DE19708537A1 - New surface protein (SpsA protein) from Streptococcus pneumoniae etc. - Google Patents
New surface protein (SpsA protein) from Streptococcus pneumoniae etc.Info
- Publication number
- DE19708537A1 DE19708537A1 DE19708537A DE19708537A DE19708537A1 DE 19708537 A1 DE19708537 A1 DE 19708537A1 DE 19708537 A DE19708537 A DE 19708537A DE 19708537 A DE19708537 A DE 19708537A DE 19708537 A1 DE19708537 A1 DE 19708537A1
- Authority
- DE
- Germany
- Prior art keywords
- spsa
- protein
- streptococcus pneumoniae
- surface protein
- positions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 41
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 32
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 24
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 24
- 241000193998 Streptococcus pneumoniae Species 0.000 title abstract description 56
- 229940031000 streptococcus pneumoniae Drugs 0.000 title abstract description 56
- 229960005486 vaccine Drugs 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims abstract description 5
- 230000003248 secreting effect Effects 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 19
- 241000194017 Streptococcus Species 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 108010040473 pneumococcal surface protein A Proteins 0.000 description 21
- 101150037205 spsA gene Proteins 0.000 description 16
- 150000004676 glycans Chemical class 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 101710183389 Pneumolysin Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 238000010367 cloning Methods 0.000 description 9
- 229920001282 polysaccharide Polymers 0.000 description 9
- 239000005017 polysaccharide Substances 0.000 description 9
- 101150080370 pspA gene Proteins 0.000 description 9
- 239000012634 fragment Substances 0.000 description 8
- 102220472274 Eukaryotic translation initiation factor 4E transporter_R36A_mutation Human genes 0.000 description 7
- 210000002919 epithelial cell Anatomy 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 239000002775 capsule Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 102000028556 IgA binding proteins Human genes 0.000 description 5
- 108091009322 IgA binding proteins Proteins 0.000 description 5
- 108090000301 Membrane transport proteins Proteins 0.000 description 5
- 102000003939 Membrane transport proteins Human genes 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 230000001018 virulence Effects 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 102400001107 Secretory component Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 4
- 239000013611 chromosomal DNA Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- LVSPDZAGCBEQAV-UHFFFAOYSA-N 4-chloronaphthalen-1-ol Chemical compound C1=CC=C2C(O)=CC=C(Cl)C2=C1 LVSPDZAGCBEQAV-UHFFFAOYSA-N 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- 101000652830 Arabidopsis thaliana Sigma factor binding protein 1, chloroplastic Proteins 0.000 description 3
- 208000031729 Bacteremia Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000606768 Haemophilus influenzae Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 206010033078 Otitis media Diseases 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 229940099472 immunoglobulin a Drugs 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 210000002345 respiratory system Anatomy 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 108010002231 IgA-specific serine endopeptidase Proteins 0.000 description 2
- 101710087657 Manganese ABC transporter substrate-binding lipoprotein Proteins 0.000 description 2
- 102000005348 Neuraminidase Human genes 0.000 description 2
- 108010006232 Neuraminidase Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- 208000035109 Pneumococcal Infections Diseases 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 108010046644 Polymeric Immunoglobulin Receptors Proteins 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 210000000224 granular leucocyte Anatomy 0.000 description 2
- 229940045808 haemophilus influenzae type b Drugs 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 210000005265 lung cell Anatomy 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000001989 nasopharynx Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 108010082406 peptide permease Proteins 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- 239000002132 β-lactam antibiotic Substances 0.000 description 2
- 229940124586 β-lactam antibiotics Drugs 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- 101100130893 Alkalihalobacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) mntA gene Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241001301450 Crocidium multicaule Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 101150105088 Dele1 gene Proteins 0.000 description 1
- 101100084597 Dictyostelium discoideum pspA gene Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001596967 Escherichia coli M15 Species 0.000 description 1
- 101000840258 Homo sapiens Immunoglobulin J chain Proteins 0.000 description 1
- 101000851058 Homo sapiens Neutrophil elastase Proteins 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100029571 Immunoglobulin J chain Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 241000588650 Neisseria meningitidis Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102100035187 Polymeric immunoglobulin receptor Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 235000003407 Sigesbeckia orientalis Nutrition 0.000 description 1
- 240000003801 Sigesbeckia orientalis Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 description 1
- 101900040393 Streptococcus pneumoniae Immunoglobulin A1 protease Proteins 0.000 description 1
- 101900032831 Streptococcus pneumoniae Pneumolysin Proteins 0.000 description 1
- 241000194022 Streptococcus sp. Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 101800003447 VP53 Proteins 0.000 description 1
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001147 anti-toxic effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000006931 brain damage Effects 0.000 description 1
- 231100000874 brain damage Toxicity 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000005255 gram-positive cell Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 102000052502 human ELANE Human genes 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000005264 laryngeal carcinoma Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 230000001459 mortal effect Effects 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229940124733 pneumococcal vaccine Drugs 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229940031937 polysaccharide vaccine Drugs 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 101150043479 psaA gene Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000001533 respiratory mucosa Anatomy 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000031998 transcytosis Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
Description
Streptococcus pneumoniae (Pneumokokken) sind gram-positive Bak terien, die von einem Kapselpolysaccharid umgeben sind und erstmals 1881 von einem gesunden Träger isoliert werden konnten (1). Pneumokokken gehören zur natürlichen Flora des oberen men schlichen Respirationstrakts und kolonisieren den Nasopharynx in bis zu 40% der gesunden Erwachsenen, wobei bis zu vier verschiedene Serotypen über mehrere Monate nachgewiesen werden konnten (2). Streptococcus pneumoniae ist in den USA und vielen anderen Teilen der Welt ein häufiger Verursacher von Infektio nen. Die Infektionen, die meist endogen erfolgen, und Todesfälle treten vor allem bei sehr jungen Kindern unter 2 Jahren, bei Per sonen über 60 Jahren sowie bei immungeschwächten Personen auf (3). Streptococcus pneumoniae (pneumococci) are gram-positive Bak series surrounded by a capsule polysaccharide and could be isolated from a healthy carrier for the first time in 1881 (1). Pneumococci are part of the natural flora of the upper men crept respiratory tract and colonize the nasopharynx up to 40% of healthy adults, with up to four different serotypes can be detected over several months could (2). Streptococcus pneumoniae is in the US and many a common cause of infection in other parts of the world nen. The infections, which are mostly endogenous, and deaths occur especially in very young children under 2 years, in Per people over the age of 60 as well as in immunocompromised people (3).
Des weiteren sind Pneumokokken die zweithäufigsten Verursacher von bakterieller Meningitis (nach Haemophilus influenzae Typ b) (4) und Otitis Media (Mittelohrentzündung) bei Kindern (5). So verursachten Pneumokokken allein in den USA innerhalb eines Jahres 500 000 Pneumonien, 7 Millionen Fälle von Otitis Media und 40 000 Todesfälle (6, 7).Pneumococci are also the second most common cause bacterial meningitis (Haemophilus influenzae type b) (4) and otitis media (otitis media) in children (5). For example, pneumococci caused within one in the United States alone 500,000 annual pneumonia, 7 million cases of otitis media and 40,000 deaths (6, 7).
Auch die Sterblichkeitsrate bei den durch Streptococcus pneu moniae verursachten Erkrankungen liegt, trotz der zur Verfügung stehenden Antibiotika, auf einem konstant hohen Niveau. Zum Bei spiel betrug die Sterblichkeit bei Bakteriämien, die durch Pneu mokokken verursacht wurden, in den letzten vier Jahrzehnten zwi schen 25 und 29% (8).The mortality rate among those due to Streptococcus pneu Diseases caused by moniae lie, despite the available ones standing antibiotics, at a consistently high level. For the case The mortality rate from bacteremia caused by pneumonia mococci have been caused in the past four decades between 25 and 29% (8).
Aufgrund der Zusammensetzung der Kapselpolysaccharidstrukturen werden Pneumokokken in 90 Serotypen unterteilt (9). Das Kapsel polysaccharid schützt die Pneumokokken vor der Phagozytose durch polymorphkernige Leukozyten (PMN's), verhindert die Aktivierung des alternativen Weges des Komplements (10, 11, 12) und ist daher ein wichtiger Virulenzfaktor von Streptococcus pneumoniae (13, 14, 15).Due to the composition of the capsule polysaccharide structures pneumococci are divided into 90 serotypes (9). The capsule polysaccharide protects the pneumococci from phagocytosis polymorphonuclear leukocytes (PMN's), prevents activation the alternative way of complement (10, 11, 12) and is therefore an important virulence factor of Streptococcus pneumoniae (13, 14, 15).
Die Virulenz des Stammes ist in Bezug auf die Kapsel abhängig von der chemischen Zusammensetzung des Polysaccharids und nicht von der Größe des Kapselpolysaccharids (16).The virulence of the strain is dependent on the capsule on the chemical composition of the polysaccharide and not the size of the capsule polysaccharide (16).
Der zur Zeit verwendete Impfstoff (Pneumokokken-Vakzin) zur ak tiven Immunisierung enthält die unkonjugierte Form von 23 Kap selpolysacchariden von Streptococcus pneumoniae. Dieser Impf stoff enthält die Polysacccharide der Serotypen, die 85-90 Pro zent der bakteriämischen Infektionen verursachen. Da bei Kindern und älteren Menschen keine Stimulation der T-Helferzellen durch ein Polysaccharidantigen erfolgt (17), ist die Anwendung dieses Impfstoffes ausschließlich auf Erwachsene beschränkt. The currently used vaccine (pneumococcal vaccine) for the ac The immunization contains the unconjugated form of 23 ch selpolysaccharides from Streptococcus pneumoniae. This vaccination fabric contains the polysacccharides of the serotypes, the 85-90 Pro cause of bacteraemic infections. Because with children and older people did not stimulate the T helper cells a polysaccharide antigen occurs (17), the application of this is Vaccine limited to adults only.
Ein weiteres Problem der Behandlung bei Pneumokokken-Infektionen ist das weltweit verstärkte Auftreten von antibiotikaresistenten Stämmen (Penicillin-Resistenz) (18). Außerdem führt der Einsatz von β-Laktam-Antibiotika bei empfindlichen Stämmen von Strepto coccus pneumoniae in 30% der behandelten Kindern zum Tod oder es verbleiben permante, irreversible Gehirnschäden. Die Bakte rien werden durch β-Laktam-Antibiotika lysiert und die freiwer denden Zellwandbestandteile verursachen eine Anschwellung des Gehirns. Die Schwellung des Gehirns führt dann zu einem gefähr lich hohen interkranialen Druck, der einen irreversiblen Schaden auslöst (19).Another treatment problem with pneumococcal infections is the worldwide increased occurrence of antibiotic-resistant Strains (penicillin resistance) (18). In addition, the application leads of β-lactam antibiotics in sensitive strains of Strepto coccus pneumoniae in 30% of treated children to death or permanent, irreversible brain damage remains. The bacts Rien are lysed by β-lactam antibiotics and frewer end cell components cause a swelling of the Brain. The swelling of the brain then leads to a dangerous one high intercranial pressure, causing irreversible damage triggers (19).
Aufgrund der steigenden Antibiotikaresistenz und der einge schränkten Wirkung des Polysaccharid-Vakzins, besteht ein großes Interesse am Auffinden eines Pneumokokken-Proteins, das als Trä ger für die Kapselpolysaccharide fungieren kann oder das auf grund seiner eigenen starken Immunogenität und des ubiquitären Vorkommens bei Streptococcus pneumoniae Isolaten all präventiver Impfstoff Verwendung finden kann.Due to the increasing resistance to antibiotics and the limited effect of the polysaccharide vaccine, there is a big one Interest in finding a pneumococcal protein that is known as Trä can act for the capsule polysaccharides or that due to its own strong immunogenicity and ubiquitous Occurrence of Streptococcus pneumoniae isolates all preventive Vaccine can be used.
Die zur Zeit als Vakzin-Kandidaten in Frage kommenden Proteine sind das Autolysin (20), die Neuraminidase (21), das pneumo coccal surface adhesin A (PsaA) (22), das pneumococcal surface protein A (PspA) (23) sowie das Pneumolysin (24).The proteins currently considered as vaccine candidates are the autolysin (20), the neuraminidase (21), the pneumo coccal surface adhesin A (PsaA) (22), the pneumococcal surface protein A (PspA) (23) and pneumolysin (24).
Pneumolysin ist ein intrazelluläres Protein und gehört zu der Familie der Thiol-aktivierten Toxine (25). Dieses cytoplasmati sche 53 kDa große Protein wird freigesetzt, wenn die Pneumokoc ken unter dem Einfluß des Autolysins spontan lysieren. In hohen Konzentrationen bilden sich auf Säugetierzellen Oligomere des Pneumolysins und verursachen durch einen Anstieg der transmem branen Poren eine Zellyse. In geringeren Konzentrationen stimu liert Pneumolysin die Produktion von inflammatorischen Cytokinen (26), zerstört in vitro Monolayer der Epithelzellen des oberen respiratorischen Trakts (27) und vermindert die bakterizide Ak tivität und Wanderung der Neutrophilen (28). Des weiteren akti viert Pneumolysin bei Abwesenheit von anti-Toxin Antikörpern den klassischen Komplementweg (29).Pneumolysin is an intracellular protein and belongs to that Family of thiol-activated toxins (25). This cytoplasmati 53 kDa protein is released when the pneumococ lyse spontaneously under the influence of autolysin. In high Concentrations form on oligomers of mammalian cells Pneumolysins and cause an increase in transmem branen pores a cell lysis. In lower concentrations stimu Pneumolysin reduces the production of inflammatory cytokines (26), destroys in vitro monolayer of the upper epithelial cells respiratory tract (27) and reduces the bactericidal Ak activity and migration of neutrophils (28). Furthermore acti fourth pneumolysin in the absence of anti-toxin antibodies classic complement path (29).
Das pneumoccocal surface protein A (PspA) ist ein Oberflächen protein mit struktureller und antigenetischer Variabilität zwi schen den verschiedenen Pneumokokken-Stämmen, dessen Funktion bisher nicht aufgeklärt ist. PspA kommt in den meisten klini schen Isolaten vor (30) und ist auch wichtig für die Entwicklung der vollen Virulenz der Pneumokokken (31, 32).The pneumoccocal surface protein A (PspA) is a surface protein with structural and antigenetic variability between the different pneumococcal strains, their function has not yet been clarified. PspA comes in most clinics isolates (30) and is also important for development full pneumococcal virulence (31, 32).
Weitere bekannte Virulenzfaktoren sind eine IgA1-Protease (33), deren Gen kloniert und charakterisiert wurde (34, 35), ein Inhi bitor der Elastase (36) und Peptidpermeasen, die homolog zu Permeasen von anderen Streptokokken, die ebenfalls den Naso pharynx besiedeln, sind. Von besonderem Interesse sind dabei die Permeasen, bei denen der Verlust der Funktion eine verminderte Adhärenz der Pneumokokken an eukaryotische Zellen zur Folge hat (37). Der Nachweis für eine direkte Beteiligung dieser Permea sen, von denen die AmiA-Permease und das PlpA (permease-like protein A) zur Familie der Protein-abhängigen Permeasen gehören, die für den Transport von kleinen Peptiden verantwortlich sind (38, 39) an der Adhärenz an eukaryotische Zellen konnte bisher aber nicht erbracht werden. Als ein weiterer potentieller Regu lator der Pneumokokken-Adhärenz wird die Pyruvatoxidase, SpxB, angesehen, die die Konzentration des Acetylphosphats beeinflußt (40).Other known virulence factors are an IgA1 protease ( 33 ) whose gene has been cloned and characterized (34, 35), an inhibitor of elastase (36) and peptide permeases that are homologous to permeases from other streptococci that also colonize the nasopharynx, are. Of particular interest are the permeases, in which the loss of function results in reduced adherence of the pneumococci to eukaryotic cells (37). Evidence of direct involvement of these permeas, of which the AmiA permease and the PlpA (permease-like protein A) belong to the family of protein-dependent permeases that are responsible for the transport of small peptides (38, 39) however, adherence to eukaryotic cells has not yet been achieved. Another potential regulator of pneumococcal adherence is pyruvate oxidase, SpxB, which affects the concentration of acetyl phosphate (40).
Gemäß einer Ausführungsform der Erfindung wird ein Oberflächenprotein von Streptococcus pneunomiae (SpsA-Protein) vorgesehen, das dadurch gekennzeichnet ist, daß es an sekretorisches IgA (sIgA) bindet.According to one embodiment of the invention, a Streptococcus pneunomiae surface protein (SpsA protein) provided, which is characterized in that it secretory IgA (sIgA) binds.
Das erfindungsgemäße Oberflächenprotein kann gekennzeichnet sein durch 523 Aminosäuren gemäß Fig. 2 (Positionen 1 bis 523). The surface protein according to the invention can be characterized by 523 amino acids according to FIG. 2 (positions 1 to 523).
Gemäß einer weiteren Ausführungsform wird ein C-terminal
deletierter Abkömmling des Oberflächenproteins gemäß der
Erfindung vorgesehen, der gekennzeichnet ist durch 324
Aminosäuren gemäß Fig. 2 (Positionen 1 bis 324) und
Repeats 1 bis 6 (Positionen 325 bis 444) oder
Repeats 1 bis 7 (Positionen 325 bis 464) oder
Repeats 1 bis 8 (Positionen 325 bis 484 oder bis 485).According to a further embodiment, a C-terminally deleted derivative of the surface protein according to the invention is provided, which is characterized by 324 amino acids according to FIG. 2 (positions 1 to 324) and
Repeats 1 to 6 (positions 325 to 444) or
Repeats 1 to 7 (positions 325 to 464) or
Repeats 1 to 8 (positions 325 to 484 or to 485).
Gemäß einer weiteren Ausführungsform wird ein N-terminal
deletierter Abkömmling des Oberflächenproteins gemäß der
Erfindung vorgesehen, der dadurch gekennzeichnet ist, daß er
According to a further embodiment, an N-terminally deleted derivative of the surface protein according to the invention is provided, which is characterized in that it
- (i) im Bereich der Positionen 1 bis 159 gemäß Fig. 2 um 1 bis maximal 159 Aminosäuren deletiert ist,(i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,
- (ii) nicht jedoch im Bereich der Positionen 160 bis 523 dele tiert ist.(ii) but not in the range of items 160 to 523 dele is.
Gemäß einer weiteren Ausführungsform wird ein N-terminal und C-
terminal deletierter Abkömmling des Oberflächenproteins gemäß
der Erfindung vorgesehen, der dadurch gekennzeichnet ist, daß er
According to a further embodiment, an N-terminal and C-terminal deleted derivative of the surface protein according to the invention is provided, which is characterized in that it
- (i) im Bereich der Positionen 1 bis 159 gemäß Fig. 2 um 1 bis maximal 159 Aminosäuren deletiert ist,(i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,
- (ii) nicht jedoch im Bereich der Positionen 160 bis 324 dele tiert ist und(ii) is not, however, deleted in the range of items 160 to 324 and
- (iii) Repeats 1 bis 8 (Positionen 325 bis 484 oder bis 485) oder Repeats 1 bis 7 (Positionen 325 bis 464) oder Repeats 1 bis 6 (Positionen 325 bis 444) aufweist.(iii) repeats 1 to 8 (positions 325 to 484 or to 485) or Repeats 1 to 7 (positions 325 to 464) or Repeats 1 to 6 (positions 325 to 444).
Gemäß einer weiteren Ausführungsform wird ferner ein Expressionssystem zur Expression eines Oberflächenproteins oder eines Abkömmlings gemäß der Erfindung vorgesehen, umfassend eine DNA-Sequenz, die das Oberflächenprotein oder den Abkömmling kodiert.According to a further embodiment, a Expression system for expressing a surface protein or of a descendant according to the invention, comprising a DNA sequence that is the surface protein or the descendant encoded.
Gemäß einer weiteren Ausführungsform wird schließlich eine Vaccine zur Bekämpfung von Erkrankungen durch Streptococcus pneunomiae vorgesehen, herstellbar mit Hilfe eines Oberflächenproteins oder eines Abkömmlings gemäß der Erfindung.According to a further embodiment, a Vaccine to fight diseases caused by Streptococcus pneunomiae provided, producible with the help of a Surface protein or a derivative according to the invention.
Nachstehend wird die Erfindung anhand von Figuren, experimenteller Ergebnisse und Beispielenäher erläutert. Es zeigen:The invention is explained below with reference to figures, experimental results and examples explained in more detail. It demonstrate:
Fig. 1 Western blot Analyse von Streptococcus pneumoniae ATCC 33400 [Serotyp 1] (Spur 1), NCTC 10319 [Serotyp 47, R36A] (Spur 2), ATCC 11733 [Serotyp 2, R36A] (Spur 3) und ATCC 12213 [Serotyp 1, I-192R) (Spur 4) mit sekretorischem Immunglobulin A. Zur Detektion der Bindung wurde ein peroxidase markierter anti-human IgA Antikörper eingesetzt. Fig. 1 Western blot analysis of Streptococcus pneumoniae ATCC 33400 [serotype 1] (lane 1), NCTC 10319 [serotype 47, R36A] (lane 2), ATCC 11733 [serotype 2, R36A] (lane 3) and ATCC 12213 [serotype 1, I-192R) (lane 4) with secretory immunoglobulin A. A peroxidase-labeled anti-human IgA antibody was used to detect the binding.
Fig. 2 Nukleinsäuresequenz des Gens spsA und die Aminosäure sequenz des Proteins SpsA (Streptococcus pneumoniae secretory IgA binding protein) von Streptococcus pneumoniae ATCC 33400 Serotyp 1. RBS: Ribosomale Bindungsstelle; Leader: Signalsequenz von SpsA (Aminosäure 1-37); mature Protein: SpsA nach der Pro zessierung; Repeats: 9 jeweils 20 Aminosäuren lange, sich wie derholende Sequenzen. Fig. 2 nucleic acid sequence of the gene SPSA and the amino acid sequence of the protein SpsA (Streptococcus pneumoniae secretory IgA binding protein) of Streptococcus pneumoniae ATCC 33400 serotype 1. RBS: ribosome binding site; Leader: signal sequence of SpsA (amino acid 1-37); mature protein: SpsA after processing; Repeats: 9 each 20 amino acids long, like repeating sequences.
Fig. 3 Western blot Analyse mit sIgA nach Klonierung von spsA und spsA-Fragmenten in den Expressionsvektor pQE (Pharmacia) und Überexpression der Proteine in Escherichia coli M15 [pREP4). Spur 1: SpsA (AS1-523; pQSHA12); Spur 2: N-Terminus von SpsA (AS1-324; pQSHA14); Spur 3: verkürzter N-Terminus von SpsA (AS1- 159; pSHA3); Spur 4: Repeats von SpsA (AS325-523; pQSHA30). Fig. 3 Western blot analysis with sIgA after cloning of SPSA and the SPSA fragments into the expression vector pQE (Pharmacia) and overexpression of the proteins in Escherichia coli M15 [pREP4). Lane 1: SpsA (AS1-523; pQSHA12); Lane 2: N-terminus of SpsA (AS1-324; pQSHA14); Lane 3: truncated N-terminus of SpsA (AS1-159; pSHA3); Lane 4: Repeats from SpsA (AS325-523; pQSHA30).
Fig. 4 Southern blot Analyse von Streptococcus pneumoniae ATCC 33400 [Serotyp 1] (Spur 1) und Streptococcus pneumoniae ATCC 11733 [Serotyp 2, R36A) (Spur 2) mit einer 32Phosphor radioaktiv markierten DNA-Sonden von spsA (A), von einem 5'-spsA Fragment [nt1-nt476] (B) und pspA (C). nt: Nukleotid. Fig. 4 Southern blot analysis of Streptococcus pneumoniae ATCC 33400 [serotype 1] (lane 1) and Streptococcus pneumoniae ATCC 11733 [serotype 2, R36A) (lane 2) with a 32 phosphorus radioactively labeled DNA probe from spsA (A), from a 5'-spsA fragment [nt1-nt476] (B) and pspA (C). nt: nucleotide.
In Bindungsstudien mit radioaktiv markiertem humanem sekretori schen Immunglobulin A (sIgA) und in Western-blots mit humanem sIgA konnte nachgewiesen werden, daß Streptococcus pneumoniae sekretorisches IgA bindet. Auf die Fähigkeit sIgA zu binden, wurden sowohl klinische Isolate als auch Stämme aus den Type Culture Collections (ATCC bzw. NCTC) der häufigsten Serotypen klinischer Isolate getestet. Von den untersuchten Pneumokokken- Stämmen konnten im Western-blot 73% sIgA binden (Fig. 1). Des weiteren konnte in Bindungsstudien gezeigt werden, nach Verdau der Pneumokokken-Proteine mit Protease, daß die Bindung von se kretorischem IgA durch ein Protein von Streptococcus pneumoniae erfolgt.In binding studies with radioactively labeled human secretory immunoglobulin A (sIgA) and in Western blots with human sIgA, it was demonstrated that Streptococcus pneumoniae binds secretory IgA. Both clinical isolates and strains from the Type Culture Collections (ATCC or NCTC) of the most common serotypes of clinical isolates were tested for the ability to bind sIgA. Of the pneumococcal strains examined, 73% could bind sIgA in the Western blot ( FIG. 1). Furthermore, after digestion of the pneumococcal proteins with protease, it could be shown in binding studies that the binding of secret IgA is carried out by a protein from Streptococcus pneumoniae.
Die Detektion des Gens, das für das sIgA-Bindungsprotein ko diert, erfolgte durch Screening einer LambdaZAP-Expressionsbank von Streptococcus pneumoniae ATCC 33400 (Serotyp 1) mit sekreto rischem IgA. Der ausgewählte positive Klon, pSHA1, hat ein 5085 bp großes Insert von Streptococcus pneumoniae ATCC 33400 im Pha gemid pBK-CMV. Durch die Konstruktion von Deletionsklonen von pSHA1 konnte das für das sIgA-Bindungsprotein kodierende Gen im Subklon pSHA2 nachgewiesen werden. Der Subklon pSHA2 zeigte im Gegensatz zu den beiden anderen Subklonen pSHA3 und pSHA4 eine Bindung des sekretorischen IgA im Western-blot. Die Sequenzie rung und anschließende Analyse der Sequenz des 2204 großen In serts zeigte ein offenes Leseraster von Nukleotid 282 bis 1853 in pSHA2 (Fig. 2). Dieses 1572 bp große Leseraster kodiert für ein 523 Aminosäuren großes sIgA-Bindungsprotein von Pneumokokken des Serotyps 1. Das Molekulargewicht des SpsA (Btreptococcus pneumoniae secretory IgA binding-protein) genannten Proteins be trägt 59151 Da (Fig. 2).The detection of the gene coding for the sIgA binding protein was carried out by screening a LambdaZAP expression bank of Streptococcus pneumoniae ATCC 33400 (serotype 1) with secretory IgA. The selected positive clone, pSHA1, has a 5085 bp insert of Streptococcus pneumoniae ATCC 33400 in the pha gemid pBK-CMV. By constructing deletion clones of pSHA1, the gene coding for the sIgA binding protein could be detected in the subclone pSHA2. In contrast to the two other subclones pSHA3 and pSHA4, the subclone pSHA2 showed a binding of the secretory IgA in the Western blot. The sequencing and subsequent analysis of the sequence of the 2204 large insert showed an open reading frame from nucleotide 282 to 1853 in pSHA2 ( FIG. 2). This 1572 bp reading frame codes for a 523 amino acid sIgA binding protein of pneumococci of serotype 1. The molecular weight of the protein called SpsA (Btreptococcus pneumoniae secretory IgA binding protein) is 59151 Da ( FIG. 2).
Der Vergleich der Nukleotidsäuresequenz von spsA mit den in der EMBL-Datenbank gespeicherten Sequenzen zeigte eine 78,8%-ige Identität zum pspA von Streptococcus pneumoniae. Auf Pro teinebene wurde eine 64,1%-ige Identität zum PspA nachgewiesen. Die Identität beschränkt sich dabei vor allem auf den C-Terminus der beiden Proteine. Der C-Terminus des PspA besteht aus zehn Repeats, die jeweils 20 Aminosäuren lang sind (41). Die Identi tät zum C-Terminus von SpsA, der aus neun Repeats besteht, be trägt 92,5%.The comparison of the nucleotide sequence of spsA with that in the EMBL database stored sequences showed a 78.8% Identity to the pspA of Streptococcus pneumoniae. On pro At the level, a 64.1% identity to the PspA was demonstrated. The identity is primarily limited to the C-terminus of the two proteins. The C-terminus of the PspA consists of ten Repeats, each 20 amino acids long (41). The ident to the C-terminus of SpsA, which consists of nine repeats, be carries 92.5%.
Die Repeats von PspA sind an der Anheftung des Proteins an der Pneumokokken-Zellwand beteiligt. Dieser Mechanismus erfordert eine Cholin vermittelte Interaktion zwischen der membranassozi ierten Lipoteichonsäure und der Repeatregion von PspsA (42). Zur Anheftung an die Zellwand müssen mindestens sechs der 20 Amino säuren langen Repeats exprimiert werden, ansonsten kommt es zu einer Sekretion des PspA (42, 43).The repeats of PspA are attached to the protein on the Pneumococcal cell wall involved. This mechanism requires a choline mediated interaction between the membrane associ ized lipoteichoic acid and the repeat region of PspsA (42). For Attachment to the cell wall must be at least six of the 20 amino acids long repeats are expressed, otherwise it happens secretion of PspA (42, 43).
Da der C-Terminus von SpsA, d. h. die Region der Repeats von SpsA, zu 92,5% identisch mit den Repeats von PspA ist, kann eine Beteiligung der SpsA-Repeats an der Anheftung an die Zell wand der Pneumokokken angenommen werden. Dieser Mechanismus der Anheftung von SpsA wird unterstützt durch die Tatsache, daß eine Computeranalyse der Sekundärstruktur von SpsA keine helikale transmembranöse Struktur detektierte, die für eine Verankerung des Proteins in der gram-positiven Zellwand notwendig wäre.Since the C-terminus of SpsA, i.e. H. the region of the repeats of SpsA, which is 92.5% identical to the repeats of PspA, can participation of the SpsA repeats in the attachment to the cells wall of the pneumococcus. This mechanism of SpsA attachment is supported by the fact that a Computer analysis of the secondary structure of SpsA no helical transmembrane structure detected for anchoring of the protein in the gram-positive cell wall would be necessary.
Des weiteren deuteten frühere Ergebnisse darauf hin, daß das 3'- Ende von pspA eine konservierte Sequenz ist, die homolog zu an deren Sequenzen bei Streptococcus pneumoniae ist. Diese homolo gen Sequenzen könnten für Proteine kodieren, die einen ähnlichen Anheftungsmechanismus an die Zellwand haben wie das pneumococcal surface protein A (PspA) (44).Furthermore, previous results indicated that the 3'- End of pspA is a conserved sequence that is homologous to whose sequences is in Streptococcus pneumoniae. This homolo gene sequences could code for proteins that have a similar Attachment mechanisms to the cell wall have like the pneumococcal surface protein A (PspA) (44).
Die Identität im N-Terminus der beiden Proteine SpsA und PspA
beträgt nur von 34,5%. Die Signalsequenz (Leader) von SpsA ist
37 Aminosäuren lang und zeigt eine 75,7%-ige Identität zur Si
gnalsequenz des IgA-Rezeptors von Streptococcus agalactiae (45,
46)
Zur Aufklärung der Bindungsdomaine wurden i) spsA (pQSH12), ii)
der N-Terminus (pQSH14) und iii) die Nukleinsäuresequenz, die
für die neun Repeats von SpsA kodiert (pQSH30), in den Expres
sionsvektor pQE30 kloniert und die sIgA-Bindung im Western-blot
untersucht. Die Analyse im Western-blot zeigte eine Bindung des
sekretorischen IgA im N-Terminus von SpsA. Zusätzlich konnte die
Bindungsdomaine von SpsA durch einen weiteren Subklon von pSHA1,
der nur die Aminosäuren 1 bis 159 des N-Terminus von SpsA expri
miert (pSHA3) und sIgA nicht bindet, auf die Aminosäuren 160 bis
324 eingeengt werden (Fig. 3).The identity in the N-terminus of the two proteins SpsA and PspA is only 34.5%. The signal sequence (leader) of SpsA is 37 amino acids long and shows a 75.7% identity to the signal sequence of the IgA receptor from Streptococcus agalactiae (45, 46)
To clarify the binding domain, i) spsA (pQSH12), ii) the N-terminus (pQSH14) and iii) the nucleic acid sequence which codes for the nine repeats of SpsA (pQSH30) were cloned into the expression vector pQE30 and the sIgA binding examined in a Western blot. Western blot analysis showed binding of the secretory IgA in the N-terminus of SpsA. In addition, the binding domain of SpsA could be restricted to amino acids 160 to 324 by a further subclone of pSHA1, which only expresses amino acids 1 to 159 of the N-terminus of SpsA (pSHA3) and does not bind sIgA ( FIG. 3).
Durch Southern-blot Analyse von Streptococcus pneumoniae ATCC 33400 (Serotyp1) und S. pneumoniae ATCC 11733 (Serotyp 2, R36A) konnte mit einer pspA-, spsA und einer 5'-spsA spezifischen DNA- Sonde nachgewiesen werden, daß spsA nicht identisch mit pspA ist (Fig. 4).Southern blot analysis of Streptococcus pneumoniae ATCC 33400 (serotype 1) and S. pneumoniae ATCC 11733 (serotype 2, R36A) with a pspA, spsA and a 5'-spsA specific DNA probe showed that spsA is not identical to pspA is ( Fig. 4).
Mit diesen Untersuchungen konnte nachgewiesen werden, daß die C- terminale Sequenz von spsA eine der beschriebenen konservierten Sequenzen ist, die zu pspA homolog sind und bei Streptococcus pneumoniae an der Anheftung der Proteine an die Zellwand betei ligt ist. These investigations have shown that the C- terminal sequence of spsA one of the conserved described Is sequences that are homologous to pspA and in Streptococcus pneumoniae on the attachment of the proteins to the cell wall is.
SpsA kodiert daher für ein neues Oberflächenprotein von Strepto coccus pneumoniae, dessen biologische Funktion die Bindung von sekretorischem Immunglobulin A im N-Terminus von SpsA ist.SpsA therefore codes for a new surface protein from Strepto coccus pneumoniae, whose biological function is the binding of secretory immunoglobulin A in the N-terminus of SpsA.
Das sekretorische IgA, das aus einem IgA-J-IgA-SC (SC: sekreto rische Komponente) Komplex besteht, ist das wichtigste Immuno globulin im humanen respiratorischen und gastrointestinalen Trakt. Die Vorstufe des sekretorischen IgA, ein IgA-Dimer ver bunden durch die 15,6 kDa große J-Kette, wird in B-Lymphozyten synthetisiert und bindet an einen auf der basolateralen Oberflä che der Epithelzellen lokalisierten poly-Immunglobulin Rezeptor. Dieser poly-Ig-Rezeptor vermittelt die Transzytose durch die Zellen (47). Dabei wird der c-terminale Teil des Rezeptors abge trennt und zur sekretorischen Komponente des (IgA)2-J-Ketten Komplex. Nach Transport an die apikale Membran der Zellen, fu sioniert der Komplex mit der Membran und wird als sekretorisches IgA freigesetzt (48). Die sekretorische Komponente, die durch eine Disulfidbrücke kovalent an den Komplex gebunden ist, schützt das synthetisierte sIgA in externen Flüssigkeiten vor Denaturierung und Proteolyse.The secretory IgA, which consists of an IgA-J-IgA-SC (SC: secretory component) complex, is the most important immunoglobulin in the human respiratory and gastrointestinal tract. The precursor of the secretory IgA, an IgA dimer linked by the 15.6 kDa J chain, is synthesized in B lymphocytes and binds to a poly-immunoglobulin receptor located on the basolateral surface of the epithelial cells. This poly-Ig receptor mediates transcytosis through the cells (47). The c-terminal part of the receptor is separated and becomes the secretory component of the (IgA) 2 -J chain complex. After transport to the apical membrane of the cells, the complex fuses with the membrane and is released as secretory IgA (48). The secretory component, which is covalently bound to the complex by a disulfide bridge, protects the synthesized sIgA in external liquids from denaturation and proteolysis.
Untersuchungen zur Adhärenz der Pneumokokken an humane Epithel zellen zeigten eine Adhärenz von größer als 100 Pneumokokken pro Epithelzelle für Stämme, die in den Bindungsstudien sIgA gebun den hatten, aber nur eine Adhärenz von ≦ 2 Pneumokokken für in Bindungsstudien negativ getestete Stämme.Studies on the adherence of pneumococci to human epithelium cells showed an adherence of more than 100 pneumococci per Epithelial cell for strains that sIgA bound in the binding studies had, but only an adherence of ≦ 2 pneumococci for in Binding studies negative strains tested.
Diese Ergebnisse weisen darauf hin, daß das SpsA an der Adhärenz der Pneumokokken an die Epithelzellen und an der Invasion in die Epithelzellen beteiligt ist.These results indicate that the SpsA is adherent the pneumococcal to the epithelial cells and to the invasion into the Epithelial cells is involved.
Das sekretorische IgA-Bindungsprotein, SpsA, von Streptococcus pneumoniae ist damit ein aussichtsreicher neuer Kandidat für die Impfstoffentwicklung. The Streptococcus secretory IgA binding protein, SpsA pneumoniae is therefore a promising new candidate for the Vaccine development.
Dieses Beispiel beschreibt die sekretorische IgA-Bindung von Streptococcus pneumoniae Stämmen im Western-blot.This example describes the secretory IgA binding of Streptococcus pneumoniae strains in Western blot.
Die Proteine der Streptococcus pneumoniae Lysate (OD600 von 1,0 eingestellt und nach Aufnahme der Bakterien in 100 µl Aufschluß lösung (20% Glycerin, 3% SDS, 3% β-Mercaptoethanol, 0,05% Bromphenolblau) 10 Minuten bei 94°C gekocht) wurden im SDS-PAGE aufgetrennt und anschließend auf eine Nitrozellulosemembran transferriert. Nach Absättigung mit 10%-iger Magermilch in 0,1 M PBS wurden die Filter mit sekretischem IgA [1 µg/ml] (Sigma, München, Germany) in 0,1 M PBS für 1 Stunde bei Raumtemperatur unter Schütteln inkubiert. Nach dreimaligem Waschen mit 0,1 M PBS wurden die Filter 1 Stunde mit einem Goat-Anti Human IgA- HRP-Konjugat Antikörper (ICN, Eschwege, Germany) inkubiert. Die Farbentwicklung erfolgte nach dreimaligem Waschen mit 1 mg 4- Chloro-1-Naphthol und 0,1% H2O2 pro 1 ml PBS.The proteins of the Streptococcus pneumoniae lysate (OD 600 set at 1.0 and after absorption of the bacteria in 100 µl digestion solution (20% glycerol, 3% SDS, 3% β-mercaptoethanol, 0.05% bromophenol blue) for 10 minutes at 94 ° C cooked) were separated in the SDS-PAGE and then transferred to a nitrocellulose membrane. After saturation with 10% skim milk in 0.1 M PBS, the filters were incubated with secretive IgA [1 μg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour at room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-HRP conjugate antibody (ICN, Eschwege, Germany). The color developed after washing three times with 1 mg of 4-chloro-1-naphthol and 0.1% H 2 O 2 per 1 ml of PBS.
Dieses Beispiel beschreibt die Bindung von 125Jod-markiertem se kretorischem IgA von Streptococcus pneumoniae Stämmen.This example describes the binding of 125 iodine-labeled Se cretory IgA from Streptococcus pneumoniae strains.
100 ng sIgA in 1 ml 0,05 M Phosphatpuffer pH 7,5 wurden nach Zugabe von 20 ug Chloramin T mit 350 µCi 125Jod inkubiert und die Reaktion nach 5 Minuten durch Zugabe von 20 -g Na-Metabisul fit gestoppt. Die markierten Proteine wurden von den nicht mar kierten Proteinen mit einer PD10-Säule (Pharmacia, Freiburg, Germany) abgetrennt und bei -20 °C eingefroren. 250 µl einer Pneumokokkensuspension (Transmission 10% bei 600 nm) in PBST (PBS mit 0,05% Tween 20) wurden mit 0,023 µCi 125Jod-markiertem sIgA 45 Minuten inkubiert und die Reaktion mit 1 ml PBST abge stoppt. Die Messung der sIgA-Bindung an die Streptococcus pneu moniae Stämme erfolgte durch Messung der Aktivität der Bakterien im Gamma-Counter (Packard, Dreieich, Germany). 100 ng sIgA in 1 ml 0.05 M phosphate buffer pH 7.5 were added after the addition of 20 μg chloramine T with 350 μCi 125 iodine and the reaction was stopped after 5 minutes by adding 20 μg Na-Metabisul fit. The labeled proteins were separated from the unmarked proteins with a PD10 column (Pharmacia, Freiburg, Germany) and frozen at -20 ° C. 250 μl of a pneumococcal suspension (transmission 10% at 600 nm) in PBST (PBS with 0.05% Tween 20) were incubated with 0.023 μCi 125 iodine-labeled sIgA for 45 minutes and the reaction was stopped with 1 ml PBST. The sIgA binding to the Streptococcus pneu moniae strains was measured by measuring the activity of the bacteria in the gamma counter (Packard, Dreieich, Germany).
Dieses Beispiel beschreibt die Klonierung der chromosomalen DNA von Streptococcus pneumoniae ATCC 33400 in den Vektor Lambda ZAP Express™ und das Screening der Genbank nach einem Streptococcus pneumoniae secretory IgA binding protein (SpsA).This example describes the cloning of the chromosomal DNA from Streptococcus pneumoniae ATCC 33400 into the vector Lambda ZAP Express ™ and screening of the gene bank for a streptococcus pneumoniae secretory IgA binding protein (SpsA).
Die chromosomale DNA von Streptococcus pneumoniae ATCC 33400 wurde isoliert, mit Sau3A partiell verdaut und in einem Natrium chloridgradienten, der durch Einfrieren und Auftauen einer 20% igen Natriumchloridlösung gebildet wurde, nach Größe der DNA- Fragmente fraktioniert. Die Ligation der 2,0 kb bis 6,0 kb großen DNA-Fragmente der chromosomalen DNA in den BamHI-ge schnittenen Lambda ZAP Express™ und die in vitro Verpackung er folgte mit einem komerziellen Kit nach Angaben des Herstellers (Stratagene, Heidelberg, Germany). Die Phagengenbank wurde ohne weitere Amplifikation ausplattiert und die rekombinanten Plaques auf die Expression eines sekretorischen IgA-Bindungsprotein un tersucht. Der Transfer der Proteine erfolgte auf Nitrozellulose filter, und nach Absättigung mit 10%-iger Magermilch in 0,1 M PBS wurde mit sekretorischem IgA [1 µg/ml] (Sigma, München, Germany) in 0,1 M PBS für 1 Stunde bei Raumtemperatur unter Schütteln inkubiert. Nach dreimaligem Waschen mit 0,1 M PBS wur den die Filter 1 Stunde mit einem Goat-Anti Human IgA-HRP-Konju gat Antikörper inkubiert. Die Farbentwicklung erfolgte nach dreimaligem Waschen mit 1 mg 4-Chloro-1-Naphthol und 0,1% H2O2 in 1 ml PBS. Positive Plaques wurden isoliert und nach Amplifi kation die in vivo Excision des pBK-CMV Phagmids mit Hilfe des Exassist He1ferphagen und XLOLR System nach den Angaben des Her stellers (Stratagene, Heidelberg, Germany) durchgeführt. The chromosomal DNA of Streptococcus pneumoniae ATCC 33400 was isolated, partially digested with Sau3A and fractionated according to the size of the DNA fragments in a sodium chloride gradient which was formed by freezing and thawing a 20% sodium chloride solution. The ligation of the 2.0 kb to 6.0 kb DNA fragments of the chromosomal DNA in the BamHI-cut Lambda ZAP Express ™ and the in vitro packaging was carried out using a commercial kit according to the manufacturer (Stratagene, Heidelberg, Germany ). The phage library was plated out without further amplification and the recombinant plaques were examined for the expression of a secretory IgA binding protein. The proteins were transferred to nitrocellulose filters, and after saturation with 10% skim milk in 0.1 M PBS, secretory IgA [1 μg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour incubated at room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-HRP conjugate antibody. The color developed after washing three times with 1 mg of 4-chloro-1-naphthol and 0.1% H 2 O 2 in 1 ml of PBS. Positive plaques were isolated and, after amplification, the in vivo excision of the pBK-CMV phagmid was carried out using the exassist He1ferphagen and XLOLR system according to the manufacturer's instructions (Stratagene, Heidelberg, Germany).
Dieses Beispiel beschreibt die DNA-Sequenzierung und die Ablei tung der Aminosäuresequenz. Die Sequenzierung des 5,085 kb In serts von Streptococcus pneumoniae ATCC 33400 im Phagemid pBK- CMV, pSHAI genannt, erfolgte durch das ABI PRISM™ Dye Termina tor Cycle Sequencing nach Angaben des Herstellers (Perkin-Elmer, Germany) mit den Vektorprimern T3X (5'-AATTAACCCTCACTAAAGGG-3') und T7X (5'-TAATACGACTCACTATCGGG-3') sowie den von der erhalte nen Sequenz abgeleiteten Primern (siehe Tabelle). Die Transla tion der Nukleinsäuresequenz in die Aminosäuresequenz erfolgte mit Hilfe des Programms GeneWorks, Version 2.45 (Intelligene tics, Montain View, CA).This example describes DNA sequencing and derivation tion of the amino acid sequence. The sequencing of the 5.085 kb In serts of Streptococcus pneumoniae ATCC 33400 in phagemid pBK- CMV, called pSHAI, was done by the ABI PRISM ™ Dye Termina Tor cycle sequencing according to the manufacturer (Perkin-Elmer, Germany) with the vector primers T3X (5'-AATTAACCCTCACTAAAGGG-3 ') and T7X (5'-TAATACGACTCACTATCGGG-3 ') as well as that received a sequence derived primers (see table). The Transla tion of the nucleic acid sequence into the amino acid sequence with the help of the program GeneWorks, version 2.45 (intelligent tics, Montain View, CA).
Tabelle 1Table 1
Dieses Beispiel beschreibt die Konstruktion der Subklone von pSHA1 und deren Charakterisierung im Western-blot. Subklon pSHA2 (nt1-nt2203 von pSHA1) wurde durch Deletion eines 2882 bp großen EcoRI-Fragments erhalten und Subklon pSHA3 (nt1- nt757) durch Deletion eines 4328 bp großen HindIII-Fragments von pSHA1. Ein weiterer Subklon, genannt pSHA4 (nt2952-nt5085 von pSHA1), wurde durch Deletion eines 2133 bp großen SacI-Fragments erhalten.This example describes the construction of the subclones of pSHA1 and its characterization in the Western blot. Subclone pSHA2 (nt1-nt2203 from pSHA1) was deleted by deleting one 2882 bp EcoRI fragment obtained and subclone pSHA3 (nt1- nt757) by deletion of a 4328 bp HindIII fragment from pSHA1. Another subclone called pSHA4 (nt2952-nt5085 from pSHA1), was obtained by deleting a 2133 bp SacI fragment receive.
Die Charakterisierung der Klone erfolgte nach Auftrennung der Proteine des Zellysats [OD600 von 1,0 eingestellt und nach Auf nahme der Bakterien in 100 µl Aufschlußlösung (20% Glycerin, 3 % SDS, 3% β-Mercaptoethanol, 0,05% Bromphenolblau) 10 Minuten bei 94°C gekocht] der rekombinanten E.coli Zellen im SDS-PAGE und Transfer der Proteine auf eine Nitrozellulosemembran im Western-blot mit sekretorischem IgA. Nach Absättigung mit 10% iger Magermilch in 0,1 M PBS wurden die Filter mit sekretori schem 1gA [1 µg/ml] (Sigma, München, Germany) in 0,1 M PBS für 1 Stunde bei Raumtemperatur unter Schütteln inkubiert. Nach drei maligem Waschen mit 0,1 M PBS wurden die Filter 1 Stunde mit einem Goat-Anti Human IgA-MRP-Konjugat Antikörper inkubiert. Die Farbentwicklung erfolgte nach dreimaligem Waschen mit 1 mg 4- Chloro-1-Naphthol und 0,1% H2O2 pro 1 ml PBS.The clones were characterized after separation of the proteins of the cell lysate [OD 600 set to 1.0 and after absorption of the bacteria in 100 μl digestion solution (20% glycerol, 3% SDS, 3% β-mercaptoethanol, 0.05% bromophenol blue) Boiled for 10 minutes at 94 ° C.] of the recombinant E. coli cells in SDS-PAGE and transfer of the proteins to a nitrocellulose membrane in a Western blot with secretory IgA. After saturation with 10% skim milk in 0.1 M PBS, the filters were incubated with secretory 1 gA [1 µg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour at room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-MRP conjugate antibody. The color developed after washing three times with 1 mg of 4-chloro-1-naphthol and 0.1% H 2 O 2 per 1 ml of PBS.
Dieses Beispiel beschreibt die PCR-Amplifikation und Klonierung von spsA, des 5'-Bereichs von spsA (nt1-nt972) und des 3'-Be reichs (nt973-nt1572) von spsA in den Expressionsvektor pQE30 (Pharmacia).This example describes PCR amplification and cloning of spsA, the 5 'region of spsA (nt1-nt972) and the 3'-Be reichs (nt973-nt1572) from spsA into the expression vector pQE30 (Pharmacia).
Die PCR Primer für spsA und der spsA-Fragmente wurden abgeleitet von der in pSHA1 erhaltenen spsA-Sequenz von Streptococcus pneu moniae ATCC 33400 Serotyp 1. Der 5'-Primer SH22 (5'-GCGCGCG CGCGGATCCTTGTTTGCATCAAAAAGCGAAAG-3') ist 39 bp lang und beginnt mit einem veränderten Startkodon des spsA-Gens (TTG statt ATG). Der 5'-Primer für die Repeats, SH24 (5'-GCGCGCGGGCGGATCCACAGGCT GGAAACAAGAAAAC-3'), beginnt mit der Anfangssequenz des ersten Repeats bei Nukleotid 973 des spsA-Gens. Der 3'-Primer von spsASH23 (CTCAGCTAATTAAGCTTGTTTAGTTTACCCATTCACCATTGGC-3'), be ginnt mit dem Stopkodon und der 3'-Primer des N-Terminus, SH25 (5'- CTCAGCTAATTAAGCTTTTTTGGAGTAGATGGTTGTGCTGG-3'), beginnt bei Nukleotid 972 des spsA-Gens. Die Primer SH22-SH23 wurden zur Konstruktion von pQSH12, die Primer SH22-SH25 zur Konstruktion von pQSH14 und die Primer SH24-SH23 zur Konstruktion von pQSH30 genutzt. Die 5'-Primer enthielten zur Klonierung eine BamHI Re striktionsschnittstelle, die 3'-Primer eine HindIII Restrik tionsschnittstelle.The PCR primers for spsA and the spsA fragments were derived from the Streptococcus pneu spsA sequence obtained in pSHA1 moniae ATCC 33400 serotype 1. The 5'-primer SH22 (5'-GCGCGCG CGCGGATCCTTGTTTGCATCAAAAAGCGAAAG-3 ') is 39 bp long and begins with a modified start codon of the spsA gene (TTG instead of ATG). The 5 'primer for the repeats, SH24 (5'-GCGCGCGGGCGGATCCACAGGCT GGAAACAAGAAAAC-3 '), begins with the initial sequence of the first Repeats at nucleotide 973 of the spsA gene. The 3 'primer from spsASH23 (CTCAGCTAATTAAGCTTGTTTAGTTTACCCATTCACCATTGGC-3 '), be starts with the stop codon and the 3 'primer of the N-terminus, SH25 (5'- CTCAGCTAATTAAGCTTTTTTGGAGTAGATGGTTGTGCTGG-3 ') starts at Nucleotide 972 of the spsA gene. The primers SH22-SH23 were used Construction of pQSH12, the primers SH22-SH25 for construction of pQSH14 and the primers SH24-SH23 for the construction of pQSH30 utilized. The 5 'primers contained a BamHI Re for cloning restriction interface, the 3 'primer is a HindIII restriction tion interface.
Die Amplifikation der genomischen Pneumokokken-DNA mit den 5'- und 3'-Primern (20 pmol jeweils) erfolgte in einem Thermocycler (MWG-Biotech, Ebersberg, Germany) in einem 100 µl Volumen mit 2,5 Units der Goldstar Taq-Polymerase nach den Angaben des Her stellers (Eurogentec, Seraing, Belgien) und 50 ng chromosomaler DNA. Die Proben wurden bei 94°C zwei Minuten denaturiert und die Amplifikation erfolgte in 35 Zyklen bestehend aus 1 Minute Denaturierung der DNA bei 94°C, 1 Minute Annealing des Primers bei 55°C und 2 Minuten Extension bei 72°C.The amplification of the genomic pneumococcal DNA with the 5'- and 3 'primers (20 pmol each) were carried out in a thermal cycler (MWG-Biotech, Ebersberg, Germany) in a 100 µl volume 2.5 units of the Goldstar Taq polymerase according to the manufacturer stellers (Eurogentec, Seraing, Belgium) and 50ng chromosomal DNA. The samples were denatured at 94 ° C for two minutes and the amplification was carried out in 35 cycles consisting of 1 minute Denaturation of the DNA at 94 ° C, 1 minute annealing of the primer at 55 ° C and 2 minutes extension at 72 ° C.
Die Primer SH22 bis SH23 konnten auch für die Amplifikation und Klonierung der spsA-Gene von Streptococcus pneumoniae Serotyp 2 (R36A smooth, ATCC 11733 und D39, NCTC 7466) und Serotyp 47 (R36A rough, NCTC 10319) verwendet werden.The primers SH22 to SH23 could also be used for amplification and Cloning of the Streptococcus pneumoniae serotype 2 spsA genes (R36A smooth, ATCC 11733 and D39, NCTC 7466) and serotype 47 (R36A rough, NCTC 10319) can be used.
Die Primer SH22 bis SH25 konnten auch für die Amplifikation und Klonierung des 5'-Bereichs von Streptococcus pneumoniae Serotyp 47 (R36A rough, NCTC 10319) verwendet werden.The primers SH22 to SH25 could also be used for amplification and Cloning of the 5 'region of Streptococcus pneumoniae serotype 47 (R36A rough, NCTC 10319) can be used.
Dieses Beispiel beschreibt die Untersuchung der Adhärenz von Streptococcus pneumoniae Stämmen an humane Epithelzellen. This example describes the study of the adherence of Streptococcus pneumoniae strains on human epithelial cells.
Konfluente HEp-2 Larynxkarzinom Zellen oder A549 alveolare Lun
genzellen (2×105), die in DMEM/5% fetales Kälberserum (FCS)
wuchsen, wurden nach Waschen der Zellen mit DMEM/1mM HEPES mit
107 Pneumokokken in DMEM/1 mM HEPES für 1 Stunde bei 37°C infi
ziert. Anschließend wurden die Zellen dreimal mit PBS gewaschen
und mit Methanol fixiert (-20°C, 30 Minuten). Die extrazellulä
ren Pneumokokken wurden zur mikroskopischen Auszählung mit
Giemsa nach den Angaben des Herstellers (Sigma Diagnostic, Mün
chen, Germany) angefärbt. Die Anzahl der adhärenten Pneumokokken
wurde durch Auszählen von mindestens 100 Epithelzellen bestimmt.
1 Sternberg, G. M. A fatal form of septicaemia in the rabbit,
produced by the subcutaneous injection of human saliva. National
Board of Health Bulletin 1881; 2: 781-783
2 Austrian, R. Some aspects of the pneumococcal carrier
state. J. Antimicrob. Chemother. 1986; 18 (suppl.A) : 35-45
3 Gray, B. A., G. M. Converse III, and H. C. Dillon, Jr.
1979. Serotypes of Streptococcus pneumonmiaecausing disease. J.
Infect. Dis. 140: 979-983
4 Dagan, R., M. Isaachson, R. Lang, J. Karpuch, C. Block, and
J. Amir from the Israeli Pediatric Bacteremia and eningitis
Group. 1994. Epidemiology of pediatric meningitis caused by
Haemophilus influenzae type b, Streptococcus pneumoniae, and
Neisseria meningitidis in Israel: a 3-year nationwide study. J.
Infect. Dis. 169: 912-916
5 Musher, D. M. 1992. infections caused by Streptococcus
pneumoniae: clinical spectrum, pathogenesis, immunity, and
treatment. Clin. Infect. Dis. 14: 801-809
6 Centers for Disease Control, 1984 [update: pneumococcal
polysaccharide O vaccine usage-United states. Morbid. Mortal.
Weekly Rep. 33: 273-276];
7 Filice, G. A., L. L. Van Etta, C. P. Darby, and D. W. Fra
ser. 1986. Bacteremia in Charlston County, South Carolina. Am.
J. Epidem. 123: 128-136]
8 Gillespe, S. H. 1989. Aspects of pneumococcal infection
including bacterial virulence, host response and vaccination. J.
Med. Microbiol. 28: 237-248
9 Henrichsen, J. 1995. Six newly recognized types of Strepto
coccus pneumoniae. J. Clin. Microbiol. 33: 2759-2762
10 Fine, D. P. 1975 Pneumococcal type associated variability
in alternate complement pathway activation. Infect. Immun. 12:
772-778
11 Chudwin, D. S., Artrip, S. G., Korenblit, A., Schiffman,
G., and Rao, S. 1985 Correlation of serum opsonins with in vitro
phagocytosis of Streptococcus pneumoniae. Infect. Immun. 50:
213-217;
12 Silvenoinen-Kasinen, S., and Koskela, M. 1986. Optimal con
ditions for the opsonophagocytosis test with Streptococcus pneu
moniae serotypes 3, 6A, 7F and 19F and human granulocytes. Acta
Pathol. Microbiol. Scand. Sect. C 94: 105-111
13 Stryker, L. Variations in the pneumococcus induced by
growth in immune serum. J. Exp. Med 1916; 24: 49-68
14 Griffith, F. The significance of pneumococcal types. J.
Hyg 1928; 27: 113-159
15 Avery, O. T., MacLeod, C. M., McCarthy, M. 1944. Studies on
the chemical nature of the substance inducing transformation of
pneumococcal types. J. Exp. Med. 79: 137-158
16 Knecht J. C., Schiffmann, G., Austrian, R. Some biological
properties of Pneumococcus type 37 and the chemistry of its cap
sular polysaccharide. J. Exp. Med. 1970; 132: 475-487
17 Stein, K. E. 1992. Thymus-independent and thymus-dependent
responses to polysaccharide antigens. J. Infect. Dis. 165
(Suppl. 1): 549-552
18 Appelbaum, P. C. 1992. Clin. Infect. Dis. 15: S. 77
19 Tuomanen, E. 1993. Breaching the blood-brain barrier. Sci.
Ami. 268 (2): 80-84
20 Lock, R. A., Hansman, D., and Paton, J. C. 1992 Comparative
efficacy of autolysin and pneumolysin as immunogens protecting
mice against infection by Streptococcus pneumoniae. Microb. Pa
thog. 12: 137-143
21 Lock, R. A., Paton, J. C., and Hansman, D., 1988 Compara
tive efficacy of pneumococcal neuraminidase and pneumolysin as
immunogens protective against Streptococcus pneumoniae. Microb.
Pathog. 5: 461-467
22 Sampson, J. S., O'Connor, S. P., Stinson, A. R., Tharpe, J.
A., and Russell, H. 1994. Cloning and nucleotide sequence analy
sis of psaA, the Streptococcus pneumoniae gene encoding a 37-
kilodalton protein homologius to previously reported Streptococ
cus sp. adhesins. Infect. Immun. 62: 319-324
23 Tart, R. C., McDaniel, L. S., Ralph, B. A., and Briles, D.
E. 1996. Truncated Streptococcus pneumoniae pspA molecules
elicit cross-protective immunity against pneumococcal challenge
in mice. J. Infect. Dis. 173: 380-386
24 Alexander, J. E., bock, R. A., Peeters, C. C. A. M., Pool
man, J. T., Andrew, P. W., Mitchell, T. J., Hansman, D., and
Paton, J. C. 1994. Immunization of mice with pneumolysin toxoid
confers a significant degree of protection against at least nine
serotypes of Streptococcus pneumoniae. Infect. Immun. 62: 5683-
5688
25 Boulnois, G. J. 1992. Pneumococcal proteins and the patho
genesis of disease caused by Streptococcus pneumoniae. J. Gen.
Microbiol. 138: 249-259
26 Houldsworth, S., Andrew, P. W., and Mitchell, T. J. 1994.
Pneumolysin stimulates production of tumor necrosis factor alpha
and interleukin-1β by human mononuklear phagocytes. Infect.
Immun. 62: 1501-1503
27 Feldman, C., Mitchell, T. J., Andrew, P. W., Boulnois, G.
J., Read, R. C., Todd, H. C., Cole, P. J., and Wilson, R. 1990.
The effect of Streptococcus pneumoniae pneumolysin on human
respiratory epithelium in vitro. Micob. Pathog. 9: 275-284
28 Paton, J. C., and Ferrante, A. 1983. Inhibition of human
polymorphonuclear leukocyte respiratory burst, bactericidal
activity, and migration by pneumolysin. Infect. Immun. 41: 1212-
1216
29 Paon, J. C., Andrew, P. W., Bounois, G. J., and Mitchell,
T. J. 1993. Molecular analysis of the pathogenicity of Strepto
coccus pneumoniae: the role of pneumococcal proteins. Annu. Rev.
Microbiol. 47: 89-115
30 Crain, M. J., Waltman II, W. D., Turner, J. S., Yother, J.
Talkington, D. F., McDaniel, L. S., Gray, B. M., and Briles, D.
E. 1990. Pneumococcal surface protein A (PspA) is serologically
highly variable and is expressed by all clinically important
capsular serotypes of Streptococcus pneumoniae. Infect. Immun.
58: 3293-3299
31 Briles, D. E., Yother, J., and McDaniel, L. S. 1988. Role
of pneumococcal surface protein A in the virulence of Strepto
coccus pneumoniae. Rev. Infect. Dis. 10 (Suppl. 4): 797-805
32 McDaniel, IJ. S, Yother, J., Vijayakumar, M., McGarry, L.,
Guild, W. R., and Briles, D. E: 1987. Use of insertional activa
tion to facilitate studies of biological properties of pneumo
coccal surface protein A (PspA). J. Exp. Med. 165: 381-394
33 Male, C. J. Immunglobulin A1 protease production by Haemo
philus influenzae und Streptococcus pneumoniae. Infect. Immun.
1979; 26: 254-261
34 Wani, J. H., Gilbert, J. V., Plaut, A. G., and Weiser, J.
N. 1996. Identfication, cloning, and sequencing of the
Immunoglobulin Al protease gene of Streptococcus pneumoniae.
Infect. Immun. 64 (10): 3967-3974
35 Poulsen, K., Reinholdt, J., and Kilian, M. Characterization
of the Streptococcus pneumoniae Immunglobulin A1 protease gene
(iga) and ist translation product. Infect. Immun. 64 (10): 3957-
3966
36 Vered, M., Schutzbank, T., Janof f, A. Inhibitors of human
neutrophil elastase in extracts of Streptococcus pneumoniae. Am.
Rev. Respir. Dis 1984; 130: 1118-1124
37 Cundell, C. R., Pearce, B. J., Young, A., Tuomanen, E. I.,
Masure, H. R. Protein dependent peptide permeases from Strepto
coccus pneumoniae medite cytoadherence to type II lung cells and
to human endothelial cells [abstract no. B107). J. Cell Biochem.
Suppl. 1994; 18A: 45
38 Lacks, S. A. Purification and properties of the complemen
tary endonucleases DpnI and DpnII. Methods Enzymol. 1980; 65:
138-146
39 Pearce, B. J., Naughton, A. M., Masure, H. R. Peptide per
meases modulate transformation in Streptococcus pneumoniae. Mol.
Microbiol. 1994; 12: 881-892
40 Masure, H. R., Campbell, E. A., Cundell, D. R., Pearce, B.
J., Sandros, J., Spellerberg, B. A. 1995. New genetic strategy
for the analysis of virulence and transformation in Streptococ
cus pneumoniae. Dev. Biol. Stand. 85: 251-260
41 Yother, J., Briles, D. E. 1992. Structural and evolutionary
relationships of PspA, a surface protein of Streptococcus pneu
moniae, as revealeds by sequence analysis. J. Bacteriol. 174:
601-609
42 Yother, J., and White, J. M. . 1994. Novel surface attach
ment mechanism of the Streptococcus pneumoniae Protein PspA. J.
Bacteriol. 176: 2976-2985
43 Yother, J., Handsome, G. I., and Briles, D. E. 1992. Trun
cated forms of PspA that are secreted from Streptococcus pneumo
niae and their use in functional studies and cloning of the pspA
gene. J. Bacteriol. 174: 610-618;
44 McDaniel, b. S., Sheffield, J. S., Swiatlo, E., Yother, J.,
Crain, M. C., and Briles, D. E. 1992. Molecular localization of
variable and conserved region of pspA and identification of
additional pspA homologous sequences in Streptococcus pneumo
niae. Micro. Pathog. 13: 261-269
45 Jerlström, P. G., Chhatwal, G. S., Timmis, K. N. 1991. The
IgA-binding B antigen of the c protein complex of group B strep
tococci: sequence determination of its gene and detection of two
binding regions. Mol. Microbiol. 5: 843-849
46 Heden, b., Frithz, E. bindhal, G. 1991. Molecular characte
rization of an 1gA receptor from group B streptococci: Sequence
of the gene, identification of a proline-rich region with unique
structure and isolation of N-terminal fragments with IgA-binding
capacity. Eur. J. Immun. 21: 1481-1490
47 Solary, R., and Krähenbühl, J. P. 1985. Immunol. Today, 17-
20
48 Brown, W. R., Isobe, Y., Nakane, P. K. 1976. Studies on
translocation of immunoglobulins across intestinal epithilium.
II. Immunelectron-microscopic localization of immunoglobulins
and secretory component in human intestinal mucosa. Gastro
enterology 71: 985-995Confluent HEp-2 laryngeal carcinoma cells or A549 alveolar lung cells (2 × 10 5 ), which grew in DMEM / 5% fetal calf serum (FCS), were washed with DMEM / 1mM HEPES with 10 7 pneumococci in DMEM / 1mM after washing the cells HEPES infected for 1 hour at 37 ° C. The cells were then washed three times with PBS and fixed with methanol (-20 ° C., 30 minutes). The extracellular pneumococci were stained for microscopic counting with Giemsa according to the manufacturer's instructions (Sigma Diagnostic, Munich, Germany). The number of adherent pneumococci was determined by counting at least 100 epithelial cells.
1 Sternberg, GM A fatal form of septicaemia in the rabbit, produced by the subcutaneous injection of human saliva. National Board of Health Bulletin 1881; 2: 781-783
2 Austrian, R. Some aspects of the pneumococcal carrier state. J. Antimicrob. Chemother. 1986; 18 (suppl.A): 35-45
3 Gray, BA, GM Converse III, and HC Dillon, Jr. 1979. Serotypes of Streptococcus pneumonmiaecausing disease. J. Infect. Dis. 140: 979-983
4 Dagan, R., M. Isaachson, R. Lang, J. Karpuch, C. Block, and J. Amir from the Israeli Pediatric Bacteremia and eningitis Group. 1994. Epidemiology of pediatric meningitis caused by Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis in Israel: a 3-year nationwide study. J. Infect. Dis. 169: 912-916
5 Musher, DM 1992. infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin. Infect. Dis. 14: 801-809
6 Centers for Disease Control, 1984 [update: pneumococcal polysaccharide O vaccine usage-United states. Morbid. Mortal. Weekly Rep. 33: 273-276];
7 Filice, GA, LL Van Etta, CP Darby, and DW Fra ser. 1986. Bacteremia in Charlston County, South Carolina. At the. J. Epidem. 123: 128-136]
8 Gillespe, SH 1989. Aspects of pneumococcal infection including bacterial virulence, host response and vaccination. J. Med. Microbiol. 28: 237-248
9 Henrichsen, J. 1995. Six newly recognized types of Strepto coccus pneumoniae. J. Clin. Microbiol. 33: 2759-2762
10 Fine, DP 1975 Pneumococcal type associated variability in alternate complement pathway activation. Infect. Immune. 12: 772-778
11 Chudwin, DS, Artrip, SG, Korenblit, A., Schiffman, G., and Rao, S. 1985 Correlation of serum opsonins with in vitro phagocytosis of Streptococcus pneumoniae. Infect. Immune. 50: 213-217;
12 Silvenoinen-Kasinen, S., and Koskela, M. 1986. Optimal conditions for the opsonophagocytosis test with Streptococcus pneu moniae serotypes 3, 6A, 7F and 19F and human granulocytes. Acta Pathol. Microbiol. Scand. Sect. C 94: 105-111
13 Stryker, L. Variations in the pneumococcus induced by growth in immune serum. J. Exp. Med 1916; 24: 49-68
14 Griffith, F. The significance of pneumococcal types. J. Hyg 1928; 27: 113-159
15 Avery, OT, MacLeod, CM, McCarthy, M. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 79: 137-158
16 Knecht JC, Schiffmann, G., Austrian, R. Some biological properties of Pneumococcus type 37 and the chemistry of its capsular polysaccharide. J. Exp. Med. 1970; 132: 475-487
17 Stein, KE 1992. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J. Infect. Dis. 165 (Suppl. 1): 549-552
18 Appelbaum, PC 1992. Clin. Infect. Dis. 15: p. 77
19 Tuomanen, E. 1993. Breaching the blood-brain barrier. Sci. Ami. 268 (2): 80-84
20 Lock, RA, Hansman, D., and Paton, JC 1992 Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae. Microb. Pa thog. 12: 137-143
21 Lock, RA, Paton, JC, and Hansman, D., 1988 Compara tive efficacy of pneumococcal neuraminidase and pneumolysin as immunogens protective against Streptococcus pneumoniae. Microb. Pathog. 5: 461-467
22 Sampson, JS, O'Connor, SP, Stinson, AR, Tharpe, JA, and Russell, H. 1994. Cloning and nucleotide sequence analyze sis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologius to previously reported Streptococ cus sp. adhesins. Infect. Immune. 62: 319-324
23 Tart, RC, McDaniel, LS, Ralph, BA, and Briles, DE 1996. Truncated Streptococcus pneumoniae pspA molecules elicit cross-protective immunity against pneumococcal challenge in mice. J. Infect. Dis. 173: 380-386
24 Alexander, JE, bock, RA, Peeters, CCAM, Pool man, JT, Andrew, PW, Mitchell, TJ, Hansman, D., and Paton, JC 1994. Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae. Infect. Immune. 62: 5683-5688
25 Boulnois, GJ 1992. Pneumococcal proteins and the patho genesis of disease caused by Streptococcus pneumoniae. J. Gen. Microbiol. 138: 249-259
26 Houldsworth, S., Andrew, PW, and Mitchell, TJ 1994. Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1β by human mononuclear phagocytes. Infect. Immune. 62: 1501-1503
27 Feldman, C., Mitchell, TJ, Andrew, PW, Boulnois, GJ, Read, RC, Todd, HC, Cole, PJ, and Wilson, R. 1990. The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro. Micob. Pathog. 9: 275-284
28 Paton, JC, and Ferrante, A. 1983. Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin. Infect. Immune. 41: 1212-1216
29 Paon, JC, Andrew, PW, Bounois, GJ, and Mitchell, TJ 1993. Molecular analysis of the pathogenicity of Strepto coccus pneumoniae: the role of pneumococcal proteins. Annu. Rev. Microbiol. 47: 89-115
30 Crain, MJ, Waltman II, WD, Turner, JS, Yother, J. Talkington, DF, McDaniel, LS, Gray, BM, and Briles, DE 1990. Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae. Infect. Immune. 58: 3293-3299
31 Briles, DE, Yother, J., and McDaniel, LS 1988. Role of pneumococcal surface protein A in the virulence of Strepto coccus pneumoniae. Rev. Infect. Dis. 10 (Suppl. 4): 797-805
32 McDaniel, IJ. S, Yother, J., Vijayakumar, M., McGarry, L., Guild, WR, and Briles, D. E: 1987. Use of insertional activation to facilitate studies of biological properties of pneumo coccal surface protein A (PspA) . J. Exp. Med. 165: 381-394
33 times, CJ immunoglobulin A1 protease production by Haemo philus influenzae and Streptococcus pneumoniae. Infect. Immune. 1979; 26: 254-261
34 Wani, JH, Gilbert, JV, Plaut, AG, and Weiser, JN 1996. Identfication, cloning, and sequencing of the Immunoglobulin Al protease gene of Streptococcus pneumoniae. Infect. Immune. 64 (10): 3967-3974
35 Poulsen, K., Reinholdt, J., and Kilian, M. Characterization of the Streptococcus pneumoniae Immunoglobulin A1 protease gene (iga) and is translation product. Infect. Immune. 64 (10): 3957- 3966
36 Vered, M., Schutzbank, T., Janof f, A. Inhibitors of human neutrophil elastase in extracts of Streptococcus pneumoniae. At the. Rev. Respir. Dis 1984; 130: 1118-1124
37 Cundell, CR, Pearce, BJ, Young, A., Tuomanen, EI, Masure, HR Protein dependent peptide permeases from Strepto coccus pneumoniae medite cytoadherence to type II lung cells and to human endothelial cells [abstract no. B107). J. Cell Biochem. Suppl. 1994; 18A: 45
38 Lacks, SA Purification and properties of the complemen tary endonucleases DpnI and DpnII. Methods Enzymol. 1980; 65: 138-146
39 Pearce, BJ, Naughton, AM, Masure, HR Peptides per meases modulate transformation in Streptococcus pneumoniae. Mol. Microbiol. 1994; 12: 881-892
40 Masure, HR, Campbell, EA, Cundell, DR, Pearce, BJ, Sandros, J., Spellerberg, BA 1995. New genetic strategy for the analysis of virulence and transformation in Streptococ cus pneumoniae. Dev. Biol. Stand. 85: 251-260
41 Yother, J., Briles, DE 1992. Structural and evolutionary relationships of PspA, a surface protein of Streptococcus pneu moniae, as revealeds by sequence analysis. J. Bacteriol. 174: 601-609
42 Yother, J., and White, JM. 1994. Novel surface attach ment mechanism of the Streptococcus pneumoniae Protein PspA. J. Bacteriol. 176: 2976-2985
43 Yother, J., Handsome, GI, and Briles, DE 1992. Trun cated forms of PspA that are secreted from Streptococcus pneumo niae and their use in functional studies and cloning of the pspA gene. J. Bacteriol. 174: 610-618;
44 McDaniel, b. S., Sheffield, JS, Swiatlo, E., Yother, J., Crain, MC, and Briles, DE 1992. Molecular localization of variable and conserved region of pspA and identification of additional pspA homologous sequences in Streptococcus pneumo niae. Micro. Pathog. 13: 261-269
45 Jerlström, PG, Chhatwal, GS, Timmis, KN 1991. The IgA-binding B antigen of the c protein complex of group B strep tococci: sequence determination of its gene and detection of two binding regions. Mol. Microbiol. 5: 843-849
46 Heden, b., Frithz, E. bindhal, G. 1991. Molecular characte rization of an 1gA receptor from group B streptococci: Sequence of the gene, identification of a proline-rich region with unique structure and isolation of N-terminal fragments with IgA binding capacity. Eur. J. Immun. 21: 1481-1490
47 Solary, R., and Krähenbühl, JP 1985. Immunol. Today, 17-20
48 Brown, WR, Isobe, Y., Nakane, PK 1976. Studies on translocation of immunoglobulins across intestinal epithilium. II. Immunelectron-microscopic localization of immunoglobulins and secretory component in human intestinal mucosa. Gastro Enterology 71: 985-995
Claims (7)
Repeats 1 bis 6 (Positionen 325 bis 444) oder
Repeats 1 bis 7 (Positionen 325 bis 464) oder
Repeats 1 bis 8 (Positionen 325 bis 484 oder bis 485). 3. C-terminally deleted derivative of the surface protein according to claim 1 or 2, characterized by 324 amino acids according to FIG. 2 (positions 1 to 324) and
Repeats 1 to 6 (positions 325 to 444) or
Repeats 1 to 7 (positions 325 to 464) or
Repeats 1 to 8 (positions 325 to 484 or to 485).
- (i) im Bereich der Positionen 1 bis 159 gemäß Fig. 2 um 1 bis maximal 159 Aminosäuren deletiert ist,
- (ii) nicht jedoch im Bereich der Positionen 160 bis 523 dele tiert ist.
- (i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,
- (ii) is not, however, deleted in the range of items 160 to 523.
- (i) im Bereich der Positionen 1 bis 159 gemäß Fig. 2 um 1 bis maximal 159 Aminosäuren deletiert ist,
- (ii) nicht jedoch im Bereich der Positionen 160 bis 324 dele tiert ist und
- (iii) Repeats 1 bis 8 (Positionen 325 bis 484 oder bis 485) oder
Repeats 1 bis 7 (Positionen 325 bis 464) oder
Repeats 1 bis 6 (Positionen 325 bis 444) aufweist.
- (i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,
- (ii) is not, however, deleted in the range of items 160 to 324 and
- (iii) repeats 1 to 8 (positions 325 to 484 or to 485) or
Repeats 1 to 7 (positions 325 to 464) or
Repeats 1 to 6 (positions 325 to 444).
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19708537A DE19708537A1 (en) | 1997-03-03 | 1997-03-03 | New surface protein (SpsA protein) from Streptococcus pneumoniae etc. |
| JP53813798A JP2001524073A (en) | 1997-03-03 | 1998-03-02 | Novel S. pneumoniae surface protein (SpsA-protein), deleted derivative, expression system for the protein and vaccine comprising the protein |
| PCT/EP1998/001149 WO1998039450A2 (en) | 1997-03-03 | 1998-03-02 | SURFACE PROTEIN (SPsA PROTEIN) OF STREPTOCOCCUS PNEUMONIAE, DELETED DERIVATIVES, EXPRESSION SYSTEM FOR SAID PROTEINS AND VACCINE SYSTEM WITH SAID PROTEINS |
| EP98916880A EP0991762A2 (en) | 1997-03-03 | 1998-03-02 | SURFACE PROTEIN (SPsA PROTEIN) OF STREPTOCOCCUS PNEUMONIAE, DELETED DERIVATIVES, EXPRESSION SYSTEM FOR SAID PROTEINS AND VACCINE SYSTEM WITH SAID PROTEINS |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19708537A DE19708537A1 (en) | 1997-03-03 | 1997-03-03 | New surface protein (SpsA protein) from Streptococcus pneumoniae etc. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE19708537A1 true DE19708537A1 (en) | 1998-09-10 |
Family
ID=7822047
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE19708537A Withdrawn DE19708537A1 (en) | 1997-03-03 | 1997-03-03 | New surface protein (SpsA protein) from Streptococcus pneumoniae etc. |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0991762A2 (en) |
| JP (1) | JP2001524073A (en) |
| DE (1) | DE19708537A1 (en) |
| WO (1) | WO1998039450A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000029434A2 (en) * | 1998-11-19 | 2000-05-25 | St. Jude Children's Research Hospital | PNEUMOCOCCAL CHOLINE BINDING PROTEINS, CbpG AND CbpD, DIAGNOSTIC AND THERAPEUTIC USES THEREOF |
| US7790412B2 (en) | 1997-07-02 | 2010-09-07 | sanofi pasteur limited/sanofi pasteur limitée | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5928895A (en) * | 1996-09-24 | 1999-07-27 | Smithkline Beecham Corporation | IgA Fc binding protein |
| KR20060126844A (en) | 1998-04-07 | 2006-12-08 | 메디뮨 인코포레이티드 | Derivatives of pneumococcal choline binding proteins for vaccines |
| WO2002008426A2 (en) * | 2000-07-20 | 2002-01-31 | Hansa Medical Ab | Fh-binding protein of streptococcus pneumoniae |
| GB0022742D0 (en) | 2000-09-15 | 2000-11-01 | Smithkline Beecham Biolog | Vaccine |
| TWI457133B (en) | 2005-12-13 | 2014-10-21 | Glaxosmithkline Biolog Sa | Novel composition |
| GB0607088D0 (en) | 2006-04-07 | 2006-05-17 | Glaxosmithkline Biolog Sa | Vaccine |
| AR058707A1 (en) | 2005-12-22 | 2008-02-20 | Glaxosmithkline Biolog Sa | VACCINE, PROCEDURE TO MANUFACTURE AND USE |
| EA200901578A1 (en) | 2007-06-26 | 2010-08-30 | Глаксосмитклайн Байолоджикалс С.А. | VACCINE CONTAINING CONJUGATES OF CAPSULAR POLYSACCHARIDES STREPTOCOCCUS PNEUMONIAE |
| WO2009127676A1 (en) | 2008-04-16 | 2009-10-22 | Glaxosmithkline Biologicals S.A. | Vaccine |
| GB201003924D0 (en) | 2010-03-09 | 2010-04-21 | Glaxosmithkline Biolog Sa | Immunogenic composition |
| AR086405A1 (en) | 2011-05-17 | 2013-12-11 | Glaxosmithkline Biolog Sa | STREPTOCOCCUS PNEUMONIAE VACCINE |
| US9815886B2 (en) | 2014-10-28 | 2017-11-14 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
| GB201518684D0 (en) | 2015-10-21 | 2015-12-02 | Glaxosmithkline Biolog Sa | Vaccine |
| US10259865B2 (en) | 2017-03-15 | 2019-04-16 | Adma Biologics, Inc. | Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996039113A2 (en) * | 1995-06-02 | 1996-12-12 | Uab Research Foundation | Oral administration of pneumococcal antigens |
| CA2253252A1 (en) * | 1996-05-01 | 1997-11-06 | The Rockefeller University | Choline binding proteins for anti-pneumococcal vaccines |
| DE69737125T3 (en) * | 1996-10-31 | 2015-02-26 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
| AU5355398A (en) * | 1996-11-12 | 1998-06-03 | Regents Of The University Of Minnesota | C3 binding protein of (streptococcus pneumoniae) |
-
1997
- 1997-03-03 DE DE19708537A patent/DE19708537A1/en not_active Withdrawn
-
1998
- 1998-03-02 JP JP53813798A patent/JP2001524073A/en active Pending
- 1998-03-02 EP EP98916880A patent/EP0991762A2/en not_active Withdrawn
- 1998-03-02 WO PCT/EP1998/001149 patent/WO1998039450A2/en not_active Application Discontinuation
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7875437B2 (en) | 1997-07-02 | 2011-01-25 | Sanofi Pasteur Limited/Sanofi Pasteur Limitee | Nucleic acid and amino acid sequences relating to streptococcus pneumoniae for diagnostics and therapeutics |
| US7893230B2 (en) | 1997-07-02 | 2011-02-22 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US7790412B2 (en) | 1997-07-02 | 2010-09-07 | sanofi pasteur limited/sanofi pasteur limitée | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US7834166B2 (en) | 1997-07-02 | 2010-11-16 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US7867501B2 (en) | 1997-07-02 | 2011-01-11 | Sanofi Pasteur Limited/Sanofi Pasteur Limitee | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US7875438B2 (en) | 1997-07-02 | 2011-01-25 | Sanofi Pasteur Limited/Sanofi Pasteur Limitee | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8609107B2 (en) | 1997-07-02 | 2013-12-17 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US7875439B2 (en) | 1997-07-02 | 2011-01-25 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8298788B2 (en) | 1997-07-02 | 2012-10-30 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8003775B2 (en) | 1997-07-02 | 2011-08-23 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8288519B2 (en) | 1997-07-02 | 2012-10-16 | Sanofi Pasteur Limited | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8293884B2 (en) | 1997-07-02 | 2012-10-23 | sanofi pasteur limited/sanofi pasteur limitée | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US8293249B2 (en) | 1997-07-02 | 2012-10-23 | Sanofi Pasteur Limited/Sanofi Pasteur Limitee | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| WO2000029434A2 (en) * | 1998-11-19 | 2000-05-25 | St. Jude Children's Research Hospital | PNEUMOCOCCAL CHOLINE BINDING PROTEINS, CbpG AND CbpD, DIAGNOSTIC AND THERAPEUTIC USES THEREOF |
| WO2000029434A3 (en) * | 1998-11-19 | 2001-03-01 | St Jude Childrens Res Hospital | PNEUMOCOCCAL CHOLINE BINDING PROTEINS, CbpG AND CbpD, DIAGNOSTIC AND THERAPEUTIC USES THEREOF |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2001524073A (en) | 2001-11-27 |
| WO1998039450A3 (en) | 1998-11-05 |
| WO1998039450A2 (en) | 1998-09-11 |
| EP0991762A2 (en) | 2000-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| BRILES et al. | PspA and PspC: their potential for use as pneumococcal vaccines | |
| DE19708537A1 (en) | New surface protein (SpsA protein) from Streptococcus pneumoniae etc. | |
| Hammerschmidt et al. | SpsA, a novel pneumococcal surface protein with specific binding to secretory immunoglobulin A and secretory component | |
| US7820789B2 (en) | Mutant pneumolysin proteins | |
| Briles et al. | PspA, a protection-eliciting pneumococcal protein: immunogenicity of isolated native PspA in mice | |
| Tai | Streptococcus pneumoniae protein vaccine candidates: properties, activities and animal studies | |
| DE69735715T2 (en) | Choline-binding protein used as a pneumococcal vaccine | |
| DE69934299T2 (en) | GROUP B-STREPTOCOCCUS ANTIGENE | |
| PATON et al. | Molecular analysis of putative pneumococcal virulence proteins | |
| DE60027890T2 (en) | STREPTOCOCCUS PNEUMONIAE PROTEINS AND VACCINES | |
| Mitchell et al. | Molecular analysis of virulence factors of Streptococcus pneumoniae | |
| EP0866133A2 (en) | A group b streptococcus vaccine | |
| JPH0710774A (en) | Vaccine for sprepto coccus switzerland infection | |
| US20040265933A1 (en) | Proteins | |
| CH672072A5 (en) | ||
| AU776735B2 (en) | Genes and proteins, and their use | |
| Fontana et al. | Characterization of preparations enriched for Streptococcus mutans fimbriae: salivary immunoglobulin A antibodies in caries-free and caries-active subjects | |
| DE69928925T2 (en) | ANTIGENIC POLYPEPTIDES FROM NEISSERIA MENINGITIDIS, POLYNUCLEOTIDES CODED THEREFOR AND CORRESPONDING PROTECTIVE ANTIBODIES | |
| DE69733067T2 (en) | TREATMENT AND DETERMINATION OF GRAM-POSITIVE COCKIN INFECTIONS | |
| DE69918446T2 (en) | POLYNUCLEOTIDE AND POLYPEPTIDE BASB033 FROM NEISSERIA MENINGITIDIS AND ITS USES | |
| Davies et al. | Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA | |
| DE69935213T2 (en) | BASB019 PROTEINS AND GENES FROM MORAXELLA CATARRHALIS, ANTIGENE, ANTIBODIES, AND USES | |
| DE69925866T2 (en) | AUSSEMBRBRANPROTEINE, HER GENE, AND ITS USE | |
| US20030104000A1 (en) | Genes and proteins, and their use | |
| JP2001504335A (en) | Lactoferrin-binding protein of Streptococcus uberis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 8130 | Withdrawal |