CA2288211A1 - Antigenic composition and method of detection for helicobacter pylori - Google Patents
Antigenic composition and method of detection for helicobacter pylori Download PDFInfo
- Publication number
- CA2288211A1 CA2288211A1 CA002288211A CA2288211A CA2288211A1 CA 2288211 A1 CA2288211 A1 CA 2288211A1 CA 002288211 A CA002288211 A CA 002288211A CA 2288211 A CA2288211 A CA 2288211A CA 2288211 A1 CA2288211 A1 CA 2288211A1
- Authority
- CA
- Canada
- Prior art keywords
- seq
- cluster
- pylori
- antigen
- nos
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 64
- 230000000890 antigenic effect Effects 0.000 title claims abstract description 51
- 239000000203 mixture Substances 0.000 title claims description 21
- 238000001514 detection method Methods 0.000 title description 14
- 241000590002 Helicobacter pylori Species 0.000 title description 2
- 229940037467 helicobacter pylori Drugs 0.000 title description 2
- 108091007433 antigens Proteins 0.000 claims abstract description 359
- 102000036639 antigens Human genes 0.000 claims abstract description 359
- 239000000427 antigen Substances 0.000 claims abstract description 356
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 129
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 102
- 229920001184 polypeptide Polymers 0.000 claims abstract description 77
- 239000002773 nucleotide Substances 0.000 claims description 69
- 125000003729 nucleotide group Chemical group 0.000 claims description 69
- 108091033319 polynucleotide Proteins 0.000 claims description 66
- 102000040430 polynucleotide Human genes 0.000 claims description 66
- 239000002157 polynucleotide Substances 0.000 claims description 66
- 150000001413 amino acids Chemical class 0.000 claims description 58
- 208000015181 infectious disease Diseases 0.000 claims description 43
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 35
- 238000009396 hybridization Methods 0.000 claims description 28
- 229960005486 vaccine Drugs 0.000 claims description 24
- 238000012216 screening Methods 0.000 claims description 18
- 239000013060 biological fluid Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 230000000845 anti-microbial effect Effects 0.000 claims description 7
- 230000004044 response Effects 0.000 claims description 6
- 238000009007 Diagnostic Kit Methods 0.000 claims description 5
- 241000282560 Macaca mulatta Species 0.000 claims description 4
- 230000005923 long-lasting effect Effects 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 248
- 102000004169 proteins and genes Human genes 0.000 abstract description 232
- 230000002163 immunogen Effects 0.000 abstract description 27
- 238000002955 isolation Methods 0.000 abstract description 10
- 238000012512 characterization method Methods 0.000 abstract description 8
- 235000018102 proteins Nutrition 0.000 description 216
- 108020004414 DNA Proteins 0.000 description 168
- 239000012634 fragment Substances 0.000 description 123
- 239000000499 gel Substances 0.000 description 57
- 108700026244 Open Reading Frames Proteins 0.000 description 49
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 44
- 235000001014 amino acid Nutrition 0.000 description 44
- 229940024606 amino acid Drugs 0.000 description 41
- 230000035945 sensitivity Effects 0.000 description 34
- 239000013598 vector Substances 0.000 description 34
- 210000002966 serum Anatomy 0.000 description 30
- 208000007882 Gastritis Diseases 0.000 description 27
- 239000000047 product Substances 0.000 description 26
- 238000012360 testing method Methods 0.000 description 25
- 239000000523 sample Substances 0.000 description 24
- 239000004202 carbamide Substances 0.000 description 23
- 238000001262 western blot Methods 0.000 description 23
- 238000003556 assay Methods 0.000 description 22
- 102000037865 fusion proteins Human genes 0.000 description 22
- 108020001507 fusion proteins Proteins 0.000 description 22
- 230000001684 chronic effect Effects 0.000 description 21
- 239000013604 expression vector Substances 0.000 description 21
- 238000012163 sequencing technique Methods 0.000 description 20
- 241001465754 Metazoa Species 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 18
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 17
- 239000000872 buffer Substances 0.000 description 17
- 125000005647 linker group Chemical group 0.000 description 17
- 230000014616 translation Effects 0.000 description 17
- 108010046334 Urease Proteins 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 16
- 108091026890 Coding region Proteins 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000000746 purification Methods 0.000 description 15
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 14
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 13
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 13
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 13
- 125000003275 alpha amino acid group Chemical group 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 12
- 230000004927 fusion Effects 0.000 description 12
- 230000007017 scission Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 238000013519 translation Methods 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 11
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 description 11
- 239000007983 Tris buffer Substances 0.000 description 11
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 150000007523 nucleic acids Chemical group 0.000 description 11
- 239000008188 pellet Substances 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000001154 acute effect Effects 0.000 description 9
- 230000003321 amplification Effects 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 8
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 8
- 102100024407 Jouberin Human genes 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 230000036961 partial effect Effects 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000003656 tris buffered saline Substances 0.000 description 8
- 101000768957 Acholeplasma phage L2 Uncharacterized 37.2 kDa protein Proteins 0.000 description 7
- 101000823746 Acidianus ambivalens Uncharacterized 17.7 kDa protein in bps2 3'region Proteins 0.000 description 7
- 101000916369 Acidianus ambivalens Uncharacterized protein in sor 5'region Proteins 0.000 description 7
- 101000769342 Acinetobacter guillouiae Uncharacterized protein in rpoN-murA intergenic region Proteins 0.000 description 7
- 101000823696 Actinobacillus pleuropneumoniae Uncharacterized glycosyltransferase in aroQ 3'region Proteins 0.000 description 7
- 101000786513 Agrobacterium tumefaciens (strain 15955) Uncharacterized protein outside the virF region Proteins 0.000 description 7
- 101000618005 Alkalihalobacillus pseudofirmus (strain ATCC BAA-2126 / JCM 17055 / OF4) Uncharacterized protein BpOF4_00885 Proteins 0.000 description 7
- 102100020724 Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Human genes 0.000 description 7
- 101000967489 Azorhizobium caulinodans (strain ATCC 43989 / DSM 5975 / JCM 20966 / LMG 6465 / NBRC 14845 / NCIMB 13405 / ORS 571) Uncharacterized protein AZC_3924 Proteins 0.000 description 7
- 101000823761 Bacillus licheniformis Uncharacterized 9.4 kDa protein in flaL 3'region Proteins 0.000 description 7
- 101000819719 Bacillus methanolicus Uncharacterized N-acetyltransferase in lysA 3'region Proteins 0.000 description 7
- 101000789586 Bacillus subtilis (strain 168) UPF0702 transmembrane protein YkjA Proteins 0.000 description 7
- 101000792624 Bacillus subtilis (strain 168) Uncharacterized protein YbxH Proteins 0.000 description 7
- 101000790792 Bacillus subtilis (strain 168) Uncharacterized protein YckC Proteins 0.000 description 7
- 101000819705 Bacillus subtilis (strain 168) Uncharacterized protein YlxR Proteins 0.000 description 7
- 101000948218 Bacillus subtilis (strain 168) Uncharacterized protein YtxJ Proteins 0.000 description 7
- 101000718627 Bacillus thuringiensis subsp. kurstaki Putative RNA polymerase sigma-G factor Proteins 0.000 description 7
- 101000641200 Bombyx mori densovirus Putative non-structural protein Proteins 0.000 description 7
- 102000016938 Catalase Human genes 0.000 description 7
- 108010053835 Catalase Proteins 0.000 description 7
- 101000947633 Claviceps purpurea Uncharacterized 13.8 kDa protein Proteins 0.000 description 7
- 102100031725 Cortactin-binding protein 2 Human genes 0.000 description 7
- 101000948901 Enterobacteria phage T4 Uncharacterized 16.0 kDa protein in segB-ipI intergenic region Proteins 0.000 description 7
- 101000805958 Equine herpesvirus 4 (strain 1942) Virion protein US10 homolog Proteins 0.000 description 7
- 101000790442 Escherichia coli Insertion element IS2 uncharacterized 11.1 kDa protein Proteins 0.000 description 7
- 101000788354 Escherichia phage P2 Uncharacterized 8.2 kDa protein in gpA 5'region Proteins 0.000 description 7
- 108010040721 Flagellin Proteins 0.000 description 7
- 101000770304 Frankia alni UPF0460 protein in nifX-nifW intergenic region Proteins 0.000 description 7
- 101000797344 Geobacillus stearothermophilus Putative tRNA (cytidine(34)-2'-O)-methyltransferase Proteins 0.000 description 7
- 101000748410 Geobacillus stearothermophilus Uncharacterized protein in fumA 3'region Proteins 0.000 description 7
- 101000772675 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) UPF0438 protein HI_0847 Proteins 0.000 description 7
- 101000631019 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Uncharacterized protein HI_0350 Proteins 0.000 description 7
- 101000768938 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.9 kDa protein in int-C1 intergenic region Proteins 0.000 description 7
- 241000589989 Helicobacter Species 0.000 description 7
- 101000785414 Homo sapiens Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Proteins 0.000 description 7
- 101000782488 Junonia coenia densovirus (isolate pBRJ/1990) Putative non-structural protein NS2 Proteins 0.000 description 7
- 101000811523 Klebsiella pneumoniae Uncharacterized 55.8 kDa protein in cps region Proteins 0.000 description 7
- 101000818409 Lactococcus lactis subsp. lactis Uncharacterized HTH-type transcriptional regulator in lacX 3'region Proteins 0.000 description 7
- 101000878851 Leptolyngbya boryana Putative Fe(2+) transport protein A Proteins 0.000 description 7
- 101000758828 Methanosarcina barkeri (strain Fusaro / DSM 804) Uncharacterized protein Mbar_A1602 Proteins 0.000 description 7
- 101001122401 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF3 Proteins 0.000 description 7
- 101001055788 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) Pentapeptide repeat protein MfpA Proteins 0.000 description 7
- 101000740670 Orgyia pseudotsugata multicapsid polyhedrosis virus Protein C42 Proteins 0.000 description 7
- 239000002033 PVDF binder Substances 0.000 description 7
- 101000769182 Photorhabdus luminescens Uncharacterized protein in pnp 3'region Proteins 0.000 description 7
- 101710197985 Probable protein Rev Proteins 0.000 description 7
- 101000961392 Pseudescherichia vulneris Uncharacterized 29.9 kDa protein in crtE 3'region Proteins 0.000 description 7
- 101000731030 Pseudomonas oleovorans Poly(3-hydroxyalkanoate) polymerase 2 Proteins 0.000 description 7
- 101001065485 Pseudomonas putida Probable fatty acid methyltransferase Proteins 0.000 description 7
- 101000711023 Rhizobium leguminosarum bv. trifolii Uncharacterized protein in tfuA 3'region Proteins 0.000 description 7
- 101000948156 Rhodococcus erythropolis Uncharacterized 47.3 kDa protein in thcA 5'region Proteins 0.000 description 7
- 101000917565 Rhodococcus fascians Uncharacterized 33.6 kDa protein in fasciation locus Proteins 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- 101000790284 Saimiriine herpesvirus 2 (strain 488) Uncharacterized 9.5 kDa protein in DHFR 3'region Proteins 0.000 description 7
- 101000936719 Streptococcus gordonii Accessory Sec system protein Asp3 Proteins 0.000 description 7
- 101000788499 Streptomyces coelicolor Uncharacterized oxidoreductase in mprA 5'region Proteins 0.000 description 7
- 101001102841 Streptomyces griseus Purine nucleoside phosphorylase ORF3 Proteins 0.000 description 7
- 101000708557 Streptomyces lincolnensis Uncharacterized 17.2 kDa protein in melC2-rnhH intergenic region Proteins 0.000 description 7
- 101000649826 Thermotoga neapolitana Putative anti-sigma factor antagonist TM1081 homolog Proteins 0.000 description 7
- 101000827562 Vibrio alginolyticus Uncharacterized protein in proC 3'region Proteins 0.000 description 7
- 101000778915 Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) Uncharacterized membrane protein VP2115 Proteins 0.000 description 7
- 238000001042 affinity chromatography Methods 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 230000002496 gastric effect Effects 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 7
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 101000977023 Azospirillum brasilense Uncharacterized 17.8 kDa protein in nodG 5'region Proteins 0.000 description 6
- 101000961984 Bacillus thuringiensis Uncharacterized 30.3 kDa protein Proteins 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 101000644901 Drosophila melanogaster Putative 115 kDa protein in type-1 retrotransposable element R1DM Proteins 0.000 description 6
- 101000747702 Enterobacteria phage N4 Uncharacterized protein Gp2 Proteins 0.000 description 6
- 101000758599 Escherichia coli Uncharacterized 14.7 kDa protein Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101000768930 Lactococcus lactis subsp. cremoris Uncharacterized protein in pepC 5'region Proteins 0.000 description 6
- 101000976302 Leptospira interrogans Uncharacterized protein in sph 3'region Proteins 0.000 description 6
- 101000778886 Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain 56601) Uncharacterized protein LA_2151 Proteins 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 101001121571 Rice tungro bacilliform virus (isolate Philippines) Protein P2 Proteins 0.000 description 6
- 101000818098 Spirochaeta aurantia Uncharacterized protein in trpE 3'region Proteins 0.000 description 6
- 101001026590 Streptomyces cinnamonensis Putative polyketide beta-ketoacyl synthase 2 Proteins 0.000 description 6
- 101000750896 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized protein Synpcc7942_2318 Proteins 0.000 description 6
- 101000916321 Xenopus laevis Transposon TX1 uncharacterized 149 kDa protein Proteins 0.000 description 6
- 101000760088 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 20.9 kDa protein Proteins 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000002405 diagnostic procedure Methods 0.000 description 6
- 208000000718 duodenal ulcer Diseases 0.000 description 6
- 238000005194 fractionation Methods 0.000 description 6
- 238000001502 gel electrophoresis Methods 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- BPHPUYQFMNQIOC-MBOVONDJSA-N (2r,3r,4s,5r)-2-(hydroxymethyl)-6-propan-2-ylsulfanyloxane-3,4,5-triol Chemical compound CC(C)SC1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-MBOVONDJSA-N 0.000 description 5
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108050001186 Chaperonin Cpn60 Proteins 0.000 description 5
- 102000052603 Chaperonins Human genes 0.000 description 5
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 239000001569 carbon dioxide Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 206010013864 duodenitis Diseases 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000001839 endoscopy Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- IBIKHMZPHNKTHM-RDTXWAMCSA-N merck compound 25 Chemical compound C1C[C@@H](C(O)=O)[C@H](O)CN1C(C1=C(F)C=CC=C11)=NN1C(=O)C1=C(Cl)C=CC=C1C1CC1 IBIKHMZPHNKTHM-RDTXWAMCSA-N 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 210000002381 plasma Anatomy 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 238000004007 reversed phase HPLC Methods 0.000 description 5
- 229910052709 silver Inorganic materials 0.000 description 5
- 239000004332 silver Substances 0.000 description 5
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108010052418 (N-(2-((4-((2-((4-(9-acridinylamino)phenyl)amino)-2-oxoethyl)amino)-4-oxobutyl)amino)-1-(1H-imidazol-4-ylmethyl)-1-oxoethyl)-6-(((-2-aminoethyl)amino)methyl)-2-pyridinecarboxamidato) iron(1+) Proteins 0.000 description 4
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 4
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 4
- 101000666833 Autographa californica nuclear polyhedrosis virus Uncharacterized 20.8 kDa protein in FGF-VUBI intergenic region Proteins 0.000 description 4
- 101000977027 Azospirillum brasilense Uncharacterized protein in nodG 5'region Proteins 0.000 description 4
- 101000962005 Bacillus thuringiensis Uncharacterized 23.6 kDa protein Proteins 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 101000785191 Drosophila melanogaster Uncharacterized 50 kDa protein in type I retrotransposable element R1DM Proteins 0.000 description 4
- 101000747704 Enterobacteria phage N4 Uncharacterized protein Gp1 Proteins 0.000 description 4
- 101000861206 Enterococcus faecalis (strain ATCC 700802 / V583) Uncharacterized protein EF_A0048 Proteins 0.000 description 4
- 101000769180 Escherichia coli Uncharacterized 11.1 kDa protein Proteins 0.000 description 4
- 241000701959 Escherichia virus Lambda Species 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 101000976301 Leptospira interrogans Uncharacterized 35 kDa protein in sph 3'region Proteins 0.000 description 4
- 101000658690 Neisseria meningitidis serogroup B Transposase for insertion sequence element IS1106 Proteins 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- 208000008469 Peptic Ulcer Diseases 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 101000748660 Pseudomonas savastanoi Uncharacterized 21 kDa protein in iaaL 5'region Proteins 0.000 description 4
- 101000584469 Rice tungro bacilliform virus (isolate Philippines) Protein P1 Proteins 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 101000818096 Spirochaeta aurantia Uncharacterized 15.5 kDa protein in trpE 3'region Proteins 0.000 description 4
- 101000766081 Streptomyces ambofaciens Uncharacterized HTH-type transcriptional regulator in unstable DNA locus Proteins 0.000 description 4
- 101000804403 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HIT-like protein Synpcc7942_1390 Proteins 0.000 description 4
- 101000750910 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HTH-type transcriptional regulator Synpcc7942_2319 Proteins 0.000 description 4
- 101000644897 Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) Uncharacterized protein SYNPCC7002_B0001 Proteins 0.000 description 4
- 101000916336 Xenopus laevis Transposon TX1 uncharacterized 82 kDa protein Proteins 0.000 description 4
- 101001000760 Zea mays Putative Pol polyprotein from transposon element Bs1 Proteins 0.000 description 4
- 101000678262 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 65 kDa protein Proteins 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000001574 biopsy Methods 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000009260 cross reactivity Effects 0.000 description 4
- 238000000265 homogenisation Methods 0.000 description 4
- 238000001155 isoelectric focusing Methods 0.000 description 4
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 4
- 230000007170 pathology Effects 0.000 description 4
- 208000011906 peptic ulcer disease Diseases 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 238000005063 solubilization Methods 0.000 description 4
- 230000007928 solubilization Effects 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229940125575 vaccine candidate Drugs 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 208000002109 Argyria Diseases 0.000 description 3
- 102100026189 Beta-galactosidase Human genes 0.000 description 3
- 108091035707 Consensus sequence Proteins 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102000005720 Glutathione transferase Human genes 0.000 description 3
- 108010070675 Glutathione transferase Proteins 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 208000007107 Stomach Ulcer Diseases 0.000 description 3
- 108020005038 Terminator Codon Proteins 0.000 description 3
- 208000025865 Ulcer Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 201000005917 gastric ulcer Diseases 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000734 protein sequencing Methods 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 230000000405 serological effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 231100000397 ulcer Toxicity 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 240000005020 Acaciella glauca Species 0.000 description 2
- 101000787132 Acidithiobacillus ferridurans Uncharacterized 8.2 kDa protein in mobL 3'region Proteins 0.000 description 2
- 101000827262 Acidithiobacillus ferrooxidans Uncharacterized 18.9 kDa protein in mobE 3'region Proteins 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 101000811747 Antithamnion sp. UPF0051 protein in atpA 3'region Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BXTVQNYQYUTQAZ-UHFFFAOYSA-N BNPS-skatole Chemical compound N=1C2=CC=CC=C2C(C)(Br)C=1SC1=CC=CC=C1[N+]([O-])=O BXTVQNYQYUTQAZ-UHFFFAOYSA-N 0.000 description 2
- 101000827607 Bacillus phage SPP1 Uncharacterized 8.5 kDa protein in GP2-GP6 intergenic region Proteins 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 101000961975 Bacillus thuringiensis Uncharacterized 13.4 kDa protein Proteins 0.000 description 2
- 101000964407 Caldicellulosiruptor saccharolyticus Uncharacterized 10.7 kDa protein in xynB 3'region Proteins 0.000 description 2
- 102000003846 Carbonic anhydrases Human genes 0.000 description 2
- 108090000209 Carbonic anhydrases Proteins 0.000 description 2
- 229920000298 Cellophane Polymers 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 208000002064 Dental Plaque Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010051815 Glutamyl endopeptidase Proteins 0.000 description 2
- 241000606768 Haemophilus influenzae Species 0.000 description 2
- 101000768777 Haloferax lucentense (strain DSM 14919 / JCM 9276 / NCIMB 13854 / Aa 2.2) Uncharacterized 50.6 kDa protein in the 5'region of gyrA and gyrB Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- 101000607404 Infectious laryngotracheitis virus (strain Thorne V882) Protein UL24 homolog Proteins 0.000 description 2
- 101000735632 Klebsiella pneumoniae Uncharacterized 8.8 kDa protein in aacA4 3'region Proteins 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000282339 Mustela Species 0.000 description 2
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108700020474 Penicillin-Binding Proteins Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 108010065084 Phosphorylase a Proteins 0.000 description 2
- 101710118538 Protease Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 101000818100 Spirochaeta aurantia Uncharacterized 12.7 kDa protein in trpE 5'region Proteins 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 101001037658 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) Glucokinase Proteins 0.000 description 2
- 101000623262 Trypanosoma brucei brucei Uncharacterized 22 kDa protein in aldolase locus Proteins 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000012411 cloning technique Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000007621 cluster analysis Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 2
- 230000006240 deamidation Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- -1 e.g. Proteins 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 108010003914 endoproteinase Asp-N Proteins 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 210000001156 gastric mucosa Anatomy 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 230000000155 isotopic effect Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 230000007505 plaque formation Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 235000003499 redwood Nutrition 0.000 description 2
- 150000003335 secondary amines Chemical class 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- CXLUIRPQSBYREP-WDSKDSINSA-N (3s)-3-[[(2s)-2-amino-4,4-dicarboxybutanoyl]amino]propane-1,1,3-tricarboxylic acid Chemical compound OC(=O)C(C(O)=O)C[C@H](N)C(=O)N[C@@H](CC(C(O)=O)C(O)=O)C(O)=O CXLUIRPQSBYREP-WDSKDSINSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101000787133 Acidithiobacillus ferridurans Uncharacterized 12.3 kDa protein in mobL 3'region Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 208000036170 B-Cell Marginal Zone Lymphoma Diseases 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 101000827603 Bacillus phage SPP1 Uncharacterized 10.2 kDa protein in GP2-GP6 intergenic region Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 101100224931 Caenorhabditis elegans dhc-1 gene Proteins 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 1
- 102000004654 Cyclic GMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010003591 Cyclic GMP-Dependent Protein Kinases Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 230000003682 DNA packaging effect Effects 0.000 description 1
- 230000007023 DNA restriction-modification system Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 101000947616 Enterobacteria phage PRD1 Putative uncharacterized 4.5 kDa protein in genes IX-XX intergenic region Proteins 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 101000599573 Homo sapiens InaD-like protein Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 101710126256 Hydrolase in agr operon Proteins 0.000 description 1
- 102100037978 InaD-like protein Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 101710172804 K protein Proteins 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 101000977786 Lymantria dispar multicapsid nuclear polyhedrosis virus Uncharacterized 9.7 kDa protein in PE 3'region Proteins 0.000 description 1
- 201000003791 MALT lymphoma Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000036675 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 206010030201 Oesophageal ulcer Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102100032709 Potassium-transporting ATPase alpha chain 2 Human genes 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 101001113905 Rice tungro bacilliform virus (isolate Philippines) Protein P4 Proteins 0.000 description 1
- 101100175496 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) gdh1 gene Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 238000010266 Sephadex chromatography Methods 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 108010029287 Threonine-tRNA ligase Proteins 0.000 description 1
- 102100034997 Threonine-tRNA ligase, mitochondrial Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 101100357018 Trypanosoma brucei brucei RNR2 gene Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009833 antibody interaction Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- VTTONGPRPXSUTJ-UHFFFAOYSA-N bufotenin Chemical compound C1=C(O)C=C2C(CCN(C)C)=CNC2=C1 VTTONGPRPXSUTJ-UHFFFAOYSA-N 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004850 capillary HPLC Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 208000016644 chronic atrophic gastritis Diseases 0.000 description 1
- 238000001360 collision-induced dissociation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010205 computational analysis Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000003241 endoproteolytic effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000010265 fast atom bombardment Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 108010004609 gamma-carboxyglutamyl-gamma-carboxyglutamic acid Proteins 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 244000000075 gastric pathogen Species 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 125000000487 histidyl group Chemical class [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 101150103775 hofC gene Proteins 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000010365 information processing Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 210000003125 jurkat cell Anatomy 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000028744 lysogeny Effects 0.000 description 1
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000012134 rapid urease test Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000009589 serological test Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- MUUHXGOJWVMBDY-UHFFFAOYSA-L tetrazolium blue Chemical class [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 MUUHXGOJWVMBDY-UHFFFAOYSA-L 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 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
- C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention pertains to the characterization and isolation of newly discovered polypeptide antigens of H. pylori. Disclosed herein are cluster families of DNA replicas of portions of the genome of H. pylori encoding antigens which are highly immunogenic. Also disclosed are new antigenic proteins recovered from H. pylori. The invention also provides methods employing the above-described antigens.
Description
f DEMANDES OU BREVETS VOLUMlNEUX
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE~
NOTE: Pour les tomes additionels, veuillez contacter to Bureau canadien des brevets JUMBO APPLICA'fIONSIPATEtIITS .
THiS SECTION OF THE APPl..lCATIONIPATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ,~ OF
' NOTE: For additional volumes-pl~ase~contact. the Canadian Patent Office .
WO 98/49314 ' PCT/US98/0848'7 ANTIGENIC COMPOSITION AND METHOD OF DETECTION
FOR HELICOBACTER PYLORI
s Field of the Invention The present invention relates to one or more antigens of H. pylori, to polynucleotide sequences coding for the antigens, and to diagnostic and therapeutic methods employing such antigens and polynucleotides.
References Allen, G., LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY' SEQUENCING of PROTEINS AND PElrrmES, Elsevier Science/North-Holland Biomedical Press, New York (1981).
Altschul, S., et al., J. Mol. Biol. 21s:403-410 (1990).
Atherton, J.C.; Spiller, R.A., Gut. 35:723-725 (1994).
Ausubel, F.M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media PA (1988).
Bauer, A. W . , et al. , Anal. Biochem. 137:134-142 ( 1984).
Beames, et al. , Biotechniques 11:378 ( 1991 ).
Blaser, M., Scientific American, p. 104 (1996).
Cover, T., et al., Adv. Int. Med. 41:8s-117 (1996).
2s Cover, T . , ASM News 61:21 ( 199s).
i Crea, R., U.S. Patent No. 4,888,286, issued December 19, 1989.
Crimmins, D.L. and Mische, S.M., in CURRENT PROTOCOLS IN PROTEIN SCIENCE, Units 11.5, John Wiley and Sons, lnc., New York (1996).
Dubois, A., et al., Infection and Immunity 64(8):2885-2891 {1996).
Earl, P.L., et al., "Expression of proteins in mammalian cells using vaccinia"
In CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al., Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991).
Eaton, M.A.W., et al., U.S. Patent No. 4,719,180, issued Jan. 12, 1988.
Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93 (1992).
Goeddel, D.V., Methods in Enzymology 185 (1990).
Guthrie, C., and Fink, G.R., Methods in Enrymology 194 (1991).
Harlow, E., et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).
Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983).
Kaufman, R.J., "Selection and coamplification of heterologous genes in mammalian cells,"
in METHODS IN ENZYMOLOGY, vol. 185, pp 537-566. Academic Press, Inc., San Diego CA (1991).
Lau, Y.F., et al., Mol. Cell. Biol. 4:1469-1475 (1984).
Maniatis, T., et al., MOLECULAR CLOMNG: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982).
McAtee, C.P., et al., J. Chromatog. B. 685:91-104 (1996).
Miller, C.G., Methods: A Companion to Methods in Enzymology 6:315-333 (1994).
Moss, B., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Section IV, Unit 16) (1991).
Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987.
Mullis, K.B., et al., U.S. Patent No. 4,683,195, issued 28 July 1987.
Nakayama, H., et al. , J. Chromatog. A 730:279-287 ( 1996).
Oakley, B.R., et al., Anal. Biochem. 105:361-363 (1980).
O'Farrell, P.H., J. Biol. Chem. 250:4007-4021 (1975).
O'Farrell, P.H., et al., Cell 12:1133-1142 (1977).
Parsonnet, J., et al., N. Engl. J. Med. 325:1127-1131 (1991).
Porath, J., Protein Exp. and Purif. 3:263 (1992).
Pearson, W.R. and Lipman, D.J., PNAS 85:2444-2448 (1988).
Pearson, W.R., Methods in Enrymology 183:63-98 (1990).
Reilly, P.R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL
(1992).
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE~
NOTE: Pour les tomes additionels, veuillez contacter to Bureau canadien des brevets JUMBO APPLICA'fIONSIPATEtIITS .
THiS SECTION OF THE APPl..lCATIONIPATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ,~ OF
' NOTE: For additional volumes-pl~ase~contact. the Canadian Patent Office .
WO 98/49314 ' PCT/US98/0848'7 ANTIGENIC COMPOSITION AND METHOD OF DETECTION
FOR HELICOBACTER PYLORI
s Field of the Invention The present invention relates to one or more antigens of H. pylori, to polynucleotide sequences coding for the antigens, and to diagnostic and therapeutic methods employing such antigens and polynucleotides.
References Allen, G., LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY' SEQUENCING of PROTEINS AND PElrrmES, Elsevier Science/North-Holland Biomedical Press, New York (1981).
Altschul, S., et al., J. Mol. Biol. 21s:403-410 (1990).
Atherton, J.C.; Spiller, R.A., Gut. 35:723-725 (1994).
Ausubel, F.M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media PA (1988).
Bauer, A. W . , et al. , Anal. Biochem. 137:134-142 ( 1984).
Beames, et al. , Biotechniques 11:378 ( 1991 ).
Blaser, M., Scientific American, p. 104 (1996).
Cover, T., et al., Adv. Int. Med. 41:8s-117 (1996).
2s Cover, T . , ASM News 61:21 ( 199s).
i Crea, R., U.S. Patent No. 4,888,286, issued December 19, 1989.
Crimmins, D.L. and Mische, S.M., in CURRENT PROTOCOLS IN PROTEIN SCIENCE, Units 11.5, John Wiley and Sons, lnc., New York (1996).
Dubois, A., et al., Infection and Immunity 64(8):2885-2891 {1996).
Earl, P.L., et al., "Expression of proteins in mammalian cells using vaccinia"
In CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al., Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991).
Eaton, M.A.W., et al., U.S. Patent No. 4,719,180, issued Jan. 12, 1988.
Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93 (1992).
Goeddel, D.V., Methods in Enzymology 185 (1990).
Guthrie, C., and Fink, G.R., Methods in Enrymology 194 (1991).
Harlow, E., et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).
Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983).
Kaufman, R.J., "Selection and coamplification of heterologous genes in mammalian cells,"
in METHODS IN ENZYMOLOGY, vol. 185, pp 537-566. Academic Press, Inc., San Diego CA (1991).
Lau, Y.F., et al., Mol. Cell. Biol. 4:1469-1475 (1984).
Maniatis, T., et al., MOLECULAR CLOMNG: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982).
McAtee, C.P., et al., J. Chromatog. B. 685:91-104 (1996).
Miller, C.G., Methods: A Companion to Methods in Enzymology 6:315-333 (1994).
Moss, B., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Section IV, Unit 16) (1991).
Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987.
Mullis, K.B., et al., U.S. Patent No. 4,683,195, issued 28 July 1987.
Nakayama, H., et al. , J. Chromatog. A 730:279-287 ( 1996).
Oakley, B.R., et al., Anal. Biochem. 105:361-363 (1980).
O'Farrell, P.H., J. Biol. Chem. 250:4007-4021 (1975).
O'Farrell, P.H., et al., Cell 12:1133-1142 (1977).
Parsonnet, J., et al., N. Engl. J. Med. 325:1127-1131 (1991).
Porath, J., Protein Exp. and Purif. 3:263 (1992).
Pearson, W.R. and Lipman, D.J., PNAS 85:2444-2448 (1988).
Pearson, W.R., Methods in Enrymology 183:63-98 (1990).
Reilly, P.R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL
(1992).
Reyes, G., et al., Molecular and Cellular Probes 5:473-481 (1991).
Romanos, M.A., et al., Yeast 8(6):423-488 (1992).
Sambrook, J., et al., In MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger, et al. , Proc. Natl. Acad. Sci . 74:5463-5467 ( 1977).
Scharf, S.J., et al., Science 233:1076 (1986).
Smith, D.B., et al., Gene 67:31 (1988).
Spiecher, D.W. in TECHNIQUES IN PROTEIN CHEMISTRY, (T. Hugli, Ed.) Academic Press, San Diego, CA, pp. 24-35 (1989).
Tomb, et al., Nature 388:539 (1997).
Warren, J.R., and Marshall, B., Lancet 1:1273-1275 (1983).
Yoshio, T., et al., U.S. Patent No. 4,849,350, issued July 18, 1989.
Background of the Invention H. pylori is a human gastric pathogen associated with chronic superficial gastritis, peptic ulcer disease, and chronic atrophic gastritis leading to gastric adenocarcinoma, although for most of this century, peptic ulcer disease was thought to be stress-related rather than caused by H. pylori infection.
Acceptance for the causal role of H. pylori in peptic ulcer disease and gastric inflammation developed when studies showed that human subjects who ingested H. pylori developed gastritis, a condition that was resolved after the infection was eliminated by antibiotic treatment (Warren and Marshall, 1983).
H. pylori is a micro-aerophilic, Gram negative, slow-growing, flagellated organism with a spiral or S-shaped morphology which infects the lining of the stomach. H.
pylori was originally cultured from gastric biopsy in 1982 and was placed in the Campylobacter genus based upon gross morphology. In 1989, the new genera Helicobacteracea was proposed and accepted, with H. pylori being its sole member (Blaser, 1996). Currently, 13 members of the Helicobacter genus are recognized.
One unusual feature shared by all members of the genus is the presence of a urease operon which encodes for a 540 kD cell surface enzyme, with urease being one of the most abundant surface proteins produced by the bacteria. This enzyme is a multisubunit urease which functions to hydrolyse urea into carbon dioxide and ammonia (Cover, 1995). The resulting ammonia molecules surround the bacteria, thereby neutralizing the acid in the immediate vicinity of the bacteria. Thus, urease is crucial for the survival of H. pylori at acidic pH and for its successful colonization of the gastric environment.
H. pylori is one of the most common chronic bacterial infections in humans. H.
pylori infection is found in over 90 % of patients with active gastritis, and the presence of H. pylori in the gastric mucosa has been associated with mucosa-associated lymphoid tissue lymphomas (Cover, et al, 1996). In developed countries, about half of the population has been colonized with H. pylori by age 50, and in developing countries, colonization is common even among children.
Further, one to two out of ten infected individuals will develop peptic ulcer disease in the course of a lifetime.
Current approaches for assessing H. pylori infection typically employ invasive techniques such as collection of gastric biopsy specimens, culture, histology and detection of pre-formed bacterial enzymes. These approaches suffer from numerous drawbacks including low sensitivity, inconvenience, and high-cost. Non-invasive approaches include the urea breath test, UBT (Atherton, _ et al., 1994) and serological tests which utilize various H. pylori antigens for detecting anti-H. pylori antibodies. The urea breath test relies upon the presence of the urease enzyme from H. pylori to convert isotopic urea to isotopic carbon dioxide (the analyte) and ammonia.
Although the'3C (or "C}-urea breath test is fairly accurate, it suffers from its reliance upon a mass spectrometer for analyzing patient breath samples, a piece of equipment absent from most clinical settings. Moreoever, existing serological assays are not yet reliable enough for routine diagnostic use, and are typically utilized in epidemiological studies for retroactively assessing infection.
In view of its importance as a human pathogen, and the persistence of H.
pylori infection amongst the world population, a clear need exists for methods and compositions effective for reliably detecting and treating infection by genetically diverse strains of H. pylori.
Ideally, the reagents for such an assay should be readily and reproducibly prepared, in addition to being highly selective and specific for H. pylori. Moreover, the method should be accurate and exhibit high sensitivity, in addition to being simple, convenient and cost-effective.
Summary of the Invention The invention pertains to the discovery and characterization of new, highly immunogenic polypeptide antigens of H. pylori. Also forming part of the invention are 69 heretofore unrecognized immunogenic cluster families. The sequence and location of these cluster families within the H. pylori genome were determined on the basis of the over 250 disclosed DNA replicas of portions of the genome of H. pylori discovered to encode highly immunogenic antigens. Also disclosed are native antigenic proteins recovered from H. pylori using a proteomics methodology.
The invention further provides methods employing one, several, many or each of the above-described antigens. Also forming part of the invention is a diagnostic kit and method employing one, several, many or each of the herein described antigenic proteins to detect H. pylori infection, where the assay is effective for detecting active infective status H. pylori.
In one aspect, the present invention includes H. pylori genomic polynucleotides encoding one or more of the polypeptide antigens described herein. With respect to the polynucleotides, some _..__-_-_ ._._ aspects of the invention include H. pylori derived RNA and DNA
polynucleotides, recombinant H.
pylori polynucleotides, a recombinant vector including any of the above polynucleotides, and a host cell transformed with any of these vectors.
These polynucleotides encode H. pylori-specific polypeptide antigens. The corresponding coding sequences allow for the production of polypeptides which are useful, for example, as reagents in diagnostic tests and/or as components of vaccines.
Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment identified by SEQ ID N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Polynucleotides encoding these antigens are particularly preferred due to the high sensitivity and specificity exhibited by the resulting antigens.
Other polynucleotides contemplated by the invention are antigen-coding DNA
fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment (typically at least about 18 nucleotides in length) spanning one of the following DNA fragment clusters corresponding to SEQ ID NOs:469-547:
{1). the DNA fragment having the sequence spanning SEQ ID NOS:222 and 223 (cluster 1, corresponding to SEQ ID NOS:469, 470);
(2). the DNA fragment having the sequence spanning SEQ ID NOS:B, 135, 161, 162, 218 (cluster 2, SEQ ID N0:471);
(3). the DNA fragment having the sequence spanning SEQ ID NOS:11, 60, 73 (cluster 3, SEQ ID N0:472);
(4). the DNA fragment having the sequence spanning SEQ ID NOS:75, 12?, 20, 169 (cluster 4, SEQ ID N0:473);
Romanos, M.A., et al., Yeast 8(6):423-488 (1992).
Sambrook, J., et al., In MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger, et al. , Proc. Natl. Acad. Sci . 74:5463-5467 ( 1977).
Scharf, S.J., et al., Science 233:1076 (1986).
Smith, D.B., et al., Gene 67:31 (1988).
Spiecher, D.W. in TECHNIQUES IN PROTEIN CHEMISTRY, (T. Hugli, Ed.) Academic Press, San Diego, CA, pp. 24-35 (1989).
Tomb, et al., Nature 388:539 (1997).
Warren, J.R., and Marshall, B., Lancet 1:1273-1275 (1983).
Yoshio, T., et al., U.S. Patent No. 4,849,350, issued July 18, 1989.
Background of the Invention H. pylori is a human gastric pathogen associated with chronic superficial gastritis, peptic ulcer disease, and chronic atrophic gastritis leading to gastric adenocarcinoma, although for most of this century, peptic ulcer disease was thought to be stress-related rather than caused by H. pylori infection.
Acceptance for the causal role of H. pylori in peptic ulcer disease and gastric inflammation developed when studies showed that human subjects who ingested H. pylori developed gastritis, a condition that was resolved after the infection was eliminated by antibiotic treatment (Warren and Marshall, 1983).
H. pylori is a micro-aerophilic, Gram negative, slow-growing, flagellated organism with a spiral or S-shaped morphology which infects the lining of the stomach. H.
pylori was originally cultured from gastric biopsy in 1982 and was placed in the Campylobacter genus based upon gross morphology. In 1989, the new genera Helicobacteracea was proposed and accepted, with H. pylori being its sole member (Blaser, 1996). Currently, 13 members of the Helicobacter genus are recognized.
One unusual feature shared by all members of the genus is the presence of a urease operon which encodes for a 540 kD cell surface enzyme, with urease being one of the most abundant surface proteins produced by the bacteria. This enzyme is a multisubunit urease which functions to hydrolyse urea into carbon dioxide and ammonia (Cover, 1995). The resulting ammonia molecules surround the bacteria, thereby neutralizing the acid in the immediate vicinity of the bacteria. Thus, urease is crucial for the survival of H. pylori at acidic pH and for its successful colonization of the gastric environment.
H. pylori is one of the most common chronic bacterial infections in humans. H.
pylori infection is found in over 90 % of patients with active gastritis, and the presence of H. pylori in the gastric mucosa has been associated with mucosa-associated lymphoid tissue lymphomas (Cover, et al, 1996). In developed countries, about half of the population has been colonized with H. pylori by age 50, and in developing countries, colonization is common even among children.
Further, one to two out of ten infected individuals will develop peptic ulcer disease in the course of a lifetime.
Current approaches for assessing H. pylori infection typically employ invasive techniques such as collection of gastric biopsy specimens, culture, histology and detection of pre-formed bacterial enzymes. These approaches suffer from numerous drawbacks including low sensitivity, inconvenience, and high-cost. Non-invasive approaches include the urea breath test, UBT (Atherton, _ et al., 1994) and serological tests which utilize various H. pylori antigens for detecting anti-H. pylori antibodies. The urea breath test relies upon the presence of the urease enzyme from H. pylori to convert isotopic urea to isotopic carbon dioxide (the analyte) and ammonia.
Although the'3C (or "C}-urea breath test is fairly accurate, it suffers from its reliance upon a mass spectrometer for analyzing patient breath samples, a piece of equipment absent from most clinical settings. Moreoever, existing serological assays are not yet reliable enough for routine diagnostic use, and are typically utilized in epidemiological studies for retroactively assessing infection.
In view of its importance as a human pathogen, and the persistence of H.
pylori infection amongst the world population, a clear need exists for methods and compositions effective for reliably detecting and treating infection by genetically diverse strains of H. pylori.
Ideally, the reagents for such an assay should be readily and reproducibly prepared, in addition to being highly selective and specific for H. pylori. Moreover, the method should be accurate and exhibit high sensitivity, in addition to being simple, convenient and cost-effective.
Summary of the Invention The invention pertains to the discovery and characterization of new, highly immunogenic polypeptide antigens of H. pylori. Also forming part of the invention are 69 heretofore unrecognized immunogenic cluster families. The sequence and location of these cluster families within the H. pylori genome were determined on the basis of the over 250 disclosed DNA replicas of portions of the genome of H. pylori discovered to encode highly immunogenic antigens. Also disclosed are native antigenic proteins recovered from H. pylori using a proteomics methodology.
The invention further provides methods employing one, several, many or each of the above-described antigens. Also forming part of the invention is a diagnostic kit and method employing one, several, many or each of the herein described antigenic proteins to detect H. pylori infection, where the assay is effective for detecting active infective status H. pylori.
In one aspect, the present invention includes H. pylori genomic polynucleotides encoding one or more of the polypeptide antigens described herein. With respect to the polynucleotides, some _..__-_-_ ._._ aspects of the invention include H. pylori derived RNA and DNA
polynucleotides, recombinant H.
pylori polynucleotides, a recombinant vector including any of the above polynucleotides, and a host cell transformed with any of these vectors.
These polynucleotides encode H. pylori-specific polypeptide antigens. The corresponding coding sequences allow for the production of polypeptides which are useful, for example, as reagents in diagnostic tests and/or as components of vaccines.
Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment identified by SEQ ID N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Polynucleotides encoding these antigens are particularly preferred due to the high sensitivity and specificity exhibited by the resulting antigens.
Other polynucleotides contemplated by the invention are antigen-coding DNA
fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment (typically at least about 18 nucleotides in length) spanning one of the following DNA fragment clusters corresponding to SEQ ID NOs:469-547:
{1). the DNA fragment having the sequence spanning SEQ ID NOS:222 and 223 (cluster 1, corresponding to SEQ ID NOS:469, 470);
(2). the DNA fragment having the sequence spanning SEQ ID NOS:B, 135, 161, 162, 218 (cluster 2, SEQ ID N0:471);
(3). the DNA fragment having the sequence spanning SEQ ID NOS:11, 60, 73 (cluster 3, SEQ ID N0:472);
(4). the DNA fragment having the sequence spanning SEQ ID NOS:75, 12?, 20, 169 (cluster 4, SEQ ID N0:473);
(5). the DNA fragment having the sequence spanning SEQ ID NOS:175 and 176 (cluster 5, SEQ ID NOs:474, 475);
(6). the DNA fragment having the sequence spanning SEQ ID NOS:115, 3, and 227 (cluster 6, SEQ ID N0:476);
(7). the DNA fragment having the sequence spanning SEQ ID N0:206 (cluster 7, SEQ ID
N0:477);
N0:477);
(8). the DNA fragment having the sequence spanning clones Y291/T7 and Y291/T3 (cluster . 8, SEQ ID N0:478);
(9). the DNA fragment having the sequence spanning SEQ ID N0:35 (cluster 9, SEQ ID
N0:479);
N0:479);
(10). the DNA fragment having the sequence spanning SEQ ID NOS:160, 100, 198, 159, 134, 197, 99 (cluster 10, SEQ ID N0:480);
(11). the DNA fragment having the sequence spanning SEQ ID NOS:201, 155, 202, and 156 (cluster 11, SEQ ID N0:481);
(12). the DNA fragment having the sequence spanning SEQ ID NOS:1, 114, 192, 130, 113, 191, 129 (cluster 12, SEQ ID N0:482);
(13). the DNA fragment having the sequence spanning SEQ ID NOS:117, 116, 214, 203, 213, and 110 (cluster 13, SEQ ID NO: 483);
(14). the DNA fragment having the sequence spanning SEQ ID NOS:158, 95, 94, (cluster 14, SEQ ID N0:484);
(15). the DNA fragment having the sequence spanning SEQ ID NOS:153 and 154 (cluster 15, SEQ ID NOs:485, 486);
(16). the DNA fragment having the sequence spanning SEQ ID NOS:140 and 141 (cluster 16, SEQ ID N0:487);
(17). the DNA fragment having the sequence spanning SEQ ID NOS:181, 182 (cluster 17, SEQ ID N0:488);
(18). the DNA fragment having the sequence spanning SEQ ID NOS:138, 144, 15, and 137 (cluster 18, SEQ ID N0:489);
(19). the DNA fragment having the sequence spanning SEQ ID NOS:51 and 80 (cluster 19, SEQ ID N0:490);
(20). the DNA fragment having the sequence spanning SEQ ID NOS:98 and 125 (cluster 20, SEQ ID N0:491);
(21). the DNA fragment having the sequence spanning SEQ ID NOS:43, 28 (cluster 21, SEQ
ID NO: 492);
ID NO: 492);
(22). the DNA fragment having the sequence spanning SEQ ID N0:6 (cluster 22, SEQ ID
N0:493);
N0:493);
(23). the DNA fragment having the sequence spanning SEQ ID NOS:78; 212, 142, 79 (cluster 23, SEQ ID N0:494);
(24). the DNA fragment corresponding to SEQ ID N0:495 (25). the DNA fragment having the sequence spanning SEQ ID N0:259 (cluster 25, SEQ ID
N0:496);
N0:496);
(26). the DNA fragment having the sequence spanning SEQ ID N0:164 (cluster 26, SEQ ID
N0:497);
N0:497);
(27). the DNA fragment having the sequence spanning SEQ ID N0:174 (cluster 27, SEQ ID
N0:498);
N0:498);
(28). the DNA fragment having the sequence spanning SEQ ID NOS:119, 83, 85, 136, 84, 120, 24 (cluster 28, SEQ ID N0:499);
(29). the DNA fragment having the sequence spanning SEQ ID NOS:194, 108, 163, and 193 (cluster 29, SEQ ID N0:500);
(30). the DNA fragment having the sequence spanning SEQ ID N0:224 (cluster 30, SEQ ID
NO:501 );
NO:501 );
(31). the DNA fragment having the sequence spanning SEQ ID NOs:150, 173, 199, 183, 230, and 253 (cluster 31, SEQ ID N0:502);
(32). the DNA fragment having the sequence spanning SEQ ID NOS:122, 77, 187 (cluster 32, SEQ ID N0:503);
{33). the DNA fragment having the sequence spanning SEQ ID NOS:88, 184, 89 (cluster 33, SEQ ID N0:504);
(34). the DNA fragment having the sequence spanning SEQ ID N0:91 (cluster 34, SEQ ID
N0:505);
N0:505);
(35). the DNA fragment having the sequence spanning SEQ ID NOS:101, 92, 102, and 93 (cluster 35, SEQ ID NOs:506, 507, 508);
(36). the DNA fragment having the sequence spanning SEQ ID NOS:170 and 118 (cluster 36, SEQ ID N0:509);
(37). the DNA fragment having the sequence spanning SEQ ID NOS:205, 104, 87, 204, I03, 126, 86, 264, 271, 151, and 152 (cluster 37, SEQ ID N0:510);
(38). the DNA fragment having the sequence spanning SEQ ID NOS:151, 152, 271 and 264 (cluster 38, SEQ ID N0:511);
(39). the DNA fragment having the sequence spanning SEQ ID NOS:143 and 225 (cluster 39, SEQ ID N0:512);
(40). the DNA fragment having the sequence spanning SEQ ID NOS:165 and 166 (cluster 40, SEQ ID NOs:513, 514);
(41). the DNA fragment having the sequence spanning SEQ ID N0:171 (cluster 41, SEQ ID
N0:515);
N0:515);
(42). the DNA fragment having the sequence spanning SEQ ID NOS:209, 216, 217, 208, 215, 38 (cluster 42, SEQ ID N0:516);
(43). the DNA fragment having the sequence spanning SEQ ID NOS:177, 178, 179, (cluster 43, SEQ ID NOs:517, 518);
. (44). the DNA fragment having the sequence spanning cluster 44 (SEQ ID
N0:519);
(45). the DNA fragment having the sequence spanning SEQ ID NOS:186, 195, 185, (cluster 45, SEQ ID N0:520);
(46). the DNA fragment having the sequence spanning SEQ ID N0:251 (cluster 46, SEQ ID
N0:521 );
(47). the DNA fragment having the sequence spanning cluster 47 (SEQ ID NOs:522, 523);
(48). the DNA fragment having the sequence spanning SEQ ID
NOS:190, 189 (cluster 48, SEQ ID
N0:524);
(49). the DNA fragment having the sequence spanning SEQ ID
N0:200 (cluster 49, SEQ ID
N0:525);
(50). the DNA fragment having the sequence spanning SEQ ID
NOS:211, 210 (cluster 50, SEQ ID
N0:526);
(51), the DNA fragment having the sequence spanning SEQ ID
NOS:168, 81, 132, 82, 121, 133, 167, 228 (cluster 51, SEQ
ID N0:527);
(52). the DNA fragment having the sequence spanning SEQ ID
N0:146 (cluster 52, SEQ ID
N0:528);
(53). the DNA fragment having the sequence spanning cluster 53 (SEQ ID N0:529);
(54). the DNA fragment having the sequence spanning SEQ ID
NOS:221 and 220 (cluster 54, SEQ
ID N0:530);
(55). the DNA fragment having the sequence spanning SEQ ID
N0:74 (cluster 55, SEQ ID
N0:531);
(56). the DNA fragment having the sequence spanning SEQ ID
N0:72 (cluster 56, SEQ ID
N0:532);
(57). the DNA fragment having the sequence spanning SEQ ID
N0:70 and 71 (cluster 57, SEQ ID
N0:533);
(58). the DNA fragment having the sequence spanning SEQ ID
NOS:96, 97 (cluster 58, SEQ
ID NOs:534,535);
(59). the DNA fragment having the sequence spanning SEQ ID
NOS:105 and 106 (cluster 59, SEQ
ID NOs:536, 537);
(60). the DNA fragment having the sequence spanning SEQ ID
N0:107 (cluster 60, SEQ ID
N0:538);
(61). the DNA fragment having the sequence spanning SEQ ID
N0:109 (cluster 61, SEQ ID
N0:539);
(62). the DNA fragment having the sequence spanning SEQ ID
NOS:111, and 112 (cluster 62, SEQ
ID N0:540);
(63). the DNA fragment having the sequence spanning cluster 63 (SEQ ID N0:541);
(64). the DNA fragment having the sequence spanning SEQ ID
N0:58 (cluster 64, SEQ ID
N0:542);
(65). the DNA fragment having the sequence spanning cluster 65 (SEQ ID
N0:543);
(66). the DNA fragment having the sequence spanning SEQ ID N0:90 (cluster 66, SEQ ID
N0:544);
(67). the DNA fragment having the sequence spanning SEQ ID N0:13 (cluster 67.
SEQ ID
N0:545);
(68). the DNA fragment having the sequence spanning SEQ ID N0:47 (ciuster 68, SEQ ID
N0:546);
- (69). the DNA fragment having the sequence spanning SEQ ID N0:16 (cluster 69, SEQ ID
N0:547).
According to another embodiment, a H. pylori polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:6, SEQ ID N0:8, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:20, SEQ ID N0:24, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:43, SEQ ID N0:47, SEQ ID N0:51, SEQ ID N0:54, SEQ ID
N0:58, SEQ ID N0:60, SEQ ID NOs:70-230, SEQ ID N0:248, SEQ ID N0:251, SEQ ID
N0:253, SEQ ID N0:255, SEQ ID N0:257, SEQ ID N0:259, SEQ ID N0:264, SEQ ID N0:270, SEQ
ID
N0:271, SEQ ID N0:322, SEQ ID N0:549, where these DNA sequences correspond to cloned and sequenced regions of the H. pylori genome encoding highly immunogenic proteins.
In a preferred embodiment, a H. pylori antigen coding polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D}.
Alternatively, a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
In yet another embodiment, a H. pylori antigen coding polynucleotide according to the invention is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:6, SEQ ID N0:8, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:20, SEQ ID N0:24, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:43, SEQ ID N0:47, SEQ ID N0:51, SEQ ID N0:54, SEQ ID
N0:58, SEQ ID N0:60, SEQ ID NOs:70-230, SEQ ID N0:248, SEQ ID N0:251, SEQ ID
N0:253, SEQ ID N0:255, SEQ ID N0:257, SEQ ID N0:259, SEQ ID N0:264, SEQ ID N0:270, SEQ
ID
N0:271, SEQ ID N0:322, SEQ ID N0:549.
In yet another embodiment, a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID
N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
In another aspect, the invention includes a H. pylori polypeptide antigen, in substantially purified form, characterized by immunoreactivity with H. pylori positive anti-sera. An antigen in accordance with the invention is, in one embodiment, encoded by a polynucleotide that is typically at least 18 nucleotides in length, having the features described above. More specifically, the antigen is encoded by all or a portion of a polynucleotide sequence at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
Additional antigens according to the invention are encoded by H. pylori antigen coding sequences as described above.
In a preferred embodiment, an antigen is encoded by a polynucleotide at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA
sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID
N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5}, SEQ ID
N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Alternatively, the invention encompasses a H. pylori antigen comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ
ID NOS:340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
In a particular embodiment, a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554.
In yet another embodiment, a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS:555-602.
Alternatively, a H. pylori antigen comprises a polypeptide sequence selected from the group consisting of SEQ ID NOs:555-602.
In one particularly preferred embodiment, a H. pylori antigen is one comprising at least 6 contiguous amino acids contained within a sequence selected from the group consisting of SEQ ID
N0:44 (A22), SEQ ID N0:39 (C1), SEQ ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID
N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ
ID N0:573 (Y128D).
Also forming part of the invention is a diagnostic kit for use in screening a biological fluid such as sera for the presence of anti-H. pylori antibodies. The kit includes a substantially purified H.
pylori antigen of the type characterized above that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of the antibody to the antigen.
The polypeptide antigen may be attached to a solid support, and the kit may further include a non-attached reporter-labelled anti-human antibody, where binding of the anti-H. pylori antibodies to the polypeptide antigen can be detected by binding of the reporter-labelled antibody to the anti-H. pylori antibodies.
According to one embodiment, the kit includes at least two H. pylori antigens having different antibody specificities.
In a related aspect, the invention includes a method of detecting H. pylori infection in a subject, or detecting the eradication of the bacteria in a previously infected subject. The method involves reacting a biological fluid sample from a subject with a purified H.
pylori polypeptide antigen of the type described above, and examining the antigen for the presence of bound antibody.
Preferred antigens for use in the method correspond to one of the following polypeptide I5 sequences or a contiguous region contained therein: SEQ ID N0:44 (A22), SEQ
ID N0:39 (C1), SEQ
ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ
ID N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ ID N0:573 (Y128D).
In still another aspect, the invention includes a H. pylori vaccine composition containing a H.
pylori polypeptide antigen of the type described above. The antigen is characterized by its ability to reduce the level of H. pylori infection in a mammalian model system, such as a mouse or rhesus monkey challenged with the peptide, and then infected with H. pylori.
Preferred antigens for use in a vaccine are those which invoke a long-lasting antigenic response, as evidenced by the persistence of antibodies for an extended period of time subsequent to antimicrobial treatment. Representative antigens for use in a vaccine composition are selected from the group consisting of SEQ ID N0:565 (Y139), SEQ ID N0:575 (Y146B), SEQ ID
N0:555 (Y175A), SEQ ID N0:44 (A22), SEQ ID N0:569 (Y184A), SEQ ID N0:578 (Z9A), SEQ
ID
N0:557 (Y261A) and SEQ ID N0:575 (Y146B).
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.
Brief Description of the Fi ,gores Fig. 1 is a computer scanned image of a Western blot of a 2-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis (high pH
conditions) of native H.
pylori antigens blotted with Roost pooled sera (H. pylori positive);
Fig. 2 is a computer scanned image illustrating a Western blot of a sodium dodecyl sulfate polyacrylamide gel 2-dimensional electrophoretic (SDS-PAGE) analysis (low pH
conditions) of native H. pylori antigens blotted with Roost pool;
Fig. 3 shows a schematic representation of the amino acid translation of ORF3 of clone Y 104-l.asm (299 amino acids). Regions of sequence indicated in the figure were confirmed by amino acid sequence analysis of the Y104-l.asm protein expressed in E. coli strain XLlBlue, and correspond to (i) the 36 kD protein of H. pylori (indicated by underline), (ii) "spot 15"
peptides (indicated by a box), and (iii) peptides encoded by clone d7 (bracketed);
Fig. 4 is a schematic representation of the in vivo processing pathway of the 36 kD protein of H. pylori and its relationship to the "spot 15" antigen disclosed herein;
Fig. 5 is a reverse phase HPLC peptide profile corresponding to the "spot 15"
antigen isolated from H. pylori (ATCC 43504) which illustrates the presence of numerous identifiable protein peaks;
Fig. 6 presents the amino acid sequence of the 36-kD protein of H. pylori, where underlining indicates the 28 kD protein region;
Fig. 7 is a schematic representation of the proteome methodology employed to identify several native antigens of H. pylori;
Fig. 8 is a graphical representation summarizing percent sensitivity of various clones against representative H. Pylori-immunopositive sera panels;
Fig. 9 is a linear representation of the H. pylori genome, indicating the approximate positions of immunogenic cluster regions forming one aspect of the present invention;
Figs. 10-63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5}, (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (53), (54), (58), (59), (61), (62), (68), and (69), respectively;
Figs. b4A-D present a summary of the immunogenic clone clusters of the present invention including (i) cluster number, (ii) clones defining the start and end regions of each cluster, coordinates of the cluster consensus region within the H. pylori genome, and expression data;
Fig. 65 is a graphical representation of comparative sensitivities of H.
pylori recombinant antigens against various sera panels. Sensitivity values were calculated using various gold reference standards as described in Example 6B;
Fig. 66 is a graphical representation of comparative specificities of H.
pylori recombinant antigens against various sera panels, computed against gold standards as indicated; and _.. __. .....__._~ _T.. _..._.. .~_ ._ _- ,~.._ Figs. 67A and 67B provide a tabular summary of (i) immunopositive clones forming the basis of the invention, (ii) their corresponding cluster numbers (indicating the relative position of each clone and cluster within the H. pylori genome), and relative (iii) sensitivity and (iv) specificity values.
Detailed Description of the Invention I. Definitions The following terms, as used herein, have the meanings as indicated:
- A polypeptide sequence or fragment is "derived" from another polypeptide sequence or fragment when it has the same sequence of amino acid residues as the corresponding region of the fragment from which it is derived.
A polynucleotide sequence or fragment is "derived" from another polynucleotide sequence or fragment when it has the same sequence of nucleic acid residues as the corresponding region of the fragment from which it is derived.
A first polynucleotide fragment is "selectively-hybridizable" to a second polynucleotide fragment if the first fragment or its complement can form a double-stranded polynucleotide hybrid with the second fragment under selective (stringent) hybridization conditions.
The first and second fragments are typically at least 15 nucleotides in length, preferably at least 18-20 nucleotides in length.
Selective (stringent) hybridization conditions are defined herein as hybridization at -45°C in --1.1M
salt followed by at least one wash at 37°C in 0.3M salt. Such conditions typically allow at most about 25-30 % basepair mismatches.
Two or more polynucleotide or polypeptide fragments have at least a given percent "sequence identity" if their nucleotide bases or amino acid residues are identical, respectively, in at least the specified percent of total base or residue position, when the two or more fragments are aligned such that they correspond to one another using a computer program such as ALIGN.
(The ALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs, Pearson and Lipman, 1988; Pearson, 1990).
An "H. pylori polynucleotide" as used herein, refers to a polynucleotide sequence derived from the genome of H. pylori and variants thereof. H. pylori polynucleotides of the type disclosed herein encode H. pylori polypeptide antigens, where the resulting antigen is characterized by immunoreactivity with H. pylori positive anti-sera. Generally, a H. pylori polynucleotide of the . invention will be at least about 18 or more nucleotides in length (i.e., encoding a 6 peptide-antigen).
In an alternative embodiment, the H. pylori polynucleotide will be at least about 24 nucleotides in length (i. e. , encoding an 8 peptide-antigen). In yet another embodiment, the H. pylori polynucleotide will be at least about 30 nucleotides in length. In some instances, the H.
pylori polynucleotide will range from about 45 to 75 nucleotides in length, but may of course be longer.
The polynucleotides WO 98/49314 ' PCT/US98/08487 of the invention may be obtained from natural or synthetic sources, or, may be prepared recombinantly. The polynucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, by deletions, by insertions and inversions, to a particular naturally-occurring sequence, provided that the subject polynucleotide is capable of expressing a H. pylori antigen as described herein. The polynucleotide sequence may optionally contain expression control sequences (typically from a heterologous source) positioned adjacent to the coding region.
The nucleotide sequences described herein are meant to encompass variants possessing essentially the same "sequence identity" as defined above. Nucleotide sequences having essentially the same sequence identity are typically selectively hybridizable to one another under selective (stringent) hybridization conditions. This is to say, a nucleic acid fragment is considered to be selectively hybridizable to a H. pylori polynucleotide if it is capable of specifically hybridizing to the H. pylori polynucleotide sequence or a variant thereof (e.g., a probe that hybridizes to a H. pylori polynucleotide but not to polynucleotides from other members of the Helicobacter family) under stringent hybridization and wash conditions.
An "H. pylori antigenic polypeptide" is meant to encompass immunoreactive variants of the polypeptide, or regions, or parts thereof, provided that the variant is immunogenic. A suitable variant is defined as any polypeptide having a sequence that is identical (i. e. , shares sequence identity) to that of a H. pylori polypeptide.
For example, an antigenic polypeptide that is essentially identical to a H.
pylori polypeptide antigen is (i) encoded by a nucleic acid that selectively hybridizes to sequences of H. pylori or its variants or (ii) is encoded by H. pylori or its variants.
A sequence comparison may also be employed for the purpose of determining "polypeptide homology", e.g., by using the local aligtnnent program LALIGN. In carrying out such a determination, a polypeptide sequence is typically compared against a selected H. pylori amino acid sequence or any of its variants, as defined above, using the LALIGN program with a ktup of 1, default parameters and the default PAM.
Any polypeptide with an optimal alignment longer than about 6 to 8 amino acids and greater than 70 % , or more preferably 75 % to 80 % of identically aligned amino acids is considered to be a "homologous polypeptide. " The LALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson, et al. , 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 44.0, Jordan Hall, Charlottesville, VA).
Sequence variations among antigens will depend upon a number of factors, such as the strain of H.
pylori, the location of the gene or gene family encoding the antigen within the H. pylori genome (i.e., whether the gene is highly conserved or prone to recombination) and the like.
_ _ _ __ _..... _.__~__ An immunogenic polypeptide or polypeptide "fragment" is one that is (i) encoded by an open reading frame of a H. pylori polynucleotide, or (ii) displays sequence identity to H. pylori polypeptides as defined above, and is immunoreactive with a H. pylori immunopositive sample, such as sera.
Typically, the immunogenic fragment will comprise at least about 6 to 8 amino acids, and preferably at least about 10 to 12 contiguous amino acid residues of a particular antigen.
By immunoreactive variant of a H. pylori polypeptide antigen is meant an amino acid substitution, deletion, and/or addition variant of a particular H. pylori polypeptide antigen sequence disclosed herein, having substantially the same or increased binding affinity to a given antibody as the particular polypeptide antigen, as determined by conventional methods, e.g., a competition assay or a two antibody sandwich assay.
A representative H. pylori antigen is composed of at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
"Cluster sequences" correspond to regions within the H. pylori genome encoding highly immunogenic polypeptides. The cluster sequences were determined on the basis of DNA sequence information for the over 250 immunogenic clones described herein. As a result, 69 unique clusters were identified, and antigens in accordance with the invention are those encoded by a contiguous series of nucleotides contained within any of the 69 clusters. Representative antigen coding sequences falling within each of the clusters are summarized in Figs. 64A-D. The cluster regions are referred to herein as clusters 1 to 69, corresponding to SEQ ID NOs: 340-468.
The location of each of the cluster regions within the H. pylori genome is llustrated pictorially in Fig. 9. In some instances, a cluster is defined by various regions within the H. pylori genome rather than by a single sequence. For example, a particular cluster may be defined by a "start"
sequence, an "end" sequence, and perhaps an invervening "middle" sequence, and thus may correspond to more than one sequence contained in the Sequence Listing. In such cases, the descriptor for the cluster sequence will indicate its relationship to a given cluster (e.g., start, middle, end).
Typically, a defining cluster sequence will code for one or more antigens, and the remainder of the sequence for a particular cluster, if not explicitly provided, can be readily determined based upon the information provided herein, when considered along with the information, e.g., provided in Tomb, et al., 1997. Cluster sequences defined by more than one representative sequence are cluster 1 (SEQ
ID NOs: 469, 470), cluster 5 {SEQ ID NOs:474,475), cluster 7 (SEQ ID N0:477, end), cluster 15 (SEQ ID NOs:485, 486), cluster 35 (SEQ ID NOs:506-508), cluster 40 (SEQ ID
NOs:513, 514), cluster 41 {SEQ ID NO:515, end), cluster 43 (SEQ ID NOs:517, 518), cluster 47 (SEQ ID NOs:522, 523), cluster 49 (SEQ ID N0:525, end), cluster 58 (SEQ ID NOs:534, 535), and cluster 59 (SEQ ID
N0:536, 537).
"Substantially purified" and "in substantially purified form" are used in several contexts and typically refer to at least partial purification of a H. pylori polynucleotide or polypeptide away from unrelated or contaminating components (e.g., serum, cells, proteins, non-H.
pylori polynucleotides, etc.) by at least one purification or isolation step. Methods and procedures for the isolation or purification of compounds or components of interest are described herein (e.g., SDS-PAGE, affinity purification of fusion proteins, blotting, and recombinant production of H.
pylori polypeptides).
An antigen is "specifically immunoreactive" with H. pylori positive anti-sera or a biological fluid sample when, under optimal conditions, the antigen binds to antibodies present in the H. pylori infected sample but does not bind to antibodies present in the majority (greater than about 60 to 65 %, preferably from about 70% to 80%, even more preferably greater than about 85%) of fluid samples from subjects who are not or have not been infected with H. pylori.
"Specifically immunoreactive"
antigens may be immunoreactive with monoclonal or polyclonal antibodies generated against specific H. pylori antigens.
By biological fluid is meant any fluid derived from the body of a mammal, particularly a human. Representative biological fluids include blood, serum, plasma, urine, faeces, mucous, gastric secretions, dental plaques, or saliva.
"Immunologically effective amount" refers to an amount administered to a mammalian host, either as a single dose or as part of a series, that is effective for treatment or prevention of infection by H. pylori. The amount will vary depending upon the health and physical condition of the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, and the like. Such an amount will typically fall within a relatively broad range, and can be determined in routine trials.
II. Isolation of H. pylori Anti enic Se9uences The present invention is based on the identification and isolation of a number of highly immunogenic H. pylori polypeptides, resulting from the screening of over a million individual H.
pylori compositions. The antigens of the present invention were either produced recombinantly, or, were separated from a mixture of soluble proteins obtained from pelleted and lysed H. pylori.
Moreover, as a result of an intensive computational analysis effort, disparate sequence information corresponding to all of the disclosed immunogenic clones was compiled to provide a collection of heretofore unrecognized antigenic cluster regions contained within the genome of H. pylori The preparation and identification of recombinant H. pylori antigens and their classification into clustered families will now be described.
__ T . -_~___ __ 1. Method for Screening Recombinant Sublibraries The H. pylori antigens of the invention can be obtained from phage libraries using conventional screening methods described below. Unless otherwise stated, the DNA lambda libraries described herein have been deposited in the Genelabs Technologies, Inc.
Culture Collection, 505 Penobscot Drive, Redwood City, CA, 94063, or in the Genelabs Diagnostics PTE
LTD Culture Collection, 85 Science Park Drive #04-O1, The Cavendish, Singapore Science Park, Singapore 118259.
E. toll XL-1 Blue MRF plasmids containing inserts corresponding to the following H. pylori clones were accepted for deposit by the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on September 9, 1997 and assigned the following designation numbers: clone dHC1S.11 (ATCC 98525), clone dHGla2S.5 (ATCC 98526), clone Y212A (ATCC 98527), clone dHC7.2cd6 (ATCC 96528), clone y175A (ATCC 98529), clone y146B (ATCC 98530), clone dHA22.8 (ATCC 98531), Y184A (ATCC 98532), clone dHB2d1 (ATCC 98533), clone y104B (ATCC
98534). The bacteriophage lambda containing H. pylori DNA insert Y-library was accepted for deposit by the American Type Culture Collection on September 3, 1997 and assigned designation number ATCC 209234.
The antigen-encoding DNA fragments of the invention can be identified by (i) immunoscreening, as described above, and/or (ii) computer analysis of coding sequences using an algorithm (such as, "ANTIGEN," Intelligenetics, Mountain View, CA) to identify potential antigenic regions. For example, an antigen-encoding DNA fragment is subcloned, and the subcloned insert is then fragmented by partial DNase digestion to generate random fragments or by specific restriction endonuclease digestion to produce specific subfragments. The resulting DNA
fragments are then inserted into an expression vector, such as the lambda gtl l vector, and subjected to immunoscreening in order to provide an epitope map of the cloned insert.
In addition, DNA fragments of the type described herein can be employed as probes in hybridization experiments to identify overlapping H. pylori sequences, and these in turn can be further used as probes to identify additional sets of contiguous clones.
Any of the herein-described clone sequences can be used to probe a DNA
library, generated in a vector such as lambda gtl0 or "LAMBDA ZAP II" (Stratagene, La Jolla, CA).
Specific subfragments of known sequence may be isolated using the polymerase chain reaction or after restriction endonuclease cleavage of vectors carrying such sequences. The resulting DNA fragments can be used as radiolabelled probes against any selected library. In particular, the 5' and 3' terminal sequences of the clone inserts are useful as probes to identify additional clones.
Further, the sequences provided by the 5' end of cloned inserts are useful as sequence specific primers in first-strand DNA synthesis reactions (Maniatis, et al., 1982;
Scharf, et al., 1986). For example, specifically primed H. pylori DNA libraries can be prepared by using specific primers derived from one of the cloned DNA sequences described herein as a template.
The second-strand of the new DNA is synthesized using RNase H and DNA polymerase I. The above procedures identify or produce DNA molecules corresponding to nucleic acid regions that are 5' adjacent to the known clone insert sequences. These newly isolated sequences can in turn be used to identify further flanking sequences, and so on. After new H. pylori sequences are isolated, the polynucleotides can be cloned and immunoscreened to identify specific sequences encoding H. pylori antigens.
2. Recombinant Anti ens In order to identify new and highly antigenic polypeptides useful for detection of H. pylori, DNA libraries were prepared from commercially available strains of H. pylori (American Type Culture Collection, Rockville, MD; ATCC Designation No. 43504, ATCC
Designation No. 43526) in either the expression vector lambda gtll or "ZAPII" (Stratagene, La Jolla, CA; Example 1).
Polynucleotide sequences were then selected for the expression of peptides which were immunoreactive with pooled sera obtained from 11 patients identified by endoscopy as H.
pylori-positive, herein identified as "Roost pool" sera, or another pool of 4 H. pylori-immunopositive sera samples identified as "SFA001 ". It is also possible to screen with other sources of H. pylori-infected samples, such as those described in Examples 6A and 6B, and referred to in Figs. 65 and 66, or described in Example 13. The samples may be individual samples, i. e. , derived from a single subject, or may be pooled samples, such as those described herein.
Recombinant proteins identified by this approach provided candidates for polypeptides that can serve, either singly or in combination, as substrates in diagnostic tests for detecting infection by H. pylori. Further, the corresponding nucleic acid coding sequences serve as useful hybridization probes for the identification of additional H. pylori antigen coding sequences.
The H. pylori strains described above were used to generate DNA libraries in lambda vectors (Example 1). Other commonly available strains of H. pylori include, for example, H. pylori samples identified as strain # 29995 and J-170. Alternatively, libraries can be constructed from H. pylori isolated from a sample confirmed as H. pylon-positive. In the method illustrated in Example 1, the libraries were generated from genomic DNA isolated from the pelleted bacteria.
Alternatively, centrifugation can be used to pellet bacteria from infected biological specimens such as gastric mucosa.
In reference to Example 1, H. pylori DNA libraries were generated using DNase-digested genomic DNA fragments isoiated from H. pylori as starting material. For preparing the "short antigen clone" library, the resulting molecules were ligated to Sequence Independent Single Primer Amplification (SISPA; Reyes, et al., 1991) linker primers and expanded in a non-selective manner, and then cloned into a suitable vector, for example, lambda gtll or ZAPII, for expression and _ ____.___ _ _.T ._ ....._ __-~_ screening of peptide antigens. The libraries disclosed herein have been designated as the "short antigen clone" library, typically designated by a prefix beginning with the letters Y or Z; and the "long antigen clone" library, designated as libraries 1 and 2.
The ZAPII libraries 1 and 2 were similarly constructed, with the following exceptions. The ZAPII libraries 1 and 2 were generated from longer H. pylori DNA fragments, i.e., either EcoRI or HindIII-digested genomic DNA which had not undergone Sequence Independent Single Primer Amplification. Library 1 clones (designated herein by upper case letters) were generated by ligating EcoRI-digested H. pylori DNA directly into the EcoRI sites of the lambda "ZAPII" vector. Library 2 clones (lower case designations) were obtained by digesting H. pylori genomic DNA with HindIII, then blunt ended with E. coli Klenow enzyme or T4 DNA polymerase enzyme and the resulting blunt ended fragments were then ligated to SISPA primers. The linker-ligated DNAs were then treated with EcoRI and ligated into the EcoRI sites of "ZAPII" lambda arms.
Lambda gtl l is a particularly useful expression vector for producing H.
pylori antigens. The vector contains a unique EcoRI insertion site, located 53 base pairs upstream of the translation termination codon of the /3-galactosidase gene. Thus, an inserted sequence is expressed as a /3-galactosidase fusion protein which contains the N-terminal portion of the /3-galactosidase gene product, the heterologous peptide, and optionally the C-terminal region of the a-galactosidase peptide (the C--terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon).
The lambda gtll vector also produces a temperature-sensitive repressor (cI857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C. Advantages of lambda gtl l include: (1) highly efficient recombinant clone generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) production of recombinant fusion protein. Further, since phage containing a heterologous insert produces an inactive /3-galactosidase enzyme, phage with inserts are typically identified using a colorimetric substrate conversion reaction employing /3-galactosidase.
In addition to the lambda gtll vector, numerous E. coli expression vectors are useful for expression of antigens. Alternative microbial hosts suitable for expression include bacilli, such as B.
subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In such hosts, the expression vectors will typically contain control sequences compatible with the host cell. Other known promoter sequences may be present in the expression vector, such as the lactose promoter system, a tryptophan promoter system, or a promoter system from phage lambda.
An amino terminal methionine can be provided, if necessary, by insertion of a Met codon in-frame with the antigen. The carboxy terminal extension of an antigen can be removed by conventional mutagenesis procedures.
Additionally, yeast expression systems can be used, such as the Saccharomyces cerevisiae pre-pro-alpha-factor leader region used to direct protein expression from yeast.
The antigen coding sequence can be fused in frame to the leader region. This construct is then typically put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as ~-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. For constructs used for expression in yeast, protease cleavage sites may be inserted to facilitate separation of the fusion protein components.
Additionally, mammalian cells can be used for expression of the antigens of the invention.
Vectors useful for expression in mammalian cells are typically characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation (polyA) signal. The vectors may optionally include selectable marker genes, such as those conferring antibiotic resistance.
Suitable host cells include Chinese hamster ovary cells (CHO) cell lines, HeLa cells, myeloma cells, Jurkat cell lines, and the like. The expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer, information processing sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
The DNA sequences are expressed in hosts after the sequences of interest have been operably linked to (i. e. , positioned to enable the functioning of) an expression control sequence.
Experiments carried out in support of the present invention will now be described, and in particular, the generation of various phage libraries and characterization of inserts coding for the H.
pylori antigens of the invention.
a. Representative H. pylori Antigens: Short Antigen Clone Library. Example 1 describes the preparation of a DNA library for H. pylori (ATCC No. 43504).
The library was immunoscreened using H. pylori-positive pooled sera (Example 2). A number of lambda clones were identified which were immunoreactive with anti-H. pylori antibodies present in the pooled sera.
Selected immunopositive clones were plaque-purified and their immunoreactivity retested. The immunoreactivity of the clones with normal human sera (control, H. pylori-negative) was also tested.
Numerous clones were identified by immunosceening and further characterized, as described in Examples 3 and 4. Immunoreactive clones further described in Example 3 and referred to herein _. _ ____ -... __T _... -T--. _ _._ as the Y and Z families of clones include clone Y-104-1, and the clones summarized in Table 2, corresponding to SEQ ID NOs: 70-230.
To obtain the polypeptide antigens of the invention, and their respective coding sequences, the DNA inserts of the immunoreactive recombinant lambda clones were PCR
amplified, using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors, and utilizing each immunoreactive clone as template. For the lambda gtl l clones, gtllF (SEQ ID N0:65) and gtl l R (SEQ ID N0:66) primers were used. For the lambda ZAPII clones, T3 (SEQ ID N0:67) and T7 (SEQ ID N0:68) primers were used.
The resulting amplification products were agarose gel purified and eluted from the gel (Ausubel, et al., 1988) to remove primers and other components. The purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced. Clones exhibiting immunoreactivity against H. pylori-positive pooled sera are identified in Example 3, and more specifically in Table 2.
Sequencing was carried out using "DYEDEOXY TERMINATOR CYCLE SEQUENCING"
(a modification of the procedure of Sanger, et al., 1977) on a Perkin Elmer Applied Biosystems model 373A DNA sequencing system according to the manufacturer's recommendations (Perkin Elmer Applied Biosystems, Foster City, CA). Sequence data is presented in the accompanying Sequence Listing.
Sequences for the Y and Z families of clones were compared with "GENBANK", EMBL
database and dbEST (National Library of Medicine) sequences at both nucleic acid and amino acid levels. Search programs FASTA, BLASTP, BLASTN and BLASTX (Altschul, et al., 1990) indicated that these sequences were unique as both nucleic acid and amino acid sequences.
In instances where the clones were determined to be significantly longer than the predicted coding regions of the proteins, the genomic clones were digested by restriction enzyme treatment, and the resulting subfragments were inserted into a suitable expression vector.
The resulting subclones, containing the specific digested DNA fragments, were then screened for immunogenicity. Clones identified as immunoreactive towards H. pylori-positive pooled sera were plaque purified. Plasmid DNA containing inserts obtained by recovery from phage were sequenced as described above and also in Example 4.
b. Representative H. pylori Antigens: ZAPII Libraries 1 and 2. Library 1 and library 2 clones isolated by immunoscreening with H. pylori immunopositive pooled sera include the following: LIBRARY 1: A3, A22, B2, B9, B17, B23, C1, C3, C7, DH, and Ltsx~lty 2: al, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, dl l, d7, e6, f3, f8, fl l, g2, g9, gll, k4.
Clone Gla was isolated by screening with monoclonal antibody 1G6 from Biogenesis, Inc. (Bournemouth, England). Sequence data for these clones is presented in the sequence listing herein.
Exemplary procedures for generating and isolating the library 1 and 2 subclones for preparing the antigens of the invention, including features of the derived coding sequences and putative proteins, are provided above and described in detail in Example 3.
c. Clone d7 and its Relationship to the 36 kD Protein of H. ~,ylori. Clone d7, another immunoreactive clone of the present invention, is a HindIII clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII to produce a beta-galactosidase fusion protein. The nucleotide sequence is presented as SEQ ID
NO:11.
Polynucleotides and polypeptides derived from this clone represent preferred embodiments of the invention.
As will be described in further detail below, this clone codes for about 70%
of the carboxy-end of the 36 Kd native protein of H. pylori, while clone Y104 codes for the entire 36 K protein.
Based upon the results of sequencing experiments carried out in support of the invention (Examples 8 and 9), portions of the amino acid sequence of the native 36 Kd protein of H. pylori have been determined. The 36 kD protein (encoded in part by clone d7) appears to be a precursor to a highly antigenic H. pylori protein referred to herein as the "spot 15" protein (Examples 8,9, to be described in more detail below). This band appears to be Western Blot positive in the majority of H. pylori-positive samples, and is typically absent in normal samples. This antigenic protein thus appears to be highly indicative of infection by H. pylori. The in-vitro processing of the 36 kD protein (i.e, the apparent molecular weight) is illustrated schematically in Fig. 4.
As shown in Fig. 4, the native H. pylori translation product, a 34 kD protein (i.e, calculated molecular weight) composed of 299 amino acids, appears to be cleaved in vivo at amino acid position 23. This results in a 31.6 kD cleavage product commonly referred to as "the 36 kD protein" of H.
pylori. The differences in molecular weight terminology in referring to this protein arise due to the following: 36-kD is observed from SDS-PAGE; 34-kD is calculated from the corresponding DNA
sequence (Fig. 6); and 31 kD is determined from experimental sequence data, as determined starting from residue 23 of SEQ ID N0:60.
A post translation modification of the 36 kD protein, i. e. , acetylation at the amino acid terminus (position 23) and cleavage at the carboxy end, results in the "spot 15" antigen, having a molecular weight of 28 kD. Referring to Fig. 4, "X"s correspond to positions where deamidations (i. e. , asparagine or glutamine) or point mutations have occurred. Further proposed modifications leading to the minor spot 15 protein are also indicated. The antigenic polypeptide corresponding to spot 15, and sequences coding for this protein, e.g., clones Y-104 and d7, represent one particularly _____- _T __ ___-. ___-~-_ WO 98/49314 PG"T/US98/08487 preferred embodiment of the present invention. This is due to the strong antigenicity of the peptide.
Moreover, the spot 15 peptide has been detected in various strains of H.
pylori, as indicated in Table 9 and in Example I0.
The results discussed above and in the Examples indicate the isolation and identification of numerous new H. pylori polypeptide antigens, and polynucleotide sequences encoding such antigens.
d. Cluster Analysis and Identification of Anti enic Regions within the H
pylori Genome. The antigenic sequences described herein were determined based upon the screening of over a million discrete H. pylori antigenic compositions. DNA sequencing was carried out for H. pylori immunopositive clones, and open reading frames coding for antigenic proteins were identified.
Nucleic acid sequences coding for the thus-identified antigens were then inserted into expression vectors and expression of the desired antigenic protein was confirmed. As a result of this work, over 250 antigenic clone sequences have been identified and are disclosed herein.
In an effort to further correlate the sequence information for the various immunogenic clones, the clones were organized into cluster groups (1-69). The antigenic polynucleotide fragments isolated herein map into 69 clusters which are identified herein as clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69). The position of these antigenic clusters within the complete H. pylori genome is shown in Fig. 9. As can be seen from Fig. 9, the locations of the clusters within the genome (and clones within the clusters) are highly random, and represent only a small portion (i.e., approximately 2-3 % ) of the overall nucleotides contained within the entire genome.
Prior to this work, a comprehensive guide to the unique and highly antigenic regions within the genome of H. pylori was unknown.
For cluster analysis, all of the immunoclone sequences were combined into a FASTA database as defined by Pearson and Lipman (1988). This database was converted to a BLAST database for high speed searching (Altschul, et al., 1990). The sequence of each immunoclone was then searched against this database using the program BLASTN to define clusters. Clusters are an assembly of clones that contain identical sequences with other clones in the group. The sequences in each group or cluster were then combined in separate database files and formatted for entry in the GEL program of IG-Suite (Oxford Molecular). GEL then assembled the sequences and suggested a consensus sequence with ambiguities. A non-ambiguous sequence was determined for each cluster by editing the consensus sequence. When the cluster contained non-contiguous sequence, the genome sequence of H. pylori (TIGR World Wide Web site) was used merely to guide assembly.
Clusters 1 to 69 correspond to SEQ ID NOa 469-547. Open reading frames for the antigens were then determined from the cluster consensus sequence. Single clone sequences were translated directly to provide antigen sequences. Antigenic regions contained within each of the clusters as determined by translation of cluster open reading frames are provided as SEQ ID NOa 340-468.
Figs. 10 to 63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), {21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (SO), (51), (53), (54), (58), (59), (6I), (62), (68), and (69). Clusters not shown on the linear maps are clusters defined by one clone, i.e., clusters (22), (24), (26), (31), (34), (52), (55), (56), (57), (60), (63}, (64), (65), (66), (67).
Figs. 64A-D present a tabular summary of clusters 1-69, clones contained within each cluster, and coordinates of each cluster consensus region within the H. pylori genome.
During the course of this work, the complete genome sequence of H. pylori was reported (Tomb, et al., 1997), and is incorporated herein by reference. H. pylori possesses a circular genome of 1,667,867 base pairs and 1590 predicted coding sequences. The genome sequence reported on the TIGR Web site was used as a reference for reporting nucleotide positions of the clusters and immunogenic clones of the invention. Based upon the present work, it can be seen that a relatively small number of the predicted open reading frames reported for the H. pylori genome encode the antigenic proteins or cluster regions forming the basis of the present invention.
Each cluster defines a continuous DNA sequence that spans, i. e. , extends, from the 5' end of the most upstream clone in the cluster to the 3' end of the most downstream clone in the cluster.
Where the spanning sequence is incomplete, it has been filled in with sequence from the reported H.
pylori genomic sequences (TIGR Web site). For exampie, and with reference to Fig. 10, the sequence defined by cluster 1 includes the sequence beginning at the 5' end of clone Y92 (SEQ ID N0:222) and ending at the 3' end of clone Y92 (SEQ ID N0:223), including the short (about 730 bases) genomic sequence connecting the two clone sequences. The positions of the individual clones in each cluster are shown in Figs. 10-63, along with corresponding SEQ ID NOs.
The invention includes antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40}, (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51}, (52), (53), (54), (55), {56), (57), {58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69).
._~__- _._T..._.._.~_....___-..T-__..__ Preferably the antigen-coding regions are the spanning sequences themselves from the clusters.
Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID N0:38 (CI), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Thus, the clusters disclosed herein provide a non-random collection of H.
pylori antigen coding sequences and resulting antigens which have been shown to react with H.
pylori immunopositive samples and are useful in a variety of diagnostic applications.
IO
3. Isolation and Mappin;~ of Native Anti;e Experiments carried out in support of the invention have resulted in the concurrent identification of several additional native antigens of H. pylori (Table 6) using a proteomics approach.
An overview of the proteome methodology disclosed herein and utitilized to separate and identify over twenty H. pylori antigens is provided in Fig. 7 (right-hand side). In turning now to this figure, bacterial proteins are separated by two-dimensional (2D) electrophoresis. Immunoreactive spots (i. e. , reactive in Western blots with pooled sera from H. pylori infected patients) are then selected and subsequently characterized by endoproteolytic digestion, chromatography, and mass spectrometry (e.g., matrix assisted laser desorption time of flight mass spectrometry, MALDI-TOF).
Box A indicates the use of electrospray mass spectrometry to determine the mass of the intact protein.
Box B indicates the use of MALDI-TOF mass spectrometry to evaluate the number and mass of Lys-C
peptides. Box C indicates the use of MALDI-MS to evaluate chromatographically separated Lys-C
peptides and provide sequence information by post source decay (when peptides are pure). Box D
indicates the use of electrospray mass spectrometry to evaluate and sequence peptides through collision induced dissociation. These procedures can be used as an alternative to, or in parallel with, data obtained from conventional cloning techniques to derive genomic data corresponding to antigens of H. pylori, as shown by experiments carried out in support of the invention.
Antigens thus obtained are highly immunogenic, and may be isolated and characterized as illustrated in Examples 7-9 and described as follows.
To obtain soluble H. pylon proteins, pelleted H. pylori is typically lysed in a French press at > 10,000 PSI, followed by centrifugation. Alternative lysis methods include mechanical douncing or detergent disruption.
Antigens are generally obtained from whole lysates as follows. Fractionation of H. pylori soluble proteins is carried out by SDS-PAGE, preferably utilizing 2-dimensional electrophoresis (O'Farrell, 1975; O'Farrell, et al., 1977).
WO 98/49314 ' PCT/CJS98/08487 In performing a typical 2-dimensional electrophoretic separation, an isoelectric focusing gel separation is first carried out (first dimension gel). Isoelectric focusing is carried out, e.g., on a acrylamide/bisacrylamide gel, for an appropriate number of volt-hrs, determined as described in ~~CURRENT PROTOCOLS IN PROTEIN SCIENCE", Units 10.46, John Wiley and Sons, Iric., New York (1996). The final tube gel pH gradient is then measured by a surface pH
electrode. Protein components in a sample mixture undergo a first separation according to pI
value, as indicated on the horizontal axes of Figs. 1 and 2. The first dimensional separation can be carried under either acidic (Fig. 2) or basic (Fig. 1) conditions, depending upon the nature of the proteins to be separated.
A second dimensional, sized-based polyacrylamide slab gel separation is then carried out, to further separate the proteins on the basis of molecular weight. Suitable molecular weight markers are typically added to the gel, for determining corresponding molecular weights of eluted proteins.
Detection is typically carried out by Coomassi blue staining or by silver staining. Silver staining may be preferred, in some cases, since silver staining methods are considerably more sensitive and can be used to detect smaller amounts of protein.
Figs. l and 2 illustrate H. pylori antigens obtained generally as described above, which have been Western blotted with Roost H. pylori-positive serum pool and a negative serum pool, respectively. The numbers in each figure correspond to spots representing H.
pylori proteins.
Features of these proteins (including approximate molecular weights and pI
values), as identified by spot number, are summarized in Table 6. The polypeptides were confirmed to be immunoreactive when tested against anti-H. pylori-antibodies contained in the Roost pool sera. The identities of the spots indicated numerically on the gels were determined by a variety of protein sequencing techniques as described in Example 9.
To further identify these peptides, immunoreactive spots were excised from the gels, digested, and sequenced. As mentioned above, the native H. pylori antigens were further characterized by a combination of sequencing methodologies, including N-terminal sequencing, liquid-chromatography-mass spectrometry, and determination of internal sequences by amino-acid specific chemical cleavage, followed by Edman sequencing (Example 9).
Internal amino acid sequences can be determined by utilizing a combination of various site specific cleavage reagents, such as ortho-pthalaldehyde {OPA)/cyanogen bromide (CNBr), hydroxylamine, formic acid, and BNPS-skatole, which cleave as follows: CNBr (cleaves at C
terminus of methionine), BNPS-skatole (cleaves at C-terminus of tryptophan), formic acid (cleaves at Asp-Pro peptide bond), hydroxylamine (cleaves at Asn-Gly bond), and OPA, which distinguishes between secondary and primary amines; and enzymatic reagents such as Endo Protease L~rs-C, Endoproteinase ASP-N, Endoproteinase GLU-C, and T sin, which cleave respectively as follows:
Endo Protease Lvs-CC (cleaves at C-terminus of lysine), Endoproteinase ASP-N
(cleaves at N-terminus _. _. _ ~ _ ...__._ ~-__.. _ _ __~_ WO 98/49314 ~ PCT/US98/08487 of aspartic acid and cysteic acid), Endoproteinase GLU-C (cleaves at C-terminus of glutamic acid), and Trysin (cleaves at C-terminus of arginine and lysine).
The N-terminal sequence corresponding to each spot was determined (Table 6).
Utilizing the approaches described above and in Examples 7-9, the spots indicated in the 2-D
blots (Figs. 1 and 2) were identified as indicated in Table 6.
Referring to the low pH blot presented as Fig. 2, spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens. Spot 9 represents the native H.
pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al, while spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
Looking at the high pH Western blot in Fig. 1, spots 9 (major and minor), 10, 12, 13, and (major and minor) represent new antigens. The relationship between basic spot 15 and clones Y104 and d7 is discussed above and also illustrated in Fig. 3.
Mass spectral profiles of selected Lys-C digested H. pylori proteins corresponding to Western positive spots are provided in Tables 11 and 12. The mass "fingerprints" of the peptide digests were 15 then used as a basis for comparison to proteins predicted from various genomics databases (details are provided in Example 12) to further confirm the identify of selected H. pylori antigens described herein.
The above-described proteomic approach is useful for analyzing the genetic diversity of H.
pylori, examining antigen-antibody responses during acute and chronic infection, and with particular gastroduodenal pathologies and possible autoimmune components to H. pylori-associated disease.
Moreover, 2-D gel electrophoresis and in-situ proteolytic digestion in conjunction with MALDI-TOF
MS provides an extremely sensitive technique for the rapid identification of H. pylori antigens and for rapid screening for preferred vaccine and diagnostic candidates.
4. Exuression and Purification of Antieenic Polypentides and Preparation of Their Respective Antibodies a. Expression and Purification of Antigenic Polypeptides. The recombinant antigenic peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and amity chromatography.
H. pylori antigens in accordance with the invention comprise at least 6 contiguous amino acids contained within one of the following cluster antigen sequences: SEQ ID
NOS:340-468. In a particular instance, a H. pylori antigen may comprise at least 6 contiguous amino acids contained within a polypeptide sequence selected from one of the following SEQ ID NOS:
2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 2S-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and S50-SS4.
Preferably, a H. pylori antigen corresponds to at least 6 contiguous amino acids contained within a poIypeptide sequence selected from the group consisting of SEQ ID
NOS:555-602, where S these sequences represent illustrative expression proteins corresponding to H. pylori antigens. Even more preferably, a H. pylori antigen is identified by at least 6 contiguous amino acids contained within one of the following sequences: SEQ ID N0:44 (A22), SEQ ID N0:39 (Cl), SEQ ID
N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID
N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ ID N0:573 (Y128D).
Polynucleotide sequences encoding the antigens of the present invention have been cloned in the plasmid p-GEX (Example 5) or various derivatives thereof (pGEX-de165). The plasmid pGEX
(Smith, et al. , 1988) and its derivatives express the polypeptide sequences of a cloned insert fused in-frame to the protein glutathione-S-transferase (sj26). In one vector construction, plasmid pGEX-hisB, an amino acid sequence of 6 histidines, is introduced at the carboxy terminus of the fusion protein.
The various recombinant pGEX plasmids can be transformed into appropriate strains of E.
coli and fusion protein production can be induced by the addition of IPTG
(isopropyl-thio galactopyranoside) as described in Example S. Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using Ni-NTA + + affinity chromatography (Example 5).
Insoluble fusion protein expressed by the plasmids can be purified by means of immobilized metal ion affinity chromatography (Porath, 1992) in buffers containing 6 M
Urea or 6 M guanidinium isothiocyanate, both of which are useful for the solubilization of proteins.
Alternatively, insoluble proteins expressed in pGEX-GLI or derivatives thereof can be purified using combinations of centrifugation to remove soluble proteins followed by solubilization of insoluble proteins and standard chromatographic methodologies, such as ion exchange or size exclusion chromatography, and other such methods are known in the art.
In the case of /3-galactosidase fusion proteins (such as those produced by lambda gtl 1 clones), the fused protein can be isolated readily by affinity chromatography, or by passing cell lysis material over a solid support having surface-bound anti-~i-galactosidase antibody.
Also included in the invention is an expression vector, such as the lambda gtll or pGEX
vectors described above, containing H. pylori antigen coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include a promoter, a translation initiation codon, translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.
_._.__ _ _ _ T ___. ___ __. __ . _._~...
WO 98/49314 PCT/US98/0848?
The DNA encoding the desired antigenic polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include, but are not limited to the following:
baculovirus expression (Reilly, et al., 1992; Beames, et al., 1991; Pharmingen, San Diego, CA; Clontech, Palo Alto, CA), vaccinia expression (Earl, et al., 1991; Moss, et al., 1991), expression in bacteria (Ausubel, et al., 1988;
Clontech), expression in yeast (Gellissen, et al. , 1992; Romanos, et al. , 1992; Goeddel, 1990; Guthrie and Fink, 1991), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, NY), e.g., Chinese hamster ovary (CHO} cell lines (Haynes, et al., 1983; Lau, et al., 1984; Kaufman, 1990).
These recombinant polypeptide antigens can be expressed directly or as fusion proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.
Expression of large polypeptide antigens is described in Example 5. Several of the long antigen clone sequences were cloned into expression vectors and successfully expressed in E. coli, as described in Example 5 and indicated in Tables 3a and 3b.
Expression in yeast systems has the advantage of commercial production.
Recombinant protein production by vaccinia and CHO cell line have the advantage of being mammalian expression systems. Further, vaccinia virus expression has several advantages including the following: (i) a wide host range; (ii) faithful post-transcriptionai modification, processing, folding, transport, secretion, and assembly of recombinant proteins; (iii) high level expression of relatively soluble recombinant proteins; and (iv) a large capacity to accommodate foreign DNA.
The recombinant expressed polypeptide-produced H. pylori polypeptide antigens are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography.
Immunoaffinity chromatography can be employed using antibodies generated based on the H. pylori antigens identified by the methods of the present invention.
The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides. In addition, amino acid sequences can be readily chemically synthesized using commercially available synthesizer (Applied Biosystems, Foster City, CA) or "PIN" technology {Applied Biosystems).
Antigens obtained by any of these methods can be used for antibody generation, diagnostic tests and vaccine development.
Exemplary amino acid sequences corresponding to expressed antigenic proteins of H. pylori are provided herein as SEQ ID NOs:555-602.
b. Antibodv Production. In another aspect, the invention includes specific antibodies directed against the polypeptide antigens of the present invention.
Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e.g., Pierce, Rockford, IL). Typically, to prepare antibodies, a host animal, such as a rabbit or a goat, is immunized with the purified antigen or fused protein antigen. Hybrid or fused proteins may be generated using a variety of coding sequences derived from other proteins, such as glutathione-S-transferase or /3-galactosidase. The host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen. These techniques are equally applicable to all immunogenic sequences described herein.
The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAF
Sephadex chromatography, amity chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
Alternatively, purified antigen or fused antigen protein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-derived hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a H.
pylori may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a suitable fusion partner can be used to produce human-derived hybridomas. Primary in vitro sensitization with viral specific polypeptides can also be used in the generation of human monoclonal antibodies.
Antibodies secreted by the immortalized cells are screened to determine the clones that secrete anti-bodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al., 1988).
Purified polyclonal or monoclonal antibodies directed against H. pylori antigens can then be used in any of a number of standard immunoassay formats to detect the presence of antigen, such as described in Harlow, et al., 1988. One representative assay format is an antigen capture sandwich assay. In a typical sandwich assay, antibody is immobilized on a solid support. H. pylori infected samples (e.g., feces, dental plaque, gastric biopsies, culture suspension from a biopsy sample) are then allowed to react with the immobilized antibodies, followed by incubation with a different antibody directed against H. pylori (e.g., whole lysate) and subsequent reaction with a secondary antibody carrying a reporter label. Detection of label in a testing region is indicative of the presence of H.
pylori antigen in a test, sample. The above-described assay is representative of any of an antigen-based __ ~__ ..T _~ - ___.___..__.._ T _ WO 98/49314 PCT/ITS98i08487 assay based on antibodies prepared as described above, useful for the early detection of H. pylori antigens in a sample suspected of infection by H. pylori.
5. ELISA and Protein Blot Screenin H. pylori antigens are first identified, typically through plaque immunoscreening as described above, and expressed and purified (as previously described). The antigens are then screened rapidly against a large number of suspected H. pylori positive anti-sera using alternative immunoassays, such as, ELISAs or Protein Blot Assays (Western blots) employing the isolated antigen peptide. The antigen polypeptide fusion protein is then isolated as described above, usually by affinity chromatography to the fusion partner such as ~i-galactosidase or glutathione-S-transferase.
Alternatively, the antigen itself is purified using antibodies generated against it (see below).
A general ELISA assay format may be employed, such as those described in Harlow, et al.
(1988). The purified antigen polypeptide or fusion polypeptide containing the antigen of interest, is attached to a solid support, for example, a multiwell polystyrene plate.
Biological fluid (e.g., sera) to be tested are diluted and added to the wells. After a period of time sufficient for the binding of antibodies to the immobilized antigens, the sera are washed out of the wells.
A labelled reporter antibody is added to each well along with an appropriate substrate: wells containing antibodies bound to the purified antigen polypeptide or fusion polypeptide containing the antigen are detected by a positive signal.
A typical format for protein blot analysis using one, any, several, or each of the polypeptide antigens of the present invention is presented in Example 6. General protein blotting methods are described by Ausubel, et al. (1988).
In Example 6A, the antigenic protein expressed by clone dHA22.8 (A22) was used to screen a number of sera samples (both pooled sera and discrete samples). The high percentage of sera reacting to antigen produced from recombinant clone dHA22.8 indicates that it is a dominant epitope, and that it is a suitable infection marker for H. pylori. The results presented in Example 6A
demonstrate that several different source H. pylori-positive anti-sera are immunoreactive with this representative polypeptide antigen. Similar results are described for recombinant protein expressed by clone dHCIS.II (C1). .
Additional experiments carried out in support of the invention as described in Example 6B
reveal, on the basis of sera paneling data using both single antigens and antigen combinations, that preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, C1, Y124A, Y261A, c5, C7, B2, Y104B, and Y128D.
These antigens are effective as serological markers for detecting active infection by H. pylori, based upon favorable sensitivity and selectivity features.
In Example 8, native proteins from H. pylori are shown to be immunoreactive with anti-H.
pylori primary antibodies obtained from "Roost" pooled serum.
The results presented above demonstrate that the polypeptide antigens of the present invention can, by these methods, be rapidly screened against panels of suspected H. pylori infected serum samples for the detection of H. pylori.
A. Protective Antibodies, Vaccines and the Generation of Protective Immunity a. Protective Antibodies. Protective antibodies can be identified using, for example, an animal model system (DuBois, et al. , 1996). To identify protective antibodies, polyclonal or monoclonal antibodies are generated against the antigens of the present invention, where the antigens may be used as the immune-stimulation component arm in conjunction with cholera toxic (CT). Antibodies thus generated are then used to pre-treat an infectious H.
pylori-containing inoculum (e.g., serum) before infection of cell cultures or animals. The ability of a single antibody or mixtures of antibodies to protect the cell culture or animal from infection is evaluated. For example, in cell culture and animals, the absence of antigen and/or nucleic acid production serves as a screen.
Further, in animals, the absence of H. pylori disease symptoms, e.g., elevated carbon dioxide/ammonia levels in a urea breath test (UBT) is also indicative of the presence of protective antibodies. The urea breath test takes advantage of the action of the urease enzyme of H. pylori to decompose ingested '3C or '°C urea to radioactive carbon dioxide and ammonia, and radioactive carbon dioxide is then measured.
Animal models for investigating H. infection include: (i) gnotobiotic newborn piglets (easily infected by H. pylori of human origin, but preferred for short term studies), (ii) mice and ferrets, which can be colonized for months (mice) or years (ferrets), and (iii) certain domestic cats, which can carry H. pylori (DuBois, et al., 1996). H. pylori causes ulcers in gnotobiotic piglets, and in mice using "THE SYDNEY STRAIN" of H. pylori, and gastritis in mouse strains SJL, C3H/HZ, DBA, C56BL/b, and Balb/C. A rhesus monkey infection model has also been developed (DuBois, et al., 1996).
Alternatively, convalescent sera can be screened for the presence of protective antibodies and then these sera used to identify H. pylori antigens that bind with the antibodies. The identified H.
pylori antigen is then recombinantly or synthetically produced. The ability of the antigen to generate protective antibodies is tested as above.
b. Vaccines. After initial screening, the antigen or antigens identified as capable of generating protective antibodies, either singly or in combination, can be used as a vaccine to inoculate test animals (to be described in greater detail below). The animals are then challenged _.._ T__w.. __ ____.-___..___-T-__ with infectious H. pylori. Protection from infection indicates the ability of the animals to generate antibodies that protect them from infection. Further, use of the animal models allows identification of antigens that activate cellular immunity.
In animal model studies, a protective immune response in response to challenge by a bacterial S preparation (e.g., infected serum) (i) protects the animal from infection or (ii) prevents manifestation of disease.
Vaccines can be prepared from one or more of the immunogenic polypeptides identified herein. Numerous H. pylori polypeptides of the invention (e.g., spot 15) have been shown to be extremely antigenic, i.e., they react strongly, with antibodies present in sera pooled from a number of confirmed H. pylori-positive donors. In a typical screening method, the intensity of color development is representative of the strength (binding affinity) of the antigen-anti-H. pylori antibody interaction.
Representative serum paneling results (Example 6) for proteins expressed by two of the clones, dHA22.8 (A22) and dHC1S.11 (C1), indicate that both of these recombinant proteins are highly immunogenic. Protein produced by clone dHA22.8 reacted with antibodies present in both of the H. pylori-positive pooled sera sources, Roost pool and SFA 001, and exhibited no cross reactivity with antibodies present in H. pylori-negative samples. Similar results were observed for protein produced by clone dHC1S.11 (C1).
In looking now at antigen reactivity with individual H. pylori-positive serum samples, antigenic protein expressed by each of the clones dHA22.8 and dHC1S.11 reacted with anti-H. pylori antibodies in 100 % and 95 % of the samples, respectively, indicating the ability of the antigens of the present invention to detect H. pylori infection, and to provide components for vaccines against H.
pylori .
Preferred antigens for use in vaccine compositions are described in Example 13. Exemplary antigens are those capable of invoking a long-lasting antigenic response, as evidenced by the persistant presence of antibodies whose titre remains high for an extended period of time subsequent antimicrobial treatment for H. pylori. On this basis, particularly preferred antigens for use in vaccine compositions include Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261Ains and Y146B.
Other peptides, among the unique H. pylori peptides disclosed herein, can be identified as useful in a vaccine for H. pylori as follows. The individual test peptide is formulated in a suitable carrier, e.g., adjuvant, at a concentration suitable for injection, e.g., 5-500 mg/ml. One suitable test animal for vaccination is the Rhesus monkey, as detailed in DuBois (DuBois, et al., 1996). The . animal is vaccinated, e.g., by oral, intramuscular or intravenous injection, in an amount typically between 0.2 to 2.0 mg/kg body weight. After a suitable period, e.g., 2 weeks, the animal may be given a booster by the same route and typically in the same amount. Two to three weeks later, the animal is challenged with H. pylori in a known manner, e.g., as described in DuBois (DuBois, et al., 1996). As an example, the inoculated animal is challenged with H. pylori (e.g., a suspension of approximately i0g-109 CFU of H. pylori, 1 ml) to test the potential vaccine effect of the specific immunogenic fragment. Following the challenge with-H. pylori, the subject is typically monitored S by endoscopy, histologic examination, microbiological methods, and/or measurement of H. pylori-specific plasma IgG, as described in DuBois (DuBois, et al., 1996).
The level of infection in the vaccinated animal is then compared with that of a control animal to assess the degree of protection. Those peptides which provide a measurable degree of protection against H. pylori infection are suitable for vaccine use, either alone or in combination with other peptide vaccine agents, such as the above noted dHA22.8 (A22), dHCIS.11 (C1) and spot 15 peptides.
The selected peptide is formulated according to known vaccine formulations.
Typically, the peptide is conjugated to a carrier protein, e.g. , keyhole limpet hemocyanin or human serum albumen, and/or suspended in a suitable adjuvant, such as Freund's adjuvant. The vaccine is administered by conventional routes, typically IM or IV routes, as above, at peptide levels preferably in the range of IS 0.2 to 2.0 mg/kg. If necessary, one or more booster injections is given.
The specificity of a putative immunogenic fragment can be assessed by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other related bacteria.
B. Synthetic Peptides Using the coding sequences of H. pylori polypeptide antigens disclosed herein, synthetic peptides can be generated which correspond to these polypeptides. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA).
Alternatively, oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea, 1989; Yoshio, et al., 1989; Eaton, et al., 1988).
Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis, et al., 1982; Ausubel, et al., 1988).
C. Immunoassays for H. oylori One utility for one, several, many, or each of the antigens herein is their use as diagnostic reagents for the effective and reliable detection of antibodies present in the sera of test subjects infected with H. pylori, to thereby provide an indication of infection in a test subject. Preferred antigens which can be employed either singly or in combination in such a method include, e.g., . T __ '_ _.._. ._______. ___T-._ antigens identified by or derived from SEQ ID N0:44 (A22), SEQ ID N0:39 (C1), SEQ ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID
NO:55 (B2), SEQ ID N0:61 (Y104B), or SEQ ID N0:573 (Y128D). Alternatively, preferred antigens are defined in terms of their DNA coding sequences, corresponding to or derived from SEQ iD N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ
ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), or SEQ
ID N0:98 (Y128D).
Alternatively, the antigen used for the detection of antibodies present in the sera of test subjects infected with H. pylori is encoded by a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (S), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), {34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (SO), (S1), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69), or immunoreactive variants thereof. Preferably the antigen-coding regions are the spanning sequences themselves from the clusters.
The antigens of the present invention can be used singly, or in combination with each other, in order to detect H. pylori, as illustrated by the results in Example 6B. The antigens of the present invention may also be coupled with diagnostic assays for other infectious agents.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention, e.g., the "spot 15" antigen.
Exemplary antigens are A22 and C1, which both show high sensitivity (as indicated in Figs. 65 and 66, and in Example 6B). After binding anti-H. pylori antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-H.
pylori antibody on the solid support. The reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate, e. g. , 5-bromo-4-chloro-3-indoyl-phosphate (BLIP) and nitroblue tetrazolium (NBT). (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material.
These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group binding to a chemically reactive group on the solid support, e.g., an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Also forming part of the invention is an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigen (e.g., antigens such as those described in Tables 3a and 3b, or encoded by the representative clones summarized in Table 2) or native H. pylori antigen (such as those identified in Table 6 and in Fig. 1 and Fig. 2, as above), and a reporter-labelled anti-human antibody for detecting surface-bound anti-H. pylori antigen antibody.
In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected. Known general types of homogeneous assays proposed heretofore include (a) spin-labelled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency or polarization, (c) enzyme reporters, where antibody binding causes enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents.
In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
The examining may involve attaching a labelled anti-human antibody to the subject antibody (for example from acute, chronic or convalescent phase) and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method. Also contemplated is an antigen capture assay as previously described in Section D.2.
The following examples illustrate, but in no way are intended to limit the scope of the present invention.
Materials and Methods 1. General Procedures Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased from commercial suppliers.
Standard molecular biology and cloning techniques were performed essentially as previously described in Ausubel, et al., 1988; Sambrook, et al., 1989; and Maniatis, et al., 1982.
Common manipulations relevant to employing antisera and/or antibodies for screening and detection of immunoreactive protein antigens were performed essentially as described in Harlow, et _ _ __ ~_ _ _._ ...____.. .. ~
al. (1988). Antibody screening of H. pylori genomic libraries was carried out by plaque immunoblot assay.
Similarly Western blot assays were performed either as described by their manufacturer (Abbott, N. Chicago, IL; Genelabs Diagnostics, Singapore) or using standard techniques known in the art (Harlow, et al, 1988).
2. Bacterial Strains Commercially available H. pylori strains corresponding to ATCC Designation Nos. 43504 (short antigen clone set) and 43526 (Libraries 1 and 2) were used to generate the DNA libraries. H.
pylori strain ATCC 43504 was used for isolation of native proteins produced by H. pylori.
Escherichia toll strains) Y1088, Y1089,and XLI-Blue for libraries 1 and 2 (Stratagene, La Jolla, CA) was the host used for phage infection. E. toll strains XL1-Blue, XLOLR
(Stratagene, La Jolla, CA) were used for protein expression of cloned genes.
Example 1 Construction of H pylori Lambda gtl l and ZAPII DNA Libraries 1. Isolation of Genomic DNA
H. pylori (American Type Culture Collection, Rockville, MD; ATCC Designation Nos.
43504) was streaked on blood agar plates and incubated in a microaerophile environment at room temperature for 7 days. Cells were harvested by scraping the bacterial cells from 10 plates, followed by washing once with phosphate buffered saline (Dulbecco's, Gibco BRL, Gaithersburg, MD).
Genomic DNA was prepared as described in Ausubel et al. 1988, with minor modifications.
Cell pellets from 5 plates were resuspended in 510 ~.1 of TE (Ausubel et al. , 1988), to which was added 60 ~1 of 10 % SDS and 30 /d of 20 mg/ml of proteinase K. The suspension was mixed, followed by incubation for a period from 4 - 8 hours at 37°C. To the suspension was added 80 ~,1 of CTAB/NaCI (10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCI), and the resulting solution was then mixed and incubated for 10 minutes at 65°C. The solution was then extracted with an equal volume of chloroform/ isoamyl alcohol and spun in a microcentrifuge for 5 minutes. The separated aqueous phase was transferred to a new tube, and the DNA was precipitated by addition of 0.6 volumes of isopropanol, followed by centrifugation. The DNA pellet was washed with 70 %
ethanol, dried briefly under vacuum and solubilized in 100 /d of distilled water. The DNA solution was then treated with DNase-free Rnase (Boehringer Mannheim, Indianapolis, IN) (Ausubel, et al., 1988; Maniatis, et al., 1982) to selectively degrade any RNA present in the sample.
WO 98/49314 ' PCT/US98/08487 2. DNase Digestion and DNA Amplification a. Short Antigen Clone Libraries. H. pylori genomic DNA as described above was digested with pancreatic DNase I (Boehringer Mannheim) essentially as described in Ausubel, et al. (1988) and Sambrook, et al. (1989). Aliquots of the digested DNA were taken at various time points. The DNA digests were resolved by preparative agarose gel electrophoresis. Product bands containing the desired size range of DNA (200-2000 base pairs) were excised from the gel, and recovered using the "GENE CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID" kit (Bio 101 Inc., La Jolla, CA), according to the manufacturer's instructions.
To generate blunt-ends, the recovered DNA fragments were incubated with E.
coli Klenow fragment of DNA polymerise (Ausubel, et al., 1988; Sambrook, et al., 1989).
The reaction mixture was incubated at room temperature for 30 minutes, followed by extraction with phenol/chloroform.
The resulting molecules were ligated to Sequence Independent Single Primer Amplification (SISPA; Reyes, et al., 1991) linker primers according to the method of Reyes (Reyes, et al., 1991).
To the blunt-ended DNA were added the following: phosphorylated SISPA
(Sequence-Independent Single Primer Amplification) linker AB, a double strand linker comprised of SEQ ID N0:63 and SEQ
ID N0:64, where SEQ ID N0:64 is in a 3' to 5' orientation relative to SEQ ID
N0:63 as a partially complementary sequence to SEQ ID N0:63, 2 ~.l 10 x ligation buffer (0.66 M
Tris.Cl pH = 7.6, SO mM MgCl2, 50 mM DTT, 10 mM ATP) and 1 ~,I T4 DNA ligase (0.3 to 0.6 Weiss Units).
Typically, the DNA and linker were mixed at a 1:100 ratio. The reaction was incubated at 14°C
overnight. The reaction was then incubated at 70°C for 3 minutes to inactivate the Iigase. Unligated linkers were removed by gel filtration using a Chromaspin column (Clonetech, Mountain View, CA) or a Sephadex G spin column (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions.
The linker ligated DNAs were then amplified by SISPA (Reyes, et al., 1991). To 100 /d of 10 mM Tris-Cl buffer, pH 8.3, containing 1.5 mM MgCl2 and 50 mM KCl (Buffer A) was added about 1 ~.1 of the linker-ligated DNA preparation, 2 /cM of a primer having the sequence shown as SEQ ID N0:63, 200 ~,M each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of Amplitaq DNA
polymerise (Applied Biosystems Division, Perkin Elmer, Foster City, CA). The reaction mixture was heated to 94°C for 30 seconds for denaturation, allowed to cool to 50°C for 30 seconds for primer annealing, and then heated to 72°C for 0.5-3 minutes to allow for primer extension by Taq polymerise. The amplification reaction, involving successive heating, cooling, and polymerise reaction, was repeated an additional 25-40 times with the aid of a Perkin-Elmer Cetus DNA thermal cycler (Mullis, 1987; Mullis, et al., 1987; Reyes, et al., 1991; Perkin-Elmer Cetus, Norwalk, CT).
After the amplification reactions, the solution was then extracted with phenol/chloroform and precipitated with two volumes of ethanol.
__ _~ ~ .____ _._.._ WO 98/49314 ' PCT/ITS98/08487 3. Cloning of the DNA into Lambda Vectors a. Short Antigen Clone Libraries. The linkers used in the ligation to the DNA
contained an EcoRI site which allowed for direct insertion of the amplified DNAs into the lambda vectors (gtll or ZAP II, Stratagene, La Jolla). The lambda vectors as purchased from the S manufacturer had been digested with EcoRI and treated with alkaline phosphatase to remove the terminal S' phosphate and prevent self ligation of the vector. The amplified DNAs from 1.B. were digested with EcoRI and short nucleotides were removed by gel filtration. The digested DNA
preparations were then ligated into lambda gtll or "ZAPII" using T4 DNA iigase (Boehringer Mannheim, Indianapolis, IN). The conditions of the ligation reactions were as follows: 1 /d vector DNA (Stratagene (La Jolla, CA) O.S mg/ml); O.S or 3 ~.1 of the PCR amplified insert DNA; O.S ul 10 x ligation buffer (O.S M Tris-HCI, pH = 7.8; 0.1 M MgCl2; 0.2 M DTT; 10 mM
ATP; O.S
mg/ml bovine serum albumin (BSA)), O.S ~,1 T4 DNA ligase (0.3 to 0.6 Weiss units) and distilled water to a final reaction volume of S wl. The ligation reactions were incubated at 14°C overnight (12-18 hours). The ligated DNA was packaged by standard procedures using a lambda DNA packaging 1S system (GIGAPAK, Stratagene, La Jolla), and then plated at various dilutions to determine the titer.
The titer of the DNA-insert phage libraries and percent recombination were determined by a standard X-gal blue/white assay (Miller, 1994; Maniatis et al., 1982).
Typically, the titer of the recombinant libraries ranged from l.S x 104 to 3 x 106 PFU/ml.
Percent recombination in each library can also be confirmed by selecting a number of random clones and isolating the corresponding phage DNA. Polymerise chain reaction (Mullis, 1987; Mullis, et al., 1987) is then performed using isolated phage DNA as template and lambda DNA sequences, derived from lambda sequences flanking the EcoRI insert site for the DNA
molecules, as primers.
The presence or absence of insert is then evident from gel analysis of the polymerise chain reaction products.
b. Lambda ZAPII Libraries (Libraries 1 and 2). The lambda ZAPII libraries (generated from H. pylori sample ATCC No. 43526) were similarly prepared with the following exceptions. The ZAPII libraries were generated from genomic DNA which had not undergone Sequence Independent Single Primer Amplification.
Library 1 clones (designated herein by upper case letters) were generated by ligating EcoRI-digested H. pylori DNA directly into the EcoRI sites of the lambda "ZAPII"
vector. Library 2 clones (lower case designations) were obtained by digesting H. pylori genomic DNA
with HindIII, blunt-ending the HindIII-fragments, and ligating the resulting molecules to SISPA
primers AB as described above. The linker-ligated DNAs were then treated with EcoRI and ligated into the EcoRI sites of 3S ZAPII lambda arms.
Unless otherwise indicated, the DNA-insert gtll and ZAPII phage libraries generated from H. pylori samples ATCC Designation No. 43504 and ATCC No. 43526 have been deposited in the Genelabs Technologies, Inc. Culture Collection, 505 Penobscot Drive, Redwood City, CA, 94063 or in the Genelabs Diagnostics PTE LTD Culture Collection, 85 Science Park Drive X04-Ol , The Cavendish, Singapore Science Park, Singapore 118259.
Example 2 ImmunoscreeninQ of Recombinant Libraries The lambda gtl l and ZAPII phage libraries described in 1.C. above were immunoscreened for the production of antigens recognizable by a pool of sera from 11 patients (designated herein as "Roost pool" sera) identified as H. pylori-positive using endoscopy, or by another pool of 4 H. pylori immunopositive sera samples identified as SFA001. The lambda short antigen clone library and ZAPII
library 2 were immunoscreened against Roost pool sera; the ZAPII library 1 was immunoscreened against sera pool SFA001. The lambda gtl l libraries were plated for plaque formation using E.
coli Y1089 bacterial plating strain, while the lambda ZAPII libraries were plated for plaque formation using E. coli XLl-Blue bacterial plating strain.
The fusion proteins expressed by the recombinant lambda phage clones were screened with serum antibodies essentially as described by Ausubel, et al. (1988).
Each library was plated at approximately 1.5 to 2 x 10' phages per 150 mm plate. Plates were overlaid with nitrocellulose filters overnight. Filters were washed with TBS (10 mM, Tris pH
7.5; 150 mM NaCI), blocked with AIB (TBS buffer with 1 % gelatin) and incubated with a primary antibody diluted 100 times in AIB.
After washing with TBS, filters were incubated with a second antibody consisting of goat-anti-human IgG conjugated to alkaline phosphatase at a concentration of 1:1000.
Reactive plaques were developed with a substrate (for example, BCIP, 5-bromo-4-chloro-3-indolyl-phosphate), with NBT
(nitro blue tetrazolium salt; Sigma Chemical Co., St. Louis, MO). Positive areas from the primary screening were replated and immunoscreened until pure plaques were obtained.
The results of the screening are presented in Tables la and lb.
Table la Roost Pooled Sera SerutstEndoscopy Culture Pathology (Gastritis) () A1 Mild antrum gastritis,Moderate Acute/chronic/moderate deep duodenal ulcer A4 None Heavy Acute/chronic/severe with prior ulcer A8 Mild annum gastritis,Moderate Chronic/mild moderate duodenitis Al l Mild antrum and Heavy Chronic/moderate body gastritis B 1 Moderate antrum Heavy Chronic/severe with gastritis, moderate prior Hp ( > 4 duodenitis years with therapy) 9 Deep gastric ulcerNA NA
14 Moderate antrum Moderate Acute/mild gastritis Gastritis in anttumHeavy Acute/chroniclsevere and body with prior ulcer 16 Mild anttvm gastritis,Moderate Chronic/moderate deep gastric ulcer 19 Superficial gastricRare Chronic/moderate and duodenal ulcers 15 20 Mild antrum gastritisModerate Acute/chronic NA = not available The above sera have all been tested with HelicoBlot 2.0 (Genelabs Diagnostics Western blot product) and were found to be positively infected samples using the criteria of the product.
SFA001 is a pool of 4 donor sera (plasma packs) showing strong reactive bands on Western blot format with a crude lysate antigen preparation from Helicobacter lysate using in HelicoBlot 2Ø
NAA121 is a pool of 4 donor sera (plasma packs) showing no reactive bands on Western blot format with the crude lysate antigen preparation from Helicobacter lysate used in HelicoBlot 2Ø
Table lb The lambda ~~tll and ZAPII libraries # Screenedfi Clones Library % Recomb.Antibody pfu Plaque-Purified gtll Y 90-95% Roost pool2 x I05 21 EcoRI/XbaI94% 1Gb monoclonal2 x 10' 1 antibody against H. pylori ZAPII Z 90-95 Roost pool2 x 105 300 %
library 99% SFA001 3.9 x 52 library 98 % Roost pool9 x 10' 86 OTALS: > 462 Example 3 Characterization of Immunoreactive Lambda gtll Z and Lambda ZapII Y library Clones 1. PCR Amplification. Purification and Sequence Determination for DNA Inserts of Immuno-reactive Recombinant Lambda Clones (Short Antigen Clone Set H pylori Cioned Families Y and Z) The specific antigen coding sequences from H. pylori cloned families were isolated by PCR
amplification of representative clones. Cloned sequences having an .ASM
extension were sequenced completely; sequences with a .SEQ extension were partially sequenced.
The DNA inserts of the immunoreactive recombinant lambda clones were PCR
amplified using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors.
Amplification was carried out by polymerase chain reactions utilizing each immunoreactive clone as template. For the lambda gtl l clones, gtllF (SEQ ID N0:65) and gtl l R (SEQ
ID N0:66) primers were used. For the lambda ZAPII clones, primers T3 (SEQ ID N0:67) and T7 (SEQ
ID N0:68) were used.
The resulting amplified fragments were then agarose gel purified and eluted from the gel (Ausubel, et al., 1988). The PCR products were further purified by "WIZARD PCR
PREPS"
(Promega, Madison, WI) or "CHROMASPIN" columns (Clonetech, Mountain View, CA) to remove primers and other ingredients. The purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced.
Sequence determination for the DNA inserts was carried out using a Perkin Eimer Applied Biosystems 373A DNA sequencer (Perkin Elmer, Applied Biosystems Division, Foster City, CA) according to the manufacturer's protocol (dideoxy chain terminator sequencing methodology. Sanger, et al. , 1977).
Sequence data is presented in the accompanying Sequence Listing. Table 2 below presents a partial summary of recombinant H. pylori nucleotide sequences encoding immunogenic proteins, i. e. , proteins shown to be reactive with H. pylori-positive sera, corresponding to cloned families Y and Z.
The EcoRI site of the linkers has typically been deleted for the corresponding sequences presented in the figures.
Table 2 Clone SEQ InsertSequencedCluster No.
ID (N'I~ (NT) NO:
Y103-1T3.SEQ70 -900 250 57 Y103-1T7.SEQ71 -900 220 57 Y103-2.ASM72 -800 820 57 Y104-1.ASM73 1080 3 YIOS.ASM 74 730 55 T3. SEQ
Y107-1T7.SEQ76 -1300 310 4 Y108-1.ASM77 790 32 Y109-1T3.SEQ78 -400 205 23 Y109-1T7.SEQ79 -400 230 23 Y111.ASM 80 1565 19 Y114-2T3.SEQ81 -900 205 51 YI14-2T7.SEQ82 -900 220 15 Y115-1T3.SEQ83 -800 205 28 Y115-1T7.SEQ84 -800 270 28 Y116-1.ASM85 510 28 Y1I7-2T3.SEQ86 -100 195 37 Y117-2T7.SEQ87 -1000 235 37 Y119-1T3.SEQ88 -600 225 33 - Y119-1T7.SEQ89 -600 245 33 Y123-IT3.SEQ92 -1300 265 35 Clone SEQ InsertSequencedCluster No.
m (NT) (NT) NO:
Y123-iT7.SEQ93 -1300335 35 Y124-2T3.SEQ94 ~--60085 14 Y124-2T7.SEQ95 -600 280 14 Y126-2T3.SEQ96 -1100278 58 S Y 126-2T7.97 -1100290 58 SEQ
Y128.ASM 98 930 20 Y 12T3. 99 -1000245 10 SEQ
Y12T7.SEQ 100 --1000305 10 Y130-1T3.SEQ101 -1000270 35 Y130-1T7.SEQI02 -1000195 35 Y131-1T3.SEQ103 --800275 37 Y131-1T7.SEQ104 -800 285 37 Y 132-2T3.105 -1100240 59 SEQ
YI32-2T7.SEQ106 -1100265 59 Y133.ASM 107 410 60 Y 135. 108 540 29 ASM
Y136-1.ASM109 380 61 YI39.ASM 110 685 13 Y13T3.SEQ 111 -800 230 62 Y13T7.SEQ 112 -800 290 62 Y140-IT3.SEQ113 -700 215 12 Y140-1T7.SEQ114 -700 240 12 Y141.ASM 115 750 6 Y 143-2T3.116 - 240 13 YI43-2T7.SEQ117 --800290 13 Y144.ASM 118 615 36 Y145-2T3.SEQ119 -700 220 28 Y146-I.ASM120 870 28 Y 147. 121 1025 51 ASM
Y150-2.ASM122 375 32 Y151-2T3.SEQ123 -700 290 Y151-2T7.SEQ124 -700 325 ~. _.._. ___.__.._ _.
WO 98/49314 , PCT/US98/08487 Clone SEQ InsertSequencedCluster No.
>D (N'I~ (NT) I
NO:
Y152-2.ASM125 615 20 Y153.ASM 126 1030 37 Y 154-2T3.127 - 800 265 4 SEQ
Y154-2T7.SEQ128 -800 305 4 Y160-1T3.SEQ129 -1000 260 12 T7. SEQ
Y163.ASM 131 610 38 Y16T3.SEQ 132 -1000 240 51 Y16T7.SEQ 133 -1000 310 51 Y173.ASM 134 765 10 Y175.ASM 135 770 2 Y176-1.ASM136 425 28 Y178-1T3.SEQ137 -900 300 18 Y178-1T7.SEQ138 --900 265 18 Y180.ASM 139 810 29 Y184-IT3.SEQ140 --600 230 16 Y184-IT7.SEQ141 -600 290 16 Y187-2.ASM142 480 23 Y190.ASM 143 760 39 YI94-2T3.SEQ137 -600 300 18 Y194-2T7.SEQ144 -600 240 18 Y195-1.ASM145 540 21 Y196-1.ASM146 425 52 Y19T7.SEQ 147 --1100320 2 Y1T3.SEQ 148 -500 295 53 YIT7.SEQ 149 -500 235 53 Y200-I.ASM150 500 31 Y203T3.SEQ151 - 700 230 38 Y203T7.SEQ152 -700 230 38 Y208T3.SEQ153 -800 260 15 Y208T7.SEQ154 -800 270 15 Y20T3.SEQ 155 -1100 320 11 Clone SEQ InsertSequencedCluster No.
ID (NT) (N'I~
NO:
Y20T"l.SEQ156 -1000315 11 Y212T3. 157 - 260 14 Y212T7.SEQ158 ~700 285 14 Y220T3. 159 -1000220 10 SEQ
S Y220T7.SEQ160 -1000260 10 Y221 T3. 161 -- 255 2 Y221T7.SEQ162 - 240 2 Y224.ASM 163 840 29 Y237.ASM 164 390 26 Y238T3.SEQ165 -1100200 40 Y238T7.SEQ166 -1100220 40 Y23T3.SEQ 167 -1100195 51 Y23T7.SEQ 168 - 320 51 Y241.ASM 169 685 4 Y247.ASM 170 490 36 Y250T7.SEQ171 -1200215 41 Y261T3.SEQ172 -700 270 4 Y261T7.SEQ169 -600 4 Y264T7.SEQ173 -500 215 31 Y268T3.SEQI74 -800 245 27 Y269T3.SEQ175 -900 220 5 Y269T7.SEQ176 ~900 240 5 Y26T3.SEQ 177 -1400140 43 Y26T7.SEQ 178 -1400230 43 Y270T3.SEQ179 ~ 280 43 Y270T7.SEQ180 -1300255 43 Y28T3.SEQ 181 - 330 17 I
Y28T7.SEQ 182 --1100330 17 Y29.ASM 183 500 31 Y30.ASM 184 545 33 Y33T3.SEQ 185 -1200335 45 Y33T7.SEQ 186 -1200345 45 ___. __ T __ _._._ __ . _ _T.
Clone SEQ InsertSequencedCluster No.
ID (NT) (1VT) NO:
Y37.ASM 187 490 32 I
Y3T3.SEQ 188 - 250 37 Y40T3.SEQ 189 -1200355 48 Y40T7.SEQ 190 - 330 4$
Y42T3.SEQ 191 - 295 12 Y42T7.SEQ 192 - 355 12 Y43T3.SEQ 193 -900 265 29 Y43T7.SEQ 194 --900310 29 Y46T3.SEQ 195 -900 280 45 Y46T7.SEQ 196 -900 290 45 Y47T3.SEQ 197 -800 250 10 Y47T7.SEQ 198 -800 290 10 Y48.ASM 199 490 31 Y49T7.SEQ 200 --900335 49 Y53T3.SEQ 201 -1200230 11 Y53T7.SEQ 202 -1200320 11 YSS.ASM 203 610 13 Y56T3.SEQ 204 -700 220 37 Y56T7.SEQ 205 -700 290 37 Y60T3.SEQ 206 --1300265 7 Y60T7.SEQ 207 -1300290 7 Y61T3.SEQ 208 --800265 42 Y61T7.SEQ 209 -800 240 42 Y69T7.SEQ 200 -500 335 49 Y73T3.SEQ 210 -- 285 50 I
Y73T7.SEQ 211 - 285 50 YSI.ASM 212 550 23 Y82T3.SEQ 213 -700 230 13 Y82T7.SEQ 214 -700 325 13 Y84T3.SEQ 215 -1100265 42 Y84T7.SEQ 216 -1100275 42 Y87.ASM 217 660 42 Y89T3.SEQ 218 -1200260 2 Clone SEQ InsertSequencedCluster No.
ID (NT~ (NT) NO:
Y89T7.SEQ 219 -1200 280 ' 2 Y91 T3. 220 - 800 250 54 i SEQ
Y91T7.SEQ 221 -800 325 54 Y92T3.SEQ 222 --1300235 1 Y92T7.SEQ 223 -1300 310 1 Z14.ASM 224 970 30 Z21.ASM 225 280 39 Z24.11f.SEQ226 195 23 Z25.ASM 227 1185 6 Z3.ASM 228 1560 51 z4l.ASM 229 518 Z9.ASM 230 525 31 Clone Y104-1 (SEQ ID N0:60) contains the entire d7 clone, and encodes all of the 36K
peptides and all of the spot 15 peptides, as indicated in Fig. 3.
The production of expressed antigenic proteins and their subsequent purification is generally described in Examples SA and SB. A tabular summary indicating clone name, expression, purification, and panelling data is provided in Table 3b, and a summary of the immunoreactivy of various recombinant antigens is provided in Table Sb. Expression profiles were obtained, and the immunoreactivity of various clones to H. pylori positive pooled sera such as "Roost" was confirmed.
Briefly, amplified products corresponding to a particular ORF were typically cloned into a pGEXhisB vector and expressed in E. toll. The size of the expression product was then determined, followed by confirmation of immunoreactivity (e.g., with Roost pool sera).
2. Sequence Comparison Sequences were compared with "GENBANK" (versions 1998, 1996), EMBL database and dbEST (National Library of Medicine) sequences at both nucleic acid and amino acid levels. Search programs FASTA, BLASTP, BLASTN and BLASTX (Altschul, et al., 1990) indicated that most of the antigen-coding polynucleotide sequences disclosed herein were not greater than 95 % identical to any sequence contained in the above-mentioned public databases prior to the publication of the genome of H. pylori (Tomb, et al., 1997).
Example 4 Characterization of Immunoreactive Lambda Library 1 and Library 2 Clones 1. Subclonin~, Purification Identification and Sequence Determination for H
pylori Anti ens Additional immunopositive clones, as described in Example 2 above, were purified and analyzed for DNA insert size and expressed protein size as in Example 3 above.
The ZAPII clones were rescued into plasmids from phagemids (as per protocol in Stratagene, La Jolla, U.S.A.); the resulting insert DNA was excised with EcoRI and ran on agarose gel to determine size. Slightly longer versions of T3 and T7 (GD 60 and GD 61, corresponding to SEQ ID N0:231 and SEQ
ID N0:232 respectively) were then used as primers for sequencing. These libraries correspond to the "library 1"
series (EcoRI-cut), i.e., clones A3, A22, B2, B9, B17, B23, C1, C3, C7, and "library 2" series clones (HindIII cut), i.e., clones al, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, d7, dll, fll, e6, f3, f8, g2, g9, gl l, and k4; and also clone Gla from the EcoRIlXbaI cut library. Many clones were determined to be larger than the predicted coding regions of the proteins.
The specific antigen coding sequences from H. pylori library 1 and library 2 clones were subcloned. The subclones were typically prepared by fragmenting the corresponding genomic clone by specific restriction endonuclease digestion to produce a specific subfragment or subfragments, which were purified using "GENE CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID" kit (Bio 101 Inc., La Jolla, CA). The resulting DNA fragments were then inserted into a suitable expression vector, i.e., the PB/Bluescript (SK) vector (Stratagene, La Jolla, CA). Expression clones were induced with 0.5 mM IPTG (isopropylthio-beta-D-galactoside) for 4 hours at 37 degrees C, and whole bacterial cell lysate was run on SDS gel. Immunoreactivity of the expressed proteins was confirmed using Roost, SFA001 and NAA001 pooled sera. Alternatively, original genomic clones were subjected to nested deletion using the "Erase-a-Base"" kit (Promega, Wisconsin, U.S.A.), and the resulting smaller nested clones were tested for immunoreactivity to locate the position of the coding antigen.
Immunoreactive DNA regions were then sequenced and locations of the open reading frames were determined. Unless otherwise indicated as a /3-galactoside fusion product, for each of the clones in Tables 3a and 3b below, expression of coding regions was determined to be driven by the corresponding H. pylon promoter rather than by the /3-galactosidase gene promoter in lambda gtll.
WO 98/49314 ' PCT/US98108487 2. Clone A3 Subclone A3CON3, originating from a genomic clone having a size of approximately 6.0 kb, was obtained from nested deletion clones. The corresponding nucleotide sequence (1878 bp) is presented herein as SEQ ID N0:16. The open reading frame (ORF) extends from nucleotides 399-1743, and codes for a putative protein containing 448 amino acids (443 amino acids when calculated from the first methionine). The protein sequence corresponding to the translation of open reading frame (ORF) 399-1743 is presented herein as SEQ ID N0:17.
3. Clone A22 Subclone A22210DMIC was obtained from an original genomic clone having an insert size of about 2.2 kb, to produce a /3-galactosidase fusion protein in E. coli. As indicated in Tables 3a and 3b below, the open reading frame (ORF) extends from nucleotides 12-599 of SEQ
ID N0:43, where bases 12-121 correspond to the R-galactosidase fusion peptide and bases 122-599 code for a unique A22 antigenic sequence. The translation sequence for the corresponding protein is presented as SEQ
ID N0:44.
4. Clone B2 B3C19 is a subclone obtained from the 5.0 kb insert in genomic clone B3 as follows. The insert was excised, digested with DNase, followed by treatment with T4 DNA
ligase and Kienow enzyme to produce blunt-ends. The blunt-ended short DNA pieces were then ligated to kinased A/B
linkers (linker A, SEQ ID N0:63, corresponding to the top strand of AB SISPA
linker; linker B, SEQ
ID N0:64, corresponding to the bottom strand of AB SISPA linker), PCR
amplified with primer A, and the amplified products digested with EcoRI. The digested products were then ligated into EcoRI-digested lambda vector ZAPII. The DNA sequence of B3C19 is presented as SEQ ID
NO:51.
Based upon the sequence of B3C19, primers GD77 (5' primer, SEQ ID N0:52) and (3' primer, SEQ ID N0:53) were designed to walk back and forward the sequence of B3C19 using genomic B2 DNA as the template. The sequence of clone B3C19 was extended in both the S' and 3' directions, resulting in B2 extension clone, B2197780 (SEQ ID N0:54).
A computer-generated protein translation of the B2197780 sequence resulted in a corresponding 291 amino acid putative protein, with a predicted transmembrane segment from amino acids 9-25 (predictions obtained using "SOAP" program from "PCGENE"). Based upon the results of the analysis, a Shine Dalgarno AGGA sequence was determined to be situated 8 b.p. in front of an ATG codon. This ATG codon at nucleotide position 278 appears to be the first "met" amino acid of the gene. The translated protein sequence for clone B2 is presented as SEQ
ID NO:55. The WO 98/49314 ' PCT/IJS98/08487 predicted molecular weight of full size protein is 31,682. There are 2 potential cleavage sites between (i) amino acids 91 and 92; and (ii) between amino acids 25 and 26 ("PCGENE" --Prediction of prokaryotic secretory signal sequence). The pI of the protein is predicted as 8.3.
5. Clone B9 Subclone B9.4C is a 1.2 kb HindIII fragment obtained from original genomic clone B9 (4.5 kb insert) as follows.
Induction of genomic clone B9 produced an immunoreactive protein with sizes of 32 kd and 14 kd in SFA001 pooled sera and 14 kd in Roost pooled sera. B9 was digested with HindIII to form several HindIII subfragments, which were each subcloned into the pBKS vector.
Protein production was induced in the resulting subclones, and the sizes of the corresponding proteins were determined.
Subclone B9.4C, corresponding to a 1.2 kb HindIII subfragment of genomic clone B9, produced an immunoreactive protein of 32 kd. The nucleotide sequence of subclone B9.4C is presented as SEQ ID N0:47; the translated protein sequence (i.e., nucleotides 230-931 of SEQ ID
N0:47) is presented as SEQ ID N0:48. Referring to SEQ ID N0:47, an AGGA Shine-Dalgarno sequence occurs at nucleotide 218, and the first amino acid of the translated protein sequence begins with a GTG codon for valine at position 230. Based upon PCGENE calculations (CHARGPRO
program) as described above, the predicted molecular weight of the corresponding protein is 25.2 kd, with a pI of 10.35. A potential cleavage site occurs between amino acid positions 225 and 226.
Based upon a FASTA sequence identity analysis of both nucleic acids and proteins, clone B9 appears to code for the SOA L1 protein of H. pylon.
6. Clone BI7 B17CON4 (2006 bases) was subcloned from a genomic clone having an insert size of about 4.0 kb. The corresponding nucleotide sequence is presented as SEQ ID N0:24.
The open reading frames (ORF) correspond to the following regions of SEQ ID N0:24: ORF1 (nucleotides 500-700);
ORF2 (nucleotides 870-1406); ORF3 (nucleotides 1410-2000); and ORF4 (nucleotides 142-705). The corresponding translated protein sequences are presented herein as SEQ ID
N0:25 (B170RF4), SEQ
ID N0:26 (B170RF1), SEQ ID N0:27 (B170RF2) and SEQ ID N0:28 (B170RF3). Based upon nested deletion experiments, it appears that the immunoreactive protein corresponds to B170RF4.
On the basis of computer-generated protein translation analysis as previously described, B 170RF4, B 170RF2 and B 170RF3 encode putative proteins having predicted sizes of 20.9 kd, 20 kd and 22.2 kd respectively. The predicted size of the immunoreactive genomic protein is a doublet of 22 kd and 23 kd.
7. Clone B23 Immunoreactivity was determined to reside in a 3.5 kb PstI subclone {one PstI
site is derived from vector pBSK) of original genomic clone B23 (5.5 kb). The 3.5 kb subclone was further sequenced, to determine the immunoreactive coding sequence presented in SEQ ID
N0:13. The nucleotide 1078 base pair sequence (SEQ ID N0:13) was determined to be open all the way (bases 3-1076). The translated protein sequence is presented as SEQ ID N0:14.
8. Clone C 1 C1CON6V2 was subcloned from an original genomic clone having an insert size of about 4.0 kb. The sequence information obtained from exo-mung deletion clones is presented as SEQ ID N0:38.
The open reading frame (ORF) extends from nucleotides 868-1926. The subcloning of the antigen coding sequence from the C1 clone into an expression vector, and characteristics of the corresponding protein product are presented in Example 5 below. The translated protein sequence is presented herein as SEQ ID N0:39.
9. Clone C3 To determine the immunoreactive coding region of original genomic clone, C3 (2.9 kb insert size), several different subfragments of the genomic clone were obtained and sequenced. The immunoreactive clone was determined to reside in an EcoRI/Kpn 2.2 kb subclone.
Based upon a number of subcloning experiments and subfragments, the C3 DNA sequence is presented as SEQ ID
NO:15.
10. Clone C7 Clone C7.2C is a EcoRIlHindIII subclone obtained from genomic clone C7. The original genomic C7 clone is a EcoRI clone of approximately 3.8kb size that produces an immunoreactive protein having a molecular weight of approximately 30 kd.
Subclone C7.2C was obtained as follows. The genomic C7 clone was digested into individual fragments using HindIII. Each DNA restriction fragment was then subcloned into the pBKS vector via either the HindIII site, or alternatively, for end-piece fragments, via the EcoRIIHindIII sites. The corresponding nucleotide sequence of C7.2C (616 base pairs) is presented as SEQ ID N0:20.
Subclone C7.2C produces an immunoreactive protein which is the same size as that produced by the genomic clone. The translated protein sequence for bases 1-561 of clone C7.2C
is presented herein as SEQ ID N0:21.
11. Clone B8 _. . T _. ~ ~_.__~_.__ T
Clone B8 (SEQ ID N0:549) is a genomic clone with an insert size of 3.5 kb that produces an immunoreactive protein of about 50 kd on SDS Western blot. The DNA sequence of B8 overlaps with that of clone C7 (clone C7.2C) which was a fusion protein with beta-galactosidase. Clone B8 added on 266 additional amino acids 5' (upstream) to C7 and contains the beginning of the gene.
However, B8 ends 64 amino acids before the C-terminus of the gene, which is also encoded by C7.
Based on the above, the complete sequence of the full gene was compiled (SEQ
ID N0:549). The corresponding protein sequence from the compiled gene sequence codes for a putative protein of 48.9 kd, and is presented herein as SEQ ID NO:550.
I2. Clone a1 Clone al is a HindIII clone that produces an inununoreactive protein of 28-30 kd on SDS gel.
The corresponding DNA sequence encoding an antigenic protein contains 1208 nucleotides (SEQ ID
N0:35). There are two open reading frames.
ORF1 extends from bases 53 - 801 of SEQ ID N0:35, and encodes a putative protein containing 249 amino acids (SEQ ID N0:36, translated protein). ORF1 contains a Shine-Dalgarno sequence extending from nucleotides 43-46 ("AGGA" sequence). The predicted molecular weight of the protein is 27.5 kd, which is in agreement with the expected protein size based upon SDS-PAGE
(above). The putative protein for ORF1 has a calculated pK of 8.42 and a predicted transmembrane region extending from amino acids 1-19.
The second ORF contained in clone al extends from nucleotides 880-1206 and ends at one of the HindIII sites, which indicates that clone al is a partial clone. The predicted size of the partial protein is 109 amino acids (SEQ ID N0:37).
13. Clone a3 Immunoreactive clone a3 (not to be confused with clone A3 from library 1) is a (3-galactosidase fusion clone with an insert size of 1975 base pairs. The corresponding nucleotide sequence corresponds to SEQ ID N0:248. The clone contains two open reading frames (ORFs):
ORFI (nucleotides 3-608, SEQ ID N0:249) and ORF2 (nucleotides 613-1266, SEQ ID
N0:250).
Based upon the observed size of the immunoreactive protein, i.e., 30 kd, the expected immunoreactive-expressing open reading frame is ORFl . Expression of a3 protein is described in the following section.
14. Clone a5 Clone a5 is an original HindIIIlEcoRI genomic clone (I kb) that produces a 25 kd im-munoreactive protein. The corresponding nucleotide sequence is presented as SEQ ID N0:3. The clone contains two open reading frames, (ORFs): ORFI (nucleotides 3-545, SEQ
ID NO:S) which codes for a beta-galactosidase fusion protein, and ORF2 (nucleotides 569-1021, SEQ ID N0:4). A
Shine Delgarno sequence occurs at nucleotides 559-562 of ORF 2. Based upon the observed size of the immunoreactive protein, i. e. , 28 kd, the expected immunoreactive expressing open reading frame is ORF 1.
15. Clone b5 Clone b5 contains a double insert with an internal EcoRI site. The individual inserts are about 0.3 and 0.2 kb in length. The combined nucleotide sequence for clone b5 corresponds to SEQ ID
N0:6, with the open reading frame (ORF) extending from nucleotides I through 414. The corresponding protein sequence translation is presented as SEQ ID N0:7.
16. Clone c2 Clone c2 is a library 2 clone with an insert size of 2077 nucleotides (SEQ ID
N0:25I). The size of the immunoreactive protein on SDS-gel is 30kd. There is one ORF
extending from nucleotides 3-877, with a possible initial start codon at nucleotide 45. The corresponding protein sequence translation is presented as SEQ ID N0:253. The protein has a predicted pI of 9.54, with a potential transmembrane region between amino acids 2 through 24. The predicted molecular size of this c2 protein is 30.2kd, which agrees with the observed size on SDS gel.
17. Clone c5 Clone c5 is a 650 base pair HindIIIIHindIII clone (SEQ ID N0:253). Clone c5 is a partial clone which is not in phase with the /3-galactosidase gene, and contains a 29 amino acid stretch extending into pBluescript at the N-terminus. The ORF extends from bases 2-604, with a predicted protein size of 23.Skd (SEQ ID N0:254). The observed protein size on SDS-gel is 30kd. The putative protein has a pI of 9.6.
18. Clone c13 Clone c13 is a HindIIIlHindIII clone with an insert size of 1742 b.p. (SEQ ID
N0:255). It is a ~-galactosidase fusion protein with an observed size of SSkd on SDS gel.
The ORF extends from bases 2 to 1420. The corresponding protein possesses a predicted molecular weight of 54.6kd (SEQ
ID N0:256). A sequence search using SWISS pro database indicates that the sequence possesses homologies with threonyl-tRNA synthetase of various organisms, from bacteria to yeast and human.
19. Clone d5 Clone d5 is an EcoRI clone that produces an immunoreactive protein (a-galactosidase fusion protein) of about 70 kd on SDS-PAGE gel. The size of the cloned insert was determined to be 1795 bases (SEQ ID N0:58), with an ORF from extending from nucleotides 1-1704. The open reading frame codes for a putative protein composed of 568 amino acids (SEQ ID N0:59), and having a predicted molecular weight of 62.1 kd. The putative protein has a predicted pK
value of 5.1, and the predicted antigenic determinant lies in the N-terminus region of the protein.
. A computer-based analysis was carried out using the "PROSITE" program on "PCGENE".
Based upon this study, several signature sites were identified within the protein, which included a cAMP and cGMP dependent protein kinase phosphorylation site and a ATP-GTP-binding motif A
(P-loop).
20. Clone d7 Clone d7 is a HindIII clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII as described above for Example 4.1. The product is a /3-galactosidase fusion protein. The nucleotide sequence is presented as SEQ ID
NO:11.
The induced protein was determined to have sizes of 40 kd and 35 kd. The corresponding protein sequence translated from SEQ ID NO:11 is presented as SEQ ID N0:12.
21. Clone f3 Clone f3 is a EcoRI clone having an insert size of 2274 nucleotides (SEQ ID
N0:257). Clone f3 produces a (3-galactosidase fusion protein. The ORF extends from nucleotides 1-1788 and codes for a putative protein having a predicted molecular size of 67 kd (excluding the a-galactosidase portion) (SEQ ID N0:258). The calculated pI of the protein is 9.66.
A sequence search against both the SWISS pro data base and the DNA data base reveals a 30% homology to penicillin binding proteins of Haemophilus influenza, E.coli, Bacillus subtilis and Streptococcus pneumoniae. A search against the EMBL DNA data base reveals a 56 % homology with penicillin binding proteins of Haemophilus influenza.
The size of the protein observed on SDS-gel is 25kd (major band), with a minor band of around 67kd. This size discrepancy may possibly be due to cleavage of the protein to produce a smaller fragment.
22. Clone f8 FBCON i is a HindIII subclone that produces an immunoreactive protein of 33-35 kd on SDS
gei. The DNA sequence corresponding to the f8 subclone is presented herein as SEQ ID NO:1. The cloned 1459 base DNA subfragment contains an open reading frame (ORF) from nucleotides 134 -1042.
The putative protein encoded by the f8 subclone contains 303 amino acids, with a predicted molecular weight of 33.9 kd (which is in agreement with the expected protein size). The predicted antigenic determinant is at the 3' end of the putative protein - extending from amino acid 270 to amino acid 275 (nucleotides 941-958). The putative protein has a pK of 9.75. The corresponding protein sequence based upon a translation of SEQ ID NO:I is presented as SEQ ID N0:2.
W. Clone ~2 Clone g2 is a EcoRIlHindIII clone with an insert size of 2474 nucleotides (SEQ
ID N0:259).
The size of the immunoreactive protein observed on SDS gel was 25kd. The clone contains 4 ORFs.
The first and last ORFs encode partial proteins, i. e. , the complete ORF was not contained in the clone. ORF1 extends from nucleotide 2 to 445 and codes for the carboxyl end of a putative protein of 148 amino acids (SEQ ID N0:260). ORF2 extends from bases 461-1156, coding for a putative protein of molecular size 25.4 kd (SEQ ID N0:261). ORF3 extends from bases 1156 to 1776 and codes for a protein having a size of about 22.2 kd (SEQ ID N0:262). ORF4 (nucleotides 1798-2472) codes for the amino terminus of a putative protein of 24.4 kd (SEQ ID N0:263).
The predicted pIs of ORF2 and ORF3 are 7.82 and 5.26, respectively. While ORF2 and ORF3 putative protein does not contain predicted transmembrane regions, ORF4 putative protein contains 3 predicted transmembrane regions with 6 predicted transmembrane helices.
23. Clone ~9 Clone g9 possesses an insert size of 4292 bases. Two subclones, k4 and gl l, are contained within clone g9. Clone g9 produces an immunoreactive protein band of about 85kd, whilst k4 has immunoreactive bands of 55 kd, 46 kd, 35 kd and 30 kd and gl l produces a weakly immunoreactive band of 22 kd.
The complete sequence of g9 was obtained by walking in the 5' direction from the gll C-terminus sequence, and walking in the 3' direction from k4 N-terminus sequence (SEQ ID N0:264).
Clone g9 has 5 ORFs. ORF1 encodes a partial protein.in the region extending from bases 2-349 (SEQ
ID N0:265). The corresponding predicted molecular size of the protein is 12.3 kd. ORF2 extends from bases 495-1403 (SEQ ID N0:266), and the corresponding predicted protein possesses a molecular size of 33.8 kd. ORF2 is followed by a "AGGA" Shine-Dalgarno sequence, positioned in front of the ATG of ORF3 (bases 1418-2209). The predicted molecular size for the putative protein encoded by ORF3 is 29.7 kd (SEQ ID N0:267). ORF4 corresponds to bases 2223-3719. The protein _T ___ ._ ___._.___. _.___.___ ~
encodes by ORF4 has a predicted molecular size of 56.8 kd (SEQ ID N0:268).
ORFS extends from nucleotides 3133 to 4236, with a predicted protein molecular size of 19.9 kd (SEQ ID N0:269).
24. Clone Gla An immunoreactive 2.8 kb HindIiI fragment of the corresponding genomic EcoRI
clone, having an insert size of 6.4kb, was treated with exo-mung nuclease to generate nested deletion clones for sequencing. The DNA sequence of a 1610 base pair region was determined (SEQ ID N0:8).
Two open reading frames (ORFs) were determined: ORF 1, nucleotides 32-964; and ORF 2, nucleotides 1034-1570. The corresponding translated protein sequences are presented herein as SEQ
ID NO: 9 and SEQ ID NO:10, respectively.
25. Details of the characterization of additional library 2 clones, e6, dll, fll, and d6 are provided in the Tables below.
1 S Table 3a Summary of Library 1 and Library 2 Clones Size of ClusterPredicted Clone Sequenced ORF (b.p:) . Protein DNA (b:p.) No. Size ' (Icd) Actual Size;on B2 1204 278-1150 19 31.6/30-32 A22 1009 122-599 1 18.2/_25 ~-gal fusion protein C1 2534 868-1926 42 39.I/35 B17 2006 500-700 (ORF1) 28 20.9/_22 870-1406 (OltF2) 1410-2006 (OItF 3) 142-705 (OltF4) A3 1878 399-1743 69 50.7/52 B9 984 230-931 68 25.3/30 C3 2705 727-1281 18 n.d.
B23 1078 3-1076 67 38.6/30 d7 616 3-614 /3-gal fusion 3 23.4/35-40 protein f8 1459 134-1042 12 33.9/33-35 'Size of Clusterpredicted Sequenced DNA (b.p.)ORIt (b.p.) No: Protein.
Size Clone' Actual Size on GeI
a5 1023 '3-545 (OItF 1) /3-gal 6 21.3/28 fusion protein, 569-1021 (OItF 2) b5 414 1-414 /3-gal fusion 22 16.6/22 protein a1 1208 'S3-801 (OItF 1) 9 27.5/30 880-1206 (ORF 2) d5 1795 1-1704 a-gal fusion 64 62.1/80 protein c5 650 2-604 31 23.3/30 a3 1975 '3-608 (a-gal) (ORF 50 23.1/30 1) 613-1266 (OItF 2) d6 1960 1-1845 24 70.2/n.d.
fll 1495 475-1224 30 30.9/30 e6 1458 96-1325 63 46.4/55 1~ dl 2059 696-1514 (ORF 2) 50 30.4/30.4 l b8c7 1484 77-1234 4 48.9/n.d.
c2 2077 45-857 46 30/30.2 c13 1742 2-1420 (/3-gal) n.d. 54.5/55 f3 2274 1-1788 (/3-gal fusion) 21 67/25 g2 2474 245 (OItF 1) 25 25.4/_25 '461-1156(OItF 2) 1156-1776 (ORF 3) 1798-2472 (OItF 4) g9 4292 5 012Fs 37, 2-349 (ORF 1 ) 123 495-1403 {OItF 2) 33.8 1418-2209 (ORF 3) 29.7 '2223-3719 (ORF 4) 56.8/85 3733-4236 (ORF 5) 19.9 k4 1624 part of g9 37 cluster Gla 1610 32-964 (ORF 1) 2 '"1034-1570 (ORF 2) gll 1899 part of g9 38 cluster n.d. = not determined, underlined numbers for protein size is observed protein size on SDS geI.
Predicted protein size does not include (3-galactosidase fusion peptide.
_ _T.._. _ . _. ~..___ _ _ Table 3b Expressed Pur- Paneled Clonein ifed pGEX Plasmid B2 Yes Yes Yes A22 Yes Yes Yes C Yes Yes Yes B Yes Yes Yes (ORE
4) A3 Yes Yes Yes C7 Yes Yes Yes Gla Yes Yes Yes (small (O~ panel) 2) B9 Yes No No C3 No No No B23 No No No d7 Yes Yes Yes (Small panel) f8 Yes No No a5 Yes Yes Yes (ORE
1) b5 Yes No No al Yes No No (ORE
1) d5 No No No c5 Yes Yes Yes a3 Yes No No d6 No No No fll No No No e6 No No No d1I Yes Yes Yes (ORE
2) Expressed Pur- Paneled Clone . in ified pGEX Plasmid' b8C7 Yes Yes Yes c2 Yes Yes No c 13 Yes No No f3 No No No g2 Yes No No (ORF
2) g9 Yes Yes Yes (small (O~ panel) 4) k4 No No No gll No No No n.d. = not determined, underlined numbers for protein size is observed protein size on SDS gel.
where there are more than 2 ORF's, * indicates ORG expressed that is immunoreactive.
Table 4 Genomic Glone/ Subclone Protein SEQ ~Q m NO
' 1D NO:
approx. insert (Subcione) size N0:51 5.0 kb B2 2197780 EQ ID N0:55 SEQ ID
N0:54 .2kb A22 2210DMIC EQ ID N0:44 EQ ID N0:43 .2 kb C1 C1CON6V2 EQ ID N0:39 EQ ID N0:38 .0 kb B17 B17CON4 EQ ID N0:26,SEQ ID
ORF N0:24 .0 kb 1 EQ ID N0:27 EQ ID N0:28, ORF
EQ ID N0:25, ORF
A3 3CON3 EQ ID N0:17 EQ ID N0:16 6.0 kb C7 C7.2C SEQ ID N0:21SEQ ID
N0:20 3.8 kb 8 (3.5 kb) 8c7 SEQ ID N0:550SEQ ID
N0:549 ____- _ __T ~ __ __ Genomic Clone/ Subclone Protein SEQ SEQ ID NO:
ID NO:
aPProx. insert (Subclone) size Gla Gla EQ ID N0:9, SEQ ID N0:8 6.4 kb EQ ID NO:10, OItF
9 9.4C EQ ID N0:48 EQ ID N0:47 .2 kb S C3 C3CONFIC EQ ID N0:15 .9 kb 23 23CON EQ ID N0:14 SEQ ID N0:13 .5 kb d7 D7CON1 EQ ID N0:12 SEQ ID NO:11 .7 kb FSCONI EQ ID N0:2 EQ ID NO:1 1.3 kb A5CON1 EQ ID N0:4, SEQ ID N0:3 1 kb EQ ID N0:5, b5 BSCON EQ ID N0:7 SEQ ID N0:6 .5 kb I 1CON1 EQ ID N0:36,SEQ ID N0:35 OItF
1.2 kb I
EQ ID N0:37, OltF
D5CON1 EQ ID N0:59 SEQ ID N0:58 1.7 kb c5 C5CON5 EQ ID N0:254SEQ ID N0:253 .7 kb d6 D6CON1V2 SEQ ID N0:495 3.8 kb fl l Fl l EQ ID N0:323part of cluster 41, 3.0 kb SEQ ID N0:322 e6 E6CON1 EQ ID N0:554SEQ ID N0:541 .5 kb dll DI1CON EQ ID N0:551,SEQ ID N0:526 OItF
.1 kb 1 EQ ID N0:552, OItF
EQ ID N0:553, 1tF 3 2 C2CON EQ ID N0:252SEQ ID N0:251 .2 kb 13 C 13CON EQ ID N0:256SEQ ID N0:255 I.7 kb f3 F3CON1 EQ ID N0:258SEQ ID N0:257 .3 kb Genomic Clonel 5ubcloneProtein SEQ'mSEQ TD
NO NO
approx. insert (Subclone) size g2 G2CON EQ ID N0:260,SEQ ID
I OItF N0:259 2.5 kb 1 SEQ ID N0:261, EQ ID N0:262, EQ ID N0:263, g9 G9CON1 EQ ID N0:265,SEQ ID
ORF N0:264 kb 1 EQ ID N0:266, OltF
EQ ID N0:267, 1tF 3 SEQ ID N0:268, 1tF 4 SEQ ID N0:269, 1tF 5 gll SEQ ID
N0:271 1.9 kb k4 SEQ ID
N0:270 .9 kb a3 3L2CON1 EQ ID N0:249,SEQ ID
OI2F N0:248 I O .2 kb 1 EQ ID N0:250, ORF
Example SA
Expression of H. pylori antigen coding regions Amplified products from various clone families, e.g., A3, A22, B2, B9, C1, C5, C7, and Gla, were cloned into pGEX vectors (Pharmacia). The cloned constructs were expressed in E.
coli strain (Stratagene, LaJolla, CA).
1. Clone B2 Expression primers GD96 (forward primer, SEQ ID N0:56) and GD97 (reverse primer, SEQ
ID N0:57) were designed to introduce NcoI and BamHI sites for subcloning the B2 fragment into expression vectors pGEXde165 and pGEXGLI. The expression product lacked the predicted transmembrane region.
Based upon the insolubility of the expressed proteins, only the expression product from pGEXde165 was further purified. The protein was purified by affinity purification chromatography, employing Ni-NTA++ resin from Qiagen (Chatsworth, CA).
_ _ _ __~. _ __ __ _ _r _ WO 98/49314 ' PCT/US98/08487 The purified protein was paneled against various sources of H. pylori-infected sera. The serum paneling was carried out twice, with differing results: (i) 75 %
sensitivity, based upon 35 sera samples, and (ii) sensitivity of about 30% , based upon 183 sera. The serum panel was performed using protein that had undergone further SDS gel eiectroelution following affinity chromatography purification.
2. Clone C1 Amplified products from clone C1CON6V2 were cloned into expression vector pGEXd165 using primers BF (SEQ ID N0:40) and BG (SEQ ID N0:41) as outlined in Table 5 below. The expressed protein was confirmed to be immunoreactive; however, it was cleaved beyond the transmembrane region to form a smaller immunoreactive band. Based upon this observation, a new forward primer was designed for the expression of the smaller protein, primer BO (SEQ ID N0:42), as indicated in Table 5.
The resulting protein was determined to be immunoreactive, and formed in much higher yields than in the previous construct.
3. Clone B17 Expression primers CA (forward primer, SEQ ID N0:29) and CB (reverse primer, SEQ ID
N0:30) were designed to introduce NcoI and BamHI sites for subcloning the B17 ORF4 fragment into expression vector GEXdeIGS.
Expression primers CC (forward primer, SEQ ID N0:31) and CD (reverse primer, SEQ ID
N0:32) were designed to introduce NcoI and BamHI sites for subcloning the B 17 ORF2 fragment into the expression vector.
4. Clone A3 Amplified products from the genomic A3 DNA were digested with NcoIlBamHI
restriction enzymes and subcloned into the NcoI and BamHI sites of expression vector pGEXd165 polyHis, using primers BP (SEQ ID N0:18) and BQ (SEQ ID N0:19) respectively. Expression of protein was induced at 0.5 mM IPTG (isopropylthiogalacto-pyranoside). The expressed protein was of the expected size, and was confirmed to be immunoreactive against Roost pooled sera.
5. Clone C7 PCR amplified products from H. pylon genomic DNA produced a DNA fragment of the expected size, which was then subcloned into the NcoIlBamHI sites of expression vector pGEXde165 using primers GD100 (SEQ ID N0:22) and GD101 (SEQ ID N0:23) respectively. The expressed protein was of the expected size, and was highly insoluble. The protein was readily purifiable by Ni-NTA column chromatography as described above. Immunogenic screening confirmed the expressed protein to be immunoreactive against Roost pooled sera.
6. Clone Gla PCR amplified products were subcloned into expression vector pde1165polyHis using primers BW (SEQ ID N0:33) and BX (SEQ ID N0:34) as outlined in Table 5 below. The expressed protein was confirmed to be immunoreactive. However, the expression product was internally cleaved, with cleavage most likely occurring at the transmembrane region.
Expression of additional cloned constructs was similarly carried out.
Table Sa Primers Used for PCR Expression of DNA Sequences Encodin Antigens of H. pylori Clone Forward Primer/SEQ Reverse Primer/SEQ
Family 1D NO: >D NO:
SEQ ID N0:56 SEQ ID N0:57 2 U v EQ ID N0:45 SEQ ID N0:46 SEQ ID N0:40 SEQ ID N0:41 O
SEQ ID N0:42 EQ ID N0:29 SEQ ID N0:30 SEQ ID N0:18 SEQ ID N0:19 SEQ ID N0:22 SEQ ID N0:23 Gla ORF2 O X
SEQ ID N0:62 SEQ ID N0:34 W
SEQ ID N0:33 SEQ ID N0:49 SEQ ID NO:SO
d7 CM CP
SEQ ID N0:233 SEQ ID N0:548 CN
SEQ ID N0:234 _~__ _... _~_ ..T _. _._. _ i __.
Clone Forward Primer/SEQ Reverse.Primer/SEQ
Family >D NO: m N
DK
f8 SEQ ID N0:235 DI, Dp SEQ ID N0:237 SEQ ID N0:236 a5 (ORFi) DH
EQ ID N0:238 SEQ ID N0:239 b5 I DJ
EQ ID N0:240 SEQ ID N0:241 al (ORF1)DQ DR
SEQ ID N0:242 SEQ ID N0:243 a3 B GC
EQ ID N0:244 SEQ ID N0:245 c5 GP GQ
EQ ID N0:325 SEQ ID N0:326 g2 (ORF2)GR GS
S EQ ID N0:327 SEQ ID N0:328 c2 GJ GI
S EQ ID N0:329 SEQ ID N0:330 13 V Gqr S EQ ID N0:331 SEQ ID N0:332 GM
S EQ ID N0:333 SEQ ID N0:334 g9 (ORF4)N GO
S EQ ID N0:335 SEQ ID N0:336 dll (ORF2)O p EQ 1D N0:337 SEQ ID N0:338 8c7 Y GDI01 S EQ ID N0:339 SEQ ID N0:23 Table Sb Immunoreactivitv of E~ressed Recombinant Antigens of H. pylori to H. pylori Infected Sera Expression I~unoreactivity Immunoreactivity.to Ctone toRoost SFA001 Pooled Sera Pooled Sera dHB2d1 + +
dHA22.8 + +
dHC 1s.11 + +
dHB 170RF4.4 + _ dHA3.5 + +
WO 98/49314 PCTlITS98/08487 Expression CloneImmunoreactivityImmunoreactivity ao Roosf to SFA001 Pooled Sera Pooled Sera dHC7.D6 + +
dHG 1 a2S.5 + +
dHB9.4.2 + +
dHd7S1 + +
dHf8u.2 + +
dHa5.2 ~- _ dHb5.4 t dHa 1.2 -f. _ dHa301tF 1.5 + _ dHc5.2 + +
dHd11.8 + +
dHb8c7.7 + +
dHc2 + +
dHc 13 dHg90RF4.1 + _ dHGla25.5 + +
t t All protein expression products were determined to be immunoreactive against Roost pooled sera, except for dHB9.4.2.
Example 5B
Purification of Illustrative Antigenic Proteins Expressed by H. yylori Clones Y175A Y212A and Y146B
Clones Y104B, Y175A, Y212A and Y146B were expressed as E. coli recombinant proteins according to the protocol described above. Primer sequences for the E. coli expression clones are presented in the Sequence Listing. The recombinant proteins were purified and their immunogenicity was confirmed according to the general procedure described below.
A pellet from a shake flask was spun and then submitted to differential solubilization. Briefly, the pellet was homogenized in a solution containg PBS/SmM PMSF, and spun for 60 minutes at 4°C, 30k. Subsequent rounds of homogenization/centrifugation were as follows: (i) 100 mM Tris/2 Triton/2M Urea/SmM EDTA/0.5 mM DTT (homogenization step)/60 minutes at 4°C, 30k (centrifugation); (ii) PBS pH 7.8/2M Urea/0.5 mM DTT/ 60 minutes at 4°C, 30k; (iii) PBS pH
_. _..._...___ ___._- .~__ __._. _....._ ._ T
7.8/4M Urea/0.5 mM DTT/60 minutes at 4°C, 30k, (iv) PBS pH 8.0/6M
Urea/0.5 mM DTT/60 minutes at 4°C, 30k; (v) PBS pH 8.0/6M urea/2M guanidine HCL/2mM BME
/60 minutes at 4°C, 30k.
The supernatant from step (iv) above was then dialyzed into PBS pH 8.0/6M
Urea/2mM
BME, followed by chromatographic separation using a pre-packed column of Chelating Sepharose (Pharmacia) loaded with five column volumes of 0.2M NiCl2. A 10 Column Volume (C.V.) gradient of Buffer A into 100% of Buffer B was utilized, where Nickel IMAC Buffer A
contained PBS pH
8.0/6M Urea/2mM BME and Nickel IMAC Buffer B contained Buffer A and 250 mM
imidazole.
The appropriate Nickel IMAC fractions were pooled and dialyzed overnight into PBS pH
8.0/6M Urea/0.5mM DTT, and the final product was then vialed and stored at -80°C.
Alternatively, glutathione-sepharose packed columns (Sigma) were utilized.
Fractions were typically analyzed by 1) Pierce Coomassie protein assay, 2) SDS-PAGE (on 12% gel), 3) Western blot using H. pylori Roost Pool fi3, 4) GLT H.pylori negative pool and 5) Anti-E.coli polyclonal antibodies. The results of these analyses confirmed the immunogenicity of the expressed proteins.
Example 6A
Optimization of Antigen Concentration and Small Scale Serum Paneling for Purified Antigen from Clones dHA22.8 (A22) and dHCIS 11 (C1) 1. Clone dHA22.8 Protein expressed by clone dHA22.8 (corresponding to clone A22) was isolated and purified as follows.
2000 ml of culture pellet was suspended in 200 ml of Buffer B (48g urea, 1.2 g NaH2P0,, 0.12 g Tris-HCI, and 90 ml deionized water, adjusted to pH 8.0 and to a total volume of 100 ml), and sonicated for 10 minutes to effect cell lysis. The sonicated mixture was then rocked at room temperature for 1 hour, and spun at 15,000 rpm for 15 minutes at 10°C
to remove the cell debris.
To the supernatant was then added 1 ml of Ni-NTA resin (Qiagen GmgH, Hilden, Germany) which was then mixed at room temperature for 1-2 hours. To pellet the resin, the mixture was centrifuged at 5,000 rpm for 15 minutes at 4°C and the supernatant discarded. The resin was washed with 50 ml of Buffer C (48 g urea, 1.2 g NaH2P04, 0.12 Tris-HCl and 90 ml deionized water, adjusted to pH
6.3 and brought to a total volume of 100 rnl), centrifuged, and the supernatant discarded. The resin was then loaded into a disposable column, and the wash step repeated with remaining Buffer C. The WO 98!49314 PCT/US98/08487 protein was then eluted with 50 ml of Buffer III (50 mM sodium phosphate, pH
8.0, 300 mM NaCI, 250 imidazole). Fractions were collected (2 ml) and analyzed on 12 % SDS-PAGE
gel.
The purified protein was then screened with serum antibodies using conventional techniques (Ausubel, et al., 1988). The purified protein was slotted at concentrations of 0.1-20 ug/mI O.1M
carbonate buffer, blocked with 5 % skim milk in TBS buffer, and reacted with H. pylori positive and H. pylon negative sera diluted 100 times. A serum panel consisting of 36 total sera was used as indicated above. Positive sera was from Roost pool #2 or SFA 001 pool;
negatives were from donor packs.
Following a 1 hour incubation period, the strips were washed 3 times with TBS
buffer, and incubated with alkaline phosphatase-conjugated anti-human Ig secondary antibodies (Promega Biotech, Madison, WI) diluted 1000 times in 5 % skim milk in TBS buffer. The strips were then washed 3 times with TBS wash buffer. Immunoreactive proteins were developed with a substrate, e.g., BCIP, (5-bromo-4-chloro-3-indolyl-phosphate), and NBT (vitro blue tetrazolium salt (Sigma)). The development of color indicated the highly antigenic nature of the purified A22 protein (i.e., reactive with H. pylori-positive sera).
The optimum concentration of antigen was determined to be 2.0 mg/ml. The dHA22.8 (A22) expression product reacted with anti-H. pylori antibodies present in both pooled sera sources (Roost pool #2 and SFA 001 pool), and with 100% of the individual H. pylori-positive sera sampies tested.
The antigen exhibited no detectable cross reactivity with normal sera.
2. Clone dHC1S.11 (C1) Protein expressed by clone dHC1S.11 (C1) was isolated and purified according to the following protocol.
Protein was extracted from a culture pellet by differential solubilization using a series of homogenization/centrifugation steps. The pellet was (i) homogenized in PBS/1 mM PMSF and centrifuged for 30 minutes at 4°C, 19,000 rpm, followed by separation of the supernatant, (ii) homogenized using 1 M urea/10 mM Tris at pH 8.0/i0 mM DTT, followed by centrifugation as in (i) and separation of the supernatant; (iii) homogenized in 4 M urea/10 mM
Tris pH 8.0/2 mM BME, followed by centrifugation as in (i) and separation of the.supernatant; and finally, (iv) homogenization of the pellet with 6 M urea/IOmM Tris pH 8.0/2 mM BME, followed by centrifugation as described above. To the combined supernatants was added 150 mM NaCI. The protein was then separated from the combined supernatants by immobilized metal adsorption chromatography (IMAC) using a 5 ml prepacked column containing chelating sepharose (Pharmacia, Chelating Sepharose Fast Flow) loaded with 2 C.V. (column volumes) 0.2 M NiCl2. The protein was eluted from the column using a 20 C.V. gradient of Buffer A (4 M urea/10 mM Tris at pH 8.0/0.2mM BME/150 mM
NaCI) into 50%
_. ._.-~ ~ _ . _.._- - __ T
Buffer B (Buffer A to which was added 0.5 M imidazole). Fractions were collected (2 ml) and analyzed on 12 % SDS-PAGE gel.
The optimal concentration for immunoscreening was determined essentially as described above. Serum panelling as described above was repeated for the purified C1 protein expressed by clone dHC1S.11.
As in the above example, the expressed protein was reactive with both sources of pooled H.
pylori-positive sera, and showed no signs of cross reactivity with control sera (H. pylori negative . samples, 1 sample of pooled sera and 13 individual samples). Additionally, as can be seen in panels 20-35 and A-C, the recombinant protein exhibited immunoreactivity with 95 % of the H. pylori-positive samples.
In summary, for this small sized panel study, A22 and C1 both exhibit high sensitivity and specificity ( > 90-95 % ) Table Sc Clinical Data for Sera Used for Serum Panellin~~ of Clones dHA22.8 and dHC 1 S 11 Serum.Eudoscopy Culture 11f. Pathology (fi,g. (gastritis) ) A1 Mild antrum gastritis,Moderate Acute/chronic/moderate deep duodenal ulcer B Moderate antrum gastritis,Heavy Chronic/severe with 1 moderate prior Hp ( > 4 duodenitis years with therapy) B2 None Rare Chronic/moderate B4 None NA NA
9 Severe gastritis NA NA
44 Mild antrum gastritis Moderate Chronic/moderate 16 Mild antrum gastritis,Moderate Chronic/moderate deep gastric ulcer 19 Superficial gastric Rare Chronic/moderate and duodenal ulcer 20 Mild antrum gastritis Moderate Acute/chronic 4 Severe gastritis . NA NA
69 Mild antrum gastritis None Chronic/mild and duodenitis, supe~cial duodenal ulcer 47 Mild duodenitis, superficialNone None duodenal ulcer 58 Mild antrum gastritis,Moderate Chroniclmild oesophageal ulcer 54 Moderate gastritis Heavy Chronic/moderate 65 Severe gastritis superficialNone None gastric ulcer WO 98/49314 PC"T/US98/08487 Serum Endoscopy Culture Pathology fH.p. fps) t) C1 Moderate antrum and Heavy Chronic/severe body gastritis C3 NA Heavy ?
34 Gastritis in body None Acutelmild ND = not available.
Example 6B
Further Sera Paneling with Additional Sera Panels for Purified Antigens:
Selection of Preferred Anti ens Additional sera paneling studies were conducted with other sera panels (USC
for University of Southern California, and POW for Prince of Wales Hospital) to further examine the sensitivity and specificity of various H. pylori antigens.
Gold standard tests were employed as reference standards to compute the sensitivities and speciftcities of each individual antigen. Standards used were conventional gold standard tests including histology, CLO test, and the UBT test. UBT is the current most widely used gold standard for indication of "active infection" by non-invasive methods. Histology, CLO
(rapid urease test) and UBT
are all indicate "active infection" since these tests depend on the presence of bacteria to produce a positive result. Since serology measures both past and present infection, the aim of this study was to (i) identify serological markers that would correlate with UBT results for use as "active infection markers", and (ii) to explore both single and muliple antigen combinations to effectively screen for active infection by H. pylori.
A. Use of UBT as a Gold Standard in Single Antigen Screenints Since different antigens have different performances in different panels (this may be a reflection of the geographical differences due to different strains of H.
pylori and previous exposures of populations to other cross-reacting antigens from other bacteria endemic to the region), the use of single antigen markers for the local population was explored using UBT as a single reference standard.
1. USC Panel. Based upon this panel, the best single antigen selection appeared to be antigen Y261A or Y124A. Both of these antigens provided an overall balance of sensitivity and specificity (>80% for both sensitivity and specificity for Y261, and 100%
sensitivity with ?0%
specificity for Y124A). Other preferred antigens, A22 and C1, were 95-100%
sensitive, and 67-77%
specific, respectively in this panel.
2. POW Panel. Based upon this paneling study, the best single antigen selection appeared to be Y 128D 12S and Y 124A, where sensitivity and specificity were about 75-80 % for Y128D12S, and both about 80% for Y124A. Another preferred antigen is Y261A, which exibited a sensitivity of 70 % and a specificity of 80 % . For this paneling series, preferred clones A22 and C 1 were found to be about 95-98% sensitive, and about 60% specific.
B. Usine Other Gold Standards 1. Roost Panel. Only positive sera was tested, and antigens A22 and C1 exhibited 90%
sensitivity. Y124A exhibited about 80% sensitivity, and Y261A possessed a sensitivity of approximately 87 % .
2. UNSW Panel In this paneling series, A22 and C1 demonstated greater than 90-100%
specificity. Y 124 demonstrated 80 % sensitivity and 96 % specificity, and Y261 A demonstrated 80 %
sensitivity and 100 % specificity.
It was observed that the sensitivity and specificity values do not change significantly whether UBT is used alone or in combination with other gold standards in the USC and POW panels. Thus, the somewhat lower specificity for A22 and C1 may be a reflection of previous exposure to other cross-reacting antigens.
C. 2-4 Antigen Combination Tests In view of the high sensitivity of A22 and C1 and somewhat lower specificity for certain panels (POW, USC), a multiple antigen format was explored. The 2-4 antigen format was based on the criteria that, for any 2 -3 highly sensitive antigens, at least both or all 3 have to be positive with respect to the criteria to increase the specificity.
This "2 antigen both positive" criteria was applied to a selected 12 antigen set, where the 12 antigens were selected for a sensitivity of at least 40-50 % for consideration.
The sensitivities and specificities of the 2 antigen both positive criterion is computed, and the resulting table is examined for good performers. Additionally, 2 antigen combinations with high specificity and lower selectivity is run against the entire two antigen combination matrix to provide a result indicating "2 antigen both positive or 2 other antigen both positive". The final analysis then provides a selection of commonly occurring antigens that provide good results across the board between the USC and POW panels.
D. Results of the 2-4 Antigen Combination Test For the USC panel, there are 15 antigenic combinations that provide around 95 % sensitivity and 100% specificity.
For the POW panel, 10 different antigen combinations were shown to provide greater than 70% sensitivity and around 80-90% specificity.
Based upon these findings, exemplary combination selections for both panels are as follows:
(a) C1 and Y124A both positive (sensitivity of 76.2 and 100% and specificity of 89.1 and 95.2 % for POW and USC panels respectively), and (b) C 1 and Y 124A and A22 all positive (sensitivity of 71.4 and 94.7 % and specificity of 91.3 and 95.2 % respectively for POW and USC panels).
For the USC panel, an antigen combination of C1 and c5 was preferred, and for the POW
panel, antigens combinations Y261A and Y124A or antigens C7 and B2 also were preferred.
Thus, based upon these sera paneling data, preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, CI, Y124A, Y261A, c5, C7, B2, Y104B, and Y128D.
Table Sd Hp Recombinant Antigen Sensitivities Antigen Roost (n UNSW (n = USC (n POW (n =
= 30) 94) = 30) 40) Y175A 73.3 33.3 70 83 Y 104B 76.7 57 77 68 Y261 A 86.7 79.6 87 70 C7 80 66.7 77 48 b8 73 a5 3.3 8.6 10 18 Y212A 90 78.5 100 _.r _..__. ___ _._ __.__~__.
Antigen Roost (n UNSW (n = USC (n POW (n = 30) 94) = 30) = 40) Y 124A 80 80. 7 100 80 Y 184A 46.7 35. 5 27 13 B2 50 36.6 47 68 Y 128Dso1 36.7 15.1 57 78 Y 146B 63. 3 65 .6 67 53 c5 70 64.5 73 63 Y 150A 20 32.3 27 13 Y153A 66.7 66.7 50 28 C 1 90 90. 3 97 98 A3 46.7 43 57 25 catalase 43.3 9.7 40 13 Table 5e Hp Recombinant Antigen Specificities Antigen UNSW (n USC (n POW (n =-= 25) = 25) 48) b8 73 a5 92 96 94 Y 124A 96 b8 81 B2 100 88 gg Y 128A g9 ~igen UNSW (n = USC (n POW (n =
25) = 25) 4t~
Y 128Dso1 96 84 77 c5 96 96 73 catalase 100 92 98 A graphical summary of sensitivity and specificity data for representative purified H. pylori antigens in different sera panelling studies is presented in Figs. 65 and 66.
Example 7 Purification of 36 kD and "Spot 15" Antigens Produced by H. pylori Cultures of H. pylori (ATCC No. 43504) were grown under appropriate conditions and the cells harvested into phosphate buffered saline. This was followed by repeated centrifugation to remove cell debris and other contaminants. The resulting pellet was then lysed in a French press at > 10,000 PSI, followed by centrifugation at 10,000 x g for 25 minutes.
Prefractionation of soluble H. pylori lysate supernatant was carried out only when enriching for spot 15 (high pH), for subsequent sequence/structural investigations (e.g., mass spectrometry, peptide map).
Fractionation of the 36 kD protein was achieved by dialysis to 10 mM Tris buffer, at pH 8.0, 50 mM NaCI, followed by gradient elution over anion exchange resin, Resource Q
(Pharmacia Biotechnology, Piscataway, NJ). SDS-PAGE/Western blot analysis indicated a Western-positive doublet at 30 kD and a positive 36 kD band. Both fractions were determined to be highly immunoreactive when tested against Roost pooled sera.
Fractionation of the spot 15 antigen was carried out in a similar fashion. The sample was passed over Sephacryl S-100 ion exchange resin (Pharmacia Biotechnology, Piscataway, NJ) (to 10 mM phosphate buffer, 50 mM NaCI, pH 8.0), followed by further fractionation by gradient elution over Resource S cation exchange resin (Pharmacia Biotechnology, Piscataway, NJ). The fraction containing spot 15 antigen was analyzed by SDS-PAGE and Western blot, and was similarly confirmed to be highly antigenic in nature, as indicated by Western blot and immunoreactivity with Roost pooled sera.
Example 8 Characterization of 36 kD and Snot H;pylori Antigens Following fractionation of the antigenic proteins as described in Example 7 above, the isolated 10 fractions were further characterized by two-dimensional electrophoresis, carried out according to the methods of O'Farrell (1975, 1977).
1. 2-Dimensional Electrophoresis Under Low pH Conditions Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975).
15 Isoelectric focusing was carried out in glass tubes having an inner diameter of 2.0 mm, using 2%
ampholines (BDH, Hofer Scientific Instruments, San Francisco, CA) for 9600 volt-hrs. The final tube gel pH gradient as measured by a surface pH electrode is on the enclosed pH
gradient form.
Following equilibration for 10 minutes in Buffer O (10% glycerol, 50 mM DTT, 2.3 % SDS, and 62.5 mM tris buffer, pH 6.8), the tube gel was sealed to the top of the stacking gel, which was placed on top of a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for 4 hours at 12.5 mA/gel. The slab gels were then fixed in a solution of 10 % acetic acid-50% methanol overnight. The following proteins were added as molecular weight markers to the agarose which sealed the tube gel to the slab gel: mysin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD).
These standards appear as horizontal lines on the silver-stained (Oakley, et al., 1980) 10%
acrylamide slab gels. The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
2. Western Blot Following slab gel electrophoresis, a duplicate gel was transferred to transfer buffer (12.5 mM
Tris, pH 8.8, 86 mM glycins, 10% methanol), transblotted onto PVDF paper overnight at 200 mA
(approximately 50 volts/gel). The blot was blocked for 2 hours in 2 % bovine serum albumin (BSA) in TTBS (Tween-Tris-Buffered Saline), rinsed in TTBS, incubated in primary antibody from Roost pool or negative serum pool and diluted 1:2500 in 1 % BSA/TTBS for 2 hours, rinsed in TTBS and placed in a solution containing secondary antibody (antihuman IgG horse radish peroxidase, 1:5000 WO 98/49314 ' PCT/US98/08487 diluted in TTBS) for 1 hour. The blot was rinsed with TTBS, treated with ECL
(Amersham Corporation, Arlington Heights, IL), and exposed to X-ray film.
A computer-generated photograph of an exemplary stained membrane containing antigenic proteins from H. pylori as described above is shown in Figure 2. The "normal"
human sera was confirmed to be H. pylori-negative using "HEr.~coBLOT 2.0" (Genelabs Diagnostics (PTE) Ltd., Singapore}. The identities of the spots indicated numerically on the gel were determined by protein sequencing (described in Example 9 below).
3. 2-Dimensional Electrophoresis Under Huh pH Conditions Two-dimensional electrophoresis adapted for the resolution of basic proteins was performed according to the method of O'Farrell (O'Farrell, et al., 1977).
Nonequilibrium pH gradient electrophoresis using 1.5 % pH 3.5-10 and 0.25 % pH
ampholines (Pharmacia Biotechnology, Piscataway, NJ) was carried out for 140 volts for 12 hour.
Purified tropomyosin, with a lower spot molecular weight of 33 kD and a pI of 5.2, and purified lysozyme, with a molecular weight of 14 kD and a pI of 10.5-I1 (Merck Sharpe &
Dohme, Philadelphia, PA) were added to the samples as internal pI markers.
After equilibration for 10 minutes in buffer (10% glycerol, 50 mM DTT, 2.3%
SDS and 62.5 mM tris, pH 6.8), the tube gel was sealed to the top of the stacking gel, which was placed on top of a 10% acrylamide slab gel (0.75 mm thick), and SDS slab gel electrophoresis was carried out for 4 hours at 12.5 mA/gel. The slab gels were fixed in a solution of 10 % acetic acid/50 % methanol overnight. The following proteins were added as molecular weight standards to the agarose which sealed the tube gel to the slab gel: myosin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD) and lysozyme (14 kD). These standards appear as horizontal lines on the silver stained (Oakley, et al., 1980) 10% acrylamide slab gels.
The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
A duplicate gel was transblotted onto PVDF paper and Western blotting carried out as described in 8.1.b. above.
A computer-generated photograph of an exemplary Western blotted membrane containing antigenic proteins from H. pylori as described above is shown in Figure 1. The identities of the spots indicated numerically on the gel were determined by protein sequencing (as described in Example 9 below).
__.._._,.. _..____ _.. __.-___-._..__.. T
WO 98/49314 ' PCT/US98/08487 Example 9 Isolation and Protein Sequence Determination for Western-Positive Spots N-terminal Seguencing The PVDF blots from Example 8 above were stained with Coomassie brilliant blue. Spots corresponding to Western positive bands were excised by scalpel and sequenced directly using a Hewlett-Packard G 1005A N-terminal sequencer with conventional sequencing techniques (e. g. , Miller, 1994; Spiecher, 1989). The sequencing techniques employed gave high repetitive yields (typically ranging from 93-98%), with a detection limit of approximately 100-200 finol.
2. Internal Sequencing Four different methods were utilized to obtain internal sequence information for the above-described isolated antigens of H. pylori (Allen, 1981): Lys-C peptide map and sequencing, CNBr peptide map and sequencing, OPA/CHBr peptide map and sequencing, and LC-MS/MS
sequencing of Lys-C digests.
a. Cyano~en Bromide Cleavage. Cyanogen bromide (CNBr) cleavage was performed on PVDF membranes in 70 % formic acid (Crimmins and Mische, 1996).
The cyanogen bromide digested peptides were then either repurified by capillary HPLC and sequenced directly, or subjected to ortho-phthalaldehyde (OPA) modification.
b. Ortho-nhthalaldehvde (OPA) Modification. Digested peptides were subjected to OPA modification according to the method of Bauer (Bauer, et al., 1984).
This reagent is used to modify primary, but not secondary amines, thereby allowing the identification of sequences having proline as their N-terminal residue and the silencing of all other sequences as determined by Edman-type N-terminal sequence analysis.
c. Liauid Chromatograph /Mass Spectrome~. Internal sequence information was also obtained from liquid chromatography/mass spectrometric based analysis.
Copper stained gel slices (Nakayama, et al., 1996) and PVDF-transferred proteins were first pre-treated by extraction, preliminary purification using Sep-Pak solid phase extraction (Millipore Corporation, Bedford MA), and protease (Lys C) digestion.
Liquid chromatography-mass spectrometry (LC-MS) was performed on digested proteins to assign internal sequence residues by peptide mass and ion trapping techniques (McAtee, et al., 1996).
The digests were chromatographed on a Vydac C18 reverse phase microbore column (150 mm x 1 mm) using an ABI Model 410 B dual syringe pumping system (Applied Biosystems Division, Perkin Elmer, Foster City, CA). The flow rate was maintained at 50 microliters/minute and elution was carried out using a linear gradient from 0.1 % aqueous TFA to 0.1 % TFA in acetonitrile. A Carlo Erba Phoenix 20 CU pump was used to deliver a mixture of methoxyethanoI and isopropanol (1:1, S v/v) at a rate of 50 microliters per minute, which was combined with the column eluent in a post column mixing chamber. An in-line flow splitter was used to restrict flow to the mass spectrometer to approximately 10 microliters/minute. Detection was performed immediately following elution from the column at 214 nanometers using an ABI 759 variable wavelength detector.
Mass spectrometric detection was achieved following post column solvent addition and flow splitting by a VG BioQ triple i0 quadruple mass spectrometer with a nano-electrospray ion source. Spectra were recorded in the positive ion mode using electrospray ionization. Calibration of the instrument was performed in the range m/z 500-2000 by using direct injection analysis of myoglobin. Spectra were recorded at 1.5 seconds intervals and a drying gas of nitrogen was used to aid evaporation of the solvent. The capillary voltage was maintained at approximately 4 kV with a source temperature of 60°C.
3. Sequencing Results Utilizing the approaches described above, the spots indicated in the Western Blots (Figs. I
and 2) were identified. The identities of the spots (where known) are presented in Table 6 below.
Table 6 H. pylori Antigens Reactive Sera With H. pylori Infected Humans Spot NlW.kDpI N-Terminal Antigen if Known Number (o6s) Sequence\
SEQ Il)'NO:
IN
WESTERN
BLOTS
1 62 6.7MKKIS Urease b subunit 2 58 6.3AKEIK Urease b associated chaperonin 3-Major42 6.5AKSIELQEIE Pyruvate favidoxin reductase 3-Minor42 6.5MLTXKDIHAL HP 154-enolase 4 41 6.4XTKIFV HP0537-cagl6 5 56 5.8AFQVN flagellin a protein 6 58 6.0AFQVN flagellin a protein 3S 7-Major42 6.1XICEICFNRTICPE. coli Tuf B protein 7-Minor42 6.1MXGXIIQVLG ATPase proton pump ___.... _...-__.T... ..........__.._. ...___ .___..__.__ __.. _.T .......
Spot MW pI N-Terminal Antigen if Known Number ltD Sequence\
(obs) ~QmNO.
I
8 60 5.8SFRINTNIAA tlagellin b precursor 9 31 6.7MIDXAIIGGG/ al clone orfl, monomine.oxidase SEQ ID N0:231 10 12 6.8MKTFEILKHL neutrophil activating protein 11 6 5.7AISKEEVLEY/ HP 1199-ribosomal protein SEQ ID N0:232L7/L12 12 20 6.0MYIPYVIEN a5 clone, clp protein SEQ ID N0:233 13 159 7.1MKLI undetermined SEQ ID N0:234 14 no sequence 15 79 5.7GKVIGIDLGT HP109 Heat shock protein SEQ ID N0:23570 16-Major42 6.8MREIIXDGNE HP0589-Ferredoxin oxidoreductase SEQ ID N0:236 16-Minor MKLLE O'Toole protein 17 78 6.9MKLLEE O'Toole protein SEQ ID N0:237 tN
Wes~t~
Bt.oTs 1 180 8.2NPP na 2 180 8.3MDXY na 3 88 8.3NKITY na 4 82 8.3ALXTY na 5 62 6.7MKKIS urease b subunit 6 58 6.3AKEIK urease b associated chaperonin 7 60 6.0SFRIN tiagellin b precursor 8 58 6.0AFQVN flagellin a precursor 9-Major46 8.5AYNPK HP0027-Isocitrate dehydrogettace SEQ ID N0:238 9-Minor AVTLI possible contaminant 10 45 9.1XNIQIQNMPK HP1018 SEQ ID N0:239 11 58 9.1MVNKD catalase 12 65 9.4EDDGFYTSVG Omp 5, 8, 9, 19, 27 SEQ ID N0:240 WO 98/49314 PCT/US98/0848~
Spot MW pI N-Terminal Antigen if Known 1cD
Number (obs) Sequence\
SEQ ID NO:
13 43 9.7 KEVKEKKA HP1350 SEQ ID N0:241 14 38 8.8 XDDGGFFTVG hopC protein 15-Major28.5 9.8 HECNAAFVAI possibly blocked SEQ ID N0:242 15-Minor GPKHNXEAGD possibly blocked 16 28 9.0 MKLTP crease a subunit Referring to the low pH blot presented as Fig. 2, spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens. Spot 9 represents the native H.
pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al, while spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
Looking at the high pH Western blot in Fig. 1, spots 9 (major and minor), 10, 12, 13, and (major and minor) represent unique antigens.
15 As will become apparent from the results below, it was later determined that the N-terminus of the spot 15 antigen (high pH) was blocked, thus, the N-terminal sequence determined for spot 15 by 2-dimensional gel analysis was later shown to be inaccurate.
Example 10 Sequence Analysis of the 36 kD Antigenic Protein and Spot 15 Antigen Isolated from Various Sources of H. pylori The H. pylori spot 15 antigen as described above was isolated from 3 different sources of H.
pylori as described in Examples 8 and 9. The H. pylori samples used were the following: H. pylori, ATCC 43504 (Australian type strain); H. pylori, strain ~ 26695, and H. pylori, J-170, (both obtained from Washington University School of Medicine, St. Louis, MO). The corresponding protein isolated from each source was then analyzed by reverse phase high performance liquid chromatography (HPLC) as described in Example 9.
Minor differences in the native spot 15 proteins from various H. pylori strains were observed, particularly in the early-eluting peak regions, and the spot 15 antigens derived from the TIGR and ATCC H. pylori sources appear to be closely related. The minor differences included slight minor amino acid changes, deamidations, and perhaps a side chain modification, as suggested by a high molecular weight (e.g., Iipopolysaccharide or glycosylation) modification in the TIGR-derived sample.
__ _..-_-~... _.. _. ....._._ .._...._.. _.... _.T.
The J-170 derived spot 15 protein appears to contain additional high molecular weight modifications, as indicated by the differences in molecular weight.
However, all peaks exhibited strong antigenicity when screened against Roost pooled sera, indicating that any minor differences in amino acid sequence among the native proteins does not adversely affect the high immunoreactivity of the recovered spot 15 antigen.
Based upon the characterization data described above, the spot 15 antigen appears to be a processed product of the putative 36 kD protein, as shown in Fig. 4.
A MALDI-TOF MS comparison of peptides generated from an in situ Lys-C digest of "Spot 15", generated by the above-described strains of H. pylori, was carried out.
In examining the total digest, the data indicated that strain 26695 and strain 43504 are more conserved in their primary sequence, and possibly in their post-translational modification profiles than are strains 43504 and J-170, or strains 26695 and J 170. This data, along with that presented above, suggests that although immunoreactivity to Spot 15 is noticeably present in all strains examined, there are differences in amino acid content and protein modifications between Spot 15 in the various strains.
The Lys-C digests were also examined by reversed phase HPLC. Not all of the peptides from the total digest were observed by RP-HPLC, however, this was attributed to the presence of extremely hydrophilic and/or hydrophobic peptides, not detectable within the operating parameters of the reverse phase column. Based upon an examination of RP-HPLC elution profiles, the peptide mass fingerprints appeared to be more conserved between strains than was indicated by the MALDI-TOF MS results.
Preferentially, the MALDI-TOF and other forms of MS (electrospray, fast atom bombardment, etc.) would present a more accurate determination of structural analysis.
Table 7 Comparison of Molecular Masses of Snot 15 Lvs-C Di e~sts by MALDI-TOF MS
43504 T'IGR J170 43504 T'IGR J170 Table 8 Comparison of Molecular Masses of Spot 15 Lys-C Digests Chromato~ranhed on C,g Reverse-Phased HPLC
STRAIN
I~LC Fraction43504 TIGR J170 4s - - -49 - 1302.58 -1302.35 1202.52 1302.19 1402.37 1402.50 1402.13 1285.48 1285.46 1285.56 1276.06 - 1275.81 m STRA IN
i HPLC Fraction43504 TIGR J170 - - 1260.54 - - 1176.82 52 _ _ 54 1281.34 1281.34 1281.17 61 1255.38 1255.33 2003.60 _ 63 2007.47 2007.52 -- _ _ 75 _ _ 76 - 1985.32 -77 - 1985.32 -78 1585.21 1584.80 -1 0 79 - 1884.72 -80 1999.46 - _ 15 Example 11 Antigenic Reactivity Between Various Strains of H
pylori. Separated by 2-D Electrophoresis Various strains of H. pylori were examined to determine the extent of conservation of the proteins identified in ATCC strain 45304, as described in Examples 7 and 8 and shown in Fig. 1, 20 among other strains of H. pylori.
Exemplary H. pylori strains tested were the following: Chico (clinical isolate from ~roville Community Hospital), TIGR 26695, 96-212 (Alaskan isolate, from Aleut), 4655-1 (Gambian child), J170, A-lc (Lithuanian isolate, which contains at least 6 kB of DNA referred to as "X" segment as an insertion near cag region and not present in C-3c), Rus-95 (isolated from Russian immigrant to the 25 United States), #l9 (Peruvian isolate), C-3c (Lithuanian isolate).
These various strains of H. pylori were purified, fractioned by 2D
electrophoresis and Western blotted as described for strain ATCC No. 45304 in Examples 7 and 8, to determine whether the immunoreactive spots indicated in Figs. 1 and 2 set by strain 45304 were conserved between strains.
The results are summarized in Tables 9 and 10 below.
Table 9 Comparison of Helicobacter Strains by 2D-PAGE (pH 8-13) from ATCC HelicobacterStrains Ge/PVDF
Spof ChicoTIGR 96:212465511J170A-lc Rus-95#9 G-3c 26695 Peru ' ' Spot I + + + + + + + + +
Spot 2 + + + + + + + + +
Spot 3 + + + - + - - - +
Spot 4 + + + - + + - - +
Spot 5 + + + + + + + + +
Spot 6 + + + + + + + + +
Spot 7 + + + + + + + + +
spot s + + + + + + + + +
Spot 9 Major+ + + + + - + + +
Spot 9 Minor+ + + + + - + + +
Spot 10 + + + + + + + + +
Spot 11 + - + + + + + + +
Spot 12 + + + + + - + + +
Spot 13 + + + + + + + + +
Spot 14 + + + + + + + + +
Spot 15 ++ ++ ++ ++ ++ ++ ++ ++ ++
Major Spot 15 ++ ++ ++ ++ ++ ++ ++ ++ ++
Minor spot 1 s + + + + + + + + +
Table 10 Comparison of Helicobacter Strains by 2D-PAGE (oH 4-81 from A,~'GC Helicobacter 43504 Strains .
G~PVDF Spot TIGR96-212: . ~ p~
Cliico26695 465511,1170A-lcRus=95 G-3c y spot 1 + + + + + + + + +
Spot 2 + + + + + + + + +
Spot 3 Major+ + + + ++ + + + +
__ ~ _. _.__ _ _. _. _ T
from ATCC HelicobacterStraiia Ge/PVDF
Spot. Chico1'IGR96-2124655/1J170A-lc Rns-95I/9 C-3c 26695 ' ',' Peru Spot 3 Minor+ + + + ++ + '+ + +
Spot 4 + + + - + - + + +
Spot 5 + t + + + + + + +
Spot 6 + t + + + + + + +
Spot 7 Major+ + + + + - + + +
Spot 7 Minor+ + + + + - + + +
Spot 8 + - + + + + + + +
Spot 9 + + + - + - - + +
Spot 10 + + + + + -t + +
Spot 11 + + + + + + t + +
Spot 12 - + + + + - + + +
+
Spot 13 + + + + + + + + +
Spot 14 + + + + + - + + +
Spot 15 + + + + + + + + +
Spot 16 + + + + + + + + +
Major Spot 16 + + + + + - + + +
Minor Spot 17 + + + + + + + + +
+
As can be seen from the above, the immunogenic proteins isolated from H. pylon strain ATCC 45304 are highly conserved between various strains of H. pylori. One of the preferred antigens of the invention, the spot 15 antigen (high pH), was observed in 100 % of the representative strains examined. Western blot results further supported the highly antigenic nature of this protein.
Example 12 MALDI-TOF Mass Snectrometrv Based Mapping of Antigens of H pylori Gel pieces stained with Coomassie or compatible silver stain as described in Example 9 were _ transferred to a microcentrifuge tube and rehydrated with 10 microliters of water. The gel pieces were then washed 3 times with 500 microliters 50% acetonitrile/0.05 M Tris-HCI, pH
8.5 for 20 minutes.
. The supernatants were discarded and the washed pieces were dried for 30 minutes in a Speed-Vac concentrator. Five microliters of a solution containing 0.05 micrograms Lys-C
was added to the tube and incubated for 20 hours at 32°C. Following digestion, the gel pieces were extracted three times with 30 microliters 50% acetonitrile/0.1 % TFA. The supernatants were transferred to a 0.5 ml microcentrifuge tube and dried. The extracted peptides were redissolved in 4 microliters 4-hydroxy-alpha-cyano cinnamic acid and a 0.8 microliter sample was spotted onto a MALDI
sample plate.
MALDI mass spectroscopic analysis was then performed on a PerSeptive BioSystems Voyager DE
mass spectrometer. Peptide mass peaks were then compared to data within the PIR, NRDB, Genebank, EMBL, TIGR H. pylori and The Swiss Protein Databases using the MS-FIT program (UCSF).
The results of matching mass spectral profile peaks of particular Lys-C
digested proteins with database information is summarized in Tables 11 and 12 below.
Table 11 MALDI-TOF Peptide Mass Mapping, pH 4-8 proteins Spot pI Apparent Selected Lys-C Peptide Protein Match Masses (Da) Molecular Weight 1 7.4 68-lcDa 1078.58, 1510.2, 1809, Urease b subunit 2126.17, 2780.08 (FIP
0072) 2 7.1 65-lcDa 1293.15, 1569.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP0010) 3 major7.1 45-lcDa 1217.41, 1819.07, 2545.87,Pyruvate flavidoxin 2469.85, 2774.14 reductase (HP
1108-1111) 5 or 5.8 65-lcDa 1471.61, 1574.82, 1752.97,Flagellin 6 1798.10, a protein 2193.39 (HP 601) 7 major6.2 50-IcDa 1047.11, 1614.51, 1683.67,Elongation 2046.33, factor Tu 2079.33 (HP 1205) 8 5.9 66-lcDa 1037.12, 1588.72, 1954.28,Flagellin 2215.34, b (HP 0115) 2950.39 2 7.1 65-kDa 1293.15, 1589.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP 0010) 12 5.7 20-IcDa 1349.46, 2103.40, 3284.77,CIpP protein 3472.91, (HP
5241.07 0794) __ .T. _ _. _ _. T
Table 12 MALDI-TOF Peptide Mass Mapping, pH 8-13 Proteins Spot pI Apparent Selected Lvs-C Peptide Protein Match Molecular Masses (Da) Weight 5 7.468-kDa 1078.58, 1510.2, 1809, Urease b subunit 2126.17, 2780.08 (HP
0072) 6 7.165-kDa 1293.15, 1569.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP0010) 7 5.966-kDa 1037.12, 1588.72, 1954.28,Flagellin b 2215.34, 2950.39 (HP 0115) 8 5.965-kDa 1471.61, 1574.82, 1752.97,Flagellin a 1798.10, 2193.39 (HP 601) 11 9.250-lcDa 1189.31, 1360.57, 1397.53,1944.16,Catalase (HP
2008.33 875) 9 9.340-kDa 1088.25, 1255.50, 1341.55,HP 0027 (icd) major 1487.71, 1716.03 i 9.250-kDa 1189.31, 1360.57, 1397.53,1944.16,Catalase (HP
1 2008.33 875) 12 9.355-kDa 2118.35, 2465.85, 2868.18,HP 0912 2987.34, 3887.41 13 9.740-kDa 1001.08, 1090.28, 1155.43,HP 1350 1650.96, 1910.31 14 9.632-kDa 2118.35, 2465.85, 2868.18,HP 0912 2987.34, 3887.41 15 9.825-kDa 1255.41, 1281.63, 1302.52,HP 0175 1584.86, 1733.87 16 9.224-kDa 1016.27, 1133.20, 1246.51,Urease a subunit 1379.60, 1976.20 (HP
073) Example 13 Anti,;ens for Use in Vaccines Representative antigens described herein were evaluated as vaccine candidates on the basis of Western blot analyses (as described above) of sera obtained from patients prior to and after antimicrobial treatment.
Briefly, prior to treatment and twelve months after antimicrobial treatment, the titres of antibodies reactive against the cloned antigens were determined. Antigens corresonding to antibodies whose titre remained high for an extended period of time subsequent to treatment were determined to be good vaccine candidates.
Two serum panels, the Gasbarrinni panel and the Greenberg panel were used for these analyses. Sera from patients were obtained prior to treatment and 12 or 24 months after treatment.
Patients who tested positive by the UBT after treatment were not included in this analysis, due to - active infection by H. pylori.
WO 98/49314 ' PCT/US98/08487 The Gasbarrinni panel was obtained from male and female patients in Italy, aged 18-70, who were diagnosed with H. pylori infection by endoscopy and UBT. Prior to antimicrobial treatment and 12 months after treatment, serum was collected from the patients.
The Greenberg panel was obtained from male and female patients in California, also aged 18 70, who were diagnosed as having H. pylori infection, as confirmed in antibody tests. Prior to antimicrobial treatment and 24 months after treatment, serum was collected from the patients.
Table 13 summarizes the data obtained from the Gasbarrinni panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 12 months are indicated therein. Twelve months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y139, Y146B, Y175A, and A22.
Table 14 summarizes the data obtained from the Greenberg panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 24 months are shown. Twenty four months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y184A, Z9A, Y261Ains and Y146B.
Since antigens which invoke a long-lasting antigenic response are considered to be good vaccine candidates, and based upon the results provided in the Tables below, the following antigens are considered to be preferred vaccine candidates: Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261 Ains and Y 146B .
Table 13 Gasbarrini Panel Yi39 Y184A Y153Ac5.2 Z9A Y128D12insY261AinsY124As Total number 15 5 9 12 4 16 16 19 of reactive samples Number of decreased6 3 6 8 2 12 9 12 titer in 12 month Number of non- 9 2 3 4 2 4 7 7 decreased titer in 12 month Percent decreased40 60 67 67 50 75 56 63 titer in 12 month Percent non- 60 40 33 33 50 25 44 37 decreased titer in 12 month ..__ __-...t... ....._.-____ _..... ...._ ~..
WO 98/49314 ' PCT/US98/08487 Y146B I Y175AI I C7.2Cb8c7.7I ~ A22 CL20 C1S.11~ B2d1Y104 Total number of 16 20 22 16 16 12 18 22 8 reactive samples Number of decreased 4 8 16 11 11 5 ~ 4 5 titer in 12 month IO
Number of non-decreased12 12 6 5 5 7 8 18 3 titer in 12 month Percent decreased 25 40 73 69 69 42 56 18 63 titer in 12 month Percent non- 75 60 27 31 31 58 44 82 37 decreased titer in 12 month Table I4 Greenberg Panel Y139Y184A Y153A c5.2 Z9A Y128D12insY261AinsY124As Total number of 8 7 6 8 2 8 9 9 reactive samples Number of decreased7 3 5 6 I 7 4 7 titer in 24 month I
Number of non-decreased1 4 I 2 1 1 5 2 titer in 24 month 2S Percent decreased88 43 83 75 50 88 44 78 titer in 24 month Percent non- 12 57 17 25 50 12 56 22 decreased titer in 24 month Y146B Y175AC1S.11 ~C7.2Cb8c7.7B2d1 Y104 A22 CL20 f Total number 8 8 12 3 3 5 9 12 9 of reactive samples Number of decreased4 7 8 2 2 3 6 8 7 titer in 24 month Number of non- 4 1 4 1 1 2 3 4 2 decreased titer in 24 month Percent decreased50 88 67 67 67 60 67 67 78 titer in 24 month Percent non- 50 12 33 33 33 40 33 33 22 decreased titer in 24 month While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.
_.__ _ _~~__ ___.___ . T. ____ .. CA 02288211 1999-10-25 s DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTS PARTIE DE CETTE DE~VIANDE OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE[~
NOTE. Pour les tomes additionels, veuitlez contacter le Bureau canadien des brevets THiS SECTION OF THE APPL.ICATION/PATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ,~ OF
' NOTE: For additional volumes-phase cantaci the Canadian Patent Ofific~ .
. (44). the DNA fragment having the sequence spanning cluster 44 (SEQ ID
N0:519);
(45). the DNA fragment having the sequence spanning SEQ ID NOS:186, 195, 185, (cluster 45, SEQ ID N0:520);
(46). the DNA fragment having the sequence spanning SEQ ID N0:251 (cluster 46, SEQ ID
N0:521 );
(47). the DNA fragment having the sequence spanning cluster 47 (SEQ ID NOs:522, 523);
(48). the DNA fragment having the sequence spanning SEQ ID
NOS:190, 189 (cluster 48, SEQ ID
N0:524);
(49). the DNA fragment having the sequence spanning SEQ ID
N0:200 (cluster 49, SEQ ID
N0:525);
(50). the DNA fragment having the sequence spanning SEQ ID
NOS:211, 210 (cluster 50, SEQ ID
N0:526);
(51), the DNA fragment having the sequence spanning SEQ ID
NOS:168, 81, 132, 82, 121, 133, 167, 228 (cluster 51, SEQ
ID N0:527);
(52). the DNA fragment having the sequence spanning SEQ ID
N0:146 (cluster 52, SEQ ID
N0:528);
(53). the DNA fragment having the sequence spanning cluster 53 (SEQ ID N0:529);
(54). the DNA fragment having the sequence spanning SEQ ID
NOS:221 and 220 (cluster 54, SEQ
ID N0:530);
(55). the DNA fragment having the sequence spanning SEQ ID
N0:74 (cluster 55, SEQ ID
N0:531);
(56). the DNA fragment having the sequence spanning SEQ ID
N0:72 (cluster 56, SEQ ID
N0:532);
(57). the DNA fragment having the sequence spanning SEQ ID
N0:70 and 71 (cluster 57, SEQ ID
N0:533);
(58). the DNA fragment having the sequence spanning SEQ ID
NOS:96, 97 (cluster 58, SEQ
ID NOs:534,535);
(59). the DNA fragment having the sequence spanning SEQ ID
NOS:105 and 106 (cluster 59, SEQ
ID NOs:536, 537);
(60). the DNA fragment having the sequence spanning SEQ ID
N0:107 (cluster 60, SEQ ID
N0:538);
(61). the DNA fragment having the sequence spanning SEQ ID
N0:109 (cluster 61, SEQ ID
N0:539);
(62). the DNA fragment having the sequence spanning SEQ ID
NOS:111, and 112 (cluster 62, SEQ
ID N0:540);
(63). the DNA fragment having the sequence spanning cluster 63 (SEQ ID N0:541);
(64). the DNA fragment having the sequence spanning SEQ ID
N0:58 (cluster 64, SEQ ID
N0:542);
(65). the DNA fragment having the sequence spanning cluster 65 (SEQ ID
N0:543);
(66). the DNA fragment having the sequence spanning SEQ ID N0:90 (cluster 66, SEQ ID
N0:544);
(67). the DNA fragment having the sequence spanning SEQ ID N0:13 (cluster 67.
SEQ ID
N0:545);
(68). the DNA fragment having the sequence spanning SEQ ID N0:47 (ciuster 68, SEQ ID
N0:546);
- (69). the DNA fragment having the sequence spanning SEQ ID N0:16 (cluster 69, SEQ ID
N0:547).
According to another embodiment, a H. pylori polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:6, SEQ ID N0:8, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:20, SEQ ID N0:24, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:43, SEQ ID N0:47, SEQ ID N0:51, SEQ ID N0:54, SEQ ID
N0:58, SEQ ID N0:60, SEQ ID NOs:70-230, SEQ ID N0:248, SEQ ID N0:251, SEQ ID
N0:253, SEQ ID N0:255, SEQ ID N0:257, SEQ ID N0:259, SEQ ID N0:264, SEQ ID N0:270, SEQ
ID
N0:271, SEQ ID N0:322, SEQ ID N0:549, where these DNA sequences correspond to cloned and sequenced regions of the H. pylori genome encoding highly immunogenic proteins.
In a preferred embodiment, a H. pylori antigen coding polynucleotide is one that is capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D}.
Alternatively, a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
In yet another embodiment, a H. pylori antigen coding polynucleotide according to the invention is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:6, SEQ ID N0:8, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:20, SEQ ID N0:24, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:43, SEQ ID N0:47, SEQ ID N0:51, SEQ ID N0:54, SEQ ID
N0:58, SEQ ID N0:60, SEQ ID NOs:70-230, SEQ ID N0:248, SEQ ID N0:251, SEQ ID
N0:253, SEQ ID N0:255, SEQ ID N0:257, SEQ ID N0:259, SEQ ID N0:264, SEQ ID N0:270, SEQ
ID
N0:271, SEQ ID N0:322, SEQ ID N0:549.
In yet another embodiment, a H. pylori antigen coding polynucleotide is composed of at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID
N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
In another aspect, the invention includes a H. pylori polypeptide antigen, in substantially purified form, characterized by immunoreactivity with H. pylori positive anti-sera. An antigen in accordance with the invention is, in one embodiment, encoded by a polynucleotide that is typically at least 18 nucleotides in length, having the features described above. More specifically, the antigen is encoded by all or a portion of a polynucleotide sequence at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
Additional antigens according to the invention are encoded by H. pylori antigen coding sequences as described above.
In a preferred embodiment, an antigen is encoded by a polynucleotide at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA
sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID
N0:38 (C1), SEQ ID
NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5}, SEQ ID
N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Alternatively, the invention encompasses a H. pylori antigen comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ
ID NOS:340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
In a particular embodiment, a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554.
In yet another embodiment, a H. pylori antigen comprises at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID NOS:555-602.
Alternatively, a H. pylori antigen comprises a polypeptide sequence selected from the group consisting of SEQ ID NOs:555-602.
In one particularly preferred embodiment, a H. pylori antigen is one comprising at least 6 contiguous amino acids contained within a sequence selected from the group consisting of SEQ ID
N0:44 (A22), SEQ ID N0:39 (C1), SEQ ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID
N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ
ID N0:573 (Y128D).
Also forming part of the invention is a diagnostic kit for use in screening a biological fluid such as sera for the presence of anti-H. pylori antibodies. The kit includes a substantially purified H.
pylori antigen of the type characterized above that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of the antibody to the antigen.
The polypeptide antigen may be attached to a solid support, and the kit may further include a non-attached reporter-labelled anti-human antibody, where binding of the anti-H. pylori antibodies to the polypeptide antigen can be detected by binding of the reporter-labelled antibody to the anti-H. pylori antibodies.
According to one embodiment, the kit includes at least two H. pylori antigens having different antibody specificities.
In a related aspect, the invention includes a method of detecting H. pylori infection in a subject, or detecting the eradication of the bacteria in a previously infected subject. The method involves reacting a biological fluid sample from a subject with a purified H.
pylori polypeptide antigen of the type described above, and examining the antigen for the presence of bound antibody.
Preferred antigens for use in the method correspond to one of the following polypeptide I5 sequences or a contiguous region contained therein: SEQ ID N0:44 (A22), SEQ
ID N0:39 (C1), SEQ
ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ
ID N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ ID N0:573 (Y128D).
In still another aspect, the invention includes a H. pylori vaccine composition containing a H.
pylori polypeptide antigen of the type described above. The antigen is characterized by its ability to reduce the level of H. pylori infection in a mammalian model system, such as a mouse or rhesus monkey challenged with the peptide, and then infected with H. pylori.
Preferred antigens for use in a vaccine are those which invoke a long-lasting antigenic response, as evidenced by the persistence of antibodies for an extended period of time subsequent to antimicrobial treatment. Representative antigens for use in a vaccine composition are selected from the group consisting of SEQ ID N0:565 (Y139), SEQ ID N0:575 (Y146B), SEQ ID
N0:555 (Y175A), SEQ ID N0:44 (A22), SEQ ID N0:569 (Y184A), SEQ ID N0:578 (Z9A), SEQ
ID
N0:557 (Y261A) and SEQ ID N0:575 (Y146B).
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.
Brief Description of the Fi ,gores Fig. 1 is a computer scanned image of a Western blot of a 2-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis (high pH
conditions) of native H.
pylori antigens blotted with Roost pooled sera (H. pylori positive);
Fig. 2 is a computer scanned image illustrating a Western blot of a sodium dodecyl sulfate polyacrylamide gel 2-dimensional electrophoretic (SDS-PAGE) analysis (low pH
conditions) of native H. pylori antigens blotted with Roost pool;
Fig. 3 shows a schematic representation of the amino acid translation of ORF3 of clone Y 104-l.asm (299 amino acids). Regions of sequence indicated in the figure were confirmed by amino acid sequence analysis of the Y104-l.asm protein expressed in E. coli strain XLlBlue, and correspond to (i) the 36 kD protein of H. pylori (indicated by underline), (ii) "spot 15"
peptides (indicated by a box), and (iii) peptides encoded by clone d7 (bracketed);
Fig. 4 is a schematic representation of the in vivo processing pathway of the 36 kD protein of H. pylori and its relationship to the "spot 15" antigen disclosed herein;
Fig. 5 is a reverse phase HPLC peptide profile corresponding to the "spot 15"
antigen isolated from H. pylori (ATCC 43504) which illustrates the presence of numerous identifiable protein peaks;
Fig. 6 presents the amino acid sequence of the 36-kD protein of H. pylori, where underlining indicates the 28 kD protein region;
Fig. 7 is a schematic representation of the proteome methodology employed to identify several native antigens of H. pylori;
Fig. 8 is a graphical representation summarizing percent sensitivity of various clones against representative H. Pylori-immunopositive sera panels;
Fig. 9 is a linear representation of the H. pylori genome, indicating the approximate positions of immunogenic cluster regions forming one aspect of the present invention;
Figs. 10-63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5}, (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (53), (54), (58), (59), (61), (62), (68), and (69), respectively;
Figs. b4A-D present a summary of the immunogenic clone clusters of the present invention including (i) cluster number, (ii) clones defining the start and end regions of each cluster, coordinates of the cluster consensus region within the H. pylori genome, and expression data;
Fig. 65 is a graphical representation of comparative sensitivities of H.
pylori recombinant antigens against various sera panels. Sensitivity values were calculated using various gold reference standards as described in Example 6B;
Fig. 66 is a graphical representation of comparative specificities of H.
pylori recombinant antigens against various sera panels, computed against gold standards as indicated; and _.. __. .....__._~ _T.. _..._.. .~_ ._ _- ,~.._ Figs. 67A and 67B provide a tabular summary of (i) immunopositive clones forming the basis of the invention, (ii) their corresponding cluster numbers (indicating the relative position of each clone and cluster within the H. pylori genome), and relative (iii) sensitivity and (iv) specificity values.
Detailed Description of the Invention I. Definitions The following terms, as used herein, have the meanings as indicated:
- A polypeptide sequence or fragment is "derived" from another polypeptide sequence or fragment when it has the same sequence of amino acid residues as the corresponding region of the fragment from which it is derived.
A polynucleotide sequence or fragment is "derived" from another polynucleotide sequence or fragment when it has the same sequence of nucleic acid residues as the corresponding region of the fragment from which it is derived.
A first polynucleotide fragment is "selectively-hybridizable" to a second polynucleotide fragment if the first fragment or its complement can form a double-stranded polynucleotide hybrid with the second fragment under selective (stringent) hybridization conditions.
The first and second fragments are typically at least 15 nucleotides in length, preferably at least 18-20 nucleotides in length.
Selective (stringent) hybridization conditions are defined herein as hybridization at -45°C in --1.1M
salt followed by at least one wash at 37°C in 0.3M salt. Such conditions typically allow at most about 25-30 % basepair mismatches.
Two or more polynucleotide or polypeptide fragments have at least a given percent "sequence identity" if their nucleotide bases or amino acid residues are identical, respectively, in at least the specified percent of total base or residue position, when the two or more fragments are aligned such that they correspond to one another using a computer program such as ALIGN.
(The ALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs, Pearson and Lipman, 1988; Pearson, 1990).
An "H. pylori polynucleotide" as used herein, refers to a polynucleotide sequence derived from the genome of H. pylori and variants thereof. H. pylori polynucleotides of the type disclosed herein encode H. pylori polypeptide antigens, where the resulting antigen is characterized by immunoreactivity with H. pylori positive anti-sera. Generally, a H. pylori polynucleotide of the . invention will be at least about 18 or more nucleotides in length (i.e., encoding a 6 peptide-antigen).
In an alternative embodiment, the H. pylori polynucleotide will be at least about 24 nucleotides in length (i. e. , encoding an 8 peptide-antigen). In yet another embodiment, the H. pylori polynucleotide will be at least about 30 nucleotides in length. In some instances, the H.
pylori polynucleotide will range from about 45 to 75 nucleotides in length, but may of course be longer.
The polynucleotides WO 98/49314 ' PCT/US98/08487 of the invention may be obtained from natural or synthetic sources, or, may be prepared recombinantly. The polynucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, by deletions, by insertions and inversions, to a particular naturally-occurring sequence, provided that the subject polynucleotide is capable of expressing a H. pylori antigen as described herein. The polynucleotide sequence may optionally contain expression control sequences (typically from a heterologous source) positioned adjacent to the coding region.
The nucleotide sequences described herein are meant to encompass variants possessing essentially the same "sequence identity" as defined above. Nucleotide sequences having essentially the same sequence identity are typically selectively hybridizable to one another under selective (stringent) hybridization conditions. This is to say, a nucleic acid fragment is considered to be selectively hybridizable to a H. pylori polynucleotide if it is capable of specifically hybridizing to the H. pylori polynucleotide sequence or a variant thereof (e.g., a probe that hybridizes to a H. pylori polynucleotide but not to polynucleotides from other members of the Helicobacter family) under stringent hybridization and wash conditions.
An "H. pylori antigenic polypeptide" is meant to encompass immunoreactive variants of the polypeptide, or regions, or parts thereof, provided that the variant is immunogenic. A suitable variant is defined as any polypeptide having a sequence that is identical (i. e. , shares sequence identity) to that of a H. pylori polypeptide.
For example, an antigenic polypeptide that is essentially identical to a H.
pylori polypeptide antigen is (i) encoded by a nucleic acid that selectively hybridizes to sequences of H. pylori or its variants or (ii) is encoded by H. pylori or its variants.
A sequence comparison may also be employed for the purpose of determining "polypeptide homology", e.g., by using the local aligtnnent program LALIGN. In carrying out such a determination, a polypeptide sequence is typically compared against a selected H. pylori amino acid sequence or any of its variants, as defined above, using the LALIGN program with a ktup of 1, default parameters and the default PAM.
Any polypeptide with an optimal alignment longer than about 6 to 8 amino acids and greater than 70 % , or more preferably 75 % to 80 % of identically aligned amino acids is considered to be a "homologous polypeptide. " The LALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson, et al. , 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 44.0, Jordan Hall, Charlottesville, VA).
Sequence variations among antigens will depend upon a number of factors, such as the strain of H.
pylori, the location of the gene or gene family encoding the antigen within the H. pylori genome (i.e., whether the gene is highly conserved or prone to recombination) and the like.
_ _ _ __ _..... _.__~__ An immunogenic polypeptide or polypeptide "fragment" is one that is (i) encoded by an open reading frame of a H. pylori polynucleotide, or (ii) displays sequence identity to H. pylori polypeptides as defined above, and is immunoreactive with a H. pylori immunopositive sample, such as sera.
Typically, the immunogenic fragment will comprise at least about 6 to 8 amino acids, and preferably at least about 10 to 12 contiguous amino acid residues of a particular antigen.
By immunoreactive variant of a H. pylori polypeptide antigen is meant an amino acid substitution, deletion, and/or addition variant of a particular H. pylori polypeptide antigen sequence disclosed herein, having substantially the same or increased binding affinity to a given antibody as the particular polypeptide antigen, as determined by conventional methods, e.g., a competition assay or a two antibody sandwich assay.
A representative H. pylori antigen is composed of at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:340-468, where the antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
"Cluster sequences" correspond to regions within the H. pylori genome encoding highly immunogenic polypeptides. The cluster sequences were determined on the basis of DNA sequence information for the over 250 immunogenic clones described herein. As a result, 69 unique clusters were identified, and antigens in accordance with the invention are those encoded by a contiguous series of nucleotides contained within any of the 69 clusters. Representative antigen coding sequences falling within each of the clusters are summarized in Figs. 64A-D. The cluster regions are referred to herein as clusters 1 to 69, corresponding to SEQ ID NOs: 340-468.
The location of each of the cluster regions within the H. pylori genome is llustrated pictorially in Fig. 9. In some instances, a cluster is defined by various regions within the H. pylori genome rather than by a single sequence. For example, a particular cluster may be defined by a "start"
sequence, an "end" sequence, and perhaps an invervening "middle" sequence, and thus may correspond to more than one sequence contained in the Sequence Listing. In such cases, the descriptor for the cluster sequence will indicate its relationship to a given cluster (e.g., start, middle, end).
Typically, a defining cluster sequence will code for one or more antigens, and the remainder of the sequence for a particular cluster, if not explicitly provided, can be readily determined based upon the information provided herein, when considered along with the information, e.g., provided in Tomb, et al., 1997. Cluster sequences defined by more than one representative sequence are cluster 1 (SEQ
ID NOs: 469, 470), cluster 5 {SEQ ID NOs:474,475), cluster 7 (SEQ ID N0:477, end), cluster 15 (SEQ ID NOs:485, 486), cluster 35 (SEQ ID NOs:506-508), cluster 40 (SEQ ID
NOs:513, 514), cluster 41 {SEQ ID NO:515, end), cluster 43 (SEQ ID NOs:517, 518), cluster 47 (SEQ ID NOs:522, 523), cluster 49 (SEQ ID N0:525, end), cluster 58 (SEQ ID NOs:534, 535), and cluster 59 (SEQ ID
N0:536, 537).
"Substantially purified" and "in substantially purified form" are used in several contexts and typically refer to at least partial purification of a H. pylori polynucleotide or polypeptide away from unrelated or contaminating components (e.g., serum, cells, proteins, non-H.
pylori polynucleotides, etc.) by at least one purification or isolation step. Methods and procedures for the isolation or purification of compounds or components of interest are described herein (e.g., SDS-PAGE, affinity purification of fusion proteins, blotting, and recombinant production of H.
pylori polypeptides).
An antigen is "specifically immunoreactive" with H. pylori positive anti-sera or a biological fluid sample when, under optimal conditions, the antigen binds to antibodies present in the H. pylori infected sample but does not bind to antibodies present in the majority (greater than about 60 to 65 %, preferably from about 70% to 80%, even more preferably greater than about 85%) of fluid samples from subjects who are not or have not been infected with H. pylori.
"Specifically immunoreactive"
antigens may be immunoreactive with monoclonal or polyclonal antibodies generated against specific H. pylori antigens.
By biological fluid is meant any fluid derived from the body of a mammal, particularly a human. Representative biological fluids include blood, serum, plasma, urine, faeces, mucous, gastric secretions, dental plaques, or saliva.
"Immunologically effective amount" refers to an amount administered to a mammalian host, either as a single dose or as part of a series, that is effective for treatment or prevention of infection by H. pylori. The amount will vary depending upon the health and physical condition of the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, and the like. Such an amount will typically fall within a relatively broad range, and can be determined in routine trials.
II. Isolation of H. pylori Anti enic Se9uences The present invention is based on the identification and isolation of a number of highly immunogenic H. pylori polypeptides, resulting from the screening of over a million individual H.
pylori compositions. The antigens of the present invention were either produced recombinantly, or, were separated from a mixture of soluble proteins obtained from pelleted and lysed H. pylori.
Moreover, as a result of an intensive computational analysis effort, disparate sequence information corresponding to all of the disclosed immunogenic clones was compiled to provide a collection of heretofore unrecognized antigenic cluster regions contained within the genome of H. pylori The preparation and identification of recombinant H. pylori antigens and their classification into clustered families will now be described.
__ T . -_~___ __ 1. Method for Screening Recombinant Sublibraries The H. pylori antigens of the invention can be obtained from phage libraries using conventional screening methods described below. Unless otherwise stated, the DNA lambda libraries described herein have been deposited in the Genelabs Technologies, Inc.
Culture Collection, 505 Penobscot Drive, Redwood City, CA, 94063, or in the Genelabs Diagnostics PTE
LTD Culture Collection, 85 Science Park Drive #04-O1, The Cavendish, Singapore Science Park, Singapore 118259.
E. toll XL-1 Blue MRF plasmids containing inserts corresponding to the following H. pylori clones were accepted for deposit by the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on September 9, 1997 and assigned the following designation numbers: clone dHC1S.11 (ATCC 98525), clone dHGla2S.5 (ATCC 98526), clone Y212A (ATCC 98527), clone dHC7.2cd6 (ATCC 96528), clone y175A (ATCC 98529), clone y146B (ATCC 98530), clone dHA22.8 (ATCC 98531), Y184A (ATCC 98532), clone dHB2d1 (ATCC 98533), clone y104B (ATCC
98534). The bacteriophage lambda containing H. pylori DNA insert Y-library was accepted for deposit by the American Type Culture Collection on September 3, 1997 and assigned designation number ATCC 209234.
The antigen-encoding DNA fragments of the invention can be identified by (i) immunoscreening, as described above, and/or (ii) computer analysis of coding sequences using an algorithm (such as, "ANTIGEN," Intelligenetics, Mountain View, CA) to identify potential antigenic regions. For example, an antigen-encoding DNA fragment is subcloned, and the subcloned insert is then fragmented by partial DNase digestion to generate random fragments or by specific restriction endonuclease digestion to produce specific subfragments. The resulting DNA
fragments are then inserted into an expression vector, such as the lambda gtl l vector, and subjected to immunoscreening in order to provide an epitope map of the cloned insert.
In addition, DNA fragments of the type described herein can be employed as probes in hybridization experiments to identify overlapping H. pylori sequences, and these in turn can be further used as probes to identify additional sets of contiguous clones.
Any of the herein-described clone sequences can be used to probe a DNA
library, generated in a vector such as lambda gtl0 or "LAMBDA ZAP II" (Stratagene, La Jolla, CA).
Specific subfragments of known sequence may be isolated using the polymerase chain reaction or after restriction endonuclease cleavage of vectors carrying such sequences. The resulting DNA fragments can be used as radiolabelled probes against any selected library. In particular, the 5' and 3' terminal sequences of the clone inserts are useful as probes to identify additional clones.
Further, the sequences provided by the 5' end of cloned inserts are useful as sequence specific primers in first-strand DNA synthesis reactions (Maniatis, et al., 1982;
Scharf, et al., 1986). For example, specifically primed H. pylori DNA libraries can be prepared by using specific primers derived from one of the cloned DNA sequences described herein as a template.
The second-strand of the new DNA is synthesized using RNase H and DNA polymerase I. The above procedures identify or produce DNA molecules corresponding to nucleic acid regions that are 5' adjacent to the known clone insert sequences. These newly isolated sequences can in turn be used to identify further flanking sequences, and so on. After new H. pylori sequences are isolated, the polynucleotides can be cloned and immunoscreened to identify specific sequences encoding H. pylori antigens.
2. Recombinant Anti ens In order to identify new and highly antigenic polypeptides useful for detection of H. pylori, DNA libraries were prepared from commercially available strains of H. pylori (American Type Culture Collection, Rockville, MD; ATCC Designation No. 43504, ATCC
Designation No. 43526) in either the expression vector lambda gtll or "ZAPII" (Stratagene, La Jolla, CA; Example 1).
Polynucleotide sequences were then selected for the expression of peptides which were immunoreactive with pooled sera obtained from 11 patients identified by endoscopy as H.
pylori-positive, herein identified as "Roost pool" sera, or another pool of 4 H. pylori-immunopositive sera samples identified as "SFA001 ". It is also possible to screen with other sources of H. pylori-infected samples, such as those described in Examples 6A and 6B, and referred to in Figs. 65 and 66, or described in Example 13. The samples may be individual samples, i. e. , derived from a single subject, or may be pooled samples, such as those described herein.
Recombinant proteins identified by this approach provided candidates for polypeptides that can serve, either singly or in combination, as substrates in diagnostic tests for detecting infection by H. pylori. Further, the corresponding nucleic acid coding sequences serve as useful hybridization probes for the identification of additional H. pylori antigen coding sequences.
The H. pylori strains described above were used to generate DNA libraries in lambda vectors (Example 1). Other commonly available strains of H. pylori include, for example, H. pylori samples identified as strain # 29995 and J-170. Alternatively, libraries can be constructed from H. pylori isolated from a sample confirmed as H. pylon-positive. In the method illustrated in Example 1, the libraries were generated from genomic DNA isolated from the pelleted bacteria.
Alternatively, centrifugation can be used to pellet bacteria from infected biological specimens such as gastric mucosa.
In reference to Example 1, H. pylori DNA libraries were generated using DNase-digested genomic DNA fragments isoiated from H. pylori as starting material. For preparing the "short antigen clone" library, the resulting molecules were ligated to Sequence Independent Single Primer Amplification (SISPA; Reyes, et al., 1991) linker primers and expanded in a non-selective manner, and then cloned into a suitable vector, for example, lambda gtll or ZAPII, for expression and _ ____.___ _ _.T ._ ....._ __-~_ screening of peptide antigens. The libraries disclosed herein have been designated as the "short antigen clone" library, typically designated by a prefix beginning with the letters Y or Z; and the "long antigen clone" library, designated as libraries 1 and 2.
The ZAPII libraries 1 and 2 were similarly constructed, with the following exceptions. The ZAPII libraries 1 and 2 were generated from longer H. pylori DNA fragments, i.e., either EcoRI or HindIII-digested genomic DNA which had not undergone Sequence Independent Single Primer Amplification. Library 1 clones (designated herein by upper case letters) were generated by ligating EcoRI-digested H. pylori DNA directly into the EcoRI sites of the lambda "ZAPII" vector. Library 2 clones (lower case designations) were obtained by digesting H. pylori genomic DNA with HindIII, then blunt ended with E. coli Klenow enzyme or T4 DNA polymerase enzyme and the resulting blunt ended fragments were then ligated to SISPA primers. The linker-ligated DNAs were then treated with EcoRI and ligated into the EcoRI sites of "ZAPII" lambda arms.
Lambda gtl l is a particularly useful expression vector for producing H.
pylori antigens. The vector contains a unique EcoRI insertion site, located 53 base pairs upstream of the translation termination codon of the /3-galactosidase gene. Thus, an inserted sequence is expressed as a /3-galactosidase fusion protein which contains the N-terminal portion of the /3-galactosidase gene product, the heterologous peptide, and optionally the C-terminal region of the a-galactosidase peptide (the C--terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon).
The lambda gtll vector also produces a temperature-sensitive repressor (cI857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C. Advantages of lambda gtl l include: (1) highly efficient recombinant clone generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) production of recombinant fusion protein. Further, since phage containing a heterologous insert produces an inactive /3-galactosidase enzyme, phage with inserts are typically identified using a colorimetric substrate conversion reaction employing /3-galactosidase.
In addition to the lambda gtll vector, numerous E. coli expression vectors are useful for expression of antigens. Alternative microbial hosts suitable for expression include bacilli, such as B.
subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In such hosts, the expression vectors will typically contain control sequences compatible with the host cell. Other known promoter sequences may be present in the expression vector, such as the lactose promoter system, a tryptophan promoter system, or a promoter system from phage lambda.
An amino terminal methionine can be provided, if necessary, by insertion of a Met codon in-frame with the antigen. The carboxy terminal extension of an antigen can be removed by conventional mutagenesis procedures.
Additionally, yeast expression systems can be used, such as the Saccharomyces cerevisiae pre-pro-alpha-factor leader region used to direct protein expression from yeast.
The antigen coding sequence can be fused in frame to the leader region. This construct is then typically put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as ~-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. For constructs used for expression in yeast, protease cleavage sites may be inserted to facilitate separation of the fusion protein components.
Additionally, mammalian cells can be used for expression of the antigens of the invention.
Vectors useful for expression in mammalian cells are typically characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation (polyA) signal. The vectors may optionally include selectable marker genes, such as those conferring antibiotic resistance.
Suitable host cells include Chinese hamster ovary cells (CHO) cell lines, HeLa cells, myeloma cells, Jurkat cell lines, and the like. The expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer, information processing sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
The DNA sequences are expressed in hosts after the sequences of interest have been operably linked to (i. e. , positioned to enable the functioning of) an expression control sequence.
Experiments carried out in support of the present invention will now be described, and in particular, the generation of various phage libraries and characterization of inserts coding for the H.
pylori antigens of the invention.
a. Representative H. pylori Antigens: Short Antigen Clone Library. Example 1 describes the preparation of a DNA library for H. pylori (ATCC No. 43504).
The library was immunoscreened using H. pylori-positive pooled sera (Example 2). A number of lambda clones were identified which were immunoreactive with anti-H. pylori antibodies present in the pooled sera.
Selected immunopositive clones were plaque-purified and their immunoreactivity retested. The immunoreactivity of the clones with normal human sera (control, H. pylori-negative) was also tested.
Numerous clones were identified by immunosceening and further characterized, as described in Examples 3 and 4. Immunoreactive clones further described in Example 3 and referred to herein _. _ ____ -... __T _... -T--. _ _._ as the Y and Z families of clones include clone Y-104-1, and the clones summarized in Table 2, corresponding to SEQ ID NOs: 70-230.
To obtain the polypeptide antigens of the invention, and their respective coding sequences, the DNA inserts of the immunoreactive recombinant lambda clones were PCR
amplified, using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors, and utilizing each immunoreactive clone as template. For the lambda gtl l clones, gtllF (SEQ ID N0:65) and gtl l R (SEQ ID N0:66) primers were used. For the lambda ZAPII clones, T3 (SEQ ID N0:67) and T7 (SEQ ID N0:68) primers were used.
The resulting amplification products were agarose gel purified and eluted from the gel (Ausubel, et al., 1988) to remove primers and other components. The purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced. Clones exhibiting immunoreactivity against H. pylori-positive pooled sera are identified in Example 3, and more specifically in Table 2.
Sequencing was carried out using "DYEDEOXY TERMINATOR CYCLE SEQUENCING"
(a modification of the procedure of Sanger, et al., 1977) on a Perkin Elmer Applied Biosystems model 373A DNA sequencing system according to the manufacturer's recommendations (Perkin Elmer Applied Biosystems, Foster City, CA). Sequence data is presented in the accompanying Sequence Listing.
Sequences for the Y and Z families of clones were compared with "GENBANK", EMBL
database and dbEST (National Library of Medicine) sequences at both nucleic acid and amino acid levels. Search programs FASTA, BLASTP, BLASTN and BLASTX (Altschul, et al., 1990) indicated that these sequences were unique as both nucleic acid and amino acid sequences.
In instances where the clones were determined to be significantly longer than the predicted coding regions of the proteins, the genomic clones were digested by restriction enzyme treatment, and the resulting subfragments were inserted into a suitable expression vector.
The resulting subclones, containing the specific digested DNA fragments, were then screened for immunogenicity. Clones identified as immunoreactive towards H. pylori-positive pooled sera were plaque purified. Plasmid DNA containing inserts obtained by recovery from phage were sequenced as described above and also in Example 4.
b. Representative H. pylori Antigens: ZAPII Libraries 1 and 2. Library 1 and library 2 clones isolated by immunoscreening with H. pylori immunopositive pooled sera include the following: LIBRARY 1: A3, A22, B2, B9, B17, B23, C1, C3, C7, DH, and Ltsx~lty 2: al, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, dl l, d7, e6, f3, f8, fl l, g2, g9, gll, k4.
Clone Gla was isolated by screening with monoclonal antibody 1G6 from Biogenesis, Inc. (Bournemouth, England). Sequence data for these clones is presented in the sequence listing herein.
Exemplary procedures for generating and isolating the library 1 and 2 subclones for preparing the antigens of the invention, including features of the derived coding sequences and putative proteins, are provided above and described in detail in Example 3.
c. Clone d7 and its Relationship to the 36 kD Protein of H. ~,ylori. Clone d7, another immunoreactive clone of the present invention, is a HindIII clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII to produce a beta-galactosidase fusion protein. The nucleotide sequence is presented as SEQ ID
NO:11.
Polynucleotides and polypeptides derived from this clone represent preferred embodiments of the invention.
As will be described in further detail below, this clone codes for about 70%
of the carboxy-end of the 36 Kd native protein of H. pylori, while clone Y104 codes for the entire 36 K protein.
Based upon the results of sequencing experiments carried out in support of the invention (Examples 8 and 9), portions of the amino acid sequence of the native 36 Kd protein of H. pylori have been determined. The 36 kD protein (encoded in part by clone d7) appears to be a precursor to a highly antigenic H. pylori protein referred to herein as the "spot 15" protein (Examples 8,9, to be described in more detail below). This band appears to be Western Blot positive in the majority of H. pylori-positive samples, and is typically absent in normal samples. This antigenic protein thus appears to be highly indicative of infection by H. pylori. The in-vitro processing of the 36 kD protein (i.e, the apparent molecular weight) is illustrated schematically in Fig. 4.
As shown in Fig. 4, the native H. pylori translation product, a 34 kD protein (i.e, calculated molecular weight) composed of 299 amino acids, appears to be cleaved in vivo at amino acid position 23. This results in a 31.6 kD cleavage product commonly referred to as "the 36 kD protein" of H.
pylori. The differences in molecular weight terminology in referring to this protein arise due to the following: 36-kD is observed from SDS-PAGE; 34-kD is calculated from the corresponding DNA
sequence (Fig. 6); and 31 kD is determined from experimental sequence data, as determined starting from residue 23 of SEQ ID N0:60.
A post translation modification of the 36 kD protein, i. e. , acetylation at the amino acid terminus (position 23) and cleavage at the carboxy end, results in the "spot 15" antigen, having a molecular weight of 28 kD. Referring to Fig. 4, "X"s correspond to positions where deamidations (i. e. , asparagine or glutamine) or point mutations have occurred. Further proposed modifications leading to the minor spot 15 protein are also indicated. The antigenic polypeptide corresponding to spot 15, and sequences coding for this protein, e.g., clones Y-104 and d7, represent one particularly _____- _T __ ___-. ___-~-_ WO 98/49314 PG"T/US98/08487 preferred embodiment of the present invention. This is due to the strong antigenicity of the peptide.
Moreover, the spot 15 peptide has been detected in various strains of H.
pylori, as indicated in Table 9 and in Example I0.
The results discussed above and in the Examples indicate the isolation and identification of numerous new H. pylori polypeptide antigens, and polynucleotide sequences encoding such antigens.
d. Cluster Analysis and Identification of Anti enic Regions within the H
pylori Genome. The antigenic sequences described herein were determined based upon the screening of over a million discrete H. pylori antigenic compositions. DNA sequencing was carried out for H. pylori immunopositive clones, and open reading frames coding for antigenic proteins were identified.
Nucleic acid sequences coding for the thus-identified antigens were then inserted into expression vectors and expression of the desired antigenic protein was confirmed. As a result of this work, over 250 antigenic clone sequences have been identified and are disclosed herein.
In an effort to further correlate the sequence information for the various immunogenic clones, the clones were organized into cluster groups (1-69). The antigenic polynucleotide fragments isolated herein map into 69 clusters which are identified herein as clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69). The position of these antigenic clusters within the complete H. pylori genome is shown in Fig. 9. As can be seen from Fig. 9, the locations of the clusters within the genome (and clones within the clusters) are highly random, and represent only a small portion (i.e., approximately 2-3 % ) of the overall nucleotides contained within the entire genome.
Prior to this work, a comprehensive guide to the unique and highly antigenic regions within the genome of H. pylori was unknown.
For cluster analysis, all of the immunoclone sequences were combined into a FASTA database as defined by Pearson and Lipman (1988). This database was converted to a BLAST database for high speed searching (Altschul, et al., 1990). The sequence of each immunoclone was then searched against this database using the program BLASTN to define clusters. Clusters are an assembly of clones that contain identical sequences with other clones in the group. The sequences in each group or cluster were then combined in separate database files and formatted for entry in the GEL program of IG-Suite (Oxford Molecular). GEL then assembled the sequences and suggested a consensus sequence with ambiguities. A non-ambiguous sequence was determined for each cluster by editing the consensus sequence. When the cluster contained non-contiguous sequence, the genome sequence of H. pylori (TIGR World Wide Web site) was used merely to guide assembly.
Clusters 1 to 69 correspond to SEQ ID NOa 469-547. Open reading frames for the antigens were then determined from the cluster consensus sequence. Single clone sequences were translated directly to provide antigen sequences. Antigenic regions contained within each of the clusters as determined by translation of cluster open reading frames are provided as SEQ ID NOa 340-468.
Figs. 10 to 63 are linear maps indicating the relative positions of immunogenic subclones within the clusters (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), {21), (23), (25), (27), (28), (29), (30), (32), (33), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (SO), (51), (53), (54), (58), (59), (6I), (62), (68), and (69). Clusters not shown on the linear maps are clusters defined by one clone, i.e., clusters (22), (24), (26), (31), (34), (52), (55), (56), (57), (60), (63}, (64), (65), (66), (67).
Figs. 64A-D present a tabular summary of clusters 1-69, clones contained within each cluster, and coordinates of each cluster consensus region within the H. pylori genome.
During the course of this work, the complete genome sequence of H. pylori was reported (Tomb, et al., 1997), and is incorporated herein by reference. H. pylori possesses a circular genome of 1,667,867 base pairs and 1590 predicted coding sequences. The genome sequence reported on the TIGR Web site was used as a reference for reporting nucleotide positions of the clusters and immunogenic clones of the invention. Based upon the present work, it can be seen that a relatively small number of the predicted open reading frames reported for the H. pylori genome encode the antigenic proteins or cluster regions forming the basis of the present invention.
Each cluster defines a continuous DNA sequence that spans, i. e. , extends, from the 5' end of the most upstream clone in the cluster to the 3' end of the most downstream clone in the cluster.
Where the spanning sequence is incomplete, it has been filled in with sequence from the reported H.
pylori genomic sequences (TIGR Web site). For exampie, and with reference to Fig. 10, the sequence defined by cluster 1 includes the sequence beginning at the 5' end of clone Y92 (SEQ ID N0:222) and ending at the 3' end of clone Y92 (SEQ ID N0:223), including the short (about 730 bases) genomic sequence connecting the two clone sequences. The positions of the individual clones in each cluster are shown in Figs. 10-63, along with corresponding SEQ ID NOs.
The invention includes antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40}, (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51}, (52), (53), (54), (55), {56), (57), {58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69).
._~__- _._T..._.._.~_....___-..T-__..__ Preferably the antigen-coding regions are the spanning sequences themselves from the clusters.
Preferred polynucleotides are H. pylori antigen-coding DNA fragments, in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID N0:43 (A22), SEQ ID N0:38 (CI), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID
NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), and SEQ ID N0:98 (Y128D).
Thus, the clusters disclosed herein provide a non-random collection of H.
pylori antigen coding sequences and resulting antigens which have been shown to react with H.
pylori immunopositive samples and are useful in a variety of diagnostic applications.
IO
3. Isolation and Mappin;~ of Native Anti;e Experiments carried out in support of the invention have resulted in the concurrent identification of several additional native antigens of H. pylori (Table 6) using a proteomics approach.
An overview of the proteome methodology disclosed herein and utitilized to separate and identify over twenty H. pylori antigens is provided in Fig. 7 (right-hand side). In turning now to this figure, bacterial proteins are separated by two-dimensional (2D) electrophoresis. Immunoreactive spots (i. e. , reactive in Western blots with pooled sera from H. pylori infected patients) are then selected and subsequently characterized by endoproteolytic digestion, chromatography, and mass spectrometry (e.g., matrix assisted laser desorption time of flight mass spectrometry, MALDI-TOF).
Box A indicates the use of electrospray mass spectrometry to determine the mass of the intact protein.
Box B indicates the use of MALDI-TOF mass spectrometry to evaluate the number and mass of Lys-C
peptides. Box C indicates the use of MALDI-MS to evaluate chromatographically separated Lys-C
peptides and provide sequence information by post source decay (when peptides are pure). Box D
indicates the use of electrospray mass spectrometry to evaluate and sequence peptides through collision induced dissociation. These procedures can be used as an alternative to, or in parallel with, data obtained from conventional cloning techniques to derive genomic data corresponding to antigens of H. pylori, as shown by experiments carried out in support of the invention.
Antigens thus obtained are highly immunogenic, and may be isolated and characterized as illustrated in Examples 7-9 and described as follows.
To obtain soluble H. pylon proteins, pelleted H. pylori is typically lysed in a French press at > 10,000 PSI, followed by centrifugation. Alternative lysis methods include mechanical douncing or detergent disruption.
Antigens are generally obtained from whole lysates as follows. Fractionation of H. pylori soluble proteins is carried out by SDS-PAGE, preferably utilizing 2-dimensional electrophoresis (O'Farrell, 1975; O'Farrell, et al., 1977).
WO 98/49314 ' PCT/CJS98/08487 In performing a typical 2-dimensional electrophoretic separation, an isoelectric focusing gel separation is first carried out (first dimension gel). Isoelectric focusing is carried out, e.g., on a acrylamide/bisacrylamide gel, for an appropriate number of volt-hrs, determined as described in ~~CURRENT PROTOCOLS IN PROTEIN SCIENCE", Units 10.46, John Wiley and Sons, Iric., New York (1996). The final tube gel pH gradient is then measured by a surface pH
electrode. Protein components in a sample mixture undergo a first separation according to pI
value, as indicated on the horizontal axes of Figs. 1 and 2. The first dimensional separation can be carried under either acidic (Fig. 2) or basic (Fig. 1) conditions, depending upon the nature of the proteins to be separated.
A second dimensional, sized-based polyacrylamide slab gel separation is then carried out, to further separate the proteins on the basis of molecular weight. Suitable molecular weight markers are typically added to the gel, for determining corresponding molecular weights of eluted proteins.
Detection is typically carried out by Coomassi blue staining or by silver staining. Silver staining may be preferred, in some cases, since silver staining methods are considerably more sensitive and can be used to detect smaller amounts of protein.
Figs. l and 2 illustrate H. pylori antigens obtained generally as described above, which have been Western blotted with Roost H. pylori-positive serum pool and a negative serum pool, respectively. The numbers in each figure correspond to spots representing H.
pylori proteins.
Features of these proteins (including approximate molecular weights and pI
values), as identified by spot number, are summarized in Table 6. The polypeptides were confirmed to be immunoreactive when tested against anti-H. pylori-antibodies contained in the Roost pool sera. The identities of the spots indicated numerically on the gels were determined by a variety of protein sequencing techniques as described in Example 9.
To further identify these peptides, immunoreactive spots were excised from the gels, digested, and sequenced. As mentioned above, the native H. pylori antigens were further characterized by a combination of sequencing methodologies, including N-terminal sequencing, liquid-chromatography-mass spectrometry, and determination of internal sequences by amino-acid specific chemical cleavage, followed by Edman sequencing (Example 9).
Internal amino acid sequences can be determined by utilizing a combination of various site specific cleavage reagents, such as ortho-pthalaldehyde {OPA)/cyanogen bromide (CNBr), hydroxylamine, formic acid, and BNPS-skatole, which cleave as follows: CNBr (cleaves at C
terminus of methionine), BNPS-skatole (cleaves at C-terminus of tryptophan), formic acid (cleaves at Asp-Pro peptide bond), hydroxylamine (cleaves at Asn-Gly bond), and OPA, which distinguishes between secondary and primary amines; and enzymatic reagents such as Endo Protease L~rs-C, Endoproteinase ASP-N, Endoproteinase GLU-C, and T sin, which cleave respectively as follows:
Endo Protease Lvs-CC (cleaves at C-terminus of lysine), Endoproteinase ASP-N
(cleaves at N-terminus _. _. _ ~ _ ...__._ ~-__.. _ _ __~_ WO 98/49314 ~ PCT/US98/08487 of aspartic acid and cysteic acid), Endoproteinase GLU-C (cleaves at C-terminus of glutamic acid), and Trysin (cleaves at C-terminus of arginine and lysine).
The N-terminal sequence corresponding to each spot was determined (Table 6).
Utilizing the approaches described above and in Examples 7-9, the spots indicated in the 2-D
blots (Figs. 1 and 2) were identified as indicated in Table 6.
Referring to the low pH blot presented as Fig. 2, spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens. Spot 9 represents the native H.
pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al, while spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
Looking at the high pH Western blot in Fig. 1, spots 9 (major and minor), 10, 12, 13, and (major and minor) represent new antigens. The relationship between basic spot 15 and clones Y104 and d7 is discussed above and also illustrated in Fig. 3.
Mass spectral profiles of selected Lys-C digested H. pylori proteins corresponding to Western positive spots are provided in Tables 11 and 12. The mass "fingerprints" of the peptide digests were 15 then used as a basis for comparison to proteins predicted from various genomics databases (details are provided in Example 12) to further confirm the identify of selected H. pylori antigens described herein.
The above-described proteomic approach is useful for analyzing the genetic diversity of H.
pylori, examining antigen-antibody responses during acute and chronic infection, and with particular gastroduodenal pathologies and possible autoimmune components to H. pylori-associated disease.
Moreover, 2-D gel electrophoresis and in-situ proteolytic digestion in conjunction with MALDI-TOF
MS provides an extremely sensitive technique for the rapid identification of H. pylori antigens and for rapid screening for preferred vaccine and diagnostic candidates.
4. Exuression and Purification of Antieenic Polypentides and Preparation of Their Respective Antibodies a. Expression and Purification of Antigenic Polypeptides. The recombinant antigenic peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and amity chromatography.
H. pylori antigens in accordance with the invention comprise at least 6 contiguous amino acids contained within one of the following cluster antigen sequences: SEQ ID
NOS:340-468. In a particular instance, a H. pylori antigen may comprise at least 6 contiguous amino acids contained within a polypeptide sequence selected from one of the following SEQ ID NOS:
2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 2S-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and S50-SS4.
Preferably, a H. pylori antigen corresponds to at least 6 contiguous amino acids contained within a poIypeptide sequence selected from the group consisting of SEQ ID
NOS:555-602, where S these sequences represent illustrative expression proteins corresponding to H. pylori antigens. Even more preferably, a H. pylori antigen is identified by at least 6 contiguous amino acids contained within one of the following sequences: SEQ ID N0:44 (A22), SEQ ID N0:39 (Cl), SEQ ID
N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID
N0:55 (B2), SEQ ID N0:61 (Y104B), SEQ ID N0:573 (Y128D).
Polynucleotide sequences encoding the antigens of the present invention have been cloned in the plasmid p-GEX (Example 5) or various derivatives thereof (pGEX-de165). The plasmid pGEX
(Smith, et al. , 1988) and its derivatives express the polypeptide sequences of a cloned insert fused in-frame to the protein glutathione-S-transferase (sj26). In one vector construction, plasmid pGEX-hisB, an amino acid sequence of 6 histidines, is introduced at the carboxy terminus of the fusion protein.
The various recombinant pGEX plasmids can be transformed into appropriate strains of E.
coli and fusion protein production can be induced by the addition of IPTG
(isopropyl-thio galactopyranoside) as described in Example S. Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using Ni-NTA + + affinity chromatography (Example 5).
Insoluble fusion protein expressed by the plasmids can be purified by means of immobilized metal ion affinity chromatography (Porath, 1992) in buffers containing 6 M
Urea or 6 M guanidinium isothiocyanate, both of which are useful for the solubilization of proteins.
Alternatively, insoluble proteins expressed in pGEX-GLI or derivatives thereof can be purified using combinations of centrifugation to remove soluble proteins followed by solubilization of insoluble proteins and standard chromatographic methodologies, such as ion exchange or size exclusion chromatography, and other such methods are known in the art.
In the case of /3-galactosidase fusion proteins (such as those produced by lambda gtl 1 clones), the fused protein can be isolated readily by affinity chromatography, or by passing cell lysis material over a solid support having surface-bound anti-~i-galactosidase antibody.
Also included in the invention is an expression vector, such as the lambda gtll or pGEX
vectors described above, containing H. pylori antigen coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include a promoter, a translation initiation codon, translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.
_._.__ _ _ _ T ___. ___ __. __ . _._~...
WO 98/49314 PCT/US98/0848?
The DNA encoding the desired antigenic polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include, but are not limited to the following:
baculovirus expression (Reilly, et al., 1992; Beames, et al., 1991; Pharmingen, San Diego, CA; Clontech, Palo Alto, CA), vaccinia expression (Earl, et al., 1991; Moss, et al., 1991), expression in bacteria (Ausubel, et al., 1988;
Clontech), expression in yeast (Gellissen, et al. , 1992; Romanos, et al. , 1992; Goeddel, 1990; Guthrie and Fink, 1991), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, NY), e.g., Chinese hamster ovary (CHO} cell lines (Haynes, et al., 1983; Lau, et al., 1984; Kaufman, 1990).
These recombinant polypeptide antigens can be expressed directly or as fusion proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.
Expression of large polypeptide antigens is described in Example 5. Several of the long antigen clone sequences were cloned into expression vectors and successfully expressed in E. coli, as described in Example 5 and indicated in Tables 3a and 3b.
Expression in yeast systems has the advantage of commercial production.
Recombinant protein production by vaccinia and CHO cell line have the advantage of being mammalian expression systems. Further, vaccinia virus expression has several advantages including the following: (i) a wide host range; (ii) faithful post-transcriptionai modification, processing, folding, transport, secretion, and assembly of recombinant proteins; (iii) high level expression of relatively soluble recombinant proteins; and (iv) a large capacity to accommodate foreign DNA.
The recombinant expressed polypeptide-produced H. pylori polypeptide antigens are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography.
Immunoaffinity chromatography can be employed using antibodies generated based on the H. pylori antigens identified by the methods of the present invention.
The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides. In addition, amino acid sequences can be readily chemically synthesized using commercially available synthesizer (Applied Biosystems, Foster City, CA) or "PIN" technology {Applied Biosystems).
Antigens obtained by any of these methods can be used for antibody generation, diagnostic tests and vaccine development.
Exemplary amino acid sequences corresponding to expressed antigenic proteins of H. pylori are provided herein as SEQ ID NOs:555-602.
b. Antibodv Production. In another aspect, the invention includes specific antibodies directed against the polypeptide antigens of the present invention.
Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e.g., Pierce, Rockford, IL). Typically, to prepare antibodies, a host animal, such as a rabbit or a goat, is immunized with the purified antigen or fused protein antigen. Hybrid or fused proteins may be generated using a variety of coding sequences derived from other proteins, such as glutathione-S-transferase or /3-galactosidase. The host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen. These techniques are equally applicable to all immunogenic sequences described herein.
The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAF
Sephadex chromatography, amity chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
Alternatively, purified antigen or fused antigen protein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-derived hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a H.
pylori may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a suitable fusion partner can be used to produce human-derived hybridomas. Primary in vitro sensitization with viral specific polypeptides can also be used in the generation of human monoclonal antibodies.
Antibodies secreted by the immortalized cells are screened to determine the clones that secrete anti-bodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al., 1988).
Purified polyclonal or monoclonal antibodies directed against H. pylori antigens can then be used in any of a number of standard immunoassay formats to detect the presence of antigen, such as described in Harlow, et al., 1988. One representative assay format is an antigen capture sandwich assay. In a typical sandwich assay, antibody is immobilized on a solid support. H. pylori infected samples (e.g., feces, dental plaque, gastric biopsies, culture suspension from a biopsy sample) are then allowed to react with the immobilized antibodies, followed by incubation with a different antibody directed against H. pylori (e.g., whole lysate) and subsequent reaction with a secondary antibody carrying a reporter label. Detection of label in a testing region is indicative of the presence of H.
pylori antigen in a test, sample. The above-described assay is representative of any of an antigen-based __ ~__ ..T _~ - ___.___..__.._ T _ WO 98/49314 PCT/ITS98i08487 assay based on antibodies prepared as described above, useful for the early detection of H. pylori antigens in a sample suspected of infection by H. pylori.
5. ELISA and Protein Blot Screenin H. pylori antigens are first identified, typically through plaque immunoscreening as described above, and expressed and purified (as previously described). The antigens are then screened rapidly against a large number of suspected H. pylori positive anti-sera using alternative immunoassays, such as, ELISAs or Protein Blot Assays (Western blots) employing the isolated antigen peptide. The antigen polypeptide fusion protein is then isolated as described above, usually by affinity chromatography to the fusion partner such as ~i-galactosidase or glutathione-S-transferase.
Alternatively, the antigen itself is purified using antibodies generated against it (see below).
A general ELISA assay format may be employed, such as those described in Harlow, et al.
(1988). The purified antigen polypeptide or fusion polypeptide containing the antigen of interest, is attached to a solid support, for example, a multiwell polystyrene plate.
Biological fluid (e.g., sera) to be tested are diluted and added to the wells. After a period of time sufficient for the binding of antibodies to the immobilized antigens, the sera are washed out of the wells.
A labelled reporter antibody is added to each well along with an appropriate substrate: wells containing antibodies bound to the purified antigen polypeptide or fusion polypeptide containing the antigen are detected by a positive signal.
A typical format for protein blot analysis using one, any, several, or each of the polypeptide antigens of the present invention is presented in Example 6. General protein blotting methods are described by Ausubel, et al. (1988).
In Example 6A, the antigenic protein expressed by clone dHA22.8 (A22) was used to screen a number of sera samples (both pooled sera and discrete samples). The high percentage of sera reacting to antigen produced from recombinant clone dHA22.8 indicates that it is a dominant epitope, and that it is a suitable infection marker for H. pylori. The results presented in Example 6A
demonstrate that several different source H. pylori-positive anti-sera are immunoreactive with this representative polypeptide antigen. Similar results are described for recombinant protein expressed by clone dHCIS.II (C1). .
Additional experiments carried out in support of the invention as described in Example 6B
reveal, on the basis of sera paneling data using both single antigens and antigen combinations, that preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, C1, Y124A, Y261A, c5, C7, B2, Y104B, and Y128D.
These antigens are effective as serological markers for detecting active infection by H. pylori, based upon favorable sensitivity and selectivity features.
In Example 8, native proteins from H. pylori are shown to be immunoreactive with anti-H.
pylori primary antibodies obtained from "Roost" pooled serum.
The results presented above demonstrate that the polypeptide antigens of the present invention can, by these methods, be rapidly screened against panels of suspected H. pylori infected serum samples for the detection of H. pylori.
A. Protective Antibodies, Vaccines and the Generation of Protective Immunity a. Protective Antibodies. Protective antibodies can be identified using, for example, an animal model system (DuBois, et al. , 1996). To identify protective antibodies, polyclonal or monoclonal antibodies are generated against the antigens of the present invention, where the antigens may be used as the immune-stimulation component arm in conjunction with cholera toxic (CT). Antibodies thus generated are then used to pre-treat an infectious H.
pylori-containing inoculum (e.g., serum) before infection of cell cultures or animals. The ability of a single antibody or mixtures of antibodies to protect the cell culture or animal from infection is evaluated. For example, in cell culture and animals, the absence of antigen and/or nucleic acid production serves as a screen.
Further, in animals, the absence of H. pylori disease symptoms, e.g., elevated carbon dioxide/ammonia levels in a urea breath test (UBT) is also indicative of the presence of protective antibodies. The urea breath test takes advantage of the action of the urease enzyme of H. pylori to decompose ingested '3C or '°C urea to radioactive carbon dioxide and ammonia, and radioactive carbon dioxide is then measured.
Animal models for investigating H. infection include: (i) gnotobiotic newborn piglets (easily infected by H. pylori of human origin, but preferred for short term studies), (ii) mice and ferrets, which can be colonized for months (mice) or years (ferrets), and (iii) certain domestic cats, which can carry H. pylori (DuBois, et al., 1996). H. pylori causes ulcers in gnotobiotic piglets, and in mice using "THE SYDNEY STRAIN" of H. pylori, and gastritis in mouse strains SJL, C3H/HZ, DBA, C56BL/b, and Balb/C. A rhesus monkey infection model has also been developed (DuBois, et al., 1996).
Alternatively, convalescent sera can be screened for the presence of protective antibodies and then these sera used to identify H. pylori antigens that bind with the antibodies. The identified H.
pylori antigen is then recombinantly or synthetically produced. The ability of the antigen to generate protective antibodies is tested as above.
b. Vaccines. After initial screening, the antigen or antigens identified as capable of generating protective antibodies, either singly or in combination, can be used as a vaccine to inoculate test animals (to be described in greater detail below). The animals are then challenged _.._ T__w.. __ ____.-___..___-T-__ with infectious H. pylori. Protection from infection indicates the ability of the animals to generate antibodies that protect them from infection. Further, use of the animal models allows identification of antigens that activate cellular immunity.
In animal model studies, a protective immune response in response to challenge by a bacterial S preparation (e.g., infected serum) (i) protects the animal from infection or (ii) prevents manifestation of disease.
Vaccines can be prepared from one or more of the immunogenic polypeptides identified herein. Numerous H. pylori polypeptides of the invention (e.g., spot 15) have been shown to be extremely antigenic, i.e., they react strongly, with antibodies present in sera pooled from a number of confirmed H. pylori-positive donors. In a typical screening method, the intensity of color development is representative of the strength (binding affinity) of the antigen-anti-H. pylori antibody interaction.
Representative serum paneling results (Example 6) for proteins expressed by two of the clones, dHA22.8 (A22) and dHC1S.11 (C1), indicate that both of these recombinant proteins are highly immunogenic. Protein produced by clone dHA22.8 reacted with antibodies present in both of the H. pylori-positive pooled sera sources, Roost pool and SFA 001, and exhibited no cross reactivity with antibodies present in H. pylori-negative samples. Similar results were observed for protein produced by clone dHC1S.11 (C1).
In looking now at antigen reactivity with individual H. pylori-positive serum samples, antigenic protein expressed by each of the clones dHA22.8 and dHC1S.11 reacted with anti-H. pylori antibodies in 100 % and 95 % of the samples, respectively, indicating the ability of the antigens of the present invention to detect H. pylori infection, and to provide components for vaccines against H.
pylori .
Preferred antigens for use in vaccine compositions are described in Example 13. Exemplary antigens are those capable of invoking a long-lasting antigenic response, as evidenced by the persistant presence of antibodies whose titre remains high for an extended period of time subsequent antimicrobial treatment for H. pylori. On this basis, particularly preferred antigens for use in vaccine compositions include Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261Ains and Y146B.
Other peptides, among the unique H. pylori peptides disclosed herein, can be identified as useful in a vaccine for H. pylori as follows. The individual test peptide is formulated in a suitable carrier, e.g., adjuvant, at a concentration suitable for injection, e.g., 5-500 mg/ml. One suitable test animal for vaccination is the Rhesus monkey, as detailed in DuBois (DuBois, et al., 1996). The . animal is vaccinated, e.g., by oral, intramuscular or intravenous injection, in an amount typically between 0.2 to 2.0 mg/kg body weight. After a suitable period, e.g., 2 weeks, the animal may be given a booster by the same route and typically in the same amount. Two to three weeks later, the animal is challenged with H. pylori in a known manner, e.g., as described in DuBois (DuBois, et al., 1996). As an example, the inoculated animal is challenged with H. pylori (e.g., a suspension of approximately i0g-109 CFU of H. pylori, 1 ml) to test the potential vaccine effect of the specific immunogenic fragment. Following the challenge with-H. pylori, the subject is typically monitored S by endoscopy, histologic examination, microbiological methods, and/or measurement of H. pylori-specific plasma IgG, as described in DuBois (DuBois, et al., 1996).
The level of infection in the vaccinated animal is then compared with that of a control animal to assess the degree of protection. Those peptides which provide a measurable degree of protection against H. pylori infection are suitable for vaccine use, either alone or in combination with other peptide vaccine agents, such as the above noted dHA22.8 (A22), dHCIS.11 (C1) and spot 15 peptides.
The selected peptide is formulated according to known vaccine formulations.
Typically, the peptide is conjugated to a carrier protein, e.g. , keyhole limpet hemocyanin or human serum albumen, and/or suspended in a suitable adjuvant, such as Freund's adjuvant. The vaccine is administered by conventional routes, typically IM or IV routes, as above, at peptide levels preferably in the range of IS 0.2 to 2.0 mg/kg. If necessary, one or more booster injections is given.
The specificity of a putative immunogenic fragment can be assessed by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other related bacteria.
B. Synthetic Peptides Using the coding sequences of H. pylori polypeptide antigens disclosed herein, synthetic peptides can be generated which correspond to these polypeptides. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA).
Alternatively, oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea, 1989; Yoshio, et al., 1989; Eaton, et al., 1988).
Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis, et al., 1982; Ausubel, et al., 1988).
C. Immunoassays for H. oylori One utility for one, several, many, or each of the antigens herein is their use as diagnostic reagents for the effective and reliable detection of antibodies present in the sera of test subjects infected with H. pylori, to thereby provide an indication of infection in a test subject. Preferred antigens which can be employed either singly or in combination in such a method include, e.g., . T __ '_ _.._. ._______. ___T-._ antigens identified by or derived from SEQ ID N0:44 (A22), SEQ ID N0:39 (C1), SEQ ID N0:568 (Y124A), SEQ ID N0:557 (Y261A), SEQ ID N0:254 (c5), SEQ ID N0:21 (C7), SEQ ID
NO:55 (B2), SEQ ID N0:61 (Y104B), or SEQ ID N0:573 (Y128D). Alternatively, preferred antigens are defined in terms of their DNA coding sequences, corresponding to or derived from SEQ iD N0:43 (A22), SEQ ID N0:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ
ID N0:253 (c5), SEQ ID N0:20 (C7), SEQ ID NOs:5l, 54 (B2), SEQ ID N0:60 (Y104B), or SEQ
ID N0:98 (Y128D).
Alternatively, the antigen used for the detection of antibodies present in the sera of test subjects infected with H. pylori is encoded by a DNA fragment spanning one of the DNA fragment clusters: (1), (2), (3), (4), (S), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), {34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (SO), (S1), (52), (53), (54), (55), (56), (57), (58), (59), (61), (62), (62), (63), (64), (65), (66), (67), (68), and (69), or immunoreactive variants thereof. Preferably the antigen-coding regions are the spanning sequences themselves from the clusters.
The antigens of the present invention can be used singly, or in combination with each other, in order to detect H. pylori, as illustrated by the results in Example 6B. The antigens of the present invention may also be coupled with diagnostic assays for other infectious agents.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention, e.g., the "spot 15" antigen.
Exemplary antigens are A22 and C1, which both show high sensitivity (as indicated in Figs. 65 and 66, and in Example 6B). After binding anti-H. pylori antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-H.
pylori antibody on the solid support. The reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate, e. g. , 5-bromo-4-chloro-3-indoyl-phosphate (BLIP) and nitroblue tetrazolium (NBT). (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material.
These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group binding to a chemically reactive group on the solid support, e.g., an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Also forming part of the invention is an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigen (e.g., antigens such as those described in Tables 3a and 3b, or encoded by the representative clones summarized in Table 2) or native H. pylori antigen (such as those identified in Table 6 and in Fig. 1 and Fig. 2, as above), and a reporter-labelled anti-human antibody for detecting surface-bound anti-H. pylori antigen antibody.
In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected. Known general types of homogeneous assays proposed heretofore include (a) spin-labelled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency or polarization, (c) enzyme reporters, where antibody binding causes enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents.
In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
The examining may involve attaching a labelled anti-human antibody to the subject antibody (for example from acute, chronic or convalescent phase) and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method. Also contemplated is an antigen capture assay as previously described in Section D.2.
The following examples illustrate, but in no way are intended to limit the scope of the present invention.
Materials and Methods 1. General Procedures Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased from commercial suppliers.
Standard molecular biology and cloning techniques were performed essentially as previously described in Ausubel, et al., 1988; Sambrook, et al., 1989; and Maniatis, et al., 1982.
Common manipulations relevant to employing antisera and/or antibodies for screening and detection of immunoreactive protein antigens were performed essentially as described in Harlow, et _ _ __ ~_ _ _._ ...____.. .. ~
al. (1988). Antibody screening of H. pylori genomic libraries was carried out by plaque immunoblot assay.
Similarly Western blot assays were performed either as described by their manufacturer (Abbott, N. Chicago, IL; Genelabs Diagnostics, Singapore) or using standard techniques known in the art (Harlow, et al, 1988).
2. Bacterial Strains Commercially available H. pylori strains corresponding to ATCC Designation Nos. 43504 (short antigen clone set) and 43526 (Libraries 1 and 2) were used to generate the DNA libraries. H.
pylori strain ATCC 43504 was used for isolation of native proteins produced by H. pylori.
Escherichia toll strains) Y1088, Y1089,and XLI-Blue for libraries 1 and 2 (Stratagene, La Jolla, CA) was the host used for phage infection. E. toll strains XL1-Blue, XLOLR
(Stratagene, La Jolla, CA) were used for protein expression of cloned genes.
Example 1 Construction of H pylori Lambda gtl l and ZAPII DNA Libraries 1. Isolation of Genomic DNA
H. pylori (American Type Culture Collection, Rockville, MD; ATCC Designation Nos.
43504) was streaked on blood agar plates and incubated in a microaerophile environment at room temperature for 7 days. Cells were harvested by scraping the bacterial cells from 10 plates, followed by washing once with phosphate buffered saline (Dulbecco's, Gibco BRL, Gaithersburg, MD).
Genomic DNA was prepared as described in Ausubel et al. 1988, with minor modifications.
Cell pellets from 5 plates were resuspended in 510 ~.1 of TE (Ausubel et al. , 1988), to which was added 60 ~1 of 10 % SDS and 30 /d of 20 mg/ml of proteinase K. The suspension was mixed, followed by incubation for a period from 4 - 8 hours at 37°C. To the suspension was added 80 ~,1 of CTAB/NaCI (10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCI), and the resulting solution was then mixed and incubated for 10 minutes at 65°C. The solution was then extracted with an equal volume of chloroform/ isoamyl alcohol and spun in a microcentrifuge for 5 minutes. The separated aqueous phase was transferred to a new tube, and the DNA was precipitated by addition of 0.6 volumes of isopropanol, followed by centrifugation. The DNA pellet was washed with 70 %
ethanol, dried briefly under vacuum and solubilized in 100 /d of distilled water. The DNA solution was then treated with DNase-free Rnase (Boehringer Mannheim, Indianapolis, IN) (Ausubel, et al., 1988; Maniatis, et al., 1982) to selectively degrade any RNA present in the sample.
WO 98/49314 ' PCT/US98/08487 2. DNase Digestion and DNA Amplification a. Short Antigen Clone Libraries. H. pylori genomic DNA as described above was digested with pancreatic DNase I (Boehringer Mannheim) essentially as described in Ausubel, et al. (1988) and Sambrook, et al. (1989). Aliquots of the digested DNA were taken at various time points. The DNA digests were resolved by preparative agarose gel electrophoresis. Product bands containing the desired size range of DNA (200-2000 base pairs) were excised from the gel, and recovered using the "GENE CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID" kit (Bio 101 Inc., La Jolla, CA), according to the manufacturer's instructions.
To generate blunt-ends, the recovered DNA fragments were incubated with E.
coli Klenow fragment of DNA polymerise (Ausubel, et al., 1988; Sambrook, et al., 1989).
The reaction mixture was incubated at room temperature for 30 minutes, followed by extraction with phenol/chloroform.
The resulting molecules were ligated to Sequence Independent Single Primer Amplification (SISPA; Reyes, et al., 1991) linker primers according to the method of Reyes (Reyes, et al., 1991).
To the blunt-ended DNA were added the following: phosphorylated SISPA
(Sequence-Independent Single Primer Amplification) linker AB, a double strand linker comprised of SEQ ID N0:63 and SEQ
ID N0:64, where SEQ ID N0:64 is in a 3' to 5' orientation relative to SEQ ID
N0:63 as a partially complementary sequence to SEQ ID N0:63, 2 ~.l 10 x ligation buffer (0.66 M
Tris.Cl pH = 7.6, SO mM MgCl2, 50 mM DTT, 10 mM ATP) and 1 ~,I T4 DNA ligase (0.3 to 0.6 Weiss Units).
Typically, the DNA and linker were mixed at a 1:100 ratio. The reaction was incubated at 14°C
overnight. The reaction was then incubated at 70°C for 3 minutes to inactivate the Iigase. Unligated linkers were removed by gel filtration using a Chromaspin column (Clonetech, Mountain View, CA) or a Sephadex G spin column (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions.
The linker ligated DNAs were then amplified by SISPA (Reyes, et al., 1991). To 100 /d of 10 mM Tris-Cl buffer, pH 8.3, containing 1.5 mM MgCl2 and 50 mM KCl (Buffer A) was added about 1 ~.1 of the linker-ligated DNA preparation, 2 /cM of a primer having the sequence shown as SEQ ID N0:63, 200 ~,M each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of Amplitaq DNA
polymerise (Applied Biosystems Division, Perkin Elmer, Foster City, CA). The reaction mixture was heated to 94°C for 30 seconds for denaturation, allowed to cool to 50°C for 30 seconds for primer annealing, and then heated to 72°C for 0.5-3 minutes to allow for primer extension by Taq polymerise. The amplification reaction, involving successive heating, cooling, and polymerise reaction, was repeated an additional 25-40 times with the aid of a Perkin-Elmer Cetus DNA thermal cycler (Mullis, 1987; Mullis, et al., 1987; Reyes, et al., 1991; Perkin-Elmer Cetus, Norwalk, CT).
After the amplification reactions, the solution was then extracted with phenol/chloroform and precipitated with two volumes of ethanol.
__ _~ ~ .____ _._.._ WO 98/49314 ' PCT/ITS98/08487 3. Cloning of the DNA into Lambda Vectors a. Short Antigen Clone Libraries. The linkers used in the ligation to the DNA
contained an EcoRI site which allowed for direct insertion of the amplified DNAs into the lambda vectors (gtll or ZAP II, Stratagene, La Jolla). The lambda vectors as purchased from the S manufacturer had been digested with EcoRI and treated with alkaline phosphatase to remove the terminal S' phosphate and prevent self ligation of the vector. The amplified DNAs from 1.B. were digested with EcoRI and short nucleotides were removed by gel filtration. The digested DNA
preparations were then ligated into lambda gtll or "ZAPII" using T4 DNA iigase (Boehringer Mannheim, Indianapolis, IN). The conditions of the ligation reactions were as follows: 1 /d vector DNA (Stratagene (La Jolla, CA) O.S mg/ml); O.S or 3 ~.1 of the PCR amplified insert DNA; O.S ul 10 x ligation buffer (O.S M Tris-HCI, pH = 7.8; 0.1 M MgCl2; 0.2 M DTT; 10 mM
ATP; O.S
mg/ml bovine serum albumin (BSA)), O.S ~,1 T4 DNA ligase (0.3 to 0.6 Weiss units) and distilled water to a final reaction volume of S wl. The ligation reactions were incubated at 14°C overnight (12-18 hours). The ligated DNA was packaged by standard procedures using a lambda DNA packaging 1S system (GIGAPAK, Stratagene, La Jolla), and then plated at various dilutions to determine the titer.
The titer of the DNA-insert phage libraries and percent recombination were determined by a standard X-gal blue/white assay (Miller, 1994; Maniatis et al., 1982).
Typically, the titer of the recombinant libraries ranged from l.S x 104 to 3 x 106 PFU/ml.
Percent recombination in each library can also be confirmed by selecting a number of random clones and isolating the corresponding phage DNA. Polymerise chain reaction (Mullis, 1987; Mullis, et al., 1987) is then performed using isolated phage DNA as template and lambda DNA sequences, derived from lambda sequences flanking the EcoRI insert site for the DNA
molecules, as primers.
The presence or absence of insert is then evident from gel analysis of the polymerise chain reaction products.
b. Lambda ZAPII Libraries (Libraries 1 and 2). The lambda ZAPII libraries (generated from H. pylori sample ATCC No. 43526) were similarly prepared with the following exceptions. The ZAPII libraries were generated from genomic DNA which had not undergone Sequence Independent Single Primer Amplification.
Library 1 clones (designated herein by upper case letters) were generated by ligating EcoRI-digested H. pylori DNA directly into the EcoRI sites of the lambda "ZAPII"
vector. Library 2 clones (lower case designations) were obtained by digesting H. pylori genomic DNA
with HindIII, blunt-ending the HindIII-fragments, and ligating the resulting molecules to SISPA
primers AB as described above. The linker-ligated DNAs were then treated with EcoRI and ligated into the EcoRI sites of 3S ZAPII lambda arms.
Unless otherwise indicated, the DNA-insert gtll and ZAPII phage libraries generated from H. pylori samples ATCC Designation No. 43504 and ATCC No. 43526 have been deposited in the Genelabs Technologies, Inc. Culture Collection, 505 Penobscot Drive, Redwood City, CA, 94063 or in the Genelabs Diagnostics PTE LTD Culture Collection, 85 Science Park Drive X04-Ol , The Cavendish, Singapore Science Park, Singapore 118259.
Example 2 ImmunoscreeninQ of Recombinant Libraries The lambda gtl l and ZAPII phage libraries described in 1.C. above were immunoscreened for the production of antigens recognizable by a pool of sera from 11 patients (designated herein as "Roost pool" sera) identified as H. pylori-positive using endoscopy, or by another pool of 4 H. pylori immunopositive sera samples identified as SFA001. The lambda short antigen clone library and ZAPII
library 2 were immunoscreened against Roost pool sera; the ZAPII library 1 was immunoscreened against sera pool SFA001. The lambda gtl l libraries were plated for plaque formation using E.
coli Y1089 bacterial plating strain, while the lambda ZAPII libraries were plated for plaque formation using E. coli XLl-Blue bacterial plating strain.
The fusion proteins expressed by the recombinant lambda phage clones were screened with serum antibodies essentially as described by Ausubel, et al. (1988).
Each library was plated at approximately 1.5 to 2 x 10' phages per 150 mm plate. Plates were overlaid with nitrocellulose filters overnight. Filters were washed with TBS (10 mM, Tris pH
7.5; 150 mM NaCI), blocked with AIB (TBS buffer with 1 % gelatin) and incubated with a primary antibody diluted 100 times in AIB.
After washing with TBS, filters were incubated with a second antibody consisting of goat-anti-human IgG conjugated to alkaline phosphatase at a concentration of 1:1000.
Reactive plaques were developed with a substrate (for example, BCIP, 5-bromo-4-chloro-3-indolyl-phosphate), with NBT
(nitro blue tetrazolium salt; Sigma Chemical Co., St. Louis, MO). Positive areas from the primary screening were replated and immunoscreened until pure plaques were obtained.
The results of the screening are presented in Tables la and lb.
Table la Roost Pooled Sera SerutstEndoscopy Culture Pathology (Gastritis) () A1 Mild antrum gastritis,Moderate Acute/chronic/moderate deep duodenal ulcer A4 None Heavy Acute/chronic/severe with prior ulcer A8 Mild annum gastritis,Moderate Chronic/mild moderate duodenitis Al l Mild antrum and Heavy Chronic/moderate body gastritis B 1 Moderate antrum Heavy Chronic/severe with gastritis, moderate prior Hp ( > 4 duodenitis years with therapy) 9 Deep gastric ulcerNA NA
14 Moderate antrum Moderate Acute/mild gastritis Gastritis in anttumHeavy Acute/chroniclsevere and body with prior ulcer 16 Mild anttvm gastritis,Moderate Chronic/moderate deep gastric ulcer 19 Superficial gastricRare Chronic/moderate and duodenal ulcers 15 20 Mild antrum gastritisModerate Acute/chronic NA = not available The above sera have all been tested with HelicoBlot 2.0 (Genelabs Diagnostics Western blot product) and were found to be positively infected samples using the criteria of the product.
SFA001 is a pool of 4 donor sera (plasma packs) showing strong reactive bands on Western blot format with a crude lysate antigen preparation from Helicobacter lysate using in HelicoBlot 2Ø
NAA121 is a pool of 4 donor sera (plasma packs) showing no reactive bands on Western blot format with the crude lysate antigen preparation from Helicobacter lysate used in HelicoBlot 2Ø
Table lb The lambda ~~tll and ZAPII libraries # Screenedfi Clones Library % Recomb.Antibody pfu Plaque-Purified gtll Y 90-95% Roost pool2 x I05 21 EcoRI/XbaI94% 1Gb monoclonal2 x 10' 1 antibody against H. pylori ZAPII Z 90-95 Roost pool2 x 105 300 %
library 99% SFA001 3.9 x 52 library 98 % Roost pool9 x 10' 86 OTALS: > 462 Example 3 Characterization of Immunoreactive Lambda gtll Z and Lambda ZapII Y library Clones 1. PCR Amplification. Purification and Sequence Determination for DNA Inserts of Immuno-reactive Recombinant Lambda Clones (Short Antigen Clone Set H pylori Cioned Families Y and Z) The specific antigen coding sequences from H. pylori cloned families were isolated by PCR
amplification of representative clones. Cloned sequences having an .ASM
extension were sequenced completely; sequences with a .SEQ extension were partially sequenced.
The DNA inserts of the immunoreactive recombinant lambda clones were PCR
amplified using primers corresponding to lambda arm sequences flanking the EcoRI cloning site of the vectors.
Amplification was carried out by polymerase chain reactions utilizing each immunoreactive clone as template. For the lambda gtl l clones, gtllF (SEQ ID N0:65) and gtl l R (SEQ
ID N0:66) primers were used. For the lambda ZAPII clones, primers T3 (SEQ ID N0:67) and T7 (SEQ
ID N0:68) were used.
The resulting amplified fragments were then agarose gel purified and eluted from the gel (Ausubel, et al., 1988). The PCR products were further purified by "WIZARD PCR
PREPS"
(Promega, Madison, WI) or "CHROMASPIN" columns (Clonetech, Mountain View, CA) to remove primers and other ingredients. The purified insert DNA was then subjected to direct sequencing. In some cases, the insert DNA was first subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and then sequenced.
Sequence determination for the DNA inserts was carried out using a Perkin Eimer Applied Biosystems 373A DNA sequencer (Perkin Elmer, Applied Biosystems Division, Foster City, CA) according to the manufacturer's protocol (dideoxy chain terminator sequencing methodology. Sanger, et al. , 1977).
Sequence data is presented in the accompanying Sequence Listing. Table 2 below presents a partial summary of recombinant H. pylori nucleotide sequences encoding immunogenic proteins, i. e. , proteins shown to be reactive with H. pylori-positive sera, corresponding to cloned families Y and Z.
The EcoRI site of the linkers has typically been deleted for the corresponding sequences presented in the figures.
Table 2 Clone SEQ InsertSequencedCluster No.
ID (N'I~ (NT) NO:
Y103-1T3.SEQ70 -900 250 57 Y103-1T7.SEQ71 -900 220 57 Y103-2.ASM72 -800 820 57 Y104-1.ASM73 1080 3 YIOS.ASM 74 730 55 T3. SEQ
Y107-1T7.SEQ76 -1300 310 4 Y108-1.ASM77 790 32 Y109-1T3.SEQ78 -400 205 23 Y109-1T7.SEQ79 -400 230 23 Y111.ASM 80 1565 19 Y114-2T3.SEQ81 -900 205 51 YI14-2T7.SEQ82 -900 220 15 Y115-1T3.SEQ83 -800 205 28 Y115-1T7.SEQ84 -800 270 28 Y116-1.ASM85 510 28 Y1I7-2T3.SEQ86 -100 195 37 Y117-2T7.SEQ87 -1000 235 37 Y119-1T3.SEQ88 -600 225 33 - Y119-1T7.SEQ89 -600 245 33 Y123-IT3.SEQ92 -1300 265 35 Clone SEQ InsertSequencedCluster No.
m (NT) (NT) NO:
Y123-iT7.SEQ93 -1300335 35 Y124-2T3.SEQ94 ~--60085 14 Y124-2T7.SEQ95 -600 280 14 Y126-2T3.SEQ96 -1100278 58 S Y 126-2T7.97 -1100290 58 SEQ
Y128.ASM 98 930 20 Y 12T3. 99 -1000245 10 SEQ
Y12T7.SEQ 100 --1000305 10 Y130-1T3.SEQ101 -1000270 35 Y130-1T7.SEQI02 -1000195 35 Y131-1T3.SEQ103 --800275 37 Y131-1T7.SEQ104 -800 285 37 Y 132-2T3.105 -1100240 59 SEQ
YI32-2T7.SEQ106 -1100265 59 Y133.ASM 107 410 60 Y 135. 108 540 29 ASM
Y136-1.ASM109 380 61 YI39.ASM 110 685 13 Y13T3.SEQ 111 -800 230 62 Y13T7.SEQ 112 -800 290 62 Y140-IT3.SEQ113 -700 215 12 Y140-1T7.SEQ114 -700 240 12 Y141.ASM 115 750 6 Y 143-2T3.116 - 240 13 YI43-2T7.SEQ117 --800290 13 Y144.ASM 118 615 36 Y145-2T3.SEQ119 -700 220 28 Y146-I.ASM120 870 28 Y 147. 121 1025 51 ASM
Y150-2.ASM122 375 32 Y151-2T3.SEQ123 -700 290 Y151-2T7.SEQ124 -700 325 ~. _.._. ___.__.._ _.
WO 98/49314 , PCT/US98/08487 Clone SEQ InsertSequencedCluster No.
>D (N'I~ (NT) I
NO:
Y152-2.ASM125 615 20 Y153.ASM 126 1030 37 Y 154-2T3.127 - 800 265 4 SEQ
Y154-2T7.SEQ128 -800 305 4 Y160-1T3.SEQ129 -1000 260 12 T7. SEQ
Y163.ASM 131 610 38 Y16T3.SEQ 132 -1000 240 51 Y16T7.SEQ 133 -1000 310 51 Y173.ASM 134 765 10 Y175.ASM 135 770 2 Y176-1.ASM136 425 28 Y178-1T3.SEQ137 -900 300 18 Y178-1T7.SEQ138 --900 265 18 Y180.ASM 139 810 29 Y184-IT3.SEQ140 --600 230 16 Y184-IT7.SEQ141 -600 290 16 Y187-2.ASM142 480 23 Y190.ASM 143 760 39 YI94-2T3.SEQ137 -600 300 18 Y194-2T7.SEQ144 -600 240 18 Y195-1.ASM145 540 21 Y196-1.ASM146 425 52 Y19T7.SEQ 147 --1100320 2 Y1T3.SEQ 148 -500 295 53 YIT7.SEQ 149 -500 235 53 Y200-I.ASM150 500 31 Y203T3.SEQ151 - 700 230 38 Y203T7.SEQ152 -700 230 38 Y208T3.SEQ153 -800 260 15 Y208T7.SEQ154 -800 270 15 Y20T3.SEQ 155 -1100 320 11 Clone SEQ InsertSequencedCluster No.
ID (NT) (N'I~
NO:
Y20T"l.SEQ156 -1000315 11 Y212T3. 157 - 260 14 Y212T7.SEQ158 ~700 285 14 Y220T3. 159 -1000220 10 SEQ
S Y220T7.SEQ160 -1000260 10 Y221 T3. 161 -- 255 2 Y221T7.SEQ162 - 240 2 Y224.ASM 163 840 29 Y237.ASM 164 390 26 Y238T3.SEQ165 -1100200 40 Y238T7.SEQ166 -1100220 40 Y23T3.SEQ 167 -1100195 51 Y23T7.SEQ 168 - 320 51 Y241.ASM 169 685 4 Y247.ASM 170 490 36 Y250T7.SEQ171 -1200215 41 Y261T3.SEQ172 -700 270 4 Y261T7.SEQ169 -600 4 Y264T7.SEQ173 -500 215 31 Y268T3.SEQI74 -800 245 27 Y269T3.SEQ175 -900 220 5 Y269T7.SEQ176 ~900 240 5 Y26T3.SEQ 177 -1400140 43 Y26T7.SEQ 178 -1400230 43 Y270T3.SEQ179 ~ 280 43 Y270T7.SEQ180 -1300255 43 Y28T3.SEQ 181 - 330 17 I
Y28T7.SEQ 182 --1100330 17 Y29.ASM 183 500 31 Y30.ASM 184 545 33 Y33T3.SEQ 185 -1200335 45 Y33T7.SEQ 186 -1200345 45 ___. __ T __ _._._ __ . _ _T.
Clone SEQ InsertSequencedCluster No.
ID (NT) (1VT) NO:
Y37.ASM 187 490 32 I
Y3T3.SEQ 188 - 250 37 Y40T3.SEQ 189 -1200355 48 Y40T7.SEQ 190 - 330 4$
Y42T3.SEQ 191 - 295 12 Y42T7.SEQ 192 - 355 12 Y43T3.SEQ 193 -900 265 29 Y43T7.SEQ 194 --900310 29 Y46T3.SEQ 195 -900 280 45 Y46T7.SEQ 196 -900 290 45 Y47T3.SEQ 197 -800 250 10 Y47T7.SEQ 198 -800 290 10 Y48.ASM 199 490 31 Y49T7.SEQ 200 --900335 49 Y53T3.SEQ 201 -1200230 11 Y53T7.SEQ 202 -1200320 11 YSS.ASM 203 610 13 Y56T3.SEQ 204 -700 220 37 Y56T7.SEQ 205 -700 290 37 Y60T3.SEQ 206 --1300265 7 Y60T7.SEQ 207 -1300290 7 Y61T3.SEQ 208 --800265 42 Y61T7.SEQ 209 -800 240 42 Y69T7.SEQ 200 -500 335 49 Y73T3.SEQ 210 -- 285 50 I
Y73T7.SEQ 211 - 285 50 YSI.ASM 212 550 23 Y82T3.SEQ 213 -700 230 13 Y82T7.SEQ 214 -700 325 13 Y84T3.SEQ 215 -1100265 42 Y84T7.SEQ 216 -1100275 42 Y87.ASM 217 660 42 Y89T3.SEQ 218 -1200260 2 Clone SEQ InsertSequencedCluster No.
ID (NT~ (NT) NO:
Y89T7.SEQ 219 -1200 280 ' 2 Y91 T3. 220 - 800 250 54 i SEQ
Y91T7.SEQ 221 -800 325 54 Y92T3.SEQ 222 --1300235 1 Y92T7.SEQ 223 -1300 310 1 Z14.ASM 224 970 30 Z21.ASM 225 280 39 Z24.11f.SEQ226 195 23 Z25.ASM 227 1185 6 Z3.ASM 228 1560 51 z4l.ASM 229 518 Z9.ASM 230 525 31 Clone Y104-1 (SEQ ID N0:60) contains the entire d7 clone, and encodes all of the 36K
peptides and all of the spot 15 peptides, as indicated in Fig. 3.
The production of expressed antigenic proteins and their subsequent purification is generally described in Examples SA and SB. A tabular summary indicating clone name, expression, purification, and panelling data is provided in Table 3b, and a summary of the immunoreactivy of various recombinant antigens is provided in Table Sb. Expression profiles were obtained, and the immunoreactivity of various clones to H. pylori positive pooled sera such as "Roost" was confirmed.
Briefly, amplified products corresponding to a particular ORF were typically cloned into a pGEXhisB vector and expressed in E. toll. The size of the expression product was then determined, followed by confirmation of immunoreactivity (e.g., with Roost pool sera).
2. Sequence Comparison Sequences were compared with "GENBANK" (versions 1998, 1996), EMBL database and dbEST (National Library of Medicine) sequences at both nucleic acid and amino acid levels. Search programs FASTA, BLASTP, BLASTN and BLASTX (Altschul, et al., 1990) indicated that most of the antigen-coding polynucleotide sequences disclosed herein were not greater than 95 % identical to any sequence contained in the above-mentioned public databases prior to the publication of the genome of H. pylori (Tomb, et al., 1997).
Example 4 Characterization of Immunoreactive Lambda Library 1 and Library 2 Clones 1. Subclonin~, Purification Identification and Sequence Determination for H
pylori Anti ens Additional immunopositive clones, as described in Example 2 above, were purified and analyzed for DNA insert size and expressed protein size as in Example 3 above.
The ZAPII clones were rescued into plasmids from phagemids (as per protocol in Stratagene, La Jolla, U.S.A.); the resulting insert DNA was excised with EcoRI and ran on agarose gel to determine size. Slightly longer versions of T3 and T7 (GD 60 and GD 61, corresponding to SEQ ID N0:231 and SEQ
ID N0:232 respectively) were then used as primers for sequencing. These libraries correspond to the "library 1"
series (EcoRI-cut), i.e., clones A3, A22, B2, B9, B17, B23, C1, C3, C7, and "library 2" series clones (HindIII cut), i.e., clones al, a3, a5, b5, b8c7, c2, c5, c13, d5, d6, d7, dll, fll, e6, f3, f8, g2, g9, gl l, and k4; and also clone Gla from the EcoRIlXbaI cut library. Many clones were determined to be larger than the predicted coding regions of the proteins.
The specific antigen coding sequences from H. pylori library 1 and library 2 clones were subcloned. The subclones were typically prepared by fragmenting the corresponding genomic clone by specific restriction endonuclease digestion to produce a specific subfragment or subfragments, which were purified using "GENE CLEAN II" kit (Bio 101 Inc., La Jolla, CA) or the "MERMAID" kit (Bio 101 Inc., La Jolla, CA). The resulting DNA fragments were then inserted into a suitable expression vector, i.e., the PB/Bluescript (SK) vector (Stratagene, La Jolla, CA). Expression clones were induced with 0.5 mM IPTG (isopropylthio-beta-D-galactoside) for 4 hours at 37 degrees C, and whole bacterial cell lysate was run on SDS gel. Immunoreactivity of the expressed proteins was confirmed using Roost, SFA001 and NAA001 pooled sera. Alternatively, original genomic clones were subjected to nested deletion using the "Erase-a-Base"" kit (Promega, Wisconsin, U.S.A.), and the resulting smaller nested clones were tested for immunoreactivity to locate the position of the coding antigen.
Immunoreactive DNA regions were then sequenced and locations of the open reading frames were determined. Unless otherwise indicated as a /3-galactoside fusion product, for each of the clones in Tables 3a and 3b below, expression of coding regions was determined to be driven by the corresponding H. pylon promoter rather than by the /3-galactosidase gene promoter in lambda gtll.
WO 98/49314 ' PCT/US98108487 2. Clone A3 Subclone A3CON3, originating from a genomic clone having a size of approximately 6.0 kb, was obtained from nested deletion clones. The corresponding nucleotide sequence (1878 bp) is presented herein as SEQ ID N0:16. The open reading frame (ORF) extends from nucleotides 399-1743, and codes for a putative protein containing 448 amino acids (443 amino acids when calculated from the first methionine). The protein sequence corresponding to the translation of open reading frame (ORF) 399-1743 is presented herein as SEQ ID N0:17.
3. Clone A22 Subclone A22210DMIC was obtained from an original genomic clone having an insert size of about 2.2 kb, to produce a /3-galactosidase fusion protein in E. coli. As indicated in Tables 3a and 3b below, the open reading frame (ORF) extends from nucleotides 12-599 of SEQ
ID N0:43, where bases 12-121 correspond to the R-galactosidase fusion peptide and bases 122-599 code for a unique A22 antigenic sequence. The translation sequence for the corresponding protein is presented as SEQ
ID N0:44.
4. Clone B2 B3C19 is a subclone obtained from the 5.0 kb insert in genomic clone B3 as follows. The insert was excised, digested with DNase, followed by treatment with T4 DNA
ligase and Kienow enzyme to produce blunt-ends. The blunt-ended short DNA pieces were then ligated to kinased A/B
linkers (linker A, SEQ ID N0:63, corresponding to the top strand of AB SISPA
linker; linker B, SEQ
ID N0:64, corresponding to the bottom strand of AB SISPA linker), PCR
amplified with primer A, and the amplified products digested with EcoRI. The digested products were then ligated into EcoRI-digested lambda vector ZAPII. The DNA sequence of B3C19 is presented as SEQ ID
NO:51.
Based upon the sequence of B3C19, primers GD77 (5' primer, SEQ ID N0:52) and (3' primer, SEQ ID N0:53) were designed to walk back and forward the sequence of B3C19 using genomic B2 DNA as the template. The sequence of clone B3C19 was extended in both the S' and 3' directions, resulting in B2 extension clone, B2197780 (SEQ ID N0:54).
A computer-generated protein translation of the B2197780 sequence resulted in a corresponding 291 amino acid putative protein, with a predicted transmembrane segment from amino acids 9-25 (predictions obtained using "SOAP" program from "PCGENE"). Based upon the results of the analysis, a Shine Dalgarno AGGA sequence was determined to be situated 8 b.p. in front of an ATG codon. This ATG codon at nucleotide position 278 appears to be the first "met" amino acid of the gene. The translated protein sequence for clone B2 is presented as SEQ
ID NO:55. The WO 98/49314 ' PCT/IJS98/08487 predicted molecular weight of full size protein is 31,682. There are 2 potential cleavage sites between (i) amino acids 91 and 92; and (ii) between amino acids 25 and 26 ("PCGENE" --Prediction of prokaryotic secretory signal sequence). The pI of the protein is predicted as 8.3.
5. Clone B9 Subclone B9.4C is a 1.2 kb HindIII fragment obtained from original genomic clone B9 (4.5 kb insert) as follows.
Induction of genomic clone B9 produced an immunoreactive protein with sizes of 32 kd and 14 kd in SFA001 pooled sera and 14 kd in Roost pooled sera. B9 was digested with HindIII to form several HindIII subfragments, which were each subcloned into the pBKS vector.
Protein production was induced in the resulting subclones, and the sizes of the corresponding proteins were determined.
Subclone B9.4C, corresponding to a 1.2 kb HindIII subfragment of genomic clone B9, produced an immunoreactive protein of 32 kd. The nucleotide sequence of subclone B9.4C is presented as SEQ ID N0:47; the translated protein sequence (i.e., nucleotides 230-931 of SEQ ID
N0:47) is presented as SEQ ID N0:48. Referring to SEQ ID N0:47, an AGGA Shine-Dalgarno sequence occurs at nucleotide 218, and the first amino acid of the translated protein sequence begins with a GTG codon for valine at position 230. Based upon PCGENE calculations (CHARGPRO
program) as described above, the predicted molecular weight of the corresponding protein is 25.2 kd, with a pI of 10.35. A potential cleavage site occurs between amino acid positions 225 and 226.
Based upon a FASTA sequence identity analysis of both nucleic acids and proteins, clone B9 appears to code for the SOA L1 protein of H. pylon.
6. Clone BI7 B17CON4 (2006 bases) was subcloned from a genomic clone having an insert size of about 4.0 kb. The corresponding nucleotide sequence is presented as SEQ ID N0:24.
The open reading frames (ORF) correspond to the following regions of SEQ ID N0:24: ORF1 (nucleotides 500-700);
ORF2 (nucleotides 870-1406); ORF3 (nucleotides 1410-2000); and ORF4 (nucleotides 142-705). The corresponding translated protein sequences are presented herein as SEQ ID
N0:25 (B170RF4), SEQ
ID N0:26 (B170RF1), SEQ ID N0:27 (B170RF2) and SEQ ID N0:28 (B170RF3). Based upon nested deletion experiments, it appears that the immunoreactive protein corresponds to B170RF4.
On the basis of computer-generated protein translation analysis as previously described, B 170RF4, B 170RF2 and B 170RF3 encode putative proteins having predicted sizes of 20.9 kd, 20 kd and 22.2 kd respectively. The predicted size of the immunoreactive genomic protein is a doublet of 22 kd and 23 kd.
7. Clone B23 Immunoreactivity was determined to reside in a 3.5 kb PstI subclone {one PstI
site is derived from vector pBSK) of original genomic clone B23 (5.5 kb). The 3.5 kb subclone was further sequenced, to determine the immunoreactive coding sequence presented in SEQ ID
N0:13. The nucleotide 1078 base pair sequence (SEQ ID N0:13) was determined to be open all the way (bases 3-1076). The translated protein sequence is presented as SEQ ID N0:14.
8. Clone C 1 C1CON6V2 was subcloned from an original genomic clone having an insert size of about 4.0 kb. The sequence information obtained from exo-mung deletion clones is presented as SEQ ID N0:38.
The open reading frame (ORF) extends from nucleotides 868-1926. The subcloning of the antigen coding sequence from the C1 clone into an expression vector, and characteristics of the corresponding protein product are presented in Example 5 below. The translated protein sequence is presented herein as SEQ ID N0:39.
9. Clone C3 To determine the immunoreactive coding region of original genomic clone, C3 (2.9 kb insert size), several different subfragments of the genomic clone were obtained and sequenced. The immunoreactive clone was determined to reside in an EcoRI/Kpn 2.2 kb subclone.
Based upon a number of subcloning experiments and subfragments, the C3 DNA sequence is presented as SEQ ID
NO:15.
10. Clone C7 Clone C7.2C is a EcoRIlHindIII subclone obtained from genomic clone C7. The original genomic C7 clone is a EcoRI clone of approximately 3.8kb size that produces an immunoreactive protein having a molecular weight of approximately 30 kd.
Subclone C7.2C was obtained as follows. The genomic C7 clone was digested into individual fragments using HindIII. Each DNA restriction fragment was then subcloned into the pBKS vector via either the HindIII site, or alternatively, for end-piece fragments, via the EcoRIIHindIII sites. The corresponding nucleotide sequence of C7.2C (616 base pairs) is presented as SEQ ID N0:20.
Subclone C7.2C produces an immunoreactive protein which is the same size as that produced by the genomic clone. The translated protein sequence for bases 1-561 of clone C7.2C
is presented herein as SEQ ID N0:21.
11. Clone B8 _. . T _. ~ ~_.__~_.__ T
Clone B8 (SEQ ID N0:549) is a genomic clone with an insert size of 3.5 kb that produces an immunoreactive protein of about 50 kd on SDS Western blot. The DNA sequence of B8 overlaps with that of clone C7 (clone C7.2C) which was a fusion protein with beta-galactosidase. Clone B8 added on 266 additional amino acids 5' (upstream) to C7 and contains the beginning of the gene.
However, B8 ends 64 amino acids before the C-terminus of the gene, which is also encoded by C7.
Based on the above, the complete sequence of the full gene was compiled (SEQ
ID N0:549). The corresponding protein sequence from the compiled gene sequence codes for a putative protein of 48.9 kd, and is presented herein as SEQ ID NO:550.
I2. Clone a1 Clone al is a HindIII clone that produces an inununoreactive protein of 28-30 kd on SDS gel.
The corresponding DNA sequence encoding an antigenic protein contains 1208 nucleotides (SEQ ID
N0:35). There are two open reading frames.
ORF1 extends from bases 53 - 801 of SEQ ID N0:35, and encodes a putative protein containing 249 amino acids (SEQ ID N0:36, translated protein). ORF1 contains a Shine-Dalgarno sequence extending from nucleotides 43-46 ("AGGA" sequence). The predicted molecular weight of the protein is 27.5 kd, which is in agreement with the expected protein size based upon SDS-PAGE
(above). The putative protein for ORF1 has a calculated pK of 8.42 and a predicted transmembrane region extending from amino acids 1-19.
The second ORF contained in clone al extends from nucleotides 880-1206 and ends at one of the HindIII sites, which indicates that clone al is a partial clone. The predicted size of the partial protein is 109 amino acids (SEQ ID N0:37).
13. Clone a3 Immunoreactive clone a3 (not to be confused with clone A3 from library 1) is a (3-galactosidase fusion clone with an insert size of 1975 base pairs. The corresponding nucleotide sequence corresponds to SEQ ID N0:248. The clone contains two open reading frames (ORFs):
ORFI (nucleotides 3-608, SEQ ID N0:249) and ORF2 (nucleotides 613-1266, SEQ ID
N0:250).
Based upon the observed size of the immunoreactive protein, i.e., 30 kd, the expected immunoreactive-expressing open reading frame is ORFl . Expression of a3 protein is described in the following section.
14. Clone a5 Clone a5 is an original HindIIIlEcoRI genomic clone (I kb) that produces a 25 kd im-munoreactive protein. The corresponding nucleotide sequence is presented as SEQ ID N0:3. The clone contains two open reading frames, (ORFs): ORFI (nucleotides 3-545, SEQ
ID NO:S) which codes for a beta-galactosidase fusion protein, and ORF2 (nucleotides 569-1021, SEQ ID N0:4). A
Shine Delgarno sequence occurs at nucleotides 559-562 of ORF 2. Based upon the observed size of the immunoreactive protein, i. e. , 28 kd, the expected immunoreactive expressing open reading frame is ORF 1.
15. Clone b5 Clone b5 contains a double insert with an internal EcoRI site. The individual inserts are about 0.3 and 0.2 kb in length. The combined nucleotide sequence for clone b5 corresponds to SEQ ID
N0:6, with the open reading frame (ORF) extending from nucleotides I through 414. The corresponding protein sequence translation is presented as SEQ ID N0:7.
16. Clone c2 Clone c2 is a library 2 clone with an insert size of 2077 nucleotides (SEQ ID
N0:25I). The size of the immunoreactive protein on SDS-gel is 30kd. There is one ORF
extending from nucleotides 3-877, with a possible initial start codon at nucleotide 45. The corresponding protein sequence translation is presented as SEQ ID N0:253. The protein has a predicted pI of 9.54, with a potential transmembrane region between amino acids 2 through 24. The predicted molecular size of this c2 protein is 30.2kd, which agrees with the observed size on SDS gel.
17. Clone c5 Clone c5 is a 650 base pair HindIIIIHindIII clone (SEQ ID N0:253). Clone c5 is a partial clone which is not in phase with the /3-galactosidase gene, and contains a 29 amino acid stretch extending into pBluescript at the N-terminus. The ORF extends from bases 2-604, with a predicted protein size of 23.Skd (SEQ ID N0:254). The observed protein size on SDS-gel is 30kd. The putative protein has a pI of 9.6.
18. Clone c13 Clone c13 is a HindIIIlHindIII clone with an insert size of 1742 b.p. (SEQ ID
N0:255). It is a ~-galactosidase fusion protein with an observed size of SSkd on SDS gel.
The ORF extends from bases 2 to 1420. The corresponding protein possesses a predicted molecular weight of 54.6kd (SEQ
ID N0:256). A sequence search using SWISS pro database indicates that the sequence possesses homologies with threonyl-tRNA synthetase of various organisms, from bacteria to yeast and human.
19. Clone d5 Clone d5 is an EcoRI clone that produces an immunoreactive protein (a-galactosidase fusion protein) of about 70 kd on SDS-PAGE gel. The size of the cloned insert was determined to be 1795 bases (SEQ ID N0:58), with an ORF from extending from nucleotides 1-1704. The open reading frame codes for a putative protein composed of 568 amino acids (SEQ ID N0:59), and having a predicted molecular weight of 62.1 kd. The putative protein has a predicted pK
value of 5.1, and the predicted antigenic determinant lies in the N-terminus region of the protein.
. A computer-based analysis was carried out using the "PROSITE" program on "PCGENE".
Based upon this study, several signature sites were identified within the protein, which included a cAMP and cGMP dependent protein kinase phosphorylation site and a ATP-GTP-binding motif A
(P-loop).
20. Clone d7 Clone d7 is a HindIII clone that was blunt-ended, ligated to A/B linkers, digested with EcoRI, and subcloned into lambda vector ZAPII as described above for Example 4.1. The product is a /3-galactosidase fusion protein. The nucleotide sequence is presented as SEQ ID
NO:11.
The induced protein was determined to have sizes of 40 kd and 35 kd. The corresponding protein sequence translated from SEQ ID NO:11 is presented as SEQ ID N0:12.
21. Clone f3 Clone f3 is a EcoRI clone having an insert size of 2274 nucleotides (SEQ ID
N0:257). Clone f3 produces a (3-galactosidase fusion protein. The ORF extends from nucleotides 1-1788 and codes for a putative protein having a predicted molecular size of 67 kd (excluding the a-galactosidase portion) (SEQ ID N0:258). The calculated pI of the protein is 9.66.
A sequence search against both the SWISS pro data base and the DNA data base reveals a 30% homology to penicillin binding proteins of Haemophilus influenza, E.coli, Bacillus subtilis and Streptococcus pneumoniae. A search against the EMBL DNA data base reveals a 56 % homology with penicillin binding proteins of Haemophilus influenza.
The size of the protein observed on SDS-gel is 25kd (major band), with a minor band of around 67kd. This size discrepancy may possibly be due to cleavage of the protein to produce a smaller fragment.
22. Clone f8 FBCON i is a HindIII subclone that produces an immunoreactive protein of 33-35 kd on SDS
gei. The DNA sequence corresponding to the f8 subclone is presented herein as SEQ ID NO:1. The cloned 1459 base DNA subfragment contains an open reading frame (ORF) from nucleotides 134 -1042.
The putative protein encoded by the f8 subclone contains 303 amino acids, with a predicted molecular weight of 33.9 kd (which is in agreement with the expected protein size). The predicted antigenic determinant is at the 3' end of the putative protein - extending from amino acid 270 to amino acid 275 (nucleotides 941-958). The putative protein has a pK of 9.75. The corresponding protein sequence based upon a translation of SEQ ID NO:I is presented as SEQ ID N0:2.
W. Clone ~2 Clone g2 is a EcoRIlHindIII clone with an insert size of 2474 nucleotides (SEQ
ID N0:259).
The size of the immunoreactive protein observed on SDS gel was 25kd. The clone contains 4 ORFs.
The first and last ORFs encode partial proteins, i. e. , the complete ORF was not contained in the clone. ORF1 extends from nucleotide 2 to 445 and codes for the carboxyl end of a putative protein of 148 amino acids (SEQ ID N0:260). ORF2 extends from bases 461-1156, coding for a putative protein of molecular size 25.4 kd (SEQ ID N0:261). ORF3 extends from bases 1156 to 1776 and codes for a protein having a size of about 22.2 kd (SEQ ID N0:262). ORF4 (nucleotides 1798-2472) codes for the amino terminus of a putative protein of 24.4 kd (SEQ ID N0:263).
The predicted pIs of ORF2 and ORF3 are 7.82 and 5.26, respectively. While ORF2 and ORF3 putative protein does not contain predicted transmembrane regions, ORF4 putative protein contains 3 predicted transmembrane regions with 6 predicted transmembrane helices.
23. Clone ~9 Clone g9 possesses an insert size of 4292 bases. Two subclones, k4 and gl l, are contained within clone g9. Clone g9 produces an immunoreactive protein band of about 85kd, whilst k4 has immunoreactive bands of 55 kd, 46 kd, 35 kd and 30 kd and gl l produces a weakly immunoreactive band of 22 kd.
The complete sequence of g9 was obtained by walking in the 5' direction from the gll C-terminus sequence, and walking in the 3' direction from k4 N-terminus sequence (SEQ ID N0:264).
Clone g9 has 5 ORFs. ORF1 encodes a partial protein.in the region extending from bases 2-349 (SEQ
ID N0:265). The corresponding predicted molecular size of the protein is 12.3 kd. ORF2 extends from bases 495-1403 (SEQ ID N0:266), and the corresponding predicted protein possesses a molecular size of 33.8 kd. ORF2 is followed by a "AGGA" Shine-Dalgarno sequence, positioned in front of the ATG of ORF3 (bases 1418-2209). The predicted molecular size for the putative protein encoded by ORF3 is 29.7 kd (SEQ ID N0:267). ORF4 corresponds to bases 2223-3719. The protein _T ___ ._ ___._.___. _.___.___ ~
encodes by ORF4 has a predicted molecular size of 56.8 kd (SEQ ID N0:268).
ORFS extends from nucleotides 3133 to 4236, with a predicted protein molecular size of 19.9 kd (SEQ ID N0:269).
24. Clone Gla An immunoreactive 2.8 kb HindIiI fragment of the corresponding genomic EcoRI
clone, having an insert size of 6.4kb, was treated with exo-mung nuclease to generate nested deletion clones for sequencing. The DNA sequence of a 1610 base pair region was determined (SEQ ID N0:8).
Two open reading frames (ORFs) were determined: ORF 1, nucleotides 32-964; and ORF 2, nucleotides 1034-1570. The corresponding translated protein sequences are presented herein as SEQ
ID NO: 9 and SEQ ID NO:10, respectively.
25. Details of the characterization of additional library 2 clones, e6, dll, fll, and d6 are provided in the Tables below.
1 S Table 3a Summary of Library 1 and Library 2 Clones Size of ClusterPredicted Clone Sequenced ORF (b.p:) . Protein DNA (b:p.) No. Size ' (Icd) Actual Size;on B2 1204 278-1150 19 31.6/30-32 A22 1009 122-599 1 18.2/_25 ~-gal fusion protein C1 2534 868-1926 42 39.I/35 B17 2006 500-700 (ORF1) 28 20.9/_22 870-1406 (OltF2) 1410-2006 (OItF 3) 142-705 (OltF4) A3 1878 399-1743 69 50.7/52 B9 984 230-931 68 25.3/30 C3 2705 727-1281 18 n.d.
B23 1078 3-1076 67 38.6/30 d7 616 3-614 /3-gal fusion 3 23.4/35-40 protein f8 1459 134-1042 12 33.9/33-35 'Size of Clusterpredicted Sequenced DNA (b.p.)ORIt (b.p.) No: Protein.
Size Clone' Actual Size on GeI
a5 1023 '3-545 (OItF 1) /3-gal 6 21.3/28 fusion protein, 569-1021 (OItF 2) b5 414 1-414 /3-gal fusion 22 16.6/22 protein a1 1208 'S3-801 (OItF 1) 9 27.5/30 880-1206 (ORF 2) d5 1795 1-1704 a-gal fusion 64 62.1/80 protein c5 650 2-604 31 23.3/30 a3 1975 '3-608 (a-gal) (ORF 50 23.1/30 1) 613-1266 (OItF 2) d6 1960 1-1845 24 70.2/n.d.
fll 1495 475-1224 30 30.9/30 e6 1458 96-1325 63 46.4/55 1~ dl 2059 696-1514 (ORF 2) 50 30.4/30.4 l b8c7 1484 77-1234 4 48.9/n.d.
c2 2077 45-857 46 30/30.2 c13 1742 2-1420 (/3-gal) n.d. 54.5/55 f3 2274 1-1788 (/3-gal fusion) 21 67/25 g2 2474 245 (OItF 1) 25 25.4/_25 '461-1156(OItF 2) 1156-1776 (ORF 3) 1798-2472 (OItF 4) g9 4292 5 012Fs 37, 2-349 (ORF 1 ) 123 495-1403 {OItF 2) 33.8 1418-2209 (ORF 3) 29.7 '2223-3719 (ORF 4) 56.8/85 3733-4236 (ORF 5) 19.9 k4 1624 part of g9 37 cluster Gla 1610 32-964 (ORF 1) 2 '"1034-1570 (ORF 2) gll 1899 part of g9 38 cluster n.d. = not determined, underlined numbers for protein size is observed protein size on SDS geI.
Predicted protein size does not include (3-galactosidase fusion peptide.
_ _T.._. _ . _. ~..___ _ _ Table 3b Expressed Pur- Paneled Clonein ifed pGEX Plasmid B2 Yes Yes Yes A22 Yes Yes Yes C Yes Yes Yes B Yes Yes Yes (ORE
4) A3 Yes Yes Yes C7 Yes Yes Yes Gla Yes Yes Yes (small (O~ panel) 2) B9 Yes No No C3 No No No B23 No No No d7 Yes Yes Yes (Small panel) f8 Yes No No a5 Yes Yes Yes (ORE
1) b5 Yes No No al Yes No No (ORE
1) d5 No No No c5 Yes Yes Yes a3 Yes No No d6 No No No fll No No No e6 No No No d1I Yes Yes Yes (ORE
2) Expressed Pur- Paneled Clone . in ified pGEX Plasmid' b8C7 Yes Yes Yes c2 Yes Yes No c 13 Yes No No f3 No No No g2 Yes No No (ORF
2) g9 Yes Yes Yes (small (O~ panel) 4) k4 No No No gll No No No n.d. = not determined, underlined numbers for protein size is observed protein size on SDS gel.
where there are more than 2 ORF's, * indicates ORG expressed that is immunoreactive.
Table 4 Genomic Glone/ Subclone Protein SEQ ~Q m NO
' 1D NO:
approx. insert (Subcione) size N0:51 5.0 kb B2 2197780 EQ ID N0:55 SEQ ID
N0:54 .2kb A22 2210DMIC EQ ID N0:44 EQ ID N0:43 .2 kb C1 C1CON6V2 EQ ID N0:39 EQ ID N0:38 .0 kb B17 B17CON4 EQ ID N0:26,SEQ ID
ORF N0:24 .0 kb 1 EQ ID N0:27 EQ ID N0:28, ORF
EQ ID N0:25, ORF
A3 3CON3 EQ ID N0:17 EQ ID N0:16 6.0 kb C7 C7.2C SEQ ID N0:21SEQ ID
N0:20 3.8 kb 8 (3.5 kb) 8c7 SEQ ID N0:550SEQ ID
N0:549 ____- _ __T ~ __ __ Genomic Clone/ Subclone Protein SEQ SEQ ID NO:
ID NO:
aPProx. insert (Subclone) size Gla Gla EQ ID N0:9, SEQ ID N0:8 6.4 kb EQ ID NO:10, OItF
9 9.4C EQ ID N0:48 EQ ID N0:47 .2 kb S C3 C3CONFIC EQ ID N0:15 .9 kb 23 23CON EQ ID N0:14 SEQ ID N0:13 .5 kb d7 D7CON1 EQ ID N0:12 SEQ ID NO:11 .7 kb FSCONI EQ ID N0:2 EQ ID NO:1 1.3 kb A5CON1 EQ ID N0:4, SEQ ID N0:3 1 kb EQ ID N0:5, b5 BSCON EQ ID N0:7 SEQ ID N0:6 .5 kb I 1CON1 EQ ID N0:36,SEQ ID N0:35 OItF
1.2 kb I
EQ ID N0:37, OltF
D5CON1 EQ ID N0:59 SEQ ID N0:58 1.7 kb c5 C5CON5 EQ ID N0:254SEQ ID N0:253 .7 kb d6 D6CON1V2 SEQ ID N0:495 3.8 kb fl l Fl l EQ ID N0:323part of cluster 41, 3.0 kb SEQ ID N0:322 e6 E6CON1 EQ ID N0:554SEQ ID N0:541 .5 kb dll DI1CON EQ ID N0:551,SEQ ID N0:526 OItF
.1 kb 1 EQ ID N0:552, OItF
EQ ID N0:553, 1tF 3 2 C2CON EQ ID N0:252SEQ ID N0:251 .2 kb 13 C 13CON EQ ID N0:256SEQ ID N0:255 I.7 kb f3 F3CON1 EQ ID N0:258SEQ ID N0:257 .3 kb Genomic Clonel 5ubcloneProtein SEQ'mSEQ TD
NO NO
approx. insert (Subclone) size g2 G2CON EQ ID N0:260,SEQ ID
I OItF N0:259 2.5 kb 1 SEQ ID N0:261, EQ ID N0:262, EQ ID N0:263, g9 G9CON1 EQ ID N0:265,SEQ ID
ORF N0:264 kb 1 EQ ID N0:266, OltF
EQ ID N0:267, 1tF 3 SEQ ID N0:268, 1tF 4 SEQ ID N0:269, 1tF 5 gll SEQ ID
N0:271 1.9 kb k4 SEQ ID
N0:270 .9 kb a3 3L2CON1 EQ ID N0:249,SEQ ID
OI2F N0:248 I O .2 kb 1 EQ ID N0:250, ORF
Example SA
Expression of H. pylori antigen coding regions Amplified products from various clone families, e.g., A3, A22, B2, B9, C1, C5, C7, and Gla, were cloned into pGEX vectors (Pharmacia). The cloned constructs were expressed in E.
coli strain (Stratagene, LaJolla, CA).
1. Clone B2 Expression primers GD96 (forward primer, SEQ ID N0:56) and GD97 (reverse primer, SEQ
ID N0:57) were designed to introduce NcoI and BamHI sites for subcloning the B2 fragment into expression vectors pGEXde165 and pGEXGLI. The expression product lacked the predicted transmembrane region.
Based upon the insolubility of the expressed proteins, only the expression product from pGEXde165 was further purified. The protein was purified by affinity purification chromatography, employing Ni-NTA++ resin from Qiagen (Chatsworth, CA).
_ _ _ __~. _ __ __ _ _r _ WO 98/49314 ' PCT/US98/08487 The purified protein was paneled against various sources of H. pylori-infected sera. The serum paneling was carried out twice, with differing results: (i) 75 %
sensitivity, based upon 35 sera samples, and (ii) sensitivity of about 30% , based upon 183 sera. The serum panel was performed using protein that had undergone further SDS gel eiectroelution following affinity chromatography purification.
2. Clone C1 Amplified products from clone C1CON6V2 were cloned into expression vector pGEXd165 using primers BF (SEQ ID N0:40) and BG (SEQ ID N0:41) as outlined in Table 5 below. The expressed protein was confirmed to be immunoreactive; however, it was cleaved beyond the transmembrane region to form a smaller immunoreactive band. Based upon this observation, a new forward primer was designed for the expression of the smaller protein, primer BO (SEQ ID N0:42), as indicated in Table 5.
The resulting protein was determined to be immunoreactive, and formed in much higher yields than in the previous construct.
3. Clone B17 Expression primers CA (forward primer, SEQ ID N0:29) and CB (reverse primer, SEQ ID
N0:30) were designed to introduce NcoI and BamHI sites for subcloning the B17 ORF4 fragment into expression vector GEXdeIGS.
Expression primers CC (forward primer, SEQ ID N0:31) and CD (reverse primer, SEQ ID
N0:32) were designed to introduce NcoI and BamHI sites for subcloning the B 17 ORF2 fragment into the expression vector.
4. Clone A3 Amplified products from the genomic A3 DNA were digested with NcoIlBamHI
restriction enzymes and subcloned into the NcoI and BamHI sites of expression vector pGEXd165 polyHis, using primers BP (SEQ ID N0:18) and BQ (SEQ ID N0:19) respectively. Expression of protein was induced at 0.5 mM IPTG (isopropylthiogalacto-pyranoside). The expressed protein was of the expected size, and was confirmed to be immunoreactive against Roost pooled sera.
5. Clone C7 PCR amplified products from H. pylon genomic DNA produced a DNA fragment of the expected size, which was then subcloned into the NcoIlBamHI sites of expression vector pGEXde165 using primers GD100 (SEQ ID N0:22) and GD101 (SEQ ID N0:23) respectively. The expressed protein was of the expected size, and was highly insoluble. The protein was readily purifiable by Ni-NTA column chromatography as described above. Immunogenic screening confirmed the expressed protein to be immunoreactive against Roost pooled sera.
6. Clone Gla PCR amplified products were subcloned into expression vector pde1165polyHis using primers BW (SEQ ID N0:33) and BX (SEQ ID N0:34) as outlined in Table 5 below. The expressed protein was confirmed to be immunoreactive. However, the expression product was internally cleaved, with cleavage most likely occurring at the transmembrane region.
Expression of additional cloned constructs was similarly carried out.
Table Sa Primers Used for PCR Expression of DNA Sequences Encodin Antigens of H. pylori Clone Forward Primer/SEQ Reverse Primer/SEQ
Family 1D NO: >D NO:
SEQ ID N0:56 SEQ ID N0:57 2 U v EQ ID N0:45 SEQ ID N0:46 SEQ ID N0:40 SEQ ID N0:41 O
SEQ ID N0:42 EQ ID N0:29 SEQ ID N0:30 SEQ ID N0:18 SEQ ID N0:19 SEQ ID N0:22 SEQ ID N0:23 Gla ORF2 O X
SEQ ID N0:62 SEQ ID N0:34 W
SEQ ID N0:33 SEQ ID N0:49 SEQ ID NO:SO
d7 CM CP
SEQ ID N0:233 SEQ ID N0:548 CN
SEQ ID N0:234 _~__ _... _~_ ..T _. _._. _ i __.
Clone Forward Primer/SEQ Reverse.Primer/SEQ
Family >D NO: m N
DK
f8 SEQ ID N0:235 DI, Dp SEQ ID N0:237 SEQ ID N0:236 a5 (ORFi) DH
EQ ID N0:238 SEQ ID N0:239 b5 I DJ
EQ ID N0:240 SEQ ID N0:241 al (ORF1)DQ DR
SEQ ID N0:242 SEQ ID N0:243 a3 B GC
EQ ID N0:244 SEQ ID N0:245 c5 GP GQ
EQ ID N0:325 SEQ ID N0:326 g2 (ORF2)GR GS
S EQ ID N0:327 SEQ ID N0:328 c2 GJ GI
S EQ ID N0:329 SEQ ID N0:330 13 V Gqr S EQ ID N0:331 SEQ ID N0:332 GM
S EQ ID N0:333 SEQ ID N0:334 g9 (ORF4)N GO
S EQ ID N0:335 SEQ ID N0:336 dll (ORF2)O p EQ 1D N0:337 SEQ ID N0:338 8c7 Y GDI01 S EQ ID N0:339 SEQ ID N0:23 Table Sb Immunoreactivitv of E~ressed Recombinant Antigens of H. pylori to H. pylori Infected Sera Expression I~unoreactivity Immunoreactivity.to Ctone toRoost SFA001 Pooled Sera Pooled Sera dHB2d1 + +
dHA22.8 + +
dHC 1s.11 + +
dHB 170RF4.4 + _ dHA3.5 + +
WO 98/49314 PCTlITS98/08487 Expression CloneImmunoreactivityImmunoreactivity ao Roosf to SFA001 Pooled Sera Pooled Sera dHC7.D6 + +
dHG 1 a2S.5 + +
dHB9.4.2 + +
dHd7S1 + +
dHf8u.2 + +
dHa5.2 ~- _ dHb5.4 t dHa 1.2 -f. _ dHa301tF 1.5 + _ dHc5.2 + +
dHd11.8 + +
dHb8c7.7 + +
dHc2 + +
dHc 13 dHg90RF4.1 + _ dHGla25.5 + +
t t All protein expression products were determined to be immunoreactive against Roost pooled sera, except for dHB9.4.2.
Example 5B
Purification of Illustrative Antigenic Proteins Expressed by H. yylori Clones Y175A Y212A and Y146B
Clones Y104B, Y175A, Y212A and Y146B were expressed as E. coli recombinant proteins according to the protocol described above. Primer sequences for the E. coli expression clones are presented in the Sequence Listing. The recombinant proteins were purified and their immunogenicity was confirmed according to the general procedure described below.
A pellet from a shake flask was spun and then submitted to differential solubilization. Briefly, the pellet was homogenized in a solution containg PBS/SmM PMSF, and spun for 60 minutes at 4°C, 30k. Subsequent rounds of homogenization/centrifugation were as follows: (i) 100 mM Tris/2 Triton/2M Urea/SmM EDTA/0.5 mM DTT (homogenization step)/60 minutes at 4°C, 30k (centrifugation); (ii) PBS pH 7.8/2M Urea/0.5 mM DTT/ 60 minutes at 4°C, 30k; (iii) PBS pH
_. _..._...___ ___._- .~__ __._. _....._ ._ T
7.8/4M Urea/0.5 mM DTT/60 minutes at 4°C, 30k, (iv) PBS pH 8.0/6M
Urea/0.5 mM DTT/60 minutes at 4°C, 30k; (v) PBS pH 8.0/6M urea/2M guanidine HCL/2mM BME
/60 minutes at 4°C, 30k.
The supernatant from step (iv) above was then dialyzed into PBS pH 8.0/6M
Urea/2mM
BME, followed by chromatographic separation using a pre-packed column of Chelating Sepharose (Pharmacia) loaded with five column volumes of 0.2M NiCl2. A 10 Column Volume (C.V.) gradient of Buffer A into 100% of Buffer B was utilized, where Nickel IMAC Buffer A
contained PBS pH
8.0/6M Urea/2mM BME and Nickel IMAC Buffer B contained Buffer A and 250 mM
imidazole.
The appropriate Nickel IMAC fractions were pooled and dialyzed overnight into PBS pH
8.0/6M Urea/0.5mM DTT, and the final product was then vialed and stored at -80°C.
Alternatively, glutathione-sepharose packed columns (Sigma) were utilized.
Fractions were typically analyzed by 1) Pierce Coomassie protein assay, 2) SDS-PAGE (on 12% gel), 3) Western blot using H. pylori Roost Pool fi3, 4) GLT H.pylori negative pool and 5) Anti-E.coli polyclonal antibodies. The results of these analyses confirmed the immunogenicity of the expressed proteins.
Example 6A
Optimization of Antigen Concentration and Small Scale Serum Paneling for Purified Antigen from Clones dHA22.8 (A22) and dHCIS 11 (C1) 1. Clone dHA22.8 Protein expressed by clone dHA22.8 (corresponding to clone A22) was isolated and purified as follows.
2000 ml of culture pellet was suspended in 200 ml of Buffer B (48g urea, 1.2 g NaH2P0,, 0.12 g Tris-HCI, and 90 ml deionized water, adjusted to pH 8.0 and to a total volume of 100 ml), and sonicated for 10 minutes to effect cell lysis. The sonicated mixture was then rocked at room temperature for 1 hour, and spun at 15,000 rpm for 15 minutes at 10°C
to remove the cell debris.
To the supernatant was then added 1 ml of Ni-NTA resin (Qiagen GmgH, Hilden, Germany) which was then mixed at room temperature for 1-2 hours. To pellet the resin, the mixture was centrifuged at 5,000 rpm for 15 minutes at 4°C and the supernatant discarded. The resin was washed with 50 ml of Buffer C (48 g urea, 1.2 g NaH2P04, 0.12 Tris-HCl and 90 ml deionized water, adjusted to pH
6.3 and brought to a total volume of 100 rnl), centrifuged, and the supernatant discarded. The resin was then loaded into a disposable column, and the wash step repeated with remaining Buffer C. The WO 98!49314 PCT/US98/08487 protein was then eluted with 50 ml of Buffer III (50 mM sodium phosphate, pH
8.0, 300 mM NaCI, 250 imidazole). Fractions were collected (2 ml) and analyzed on 12 % SDS-PAGE
gel.
The purified protein was then screened with serum antibodies using conventional techniques (Ausubel, et al., 1988). The purified protein was slotted at concentrations of 0.1-20 ug/mI O.1M
carbonate buffer, blocked with 5 % skim milk in TBS buffer, and reacted with H. pylori positive and H. pylon negative sera diluted 100 times. A serum panel consisting of 36 total sera was used as indicated above. Positive sera was from Roost pool #2 or SFA 001 pool;
negatives were from donor packs.
Following a 1 hour incubation period, the strips were washed 3 times with TBS
buffer, and incubated with alkaline phosphatase-conjugated anti-human Ig secondary antibodies (Promega Biotech, Madison, WI) diluted 1000 times in 5 % skim milk in TBS buffer. The strips were then washed 3 times with TBS wash buffer. Immunoreactive proteins were developed with a substrate, e.g., BCIP, (5-bromo-4-chloro-3-indolyl-phosphate), and NBT (vitro blue tetrazolium salt (Sigma)). The development of color indicated the highly antigenic nature of the purified A22 protein (i.e., reactive with H. pylori-positive sera).
The optimum concentration of antigen was determined to be 2.0 mg/ml. The dHA22.8 (A22) expression product reacted with anti-H. pylori antibodies present in both pooled sera sources (Roost pool #2 and SFA 001 pool), and with 100% of the individual H. pylori-positive sera sampies tested.
The antigen exhibited no detectable cross reactivity with normal sera.
2. Clone dHC1S.11 (C1) Protein expressed by clone dHC1S.11 (C1) was isolated and purified according to the following protocol.
Protein was extracted from a culture pellet by differential solubilization using a series of homogenization/centrifugation steps. The pellet was (i) homogenized in PBS/1 mM PMSF and centrifuged for 30 minutes at 4°C, 19,000 rpm, followed by separation of the supernatant, (ii) homogenized using 1 M urea/10 mM Tris at pH 8.0/i0 mM DTT, followed by centrifugation as in (i) and separation of the supernatant; (iii) homogenized in 4 M urea/10 mM
Tris pH 8.0/2 mM BME, followed by centrifugation as in (i) and separation of the.supernatant; and finally, (iv) homogenization of the pellet with 6 M urea/IOmM Tris pH 8.0/2 mM BME, followed by centrifugation as described above. To the combined supernatants was added 150 mM NaCI. The protein was then separated from the combined supernatants by immobilized metal adsorption chromatography (IMAC) using a 5 ml prepacked column containing chelating sepharose (Pharmacia, Chelating Sepharose Fast Flow) loaded with 2 C.V. (column volumes) 0.2 M NiCl2. The protein was eluted from the column using a 20 C.V. gradient of Buffer A (4 M urea/10 mM Tris at pH 8.0/0.2mM BME/150 mM
NaCI) into 50%
_. ._.-~ ~ _ . _.._- - __ T
Buffer B (Buffer A to which was added 0.5 M imidazole). Fractions were collected (2 ml) and analyzed on 12 % SDS-PAGE gel.
The optimal concentration for immunoscreening was determined essentially as described above. Serum panelling as described above was repeated for the purified C1 protein expressed by clone dHC1S.11.
As in the above example, the expressed protein was reactive with both sources of pooled H.
pylori-positive sera, and showed no signs of cross reactivity with control sera (H. pylori negative . samples, 1 sample of pooled sera and 13 individual samples). Additionally, as can be seen in panels 20-35 and A-C, the recombinant protein exhibited immunoreactivity with 95 % of the H. pylori-positive samples.
In summary, for this small sized panel study, A22 and C1 both exhibit high sensitivity and specificity ( > 90-95 % ) Table Sc Clinical Data for Sera Used for Serum Panellin~~ of Clones dHA22.8 and dHC 1 S 11 Serum.Eudoscopy Culture 11f. Pathology (fi,g. (gastritis) ) A1 Mild antrum gastritis,Moderate Acute/chronic/moderate deep duodenal ulcer B Moderate antrum gastritis,Heavy Chronic/severe with 1 moderate prior Hp ( > 4 duodenitis years with therapy) B2 None Rare Chronic/moderate B4 None NA NA
9 Severe gastritis NA NA
44 Mild antrum gastritis Moderate Chronic/moderate 16 Mild antrum gastritis,Moderate Chronic/moderate deep gastric ulcer 19 Superficial gastric Rare Chronic/moderate and duodenal ulcer 20 Mild antrum gastritis Moderate Acute/chronic 4 Severe gastritis . NA NA
69 Mild antrum gastritis None Chronic/mild and duodenitis, supe~cial duodenal ulcer 47 Mild duodenitis, superficialNone None duodenal ulcer 58 Mild antrum gastritis,Moderate Chroniclmild oesophageal ulcer 54 Moderate gastritis Heavy Chronic/moderate 65 Severe gastritis superficialNone None gastric ulcer WO 98/49314 PC"T/US98/08487 Serum Endoscopy Culture Pathology fH.p. fps) t) C1 Moderate antrum and Heavy Chronic/severe body gastritis C3 NA Heavy ?
34 Gastritis in body None Acutelmild ND = not available.
Example 6B
Further Sera Paneling with Additional Sera Panels for Purified Antigens:
Selection of Preferred Anti ens Additional sera paneling studies were conducted with other sera panels (USC
for University of Southern California, and POW for Prince of Wales Hospital) to further examine the sensitivity and specificity of various H. pylori antigens.
Gold standard tests were employed as reference standards to compute the sensitivities and speciftcities of each individual antigen. Standards used were conventional gold standard tests including histology, CLO test, and the UBT test. UBT is the current most widely used gold standard for indication of "active infection" by non-invasive methods. Histology, CLO
(rapid urease test) and UBT
are all indicate "active infection" since these tests depend on the presence of bacteria to produce a positive result. Since serology measures both past and present infection, the aim of this study was to (i) identify serological markers that would correlate with UBT results for use as "active infection markers", and (ii) to explore both single and muliple antigen combinations to effectively screen for active infection by H. pylori.
A. Use of UBT as a Gold Standard in Single Antigen Screenints Since different antigens have different performances in different panels (this may be a reflection of the geographical differences due to different strains of H.
pylori and previous exposures of populations to other cross-reacting antigens from other bacteria endemic to the region), the use of single antigen markers for the local population was explored using UBT as a single reference standard.
1. USC Panel. Based upon this panel, the best single antigen selection appeared to be antigen Y261A or Y124A. Both of these antigens provided an overall balance of sensitivity and specificity (>80% for both sensitivity and specificity for Y261, and 100%
sensitivity with ?0%
specificity for Y124A). Other preferred antigens, A22 and C1, were 95-100%
sensitive, and 67-77%
specific, respectively in this panel.
2. POW Panel. Based upon this paneling study, the best single antigen selection appeared to be Y 128D 12S and Y 124A, where sensitivity and specificity were about 75-80 % for Y128D12S, and both about 80% for Y124A. Another preferred antigen is Y261A, which exibited a sensitivity of 70 % and a specificity of 80 % . For this paneling series, preferred clones A22 and C 1 were found to be about 95-98% sensitive, and about 60% specific.
B. Usine Other Gold Standards 1. Roost Panel. Only positive sera was tested, and antigens A22 and C1 exhibited 90%
sensitivity. Y124A exhibited about 80% sensitivity, and Y261A possessed a sensitivity of approximately 87 % .
2. UNSW Panel In this paneling series, A22 and C1 demonstated greater than 90-100%
specificity. Y 124 demonstrated 80 % sensitivity and 96 % specificity, and Y261 A demonstrated 80 %
sensitivity and 100 % specificity.
It was observed that the sensitivity and specificity values do not change significantly whether UBT is used alone or in combination with other gold standards in the USC and POW panels. Thus, the somewhat lower specificity for A22 and C1 may be a reflection of previous exposure to other cross-reacting antigens.
C. 2-4 Antigen Combination Tests In view of the high sensitivity of A22 and C1 and somewhat lower specificity for certain panels (POW, USC), a multiple antigen format was explored. The 2-4 antigen format was based on the criteria that, for any 2 -3 highly sensitive antigens, at least both or all 3 have to be positive with respect to the criteria to increase the specificity.
This "2 antigen both positive" criteria was applied to a selected 12 antigen set, where the 12 antigens were selected for a sensitivity of at least 40-50 % for consideration.
The sensitivities and specificities of the 2 antigen both positive criterion is computed, and the resulting table is examined for good performers. Additionally, 2 antigen combinations with high specificity and lower selectivity is run against the entire two antigen combination matrix to provide a result indicating "2 antigen both positive or 2 other antigen both positive". The final analysis then provides a selection of commonly occurring antigens that provide good results across the board between the USC and POW panels.
D. Results of the 2-4 Antigen Combination Test For the USC panel, there are 15 antigenic combinations that provide around 95 % sensitivity and 100% specificity.
For the POW panel, 10 different antigen combinations were shown to provide greater than 70% sensitivity and around 80-90% specificity.
Based upon these findings, exemplary combination selections for both panels are as follows:
(a) C1 and Y124A both positive (sensitivity of 76.2 and 100% and specificity of 89.1 and 95.2 % for POW and USC panels respectively), and (b) C 1 and Y 124A and A22 all positive (sensitivity of 71.4 and 94.7 % and specificity of 91.3 and 95.2 % respectively for POW and USC panels).
For the USC panel, an antigen combination of C1 and c5 was preferred, and for the POW
panel, antigens combinations Y261A and Y124A or antigens C7 and B2 also were preferred.
Thus, based upon these sera paneling data, preferred antigens for use in reliably and universally detecting H. pylori infection include but are not limited to the following: A22, CI, Y124A, Y261A, c5, C7, B2, Y104B, and Y128D.
Table Sd Hp Recombinant Antigen Sensitivities Antigen Roost (n UNSW (n = USC (n POW (n =
= 30) 94) = 30) 40) Y175A 73.3 33.3 70 83 Y 104B 76.7 57 77 68 Y261 A 86.7 79.6 87 70 C7 80 66.7 77 48 b8 73 a5 3.3 8.6 10 18 Y212A 90 78.5 100 _.r _..__. ___ _._ __.__~__.
Antigen Roost (n UNSW (n = USC (n POW (n = 30) 94) = 30) = 40) Y 124A 80 80. 7 100 80 Y 184A 46.7 35. 5 27 13 B2 50 36.6 47 68 Y 128Dso1 36.7 15.1 57 78 Y 146B 63. 3 65 .6 67 53 c5 70 64.5 73 63 Y 150A 20 32.3 27 13 Y153A 66.7 66.7 50 28 C 1 90 90. 3 97 98 A3 46.7 43 57 25 catalase 43.3 9.7 40 13 Table 5e Hp Recombinant Antigen Specificities Antigen UNSW (n USC (n POW (n =-= 25) = 25) 48) b8 73 a5 92 96 94 Y 124A 96 b8 81 B2 100 88 gg Y 128A g9 ~igen UNSW (n = USC (n POW (n =
25) = 25) 4t~
Y 128Dso1 96 84 77 c5 96 96 73 catalase 100 92 98 A graphical summary of sensitivity and specificity data for representative purified H. pylori antigens in different sera panelling studies is presented in Figs. 65 and 66.
Example 7 Purification of 36 kD and "Spot 15" Antigens Produced by H. pylori Cultures of H. pylori (ATCC No. 43504) were grown under appropriate conditions and the cells harvested into phosphate buffered saline. This was followed by repeated centrifugation to remove cell debris and other contaminants. The resulting pellet was then lysed in a French press at > 10,000 PSI, followed by centrifugation at 10,000 x g for 25 minutes.
Prefractionation of soluble H. pylori lysate supernatant was carried out only when enriching for spot 15 (high pH), for subsequent sequence/structural investigations (e.g., mass spectrometry, peptide map).
Fractionation of the 36 kD protein was achieved by dialysis to 10 mM Tris buffer, at pH 8.0, 50 mM NaCI, followed by gradient elution over anion exchange resin, Resource Q
(Pharmacia Biotechnology, Piscataway, NJ). SDS-PAGE/Western blot analysis indicated a Western-positive doublet at 30 kD and a positive 36 kD band. Both fractions were determined to be highly immunoreactive when tested against Roost pooled sera.
Fractionation of the spot 15 antigen was carried out in a similar fashion. The sample was passed over Sephacryl S-100 ion exchange resin (Pharmacia Biotechnology, Piscataway, NJ) (to 10 mM phosphate buffer, 50 mM NaCI, pH 8.0), followed by further fractionation by gradient elution over Resource S cation exchange resin (Pharmacia Biotechnology, Piscataway, NJ). The fraction containing spot 15 antigen was analyzed by SDS-PAGE and Western blot, and was similarly confirmed to be highly antigenic in nature, as indicated by Western blot and immunoreactivity with Roost pooled sera.
Example 8 Characterization of 36 kD and Snot H;pylori Antigens Following fractionation of the antigenic proteins as described in Example 7 above, the isolated 10 fractions were further characterized by two-dimensional electrophoresis, carried out according to the methods of O'Farrell (1975, 1977).
1. 2-Dimensional Electrophoresis Under Low pH Conditions Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975).
15 Isoelectric focusing was carried out in glass tubes having an inner diameter of 2.0 mm, using 2%
ampholines (BDH, Hofer Scientific Instruments, San Francisco, CA) for 9600 volt-hrs. The final tube gel pH gradient as measured by a surface pH electrode is on the enclosed pH
gradient form.
Following equilibration for 10 minutes in Buffer O (10% glycerol, 50 mM DTT, 2.3 % SDS, and 62.5 mM tris buffer, pH 6.8), the tube gel was sealed to the top of the stacking gel, which was placed on top of a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for 4 hours at 12.5 mA/gel. The slab gels were then fixed in a solution of 10 % acetic acid-50% methanol overnight. The following proteins were added as molecular weight markers to the agarose which sealed the tube gel to the slab gel: mysin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD).
These standards appear as horizontal lines on the silver-stained (Oakley, et al., 1980) 10%
acrylamide slab gels. The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
2. Western Blot Following slab gel electrophoresis, a duplicate gel was transferred to transfer buffer (12.5 mM
Tris, pH 8.8, 86 mM glycins, 10% methanol), transblotted onto PVDF paper overnight at 200 mA
(approximately 50 volts/gel). The blot was blocked for 2 hours in 2 % bovine serum albumin (BSA) in TTBS (Tween-Tris-Buffered Saline), rinsed in TTBS, incubated in primary antibody from Roost pool or negative serum pool and diluted 1:2500 in 1 % BSA/TTBS for 2 hours, rinsed in TTBS and placed in a solution containing secondary antibody (antihuman IgG horse radish peroxidase, 1:5000 WO 98/49314 ' PCT/US98/08487 diluted in TTBS) for 1 hour. The blot was rinsed with TTBS, treated with ECL
(Amersham Corporation, Arlington Heights, IL), and exposed to X-ray film.
A computer-generated photograph of an exemplary stained membrane containing antigenic proteins from H. pylori as described above is shown in Figure 2. The "normal"
human sera was confirmed to be H. pylori-negative using "HEr.~coBLOT 2.0" (Genelabs Diagnostics (PTE) Ltd., Singapore}. The identities of the spots indicated numerically on the gel were determined by protein sequencing (described in Example 9 below).
3. 2-Dimensional Electrophoresis Under Huh pH Conditions Two-dimensional electrophoresis adapted for the resolution of basic proteins was performed according to the method of O'Farrell (O'Farrell, et al., 1977).
Nonequilibrium pH gradient electrophoresis using 1.5 % pH 3.5-10 and 0.25 % pH
ampholines (Pharmacia Biotechnology, Piscataway, NJ) was carried out for 140 volts for 12 hour.
Purified tropomyosin, with a lower spot molecular weight of 33 kD and a pI of 5.2, and purified lysozyme, with a molecular weight of 14 kD and a pI of 10.5-I1 (Merck Sharpe &
Dohme, Philadelphia, PA) were added to the samples as internal pI markers.
After equilibration for 10 minutes in buffer (10% glycerol, 50 mM DTT, 2.3%
SDS and 62.5 mM tris, pH 6.8), the tube gel was sealed to the top of the stacking gel, which was placed on top of a 10% acrylamide slab gel (0.75 mm thick), and SDS slab gel electrophoresis was carried out for 4 hours at 12.5 mA/gel. The slab gels were fixed in a solution of 10 % acetic acid/50 % methanol overnight. The following proteins were added as molecular weight standards to the agarose which sealed the tube gel to the slab gel: myosin (220 kD), phosphorylase A (94 kD), catalase (60 kD), actin (43 kD), carbonic anhydrase (29 kD) and lysozyme (14 kD). These standards appear as horizontal lines on the silver stained (Oakley, et al., 1980) 10% acrylamide slab gels.
The silver stained gel was dried between sheets of cellophane paper with the acid edge to the left.
A duplicate gel was transblotted onto PVDF paper and Western blotting carried out as described in 8.1.b. above.
A computer-generated photograph of an exemplary Western blotted membrane containing antigenic proteins from H. pylori as described above is shown in Figure 1. The identities of the spots indicated numerically on the gel were determined by protein sequencing (as described in Example 9 below).
__.._._,.. _..____ _.. __.-___-._..__.. T
WO 98/49314 ' PCT/US98/08487 Example 9 Isolation and Protein Sequence Determination for Western-Positive Spots N-terminal Seguencing The PVDF blots from Example 8 above were stained with Coomassie brilliant blue. Spots corresponding to Western positive bands were excised by scalpel and sequenced directly using a Hewlett-Packard G 1005A N-terminal sequencer with conventional sequencing techniques (e. g. , Miller, 1994; Spiecher, 1989). The sequencing techniques employed gave high repetitive yields (typically ranging from 93-98%), with a detection limit of approximately 100-200 finol.
2. Internal Sequencing Four different methods were utilized to obtain internal sequence information for the above-described isolated antigens of H. pylori (Allen, 1981): Lys-C peptide map and sequencing, CNBr peptide map and sequencing, OPA/CHBr peptide map and sequencing, and LC-MS/MS
sequencing of Lys-C digests.
a. Cyano~en Bromide Cleavage. Cyanogen bromide (CNBr) cleavage was performed on PVDF membranes in 70 % formic acid (Crimmins and Mische, 1996).
The cyanogen bromide digested peptides were then either repurified by capillary HPLC and sequenced directly, or subjected to ortho-phthalaldehyde (OPA) modification.
b. Ortho-nhthalaldehvde (OPA) Modification. Digested peptides were subjected to OPA modification according to the method of Bauer (Bauer, et al., 1984).
This reagent is used to modify primary, but not secondary amines, thereby allowing the identification of sequences having proline as their N-terminal residue and the silencing of all other sequences as determined by Edman-type N-terminal sequence analysis.
c. Liauid Chromatograph /Mass Spectrome~. Internal sequence information was also obtained from liquid chromatography/mass spectrometric based analysis.
Copper stained gel slices (Nakayama, et al., 1996) and PVDF-transferred proteins were first pre-treated by extraction, preliminary purification using Sep-Pak solid phase extraction (Millipore Corporation, Bedford MA), and protease (Lys C) digestion.
Liquid chromatography-mass spectrometry (LC-MS) was performed on digested proteins to assign internal sequence residues by peptide mass and ion trapping techniques (McAtee, et al., 1996).
The digests were chromatographed on a Vydac C18 reverse phase microbore column (150 mm x 1 mm) using an ABI Model 410 B dual syringe pumping system (Applied Biosystems Division, Perkin Elmer, Foster City, CA). The flow rate was maintained at 50 microliters/minute and elution was carried out using a linear gradient from 0.1 % aqueous TFA to 0.1 % TFA in acetonitrile. A Carlo Erba Phoenix 20 CU pump was used to deliver a mixture of methoxyethanoI and isopropanol (1:1, S v/v) at a rate of 50 microliters per minute, which was combined with the column eluent in a post column mixing chamber. An in-line flow splitter was used to restrict flow to the mass spectrometer to approximately 10 microliters/minute. Detection was performed immediately following elution from the column at 214 nanometers using an ABI 759 variable wavelength detector.
Mass spectrometric detection was achieved following post column solvent addition and flow splitting by a VG BioQ triple i0 quadruple mass spectrometer with a nano-electrospray ion source. Spectra were recorded in the positive ion mode using electrospray ionization. Calibration of the instrument was performed in the range m/z 500-2000 by using direct injection analysis of myoglobin. Spectra were recorded at 1.5 seconds intervals and a drying gas of nitrogen was used to aid evaporation of the solvent. The capillary voltage was maintained at approximately 4 kV with a source temperature of 60°C.
3. Sequencing Results Utilizing the approaches described above, the spots indicated in the Western Blots (Figs. I
and 2) were identified. The identities of the spots (where known) are presented in Table 6 below.
Table 6 H. pylori Antigens Reactive Sera With H. pylori Infected Humans Spot NlW.kDpI N-Terminal Antigen if Known Number (o6s) Sequence\
SEQ Il)'NO:
IN
WESTERN
BLOTS
1 62 6.7MKKIS Urease b subunit 2 58 6.3AKEIK Urease b associated chaperonin 3-Major42 6.5AKSIELQEIE Pyruvate favidoxin reductase 3-Minor42 6.5MLTXKDIHAL HP 154-enolase 4 41 6.4XTKIFV HP0537-cagl6 5 56 5.8AFQVN flagellin a protein 6 58 6.0AFQVN flagellin a protein 3S 7-Major42 6.1XICEICFNRTICPE. coli Tuf B protein 7-Minor42 6.1MXGXIIQVLG ATPase proton pump ___.... _...-__.T... ..........__.._. ...___ .___..__.__ __.. _.T .......
Spot MW pI N-Terminal Antigen if Known Number ltD Sequence\
(obs) ~QmNO.
I
8 60 5.8SFRINTNIAA tlagellin b precursor 9 31 6.7MIDXAIIGGG/ al clone orfl, monomine.oxidase SEQ ID N0:231 10 12 6.8MKTFEILKHL neutrophil activating protein 11 6 5.7AISKEEVLEY/ HP 1199-ribosomal protein SEQ ID N0:232L7/L12 12 20 6.0MYIPYVIEN a5 clone, clp protein SEQ ID N0:233 13 159 7.1MKLI undetermined SEQ ID N0:234 14 no sequence 15 79 5.7GKVIGIDLGT HP109 Heat shock protein SEQ ID N0:23570 16-Major42 6.8MREIIXDGNE HP0589-Ferredoxin oxidoreductase SEQ ID N0:236 16-Minor MKLLE O'Toole protein 17 78 6.9MKLLEE O'Toole protein SEQ ID N0:237 tN
Wes~t~
Bt.oTs 1 180 8.2NPP na 2 180 8.3MDXY na 3 88 8.3NKITY na 4 82 8.3ALXTY na 5 62 6.7MKKIS urease b subunit 6 58 6.3AKEIK urease b associated chaperonin 7 60 6.0SFRIN tiagellin b precursor 8 58 6.0AFQVN flagellin a precursor 9-Major46 8.5AYNPK HP0027-Isocitrate dehydrogettace SEQ ID N0:238 9-Minor AVTLI possible contaminant 10 45 9.1XNIQIQNMPK HP1018 SEQ ID N0:239 11 58 9.1MVNKD catalase 12 65 9.4EDDGFYTSVG Omp 5, 8, 9, 19, 27 SEQ ID N0:240 WO 98/49314 PCT/US98/0848~
Spot MW pI N-Terminal Antigen if Known 1cD
Number (obs) Sequence\
SEQ ID NO:
13 43 9.7 KEVKEKKA HP1350 SEQ ID N0:241 14 38 8.8 XDDGGFFTVG hopC protein 15-Major28.5 9.8 HECNAAFVAI possibly blocked SEQ ID N0:242 15-Minor GPKHNXEAGD possibly blocked 16 28 9.0 MKLTP crease a subunit Referring to the low pH blot presented as Fig. 2, spots 9, 11, 12, 13, 15, 16 (major and minor), and 17 represent unique antigens. Spot 9 represents the native H.
pylori antigen corresponding to the recombinant protein expressed by ORF2 of clone al, while spot 12 represents the native antigen that corresponds to the antigenic protein encoded by clone a5.
Looking at the high pH Western blot in Fig. 1, spots 9 (major and minor), 10, 12, 13, and (major and minor) represent unique antigens.
15 As will become apparent from the results below, it was later determined that the N-terminus of the spot 15 antigen (high pH) was blocked, thus, the N-terminal sequence determined for spot 15 by 2-dimensional gel analysis was later shown to be inaccurate.
Example 10 Sequence Analysis of the 36 kD Antigenic Protein and Spot 15 Antigen Isolated from Various Sources of H. pylori The H. pylori spot 15 antigen as described above was isolated from 3 different sources of H.
pylori as described in Examples 8 and 9. The H. pylori samples used were the following: H. pylori, ATCC 43504 (Australian type strain); H. pylori, strain ~ 26695, and H. pylori, J-170, (both obtained from Washington University School of Medicine, St. Louis, MO). The corresponding protein isolated from each source was then analyzed by reverse phase high performance liquid chromatography (HPLC) as described in Example 9.
Minor differences in the native spot 15 proteins from various H. pylori strains were observed, particularly in the early-eluting peak regions, and the spot 15 antigens derived from the TIGR and ATCC H. pylori sources appear to be closely related. The minor differences included slight minor amino acid changes, deamidations, and perhaps a side chain modification, as suggested by a high molecular weight (e.g., Iipopolysaccharide or glycosylation) modification in the TIGR-derived sample.
__ _..-_-~... _.. _. ....._._ .._...._.. _.... _.T.
The J-170 derived spot 15 protein appears to contain additional high molecular weight modifications, as indicated by the differences in molecular weight.
However, all peaks exhibited strong antigenicity when screened against Roost pooled sera, indicating that any minor differences in amino acid sequence among the native proteins does not adversely affect the high immunoreactivity of the recovered spot 15 antigen.
Based upon the characterization data described above, the spot 15 antigen appears to be a processed product of the putative 36 kD protein, as shown in Fig. 4.
A MALDI-TOF MS comparison of peptides generated from an in situ Lys-C digest of "Spot 15", generated by the above-described strains of H. pylori, was carried out.
In examining the total digest, the data indicated that strain 26695 and strain 43504 are more conserved in their primary sequence, and possibly in their post-translational modification profiles than are strains 43504 and J-170, or strains 26695 and J 170. This data, along with that presented above, suggests that although immunoreactivity to Spot 15 is noticeably present in all strains examined, there are differences in amino acid content and protein modifications between Spot 15 in the various strains.
The Lys-C digests were also examined by reversed phase HPLC. Not all of the peptides from the total digest were observed by RP-HPLC, however, this was attributed to the presence of extremely hydrophilic and/or hydrophobic peptides, not detectable within the operating parameters of the reverse phase column. Based upon an examination of RP-HPLC elution profiles, the peptide mass fingerprints appeared to be more conserved between strains than was indicated by the MALDI-TOF MS results.
Preferentially, the MALDI-TOF and other forms of MS (electrospray, fast atom bombardment, etc.) would present a more accurate determination of structural analysis.
Table 7 Comparison of Molecular Masses of Snot 15 Lvs-C Di e~sts by MALDI-TOF MS
43504 T'IGR J170 43504 T'IGR J170 Table 8 Comparison of Molecular Masses of Spot 15 Lys-C Digests Chromato~ranhed on C,g Reverse-Phased HPLC
STRAIN
I~LC Fraction43504 TIGR J170 4s - - -49 - 1302.58 -1302.35 1202.52 1302.19 1402.37 1402.50 1402.13 1285.48 1285.46 1285.56 1276.06 - 1275.81 m STRA IN
i HPLC Fraction43504 TIGR J170 - - 1260.54 - - 1176.82 52 _ _ 54 1281.34 1281.34 1281.17 61 1255.38 1255.33 2003.60 _ 63 2007.47 2007.52 -- _ _ 75 _ _ 76 - 1985.32 -77 - 1985.32 -78 1585.21 1584.80 -1 0 79 - 1884.72 -80 1999.46 - _ 15 Example 11 Antigenic Reactivity Between Various Strains of H
pylori. Separated by 2-D Electrophoresis Various strains of H. pylori were examined to determine the extent of conservation of the proteins identified in ATCC strain 45304, as described in Examples 7 and 8 and shown in Fig. 1, 20 among other strains of H. pylori.
Exemplary H. pylori strains tested were the following: Chico (clinical isolate from ~roville Community Hospital), TIGR 26695, 96-212 (Alaskan isolate, from Aleut), 4655-1 (Gambian child), J170, A-lc (Lithuanian isolate, which contains at least 6 kB of DNA referred to as "X" segment as an insertion near cag region and not present in C-3c), Rus-95 (isolated from Russian immigrant to the 25 United States), #l9 (Peruvian isolate), C-3c (Lithuanian isolate).
These various strains of H. pylori were purified, fractioned by 2D
electrophoresis and Western blotted as described for strain ATCC No. 45304 in Examples 7 and 8, to determine whether the immunoreactive spots indicated in Figs. 1 and 2 set by strain 45304 were conserved between strains.
The results are summarized in Tables 9 and 10 below.
Table 9 Comparison of Helicobacter Strains by 2D-PAGE (pH 8-13) from ATCC HelicobacterStrains Ge/PVDF
Spof ChicoTIGR 96:212465511J170A-lc Rus-95#9 G-3c 26695 Peru ' ' Spot I + + + + + + + + +
Spot 2 + + + + + + + + +
Spot 3 + + + - + - - - +
Spot 4 + + + - + + - - +
Spot 5 + + + + + + + + +
Spot 6 + + + + + + + + +
Spot 7 + + + + + + + + +
spot s + + + + + + + + +
Spot 9 Major+ + + + + - + + +
Spot 9 Minor+ + + + + - + + +
Spot 10 + + + + + + + + +
Spot 11 + - + + + + + + +
Spot 12 + + + + + - + + +
Spot 13 + + + + + + + + +
Spot 14 + + + + + + + + +
Spot 15 ++ ++ ++ ++ ++ ++ ++ ++ ++
Major Spot 15 ++ ++ ++ ++ ++ ++ ++ ++ ++
Minor spot 1 s + + + + + + + + +
Table 10 Comparison of Helicobacter Strains by 2D-PAGE (oH 4-81 from A,~'GC Helicobacter 43504 Strains .
G~PVDF Spot TIGR96-212: . ~ p~
Cliico26695 465511,1170A-lcRus=95 G-3c y spot 1 + + + + + + + + +
Spot 2 + + + + + + + + +
Spot 3 Major+ + + + ++ + + + +
__ ~ _. _.__ _ _. _. _ T
from ATCC HelicobacterStraiia Ge/PVDF
Spot. Chico1'IGR96-2124655/1J170A-lc Rns-95I/9 C-3c 26695 ' ',' Peru Spot 3 Minor+ + + + ++ + '+ + +
Spot 4 + + + - + - + + +
Spot 5 + t + + + + + + +
Spot 6 + t + + + + + + +
Spot 7 Major+ + + + + - + + +
Spot 7 Minor+ + + + + - + + +
Spot 8 + - + + + + + + +
Spot 9 + + + - + - - + +
Spot 10 + + + + + -t + +
Spot 11 + + + + + + t + +
Spot 12 - + + + + - + + +
+
Spot 13 + + + + + + + + +
Spot 14 + + + + + - + + +
Spot 15 + + + + + + + + +
Spot 16 + + + + + + + + +
Major Spot 16 + + + + + - + + +
Minor Spot 17 + + + + + + + + +
+
As can be seen from the above, the immunogenic proteins isolated from H. pylon strain ATCC 45304 are highly conserved between various strains of H. pylori. One of the preferred antigens of the invention, the spot 15 antigen (high pH), was observed in 100 % of the representative strains examined. Western blot results further supported the highly antigenic nature of this protein.
Example 12 MALDI-TOF Mass Snectrometrv Based Mapping of Antigens of H pylori Gel pieces stained with Coomassie or compatible silver stain as described in Example 9 were _ transferred to a microcentrifuge tube and rehydrated with 10 microliters of water. The gel pieces were then washed 3 times with 500 microliters 50% acetonitrile/0.05 M Tris-HCI, pH
8.5 for 20 minutes.
. The supernatants were discarded and the washed pieces were dried for 30 minutes in a Speed-Vac concentrator. Five microliters of a solution containing 0.05 micrograms Lys-C
was added to the tube and incubated for 20 hours at 32°C. Following digestion, the gel pieces were extracted three times with 30 microliters 50% acetonitrile/0.1 % TFA. The supernatants were transferred to a 0.5 ml microcentrifuge tube and dried. The extracted peptides were redissolved in 4 microliters 4-hydroxy-alpha-cyano cinnamic acid and a 0.8 microliter sample was spotted onto a MALDI
sample plate.
MALDI mass spectroscopic analysis was then performed on a PerSeptive BioSystems Voyager DE
mass spectrometer. Peptide mass peaks were then compared to data within the PIR, NRDB, Genebank, EMBL, TIGR H. pylori and The Swiss Protein Databases using the MS-FIT program (UCSF).
The results of matching mass spectral profile peaks of particular Lys-C
digested proteins with database information is summarized in Tables 11 and 12 below.
Table 11 MALDI-TOF Peptide Mass Mapping, pH 4-8 proteins Spot pI Apparent Selected Lys-C Peptide Protein Match Masses (Da) Molecular Weight 1 7.4 68-lcDa 1078.58, 1510.2, 1809, Urease b subunit 2126.17, 2780.08 (FIP
0072) 2 7.1 65-lcDa 1293.15, 1569.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP0010) 3 major7.1 45-lcDa 1217.41, 1819.07, 2545.87,Pyruvate flavidoxin 2469.85, 2774.14 reductase (HP
1108-1111) 5 or 5.8 65-lcDa 1471.61, 1574.82, 1752.97,Flagellin 6 1798.10, a protein 2193.39 (HP 601) 7 major6.2 50-IcDa 1047.11, 1614.51, 1683.67,Elongation 2046.33, factor Tu 2079.33 (HP 1205) 8 5.9 66-lcDa 1037.12, 1588.72, 1954.28,Flagellin 2215.34, b (HP 0115) 2950.39 2 7.1 65-kDa 1293.15, 1589.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP 0010) 12 5.7 20-IcDa 1349.46, 2103.40, 3284.77,CIpP protein 3472.91, (HP
5241.07 0794) __ .T. _ _. _ _. T
Table 12 MALDI-TOF Peptide Mass Mapping, pH 8-13 Proteins Spot pI Apparent Selected Lvs-C Peptide Protein Match Molecular Masses (Da) Weight 5 7.468-kDa 1078.58, 1510.2, 1809, Urease b subunit 2126.17, 2780.08 (HP
0072) 6 7.165-kDa 1293.15, 1569.93, 1995.69,Urease b associated 2404.07, 3323.74 chaperonin (HP0010) 7 5.966-kDa 1037.12, 1588.72, 1954.28,Flagellin b 2215.34, 2950.39 (HP 0115) 8 5.965-kDa 1471.61, 1574.82, 1752.97,Flagellin a 1798.10, 2193.39 (HP 601) 11 9.250-lcDa 1189.31, 1360.57, 1397.53,1944.16,Catalase (HP
2008.33 875) 9 9.340-kDa 1088.25, 1255.50, 1341.55,HP 0027 (icd) major 1487.71, 1716.03 i 9.250-kDa 1189.31, 1360.57, 1397.53,1944.16,Catalase (HP
1 2008.33 875) 12 9.355-kDa 2118.35, 2465.85, 2868.18,HP 0912 2987.34, 3887.41 13 9.740-kDa 1001.08, 1090.28, 1155.43,HP 1350 1650.96, 1910.31 14 9.632-kDa 2118.35, 2465.85, 2868.18,HP 0912 2987.34, 3887.41 15 9.825-kDa 1255.41, 1281.63, 1302.52,HP 0175 1584.86, 1733.87 16 9.224-kDa 1016.27, 1133.20, 1246.51,Urease a subunit 1379.60, 1976.20 (HP
073) Example 13 Anti,;ens for Use in Vaccines Representative antigens described herein were evaluated as vaccine candidates on the basis of Western blot analyses (as described above) of sera obtained from patients prior to and after antimicrobial treatment.
Briefly, prior to treatment and twelve months after antimicrobial treatment, the titres of antibodies reactive against the cloned antigens were determined. Antigens corresonding to antibodies whose titre remained high for an extended period of time subsequent to treatment were determined to be good vaccine candidates.
Two serum panels, the Gasbarrinni panel and the Greenberg panel were used for these analyses. Sera from patients were obtained prior to treatment and 12 or 24 months after treatment.
Patients who tested positive by the UBT after treatment were not included in this analysis, due to - active infection by H. pylori.
WO 98/49314 ' PCT/US98/08487 The Gasbarrinni panel was obtained from male and female patients in Italy, aged 18-70, who were diagnosed with H. pylori infection by endoscopy and UBT. Prior to antimicrobial treatment and 12 months after treatment, serum was collected from the patients.
The Greenberg panel was obtained from male and female patients in California, also aged 18 70, who were diagnosed as having H. pylori infection, as confirmed in antibody tests. Prior to antimicrobial treatment and 24 months after treatment, serum was collected from the patients.
Table 13 summarizes the data obtained from the Gasbarrinni panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 12 months are indicated therein. Twelve months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y139, Y146B, Y175A, and A22.
Table 14 summarizes the data obtained from the Greenberg panel. The numbers and percentages of patients who exhibited high antibody titre against the indicated antigens at 24 months are shown. Twenty four months after treatment, a high percentage of patients continued to exhibit high antibody titre against clones Y184A, Z9A, Y261Ains and Y146B.
Since antigens which invoke a long-lasting antigenic response are considered to be good vaccine candidates, and based upon the results provided in the Tables below, the following antigens are considered to be preferred vaccine candidates: Y139, Y146B, Y175A, and A22, Y184A, Z9A, Y261 Ains and Y 146B .
Table 13 Gasbarrini Panel Yi39 Y184A Y153Ac5.2 Z9A Y128D12insY261AinsY124As Total number 15 5 9 12 4 16 16 19 of reactive samples Number of decreased6 3 6 8 2 12 9 12 titer in 12 month Number of non- 9 2 3 4 2 4 7 7 decreased titer in 12 month Percent decreased40 60 67 67 50 75 56 63 titer in 12 month Percent non- 60 40 33 33 50 25 44 37 decreased titer in 12 month ..__ __-...t... ....._.-____ _..... ...._ ~..
WO 98/49314 ' PCT/US98/08487 Y146B I Y175AI I C7.2Cb8c7.7I ~ A22 CL20 C1S.11~ B2d1Y104 Total number of 16 20 22 16 16 12 18 22 8 reactive samples Number of decreased 4 8 16 11 11 5 ~ 4 5 titer in 12 month IO
Number of non-decreased12 12 6 5 5 7 8 18 3 titer in 12 month Percent decreased 25 40 73 69 69 42 56 18 63 titer in 12 month Percent non- 75 60 27 31 31 58 44 82 37 decreased titer in 12 month Table I4 Greenberg Panel Y139Y184A Y153A c5.2 Z9A Y128D12insY261AinsY124As Total number of 8 7 6 8 2 8 9 9 reactive samples Number of decreased7 3 5 6 I 7 4 7 titer in 24 month I
Number of non-decreased1 4 I 2 1 1 5 2 titer in 24 month 2S Percent decreased88 43 83 75 50 88 44 78 titer in 24 month Percent non- 12 57 17 25 50 12 56 22 decreased titer in 24 month Y146B Y175AC1S.11 ~C7.2Cb8c7.7B2d1 Y104 A22 CL20 f Total number 8 8 12 3 3 5 9 12 9 of reactive samples Number of decreased4 7 8 2 2 3 6 8 7 titer in 24 month Number of non- 4 1 4 1 1 2 3 4 2 decreased titer in 24 month Percent decreased50 88 67 67 67 60 67 67 78 titer in 24 month Percent non- 50 12 33 33 33 40 33 33 22 decreased titer in 24 month While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.
_.__ _ _~~__ ___.___ . T. ____ .. CA 02288211 1999-10-25 s DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTS PARTIE DE CETTE DE~VIANDE OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE[~
NOTE. Pour les tomes additionels, veuitlez contacter le Bureau canadien des brevets THiS SECTION OF THE APPL.ICATION/PATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ,~ OF
' NOTE: For additional volumes-phase cantaci the Canadian Patent Ofific~ .
Claims (49)
1. A H. pylori antigen comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID NOS:340-468, where said antigen is in substantially purified form and is characterized by immunoreactivity with H. pylori positive anti-sera.
2. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:340-349.
NOS:340-349.
3. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:350-359.
NOS:350-359.
4. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:360-369.
NOS:360-369.
5. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ
ID. NOS:370-379.
ID. NOS:370-379.
6. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:380-389.
NOS:380-389.
7. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:390-399.
NOS:390-399.
8. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:400-409.
NOS:400-409.
9. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:410-419.
NOS:410-419.
10. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:420-429.
NOS:420-429.
11. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:430-439.
NOS:430-439.
12. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:440-449.
NOS:440-449.
13. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:450-459.
NOS:450-459.
14. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a cluster antigen sequence selected from the group consisting of SEQ ID
NOS:460-468.
NOS:460-468.
15. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID
NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554.
NOS: 2, 4, 5, 7, 9, 10, 12, 14, 17, 21, 25-28, 36, 37, 39, 44, 48, 55, 59, 61, 69, 249, 250, 252, 254, 256, 258, 260-263, 265-269, 323, 324, and 550-554.
16. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a polypeptide sequence selected from the group consisting of SEQ ID
NOS:555-602.
NOS:555-602.
17. A H. pylori antigen of claim 1, comprising a polypeptide sequence selected from the group consisting of SEQ ID NOs:555-602.
18. A H. pylori antigen of claim 1, comprising at least 6 contiguous amino acids contained within a sequence selected from the group consisting of SEQ ID NO:44 (A22), SEQ ID N0:39 (C1), SEQ ID NO:568 (Y124A), SEQ ID NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 (C7), SEQ ID NO:55 (B2), SEQ ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
19. A H. pylori antigen in substantially purified form and characterized by immunoreactivity with H. pylori positive anti-sera, where said antigen is encoded by a polynucleotide sequence at least 18 nucleotides in length and capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of SEQ
ID NOs:469-547.
ID NOs:469-547.
20. The H. pylori antigen of claim 19, encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 1-10, corresponding to SEQ ID
NOS:469, 470 (cluster 1); SEQ ID NO:471 (cluster 2); SEQ ID NO:472 (cluster 3); SEQ ID NO:473 (cluster 4); SEQ ID
NOs:474, 475 (cluster 5); SEQ ID NO:476 (cluster 6); SEQ ID NO:477 (cluster 7); SEQ ID NO:478 (cluster 8); SEQ ID NO:479 (cluster 9); and SEQ ID NO:480 (cluster 10).
NOS:469, 470 (cluster 1); SEQ ID NO:471 (cluster 2); SEQ ID NO:472 (cluster 3); SEQ ID NO:473 (cluster 4); SEQ ID
NOs:474, 475 (cluster 5); SEQ ID NO:476 (cluster 6); SEQ ID NO:477 (cluster 7); SEQ ID NO:478 (cluster 8); SEQ ID NO:479 (cluster 9); and SEQ ID NO:480 (cluster 10).
21. The H. pylori antigen of claim 19, encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 11-20, corresponding to SEQ ID
NO:481 (cluster 11);
SEQ ID NO:482 (cluster 12); SEQ ID NO: 483 (cluster 13); SEQ ID NO:484 (cluster 14); SEQ ID
NOs:485, 486 (cluster 15); SEQ ID NO:487 (cluster 16); SEQ ID NO:488 (cluster 17); SEQ ID
NO:489 (cluster 18); SEQ ID NO:490 (cluster 19); and SEQ ID NO:491 (cluster 20).
NO:481 (cluster 11);
SEQ ID NO:482 (cluster 12); SEQ ID NO: 483 (cluster 13); SEQ ID NO:484 (cluster 14); SEQ ID
NOs:485, 486 (cluster 15); SEQ ID NO:487 (cluster 16); SEQ ID NO:488 (cluster 17); SEQ ID
NO:489 (cluster 18); SEQ ID NO:490 (cluster 19); and SEQ ID NO:491 (cluster 20).
22. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 21-30, corresponding to SEQ
ID NO: 492 (cluster 21); SEQ ID NO:493 (cluster 22); SEQ ID NO:494 (cluster 23); SEQ ID
NO:495 (cluster 24); SEQ ID NOs:496 (cluster 25); SEQ ID NO:497 (cluster 26);
SEQ ID NO:498 (cluster 27); SEQ ID NO:499 (cluster 28); SEQ ID NO:500 (cluster 29); and SEQ
ID NO:501 (cluster 30).
ID NO: 492 (cluster 21); SEQ ID NO:493 (cluster 22); SEQ ID NO:494 (cluster 23); SEQ ID
NO:495 (cluster 24); SEQ ID NOs:496 (cluster 25); SEQ ID NO:497 (cluster 26);
SEQ ID NO:498 (cluster 27); SEQ ID NO:499 (cluster 28); SEQ ID NO:500 (cluster 29); and SEQ
ID NO:501 (cluster 30).
23. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 31-40, corresponding to SEQ
ID NO:502 (cluster 31); SEQ ID NO:503 (cluster 32); SEQ ID NO:504 (cluster 33); SEQ ID NO:505 (cluster 34); SEQ ID NOs:506, 507, 508 (cluster 35); SEQ ID NO:509 (cluster 36); SEQ ID NO:510 (cluster 37); SEQ ID NO:511 (cluster 38); SEQ ID NO:512 (cluster 39); and SEQ
ID NOs:513, 514 (cluster 40).
ID NO:502 (cluster 31); SEQ ID NO:503 (cluster 32); SEQ ID NO:504 (cluster 33); SEQ ID NO:505 (cluster 34); SEQ ID NOs:506, 507, 508 (cluster 35); SEQ ID NO:509 (cluster 36); SEQ ID NO:510 (cluster 37); SEQ ID NO:511 (cluster 38); SEQ ID NO:512 (cluster 39); and SEQ
ID NOs:513, 514 (cluster 40).
24. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 41-50, corresponding to SEQ
ID NO:515 (cluster 41); SEQ ID NO:516 (cluster 42); SEQ ID NOs:517, 518 (cluster 43); SEQ ID
NO:519 (cluster 44); SEQ ID NOs:520 (cluster 45); SEQ ID NO:521 (cluster 46);
SEQ ID NOs:522, 523 (cluster 47); SEQ ID NO:524 (cluster 48); SEQ ID NO:525 (cluster 49); and SEQ ID NO:526 (cluster 50).
ID NO:515 (cluster 41); SEQ ID NO:516 (cluster 42); SEQ ID NOs:517, 518 (cluster 43); SEQ ID
NO:519 (cluster 44); SEQ ID NOs:520 (cluster 45); SEQ ID NO:521 (cluster 46);
SEQ ID NOs:522, 523 (cluster 47); SEQ ID NO:524 (cluster 48); SEQ ID NO:525 (cluster 49); and SEQ ID NO:526 (cluster 50).
25. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 51-60, corresponding to SEQ
ID NO:527 (cluster 51); SEQ ID NO:528 (cluster 52); SEQ ID NOs:529 (cluster 53); SEQ ID
NO:530 (cluster 54); SEQ ID NO:531 (cluster 55); SEQ ID NO:532 (cluster 56);
SEQ ID NOs:533 (cluster 57); SEQ ID NOs:534, 535 (cluster 58); SEQ ID NOs:536, 537 (cluster 59); and SEQ ID
NO:538 (cluster 60).
ID NO:527 (cluster 51); SEQ ID NO:528 (cluster 52); SEQ ID NOs:529 (cluster 53); SEQ ID
NO:530 (cluster 54); SEQ ID NO:531 (cluster 55); SEQ ID NO:532 (cluster 56);
SEQ ID NOs:533 (cluster 57); SEQ ID NOs:534, 535 (cluster 58); SEQ ID NOs:536, 537 (cluster 59); and SEQ ID
NO:538 (cluster 60).
26. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence spanning a cluster region selected from the group consisting of clusters 61-69, corresponding to SEQ
ID NO:539 (cluster 61); SEQ ID NO:540 (cluster 62); SEQ ID NOs:541 (cluster 63); SEQ ID
NO:542 (cluster 64); SEQ ID NO:543 (cluster 65); SEQ ID NO:544 (cluster 66);
SEQ ID NOs:545 (cluster 67); SEQ ID NO:546 (cluster 68); and SEQ ID NO:547 (cluster 69).
ID NO:539 (cluster 61); SEQ ID NO:540 (cluster 62); SEQ ID NOs:541 (cluster 63); SEQ ID
NO:542 (cluster 64); SEQ ID NO:543 (cluster 65); SEQ ID NO:544 (cluster 66);
SEQ ID NOs:545 (cluster 67); SEQ ID NO:546 (cluster 68); and SEQ ID NO:547 (cluster 69).
27. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID
NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID
NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID
NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:264, SEQ ID
NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID NO:549.
NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID
NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID
NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:264, SEQ ID
NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID NO:549.
28. The H. pylori antigen of claim 19, where said antigen is encoded by a polynucleotide capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID
NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
29. The H. pylori antigen of claim 19, encoded by a contiguous series of nucleotides spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
30. The H. pylori antigen of claim 27, encoded by a contiguous series of nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID
NO:47, SEQ
ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID
NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ
ID
NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID NO:549.
ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID
NO:47, SEQ
ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID
NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ
ID
NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID NO:549.
31. The H. pylori antigen of claim 19, encoded by a contiguous series of nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID
NO:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID
NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID
NO:98 (Y128D).
NO:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID
NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID
NO:98 (Y128D).
32. A H. pylori antigen-coding polynucleotide in substantially purified form, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA
sequence at least 18 nucleotides in length spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
sequence at least 18 nucleotides in length spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
33. The polynucleotide of claim 32, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID
NO:47, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ
ID
NO:259, SEQ ID NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID
NO:549.
ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:43, SEQ ID
NO:47, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ
ID
NO:259, SEQ ID NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:322, SEQ ID
NO:549.
34. The polynucleotide of claim 32, capable of selectively hybridizing, under conditions of stringent hybridization, to a DNA sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ
ID NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ
ID NO:98 (Y128D).
ID NO:253 (c5), SEQ ID NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ
ID NO:98 (Y128D).
35. The polynucleotide of claim 32, comprising at least 18 contiguous nucleotides spanning a cluster region selected from the group consisting of SEQ ID NOs:469-547.
36. The polynucleotide of claim 32, comprising at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID
NO:13, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID
NO:38, SEQ
ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID
NO:60, SEQ
ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ
ID
NO:257, SEQ ID NO:259, SEQ ID NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID
NO:322, SEQ ID NO:549.
NO:13, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:35, SEQ ID
NO:38, SEQ
ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:58, SEQ ID
NO:60, SEQ
ID NOs:70-230, SEQ ID NO:248, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ
ID
NO:257, SEQ ID NO:259, SEQ ID NO:264, SEQ ID NO:270, SEQ ID NO:271, SEQ ID
NO:322, SEQ ID NO:549.
37. The polynucleotide of claim 32, comprising at least 18 contiguous nucleotides contained within a sequence selected from the group consisting of SEQ ID NO:43 (A22), SEQ ID NO:38 (C1), SEQ ID NOs:94, 95 (Y124A), SEQ ID NOs:169, 172 (Y261A), SEQ ID NO:253 (c5), SEQ ID
NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
NO:20 (C7), SEQ ID NOs:51, 54 (B2), SEQ ID NO:60 (Y104B), and SEQ ID NO:98 (Y128D).
38. A diagnostic kit for use in screening a biological fluid for the presence of an anti-H.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 1 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 1 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
39. A diagnostic kit for use in screening a biological fluid for the presence of an anti-H.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 18 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 18 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
40. A diagnostic kit for use in screening a biological fluid for the presence of an anti-H.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 19 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
pylori antibody, comprising a substantially purified H. pylori antigen of claim 19 that is immunoreactive with at least one anti-H. pylori antibody, and a reporter for detecting binding of said antibody to the antigen.
41. The kit of claim 38, which includes at least two H. pylori antigens having different antibody specificities.
42. The kit of claim 38, wherein said polypeptide antigen is attached to a solid support.
43. The kit of claim 38, further comprising a non-attached reporter-labelled anti-human antibody, wherein binding of said anti-H. pylori antibody to said polypeptide antigen can be detected by binding of the reporter-labelled antibody to said anti-H. pylori antibody.
44. A method of detecting H. pylori infection in a subject, comprising reacting a biological fluid from a subject with a purified H. pylori polypeptide antigen of claim 1, and detecting the presence of antibody bound to said antigen.
45. A method of detecting H. pylori infection in a subject, comprising reacting a biological fluid from a subject with a purified H. pylori polypeptide antigen of claim 18, and detecting the presence of antibody bound to said antigen.
46. A H. pylori vaccine composition, comprising a H. pylori polypeptide antigen of claim 1 which is characterized by its ability to reduce the level of H. pylori infection in a Rhesus monkey or mouse challenged with the peptide, and then infected with H. pylori.
47. The vaccine composition of claim 46, comprising an antigen selected from the group consisting of SEQ ID NO:44 (A22), SEQ ID NO:39 (C1), SEQ ID NO:568 (Y124A), SEQ ID
NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 (C7), SEQ ID NO:55 (B2), SEQ
ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
NO:557 (Y261A), SEQ ID NO:254 (c5), SEQ ID NO:21 (C7), SEQ ID NO:55 (B2), SEQ
ID NO:61 (Y104B), SEQ ID NO:573 (Y128D).
48. The vaccine composition of claim 46, comprising an antigen selected from the group consisting of SEQ ID NO:565 (Y139), SEQ ID NO:575 (Y146B), SEQ ID NO:555 (Y175A), SEQ
ID NO:44 (A22), SEQ ID NO:569 (Y184A), SEQ ID NO:578 (Z9A), SEQ ID NO:557 (Y261A) and SEQ ID NO:575 (Y146B).
ID NO:44 (A22), SEQ ID NO:569 (Y184A), SEQ ID NO:578 (Z9A), SEQ ID NO:557 (Y261A) and SEQ ID NO:575 (Y146B).
49. A H. pylori vaccine composition, comprising a H. pylori polypeptide antigen of claim 1, characterized by its ability to invoke a long-lasting antigenic response in a subject challenged with said antigen and subjected to antimicrobial treatment.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4510797P | 1997-04-25 | 1997-04-25 | |
US6195897P | 1997-10-14 | 1997-10-14 | |
US60/045,107 | 1997-10-14 | ||
US60/061,958 | 1997-10-14 | ||
PCT/US1998/008487 WO1998049314A2 (en) | 1997-04-25 | 1998-04-25 | ANTIGENIC COMPOSITION AND METHOD OF DETECTION FOR $i(HELICOBACTER PYLORI) |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2288211A1 true CA2288211A1 (en) | 1998-11-05 |
Family
ID=26722381
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002288211A Abandoned CA2288211A1 (en) | 1997-04-25 | 1998-04-25 | Antigenic composition and method of detection for helicobacter pylori |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0977864A2 (en) |
JP (1) | JP2001517091A (en) |
AU (1) | AU7166098A (en) |
CA (1) | CA2288211A1 (en) |
WO (1) | WO1998049314A2 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9806039D0 (en) * | 1998-03-20 | 1998-05-20 | Cortecs Ltd | Therapy |
AU3356300A (en) * | 1999-02-04 | 2000-08-25 | American Cyanamid Company | 19 kilodalton protein of (helicobacter pylori) |
US6316205B1 (en) | 2000-01-28 | 2001-11-13 | Genelabs Diagnostics Pte Ltd. | Assay devices and methods of analyte detection |
DE60124526D1 (en) * | 2000-04-27 | 2006-12-28 | Max Planck Gesellschaft | METHOD FOR IDENTIFYING HELICOBACTER ANTIGENES |
DE10194938D2 (en) * | 2000-11-15 | 2003-12-04 | Ludwig Deml | The Helicobacter cysteine rich Protein A (HcpA) and its applications |
SE0101030D0 (en) * | 2001-03-23 | 2001-03-23 | Nordic Bio Ab | Immunogenic cell surface proteins of helicobacter pylori |
WO2003085378A2 (en) * | 2002-04-03 | 2003-10-16 | Syngenta Participations Ag | Detection of wheat and barley fungal pathogens which are resistant to certain fungicides using the polymerase chain reaction |
JP3823268B2 (en) * | 2002-05-30 | 2006-09-20 | 独立行政法人科学技術振興機構 | Helicobacter pylori cell death inducer |
GB0718966D0 (en) * | 2007-09-28 | 2007-11-07 | Liverpool School Of Tropical M | Bacterial vaccine |
MY182584A (en) * | 2011-08-03 | 2021-01-25 | Univ Sains Malaysia | Helicobacter pylori proteins for diagnostic kit and vaccine |
WO2016000022A1 (en) * | 2014-06-30 | 2016-01-07 | Murdoch Childrens Research Institute | Helicobacter therapeutic |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996040893A1 (en) * | 1995-06-07 | 1996-12-19 | Astra Aktiebolag | Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics |
FR2739622B1 (en) * | 1995-10-04 | 1997-12-05 | Pasteur Merieux Serums Vacc | NEW MEMBRANE PROTEINS FROM HELICOBACTER PYLORI |
-
1998
- 1998-04-25 CA CA002288211A patent/CA2288211A1/en not_active Abandoned
- 1998-04-25 AU AU71660/98A patent/AU7166098A/en not_active Abandoned
- 1998-04-25 WO PCT/US1998/008487 patent/WO1998049314A2/en active Application Filing
- 1998-04-25 EP EP98918806A patent/EP0977864A2/en not_active Withdrawn
- 1998-04-25 JP JP54726398A patent/JP2001517091A/en not_active Ceased
Also Published As
Publication number | Publication date |
---|---|
AU7166098A (en) | 1998-11-24 |
EP0977864A2 (en) | 2000-02-09 |
WO1998049314A8 (en) | 1999-04-08 |
WO1998049314A2 (en) | 1998-11-05 |
WO1998049314A3 (en) | 1999-01-14 |
JP2001517091A (en) | 2001-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2907552B2 (en) | Haemophilus outer membrane protein | |
US8992936B2 (en) | Altered OspA of Borrelia burgdorferi | |
CA2294701C (en) | Surface antigens and proteins useful in compositions for the diagnosis and prevention of lyme disease | |
US5350842A (en) | DNAs encoding Treponema pallidum antigens | |
CA2288211A1 (en) | Antigenic composition and method of detection for helicobacter pylori | |
CA2331845A1 (en) | Compounds and methods for the diagnosis and treatment of ehrlichia infection | |
CA2112466A1 (en) | Methods and compositions for diagnosing lyme disease | |
CA2071863C (en) | Recombinant vaccine for porcine pleuropneumonia | |
WO2008084072A2 (en) | Protective proteins of s. agalactiae, combinations thereof and methods of using the same | |
AU722103B2 (en) | Toxoplasma gondii antigen Tg20 | |
US6143872A (en) | Borrelia burdorferi Osp A and B proteins and immunogenic peptides | |
CA2025230A1 (en) | Treponema hyodysenteriae antigens having a molecular weight of 39 kda and dna encoding therefor | |
US20040091901A1 (en) | Immunogenic Mycoplasma hyopneumoniae polypeptides | |
CA2006587C (en) | Membrane proteins and peptides of haemophilus influenzae type b | |
US5681934A (en) | 47-kilodalton antigen of Treponema pallidum | |
US6436399B1 (en) | Nucleic acid encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby | |
AU747124B2 (en) | Recombinant P37/FlaA as a diagnostic reagent | |
EP1038000A1 (en) | Compounds and methods for the detection and prevention of (t. cruzi) infection | |
WO1995010612A1 (en) | FIMBRILLIN PROTEIN OF $i(PORPHYROMONAS GINGIVALIS) | |
CA2190999A1 (en) | Cloned leptospira outer membrane protein | |
JP4752054B2 (en) | Secreted antigen 1 protein of Babesia gibsoni, DNA encoding this protein, and use thereof | |
US5916562A (en) | Membrane proteins and peptides of haemophilus influenzae type B | |
JP4727059B2 (en) | Utilization of nucleic acid molecule encoding Babesagibsoni P50 antigen and its expression product | |
EP0915977A1 (en) | B. burgdorferi polypeptides expressed in vivo | |
CA1341148C (en) | Methods and compositions for the preparation of treponema pallidum antigens, including purified antigens and dna sequences coding therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |