination experiments (Zhang et al, 1997; Kurar and Splitter, 1997; Anderson et al, 1996).
- the vector possesses a multiple cloning site which facilitates the cloning of multiple gene targets downstream of the human cytomegalovirus (CMV) immediate-early promoter/enhancer which permits efficient, high-level expression of the target gene in a wide variety of mammalian cells and cell types including both muscle and immune cells. This is important for optimal immune response as it remains unknown as to which cells types are most important in generating a protective response in vivo.
- the plasmid also contains the ColEl origin of replication which allows convenient high- copy number replication and growth in E. coli and the ampicillin resistance gene (B- lactamase) for selection in E. coli.
- pcDNA 3.1 possesses a T7 promoter/priming site upstream of the MCS which allows for in vitro transcription of a cloned gene in the sense orientation.
- Oligonucleotide primers were designed for each individual gene of interest derived using the LEEP system. Each gene was examined thoroughly, and where possible, primers were designed such that they targeted that portion of the gene thought to encode only the mature portion of the protein (APPENDIX I). It was hoped that expressing those sequences that encode only the mature portion of a target gene protein, would facilitate its correct folding when expressed in mammalian cells. For example, in the majority of cases primers were designed such that putative N-terminal signal peptide sequences would not be included in the final amplification product to be cloned into the pcDNA3.1 expression vector.
- the signal peptide directs the polypeptide precursor to the cell membrane via the protein export pathway where it is normally cleaved off by signal peptidase I (or signal peptidase II if a lipoprotein). Hence the signal peptide does not make up any part of the mature protein whether it be displayed on the bacterium's surface or secreted. Where a N-terminal leader peptide sequence was not immediately obvious, primers were designed to target the whole of the gene sequence for cloning and ultimately, expression in pcDNA3.1.
- All forward and reverse oligonucleotide primers incorporated appropriate restriction enzyme sites to facilitate cloning into the pcDNA3.1 MCS region. All forward primers were also designed to include the conserved Kozak nucleotide sequence 5'-gccacc-3' immediately upstream of an 'atg' translation initiation codon in frame with the target gene insert. The Kozak sequence facilitates the recognition of initiator sequences by eukaryotic ribosomes. Typically, a forward primer incorporating a BamHI restriction enzyme site the primer would begin with the sequence 5'-cgggatccgccaccatg-3', followed by a sequence homologous to the 5' end of that part of a gene being amplified. All reverse primers incorporated a Not I restriction enzyme site sequence 5' -ttgcggccgc-3'. All gene-specific forward and reverse primers were designed with compatible melting temperatures to facilitate their amplification.
- All gene targets were amplified by PCR from S. agalactiae genomic DNA template using Vent DNA polymerase (NEB) or x th DNA polymerase (PE Applied Biosystems) using conditions recommended by the manufacturer.
- a typical amplification reaction involved an initial denaturation step at 95 °C for 2 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at the appropriate melting temperature for 30 seconds, and extension at 72 °C for 1 minute (1 minute per kilobase of DNA being amplified). This was followed by a final extension period at 72°C for 10 minutes. All PCR amplified products were extracted once with phenol chloroform (2:1:1) and once with chloroform (1 :1) and ethanol precipitated.
- agalactiae serotype III (strain 97/0099) does contain the rib gene, hence the rib gene as part of a DNA vaccine would represent a potential positive control for all DNA immunisation experiments.
- Oligonucleotide primers were designed (Appendix I) that targeted only the mature portion of the rib gene and which included appropriate restriction enzyme sites for cloning into pcDNA3.1.
- rib was amplified using rTtA DNA polymerase (PE Applied Biosystems) using conditions recommended by the manufacturer. Conditions for cloning were similar to that described previously.
- S. agalactiae serotype III (strain 97/0099) is a recent clinical isolate derived from the cerebral spinal fluid of a new born baby suffering from meningitis.
- This haemolytic strain of Group B Streptococcus was epidemiologically tested and validated at the Respiratory and Systemic Infection Laboratory, PHLS Central Public health laboratory, 61 Collindale Avenue, London NW9 5HT. The strain was subcultured only twice prior to its arrival in the laboratory. Upon its arrival on a agar slope, a sweep of 4-5 colonies was immediately used to inoculate a Todd Hewitt/5%> horse blood broth which was incubated overnight statically at 37 C .
- mice were challenged with various concentrations of the pathogen ranging from 10 ⁇ to 10 ⁇ colony forming units (cfu). Mice that developed symptoms were terminally anaesthetized and cardiac punctures were performed (Only mice that had been challenged with the highest doses, i.e. 1 X 10 ⁇ cfu, developed symptoms).
- the retrieved unclotted blood was used to inoculate directly a 50ml serum broth (Todd Hewitt/20%) inactivated foetal calf serum).
- the culture was constantly monitored and allowed to grow to late logarithmic phase. The presence of blood in the medium interfered with OD600 readings as it was being increasingly lysed with increasing growth of the bacterium, hence the requirement to constantly monitor the culture.
- the culture was transferred to a fresh 50 ml tube in order to exclude dead bacterial cells and remaining blood cells which would have sedimented at the bottom of the tube.
- 0.5 ml aliquots were then transferred to sterile cryovials, frozen in liquid nitrogen and stored at -70 C .
- a viable count was carried out on a single standard inoculum aliquot in order to determine bacterial numbers. This was determined to be approximately 5 X10 ⁇ cfu per ml.
- the optimal dose was estimated to be approximately 2.5 X10 ⁇ cfu. This represented a
- mice were accomplished by the administration of DNA to 6 week old CBA/ca mice (Harlan, UK). Mice to be vaccinated were divided into groups of six and each group were immunised with recombinant pcDNA3.1 plasmid DNA containing a specific target-gene sequence derived using the LEEP system. A total of 100 ⁇ g of
- mice groups were included in all vaccine trials. These control groups were either not DNA-vaccinated or were immunised with non-recombinant pcDNA3.1 plasmid DNA only, using the same time course described above. Four weeks after the second immunisation, all mice groups were challenged intra-peritoneally with a lethal dose of S. agalactiae serotype III (strain 97/0099).
- mice were killed 3 or 4 days after infection.
- challenged mice were monitored for the development of symptoms associated with the onset of S. agalactiae induced-disease.
- Typical symptoms in an appropriate order included piloerection, an increasingly hunched posture, discharge from eyes, increased lethargy and reluctance to move which was often the result of apparent paralysis in the lower body/hind leg region. The latter symptoms usually coincided with the development of a moribund state at which stage the mice were culled to prevent further suffering.
- mice were deemed to be very close to death, and the time of culling was used to determine a survival time for statistical analysis. Where mice were found dead, a survival time was calculated by averaging the time when a particular mouse was last observed alive and the time when found dead, in order to determine a more accurate time of death.
- a positive result was taken as any DNA sequence that was cloned and used in challenge experiments as described above and gave protection against that challenge.
- DNA sequences were determined to be protective
- mice may survive for significantly longer time periods when compared with control mice.
- the time to first death may also be prolonged when compared to counterpart mice in control groups.
- p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
- p value 2 refers to statistical significance when compared to rib positive control.
- mice immunised with the '17 (ID-8)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are two outlying mice one of which survived the term of the experiment despite developing symptoms. The group also exhibited a much wider range of survival times reflected by a mean survival value which is approximately 14 hours higher than that demonstrated by the unvaccinated control group.
- mice immunised with the '20 (ID-25)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there was one outlying mouse which survived the term of the experiment despite developing symptoms.
- p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
- p value 2 refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '22 (ID-10)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group but not when compared with the pcDNA3.1 control group.
- the time to first death in this group was prolonged by approximately 12 hours when compared to the pcDNA3.1 and unvaccinated control groups.
- the mean survival time of 43.691 hours is also considerbly higher than that determined for both control groups.
- mice immunised with the '28 (ID-13)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups. However there are three outlying mice, two of which survived the term of the experiment despite showing symptoms. In addition, the time to first death in this group was prolonged by approximately 9 hours when compared to the pcDNA3.1 and unvaccinated control groups. The mean survival time of 52.449 hours is also considerbly higher than that determined for both control groups, as well demonstrating a wider range of survival times.
- p value refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '70 (ID-42)' DNA vaccine marginally did not show significantly longer survival times when compared with the unvaccinated control group.
- the first death in this group is prolonged (by approximately 3 hours ) when compared with the unvaccinated group.
- the group has a mean survival time which is approximately 8 hours longer than the unvaccinated group.
- mice immunised with the '94 (ID-48)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
- mice immunised with the '51 (ID-37)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
- p value 2 refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '32 (ID-15)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups.
- the '32 (ID-15)' group has two outlying mice one of which survived the term of the experiment despite showing symptoms. This group also exhibits a wide range of survival times.
- mice immunised with the '39 (ID-17)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group but was not significant when compared with the pcDNA3.1 control group.
- the group has a considerbly higher mean survival time of 44.016 hours than that determined for either of the control groups.
- mice immunised with the '32 (ID-15)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups.
- the '32 (ID-15)' group has one outlying mouse which survived the term of the experiment despite showing symptoms.
- p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
- p value 2 refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '13 (ID-72)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups. However, there is one outlying mouse which survived approximately 24 hours longer than the longest surviving mice in the pcDNA3.1 and unvaccinated control groups respectively. In addition, the time to first death in this group was prolonged when compared to the pcDNA3.1 and unvaccinated control groups. The mean survival time of 43.582 hours is considerbly higher than that determined for both control groups.
- p value refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '3-5 (ID-66)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
- p value refers to statistical significance when compared to unvaccinated controls
- mice immunised with the '3-40 (ID-67)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there is one outlying mouse which survived approximately 43 hours longer than the longest surviving mice in the unvaccinated control group.
- mice immunised with the '2-19 (ID-73)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there was one outlying mouse which survived approximately 32 hours longer than the longest surviving mice in the unvaccinated control group. In addition, the time to first death was prolonged (by approximately 8 hours) when compared to the unvaccinated controls.
- p value - refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '3-20 (ID-71)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there is one outlying mouse which survived approximately 10 hours longer than the longest surviving mice in the unvaccinated control group.
- mice immunised with the '2-19 (ID-73)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are three outlying mouse which survived approximately 4, 10 and 23 hours longer than the longest surviving mice in the unvaccinated control group. This is reflected in the higher mean survival time of 48.749 hours and a much wider range of survival times.
- mice immunised with the '3-6 (ID-74)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are three outlying mouse which survived approximately 4, 10 and 23 hours longer than the longest surviving mice in the unvaccinated control group. This is reflected in the higher mean survival time of 49.599 hours and a much wider range of survival times.
- p value 1 refers to statistical significance when compared to pcDNA3.1 controls
- p value 2 refers to statistical significance when compared to unvaccinated controls.
- mice immunised with the '3-51 (ID-75)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 control group but was relatively close to significant when compared with the unvaccinated control group.
- the '3-51' group has two outlying mouse one of which survived the term of the experiment despite developing symptoms.
- the mean survival time of 44.499 hours is considerbly higher than that determined for both control groups and the group also demonstrates as a much wider range of survival times.
- mice immunised with the '3-56 (ID-76)' DNA vaccine exhibited significantly longer survival times when compared with the pcDNA3.1 control group but were marginally not significant when compared with unvaccinated control group.
- Example 3 Conservation and varability of candidate vaccine antigen genes among different isolates of Group B Strepococci
- Oligonucleotide primers were designed for each individual gene of interest derived using the LEEP system. Primers were designed to target the whole of the gene being investigated (All primers are listed in APPENDIX III). Specific gene targets were amplified by PCR using Vent DNA polymerase (NEB) according to the manufacturers instructions. Typical reactions were carried out in a 100 ⁇ l volume containing 50 ng of GBS template DNA, a one tenth volume of enzyme reaction buffer, 1 ⁇ M of each primer, 250 ⁇ M of each dNTP and 2 units of Vent DNA polymerase.
- NEB Vent DNA polymerase
- a typical reaction contained an initial 2 minute denaturation at 95°C, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at the appropriate melting temperature for 30 seconds, and extension at 72°C for 1 minute (1 minute per kilobase of DNA being amplified). The annealing temperature was determined by the lower melting temperature of the two oligonucleotide primers. The reaction was concluded with a final extension period of 10 minutes at 72°C. All PCR amplified products were extracted once with phenol chloroform (2:1 :1) and once with chloroform (1 :1) and ethanol precipitated. Specific DNA fragments were isolated from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). For use as DNA probes, purified amplified gene DNA fragments were labelled with digoxygenin using the DIG Nucleic Acid Labelling Kit (Boehringer Mannheim) according to the manufacturer's instructions.
- DNA concentrations were digested using either Hin Dili, Eco RI or Bgl Ilrestriction enzymes (NEB) according to manufacturer instructions and analysed by agarose gel electrophoresis. Following agarose gel electrophoresis of DNA samples, the gel was denatured in 0.25M HCl for 20 minutes and DNA was transferred onto HybondTM N + membrane (Amersham) by overnight capillary blotting. The method is essentially as described in Sambrook et al. (1989) using Whatman 3MM wicks on a platform over a reservoir of 0.4M NaOH. After transfer, the filter was washed briefly in 2x SSC and stored at 4 C in Saran wrap (Dow chemical company).
- Filters were prehybridised, hybridised with the digoxygenin labelled DNA probes and washed using conditions recommended by Boehringer Mannheim when using their DIG Nucleic Acid Detection Kit. Filters were prehybridised at 68°C for one hour in hybridisation buffer (1% w/v supplied blocking reagent, 5x SSC, 0.1% v/v N-lauryl sarcosine, 0.02%) v/v sodium dodecyl sulphate[SDS]). The digoxygenin labelled DNA probe was denatured at 99.9°C for 10 minutes before being added to the hybridisation buffer.
- Hybridisation was allowed to proceed overnight in a rotating Hybaid tube in a Hybaid Mini-hybridisation oven. Unbound probe was removed by washing the filter twice with 2x SSC- 0.1 % SDS for 5 minutes at room temperature. For increased stringency filters were then washed twice with O.lx SSC-0.1%o SDS for 15 minutes at 68°C.
- the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) was used to immunologically detect specifically bound digoxygenin labelled DNA probes. Results of Southern blot analysis
- Genomic DNA from each strain was digested completely with Hin Dili (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8% o agarose, transferred onto Hybond ⁇ NT (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled rib gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
- rib appears to be absent from all serotype la and lb strains (lanes 2 to 5) and from strains 118/158 and 97/0057 of serotype II (lanes 8 and 9). However, rib would appear to present in strains 18RS21 and 1954/92 of serotype II (lanes 6 and 7) and in all strains of serotype III (lanes 10 to 13). This is in agreement with previously published data (Stalhammar-Carlemalm et al, 1993). rib would also appear to be present in strains representing serotypes VII and VII (lanes 17 and 18) but was absent from strains representing serotypes IV, V and V (lanes 14 to
- the rib. gene probe did hybridise with lower intensity to genomic DNA fragments from strains representing serotypes la, lb, IV, VI, VII and serotype II strains 118/158 and 97/0057. This may indicate the presence of a gene in these strains with a lower level of homology to rib.
- These hybridising DNA fragments may contain a homologue of the GBS bca gene encoding the Ca protein antigen which has been shown to be closely homologous to the Rib protein (Wastfelt et al, 1996).
- Genomic DNA from each strain was digested completely with Hin Dili (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8% agarose, transfe ⁇ ed onto Hybond N + (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 4 (ID-1) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
- Genomic DNA from each strain was digested completely with Eco RI (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N " (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled
- ID-2 gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
- Genomic DNA from each strain was digested completely with Eco RI (N ⁇ B) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N + (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 15 (ID-7) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
- the gene probe hybridised specifically with Eco RI -digested DNA fragments ranging from approximately 3.5 kb to 5.2 kb in size.
- Genomic DNA from each strain was digested completely with Hin Dili (N ⁇ B) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond NT 1" (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 17 (ID-8) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
- Genomic DNA from each strain was digested completely with Bgl II (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N 1" (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 22 (ID-10) gene probe. Specifically bound DNA probe was identified using the DIG
- Il-digested genomic DNA fragment of approximately 3.1 kb in DNA digests from all GBS representatives except serotype lb strain H36B, where the gene probe hybridised specifically to a a Bgl Il-digested genomic DNA fragment.
- Gene 22 (ID-10) was absent from both the control strains (lanes 19 and 20).
- a group A Streptococcal strain (serotype Ml, strain NCTC8198) and Streptococcus pneumoniae (serotype 14) were also included in the analysis for control pu ⁇ oses.
- Protein Rib a novel Group B Streptococcal protein that confers protective immunity and is expressed by most strains causing invasive infections: J. Exp. Med. Ill: 1593-1603
- DNA vaccination with the major outer-membrane protein genes induces acquired immunity to Chlamydia trachomatis (mouse pneumonitis) infection. Infection and Immunity, 176, 1035-40.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002337102A CA2337102A1 (en) | 1998-07-27 | 1999-07-27 | Nucleic acids and proteins from group b streptococcus |
JP2000562518A JP2002531054A (en) | 1998-07-27 | 1999-07-27 | Nucleic acids and proteins from group B streptococci |
EP99934984A EP1100920A2 (en) | 1998-07-27 | 1999-07-27 | Nucleic acids and proteins from group b streptococcus |
US09/769,736 US7098182B2 (en) | 1998-07-27 | 2001-01-26 | Nucleic acids and proteins from group B streptococcus |
US11/123,241 US20060078565A1 (en) | 1998-07-27 | 2005-05-06 | Nucleic acids and proteins from Group B Streptococcus |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9816335.5A GB9816335D0 (en) | 1998-07-27 | 1998-07-27 | Proteins |
GB9816335.5 | 1998-07-27 | ||
US12516399P | 1999-03-19 | 1999-03-19 | |
US60/125,163 | 1999-03-19 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/769,736 Continuation US7098182B2 (en) | 1998-07-27 | 2001-01-26 | Nucleic acids and proteins from group B streptococcus |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000006736A2 true WO2000006736A2 (en) | 2000-02-10 |
WO2000006736A3 WO2000006736A3 (en) | 2000-06-22 |
Family
ID=26314123
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1999/002444 WO2000006736A2 (en) | 1998-07-27 | 1999-07-27 | Nucleic acids and proteins from group b streptococcus |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1100920A2 (en) |
JP (1) | JP2002531054A (en) |
CA (1) | CA2337102A1 (en) |
WO (1) | WO2000006736A2 (en) |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000037646A2 (en) * | 1998-12-22 | 2000-06-29 | Microscience Limited | Group b streptococcus proteins, and their use |
WO2001011060A2 (en) * | 1999-08-06 | 2001-02-15 | Bioteknologisk Institut | Method of isolating secretion signals in lactic acid bacteria and novel secretion signals isolated from lactococcus lactis |
WO2001014421A1 (en) * | 1999-08-25 | 2001-03-01 | Medimmune, Inc. | Homologs of a pneumococcal protein and fragments for vaccines |
WO2002072623A1 (en) * | 2001-03-09 | 2002-09-19 | Microscience Limited | Genes and proteins, and their use |
WO2004035618A2 (en) * | 2002-10-15 | 2004-04-29 | Intercell Ag | Nucleic acids coding for adhesion factor of group b streptococcus, adhesion factors of group b streptococcus and further uses thereof |
US6833356B1 (en) | 1999-08-25 | 2004-12-21 | Medimmune, Inc. | Pneumococcal protein homologs and fragments for vaccines |
US6890539B2 (en) | 1998-12-22 | 2005-05-10 | Microscience, Ltd. | Genes and proteins, and their use |
US7128919B2 (en) | 2000-08-08 | 2006-10-31 | St. Jude Children's Research Hospital | Group B streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof |
WO2006069200A3 (en) * | 2004-12-22 | 2007-03-01 | Chiron Corp | Group b streptococcus |
US7348161B2 (en) | 2001-03-23 | 2008-03-25 | Emory University | Macrolide efflux genetic assembly |
US7709009B2 (en) | 2003-07-31 | 2010-05-04 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for streptococcus pyogenes |
EP2277894A1 (en) * | 2000-10-27 | 2011-01-26 | Novartis Vaccines and Diagnostics S.r.l. | Nucleic acids and proteins from streptococcus groups A & B |
US7892552B2 (en) | 2001-08-08 | 2011-02-22 | University Of Utah Research Foundation | Group B Streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof |
US7914794B2 (en) | 1998-02-20 | 2011-03-29 | Id Biomedical Corporation | Group B streptococcus antigens |
US8101187B2 (en) | 2001-03-30 | 2012-01-24 | Sanofi Pasteur Limited | Secreted Streptococcus pneumoniae proteins |
US8778358B2 (en) | 2004-07-29 | 2014-07-15 | Novartis Vaccines And Diagnostics, Inc. | Immunogenic compositions for gram positive bacteria such as Streptococcus agalactiae |
US8821895B2 (en) | 2000-12-21 | 2014-09-02 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
US8945589B2 (en) | 2003-09-15 | 2015-02-03 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for Streptococcus agalactiae |
US9102741B2 (en) | 2007-09-12 | 2015-08-11 | Novartis Ag | GAS57 mutant antigens and GAS57 antibodies |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0522303D0 (en) * | 2005-11-01 | 2005-12-07 | Chiron Srl | Culture method |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994010317A2 (en) * | 1992-11-02 | 1994-05-11 | The General Hospital Corporation | Conjugate vaccine against group b streptococcus |
WO1995006732A2 (en) * | 1993-09-01 | 1995-03-09 | The Rockefeller University | Bacterial exported proteins and acellular vaccines based thereon |
WO1998018930A2 (en) * | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
WO1998023631A1 (en) * | 1996-11-27 | 1998-06-04 | Smithkline Beecham Corporation | Novel bacterial polypeptides and polynucleotides |
WO1999016882A1 (en) * | 1997-09-26 | 1999-04-08 | Medimmune, Inc. | LMB GENE OF $i(STREPTOCOCCUS AGALACTIAE) |
WO1999042588A2 (en) * | 1998-02-20 | 1999-08-26 | Biochem Pharma Inc. | Group b streptococcus antigens |
-
1999
- 1999-07-27 WO PCT/GB1999/002444 patent/WO2000006736A2/en not_active Application Discontinuation
- 1999-07-27 JP JP2000562518A patent/JP2002531054A/en active Pending
- 1999-07-27 CA CA002337102A patent/CA2337102A1/en not_active Abandoned
- 1999-07-27 EP EP99934984A patent/EP1100920A2/en not_active Withdrawn
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994010317A2 (en) * | 1992-11-02 | 1994-05-11 | The General Hospital Corporation | Conjugate vaccine against group b streptococcus |
WO1995006732A2 (en) * | 1993-09-01 | 1995-03-09 | The Rockefeller University | Bacterial exported proteins and acellular vaccines based thereon |
WO1998018930A2 (en) * | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
WO1998018931A2 (en) * | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae polynucleotides and sequences |
WO1998023631A1 (en) * | 1996-11-27 | 1998-06-04 | Smithkline Beecham Corporation | Novel bacterial polypeptides and polynucleotides |
WO1999016882A1 (en) * | 1997-09-26 | 1999-04-08 | Medimmune, Inc. | LMB GENE OF $i(STREPTOCOCCUS AGALACTIAE) |
WO1999042588A2 (en) * | 1998-02-20 | 1999-08-26 | Biochem Pharma Inc. | Group b streptococcus antigens |
Non-Patent Citations (9)
Title |
---|
DATABASE SWISSPROT E.M.B.L. Databases Accession Number: P29850, 1 April 1993 (1993-04-01) PUYET A ET AL: " MALTOSE/MALTODEXTRIN-BINDING PROTEIN PRECURSOR" XP002125784 * |
DATABASE SWISSPROT E.M.B.L. Databases Accession Number: P39845, 1 February 1995 (1995-02-01) TOGNONI A ET AL: "Peptide Synthetase 1" XP002133345 * |
DATABASE SWISSPROT E.M.B.L. Databases Accession Number: P42422, 1 November 1995 (1995-11-01) YOSHIDA K ET AL: "Hypothetical sensor-like Histidine Kinase in IDH 3' region" XP002133344 * |
DATABASE TREMBL E.M.B.L. databases Accession Number: P94374, 1 May 1997 (1997-05-01) YOSHIDA K ET AL: "HOMOLOGOUS TO MANY ATP-BINDING TRANSPORT PROTEINS" XP002133346 * |
DATABASE TREMBL E.M.B.L. Databases Accession Number: Q54914, 1 November 1996 (1996-11-01) PODBIELSKI A ET AL: "ORF 1 AND ORF2 5' REGION" XP002133342 * |
LACHENAUER C S ET AL: "Cloning and expression in Escherichia coli of a protective surface protein from type V group B Streptococci" ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY,US,SPRING ST., NY, vol. 418, no. 418, 9 December 1997 (1997-12-09), page 615-618-618 XP002107261 ISSN: 0065-2598 * |
LARSSON C ET AL: "Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and alpha" INFECT. IMMUN., vol. 64, no. 9, September 1996 (1996-09), pages 3518-3523, XP002125783 cited in the application * |
MICHEL J L ET AL: "Cloned alpha and beta C-protein antigens of group B Streptococci elicit protective immunity" INFECTION AND IMMUNITY,US,AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, vol. 59, no. 6, June 1991 (1991-06), page 2023-2028-2028 XP002107260 ISSN: 0019-9567 * |
See also references of EP1100920A2 * |
Cited By (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7914794B2 (en) | 1998-02-20 | 2011-03-29 | Id Biomedical Corporation | Group B streptococcus antigens |
US8580262B2 (en) | 1998-02-20 | 2013-11-12 | Id Biomedical Corporation Of Quebec | Group B Streptococcus antigens |
US8226953B2 (en) | 1998-02-20 | 2012-07-24 | Id Biomedical Corporation Of Quebec | Group B Streptococcus antigens |
WO2000037646A3 (en) * | 1998-12-22 | 2000-10-26 | Microscience Ltd | Group b streptococcus proteins, and their use |
US7592011B2 (en) | 1998-12-22 | 2009-09-22 | Emergent Product Development Uk Limited | Genes and proteins, and their use |
US7419672B2 (en) | 1998-12-22 | 2008-09-02 | Emergent Product Development Uk Limited | Genes and proteins, and their use |
WO2000037646A2 (en) * | 1998-12-22 | 2000-06-29 | Microscience Limited | Group b streptococcus proteins, and their use |
US6812021B1 (en) | 1998-12-22 | 2004-11-02 | Microscience Limited | Genes and proteins and their use |
US6890539B2 (en) | 1998-12-22 | 2005-05-10 | Microscience, Ltd. | Genes and proteins, and their use |
WO2001011060A2 (en) * | 1999-08-06 | 2001-02-15 | Bioteknologisk Institut | Method of isolating secretion signals in lactic acid bacteria and novel secretion signals isolated from lactococcus lactis |
WO2001011060A3 (en) * | 1999-08-06 | 2001-08-23 | Biotecknologisk Inst | Method of isolating secretion signals in lactic acid bacteria and novel secretion signals isolated from lactococcus lactis |
US7186815B2 (en) | 1999-08-06 | 2007-03-06 | Bioneer A/S | Method of isolating secretion signals in lactic acid bacteria and novel secretion signals isolated from Lactococcus lactis |
US6833356B1 (en) | 1999-08-25 | 2004-12-21 | Medimmune, Inc. | Pneumococcal protein homologs and fragments for vaccines |
JP2003507054A (en) * | 1999-08-25 | 2003-02-25 | メディミューン,インコーポレイテッド | Homologs and fragments of pneumococcal proteins for vaccines |
JP4749641B2 (en) * | 1999-08-25 | 2011-08-17 | メディミューン,エルエルシー | Homologues and fragments of pneumococcal proteins for vaccine use |
WO2001014421A1 (en) * | 1999-08-25 | 2001-03-01 | Medimmune, Inc. | Homologs of a pneumococcal protein and fragments for vaccines |
US7128919B2 (en) | 2000-08-08 | 2006-10-31 | St. Jude Children's Research Hospital | Group B streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof |
US7645577B2 (en) | 2000-08-08 | 2010-01-12 | St. Jude Children's Research Hospital | Group B streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof |
EP1810978A2 (en) | 2000-08-08 | 2007-07-25 | St. Jude Children's Research Hospital | Group B streptococcus polypeptides nucleic acids and therapeutic composition and vaccines thereof |
EP1810978A3 (en) * | 2000-08-08 | 2007-10-24 | St. Jude Children's Research Hospital | Group B streptococcus polypeptides nucleic acids and therapeutic composition and vaccines thereof |
US8529912B2 (en) | 2000-08-08 | 2013-09-10 | St. Jude Children's Reseach Hospital | Group B Streptococcus polypeptides, nucleic acids and therapeutic compositions and vaccines thereof |
US9840538B2 (en) | 2000-10-27 | 2017-12-12 | Novartis Ag | Nucleic acids and proteins from Streptococcus groups A and B |
US8025890B2 (en) | 2000-10-27 | 2011-09-27 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from streptococcus groups A and B |
US8137673B2 (en) | 2000-10-27 | 2012-03-20 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from Streptococcus groups A & B |
EP2277894A1 (en) * | 2000-10-27 | 2011-01-26 | Novartis Vaccines and Diagnostics S.r.l. | Nucleic acids and proteins from streptococcus groups A & B |
US8431139B2 (en) | 2000-10-27 | 2013-04-30 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from Streptococcus groups A and B |
US9738693B2 (en) | 2000-10-27 | 2017-08-22 | Novartis Ag | Nucleic acids and proteins from streptococcus groups A and B |
US7939087B2 (en) | 2000-10-27 | 2011-05-10 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from Streptococcus groups A & B |
US7955604B2 (en) | 2000-10-27 | 2011-06-07 | Novartis Vaccines And Diagnostics, Inc. | Nucleic acids and proteins from streptococcus groups A and B |
US10428121B2 (en) | 2000-10-27 | 2019-10-01 | Novartis Ag | Nucleic acids and proteins from streptococcus groups A and B |
US8821895B2 (en) | 2000-12-21 | 2014-09-02 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
US9340586B2 (en) | 2000-12-21 | 2016-05-17 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
WO2002072623A1 (en) * | 2001-03-09 | 2002-09-19 | Microscience Limited | Genes and proteins, and their use |
US7348161B2 (en) | 2001-03-23 | 2008-03-25 | Emory University | Macrolide efflux genetic assembly |
US8101187B2 (en) | 2001-03-30 | 2012-01-24 | Sanofi Pasteur Limited | Secreted Streptococcus pneumoniae proteins |
US7892552B2 (en) | 2001-08-08 | 2011-02-22 | University Of Utah Research Foundation | Group B Streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof |
AU2003274011B2 (en) * | 2002-10-15 | 2010-03-04 | Intercell Ag | Nucleic acids coding for adhesion factor of group B streptococcus, adhesion factors of group B streptococcus and further uses thereof |
US8318908B2 (en) | 2002-10-15 | 2012-11-27 | Intercell Ag | Nucleic acids coding for adhesion factor of group B streptococcus, adhesion factors of group B streptococcus and further uses thereof |
WO2004035618A2 (en) * | 2002-10-15 | 2004-04-29 | Intercell Ag | Nucleic acids coding for adhesion factor of group b streptococcus, adhesion factors of group b streptococcus and further uses thereof |
WO2004035618A3 (en) * | 2002-10-15 | 2004-09-30 | Intercell Ag | Nucleic acids coding for adhesion factor of group b streptococcus, adhesion factors of group b streptococcus and further uses thereof |
US7960533B2 (en) | 2002-10-15 | 2011-06-14 | Intercell Ag | Nucleic acids coding for adhesion factor of group B Streptococcus, adhesion factors of group B Streptococcus and further uses thereof |
US7485710B2 (en) | 2002-10-15 | 2009-02-03 | Intercell Ag | Nucleic acids coding for adhesion factor of group B streptococcus, adhesion factors of group B streptococcus and further uses thereof |
AU2010201016B2 (en) * | 2002-10-15 | 2012-06-14 | Intercell Ag | Nucleic acids coding for adhesion factor of group B streptococcus, adhesion factors of group B streptococcus and further uses thereof |
US9056912B2 (en) | 2003-07-31 | 2015-06-16 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for Streptococcus pyogenes |
US7709009B2 (en) | 2003-07-31 | 2010-05-04 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for streptococcus pyogenes |
US8529913B2 (en) | 2003-07-31 | 2013-09-10 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for Streptococcus pyogenes |
US8128936B2 (en) | 2003-07-31 | 2012-03-06 | Novartis Vaccines And Diagnostics, S.R.L. | Immunogenic compositions for Streptococcus pyogenes |
US8945589B2 (en) | 2003-09-15 | 2015-02-03 | Novartis Vaccines And Diagnostics, Srl | Immunogenic compositions for Streptococcus agalactiae |
US8778358B2 (en) | 2004-07-29 | 2014-07-15 | Novartis Vaccines And Diagnostics, Inc. | Immunogenic compositions for gram positive bacteria such as Streptococcus agalactiae |
WO2006069200A3 (en) * | 2004-12-22 | 2007-03-01 | Chiron Corp | Group b streptococcus |
US9102741B2 (en) | 2007-09-12 | 2015-08-11 | Novartis Ag | GAS57 mutant antigens and GAS57 antibodies |
Also Published As
Publication number | Publication date |
---|---|
EP1100920A2 (en) | 2001-05-23 |
WO2000006736A3 (en) | 2000-06-22 |
JP2002531054A (en) | 2002-09-24 |
CA2337102A1 (en) | 2000-02-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060078565A1 (en) | Nucleic acids and proteins from Group B Streptococcus | |
US8101187B2 (en) | Secreted Streptococcus pneumoniae proteins | |
WO2000006736A2 (en) | Nucleic acids and proteins from group b streptococcus | |
EP1144640A2 (en) | Nucleic acids and proteins from streptococcus pneumoniae | |
Jacobs et al. | Identification, purification, and characterization of a thiol-activated hemolysin (suilysin) of Streptococcus suis | |
Brodeur et al. | Identification of group B streptococcal Sip protein, which elicits cross-protective immunity | |
Overweg et al. | The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses | |
US20100278740A1 (en) | Streptococcus pneumoniae proteins and nucleic acid molecules | |
EP2053126A1 (en) | Streptococcus pneumoniae proteins and nucleic acid molecules | |
EP0866133A2 (en) | A group b streptococcus vaccine | |
EP1214417A2 (en) | Nucleic acids and proteins from group b streptococcus | |
JP2776633B2 (en) | DNA sequence encoding Streptococcus suis virulence, part of the DNA sequence, polypeptides and antibodies derived from the DNA sequence | |
US8632784B2 (en) | Nucleic acids and proteins from Streptococcus pneumoniae | |
JPH09502604A (en) | Campylobacter jejuni antigens, and their production and use | |
Erdogan et al. | Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci | |
Fischetti | Vaccine approaches to protect against group A streptococcal pharyngitis | |
IL95578A (en) | Conjugate vaccine formed from a polysaccharide and a c protein of b-streptococcus | |
AU2004239596A1 (en) | Method for selecting and producing vaccine components and vaccines based thereon | |
JP4500615B2 (en) | Novel polypeptide having protective activity against sweptococcal infection derived from serotype 18, which is another species of the genus Erichiperotricks, its gene and production method | |
EP1624064A2 (en) | Nucleic acids and proteins from streptococcus pneumoniae | |
EP1801218A2 (en) | Nucleic acids and proteins from streptococcus pneumoniae | |
EP1790730A2 (en) | Streptococcus pneumoniae proteins and nucleic acid molecules | |
JP2001504335A (en) | Lactoferrin-binding protein of Streptococcus uberis | |
CN101108877A (en) | Nucleic acids and proteins from streptococcus pneumoniae | |
To et al. | Genetic and Antigenic Diversity of the |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 99811358.1 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): CA CN JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): CA CN JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
ENP | Entry into the national phase |
Ref document number: 2337102 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09769736 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2000 562518 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999934984 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1999934984 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999934984 Country of ref document: EP |