WO1996040893A1 - Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics - Google Patents

Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics Download PDF

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Publication number
WO1996040893A1
WO1996040893A1 PCT/US1996/009122 US9609122W WO9640893A1 WO 1996040893 A1 WO1996040893 A1 WO 1996040893A1 US 9609122 W US9609122 W US 9609122W WO 9640893 A1 WO9640893 A1 WO 9640893A1
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WO
WIPO (PCT)
Prior art keywords
seq
pylori
polypeptide
fragment
nucleic acid
Prior art date
Application number
PCT/US1996/009122
Other languages
French (fr)
Inventor
Douglas Smith
O. Thomas Berglindh
Björn L. MELLGÄRD
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Astra Aktiebolag
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Astra Aktiebolag filed Critical Astra Aktiebolag
Priority to EE9700373A priority Critical patent/EE9700373A/en
Priority to AU63278/96A priority patent/AU710880C/en
Priority to NZ311874A priority patent/NZ311874A/en
Priority to JP9501519A priority patent/JPH11504220A/en
Priority to SK1651-97A priority patent/SK165197A3/en
Priority to BR9609430A priority patent/BR9609430A/en
Priority to EP96922393A priority patent/EP0842270A4/en
Publication of WO1996040893A1 publication Critical patent/WO1996040893A1/en
Priority to IS4630A priority patent/IS4630A/en
Priority to NO975745A priority patent/NO975745L/en

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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • H. pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet 1: 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83- 88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H.
  • H pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma.
  • Boser M. J. (1993) Trends Microbiol. 1 : 255-260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age.
  • H pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
  • urease an enzyme that may play a role in neutralizing gastric acid pH
  • Ferrero R.L. and A. Lee (1991) Microb. Ecol. Hlth. Dis. 4: 121-134; Labigne et al., (1991) J. Bacteriol. 173: 1920- 1931
  • the bacterial flagellar proteins responsible for motility across the mucous layer (Hazell et al., (1986) J. Inf. Dis.
  • This invention relates to novel genes, e.g., genes encoding polypeptides such as bacterial surface proteins, from the organism Helicobacter pylori (H. pylori), and other related genes, their products, and uses thereof.
  • the nucleic acids and peptides of the present invention have utility for diagnostic and therapeutics for H. pylori and other
  • this invention features compositions of nucleic acids corresponding to entire coding sequences of H. pylori proteins, including surface or secreted proteins or parts thereof, nucleic acids capable of binding mRNA from H. pylori proteins to block protein translation, and methods for producing H. pylori proteins or parts thereof using peptide synthesis and recombinant DNA techniques.
  • This invention also features antibodies and nucleic acids useful as probes to detect H. pylori infection.
  • vaccine compositions and methods for the protection or treatment of infection by H. pylori are within the scope of this invention.
  • Figure 1 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.
  • Figure 2 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.
  • Figure 3 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
  • Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in buffer containing DOC.
  • the invention features a recombinant or substantially pure preparation of H. pylori polypeptide of SEQ ID NO: 384.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 384, such nucleic acid is contained in SEQ ID NO: 1.
  • the H. pylori polypeptide sequences of the invention described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides of the invention are contained in the Sequence Listing.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 384 through SEQ ID NO: 389, SEQ ID NO: 391 through SEQ ID NO: 400, SEQ ID NO: 402 through SEQ ID NO: 406, SEQ ID NO: 408, SEQ ID NO: 411 through SEQ ID NO: 412, SEQ ID NO: 414 through SEQ ID NO: 430, SEQ ID NO: 432 through SEQ ID NO: 434, SEQ ID NO: 436 through SEQ ID NO: 441, and SEQ ID NO: 443.
  • the invention also includes substantially pure nucleic acid encoding an H.
  • pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 384 through SEQ ID NO: 389, SEQ ID NO: 391 through SEQ ID NO: 400, SEQ ID NO: 402 through SEQ ID NO: 406, SEQ ID NO: 408, SEQ ID NO: 411 through SEQ ID NO: 412, SEQ ID NO: 414 through SEQ ID NO: 430, SEQ ID NO: 432 through SEQ ID NO: 434, SEQ ID NO: 436 through SEQ ID NO: 441, and SEQ ID NO: 443, such nucleic acids are contained in SEQ ID NO: 1 through SEQ ID NO: 50.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 444, SEQ ID NO: 446 through SEQ ID NO: 448, SEQ ID NO: 450 through SEQ ID NO: 462, SEQ ID NO: 465 through SEQ ID NO: 466, SEQ ID NO: 468 through SEQ ID NO: 469, SEQ ID NO: 471 through SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 478 through SEQ ID NO: 479, SEQ ID NO: 481 through SEQ ID NO: 484, SEQ ID NO: 486, SEQ ID NO: 488 through SEQ ID NO: 501, and SEQ ID NO: 503 through SEQ ID NO: 506.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 444, SEQ ID NO: 446 through SEQ ID NO: 448, SEQ ID NO: 450 through SEQ ID NO: 462, SEQ ID NO: 465 through SEQ ID NO: 466, SEQ ID NO: 468 through SEQ ID NO: 469, SEQ ID NO: 471 through SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 478 through SEQ ID NO: 479, SEQ ID NO: 481 through SEQ ID NO: 484, SEQ ID NO: 486, SEQ ID NO: 488 through SEQ ID NO: 501, and SEQ ID NO: 503 through SEQ ID NO: 506, such nucleic acids are contained in SEQ ID NO: 51 through SEQ ID NO: 100.
  • the invention features a recombinant or substantially pure preparation of an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 509 through SEQ ID NO: 510, SEQ ID NO: 512 through SEQ ID NO: 514, SEQ ID NO: 516, SEQ ID NO: 518 through SEQ ID NO: 520, SEQ ID NO: 522 through SEQ ID NO: 525, SEQ ID NO: 527 through SEQ ID NO: 533, SEQ ID NO: 535 through SEQ ID NO: 537, SEQ ID NO: 539 through SEQ ID NO: 540, SEQ ID NO: 542 through SEQ ID NO: 544, SEQ ID NO: 546 through SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 553 through SEQ ID NO: 556, SEQ ID NO: 558, SEQ ID NO: 560, SEQ ID NO: 562 through SEQ ID NO: 568
  • the invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 509 through SEQ ID NO: 510, SEQ ID NO: 512 through SEQ ID NO: 514, SEQ ID NO: 516, SEQ ID NO: 518 through SEQ ID NO: 520, SEQ ID NO: 522 through SEQ ID NO: 525, SEQ ID NO: 527 through SEQ ID NO: 533, SEQ ID NO: 535 through SEQ ID NO: 537, SEQ ID NO: 539 through SEQ ID NO: 540, SEQ ID NO: 542 through SEQ ID NO: 544, SEQ ID NO: 546 through SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 553 through SEQ ID NO: 556, SEQ ID NO: 558, SEQ ID NO: 560, SEQ ID NO: 562 through SEQ ID NO: 568, SEQ ID NO
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 576 through SEQ ID NO: 579, SEQ ID NO: 581 through SEQ ID NO: 583, SEQ ID NO: 585 through SEQ ID NO: 593, SEQ ID NO: 596 through SEQ ID NO: 614, SEQ ID NO: 617 through SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO: 629 through SEQ ID NO: 631, SEQ ID NO: 633, and SEQ ID NO: 635 through SEQ ID NO: 636.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 576 through SEQ ID NO: 579, SEQ ID NO: 581 through SEQ ID NO: 583, SEQ ID NO: 585 through SEQ ID NO: 593, SEQ ID NO: 596 through SEQ ID NO: 614, SEQ ID NO: 617 through SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO: 629 through SEQ ID NO: 631, SEQ ID NO: 633, and SEQ ID NO: 635 through SEQ ID NO: 636, such nucleic acids are contained in SEQ ID NO: 151 through SEQ ID NO: 200.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 638 through SEQ ID NO: 640, SEQ ID NO: 642 through SEQ ID NO: 643, SEQ ID NO: 647, SEQ ID NO: 649 through SEQ ID NO: 651, SEQ ID NO: 653 through SEQ ID NO: 661, SEQ ID NO: 663 through SEQ ID NO: 670, SEQ ID NO: 673 through SEQ ID NO: 674, SEQ ID NO: 676, SEQ ID NO: 678 through SEQ ID NO: 683, SEQ ID NO: 687 through SEQ ID NO: 692, and SEQ ID NO: 694 through SEQ ID NO: 702.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 638 through SEQ ID NO: 640, SEQ ID NO: 642 through SEQ ID NO: 643, SEQ ID NO: 647.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 705 through SEQ ID NO: 708, SEQ ID NO: 712 through SEQ ID NO: 714, SEQ ID NO: 716 through SEQ ID NO: 722, SEQ ID NO: 725 through SEQ ID NO: 730, SEQ ID NO: 732 through SEQ ID NO: 733, SEQ ID NO: 735 through SEQ ID NO: 744, SEQ ID NO: 746 through SEQ ID NO: 752, SEQ ID NO: 755 through SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761 through SEQ ID NO: 763, and SEQ ID NO: 767 through SEQ ID NO: 770.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 705 through SEQ ID NO: 708, SEQ ID NO: 712 through SEQ ID NO: 714, SEQ ID NO: 716 through SEQ ID NO: 722, SEQ ID NO: 725 through SEQ ID NO: 730, SEQ ID NO: 732 through SEQ ID NO: 733, SEQ ID NO: 735 through SEQ ID NO: 744, SEQ ID NO: 746 through SEQ ID NO: 752, SEQ ID NO: 755 through SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761 through SEQ ID NO: 763, and SEQ ID NO: 767 through SEQ ID NO: 770, such nucleic acids are contained in SEQ ID NO: 251 through SEQ ID NO: 300.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 771 through SEQ ID NO: 773, SEQ ID NO: 775, SEQ ID NO: 777, SEQ ID NO: 779 through SEQ ID NO: 784, SEQ ID NO: 786 through SEQ ID NO: 787, SEQ ID NO: 789 through SEQ ID NO: 792, SEQ ID NO: 794, SEQ ID NO: 796, SEQ ID NO: 798 through SEQ ID NO: 805, SEQ ID NO: 807 through SEQ ID NO: 811, SEQ ID NO: 813 through SEQ ID NO: 819, SEQ ID NO: 821 through SEQ ID NO: 822, SEQ ID NO: 824 through SEQ ID NO: 826, SEQ ID NO: 828 through SEQ ID NO: 832, and SEQ ID NO: 835.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 771 through SEQ ID NO: 773, SEQ ID NO: 775, SEQ ID NO: 777, SEQ ID NO: 779 through SEQ ID NO: 784, SEQ ID NO: 786 through SEQ ID NO: 787, SEQ ID NO: 789 through SEQ ID NO: 792, SEQ ID NO: 794, SEQ ID NO: 796, SEQ ID NO: 798 through SEQ ID NO: 805, SEQ ID NO: 807 through SEQ ID NO: 811, SEQ ID NO: 813 through SEQ ID NO: 819, SEQ ID NO: 821 through SEQ ID NO: 822, SEQ ID NO: 824 through SEQ ID NO: 826, SEQ ID NO: 828 through SEQ ID NO: 832, and SEQ ID NO: 835, such nucleic acids are contained in SEQ ID
  • the invention features a recombinant or substantially pure preparation of an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 836 through SEQ ID NO: 841, SEQ ID NO: 843 through SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855 through SEQ ID NO: 857, SEQ ID NO: 859 through SEQ ID NO: 862, SEQ ID NO: 866, SEQ ID NO: 868 through SEQ ID NO: 871, SEQ ID NO: 873 through SEQ ID NO: 876, and SEQ ID NO: 879.
  • the invention also includes substantially pure nucleic acid encoding an H.
  • pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 836 through SEQ ID NO: 841, SEQ ID NO: 843 through SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855 through SEQ ID NO: 857, SEQ ID NO: 859 through SEQ ID NO: 862, SEQ ID NO: 866, SEQ ID NO: 868 through SEQ ID NO: 871, SEQ ID NO: 873 through SEQ ID NO: 876, and SEQ ID NO: 879, such nucleic acids are contained in SEQ ID NO: 351 through SEQ ID NO: 383.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 385, SEQ ID NO: 390, SEQ ID NO: 401, SEQ ID NO: 407, SEQ ID NO: 409 through SEQ ID NO: 410, SEQ ID NO: 413, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 442.
  • the invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 637, SEQ ID NO: 641, SEQ ID NO: 644 through SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 652, SEQ ID NO: 662, SEQ ID NO: 671 through SEQ ID NO: 672, SEQ ID NO: 675, SEQ ID NO: 677, SEQ ID NO: 684 through SEQ ID NO: 686, SEQ ID NO: 693, SEQ ID NO: 703 through SEQ ID NO: 704, SEQ ID NO: 709 through SEQ ID NO: 711, SEQ ID NO: 715, SEQ ID NO: 723 through SEQ ID NO: 724, SEQ ID NO: 731, SEQ ID NO: 734, SEQ ID NO:
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 637, SEQ ID NO: 641, SEQ ID NO: 644 through SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 652, SEQ ID NO: 662.
  • H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 637, SEQ ID NO: 641, SEQ ID NO: 644 through SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 652, SEQ ID NO: 662.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 833 through SEQ ID NO: 834, SEQ ID NO: 842, SEQ ID NO: 852, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 863, SEQ ID NO: 864 through SEQ ID NO: 865, SEQ ID NO: 867, SEQ ID NO: 872, SEQ ID NO: 877 through SEQ ID NO: 878, and SEQ ID NO: 880.
  • the invention also includes substantially pure nucleic acid encoding an H.
  • pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 833 through SEQ ID NO: 834, SEQ ID NO: 842, SEQ ID NO: 852, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 863, SEQ ID NO: 864 through SEQ ID NO: 865, SEQ ID NO: 867, SEQ ID NO: 872, SEQ ID NO: 877 through SEQ ID NO: 878, and SEQ ID NO: 880, such nucleic acids are contained in SEQ ID NO: 981 through SEQ ID NO: 994.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1446 through SEQ ID NO: 1461, SEQ ID NO: 1463, and SEQ ID NO: 1465 through SEQ ID NO: 1495.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H.
  • pylori polypeptides SEQ ID NO: 1446 through SEQ ID NO: 1461, SEQ ID NO: 1463, and SEQ ID NO: 1465 through SEQ ID NO: 1495, such nucleic acids are contained in SEQ ID NO: 995 through SEQ ID NO: SEQ ID NO: 1010, SEQ ID NO: 1012, and SEQ ID NO: 1014 through SEQ ID NO: 1044.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1497 through SEQ ID NO: 1515, and SEQ ID NO: 1517 through SEQ ID NO: 1545.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H.
  • pylori polypeptides SEQ ID NO: 1497 through SEQ ID NO: 1515, and SEQ ID NO: 1517 through SEQ ID NO: 1545, such nucleic acids are contained in SEQ ID NO: 1046 through SEQ ID NO: 1064, and SEQ ID NO: 1066 through SEQ ID NO: 1094.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1546 through SEQ ID NO: 1595.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1546 through SEQ ID NO: 1595, such nucleic acids are contained in SEQ ID NO: 1095 through SEQ ID NO: 1144.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1596 through SEQ ID NO: 1617, SEQ ID NO: 1620 through SEQ ID NO: 1645.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H.
  • pylori polypeptides SEQ ID NO: 1596 through SEQ ID NO: 1617, SEQ ID NO: 1620 through SEQ ID NO: 1645, such nucleic acids are contained in SEQ ID NO: 1145 through SEQ ID NO: 1166, and SEQ ID NO: 1169 through SEQ ID NO: 1194.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1646 through SEQ ID NO: 1681, and SEQ ID NO: 1683 through SEQ ID NO: 1695.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H.
  • pylori polypeptides SEQ ID NO: 1646 through SEQ ID NO: 1681, and SEQ ID NO: 1683 through SEQ ID NO: 1695, such nucleic acids are contained in SEQ ID NO: 1195 through SEQ ID NO: 1230, and SEQ ID NO: 1232 through SEQ ID NO: 1244.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1696 through SEQ ID NO: 1745.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1696 through SEQ ID NO: 1745, such nucleic acids are contained in SEQ ID NO: 1245 through SEQ ID NO: 1294.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1746 through SEQ ID NO: 1783, and SEQ ID NO: 1786 through SEQ ID NO: 1795.
  • the invention also includes substantially pure nucleic acid encoding an H.
  • pylori polypeptide selected from the group consisting of H pylori polypeptides SEQ ID NO: 1746 through SEQ ID NO: 1783, and SEQ ID NO: 1786 through SEQ ID NO: 1795, such nucleic acids are contained in SEQ ID NO: 1295 through SEQ ID NO: 1332, and SEQ ID NO: 1335 through SEQ ID NO: 1344.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1796 through SEQ ID NO: 1817, SEQ ID NO: 1819, SEQ ID NO: 1821, SEQ ID NO: 1823 through SEQ ID NO: 1836, and SEQ ID NO: 1838 through SEQ ID NO: 1845.
  • the invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H.
  • pylori polypeptides SEQ ID NO: 1796 through SEQ ID NO: 1817, SEQ ID NO: 1819, SEQ ID NO: 1821, SEQ ID NO: 1823 through SEQ ID NO: 1836, and SEQ ID NO: 1838 through SEQ ID NO: 1845, such nucleic acids are contained in SEQ ID NO: 1345 through SEQ ID NO: 1366, SEQ ID NO: 1368, SEQ ID NO: 1370, SEQ ID NO: 1372 through SEQ ID NO: 1385, and SEQ ID NO: 1387 through SEQ ID NO: 1394.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1846 through SEQ ID NO: 1896.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1846 through SEQ ID NO: 1896, such nucleic acids are contained in SEQ ID NO: 1395 through SEQ ID NO: 1445.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of the invention as set forth in the Sequence Listing.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of the invention as set forth in the Sequence Listing. It should be understood that this invention encompasses each of the H. pylori polypeptides and nucleic acids encoding such polypeptides as identified in the Sequence Listing by a given sequence identification number. For example, a representative H.
  • this invention encompasses a recombinant or substantially pure preparation of an H pylori polypeptide of SEQ ID NO: 1450.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 1450.
  • the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such member from the above-identified groups of H.
  • pylori polypeptides e.g., SEQ ID NO: 1546 through SEQ ID NO: 1595
  • nucleic acids e.g., SEQ ID NO: 1095-SEQ ID NO: 1144
  • the subgroups can preferably consists of 1, 3, 5, 10, 15, 20, 30 or 40 members of any of the groups identified above, as well as any
  • H. pylori polypeptides SEQ ID NO: 1846 through SEQ ID NO: 1896 can be divided into one or more subgroups as follows: SEQ ID NO: 1846-SEQ ID NO: 1860; SEQ ID NO: 1861-SEQ ID NO: 1875; SEQ ID NO: 1876-SEQ ID NO: 1885; SEQ ID NO: 1886-SEQ ID NO: 1896; or any combinations thereof.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021, SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 1002, SEQ ID NO: 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1020
  • SEQ ID NO: 1240 SEQ ID NO: 1164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332, SEQ ID NO: 1345, SEQ ID NO: 1358, SEQ ID NO: 1375, SEQ ID NO: 1417, SEQ ID NO: 1283, SEQ ID NO: 1335, SEQ ID NO: 1368, SEQ ID NO: 1179, SEQ ID NO: 1255, SEQ ID NO: 1258, SEQ ID NO: 1044, SEQ ID NO: 1273, SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ
  • SEQ ID NO: 1052 SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069.
  • SEQ ID NO: 1005 SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1 193.
  • SEQ ID NO: 1304 SEQ ID NO: 1305.
  • SEQ ID NO: 943 SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199, SEQ ID NO: 992, SEQ ID NO: 934, SEQ ID NO: 899, SEQ ID NO: 302, SEQ ID NO: 215, SEQ ID NO: 893, SEQ ID NO: 984, SEQ ID NO: 97,SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 309, SEQ ID NO: 150, SEQ ID NO: 240, SEQ ID NO: 957, SEQ ID NO: 57, SEQ ID NO: 2, SEQ ID NO: 92, SEQ ID NO: 255, SEQ ID NO: 164, SEQ ID NO: 201, SEQ ID NO: 278, SEQ ID NO: 245, SEQ ID NO: 921, SEQ ID NO: 896, SEQ ID NO: 248, SEQ ID NO: 159, SEQ ID NO: 979, SEQ ID NO: 19
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021, SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 217, SEQ ID NO: 367, SEQ ID NO: 911,SEQ ID NO:
  • SEQ ID NO: 944 SEQ ID NO: 18, SEQ ID NO: 107, SEQ ID NO: 894, SEQ ID NO: 943, SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO : 1002, SEQ ID NO : 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1251, SEQ ID NO: 1262, SEQ ID NO: 1149, SEQ ID NO: 1220, SEQ ID NO: 1240, SEQ ID NO: 1164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ ID NO: 1331, SEQ ID NO: 1432, SEQ ID NO: 1052, SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069, SEQ ID NO: 1005, SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1193, SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1346, SEQ ID NO: 1348, SEQ ID NO: 1350, SEQ ID NO: 1219,
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1173, SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1410, SEQ ID NO: 1086, SEQ ID NO: 1322.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori cytoplasmic polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO: 1229, SEQ ID NO: 1250, SEQ ID NO:
  • SEQ ID NO: 1268 SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ
  • SEQ ID NO: 1055 SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327.
  • SEQ ID NO: 1412 SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1 1 16, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 882, SEQ ID NO: 382, SEQ ID NO: 130, and SEQ ID NO: 230.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof involved in amino acid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO:
  • SEQ ID NO: 1229 SEQ ID NO: 1250, SEQ ID NO: 1268, SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 269, SEQ ID NO: 312, SEQ ID NO: 211, SEQ ID NO: 959, SEQ ID NO: 938, SEQ ID NO: 110, SEQ ID NO: 244, SEQ ID NO: 328, SEQ ID NO: 235, SEQ ID NO: 315, SEQ ID NO: 296, SEQ ID NO: 976,
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in nucleotide metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 344, SEQ ID NO: 139, SEQ ID NO: 30, SEQ ID NO: 220, SEQ ID NO: 364, SEQ ID NO: 369, SEQ ID NO: 372, SEQ ID NO: 991, SEQ ID NO: 128, SEQ ID NO: 347, and SEQ ID NO: 52.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ ID NO: 996, SEQ ID NO: 12, SEQ ID NO: 247, SEQ ID NO: 64, SEQ ID NO: 101, SEQ ID NO: 338, SEQ ID NO: 83, SEQ ID NO: 46, SEQ ID NO: 348, SEQ ID NO: 223, SEQ ID NO: 39, SEQ ID NO: 232, and SEQ ID NO: 168.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1095, SEQ ID NO: 1156, SEQ ID NO: 1158, SEQ ID NO: 1159, SEQ ID NO: 1277, SEQ ID NO: 1038, SEQ ID NO: 65, SEQ ID NO: 952, SEQ ID NO: 341, SEQ ID NO: 69, SEQ ID NO: 924, and SEQ ID NO: 4.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1257, SEQ ID NO: 1357, SEQ ID NO: 1436, SEQ ID NO: 197, SEQ ID NO: 313, and SEQ ID NO: 119.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327, SEQ ID NO: 1412, SEQ ID NO: 188, SEQ ID NO: 956, SEQ ID NO: 935, SEQ ID NO: 246, SEQ ID NO: 196, and SEQ ID NO: 376.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1116, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188, SEQ ID NO: 1198, SEQ ID NO: 1218, SEQ ID NO: 1244, SEQ ID NO: 1306, SEQ ID NO: 1325, SEQ ID NO: 1397, SEQ ID NO: 1398, SEQ ID NO: 1407, SEQ ID NO: 1433, SEQ ID NO: 172, SEQ ID NO: 25, SEQ ID NO: 126, SEQ ID NO: 951 , SEQ ID NO: 147, SEQ ID NO: 895, SEQ ID NO: 14, SEQ ID NO:
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1216, SEQ ID NO: 1239, SEQ ID NO: 1362, SEQ ID NO: 1017, SEQ ID NO: 1019, SEQ ID NO: 1360, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 349, SEQ ID NO: 209, SEQ ID NO: 990, SEQ ID NO: 185, SEQ ID NO: 883, and SEQ ID NO: 8.
  • the H. pylori cytoplasmic polypeptide is an H. pylori chaperone polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1423, SEQ ID NO: 1425, SEQ ID NO: 1374, SEQ ID NO: 887, SEQ ID NO: 350, and SEQ ID NO: 987.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted or periplasmic polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 1004, SEQ ID NO: 1138, SEQ ID NO: 1067, SEQ ID NO: 1078, SEQ ID NO: 1314, SEQ ID NO: 1319, SEQ ID NO: 1378, SEQ ID NO: 1105, SEQ ID NO: 1114, SEQ ID NO: 1 118, SEQ ID NO: 1120, SEQ ID NO: 1123, SEQ ID NO: 1127, SEQ ID NO: 1212, SEQ ID NO: 1223, SEQ ID NO: 1225, SEQ ID NO: 1246, SEQ ID NO: 1248, SEQ ID NO: 1259, SEQ ID NO: 1264, SEQ ID NO: 1265, SEQ ID NO: 1281, SEQ ID NO: 1285, SEQ ID NO: 1294, SEQ ID NO: 100
  • SEQ ID NO: 360 SEQ ID NO: 271, SEQ ID NO: 970, SEQ ID NO: 288, SEQ ID NO: 282, SEQ ID NO: 98, SEQ ID NO: 29, SEQ ID NO: 317, SEQ ID NO: 343, SEQ ID NO: 291, SEQ ID NO: 108, SEQ ID NO: 377, SEQ ID NO: 305, SEQ ID NO: 305, SEQ ID NO: 100.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori surface or membrane polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026
  • SEQ ID NO: 1340 SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 381, SEQ ID NO: 229.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 381, SEQ ID NO: 229, SEQ ID NO: 323, SEQ ID NO: 371, SEQ ID NO: 284, SEQ ID NO: 116, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 907, SEQ ID NO: 193, SEQ ID NO: 145, SEQ ID NO: 59, SEQ ID NO: 322, SEQ ID NO: 94, SEQ ID NO:
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026, SEQ ID NO: 1048, SEQ ID NO: 1063, SEQ ID NO: 1190, SEQ ID NO: 939, SEQ ID NO: 205, SEQ ID NO: 123, SEQ ID NO: 906, SEQ ID NO: 928, SEQ ID NO: 346, SEQ ID NO: 129, SEQ ID NO
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof having at least three membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1083, SEQ ID NO: 1113, SEQ ID NO: 1222, SEQ ID NO: 1295, SEQ ID NO: 1343, SEQ ID NO: 1392, SEQ ID NO: 1443, SEQ ID NO: 33, SEQ ID NO: 331, SEQ ID NO: 175, SEQ ID NO: 200, SEQ ID NO: 292, SEQ ID NO: 23, and SEQ ID NO: 336.
  • the H pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1085, SEQ ID NO: 1093, SEQ ID NO: 1117, SEQ ID NO: 1121, SEQ ID NO: 1131, SEQ ID NO: 1287, SEQ ID NO: 1440, SEQ ID NO: 1209, SEQ ID NO: 301, SEQ ID NO: 28, SEQ ID NO: 941, SEQ ID NO: 103, SEQ ID NO: 231, SEQ ID NO: 176, SEQ ID NO: 31, SEQ ID NO: 917, SEQ ID NO: 151, and SEQ ID NO: 922.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof having at least five membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1342, SEQ ID NO: 1381, SEQ ID NO: 1390, SEQ ID NO: 1409, SEQ ID NO: 1035, SEQ ID NO: 265, SEQ ID NO: 142, SEQ ID NO: 259, SEQ ID NO: 122, SEQ ID NO: 206, and SEQ ID NO: 885.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least six membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1014, SEQ ID NO: 1088, SEQ ID NO: 1242, SEQ ID NO: 1178, SEQ ID NO: 96, SEQ ID NO: 353, SEQ ID NO: 38, SEQ ID NO: 89, SEQ ID NO: 77, SEQ ID NO: 954, SEQ ID NO: 264.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1089, SEQ ID NO: 1340, SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 937, SEQ ID NO: 226, SEQ ID NO: 283, SEQ ID NO: 88, SEQ ID NO: 125, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 81, SEQ ID NO: 901, SEQ ID NO: 82, and SEQ ID NO: 42.
  • H. pylori cell envelope polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812, SEQ ID NO: 1830, SEQ ID NO:
  • SEQ ID NO: 404 SEQ ID NO: 518, SEQ ID NO: 464, SEQ ID NO: 672, SEQ ID NO: 640, SEQ ID NO: 490, SEQ ID NO: 755, SEQ ID NO: 389, SEQ ID NO: 635, SEQ ID NO: 877, SEQ ID NO: 637, SEQ ID NO: 477, SEQ ID NO: 772, SEQ ID NO: 658, SEQ ID NO: 463, SEQ ID NO: 852, SEQ ID NO: 503, SEQ ID NO: 411, SEQ ID NO: 441, SEQ ID NO: 782, SEQ ID NO: 575, SEQ ID NO: 691, SEQ ID NO: 724, SEQ ID NO: 452, SEQ ID NO: 386, SEQ ID NO: 497, SEQ ID NO: 712, SEQ ID NO: 591, SEQ ID NO: 638, SEQ ID NO: 740, SEQ ID NO: 697, SEQ ID NO: 569, SEQ ID NO
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1453, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1685, SEQ ID NO: 1687, SEQ ID NO: 1688, SEQ ID NO: 1675, SEQ ID NO: 1702, SEQ ID NO: 1713, SEQ ID NO: 1600, SEQ ID NO: 1671, SEQ ID NO: 1691, SEQ ID NO: 1615, SEQ ID NO: 1616, SEQ ID NO: 1855, SEQ ID NO: 1595, SEQ ID NO: 1633, SEQ ID NO: 1608, SEQ ID NO: 1611, SEQ ID NO: 1751, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1780, SEQ ID NO: 1783, SEQ ID NO: 1796, SEQ ID NO
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1670, SEQ ID NO: 1725, SEQ ID NO: 1661, SEQ ID NO: 1873, SEQ ID NO: 1753, SEQ ID NO: 1759, SEQ ID NO: 1761, SEQ ID NO: 1782, SEQ ID NO: 1883, SEQ ID NO: 1503, SEQ ID NO: 1542, SEQ ID NO: 1872, SEQ ID NO: 1520, SEQ ID NO: 1456, SEQ ID NO: 1458, SEQ ID NO: 1617, SEQ ID NO: 1628, SEQ ID NO: 1644, SEQ ID NO: 1657, SEQ ID NO: 1658, SEQ ID NO: 1755, SEQ ID NO: 1756, SEQ ID NO: 1797, SEQ ID NO: 1799, SEQ ID NO: 1801, SEQ ID NO: 1483,
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1624, SEQ ID NO: 1856, SEQ ID NO: 1857, SEQ ID NO: 1861, SEQ ID NO: 1537, SEQ ID NO: 1773, SEQ ID NO: 1717, SEQ ID NO: 1733, SEQ ID NO: 1722, SEQ ID NO: 1659, SEQ ID NO: 1577, SEQ ID NO: 1721, SEQ ID NO: 1729, SEQ ID NO: 1870, SEQ ID NO: 1576, SEQ ID NO: 1632, SEQ ID NO: 1867, SEQ ID NO: 1547, SEQ ID NO: 1533, SEQ ID NO: 1597, SEQ ID NO: 1596, SEQ ID NO: 1559, SEQ ID NO: 1599, SEQ ID NO: 1788, SEQ ID NO: 1789, SEQ ID NO: 1875
  • H. pylori cytoplasmic polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485.
  • SEQ ID NO: 1459 SEQ ID NO: 1512, SEQ ID NO: 1515.
  • SEQ ID NO: 1642 SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866.
  • SEQ ID NO: 1886 SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703. SEQ ID NO: 1704.
  • SEQ ID NO: 1737 SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609.
  • SEQ ID NO: 1678 SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 390, SEQ ID NO: 876, SEQ ID NO: 547, and SEQ ID NO: 678.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in amino acid metabolism selected from the group consisting of SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485, SEQ ID NO: 1459, SEQ ID NO: 729, SEQ ID NO: 786, SEQ ID NO: 654, SEQ ID NO: 734, SEQ ID NO: 646, SEQ ID NO: 522, SEQ ID NO: 696, SEQ ID NO: 807, SEQ ID NO: 1448
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in nucleotide metabolism selected from the group consisting of SEQ ID NO: 1512, SEQ ID NO: 1515, SEQ ID NO: 1642, SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866, SEQ ID NO: 1886, SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 826, SEQ ID NO: 562, SEQ ID NO: 420, SEQ ID NO: 664, SEQ ID NO: 850, SEQ ID NO: 857, SEQ ID NO: 861, SEQ ID NO: 872, SEQ ID NO: 544, SEQ ID NO: 830, and SEQ ID NO: 446.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism selected from the group consisting of SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1737, SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 397, SEQ ID NO: 699, SEQ ID NO: 459, SEQ ID NO: 509, SEQ ID NO: 818, SEQ ID NO: 488, SEQ ID NO: 438, SEQ ID NO: 831, SEQ ID NO: 667, SEQ ID NO: 429, SEQ ID NO: 680, and SEQ ID NO: 597.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism selected from the group consisting of SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1728, SEQ ID NO: 1489, SEQ ID NO: 460, SEQ ID NO: 709, SEQ ID NO: 822, SEQ ID NO: 466, SEQ ID NO: 584, and SEQ ID NO: 388.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism selected from the group consisting of SEQ ID NO: 1708, SEQ ID NO: 1808, SEQ ID NO: 1887, SEQ ID NO: 631 , SEQ ID NO: 787, and SEQ ID NO: 532.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis selected from the group consisting of SEQ ID NO: 1498, SEQ ID NO: 1506, SEQ ID NO: 1592, SEQ ID NO: 1678, SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 619, SEQ ID NO: 723, SEQ ID NO: 641, SEQ ID NO: 698, SEQ ID NO: 630, and SEQ ID NO: 869.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair selected from the group consisting of SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636, SEQ ID NO: 1639, SEQ ID NO: 1649, SEQ ID NO: 1669, SEQ ID NO: 1695, SEQ ID NO: 1757, SEQ ID NO: 1776, SEQ ID NO: 1848, SEQ ID NO: 1849, SEQ ID NO: 1858, SEQ ID NO: 1884, SEQ ID NO: 601, SEQ ID NO: 415, SEQ ID NO: 542, SEQ ID NO: 704, SEQ ID NO: 572, SEQ ID NO: 467, SEQ ID NO: 399, SEQ ID NO: 579, SEQ ID NO: 1454
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis selected from the group consisting of SEQ ID NO: 1667, SEQ ID NO: 1690, SEQ ID NO: 1813, SEQ ID NO: 1468, SEQ ID NO: 1470, SEQ ID NO: 1811, SEQ ID NO: 574, SEQ ID NO: 596, SEQ ID NO: 832, SEQ ID NO: 651, SEQ ID NO: 867, SEQ ID NO: 614, SEQ ID NO: 401, and SEQ ID NO: 393.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori chaperone polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1874, SEQ ID NO: 1876, SEQ ID NO: 1825, SEQ ID NO: 413, SEQ ID NO: 835, and SEQ ID NO: 863.
  • polypeptide is selected from the group consisting of SEQ ID NO: 1455, SEQ ID NO: 1589, SEQ ID NO: 1518, SEQ ID NO: 1529, SEQ ID NO: 1765, SEQ ID NO: 1770, SEQ ID NO: 1829, SEQ ID NO: 1556, SEQ ID NO: 1565, SEQ ID NO: 1569, SEQ ID NO: 1571, SEQ ID NO: 1574, SEQ ID NO: 1578, SEQ ID NO: 1663, SEQ ID NO: 1674, SEQ ID NO: 1676, SEQ ID NO: 1697, SEQ ID NO: 1699, SEQ ID NO: 1710, SEQ ID NO: 1715, SEQ ID NO: 1716, SEQ ID NO: 1732, SEQ ID NO: 1736, SEQ ID NO: 1745, SEQ ID NO: 1749, SEQ ID NO: 1750
  • H. pylori surface or membrane polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731, SEQ ID NO: 1743, SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO: 1499, SEQ ID NO:
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731.
  • SEQ ID NO: 1743 SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 875, SEQ ID NO: 676, SEQ ID NO: 801, SEQ ID NO: 860, SEQ ID NO: 747, SEQ ID NO: 529, SEQ ID NO: 387, SEQ ID NO: 391, SEQ ID NO: 515, SEQ ID NO: 625, SEQ ID NO: 568, SEQ ID NO: 454, SEQ ID NO: 800, SEQ ID NO: 499, SEQ ID NO: 779, and SEQ ID NO: 385.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions selected from the group consisting of SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO:
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least three membrane spanning regions selected from the group consisting of SEQ ID NO: 1534, SEQ ID NO: 1564, SEQ ID NO: 1673, SEQ ID NO: 1746, SEQ ID NO: 1794, SEQ ID NO: 1843, SEQ ID NO: 1894, SEQ ID NO: 423, SEQ ID NO: 810, SEQ ID NO: 604, SEQ ID NO: 636, SEQ ID NO: 757, SEQ ID NO: 412, and SEQ ID NO: 816.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions selected from the group consisting of SEQ ID NO: 1536, SEQ ID NO: 1544, SEQ ID NO: 1568, SEQ ID NO: 1572, SEQ ID NO: 1582, SEQ ID NO:
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least five membrane spanning regions selected from the group consisting of SEQ ID NO: 1793, SEQ ID NO: 1832, SEQ ID NO: 1841, SEQ ID NO: 1860, SEQ ID NO: 1486, SEQ ID NO: 725, SEQ ID NO: 565, SEQ ID NO: 717, SEQ ID NO: 536, SEQ ID NO: 647, and SEQ ID NO: 409.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H.
  • the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions selected from the group consisting of SEQ ID NO: 1540, SEQ ID NO: 1791, SEQ ID NO: 1525, SEQ ID NO: 1558, SEQ ID NO: 1655, SEQ ID NO: 1517, SEQ ID NO: 645, SEQ ID NO: 670, SEQ ID NO: 746, SEQ ID NO: 493, SEQ ID NO: 540, SEQ ID NO: 612, SEQ ID NO: 629, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, and SEQ ID NO: 433.
  • the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such a member from the above-identified groups of H. pylori polypeptides.
  • the invention features nucleic acids capable of binding mRNA of H. pylori.
  • nucleic acid is capable of acting as antisense nucleic acid to control the translation of mRNA of H. pylori.
  • a further aspect features a nucleic acid which is capable of binding specifically to an H. pylori nucleic acid. These nucleic acids are also referred to herein as complements and have utility as probes and as capture reagents.
  • the invention features an expression system comprising an open reading frame corresponding to H. pylori nucleic acid.
  • the nucleic acid further comprises a control sequence compatible with an intended host.
  • the expression system is useful for making polypeptides corresponding to H. pylori nucleic acid.
  • the invention features a cell transformed with the expression system to produce H. pylori polypeptides.
  • the invention features a method of generating antibodies against H. pylori polypeptides which are capable of binding specifically to H. pylori polypeptides.
  • Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori -specific antigens.
  • the invention features a method of generating vaccines for immunizing an individual against H. pylori.
  • the method includes: immunizing a subject with an H. pylori polypeptide, e.g., a surface or secreted polypeptide. or active portion thereof, and a pharmaceutically acceptable carrier.
  • Such vaccines have therapeutic and prophylactic utilities.
  • the invention provides a method for generating a vaccine comprising a modified immunogenic H. pylori polypeptide, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmacologically acceptable carrier.
  • the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori polypeptide. The method includes: contacting the candidate compound with an H. pylori polypeptide and determining if the compound binds or otherwise interacts with an H. pylori polypeptide.
  • Compounds which bind H. pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
  • the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori nucleic acid, e.g., DNA or RNA.
  • the method includes: contacting the candidate compound with an H. pylori nucleic acid and determining if the compound binds or otherwise interacts with an H. pylori polypeptide.
  • Compounds which bind H. pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
  • the invention features H. pylori polypeptides, preferably a substantially pure preparation of an H. pylori polypeptide, or a recombinant H. pylori polypeptide.
  • the polypeptide has biological activity; the polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acid residues of
  • the H. pylori polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.
  • the subject H. pylori polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the H. pylori polypeptide exhibits an H. pylori biological activity, e.g., the H. pylori polypeptide retains a biological activity of a naturally occurring H. pylori enzyme.
  • the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
  • the H. pylori polypeptide is a recombinant fusion protein having a first H. pylori polypeptide portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to H. pylori.
  • the second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain.
  • the fusion protein can be used in a two-hybrid assay.
  • Polypeptides of the invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
  • the invention also encompasses an immunogenic component which includes an H. pylori polypeptide in an immunogenic preparation; the immunogenic component being capable of eliciting an immune response specific for the H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
  • the immunogenic component comprises at least one antigenic determinant from a polypeptide of the invention contained in the Sequence Listing.
  • the invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an H. pylori polypeptide.
  • the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.
  • the nucleic acid of the invention is that contained in the Sequence Listing; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence of the invention contained in the Sequence Listing.
  • the encoded H. pylori polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing.
  • the differences are such that: the H. pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.
  • the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
  • the subject H. pylori nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the H. pylori gene sequence, e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
  • operably linked to the H. pylori gene sequence e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
  • the nucleic acid which encodes an H. pylori polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of the invention contained in the Sequence Listing.
  • the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences of the invention contained in the Sequence Listing.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence of the invention contained in the Sequence Listing which encodes amino acids of the invention contained in the Sequence Listing.
  • the invention encompasses: a vector including a nucleic acid which encodes an H. pylori polypeptide or an H. pylori polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant H. pylori polypeptide or H. pylori polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori or H. pylori polypeptide variant, e.g., from the cell or from the cell culture medium.
  • the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence of the invention contained in the Sequence Listing.
  • the invention also provides a probe or primer which includes a substantially purified oligonucleotide.
  • the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of the invention contained in the Sequence Listing, or naturally occurring mutants thereof.
  • the probe or primer further includes a label group attached thereto.
  • the label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
  • the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
  • the invention further provides nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention.
  • nucleic acids e.g., RNA or DNA
  • the H. pylori strain from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection(ATCC # 55679) as strain ⁇ P-J99.
  • allelic variations include allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference); and, polypeptides specifically bound by antisera to H. pylori polypeptides, especially by antisera to an active site or binding domain of H. pylori polypeptide.
  • the invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as H. pylori polypeptide analogs or variants.
  • H. pylori polypeptides characterized as shown in Table 1 below, including: H pylori cell envelope proteins, H. pylori periplasmic/secreted proteins, H. pylori cytoplasmic proteins, and other H. pylori surface and membrane proteins.
  • H pylori cell envelope proteins H. pylori periplasmic/secreted proteins
  • H. pylori cytoplasmic proteins H. pylori surface and membrane proteins.
  • Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs.
  • nt represents nucleotide Seq. ID number and “aa” represents amino
  • a purified or isolated polypeptide or a substantially pure preparation of a polypeptide are used interchangeably herein and, as used herein, mean a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
  • the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it.
  • the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation.
  • the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 ⁇ g of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
  • a purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
  • a purified or isolated or a substantially pure nucleic acid is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally- occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived.
  • the term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
  • Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional H. pylori DNA sequence.
  • a "contig” as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.
  • ORF an "open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.
  • a "coding sequence” is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus.
  • a coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.
  • a "complement" of a nucleic acid as used herein referes to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
  • a “gene product” is a protein or structural RNA which is specifically encoded by a gene.
  • probe refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label.
  • a label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like.
  • Capture ligand a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand".
  • Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
  • Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions.
  • Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions.
  • Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated.
  • hybridization of two sequences at high stingency such as, for example, in a solution of 0.5X SSC, at 65° C) requires that the sequences be essentially completely homologous.
  • Conditions of intermediate stringency such as, for example, 2X SSC at 65 ° C
  • low stringency such as, for example 2X SSC at 55° C
  • (1X SSC is 0.15 M NaCl, 0.015 M Na citrate).
  • peptides, proteins, and polypeptides are used interchangeably herein.
  • surface protein refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
  • a polypeptide has H. pylori biological activity if it has one, two and preferably more of the following properties: (1) if when expressed in the course of an H. pylori infection, it can promote, or mediate the attachment of H. pylori to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an H pylori protein; (3) or the gene which encodes it can rescue a lethal mutation in an H. pylori gene.
  • a polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.
  • a biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the H. pylori polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring H. pylori polypeptides, e.g., one or more of the biological activities described herein.
  • fragments which exist in vivo e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in C ⁇ O cells. Because peptides such as H.
  • H. pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H. pylori fragment or H. pylori analog is one which exhibits a biological activity in any biological assay for H. pylori activity. Most preferably the fragment or analog possesses 10%, preferably 40%, more preferably 60%, 70%, 80% or 90% or greater of the activity of H. pylori, in any in vivo or in vitro assay. Analogs can differ from naturally occurring H. pylori polypeptides in amino acid sequence or in ways that do not involve sequence, or both.
  • Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.
  • Preferred analogs include H. pylori polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the H. pylori polypeptide.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be made in view of the table below.
  • analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs.
  • fragment as applied to an H. pylori analog, will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of H. pylori polypeptides can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of H. pylori polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are H. pylori polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
  • an "immunogenic component” as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal.
  • an "antigenic component” as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
  • transgene means a nucleic acid (encoding, e.g., one or more polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the cell's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
  • the term "transgenic cell” refers to a cell containing a transgene.
  • a "transgenic animal” is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by a process of transformation of competent cells or by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • antibody as used herein is intended to include fragments thereof which are specifically reactive with H. pylori polypeptides.
  • cell-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • Misexpression refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
  • host cells and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
  • control sequence refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers.
  • the term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.
  • operably linked refers to sequences joined or ligated to function in their intended manner.
  • a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.
  • the metabolism of a substance means any aspect of the, expression, function, action, or regulation of the substance.
  • the metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance.
  • the metabolism of a substance includes modifications, e.g., covalent or non-covalent modification, the substance induces in other substances.
  • the metabolism of a substance also includes changes in the distribution of the substance.
  • the metabolism of a substance includes changes the substance induces in the distribution of other substances.
  • sample refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • This invention provides nucleotide sequences of the genome of H. pylori which thus comprises a DNA sequence library of H. pylori genomic DNA.
  • the detailed description that follows provides nucleotide sequences of H. pylori, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are methods of using the disclosed H. pylori sequences in methods including diagnostic and therapeutic applications.
  • the library can be used as a database for identification and comparison of medically important sequences in this and other strains of H. pylori.
  • DNA sequencing was achieved using multiplex sequencing procedures essentially as disclosed in Church et al., 1988, Science 240: 185; U.S. Patents No. 4,942,124 and 5,149,625).
  • DNA was extracted from pooled cultures and subjected to chemical or enzymatic sequencing. Sequencing reactions were resolved by electrophoresis, and the products were transferred and covalently bound to nylon membranes. Finally, the membranes were sequentially hybridized with a series of labelled oligonucleotides complimentary to "tag" sequences present in the different shotgun cloning vectors. In this manner, a large number of sequences could be obtained from a single set of sequencing reactions. The cloning and sequencing procedures are described in more detail in the Exemplification.
  • Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of H. pylori genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined.
  • PCR polymerase chain reaction
  • ORFs open reading frames
  • H. pylori sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides.
  • ORFs open reading frames
  • these ORFs may not correspond to the ORF of a naturally-occurring H. pylori polypeptide.
  • These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally-occurring H.
  • Such start codons within the ORFs provided herein can be identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded H. pylori polypeptide is within the scope of this invention.
  • a codon such as AUG or GUG encoding methionine or valine
  • the predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARKTM (Borodovsky and Mclninch, 1993, Comp. Chem. 17:123).
  • the nucleic acids of this invention may be obtained directly from the DNA of the above referenced H. pylori strain by using the polymerase chain reaction (PCR). See “PCR, A Practical Approach” (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products can be checked by conventional sequencing methods.
  • PCR polymerase chain reaction
  • Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
  • nucleic acids encoding H. pylori polypeptides from a cDNA library in accordance with protocols herein described.
  • a cDNA encoding an H. pylori polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques.
  • nucleic acids of the invention can be DNA or RNA.
  • Preferred nucleic acids of the invention are contained in the Sequence Listing.
  • the nucleic acids of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4.373,071, incorporated by reference herein).
  • nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands. antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences.
  • the nucleic acid normally consists of all or part (approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below.
  • a nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect H. pylori.
  • sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to H. pylori, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules.
  • nucleic acids for use as probes, can be provided with a label to facilitate detection of a hybridization product.
  • Nucleic acid isolated and synthesized in accordance with the sequence of the invention contained in the Sequence Listing can also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using appropriate stringency hybridization conditions as described herein.
  • nucleic acid selected in the manner described above with respect to probes can be readily associated with a support.
  • the manner in which nucleic acid is associated with supports is well known.
  • Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate H. pylori nucleic acid from the nucleic acid of each other and other organisms.
  • Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Helicobacter species from each other and from other organisms.
  • the sequence will comprise at least twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques.
  • Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of H. pylori nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Helicobacter species.
  • PCR polymerase chain reaction
  • nucleic acid sequences of ⁇ 10-15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of H. pylori nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods.
  • the copies can be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be used in diagnostic assays to detect specific sequences, including genes from H. pylori
  • Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of H. pylori genes. These sequences also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
  • nucleic acid or derivatives corresponding to H. pylori nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells.
  • a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA.
  • the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA.
  • Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques.
  • nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides.
  • the nucleic acid of the invention exemplified in the Sequence Listing or fragments of said nucleic acid encoding active portions of H. pylori polypeptides can be cloned into suitable vectors or used to isolate nucleic acid.
  • the isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.
  • a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to other Helicobacter strains, or other bacterial strains such as E. coli, Norcardia, Corynebacterium, Campylobacter, and Streptomyces species. In some cases the expression host will utilize the natural
  • Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta- galactosidase promoter for expression in E. coli).
  • a promoter sequence derived from the expressing organism e.g., an E. coli beta- galactosidase promoter for expression in E. coli.
  • a procedure such as the following can be used to express a gene product using the natural H. pylori promoter.
  • a restriction fragment containing the gene of interest, together with its associated natural promoter element and regulatory sequences is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together.
  • the recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
  • the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid.
  • This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning.
  • a suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell.
  • an H. pylori polypeptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (C ⁇ O).
  • Other suitable host cells are known to those skilled in the art.
  • yeast S. cerivisae examples include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and ⁇ erskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113- 123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • COS cells Gluzman, Y., (1981) Cell 23:175-182
  • pCDM 8 are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection. or electroporation.
  • Fusion vectors usually add a number of NH 2 terminal amino acids to the expressed target gene. These NH 2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein.
  • Such enzymes include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia,
  • Piscataway, N J which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • a preferred reporter group is poly(His), which may be fused to the amino or carboxy terminus of the protein and which renders the recombinant fusion protein easily purifiable by metal chelate chromatography.
  • Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene
  • target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc
  • expression of target genes inserted into pET11d relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gn1).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur.
  • the polypeptide may be secreted and isolated from a mixture of cells and medium containing the peptide.
  • the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides. Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
  • membrane bound proteins these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be
  • a detergent suitable for solubilizing these complexes Several different criteria are used for choosing a detergent suitable for solubilizing these complexes. For example, one property considered is the ability of the detergent to solubilize the H. pylori protein within the membrane fraction at minimal denaturation of the membrane-associated protein allowing for the activity or functionality of the membrane-associated protein to return upon reconstitution of the protein. Another property considered when selecting the detergent is the critical micelle concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution. A third property considered when selecting a detergent is the hydrophobicity of the detergent.
  • CMC critical micelle concentration
  • membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g., the triton series, would be useful for solubilizing the hydrophobic proteins.
  • Another property important to a detergent can be the capability of the detergent to remove the H. pylori protein with minimal protein-protein interaction facilitating further purification.
  • a fifth property of the detergent which should be considered is the charge of the detergent. For example, if it is desired to use ion exchange resins in the purification process then preferably detergent should be an uncharged detergent. Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
  • nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
  • the nucleic acids of the invention can also be chemically synthesized using standard techniques.
  • This invention encompasses isolated H. pylori polypeptides encoded by the disclosed H. pylori genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an H. pylori polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region.
  • polypeptides of the invention can be isolated from wild-type or mutant H. pylori cells or from heterologous organisms or cells (including, but not limited to, bacteria, yeast, insect, plant and mammalian cells) into which an H. pylori nucleic acid has been introduced and expressed.
  • the polypeptides can be part of recombinant fusion proteins.
  • H. pylori polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein.
  • polypeptides of the invention are related to one another. Some of these relationships are described in Table 3 below. Most polypeptides described in Table 3 are over 90% identical to one another as noted in the last two columns; some are between 70% and 90%) identical to one another; and very few share between 60% and 10% identity with each other.
  • the polypeptides represented by the sequence identification numbers in the third column of Table 3 result from translations carried out from stop codon to stop codon in the genomic nucleotide sequence of the invention, while those in the first column result from translations carried out from the first methionine or valine codon following the prior stop codon and proceeding to the final stop codon in the nucleotide sequence.
  • the nucleotide sequence encoding the related polypeptides is slightly different, resulting in some differences in amino acid residues of the related polypeptides.
  • the related polypeptides differ significantly in length, with one polypeptide containing amino acid residues in addition to those in common between the two polypeptides.
  • the relationships described in Table 3 are highly significant, and the nucleotide sequences encoding these related polypeptides are also very similar to one another.
  • the nucleotide probes derived from the coding sequence of the polypeptides in column one can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the polypeptides of column three.
  • polypeptides shown in Table 3 can be classified in five broad categories as follows. First, for many polypeptides (designated "A" in the last column of the Table 3), the polypeptide denoted in column one is identical to the polypeptide denoted in column three except for an occasional addition of a few putative amino acid residues at the N-terminus which result from the fact that the polypeptides of column three were derived by translating from stop codon to stop codon instead of from a predicted start codon (i.e., Met or Val) to a stop codon as was done for the polypeptides in column one.
  • a predicted start codon i.e., Met or Val
  • the polypeptide of column one is at least 95% identical to the polypeptide of column three except that the polypeptide in column three is longer (at either or both ends) by one or more amino acid residues which do not result from the difference between reading from stop to stop instead of from start to stop.
  • polypeptide of column one is at least 95% identical to the polypeptide of column three except that the polypeptide in column three is shorter (at either or both ends).
  • polypeptide of column shares a high level of amino acid identity (i.e., at least 95%) with the polypeptide of column three in the region in which they overlap, but shares little or no identity (i.e., less than 95%) at one or both ends.
  • polypeptides in columns one and three in categories "B", "C” and “D” is highly significant.
  • a typical H. pylori gene product will exhibit amino acid sequence identities of between 92%) to 100% among strains of H. pylori isolated from human patients (see Table 10 below).
  • polypeptides in column one are closely related but differ significantly (i.e., less than 95% identical) from the polypeptide of column three. These polypeptides are likely "paralogs,” members of related gene families in H. pylori.
  • the disclosed H. pylori genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids.
  • H. pylori nucleic acid and polypeptide sequences Computer-assisted comparison of the disclosed H. pylori sequences with previously reported sequences present in publicly available databases is useful for identifying functional H. pylori nucleic acid and polypeptide sequences.
  • protein-coding sequences may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80-90%) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc.
  • H. pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
  • H. pylori Nucleic acids that encode proteins essential for growth or viability of H. pylori are preferred drug targets.
  • H. pylori genes can be tested for their biological relevance to the organism by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene "knockout", using techniques known to those skilled in the relevant art. In this manner, essential genes may be identified.
  • H. pylori sequences are useful for identifying, and/or discriminating between, previously known and new H. pylori strains. It is believed that other H. pylori strains will exhibit at least 70% sequence homology with the presently disclosed sequence. Systematic and routine analyses of DNA sequences derived from samples containing H. pylori strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all H. pylori strains.
  • the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of H. pylori.
  • Strain- specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more H. pylori strains.
  • the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all H. pylori strains but are not found in other bacterial species. Specific Example: Determination Of Candidate Protein Antigens For Antibody And Vaccine Development
  • the selection of candidate protein antigens for vaccine development can be derived from the nucleic acids encoding H. pylori polypeptides.
  • the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C.
  • Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases.
  • BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database.
  • ORF's with significant homology e.g. probabilities lower than 1x10 -6 that the homology is only due to random chance
  • membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to H. pylori genes based on sequence homology to genes cloned in other organisms.
  • ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
  • amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code.
  • H. pylori gene products of the invention provided in the Sequence Lsiting, one skilled in the art can alter the disclosed structure (of H. pylori genes), e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind H. pylori polypeptides. Such screens are useful for the identification of inhibitors of H. pylori.
  • Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis.
  • Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide.
  • Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments.
  • DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
  • Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length. Alteration of Nucleic Acids and Polypeptides: Random Methods
  • Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein).
  • PCR mutagenesis reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15).
  • the DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn 2+ to the PCR reaction.
  • the pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
  • Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242).
  • This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand.
  • the mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
  • a library of homologs can also be generated from a set of degenerate
  • oligonucleotide sequences Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector.
  • the synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al.
  • Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
  • Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
  • Oligonucleotide-mediated mutagenesis is a useful method for preparing
  • the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
  • the starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA to be mutated are identified.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated desired protein subunit DNA sequence.
  • Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630).
  • the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences.
  • the variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • Other Modifications of H. pylori Nucleic Acids and Polypeptides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • H. pylori polypeptide it is possible to modify the structure of an H. pylori polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo).
  • a modified H. pylori protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.
  • H. pylori peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages.
  • amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.
  • an H. pylori polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an H. pylori polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of H.
  • PEG polyethylene glycol
  • pylori proteins include reduction/alkylation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton N J 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch, of Allergy and Appl. Immunol., 41 : 199 - 215). To facilitate purification and potentially increase solubility of an H.
  • hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography ( ⁇ ochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325).
  • immobilized metal ion affinity chromatography ⁇ ochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325.
  • endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
  • canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods.
  • charged amino acid pairs such as KK or RR
  • KK or RR can be introduced between regions withpm a protein or fragment during recombinant construction thereof.
  • the resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes.
  • such charged amino acid residues can result in an increase in the solubility of the peptide.
  • Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to H. pylori polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • detection of a desired activity e.g., in this case, binding to H. pylori polypeptide or an interacting protein.
  • Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
  • Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring H. pylori polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an H. pylori protein.
  • the H. pylori domain is used as the bait protein and the library of variants are expressed as fish fusion proteins.
  • a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind a H. pylori polypeptide.
  • the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
  • the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).
  • a detectably labeled ligand can be used to score for potentially functional peptide homologs.
  • Fluorescently labeled ligands e.g., receptors
  • fluorescently labeled ligands allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a
  • a gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages M13, fd., and f1 are most often used in phage display libraries. Either of the phage gIll or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle.
  • Foreign epitopes can be expressed at the NH 2 -terminal end of pIll and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
  • E. coli the outer membrane protein, LamB
  • LamB the outer membrane protein
  • Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands. e.g., to antibodies, and can elicit an immune response when the cells are administered to animals.
  • Other cell surface proteins e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al.
  • Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment.
  • Another large surface structure used for peptide display is the bacterial motive organ, the flagellum.
  • Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083).
  • Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J.
  • the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within.
  • An alternative scheme uses the DNA- binding protein LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the LacI gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced.
  • This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO).
  • LacO operator By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
  • the associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands.
  • a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B.
  • a cohort of peptides was recovered, all related by a consensus sequence corresponding to a six- residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869)
  • This scheme sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods.
  • the peptides are attached to the C- terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini.
  • Both of the filamentous phage coat proteins, pIll and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains.
  • the phage-displayed peptides are presented right at the amino terminus of the fusion protein.
  • a second difference is the set of biological biases affecting the population of peptides actually present in the libraries.
  • the LacI fusion molecules are confined to the cytoplasm of the host cells.
  • the phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles.
  • the peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.
  • the phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage.
  • phage/phagemid or plasmid libraries Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.
  • RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening.
  • the polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector.
  • This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification.
  • a DNA sequencing vector for peptide identification.
  • the high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists.
  • the type of a secondary screen used will depend on the desired activity that needs to be tested.
  • an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
  • the invention also provides for reduction of the protein binding domains of the subject H. pylori polypeptides to generate mimetics, e.g. peptide or non-peptide agents.
  • the peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an H. pylori polypeptide binding to a naturally occurring ligand.
  • the critical residues of a subject H. pylori polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate H. pylori -derived peptidomimetics which competitively or noncompetitively inhibit binding of the H. pylori polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31,080A).
  • scanning mutagenesis can be used to map the amino acid residues of a particular H. pylori polypeptide involved in binding an interacting polypeptide
  • peptidomimetic compounds e.g. diazepine or isoquinoline derivatives
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
  • This invention also features vaccine compositions for protection against infection by H. pylori or for treatment of H. pylori infection, a gram-negative spiral microaerophilic bacterium.
  • the vaccine compositions contain one or more
  • nucleic acids within the scope of the invention are exemplified by the nucleic acids of the invention contained in the Sequence Listing which encode H. pylori surface proteins.
  • the preferred nucleic acid for a vaccine composition of the invention is isolated from the group of nucleic acids which encode cell envelope proteins as outlined in Table 1.
  • amino acids of SEQ ID NO:812, SEQ ID NO:820, SEQ ID NO:880, SEQ ID NO:658, SEQ ID NO:865, SEQ ID NO: 1729, SEQ ID NO: 1861, or fragments thereof can be used alone or in combination for the formulation of vaccine compositions of the invention, as well as, their corresponding nucleic acids of SEQ ID NO:977, SEQ ID NO:978, SEQ ID NO:994, SEQ ID NO:215, SEQ ID NO:989, SEQ ID NO:1278, and SEQ ID NO:1410.
  • any nucleic acid encoding an immunogenic H. pylori protein, or portion thereof, which is capable of expression in a cell can be used in the present invention. These vaccines have therapeutic and prophylactic utilities.
  • One aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains at least one immunogenic fragment of an H. pylori protein and a pharmaceutically acceptable carrier.
  • Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10-20 amino acid residues in length, and more preferably about 12-16 amino acid residues in length.
  • Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H. pylori protein.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • immunogenic components are identified by the ability of the peptide to stimulate T cells.
  • Peptides which stimulate T cells as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope.
  • T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate ⁇ LA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope.
  • a T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.
  • Screening immunogenic components can be accomplished using one or more of several different assays.
  • peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture.
  • Presentation of an immunogenic H. pylori peptide in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4.
  • the culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines.
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incorporated herein by reference.
  • a kit for an assay for the production of interferon is also available from
  • a common assay for T cell proliferation entails measuring tritiated thymidine incorporation.
  • the proliferation of T cells can be measured in vitro by determining the amount of 3 H-labeled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
  • Vaccine compositions of the invention containing immunogenic components preferably include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered.
  • Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of antibody administered, whether the protein or DNA is administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or DNA.
  • Vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular
  • Oral immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 91: 9866-9870.
  • Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
  • Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 11 : 637-642.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl--L-threonyl-D-isoglutamine (thr- MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn- glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835 A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion;
  • Non-toxic derivatives of cholera toxin including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial lysates, block polymers or saponins.
  • Suitable delivery methods include biodegradable microcapsules or immuno- stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue.
  • the amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 ⁇ g to 50 ⁇ g, for example 10 ⁇ g to 35 ⁇ g. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.
  • Carrier systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H. pylori polypeptide in an insoluble form as fusion proteins.
  • Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres.
  • Suitable diluents are 0.2 N Na ⁇ CO3 and/or saline.
  • Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of H. pylori in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by H. pylori.
  • the vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art.
  • a suitable dosage will be in the range of 10 ⁇ g to 10 g, preferably 10 ⁇ g to 100 mg, for example 50 ⁇ g to 50 mg.
  • a suitable dosage for adults will also be in the range of 5 ⁇ g to 500 mg. Similar dosage ranges will be applicable for children.
  • the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors.
  • appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol. 150:917-921 (1993); Boedecker et al., American
  • the number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American
  • a vaccine composition of the invention can be based on a killed whole E coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.
  • the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H. pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. pylori -caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
  • the invention also includes antibodies specifically reactive with the subject H. pylori polypeptide.
  • Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by ⁇ arlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of the subject H. pylori polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • the subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least 95% homologous).
  • the anti-H. pylori antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80% percent homologous to a sequence of the invention contained in the Sequence Listing.
  • the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no crossreactivity between bacterial and mammalian antigens.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with H. pylori polypeptides.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab') 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • the antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-H. pylori portion.
  • Both monoclonal and polyclonal antibodies (Ab) directed against H. pylori polypeptides or H. pylori polypeptide variants, and antibody fragments such as Fab' and F(ab') 2 , can be used to block the action of H. pylori polypeptide and allow the study of the role of a particular H. pylori polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention.
  • Antibodies which specifically bind H. pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H. pylori antigens.
  • Anti H. pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure.
  • the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
  • the level of an H. pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy.
  • Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections.
  • the present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
  • anti-H. pylori polypeptide antibodies of the invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gt11, ⁇ gt18-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gt11 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H.
  • H. pylori polypeptide antibodies Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H. pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
  • kits for diagnostic purposes typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels.
  • Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like.
  • Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like.
  • Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like.
  • Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular purpose. Kits further can include instructions for use.
  • labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like.
  • the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject H. pylori polypeptides, or of their role in intracellular signaling.
  • Such inhibitors or potentiators may be useful as new therapeutic agents to combat H. pylori infections in humans.
  • a variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the skilled artisan. In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time.
  • Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified H. pylori polypeptide.
  • Screening assays can be constructed in vitro with a purified H. pylori polypeptide or fragment thereof, such as an H. pylori polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive absorption, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated.
  • a variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the H. pylori polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live H. pylori cells.
  • H. pylori chromosomal DNA was isolated according to a basic DNA protocol outlined in Schleif R.F. and Wensink P.C, Practical Methods in Molecular Biology, p.98,
  • the purified DNA fragments were then blunt-ended using T4 DNA polymerase.
  • linkers are complimentary to the BstXI-cut pMPX vectors, while the overhang is not self- complimentary. Therefore, the linkers will not concatemerize nor will the cut-vector religate itself easily.
  • the linker-adopted inserts were separated from the unincorporated linkers on a 1% agarose gel and purified using GeneClean. The linker-adopted inserts were then ligated to each of the 20 pMPX vectors to construct a series of "shotgun" subclone libraries.
  • the vectors contain an out-of-frame lacZ gene at the cloning site which becomes in-frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.
  • each of the 20 vectors was then transformed into DH5 ⁇ competent cells (Gibco/BRL, DH5 ⁇ transformation protocol).
  • the libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C. DNA was purified using the Qiagen Midi-prep kits and Tip- 100 columns
  • oligonucleotides complimentary to tag sequences on the vectors (Church, supra).
  • the membranes were washed to rinse off non-specifically bound probe, and exposed to X-ray film to visualize individual sequence ladders. After autoradiography, the hybridized probe was removed by incubation at 65° C, and the hybridization cycle repeated with another tag sequence until the membrane had been probed 38 times for chemical sequencing
  • each gel produced a large number of films, each containing new sequencing information. Whenever a new blot was processed, it was initially probed for an internal standard sequence added to each of the pools.
  • Digital images of the films were generated using a laser-scanning densitometer (Molecular Dynamics, Sunnyvale, CA).
  • the digitized images were processed on computer workstations (VaxStation 4000's) using the program REPLICATM (Church et al.,
  • Image processing included lane straightening, contrast adjustment to smooth out intensity differences, and resolution enhancement by iterative gaussian deconvolution.
  • the sequences were then automatically picked in REPLICATM and displayed for interactive proofreading before being stored in a project database.
  • the proofreading was accomplished by a quick visual scan of the film image followed by mouse clicks on the bands of the displayed image to modify the base calls.
  • Many of the sequence errors could be detected and corrected because multiple sequence reads covering the same portion of the genomic DNA provide adequate sequence redundancy for editing.
  • Each sequence automatically received an identification number (corresponding to microtiter plate, probe information, and lane set number). This number serves as a permanent identifier of the sequence so it is always possible to identify the original of any particular sequence without recourse to a specialized database.
  • GelAssemble developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICATM. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICATM database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly.
  • GCG Genetics Computer Group
  • H. pylori a powerful gene expression system
  • the pET System Novagen
  • a DNA sequence encoding a peptide tag, the ⁇ is-Tag was fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products.
  • the 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence.
  • ppiB a gene cloned for use as a control in the expression studies.
  • the sequence for H. pylori ppiB contains a DNA sequence encoding a ⁇ is-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein.
  • Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR).
  • Synthetic oligonucleotide primers (Table 4) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an Ncol cloning site at the extreme 5' terminus, except for ⁇ pSeq. 4821082 (SEQ ID NO: 820) where Ndel was used.
  • the pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids (only 19 amino acids in ⁇ pSeq. 26380318 (SEQ ID NO: 658)and ⁇ pSeq.14640637 (SEQ ID NO: 447)) including six histidine residues (at the extreme C-terminus), which comprise the ⁇ is-Tag.
  • Genomic DNA prepared from the J99 strain of H. pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • genomic DNA 50 nanograms was introduced into a reaction vial containing 2 mM MgCl 2 , 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H.
  • pylori ORF 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 microliters.
  • the following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
  • Sequences 26054702 (SEQ ID NO: 649), 7116626 (SEQ ID NO: 865), 29479681 (SEQ ID NO: 677), 30100332 (SEQ ID NO: 685), 4821082 (SEQ ID NO: 820) and 978477 (SEQ ID NO: 880);
  • each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, Ncol and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of ⁇ pSeq. 4821082 (SEQ ID NO: 820), with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA)
  • the pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case of H pylori sequence 4821082 (SEQ ID NO: 820) with Ndel and EcoRI
  • cloning ppiB the pET-28a vector, which encodes a ⁇ is-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with Bam ⁇ I and Xhol restriction endonucleases.
  • Competent bacteria E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular. John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • the pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5, etc. for the purpose of cloning or plasmid preparation.
  • Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacI gene, the lacUN5 promoter and the gene for T7 R ⁇ A polymerase.
  • T7 R ⁇ A polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 R ⁇ A polymerase transcribes any target plasmid, such as pET-28b, carrying a T7 promoter and a gene of interest.
  • Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
  • H. pylori sequences 50 nanograms of plasmid D ⁇ A isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by ⁇ ovagen as part of the pET expression system kit).
  • the lacZ gene (beta-galactosidase) was expressed in the pET-System as described for the H. pylori recombinant constructions.
  • Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate.
  • bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
  • bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 milliliters of cold 10 mM Tris- ⁇ Cl, p ⁇ 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification.
  • SDS-polyacrylamide gels (12% or 4.0 to 25 % acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
  • Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
  • lysozyme 200 ⁇ g/ ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each of leupeptin, aprotinin, pepstatin, L-1-chloro-3-[4- tosylamido]-7-amino-2-heptanone (TLCK), L-1-chloro-3-[4-tosylamido]-4-phenyl-2- butanone (TPCK), and soybean trypsin inhibitor, and ruptured by several passages through a small volume micro fluidizer (Model M-110S, Microfluidics International Corporation, Newton, MA). The resultant homogenate was made 0.1 % Brij 35, and centrifuged at 100,000 x g for 1 hour to yield a clear supernatant (crude extract).
  • PMSF phenylmethylsulfonyl fluoride
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and was eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD 280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
  • Recombinant protein 14640637 (SEQ ID NO: 447) and proteins, beta-galactosidase (lacZ) and peptidyl-prolyl cis-trans isomerase (ppiB)
  • Recombinant protein 7116626 (SEQ ID NO: 865)
  • the pellets were washed with lysis buffer containing 10 % glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1 % -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol.
  • the resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials..
  • Recombinant proteins 26054702 (SEQ ID NO: 649), 16225006 (SEQ ID NO: 465), 30100332 (SEQ ID NO: 685), 4721061 (SEQ ID NO: 812)
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD 280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
  • the pellet containing the inclusion bodies was solubilized in buffer B containing 8
  • Urea was removed slowly from the protein samples by dialysis against Tris- buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M, 4M, 3M, 2M, 1M, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.
  • TBS Tris- buffered saline
  • DOC deoxycholate
  • H. pylori To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H. pylori model was used. This model mimics the human H. pylori infection in many respects. The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immunotherapy.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding center
  • H. pylori H. pylori
  • strain 244 originally isolated from an ulcer patient
  • This strain has earlier proven to be a good colonizer of the mouse stomach.
  • the bacteria were grown overnight in Brucella broth supplemented with 10 % fetal calf serum, at 37°C in a microaerophilic atmosphere (10% CO 2 , 5%O 2 ).
  • the animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and 3-5 h after this an oral inoculation of H. pylori in broth (approximately 10 8 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation.
  • Recombinant H. pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa- ⁇ IS tag for purification reasons and the non-Helicobacter pylori control protein ( ⁇ -galactosidase from E. coli; LacZ), was constructed in the same way.
  • the antigens are listed in Table 6 below.
  • mice in each group were immunized 4 times over a 34 day period (day 1, 15, 25 and 35).
  • Purified antigens in solution or suspension were given at a dose of 100 ⁇ g/mouse.
  • CT Cholera toxin
  • Omeprazole (400 ⁇ mol/kg) was given orally to the animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation.
  • Infected control animals received HEPES buffer + CT or DOC buffer + CT. Animals were sacrificed 2-4 weeks after final immunization. A general outline of the study is shown in Table 7 below.
  • Mucosal infection The mice were sacrificed by CO 2 and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. The mucosa from the antrum and corpus of an area of 25mm 2 was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
  • the urease test was performed essentially as follows.
  • Urea agar base concentrate was diluted 1:10 with water. 1 ml of if the diluted concentrate was mixed with 100-200 ⁇ l of actively growing H. pylori cells. Color change to magenta indicated that cells were urease positive.
  • the catalase test was performed essentially as follows.
  • Tetramethyl-p-Phenylenediamine was purchased from Sigma, St. Louis, MO (Catalog #
  • Serum antibodies From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.
  • Mucosal antibodies Gentle scrapings of a defined part of the corpus and of 4 cm of duodenum were performed in 50% of the mice in order to detect the presence of antibodies in the mucous.
  • the antibody titers were determined by regular ELISA technique as for serum antibodies.
  • Antibodies in mucus In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with SEQ ID NOs:685, followed by 447, 865, and 658 (see Figure 2). Therapeutic immunization effects:
  • H. pylori strains (as listed in Table 10) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD 600 of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mis of 10 mM Tris-HCl, 0.1 mM EDTA (TE).
  • BLBB 1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum
  • Lysozyme was added to a final concentration of 1 mg/ml along with, SDS to 1% and RNAse A + T1 to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55 C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1% CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes.
  • Genomic DNA prepared from twelve strains of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • genomic DNA (10 nanograms) was introduced into a reaction vial containing 2 mM MgCl 2 , 1 micromolar synthetic oligonucleotide primers
  • each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
  • TOP10F' (INVaF' in the case of ⁇ . pylori sequence 865) strain of E. coli as described below.
  • Competent bacteria E coli strain TOP10F' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Briefly, 2 microliters of 0.5 micromolar BME was added to each vial of 50 microliters of competent cells. Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes.
  • the cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of TOPI OF' or INVaF' were then picked and analyzed to evaluate cloned inserts as described below.
  • the data demonstrate that there is variation in the DNA sequence ranging from as little as 0.12 % difference (SEQ ID NO:764, J99 strain) to approximately 7% change (SEQ ID NO:649, strain A ⁇ 5).
  • the deduced protein sequences show either no variation (SEQ ID NO:764, strains AH18 and AH24) or up to as much as 7.66% amino acid changes (SEQ ID NO:649, Strain AH5).
  • Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.
  • the sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Madison, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.
  • Genomic DNA prepared from the Helicobacter pylori HpJ99 strain (ATCC 55679) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • PCR polymerase chain reaction
  • PCR amplification is carried out by introducing 10 nanograms of genomic DNA
  • each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction.
  • Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
  • PCR products are cloned into the pT7Blue T- Vector (catalog#69820-1, Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • the ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
  • Ligation products are electroporated (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) into electroporation-competent XL-1 Blue or DH5- ⁇ E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA).
  • 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose) at 37°C with shaking for 1 hour.
  • SOC medium 0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose
  • LB 10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/l sodium chloride
  • LB 10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/l sodium chloride
  • Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
  • these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 H.pylori sequence.
  • Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned.
  • Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage. To minimize errors due to PCR, plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.
  • the sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other.
  • the pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product.
  • the PCR product is a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, resultes in the deletion of a portion of the ORFs.
  • the PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified.
  • pCTB8:kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573).
  • the proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA).
  • the fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes.
  • This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides.
  • the "T” overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris p ⁇ 8.3, 50 mM KCl, 2 mM MgCl 2 , 5 units of DNA Polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP; in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C.
  • the "Kan-T” cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA).
  • the PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter 10x T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16°C.
  • the ligation products are transformed into XL-1 Blue or DH5- ⁇ E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
  • H. pylori Two strains of H. pylori are used for transformation: ATCC 55679. the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AH244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach.
  • Cells for transformation are grown at 37°C, 10% CO 2 , 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated.
  • cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf microfuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth. An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells.
  • Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO 2 .
  • Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C, 6% CO 2 , 100% humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep- Blood agar plate containing 25 micrograms/ml Kanamycin.
  • the template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1:1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
  • TEST 1 PCR with cloning primers originally used to amplify the gene/ORF.
  • a positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette.
  • a PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.
  • PCR with R3 primer designed from sequences downstream of the gene/ORF and not present on the plasmid
  • primer Kan-1 or Kan-2 depending on whether the plasmid DNA used was of "A" or "B” orientation.
  • Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3).
  • no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
  • plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3.
  • TEST 2 and TEST 3 Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST 2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation VII. High-throughput drug screen assay
  • H. pylori target gene and its protein product e.g., an H. pylori enzyme
  • H. pylori enzyme e.g., a H. pylori enzyme
  • a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase is described below as a specific example.
  • the assay is essentially as described by Fisher (Fischer, G., etal. (1984) Biomed. Biochim. Acta 43:1101-1111).
  • the assay measures the cis-trans isomerization of the Ala- Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S-7388, lot # 84H5805).
  • the assay is coupled with ⁇ -chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans.
  • test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second. Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 ⁇ M ⁇ -chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase.
  • a 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD 600 nm ⁇ 1) and resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 ⁇ g/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor.
  • the suspension is subjected to 3 cycles of freeze- thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts).
  • the lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
  • H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.

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Abstract

Recombinant or substantially pure preparations of H. pylori polypeptides are described. The nucleic acids encoding the polypeptides also are described. The H. pylori polypeptides are useful for diagnostics and vaccine compositions.

Description

NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO
HELICOBACTER PYLORI FOR DIAGNOSTICS AND THERAPEUTICS
Background of the Invention
Helicobacter pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet 1: 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83- 88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H. pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. (Blaser M. J., (1993) Trends Microbiol. 1 : 255-260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age. (Taylor, D.N. and M. J. Blaser, (1991) Epidemiol. Rev 13: 42-50). H pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
The bacterial factors necessary for colonization of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of the putative virulence factors include the following: urease, an enzyme that may play a role in neutralizing gastric acid pH (Eaton et al., (1991) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee (1991) Microb. Ecol. Hlth. Dis. 4: 121-134; Labigne et al., (1991) J. Bacteriol. 173: 1920- 1931); the bacterial flagellar proteins responsible for motility across the mucous layer. (Hazell et al., (1986) J. Inf. Dis. 153: 658-663; Leying et al., (1992) Mol. Microbiol. 6: 2863-2874; and Haas et al., (1993) Mol. Microbiol. 8: 753-760); Vac A, a bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells (Schmitt. W. and R. Haas, (1994) Molecular Microbiol. 12(2): 307-319); and several gastric tissue-specific adhesins. (Boren et al., (1993) Science 262: 1892-1895; Evans et al., (1993) J. Bacteriol. 175: 674-683; and Falk et al., (1993) Proc. Natl. Acad. Sci. USA 90: 2035-203).
Numerous therapeutic agents are currently available that eradicate H. pylori infections in vitro. (Huesca et. al., (1993) Zbl. Bakt. 280: 244-252; Hopkins, R. J. and J. G. Morris, supra). However, many of these treatments are suboptimally effective in vivo because of bacterial resistance, altered drug distribution, patient non-compliance or poor drug availabilty. (Hopkins, R. J. and J. G. Morris, supra). Treatment with antibiotics combined with bismuth are part of the standard regime used to treat H. pylori infection. (Malfertheiner, P. and J. E. Dominguez-Munoz (1993) Clinical Therapeutics 15 Supp. B: 37-48). Recently, combinations of a proton pump inhibitors and a single antibiotic have been shown to ameliorate duodenal ulcer disease. (Malfertheiner, P. and J. E. Dominguez- Munoz supra). However, methods employing antibiotic agents can have the problem of the emergence of bacterial strains which are resistant to these agents. (Hopkins, R. J. and J. G. Morris, supra). These limitations demonstrate that new more effective methods are needed to combat H. pylori infections in vivo. In particular, the design of new vaccines that may prevent infection by this bacterium is highly desirable.
Summary of the Invention
This invention relates to novel genes, e.g., genes encoding polypeptides such as bacterial surface proteins, from the organism Helicobacter pylori (H. pylori), and other related genes, their products, and uses thereof. The nucleic acids and peptides of the present invention have utility for diagnostic and therapeutics for H. pylori and other
Helicobacter species. They can also be used to detect the presence of H. pylori and other Helicobacter species in a sample; and for use in screening compounds for the ability to interfere with the H. pylori life cycle or to inhibit H. pylori infection. More specifically, this invention features compositions of nucleic acids corresponding to entire coding sequences of H. pylori proteins, including surface or secreted proteins or parts thereof, nucleic acids capable of binding mRNA from H. pylori proteins to block protein translation, and methods for producing H. pylori proteins or parts thereof using peptide synthesis and recombinant DNA techniques. This invention also features antibodies and nucleic acids useful as probes to detect H. pylori infection. In addition, vaccine compositions and methods for the protection or treatment of infection by H. pylori are within the scope of this invention.
Detailed Description of the Drawings
Figure 1 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.
Figure 2 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens. Figure 3 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in buffer containing DOC.
Detailed Description of the Invention
In one aspect, the invention features a recombinant or substantially pure preparation of H. pylori polypeptide of SEQ ID NO: 384. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 384, such nucleic acid is contained in SEQ ID NO: 1. The H. pylori polypeptide sequences of the invention described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides of the invention are contained in the Sequence Listing.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 384 through SEQ ID NO: 389, SEQ ID NO: 391 through SEQ ID NO: 400, SEQ ID NO: 402 through SEQ ID NO: 406, SEQ ID NO: 408, SEQ ID NO: 411 through SEQ ID NO: 412, SEQ ID NO: 414 through SEQ ID NO: 430, SEQ ID NO: 432 through SEQ ID NO: 434, SEQ ID NO: 436 through SEQ ID NO: 441, and SEQ ID NO: 443. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 384 through SEQ ID NO: 389, SEQ ID NO: 391 through SEQ ID NO: 400, SEQ ID NO: 402 through SEQ ID NO: 406, SEQ ID NO: 408, SEQ ID NO: 411 through SEQ ID NO: 412, SEQ ID NO: 414 through SEQ ID NO: 430, SEQ ID NO: 432 through SEQ ID NO: 434, SEQ ID NO: 436 through SEQ ID NO: 441, and SEQ ID NO: 443, such nucleic acids are contained in SEQ ID NO: 1 through SEQ ID NO: 50.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 444, SEQ ID NO: 446 through SEQ ID NO: 448, SEQ ID NO: 450 through SEQ ID NO: 462, SEQ ID NO: 465 through SEQ ID NO: 466, SEQ ID NO: 468 through SEQ ID NO: 469, SEQ ID NO: 471 through SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 478 through SEQ ID NO: 479, SEQ ID NO: 481 through SEQ ID NO: 484, SEQ ID NO: 486, SEQ ID NO: 488 through SEQ ID NO: 501, and SEQ ID NO: 503 through SEQ ID NO: 506. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 444, SEQ ID NO: 446 through SEQ ID NO: 448, SEQ ID NO: 450 through SEQ ID NO: 462, SEQ ID NO: 465 through SEQ ID NO: 466, SEQ ID NO: 468 through SEQ ID NO: 469, SEQ ID NO: 471 through SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 478 through SEQ ID NO: 479, SEQ ID NO: 481 through SEQ ID NO: 484, SEQ ID NO: 486, SEQ ID NO: 488 through SEQ ID NO: 501, and SEQ ID NO: 503 through SEQ ID NO: 506, such nucleic acids are contained in SEQ ID NO: 51 through SEQ ID NO: 100.
In another aspect, the invention features a recombinant or substantially pure preparation of an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 509 through SEQ ID NO: 510, SEQ ID NO: 512 through SEQ ID NO: 514, SEQ ID NO: 516, SEQ ID NO: 518 through SEQ ID NO: 520, SEQ ID NO: 522 through SEQ ID NO: 525, SEQ ID NO: 527 through SEQ ID NO: 533, SEQ ID NO: 535 through SEQ ID NO: 537, SEQ ID NO: 539 through SEQ ID NO: 540, SEQ ID NO: 542 through SEQ ID NO: 544, SEQ ID NO: 546 through SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 553 through SEQ ID NO: 556, SEQ ID NO: 558, SEQ ID NO: 560, SEQ ID NO: 562 through SEQ ID NO: 568, SEQ ID NO: 570, and SEQ ID NO: 572 through SEQ ID NO: 575. The invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 509 through SEQ ID NO: 510, SEQ ID NO: 512 through SEQ ID NO: 514, SEQ ID NO: 516, SEQ ID NO: 518 through SEQ ID NO: 520, SEQ ID NO: 522 through SEQ ID NO: 525, SEQ ID NO: 527 through SEQ ID NO: 533, SEQ ID NO: 535 through SEQ ID NO: 537, SEQ ID NO: 539 through SEQ ID NO: 540, SEQ ID NO: 542 through SEQ ID NO: 544, SEQ ID NO: 546 through SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 553 through SEQ ID NO: 556, SEQ ID NO: 558, SEQ ID NO: 560, SEQ ID NO: 562 through SEQ ID NO: 568, SEQ ID NO: 570, and SEQ ID NO: 572 through SEQ ID NO: 575, such nucleic acids are contained in SEQ ID NO: 101 through SEQ ID NO: 150.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 576 through SEQ ID NO: 579, SEQ ID NO: 581 through SEQ ID NO: 583, SEQ ID NO: 585 through SEQ ID NO: 593, SEQ ID NO: 596 through SEQ ID NO: 614, SEQ ID NO: 617 through SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO: 629 through SEQ ID NO: 631, SEQ ID NO: 633, and SEQ ID NO: 635 through SEQ ID NO: 636. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 576 through SEQ ID NO: 579, SEQ ID NO: 581 through SEQ ID NO: 583, SEQ ID NO: 585 through SEQ ID NO: 593, SEQ ID NO: 596 through SEQ ID NO: 614, SEQ ID NO: 617 through SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO: 629 through SEQ ID NO: 631, SEQ ID NO: 633, and SEQ ID NO: 635 through SEQ ID NO: 636, such nucleic acids are contained in SEQ ID NO: 151 through SEQ ID NO: 200.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 638 through SEQ ID NO: 640, SEQ ID NO: 642 through SEQ ID NO: 643, SEQ ID NO: 647, SEQ ID NO: 649 through SEQ ID NO: 651, SEQ ID NO: 653 through SEQ ID NO: 661, SEQ ID NO: 663 through SEQ ID NO: 670, SEQ ID NO: 673 through SEQ ID NO: 674, SEQ ID NO: 676, SEQ ID NO: 678 through SEQ ID NO: 683, SEQ ID NO: 687 through SEQ ID NO: 692, and SEQ ID NO: 694 through SEQ ID NO: 702. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 638 through SEQ ID NO: 640, SEQ ID NO: 642 through SEQ ID NO: 643, SEQ ID NO: 647. SEQ ID NO: 649 through SEQ ID NO: 651, SEQ ID NO: 653 through SEQ ID NO: 661, SEQ ID NO: 663 through SEQ ID NO: 670, SEQ ID NO: 673 through SEQ ID NO: 674, SEQ ID NO: 676, SEQ ID NO: 678 through SEQ ID NO: 683, SEQ ID NO: 687 through SEQ ID NO: 692, and SEQ ID NO: 694 through SEQ ID NO: 702, such nucleic acids are contained in SEQ ID NO: 201 through SEQ ID NO: 250.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 705 through SEQ ID NO: 708, SEQ ID NO: 712 through SEQ ID NO: 714, SEQ ID NO: 716 through SEQ ID NO: 722, SEQ ID NO: 725 through SEQ ID NO: 730, SEQ ID NO: 732 through SEQ ID NO: 733, SEQ ID NO: 735 through SEQ ID NO: 744, SEQ ID NO: 746 through SEQ ID NO: 752, SEQ ID NO: 755 through SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761 through SEQ ID NO: 763, and SEQ ID NO: 767 through SEQ ID NO: 770. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 705 through SEQ ID NO: 708, SEQ ID NO: 712 through SEQ ID NO: 714, SEQ ID NO: 716 through SEQ ID NO: 722, SEQ ID NO: 725 through SEQ ID NO: 730, SEQ ID NO: 732 through SEQ ID NO: 733, SEQ ID NO: 735 through SEQ ID NO: 744, SEQ ID NO: 746 through SEQ ID NO: 752, SEQ ID NO: 755 through SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761 through SEQ ID NO: 763, and SEQ ID NO: 767 through SEQ ID NO: 770, such nucleic acids are contained in SEQ ID NO: 251 through SEQ ID NO: 300.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 771 through SEQ ID NO: 773, SEQ ID NO: 775, SEQ ID NO: 777, SEQ ID NO: 779 through SEQ ID NO: 784, SEQ ID NO: 786 through SEQ ID NO: 787, SEQ ID NO: 789 through SEQ ID NO: 792, SEQ ID NO: 794, SEQ ID NO: 796, SEQ ID NO: 798 through SEQ ID NO: 805, SEQ ID NO: 807 through SEQ ID NO: 811, SEQ ID NO: 813 through SEQ ID NO: 819, SEQ ID NO: 821 through SEQ ID NO: 822, SEQ ID NO: 824 through SEQ ID NO: 826, SEQ ID NO: 828 through SEQ ID NO: 832, and SEQ ID NO: 835. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 771 through SEQ ID NO: 773, SEQ ID NO: 775, SEQ ID NO: 777, SEQ ID NO: 779 through SEQ ID NO: 784, SEQ ID NO: 786 through SEQ ID NO: 787, SEQ ID NO: 789 through SEQ ID NO: 792, SEQ ID NO: 794, SEQ ID NO: 796, SEQ ID NO: 798 through SEQ ID NO: 805, SEQ ID NO: 807 through SEQ ID NO: 811, SEQ ID NO: 813 through SEQ ID NO: 819, SEQ ID NO: 821 through SEQ ID NO: 822, SEQ ID NO: 824 through SEQ ID NO: 826, SEQ ID NO: 828 through SEQ ID NO: 832, and SEQ ID NO: 835, such nucleic acids are contained in SEQ ID NO: 301 through SEQ ID NO: 350.
In another aspect, the invention features a recombinant or substantially pure preparation of an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 836 through SEQ ID NO: 841, SEQ ID NO: 843 through SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855 through SEQ ID NO: 857, SEQ ID NO: 859 through SEQ ID NO: 862, SEQ ID NO: 866, SEQ ID NO: 868 through SEQ ID NO: 871, SEQ ID NO: 873 through SEQ ID NO: 876, and SEQ ID NO: 879. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 836 through SEQ ID NO: 841, SEQ ID NO: 843 through SEQ ID NO: 851, SEQ ID NO: 853, SEQ ID NO: 855 through SEQ ID NO: 857, SEQ ID NO: 859 through SEQ ID NO: 862, SEQ ID NO: 866, SEQ ID NO: 868 through SEQ ID NO: 871, SEQ ID NO: 873 through SEQ ID NO: 876, and SEQ ID NO: 879, such nucleic acids are contained in SEQ ID NO: 351 through SEQ ID NO: 383.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 385, SEQ ID NO: 390, SEQ ID NO: 401, SEQ ID NO: 407, SEQ ID NO: 409 through SEQ ID NO: 410, SEQ ID NO: 413, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 442. SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 463 through SEQ ID NO: 464, SEQ ID NO: 467, SEQ ID NO: 470, SEQ ID NO: 474, SEQ ID NO: 476 through SEQ ID NO: 477, SEQ ID NO: 480, SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO: 502, SEQ ID NO: 507 through SEQ ID NO: 508, SEQ ID NO: 511, SEQ ID NO: 515, SEQ ID NO: 517, SEQ ID NO: 521, SEQ ID NO: 526, SEQ ID NO: 534, SEQ ID NO: 538, SEQ ID NO: 541, SEQ ID NO: 545, SEQ ID NO: 549, SEQ ID NO: 551 through SEQ ID NO: 552, SEQ ID NO: 557, SEQ ID NO: 559, SEQ ID NO: 561, SEQ ID NO: 569,
SEQ ID NO: 571, SEQ ID NO: 580, SEQ ID NO: 584, SEQ ID NO: 594 through SEQ ID NO: 595, SEQ ID NO: 615 through SEQ ID NO: 616, SEQ ID NO: 624, and SEQ ID NO: 626. The invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 385, SEQ ID NO: 390, SEQ ID NO: 401, SEQ ID NO: 407, SEQ ID NO: 409 through SEQ ID NO: 410, SEQ ID NO: 413, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 442, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 463 through SEQ ID NO: 464, SEQ ID NO: 467, SEQ ID NO: 470, SEQ ID NO: 474, SEQ ID NO: 476 through SEQ ID NO: 477, SEQ ID NO: 480, SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO: 502, SEQ ID NO: 507 through SEQ ID NO: 508, SEQ ID NO: 511, SEQ ID NO: 515, SEQ ID NO: 517. SEQ ID NO: 521, SEQ ID NO: 526, SEQ ID NO: 534, SEQ ID NO: 538, SEQ ID NO: 541, SEQ ID NO: 545, SEQ ID NO: 549, SEQ ID NO: 551 through SEQ ID NO: 552, SEQ ID NO: 557, SEQ ID NO: 559, SEQ ID NO: 561, SEQ ID NO: 569, SEQ ID NO: 571, SEQ ID NO: 580, SEQ ID NO: 584, SEQ ID NO: 594 through SEQ ID NO: 595, SEQ ID NO: 615 through SEQ ID NO: 616, SEQ ID NO: 624, and SEQ ID NO: 626, such nucleic acids are contained in SEQ ID NO: 881 through SEQ ID NO: 930.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 637, SEQ ID NO: 641, SEQ ID NO: 644 through SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 652, SEQ ID NO: 662, SEQ ID NO: 671 through SEQ ID NO: 672, SEQ ID NO: 675, SEQ ID NO: 677, SEQ ID NO: 684 through SEQ ID NO: 686, SEQ ID NO: 693, SEQ ID NO: 703 through SEQ ID NO: 704, SEQ ID NO: 709 through SEQ ID NO: 711, SEQ ID NO: 715, SEQ ID NO: 723 through SEQ ID NO: 724, SEQ ID NO: 731, SEQ ID NO: 734, SEQ ID NO: 745, SEQ ID NO: 753 through SEQ ID NO: 754, SEQ ID NO: 758, SEQ ID NO: 760, SEQ ID NO: 764 through SEQ ID NO: 766, SEQ ID NO: 774, SEQ ID NO: 776, SEQ ID NO: 778, SEQ ID NO: 785, SEQ ID NO: 788, SEQ ID NO: 793, SEQ ID NO: 795, SEQ ID NO: 797, SEQ ID NO: 806, SEQ ID NO: 812, SEQ ID NO: 820, SEQ ID NO: 823, and SEQ ID NO: 827. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 637, SEQ ID NO: 641, SEQ ID NO: 644 through SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 652, SEQ ID NO: 662. SEQ ID NO: 671 through SEQ ID NO: 672, SEQ ID NO: 675, SEQ ID NO: 677, SEQ ID NO: 684 through SEQ ID NO: 686, SEQ ID NO: 693, SEQ ID NO: 703 through SEQ ID NO: 704, SEQ ID NO: 709 through SEQ ID NO: 711, SEQ ID NO: 715, SEQ ID NO: 723 through SEQ ID NO: 724, SEQ ID NO: 731, SEQ ID NO: 734, SEQ ID NO: 745, SEQ ID NO: 753 through SEQ ID NO: 754, SEQ ID NO: 758, SEQ ID NO: 760, SEQ ID NO: 764 through SEQ ID NO: 766, SEQ ID NO: 774, SEQ ID NO: 776, SEQ ID NO: 778, SEQ ID NO: 785, SEQ ID NO: 788, SEQ ID NO: 793, SEQ ID NO: 795, SEQ ID NO: 797, SEQ ID NO: 806, SEQ ID NO: 812, SEQ ID NO: 820, SEQ ID NO: 823, and SEQ ID NO: 827, such nucleic acids are contained in SEQ ID NO: 931 through SEQ ID NO: 980.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 833 through SEQ ID NO: 834, SEQ ID NO: 842, SEQ ID NO: 852, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 863, SEQ ID NO: 864 through SEQ ID NO: 865, SEQ ID NO: 867, SEQ ID NO: 872, SEQ ID NO: 877 through SEQ ID NO: 878, and SEQ ID NO: 880. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides of SEQ ID NO: 833 through SEQ ID NO: 834, SEQ ID NO: 842, SEQ ID NO: 852, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 863, SEQ ID NO: 864 through SEQ ID NO: 865, SEQ ID NO: 867, SEQ ID NO: 872, SEQ ID NO: 877 through SEQ ID NO: 878, and SEQ ID NO: 880, such nucleic acids are contained in SEQ ID NO: 981 through SEQ ID NO: 994.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1446 through SEQ ID NO: 1461, SEQ ID NO: 1463, and SEQ ID NO: 1465 through SEQ ID NO: 1495. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1446 through SEQ ID NO: 1461, SEQ ID NO: 1463, and SEQ ID NO: 1465 through SEQ ID NO: 1495, such nucleic acids are contained in SEQ ID NO: 995 through SEQ ID NO: SEQ ID NO: 1010, SEQ ID NO: 1012, and SEQ ID NO: 1014 through SEQ ID NO: 1044.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1497 through SEQ ID NO: 1515, and SEQ ID NO: 1517 through SEQ ID NO: 1545. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1497 through SEQ ID NO: 1515, and SEQ ID NO: 1517 through SEQ ID NO: 1545, such nucleic acids are contained in SEQ ID NO: 1046 through SEQ ID NO: 1064, and SEQ ID NO: 1066 through SEQ ID NO: 1094.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1546 through SEQ ID NO: 1595. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1546 through SEQ ID NO: 1595, such nucleic acids are contained in SEQ ID NO: 1095 through SEQ ID NO: 1144.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1596 through SEQ ID NO: 1617, SEQ ID NO: 1620 through SEQ ID NO: 1645. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1596 through SEQ ID NO: 1617, SEQ ID NO: 1620 through SEQ ID NO: 1645, such nucleic acids are contained in SEQ ID NO: 1145 through SEQ ID NO: 1166, and SEQ ID NO: 1169 through SEQ ID NO: 1194.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1646 through SEQ ID NO: 1681, and SEQ ID NO: 1683 through SEQ ID NO: 1695. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1646 through SEQ ID NO: 1681, and SEQ ID NO: 1683 through SEQ ID NO: 1695, such nucleic acids are contained in SEQ ID NO: 1195 through SEQ ID NO: 1230, and SEQ ID NO: 1232 through SEQ ID NO: 1244.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1696 through SEQ ID NO: 1745. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1696 through SEQ ID NO: 1745, such nucleic acids are contained in SEQ ID NO: 1245 through SEQ ID NO: 1294.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1746 through SEQ ID NO: 1783, and SEQ ID NO: 1786 through SEQ ID NO: 1795. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H pylori polypeptides SEQ ID NO: 1746 through SEQ ID NO: 1783, and SEQ ID NO: 1786 through SEQ ID NO: 1795, such nucleic acids are contained in SEQ ID NO: 1295 through SEQ ID NO: 1332, and SEQ ID NO: 1335 through SEQ ID NO: 1344.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1796 through SEQ ID NO: 1817, SEQ ID NO: 1819, SEQ ID NO: 1821, SEQ ID NO: 1823 through SEQ ID NO: 1836, and SEQ ID NO: 1838 through SEQ ID NO: 1845. The invention also includes substantially pure nucleic acid encoding an H pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1796 through SEQ ID NO: 1817, SEQ ID NO: 1819, SEQ ID NO: 1821, SEQ ID NO: 1823 through SEQ ID NO: 1836, and SEQ ID NO: 1838 through SEQ ID NO: 1845, such nucleic acids are contained in SEQ ID NO: 1345 through SEQ ID NO: 1366, SEQ ID NO: 1368, SEQ ID NO: 1370, SEQ ID NO: 1372 through SEQ ID NO: 1385, and SEQ ID NO: 1387 through SEQ ID NO: 1394.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of SEQ ID NO: 1846 through SEQ ID NO: 1896. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides SEQ ID NO: 1846 through SEQ ID NO: 1896, such nucleic acids are contained in SEQ ID NO: 1395 through SEQ ID NO: 1445.
In another aspect, the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of the invention as set forth in the Sequence Listing. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides of the invention as set forth in the Sequence Listing. It should be understood that this invention encompasses each of the H. pylori polypeptides and nucleic acids encoding such polypeptides as identified in the Sequence Listing by a given sequence identification number. For example, a representative H. pylori polypeptide is contained in SEQ ID NO: 1450. Therefore, this invention encompasses a recombinant or substantially pure preparation of an H pylori polypeptide of SEQ ID NO: 1450. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 1450. In another aspect, the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such member from the above-identified groups of H. pylori polypeptides (e.g., SEQ ID NO: 1546 through SEQ ID NO: 1595) or nucleic acids (e.g., SEQ ID NO: 1095-SEQ ID NO: 1144), as well as any subgroups from within the above-identified groups. Furthermore, the subgroups can preferably consists of 1, 3, 5, 10, 15, 20, 30 or 40 members of any of the groups identified above, as well as any
combinations thereof. For example, the group consisting of H. pylori polypeptides SEQ ID NO: 1846 through SEQ ID NO: 1896 can be divided into one or more subgroups as follows: SEQ ID NO: 1846-SEQ ID NO: 1860; SEQ ID NO: 1861-SEQ ID NO: 1875; SEQ ID NO: 1876-SEQ ID NO: 1885; SEQ ID NO: 1886-SEQ ID NO: 1896; or any combinations thereof.
Particularly perferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021, SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 1002, SEQ ID NO: 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1251, SEQ ID NO: 1262, SEQ ID NO: 1149, SEQ ID NO: 1220, SEQ ID NO:
1240, SEQ ID NO: 1164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332, SEQ ID NO: 1345, SEQ ID NO: 1358, SEQ ID NO: 1375, SEQ ID NO: 1417, SEQ ID NO: 1283, SEQ ID NO: 1335, SEQ ID NO: 1368, SEQ ID NO: 1179, SEQ ID NO: 1255, SEQ ID NO: 1258, SEQ ID NO: 1044, SEQ ID NO: 1273, SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ ID NO: 1331, SEQ ID NO: 1432. SEQ ID NO: 1052, SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069. SEQ ID NO: 1005, SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1 193. SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO:
1304, SEQ ID NO: 1305. SEQ ID NO: 1346, SEQ ID NO: 1348, SEQ ID NO: 1350, SEQ ID NO: 1032, SEQ ID NO: 1053. SEQ ID NO: 1081, SEQ ID NO: 1124, SEQ ID NO: 1382, SEQ ID NO: 1437. SEQ ID NO: 1263, SEQ ID NO: 1173, SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1410. SEQ ID NO: 1086, SEQ ID NO: 1322, SEQ ID NO: 1266, SEQ ID NO: 1282, SEQ ID NO: 1271, SEQ ID NO: 1208, SEQ ID NO: 1126, SEQ ID NO: 1270, SEQ ID NO: 1278, SEQ ID NO: 1419, SEQ ID NO: 1125, SEQ ID NO: 1181, SEQ ID NO: 1416, SEQ ID NO: 1096, SEQ ID NO: 1082, SEQ ID NO: 1146, SEQ ID NO: 1 145, SEQ ID NO: 1 108, SEQ ID NO: 1148, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1424, SEQ ID NO: 1000, SEQ ID NO: 1027, SEQ ID NO: 1175, SEQ ID NO: 1330, SEQ ID NO: 217, SEQ ID NO: 217, SEQ ID NO: 367, SEQ ID NO:
911,SEQ ID NO: 944, SEQ ID NO: 18, SEQ ID NO: 107, SEQ ID NO: 894, SEQ ID NO:
943, SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199, SEQ ID NO: 992, SEQ ID NO: 934, SEQ ID NO: 899, SEQ ID NO: 302, SEQ ID NO: 215, SEQ ID NO: 893, SEQ ID NO: 984, SEQ ID NO: 97,SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 309, SEQ ID NO: 150, SEQ ID NO: 240, SEQ ID NO: 957, SEQ ID NO: 57, SEQ ID NO: 2, SEQ ID NO: 92, SEQ ID NO: 255, SEQ ID NO: 164, SEQ ID NO: 201, SEQ ID NO: 278, SEQ ID NO: 245, SEQ ID NO: 921, SEQ ID NO: 896, SEQ ID NO: 248, SEQ ID NO: 159, SEQ ID NO: 979, SEQ ID NO: 194, SEQ ID NO: 194, SEQ ID NO: 946, SEQ ID NO: 916, SEQ ID NO: 76, SEQ ID NO: 905, SEQ ID NO: 914, SEQ ID NO: 931, SEQ ID NO: 50, SEQ ID NO: 250, SEQ ID NO: 969, SEQ ID NO: 66,SEQ ID NO: 275, SEQ ID NO: 330, SEQ ID NO: 204, SEQ ID NO: 383, SEQ ID NO: 303, SEQ ID NO: 70, SEQ ID NO: 983, SEQ ID NO: 972, SEQ ID NO: 929, SEQ ID NO: 972, SEQ ID NO: 936, SEQ ID NO: 267, SEQ ID NO: 197, SEQ ID NO: 55, SEQ ID NO: 54, SEQ ID NO: 210, SEQ ID NO: 90, SEQ ID NO: 15, SEQ ID NO: 913, SEQ ID NO: 227, SEQ ID NO: 79, SEQ ID NO: 191, SEQ ID NO: 238, SEQ ID NO: 274, SEQ ID NO: 27, SEQ ID NO: 258, SEQ ID NO: 295, SEQ ID NO: 10, SEQ ID NO: 160, SEQ ID NO: 225, SEQ ID NO: 964, SEQ ID NO: 166, SEQ ID NO: 56, SEQ ID NO: 980,SEQ ID NO: 903, SEQ ID NO: 261, SEQ ID NO: 71, SEQ ID NO: 955, SEQ ID NO: 361, SEQ ID NO: 58, SEQ ID NO: 114, SEQ ID NO: 940, SEQ ID NO: 960, SEQ ID NO: 144, SEQ ID NO: 362, SEQ ID NO: 40, SEQ ID NO: 285, SEQ ID NO: 11, SEQ ID NO: 161, SEQ ID NO: 974, SEQ ID NO: 111, SEQ ID NO: 316, SEQ ID NO: 257, SEQ ID NO: 78, SEQ ID NO: 966, SEQ ID NO: 352, SEQ ID NO: 981, SEQ ID NO: 158, SEQ ID NO: 989, SEQ ID NO: 963, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 135, SEQ ID NO: 910, SEQ ID NO: 236, SEQ ID NO: 241, SEQ ID NO: 949, SEQ ID NO: 945, SEQ ID NO: 207, SEQ ID NO: 977, SEQ ID NO: 978, SEQ ID NO: 994, SEQ ID NO: 163, SEQ ID NO: 256, SEQ ID NO: 287, SEQ ID NO: 184, SEQ ID NO: 45, SEQ ID NO: 136, SEQ ID NO: 214, SEQ ID NO: 16, SEQ ID NO: 192, SEQ ID NO: 373, SEQ ID NO: 892, SEQ ID NO: 239, SEQ ID NO: 34, SEQ ID NO: 340, SEQ ID NO: 41, SEQ ID NO: 332, SEQ ID NO: 134, and SEQ ID NO: 330.
In one embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021, SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 217, SEQ ID NO: 367, SEQ ID NO: 911,SEQ ID NO:
944, SEQ ID NO: 18, SEQ ID NO: 107, SEQ ID NO: 894, SEQ ID NO: 943, SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199. SEQ ID NO: 992, SEQ ID NO: 934, SEQ ID NO: 899, SEQ ID NO: 302, and SEQ ID NO: 215.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO : 1002, SEQ ID NO : 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1251, SEQ ID NO: 1262, SEQ ID NO: 1149, SEQ ID NO: 1220, SEQ ID NO: 1240, SEQ ID NO: 1164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332, SEQ ID NO: 1345, SEQ ID NO: 1358, SEQ ID NO: 1375, SEQ ID NO: 1417, SEQ ID NO: 1283, SEQ ID NO: 1335, SEQ ID NO: 1368, SEQ ID NO: 1179, SEQ ID NO: 1255, SEQ ID NO: 1258, SEQ ID NO: 1044, SEQ ID NO: 1273, SEQ ID NO: 893, SEQ ID NO: 984, SEQ ID NO: 97,SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 309, SEQ ID NO: 150, SEQ ID NO: 240, SEQ ID NO: 957, SEQ ID NO: 57, SEQ ID NO: 2, SEQ ID NO: 92, SEQ ID NO: 255, SEQ ID NO: 164, SEQ ID NO: 201, SEQ ID NO: 278, SEQ ID NO: 245, SEQ ID NO: 921, SEQ ID NO: 896, SEQ ID NO: 248, SEQ ID NO: 159, SEQ ID NO: 979, SEQ ID NO: 194, SEQ ID NO: 194, SEQ ID NO: 946, SEQ ID NO: 916, SEQ ID NO: 76, SEQ ID NO: 905, SEQ ID NO: 914, SEQ ID NO: 931, SEQ ID NO: 50, SEQ ID NO: 250, SEQ ID NO: 969, SEQ ID NO: 66,SEQ ID NO: 275, SEQ ID
NO: 330, SEQ ID NO: 204, SEQ ID NO: 383, SEQ ID NO: 303, SEQ ID NO: 70, SEQ ID
NO: 983, SEQ ID NO: 972, SEQ ID NO: 929, SEQ ID NO: 972, SEQ ID NO: 936, SEQ
ID NO: 267, SEQ ID NO: 197, SEQ ID NO: 55, SEQ ID NO: 54, and SEQ ID NO: 210.
In yet another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ ID NO: 1331, SEQ ID NO: 1432, SEQ ID NO: 1052, SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069, SEQ ID NO: 1005, SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1193, SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1346, SEQ ID NO: 1348, SEQ ID NO: 1350, SEQ ID NO: 1032, SEQ ID NO: 1053, SEQ ID NO: 1081, SEQ ID NO: 1124, SEQ ID NO: 1382, SEQ ID NO: 1437, SEQ ID NO: 1263, SEQ ID NO: 90, SEQ ID NO: 15, SEQ ID NO: 913, SEQ ID NO: 227, SEQ ID NO: 79, SEQ ID NO: 191, SEQ ID NO: 238, SEQ ID NO: 274, SEQ ID NO: 27, SEQ ID NO: 258, SEQ ID NO: 295, SEQ ID NO: 10, SEQ ID NO: 160, SEQ ID NO: 225, SEQ ID NO: 964, SEQ ID NO: 166, SEQ ID NO: 56, SEQ ID NO: 980,SEQ ID NO: 903. SEQ ID NO: 261, SEQ ID NO: 71, SEQ ID NO: 955, SEQ ID NO: 361, SEQ ID NO: 58, SEQ ID NO: 114, SEQ ID NO: 940, SEQ ID NO: 960, SEQ ID NO: 144, SEQ ID NO: 362, SEQ ID NO: 40, SEQ ID NO: 285, SEQ ID NO: 11, SEQ ID NO: 161, SEQ ID NO: 974, SEQ ID NO: 111, SEQ ID NO: 316, SEQ ID NO: 257, SEQ ID NO: 78, and SEQ ID NO: 966.
In yet a further embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1173, SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1410, SEQ ID NO: 1086, SEQ ID NO: 1322. SEQ ID NO: 1266, SEQ ID NO: 1282, SEQ ID NO: 1271, SEQ ID NO: 1208, SEQ ID NO: 1126, SEQ ID NO: 1270, SEQ ID NO: 1278, SEQ ID NO: 1419, SEQ ID NO: 1125, SEQ ID NO: 1181, SEQ ID NO: 1416, SEQ ID NO: 1096, SEQ ID NO: 1082, SEQ ID NO: 1146, SEQ ID NO: 1145, SEQ ID NO: 1108, SEQ ID NO: 1148, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1424, SEQ ID NO: 1000, SEQ ID NO: 1027, SEQ ID NO: 1175, SEQ ID NO: 1330, SEQ ID NO: 352, SEQ ID NO: 981, SEQ ID NO: 158, SEQ ID NO: 989, SEQ ID NO: 963, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 135, SEQ ID NO: 910, SEQ ID NO: 236, SEQ ID NO: 241, SEQ ID NO: 949, SEQ ID NO: 945, SEQ ID NO: 207, and SEQ ID NO: 977.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cytoplasmic polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO: 1229, SEQ ID NO: 1250, SEQ ID NO:
1268, SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ ID NO: 996, SEQ ID NO: 1095, SEQ ID NO: 1156, SEQ ID NO: 1158, SEQ ID NO: 1159, SEQ ID NO: 1277, SEQ ID NO: 1038, SEQ ID NO: 1257, SEQ ID NO: 1357, SEQ ID NO: 1436, SEQ ID NO: 1047. SEQ ID NO: 1055, SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327. SEQ ID NO: 1412, SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1 1 16, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188. SEQ ID NO: 1 198. SEQ ID NO: 1218, SEQ ID NO: 1244, SEQ ID NO: 1306, SEQ ID NO: 1325, SEQ ID NO: 1397, SEQ ID NO: 1398, SEQ ID NO: 1407, SEQ ID NO: 1433, SEQ ID NO: 1216. SEQ ID NO: 1239, SEQ ID NO: 1362, SEQ ID NO:
1017, SEQ ID NO: 1019, SEQ ID NO: 1360, SEQ ID NO: 1423, SEQ ID NO: 1425, SEQ ID NO: 1374, SEQ ID NO: 1028. SEQ ID NO: 1037, SEQ ID NO: 1077, SEQ ID NO: 1115, SEQ ID NO: 1232, SEQ ID NO: 1241, SEQ ID NO: 1267, SEQ ID NO: 1163, SEQ ID NO: 1068. SEQ ID NO: 1025. SEQ ID NO: 1042, SEQ ID NO: 1046, SEQ ID NO: 1056, SEQ ID NO: 1039, SEQ ID NO: 1072, SEQ ID NO: 1073, SEQ ID NO: 1092, SEQ ID NO: 1100, SEQ ID NO: 1102, SEQ ID NO: 1103, SEQ ID NO: 1104, SEQ ID NO: 1111, SEQ ID NO: 1119, SEQ ID NO: 1136, SEQ ID NO: 1137, SEQ ID NO: 1140, SEQ ID NO: 1142, SEQ ID NO: 1233, SEQ ID NO: 1238, SEQ ID NO: 1243, SEQ ID NO: 1245, SEQ ID NO: 1247, SEQ ID NO: 1249, SEQ ID NO: 1261, SEQ ID NO: 1269, SEQ ID NO: 1279, SEQ ID NO: 1284, SEQ ID NO: 1290, SEQ ID NO: 1297, SEQ ID NO: 1328, SEQ ID NO: 1370, SEQ ID NO: 1372, SEQ ID NO: 1377, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1388, SEQ ID NO: 1401, SEQ ID NO: 1402, SEQ ID NO: 1418, SEQ ID NO: 1420, SEQ ID NO: 1427, SEQ ID NO: 1070, SEQ ID NO: 1151, SEQ ID NO: 1176, SEQ ID NO: 999, SEQ ID NO: 1006, SEQ ID NO: 1012, SEQ ID NO: 1018, SEQ ID NO: 1030, SEQ ID NO: 1033, SEQ ID NO: 1041, SEQ ID NO: 1049, SEQ ID NO: 1054, SEQ ID NO: 1057, SEQ ID NO: 1090, SEQ ID NO: 1097, SEQ ID NO: 1129, SEQ ID NO: 1139, SEQ ID NO: 1143, SEQ ID NO: 1152, SEQ ID NO: 1153, SEQ ID NO: 1155, SEQ ID NO: 1161, SEQ ID NO: 1162, SEQ ID NO: 1169, SEQ ID NO: 1170, SEQ ID NO: 1171, SEQ ID NO: 1180, SEQ ID NO: 1194. SEQ ID NO: 1195, SEQ ID NO: 1199, SEQ ID NO: 1200, SEQ ID NO: 1201, SEQ ID NO: 1202, SEQ ID NO: 1205, SEQ ID NO: 1312, SEQ ID NO: 1336, SEQ ID NO: 1349, SEQ ID NO: 1355, SEQ ID NO: 1359, SEQ ID NO: 1413, SEQ ID NO: 1426, SEQ ID NO: 1430, SEQ ID NO: 882, SEQ ID NO: 382, SEQ ID NO: 130, SEQ ID NO: 230, SEQ ID NO: 269, SEQ ID NO: 312, SEQ ID NO: 211, SEQ ID NO: 959, SEQ ID NO: 938, SEQ ID NO: 110, SEQ ID NO: 244, SEQ ID NO: 328, SEQ ID NO: 235, SEQ ID NO: 315, SEQ ID NO: 296, SEQ ID NO: 976, SEQ ID NO: 321, SEQ ID NO: 43, SEQ ID NO: 281, SEQ ID NO: 326, SEQ ID NO: 272, SEQ ID NO: 344, SEQ ID NO: 139, SEQ ID NO: 30, SEQ ID NO: 220, SEQ ID NO: 364, SEQ ID NO: 369, SEQ ID NO: 372, SEQ ID NO: 991, SEQ ID NO: 128, SEQ ID NO: 347, SEQ ID NO: 52, SEQ ID NO: 12, SEQ ID NO: 247, SEQ ID NO: 64, SEQ ID NO: 101, SEQ ID NO: 338, SEQ ID NO: 83, SEQ ID NO: 46, SEQ ID NO: 348, SEQ ID NO: 223, SEQ ID NO: 39, SEQ ID NO: 232, SEQ ID NO: 168, SEQ ID NO: 65, SEQ ID NO: 952, SEQ ID NO: 341, SEQ ID NO: 69, SEQ ID NO: 924, SEQ ID NO: 4, SEQ ID NO: 197, SEQ ID NO: 313, SEQ ID NO: 119, SEQ ID NO: 188, SEQ ID NO: 956, SEQ ID NO: 935, SEQ ID NO: 246, SEQ ID NO: 196, SEQ ID NO:
376, SEQ ID NO: 172, SEQ ID NO: 25, SEQ ID NO: 126, SEQ ID NO: 951, SEQ ID NO: 147, SEQ ID NO: 895, SEQ ID NO: 14, SEQ ID NO: 154, SEQ ID NO: 277, SEQ ID NO: 363, SEQ ID NO: 342, SEQ ID NO: 378, SEQ ID NO: 130, SEQ ID NO: 198, SEQ ID NO: 243, SEQ ID NO: 19, SEQ ID NO: 9, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 349, SEQ ID NO: 209, SEQ ID NO: 990, SEQ ID NO: 185, SEQ ID NO: 883, SEQ ID NO: 8, SEQ ID NO: 887, SEQ ID NO: 350, SEQ ID NO: 987, SEQ ID NO: 63, SEQ ID NO: 249, SEQ ID NO: 118, SEQ ID NO: 132, SEQ ID NO: 47, SEQ ID NO: 106, SEQ ID NO: 324, SEQ ID NO: 155, SEQ ID NO: 121, SEQ ID NO: 153, SEQ ID NO: 87, SEQ ID NO: 986, SEQ ID NO: 262, SEQ ID NO: 333. SEQ ID NO: 36, SEQ ID NO: 982, SEQ ID NO: 180, SEQ ID NO: 84, SEQ ID NO: 900, SEQ ID NO: 20, SEQ ID NO: 7, SEQ ID NO: 61, SEQ ID NO: 253, SEQ ID NO: 120, SEQ ID NO: 268, SEQ ID NO: 299, SEQ ID NO: 942, SEQ ID NO: 173, SEQ ID NO: 187, SEQ ID NO: 187, SEQ ID NO: 234, SEQ ID NO: 112, SEQ ID NO: 324, SEQ ID NO: 971, SEQ ID NO: 62, SEQ ID NO: 308, SEQ ID NO: 74, SEQ ID NO: 1 , SEQ ID NO: 266, SEQ ID NO: 337, SEQ ID NO: 93, SEQ ID NO: 44, SEQ ID NO: 335, SEQ ID NO: 368, SEQ ID NO: 208, SEQ ID NO: 358, SEQ ID NO: 923, SEQ ID NO: 310, SEQ ID NO: 26, SEQ ID NO: 279, SEQ ID NO: 890, SEQ ID NO: 325, SEQ ID NO: 109, SEQ ID NO: 143, SEQ ID NO: 918, SEQ ID NO: 252, SEQ ID NO: 953, SEQ ID NO: 902, SEQ ID NO: 174, SEQ ID NO: 73, SEQ ID NO: 898, SEQ ID NO: 300, SEQ ID NO: 356, SEQ ID NO: 298, SEQ ID NO: 354, SEQ ID NO: 138, SEQ ID NO: 319, SEQ ID NO: 80, SEQ ID NO: 933, SEQ ID NO: 891, SEQ ID NO: 366, SEQ ID NO: 113, SEQ ID NO: 320, SEQ ID NO: 915, SEQ ID NO: 351, SEQ ID NO: 162, SEQ ID NO: 965, SEQ ID NO: 67, SEQ ID NO: 314, SEQ ID NO: 904, SEQ ID NO: 345, SEQ ID NO: 374, SEQ ID NO: 962, SEQ ID NO: 270, SEQ ID NO: 186, SEQ ID NO: 60, SEQ ID NO: 379, SEQ ID NO: 889, SEQ ID NO: 967, SEQ ID NO: 973, SEQ ID NO: 280, SEQ ID NO: 170, SEQ ID NO: 985, and SEQ ID NO: 932.
In one embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 882, SEQ ID NO: 382, SEQ ID NO: 130, and SEQ ID NO: 230.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof involved in amino acid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO:
1229, SEQ ID NO: 1250, SEQ ID NO: 1268, SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 269, SEQ ID NO: 312, SEQ ID NO: 211, SEQ ID NO: 959, SEQ ID NO: 938, SEQ ID NO: 110, SEQ ID NO: 244, SEQ ID NO: 328, SEQ ID NO: 235, SEQ ID NO: 315, SEQ ID NO: 296, SEQ ID NO: 976,
SEQ ID NO: 321, SEQ ID NO: 43, SEQ ID NO: 281, SEQ ID NO: 326, and SEQ ID NO: 272.
In yet another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in nucleotide metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 344, SEQ ID NO: 139, SEQ ID NO: 30, SEQ ID NO: 220, SEQ ID NO: 364, SEQ ID NO: 369, SEQ ID NO: 372, SEQ ID NO: 991, SEQ ID NO: 128, SEQ ID NO: 347, and SEQ ID NO: 52.
In yet a further embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ ID NO: 996, SEQ ID NO: 12, SEQ ID NO: 247, SEQ ID NO: 64, SEQ ID NO: 101, SEQ ID NO: 338, SEQ ID NO: 83, SEQ ID NO: 46, SEQ ID NO: 348, SEQ ID NO: 223, SEQ ID NO: 39, SEQ ID NO: 232, and SEQ ID NO: 168.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1095, SEQ ID NO: 1156, SEQ ID NO: 1158, SEQ ID NO: 1159, SEQ ID NO: 1277, SEQ ID NO: 1038, SEQ ID NO: 65, SEQ ID NO: 952, SEQ ID NO: 341, SEQ ID NO: 69, SEQ ID NO: 924, and SEQ ID NO: 4.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1257, SEQ ID NO: 1357, SEQ ID NO: 1436, SEQ ID NO: 197, SEQ ID NO: 313, and SEQ ID NO: 119.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327, SEQ ID NO: 1412, SEQ ID NO: 188, SEQ ID NO: 956, SEQ ID NO: 935, SEQ ID NO: 246, SEQ ID NO: 196, and SEQ ID NO: 376.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1116, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188, SEQ ID NO: 1198, SEQ ID NO: 1218, SEQ ID NO: 1244, SEQ ID NO: 1306, SEQ ID NO: 1325, SEQ ID NO: 1397, SEQ ID NO: 1398, SEQ ID NO: 1407, SEQ ID NO: 1433, SEQ ID NO: 172, SEQ ID NO: 25, SEQ ID NO: 126, SEQ ID NO: 951 , SEQ ID NO: 147, SEQ ID NO: 895, SEQ ID NO: 14, SEQ ID NO: 154, SEQ ID NO: 277, SEQ ID NO: 363, SEQ ID NO: 342, SEQ ID NO: 378, SEQ ID NO: 130, SEQ ID NO: 198, SEQ ID NO: 243, SEQ ID NO: 19, and SEQ ID NO: 9.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1216, SEQ ID NO: 1239, SEQ ID NO: 1362, SEQ ID NO: 1017, SEQ ID NO: 1019, SEQ ID NO: 1360, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 349, SEQ ID NO: 209, SEQ ID NO: 990, SEQ ID NO: 185, SEQ ID NO: 883, and SEQ ID NO: 8.
In yet another embodiment, the H. pylori cytoplasmic polypeptide is an H. pylori chaperone polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1423, SEQ ID NO: 1425, SEQ ID NO: 1374, SEQ ID NO: 887, SEQ ID NO: 350, and SEQ ID NO: 987.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted or periplasmic polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 1004, SEQ ID NO: 1138, SEQ ID NO: 1067, SEQ ID NO: 1078, SEQ ID NO: 1314, SEQ ID NO: 1319, SEQ ID NO: 1378, SEQ ID NO: 1105, SEQ ID NO: 1114, SEQ ID NO: 1 118, SEQ ID NO: 1120, SEQ ID NO: 1123, SEQ ID NO: 1127, SEQ ID NO: 1212, SEQ ID NO: 1223, SEQ ID NO: 1225, SEQ ID NO: 1246, SEQ ID NO: 1248, SEQ ID NO: 1259, SEQ ID NO: 1264, SEQ ID NO: 1265, SEQ ID NO: 1281, SEQ ID NO: 1285, SEQ ID NO: 1294, SEQ ID NO: 1298, SEQ ID NO: 1299, SEQ ID NO: 1315, SEQ ID NO: 1316, SEQ ID NO: 1317, SEQ ID NO: 1318, SEQ ID NO: 1344, SEQ ID NO: 1351, SEQ ID NO: 1353, SEQ ID NO: 1373, SEQ ID NO: 1380, SEQ ID NO: 1387, SEQ ID NO: 1389, SEQ ID NO: 1393, SEQ ID NO: 1411, SEQ ID NO: 1428, SEQ ID NO: 1431, SEQ ID NO: 1439, SEQ ID NO: 1043, SEQ ID NO: 1183, SEQ ID NO: 1184, SEQ ID NO: 1196, SEQ ID NO: 1197, SEQ ID NO: 1203, SEQ ID NO: 995, SEQ ID NO: 998, SEQ ID NO: 1001, SEQ ID NO: 1022, SEQ ID NO: 1023, SEQ ID NO: 1029, SEQ ID NO: 1040, SEQ ID NO: 1051, SEQ ID NO: 1062, SEQ ID NO: 1154, SEQ ID NO: 1320, SEQ ID NO: 1075, SEQ ID NO: 1106, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1221, SEQ ID NO: 1226, SEQ ID NO: 1235, SEQ ID NO: 1301, SEQ ID NO: 1311, SEQ ID NO: 1326, SEQ ID NO: 1341, SEQ ID NO: 1354, SEQ ID NO: 1364, SEQ ID NO: 1366, SEQ ID NO: 1376, SEQ ID NO: 1391, SEQ ID NO: 1395, SEQ ID NO: 1445, SEQ ID NO: 1079, SEQ ID NO: 1186, SEQ ID NO: 1010, SEQ ID NO: 1016, SEQ ID NO: 1172, SEQ ID NO: 1174, SEQ ID NO: 117, SEQ ID NO: 254, SEQ ID NO: 24, SEQ ID NO: 242, SEQ ID NO: 950, SEQ ID NO: 263, SEQ ID NO: 286, SEQ ID NO: 947, SEQ ID NO: 51, SEQ ID NO: 177, SEQ ID NO: 156, SEQ ID NO: 190, SEQ ID NO: 375, SEQ ID NO: 222. SEQ ID NO: 21, SEQ ID NO: 912, SEQ ID NO: 148, SEQ ID NO: 202, SEQ ID NO: 224, SEQ ID NO: 112, SEQ ID NO: 32, SEQ ID NO: 339, SEQ ID NO: 182, SEQ ID NO: 228, SEQ ID NO: 152, SEQ ID NO: 219, SEQ ID NO: 137, SEQ ID NO: 318, SEQ ID NO: 141, SEQ ID NO: 165, SEQ ID NO: 334, SEQ ID NO: 13, SEQ ID NO: 297, SEQ ID NO: 35, SEQ ID NO: 216, SEQ ID NO: 908, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 927, SEQ ID NO: 221, SEQ ID NO: 178, SEQ ID NO: 169, SEQ ID NO: 293, SEQ ID NO: 289, SEQ ID NO: 926, SEQ ID NO: 948, SEQ ID NO: 115, SEQ ID NO: 251 , SEQ ID NO: 345, SEQ ID NO: 17, SEQ ID NO: 920, SEQ ID NO: 95, SEQ ID NO: 86. SEQ ID NO: 360, SEQ ID NO: 271, SEQ ID NO: 970, SEQ ID NO: 288, SEQ ID NO: 282, SEQ ID NO: 98, SEQ ID NO: 29, SEQ ID NO: 317, SEQ ID NO: 343, SEQ ID NO: 291, SEQ ID NO: 108, SEQ ID NO: 377, SEQ ID NO: 305, SEQ ID NO: 305, SEQ ID NO: 100. SEQ ID NO: 988, SEQ ID NO: 212, SEQ ID NO: 884, SEQ ID NO: 37, SEQ ID NO: 968, SEQ ID NO: 975, SEQ ID NO: 237, SEQ ID NO: 335, SEQ ID NO: 260, SEQ ID NO: 370, SEQ ID NO: 91, SEQ ID NO: 276, SEQ ID NO: 311, SEQ ID NO: 173, SEQ ID NO: 102. SEQ ID NO: 304, SEQ ID NO: 380, SEQ ID NO: 127, SEQ ID NO: 993, SEQ ID NO: 925, SEQ ID NO: 181, and SEQ ID NO: 171.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori surface or membrane polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026, SEQ ID NO: 1048, SEQ ID NO: 1063, SEQ ID NO: 1190, SEQ ID NO: 1083, SEQ ID NO: 1113, SEQ ID NO: 1222, SEQ ID NO: 1295, SEQ ID NO: 1343, SEQ ID NO: 1392, SEQ ID NO: 1443, SEQ ID NO: 1085, SEQ ID NO: 1093, SEQ ID NO: 1117, SEQ ID NO: 1121 , SEQ ID NO: 1131 , SEQ ID NO: 1287, SEQ ID NO: 1440, SEQ ID NO: 1209, SEQ ID NO: 1342, SEQ ID NO: 1381, SEQ ID NO: 1390, SEQ ID NO: 1409, SEQ ID NO: 1035, SEQ ID NO: 1014, SEQ ID NO: 1088, SEQ ID NO: 1242, SEQ ID NO: 1178, SEQ ID NO: 1089. SEQ ID NO: 1340, SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 381, SEQ ID NO: 229. SEQ ID NO: 323, SEQ ID NO: 371, SEQ ID NO: 284, SEQ ID NO: 116, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 907, SEQ ID NO: 193, SEQ ID NO: 145, SEQ ID NO: 59, SEQ ID NO: 322, SEQ ID NO: 94, SEQ ID NO: 306, SEQ ID NO: 939, SEQ ID NO: 205, SEQ ID NO: 123, SEQ ID NO: 906, SEQ ID NO: 928, SEQ ID NO: 346, SEQ ID NO: 129, SEQ ID NO: 307, SEQ ID NO: 133, SEQ ID NO: 131, SEQ ID NO: 886, SEQ ID NO: 179, SEQ ID NO: 104, SEQ ID NO: 213, SEQ ID NO: 359, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO: 327, SEQ ID NO: 365, SEQ ID NO: 33, SEQ ID NO: 331 , SEQ ID NO: 175, SEQ ID NO: 200, SEQ ID NO: 292, SEQ ID NO: 23, SEQ ID NO: 336, SEQ ID NO: 301, SEQ ID NO: 28, SEQ ID NO: 941, SEQ ID NO: 103, SEQ ID NO: 231, SEQ ID NO: 176, SEQ ID NO: 31, SEQ ID NO: 917, SEQ ID NO: 151, SEQ ID NO: 922, SEQ ID NO: 265, SEQ ID NO: 142, SEQ ID NO: 259, SEQ ID NO: 122, SEQ ID NO: 206, SEQ ID NO: 96, SEQ ID NO: 353, SEQ ID NO: 38, SEQ ID NO: 89, SEQ ID NO: 77, SEQ ID NO: 954, SEQ ID NO: 264, SEQ ID NO: 937, SEQ ID NO: 226, SEQ ID NO: 283, SEQ ID NO: 88, SEQ ID NO: 125, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 81, SEQ ID NO: 901, SEQ ID NO: 82, SEQ ID NO: 42, SEQ ID NO: 881, and SEQ ID NO: 885.
In one embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 381, SEQ ID NO: 229, SEQ ID NO: 323, SEQ ID NO: 371, SEQ ID NO: 284, SEQ ID NO: 116, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 907, SEQ ID NO: 193, SEQ ID NO: 145, SEQ ID NO: 59, SEQ ID NO: 322, SEQ ID NO: 94, SEQ ID NO: 306, and SEQ ID NO: 881.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026, SEQ ID NO: 1048, SEQ ID NO: 1063, SEQ ID NO: 1190, SEQ ID NO: 939, SEQ ID NO: 205, SEQ ID NO: 123, SEQ ID NO: 906, SEQ ID NO: 928, SEQ ID NO: 346, SEQ ID NO: 129, SEQ ID NO: 307, SEQ ID NO: 133, SEQ ID NO: 131, SEQ ID NO: 886, SEQ ID NO: 179, SEQ ID NO: 104, SEQ ID NO: 213, SEQ ID NO: 359, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO: 327, and SEQ ID NO: 365.
In yet another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof having at least three membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1083, SEQ ID NO: 1113, SEQ ID NO: 1222, SEQ ID NO: 1295, SEQ ID NO: 1343, SEQ ID NO: 1392, SEQ ID NO: 1443, SEQ ID NO: 33, SEQ ID NO: 331, SEQ ID NO: 175, SEQ ID NO: 200, SEQ ID NO: 292, SEQ ID NO: 23, and SEQ ID NO: 336.
In yet a further embodiment, the H pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1085, SEQ ID NO: 1093, SEQ ID NO: 1117, SEQ ID NO: 1121, SEQ ID NO: 1131, SEQ ID NO: 1287, SEQ ID NO: 1440, SEQ ID NO: 1209, SEQ ID NO: 301, SEQ ID NO: 28, SEQ ID NO: 941, SEQ ID NO: 103, SEQ ID NO: 231, SEQ ID NO: 176, SEQ ID NO: 31, SEQ ID NO: 917, SEQ ID NO: 151, and SEQ ID NO: 922.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof having at least five membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1342, SEQ ID NO: 1381, SEQ ID NO: 1390, SEQ ID NO: 1409, SEQ ID NO: 1035, SEQ ID NO: 265, SEQ ID NO: 142, SEQ ID NO: 259, SEQ ID NO: 122, SEQ ID NO: 206, and SEQ ID NO: 885.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least six membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1014, SEQ ID NO: 1088, SEQ ID NO: 1242, SEQ ID NO: 1178, SEQ ID NO: 96, SEQ ID NO: 353, SEQ ID NO: 38, SEQ ID NO: 89, SEQ ID NO: 77, SEQ ID NO: 954, SEQ ID NO: 264.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1089, SEQ ID NO: 1340, SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 937, SEQ ID NO: 226, SEQ ID NO: 283, SEQ ID NO: 88, SEQ ID NO: 125, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 81, SEQ ID NO: 901, SEQ ID NO: 82, and SEQ ID NO: 42.
Particularly preferred is a purified or isolated H. pylori cell envelope polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812, SEQ ID NO: 1830, SEQ ID NO:
1850, SEQ ID NO: 1854, SEQ ID NO: 1851, SEQ ID NO: 1640, SEQ ID NO: 1453, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1685, SEQ ID NO: 1687, SEQ ID NO: 1688, SEQ ID NO: 1675, SEQ ID NO: 1702, SEQ ID NO: 1713, SEQ ID NO: 1600, SEQ ID NO: 1671, SEQ ID NO: 1691, SEQ ID NO: 1615, SEQ ID NO: 1616, SEQ ID NO: 1855, SEQ ID NO: 1595, SEQ ID NO: 1633, SEQ ID NO: 1608, SEQ ID NO: 1611, SEQ ID NO: 1751, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1780, SEQ ID NO: 1783, SEQ ID NO: 1796, SEQ ID NO: 1809, SEQ ID NO: 1826, SEQ ID NO: 1868, SEQ ID NO: 1734, SEQ ID NO: 1786, SEQ ID NO: 1819, SEQ ID NO: 1630, SEQ ID NO: 1706, SEQ ID NO: 1709, SEQ ID NO: 1495, SEQ ID NO: 1724, SEQ ID NO: 1670, SEQ ID NO: 1725. SEQ ID NO: 1661, SEQ ID NO: 1873, SEQ ID NO: 1753, SEQ ID NO: 1759. SEQ ID NO: 1761, SEQ ID NO: 1782, SEQ ID NO: 1883, SEQ ID NO: 1503, SEQ ID NO: 1542. SEQ ID NO: 1872, SEQ ID NO: 1520, SEQ ID NO: 1456, SEQ ID NO: 1458, SEQ ID NO: 1617, SEQ ID NO: 1628, SEQ ID NO: 1644, SEQ ID NO: 1657, SEQ ID NO: 1658. SEQ ID NO: 1755, SEQ ID NO: 1756, SEQ ID NO: 1797, SEQ ID NO: 1799, SEQ ID NO: 1801, SEQ ID NO: 1483, SEQ ID NO: 1504, SEQ ID NO: 1532, SEQ ID NO: 1575. SEQ ID NO: 1833, SEQ ID NO: 1888, SEQ ID NO: 1714, SEQ ID NO: 1624, SEQ ID NO: 1856, SEQ ID NO: 1857, SEQ ID NO: 1861, SEQ ID NO: 1537, SEQ ID NO: 1773, SEQ ID NO: 1717, SEQ ID NO: 1733, SEQ ID NO: 1722, SEQ ID NO: 1659, SEQ ID NO: 1577, SEQ ID NO: 1721, SEQ ID NO: 1729, SEQ ID NO: 1870, SEQ ID NO: 1576, SEQ ID NO: 1632, SEQ ID NO: 1867, SEQ ID NO:
1547, SEQ ID NO: 1533, SEQ ID NO: 1597, SEQ ID NO: 1596, SEQ ID NO: 1559, SEQ ID NO: 1599, SEQ ID NO: 1788, SEQ ID NO: 1789, SEQ ID NO: 1875, SEQ ID NO:
1451, SEQ ID NO: 1478, SEQ ID NO: 1626, SEQ ID NO: 1781, SEQ ID NO: 660, SEQ ID NO: 660, SEQ ID NO: 855, SEQ ID NO: 534, SEQ ID NO: 675. SEQ ID NO: 404, SEQ ID NO: 518, SEQ ID NO: 464, SEQ ID NO: 672, SEQ ID NO: 640, SEQ ID NO: 490, SEQ ID NO: 755, SEQ ID NO: 389, SEQ ID NO: 635, SEQ ID NO: 877, SEQ ID NO: 637, SEQ ID NO: 477, SEQ ID NO: 772, SEQ ID NO: 658, SEQ ID NO: 463, SEQ ID NO: 852, SEQ ID NO: 503, SEQ ID NO: 411, SEQ ID NO: 441, SEQ ID NO: 782, SEQ ID NO: 575, SEQ ID NO: 691, SEQ ID NO: 724, SEQ ID NO: 452, SEQ ID NO: 386, SEQ ID NO: 497, SEQ ID NO: 712, SEQ ID NO: 591, SEQ ID NO: 638, SEQ ID NO: 740, SEQ ID NO: 697, SEQ ID NO: 569, SEQ ID NO: 470, SEQ ID NO: 700, SEQ ID NO: 586, SEQ ID NO: 823, SEQ ID NO: 627, SEQ ID NO: 627, SEQ ID NO: 684, SEQ ID NO: 551, SEQ ID NO: 478, SEQ ID NO: 508, SEQ ID NO: 545, SEQ ID NO: 628, SEQ ID NO: 443, SEQ ID NO: 702, SEQ ID NO: 776, SEQ ID NO: 461, SEQ ID NO: 737, SEQ ID NO: 809, SEQ ID NO: 642, SEQ ID NO: 879, SEQ ID NO: 773. SEQ ID NO: 468, SEQ ID NO: 842, SEQ ID NO: 788, SEQ ID NO: 624, SEQ ID NO: 788, SEQ ID NO: 644, SEQ ID NO: 727, SEQ ID NO: 631, SEQ ID NO: 450, SEQ ID NO: 448, SEQ ID NO: 653, SEQ ID NO: 495, SEQ ID NO: 400, SEQ ID NO: 541, SEQ ID NO: 673, SEQ ID NO: 482, SEQ ID NO: 622, SEQ ID NO: 689, SEQ ID NO: 736, SEQ ID NO: 417, SEQ ID NO: 716, SEQ ID NO: 762, SEQ ID NO: 395, SEQ ID NO: 587, SEQ ID NO: 669, SEQ ID NO: 758, SEQ ID NO: 593, SEQ ID NO: 451, SEQ ID NO: 827, SEQ ID NO: 502, SEQ ID NO: 719, SEQ ID NO: 469, SEQ ID NO: 715, SEQ ID NO: 847, SEQ ID NO: 453, SEQ ID NO: 527, SEQ ID NO: 652, SEQ ID NO: 745, SEQ ID NO: 567, SEQ ID NO: 848, SEQ ID NO: 430, SEQ ID NO: 748, SEQ ID NO: 396, SEQ ID NO: 588, SEQ ID NO: 795, SEQ ID NO: 523, SEQ ID NO: 791, SEQ ID NO: 714, SEQ ID NO: 481, SEQ ID NO: 765, SEQ ID NO: 837, SEQ ID NO: 833, SEQ ID NO: 585, SEQ ID NO: 865, SEQ ID NO: 764, SEQ ID NO: 440, SEQ ID NO: 465, SEQ ID NO: 555, SEQ ID NO: 526, SEQ ID NO: 687, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 677, SEQ ID NO: 649, SEQ ID NO: 812, SEQ ID NO: 820, SEQ ID NO: 880, SEQ ID NO: 590, SEQ ID NO:713, SEQ ID NO:750, SEQ ID NO:613, SEQ ID NO: 437, SEQ ID NO: 556, SEQ ID NO: 657, SEQ ID NO: 402, SEQ ID NO: 623, SEQ ID NO: 862, SEQ ID NO: 449, SEQ ID NO: 690, SEQ ID NO: 424, SEQ ID NO: 821, SEQ ID NO: 432, SEQ ID NO: 811, SEQ ID NO: 554, and SEQ ID NO: 809.
In one embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812. SEQ ID NO: 1830, SEQ ID NO: 1850, SEQ ID NO: 1854, SEQ ID NO: 1851, SEQ ID NO: 1640, SEQ ID NO: 660, SEQ ID NO: 660, SEQ ID NO: 855, SEQ ID NO: 534, SEQ ID NO: 675, SEQ ID NO: 404, SEQ ID NO: 518, SEQ ID NO: 464, SEQ ID NO: 672, SEQ ID NO: 640, SEQ ID NO: 490, SEQ ID NO: 755, SEQ ID NO: 389, SEQ ID NO: 635, SEQ ID NO: 877, SEQ ID NO: 637, SEQ ID NO: 477, SEQ ID NO: 772, and SEQ ID NO: 658.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1453, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1685, SEQ ID NO: 1687, SEQ ID NO: 1688, SEQ ID NO: 1675, SEQ ID NO: 1702, SEQ ID NO: 1713, SEQ ID NO: 1600, SEQ ID NO: 1671, SEQ ID NO: 1691, SEQ ID NO: 1615, SEQ ID NO: 1616, SEQ ID NO: 1855, SEQ ID NO: 1595, SEQ ID NO: 1633, SEQ ID NO: 1608, SEQ ID NO: 1611, SEQ ID NO: 1751, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1780, SEQ ID NO: 1783, SEQ ID NO: 1796, SEQ ID NO: 1809, SEQ ID NO: 1826, SEQ ID NO: 1868, SEQ ID NO: 1734, SEQ ID NO: 1786, SEQ ID NO: 1819, SEQ ID NO: 1630, SEQ ID NO: 1706, SEQ ID NO: 1709, SEQ ID NO: 1495, SEQ ID NO: 1724, SEQ ID NO: 463, SEQ ID NO: 852, SEQ ID NO: 503, SEQ ID NO: 411, SEQ ID NO: 441, SEQ ID NO: 782, SEQ ID NO: 575, SEQ ID NO: 691, SEQ ID NO: 724, SEQ ID NO: 452, SEQ ID NO: 386, SEQ ID NO: 497, SEQ ID NO: 712, SEQ ID NO: 591, SEQ ID NO: 638, SEQ ID NO: 740, SEQ ID NO: 697, SEQ ID NO: 569, SEQ ID NO: 470, SEQ ID NO: 700, SEQ ID NO: 586, SEQ ID NO: 823, SEQ ID NO: 627, SEQ ID NO: 627, SEQ ID NO: 684, SEQ ID NO: 551, SEQ ID NO: 478, SEQ ID NO: 508, SEQ ID NO: 545, SEQ ID NO: 628, SEQ ID NO: 443, SEQ ID NO: 702, SEQ ID NO: 776, SEQ ID NO: 461, SEQ ID NO: 737, SEQ ID NO: 809, SEQ ID NO: 642, SEQ ID NO: 879, SEQ ID NO: 773, SEQ ID NO: 468, SEQ ID NO: 842, SEQ ID NO: 788, SEQ ID NO: 624, SEQ ID NO: 788, SEQ ID NO: 644, SEQ ID NO: 727, SEQ ID NO: 631, SEQ ID NO: 450, SEQ ID NO: 448, and SEQ ID NO: 653.
In yet another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1670, SEQ ID NO: 1725, SEQ ID NO: 1661, SEQ ID NO: 1873, SEQ ID NO: 1753, SEQ ID NO: 1759, SEQ ID NO: 1761, SEQ ID NO: 1782, SEQ ID NO: 1883, SEQ ID NO: 1503, SEQ ID NO: 1542, SEQ ID NO: 1872, SEQ ID NO: 1520, SEQ ID NO: 1456, SEQ ID NO: 1458, SEQ ID NO: 1617, SEQ ID NO: 1628, SEQ ID NO: 1644, SEQ ID NO: 1657, SEQ ID NO: 1658, SEQ ID NO: 1755, SEQ ID NO: 1756, SEQ ID NO: 1797, SEQ ID NO: 1799, SEQ ID NO: 1801, SEQ ID NO: 1483, SEQ ID NO: 1504, SEQ ID NO: 1532, SEQ ID NO: 1575, SEQ ID NO: 1833, SEQ ID NO: 1888, SEQ ID NO: 1714, SEQ ID NO: 495, SEQ ID NO: 400, SEQ ID NO: 541, SEQ ID NO: 673, SEQ ID NO: 482, SEQ ID NO: 622, SEQ ID NO: 689, SEQ ID NO: 736, SEQ ID NO: 417, SEQ ID NO: 716, SEQ ID NO: 762, SEQ ID NO: 395, SEQ ID NO: 587, SEQ ID NO: 669, SEQ ID NO: 758, SEQ ID NO: 593, SEQ ID NO: 451, SEQ ID NO: 827, SEQ ID NO: 502, SEQ ID NO: 719, SEQ ID NO: 469, SEQ ID NO: 715, SEQ ID NO: 847, SEQ ID NO: 453, SEQ ID NO: 527, SEQ ID NO: 652, SEQ ID NO: 745, SEQ ID NO: 567, SEQ ID NO: 848, SEQ ID NO: 430, SEQ ID NO: 748, SEQ ID NO: 396, SEQ ID NO: 588, SEQ ID NO: 795, SEQ ID NO: 523, SEQ ID NO: 791, SEQ ID NO: 714, SEQ ID NO: 481, and SEQ ID NO: 765.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1624, SEQ ID NO: 1856, SEQ ID NO: 1857, SEQ ID NO: 1861, SEQ ID NO: 1537, SEQ ID NO: 1773, SEQ ID NO: 1717, SEQ ID NO: 1733, SEQ ID NO: 1722, SEQ ID NO: 1659, SEQ ID NO: 1577, SEQ ID NO: 1721, SEQ ID NO: 1729, SEQ ID NO: 1870, SEQ ID NO: 1576, SEQ ID NO: 1632, SEQ ID NO: 1867, SEQ ID NO: 1547, SEQ ID NO: 1533, SEQ ID NO: 1597, SEQ ID NO: 1596, SEQ ID NO: 1559, SEQ ID NO: 1599, SEQ ID NO: 1788, SEQ ID NO: 1789, SEQ ID NO: 1875, SEQ ID NO: 1451, SEQ ID NO: 1478, SEQ ID NO: 1626, SEQ ID NO: 1781, SEQ ID NO: 837, SEQ ID NO: 833, SEQ ID NO: 585, SEQ ID NO: 865, SEQ ID NO: 764, SEQ ID NO: 440, SEQ ID NO: 465, SEQ ID NO: 555, SEQ ID NO: 526, SEQ ID NO: 687, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 677, SEQ ID NO: 649, and SEQ ID NO: 812.
Particularly preferred is a purified or isolated H. pylori cytoplasmic polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485. SEQ ID NO: 1459, SEQ ID NO: 1512, SEQ ID NO: 1515. SEQ ID NO: 1642, SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866. SEQ ID NO: 1886, SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703. SEQ ID NO: 1704. SEQ ID NO: 1737, SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609. SEQ ID NO: 1610, SEQ ID NO: 1728, SEQ ID NO: 1489, SEQ ID NO: 1708, SEQ ID NO: 1808, SEQ ID NO: 1887, SEQ ID NO: 1498, SEQ ID NO: 1506, SEQ ID NO: 1592. SEQ ID NO: 1678, SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636. SEQ ID NO: 1639, SEQ ID NO: 1649, SEQ ID NO: 1669, SEQ ID NO: 1695, SEQ ID NO: 1757, SEQ ID NO: 1776, SEQ ID NO: 1848, SEQ ID NO: 1849, SEQ ID NO: 1858, SEQ ID NO: 1884, SEQ ID NO: 1667, SEQ ID NO: 1690, SEQ ID NO: 1813, SEQ ID NO: 1468, SEQ ID NO: 1470, SEQ ID NO: 1811, SEQ ID NO: 1874, SEQ ID NO: 1876. SEQ ID NO: 1825, SEQ ID NO: 1479, SEQ ID NO: 1488, SEQ ID NO: 1528, SEQ ID NO: 1566, SEQ ID NO: 1683, SEQ ID NO: 1692, SEQ ID NO: 1718, SEQ ID NO: 1614, SEQ ID NO: 1519, SEQ ID NO: 1476, SEQ ID NO: 1493, SEQ ID NO: 1497, SEQ ID NO: 1507, SEQ ID NO: 1490, SEQ ID NO: 1523, SEQ ID NO: 1524, SEQ ID NO: 1543, SEQ ID NO: 1551, SEQ ID NO: 1553, SEQ ID NO: 1554, SEQ ID NO: 1555, SEQ ID NO: 1562, SEQ ID NO: 1570, SEQ ID NO: 1587, SEQ ID NO: 1588, SEQ ID NO: 1591, SEQ ID NO: 1593, SEQ ID NO: 1684, SEQ ID NO: 1689, SEQ ID NO: 1694, SEQ ID NO: 1696, SEQ ID NO: 1698, SEQ ID NO: 1700, SEQ ID NO: 1712, SEQ ID NO: 1720, SEQ ID NO: 1730, SEQ ID NO: 1735, SEQ ID NO: 1741, SEQ ID NO: 1748, SEQ ID NO: 1779, SEQ ID NO: 1821, SEQ ID NO: 1823, SEQ ID NO: 1828, SEQ ID NO: 1834, SEQ ID NO: 1835, SEQ ID NO: 1836, SEQ ID NO: 1839, SEQ ID NO: 1852, SEQ ID NO: 1853, SEQ ID NO: 1869, SEQ ID NO: 1871, SEQ ID NO: 1878, SEQ ID NO: 1521, SEQ ID NO: 1602, SEQ ID NO: 1627, SEQ ID NO: 1450, SEQ ID NO: 1457, SEQ ID NO: 1463, SEQ ID NO: 1469, SEQ ID NO: 1481, SEQ ID NO: 1484, SEQ ID NO: 1492, SEQ ID NO: 1500, SEQ ID NO: 1505, SEQ ID NO: 1508, SEQ ID NO: 1541, SEQ ID NO: 1548, SEQ ID NO: 1580, SEQ ID NO: 1590, SEQ ID NO: 1594, SEQ ID NO: 1603, SEQ ID NO: 1604, SEQ ID NO: 1606, SEQ ID NO: 1612, SEQ ID NO: 1613, SEQ ID NO: 1620, SEQ ID NO: 1621, SEQ ID NO: 1622, SEQ ID NO: 1631, SEQ ID NO: 1645, SEQ ID NO: 1646, SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, SEQ ID NO: 1653, SEQ ID NO: 1656, SEQ ID NO: 1763, SEQ ID NO: 1787, SEQ ID NO: 1800, SEQ ID NO: 1806, SEQ ID NO: 1810, SEQ ID NO: 1864, SEQ ID NO: 1877, SEQ ID NO: 1881, SEQ ID NO: 390, SEQ ID NO: 876, SEQ ID NO: 547, SEQ ID NO: 678, SEQ ID NO: 729, SEQ ID NO: 786, SEQ ID NO: 654, SEQ ID NO: 734, SEQ ID NO: 646, SEQ ID NO: 522, SEQ ID NO: 696, SEQ ID NO: 807, SEQ ID NO: 683, SEQ ID NO: 790, SEQ ID NO: 763, SEQ ID NO: 806, SEQ ID NO: 799, SEQ ID NO: 434, SEQ ID NO: 743, SEQ ID NO: 804, SEQ ID NO: 733, SEQ ID NO: 826, SEQ ID NO: 562, SEQ ID NO: 420, SEQ ID NO: 664, SEQ ID NO: 850, SEQ ID NO: 857, SEQ ID NO: 861, SEQ ID NO: 872, SEQ ID NO: 544, SEQ ID NO: 830, SEQ ID NO: 446, SEQ ID NO: 397, SEQ ID NO: 699, SEQ ID NO: 459, SEQ ID NO: 509, SEQ ID NO: 818, SEQ ID NO: 488, SEQ ID NO: 438, SEQ ID NO: 831, SEQ ID NO: 667, SEQ ID NO: 429, SEQ ID NO: 680, SEQ ID NO: 597, SEQ ID NO: 460, SEQ ID NO: 709, SEQ ID NO: 822, SEQ ID NO: 466, SEQ ID NO: 584, SEQ ID NO: 388, SEQ ID NO: 631, SEQ ID NO: 787, SEQ ID NO: 532, SEQ ID NO: 619, SEQ ID NO: 723, SEQ ID NO: 641, SEQ ID NO: 698, SEQ ID NO: 630, SEQ ID NO: 869, SEQ ID NO: 601, SEQ ID NO: 415, SEQ ID NO: 542, SEQ ID NO: 704, SEQ ID NO: 572, SEQ ID NO: 467, SEQ ID NO: 399, SEQ ID NO: 579, SEQ ID NO: 739, SEQ ID NO: 849, SEQ ID NO: 824, SEQ ID NO: 871, SEQ ID NO: 547, SEQ ID NO: 633, SEQ ID NO: 695, SEQ ID NO: 405, SEQ ID NO: 394, SEQ ID NO: 761, SEQ ID NO: 574, SEQ ID NO: 596, SEQ ID NO: 832, SEQ ID NO: 651, SEQ ID NO: 867, SEQ ID NO: 614, SEQ ID NO: 401, SEQ ID NO: 393, SEQ ID NO: 413, SEQ ID NO: 835, SEQ ID NO: 863, SEQ ID NO: 458, SEQ ID NO: 701, SEQ ID NO: 531, SEQ ID NO: 550, SEQ ID NO: 439, SEQ ID NO: 516, SEQ ID NO: 802, SEQ ID NO: 581, SEQ ID NO: 535, SEQ ID NO: 578, SEQ ID NO: 492, SEQ ID NO: 858, SEQ ID NO: 720, SEQ ID NO: 813, SEQ ID NO: 426, SEQ ID NO: 834, SEQ ID NO: 609, SEQ ID NO: 489, SEQ ID NO: 480, SEQ ID NO: 406, SEQ ID NO: 392, SEQ ID NO: 456, SEQ ID NO: 707, SEQ ID NO: 533, SEQ ID NO: 728, SEQ ID NO: 769, SEQ ID NO: 671, SEQ ID NO: 602, SEQ ID NO: 618, SEQ ID NO: 618, SEQ ID NO: 682, SEQ ID NO: 524, SEQ ID NO: 802, SEQ ID NO: 785, SEQ ID NO: 457, SEQ ID NO: 781, SEQ ID NO: 473, SEQ ID NO: 384, SEQ ID NO: 726, SEQ ID NO: 817, SEQ ID NO: 498, SEQ ID NO: 436, SEQ ID NO: 815, SEQ ID NO: 856, SEQ ID NO: 650, SEQ ID NO: 844, SEQ ID NO: 580, SEQ ID NO: 783, SEQ ID NO: 416, SEQ ID NO: 741, SEQ ID NO: 442, SEQ ID NO: 803, SEQ ID NO: 520, SEQ ID NO: 566, SEQ ID NO: 557, SEQ ID NO: 706, SEQ ID NO: 710, SEQ ID NO: 487, SEQ ID NO: 603, SEQ ID NO: 472, SEQ ID NO: 476, SEQ ID NO: 770, SEQ ID NO: 841, SEQ ID NO: 768, SEQ ID NO: 839, SEQ ID NO: 560, SEQ ID NO: 796, SEQ ID NO: 483, SEQ ID NO: 634, SEQ ID NO: 445, SEQ ID NO: 853, SEQ ID NO: 525, SEQ ID NO: 798, SEQ ID NO: 549, SEQ ID NO: 836, SEQ ID NO: 589, SEQ ID NO: 760, SEQ ID NO: 462, SEQ ID NO: 789, SEQ ID NO: 507, SEQ ID NO: 828, SEQ ID NO: 866, SEQ ID NO: 754, SEQ ID NO: 730, SEQ ID NO: 617, SEQ ID NO: 455, SEQ ID NO: 873, SEQ ID NO: 435, SEQ ID NO: 766, SEQ ID NO: 793, SEQ ID NO: 742, SEQ ID NO: 599, SEQ ID NO: 854, and SEQ ID NO: 632.
In one embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 390, SEQ ID NO: 876, SEQ ID NO: 547, and SEQ ID NO: 678.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in amino acid metabolism selected from the group consisting of SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485, SEQ ID NO: 1459, SEQ ID NO: 729, SEQ ID NO: 786, SEQ ID NO: 654, SEQ ID NO: 734, SEQ ID NO: 646, SEQ ID NO: 522, SEQ ID NO: 696, SEQ ID NO: 807, SEQ ID NO: 683, SEQ ID NO: 790, SEQ ID NO: 763, SEQ ID NO: 806, SEQ ID NO: 799, SEQ ID NO: 434, SEQ ID NO: 743, SEQ ID NO: 804, and SEQ ID NO: 733.
In yet another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in nucleotide metabolism selected from the group consisting of SEQ ID NO: 1512, SEQ ID NO: 1515, SEQ ID NO: 1642, SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866, SEQ ID NO: 1886, SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 826, SEQ ID NO: 562, SEQ ID NO: 420, SEQ ID NO: 664, SEQ ID NO: 850, SEQ ID NO: 857, SEQ ID NO: 861, SEQ ID NO: 872, SEQ ID NO: 544, SEQ ID NO: 830, and SEQ ID NO: 446.
In yet a further embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism selected from the group consisting of SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1737, SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 397, SEQ ID NO: 699, SEQ ID NO: 459, SEQ ID NO: 509, SEQ ID NO: 818, SEQ ID NO: 488, SEQ ID NO: 438, SEQ ID NO: 831, SEQ ID NO: 667, SEQ ID NO: 429, SEQ ID NO: 680, and SEQ ID NO: 597.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism selected from the group consisting of SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1728, SEQ ID NO: 1489, SEQ ID NO: 460, SEQ ID NO: 709, SEQ ID NO: 822, SEQ ID NO: 466, SEQ ID NO: 584, and SEQ ID NO: 388.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism selected from the group consisting of SEQ ID NO: 1708, SEQ ID NO: 1808, SEQ ID NO: 1887, SEQ ID NO: 631 , SEQ ID NO: 787, and SEQ ID NO: 532.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis selected from the group consisting of SEQ ID NO: 1498, SEQ ID NO: 1506, SEQ ID NO: 1592, SEQ ID NO: 1678, SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 619, SEQ ID NO: 723, SEQ ID NO: 641, SEQ ID NO: 698, SEQ ID NO: 630, and SEQ ID NO: 869.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair selected from the group consisting of SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636, SEQ ID NO: 1639, SEQ ID NO: 1649, SEQ ID NO: 1669, SEQ ID NO: 1695, SEQ ID NO: 1757, SEQ ID NO: 1776, SEQ ID NO: 1848, SEQ ID NO: 1849, SEQ ID NO: 1858, SEQ ID NO: 1884, SEQ ID NO: 601, SEQ ID NO: 415, SEQ ID NO: 542, SEQ ID NO: 704, SEQ ID NO: 572, SEQ ID NO: 467, SEQ ID NO: 399, SEQ ID NO: 579, SEQ ID NO: 739, SEQ ID NO: 849, SEQ ID NO: 824, SEQ ID NO: 871, SEQ ID NO: 547, SEQ ID NO: 633, SEQ ID NO: 695, SEQ ID NO: 405, SEQ ID NO: 394, and SEQ ID NO: 761.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis selected from the group consisting of SEQ ID NO: 1667, SEQ ID NO: 1690, SEQ ID NO: 1813, SEQ ID NO: 1468, SEQ ID NO: 1470, SEQ ID NO: 1811, SEQ ID NO: 574, SEQ ID NO: 596, SEQ ID NO: 832, SEQ ID NO: 651, SEQ ID NO: 867, SEQ ID NO: 614, SEQ ID NO: 401, and SEQ ID NO: 393.
In yet another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori chaperone polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1874, SEQ ID NO: 1876, SEQ ID NO: 1825, SEQ ID NO: 413, SEQ ID NO: 835, and SEQ ID NO: 863.
Particularly preferred is a purified or isolated H. pylori secreted or periplasmic polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1455, SEQ ID NO: 1589, SEQ ID NO: 1518, SEQ ID NO: 1529, SEQ ID NO: 1765, SEQ ID NO: 1770, SEQ ID NO: 1829, SEQ ID NO: 1556, SEQ ID NO: 1565, SEQ ID NO: 1569, SEQ ID NO: 1571, SEQ ID NO: 1574, SEQ ID NO: 1578, SEQ ID NO: 1663, SEQ ID NO: 1674, SEQ ID NO: 1676, SEQ ID NO: 1697, SEQ ID NO: 1699, SEQ ID NO: 1710, SEQ ID NO: 1715, SEQ ID NO: 1716, SEQ ID NO: 1732, SEQ ID NO: 1736, SEQ ID NO: 1745, SEQ ID NO: 1749, SEQ ID NO: 1750, SEQ ID NO: 1766, SEQ ID NO: 1767, SEQ ID NO: 1768, SEQ ID NO: 1769, SEQ ID NO: 1795, SEQ ID NO: 1802, SEQ ID NO: 1804, SEQ ID NO: 1824, SEQ ID NO: 1831, SEQ ID NO: 1838, SEQ ID NO: 1840, SEQ ID NO: 1844, SEQ ID NO: 1862, SEQ ID NO: 1879, SEQ ID NO: 1882, SEQ ID NO: 1890, SEQ ID NO: 1494, SEQ ID NO: 1634, SEQ ID NO: 1635, SEQ ID NO: 1647, SEQ ID NO: 1648, SEQ ID NO: 1654, SEQ ID NO: 1446, SEQ ID NO: 1449, SEQ ID NO: 1452, SEQ ID NO: 1473, SEQ ID NO: 1474, SEQ ID NO: 1480, SEQ ID NO: 1491, SEQ ID NO: 1502, SEQ ID NO: 1513, SEQ ID NO: 1605, SEQ ID NO: 1771, SEQ ID NO: 1526, SEQ ID NO: 1557, SEQ ID NO: 1560, SEQ ID NO: 1585, SEQ ID NO: 1672, SEQ ID NO: 1677, SEQ ID NO: 1686, SEQ ID NO: 1752, SEQ ID NO: 1762, SEQ ID NO: 1777, SEQ ID NO: 1792, SEQ ID NO: 1805, SEQ ID NO: 1815, SEQ ID NO: 1817, SEQ ID NO: 1827, SEQ ID NO: 1842, SEQ ID NO: 1846, SEQ ID NO: 1896, SEQ ID NO: 1530, SEQ ID NO: 1637, SEQ ID NO: 1461, SEQ ID NO: 1467, SEQ ID NO: 1623, SEQ ID NO: 1625, SEQ ID NO: 530, SEQ ID NO: 708, SEQ ID NO: 414, SEQ ID NO: 694, SEQ ID NO: 703, SEQ ID NO: 721, SEQ ID NO: 749, SEQ ID NO: 685, SEQ ID NO: 444, SEQ ID NO: 606, SEQ ID NO: 582, SEQ ID NO: 621, SEQ ID NO: 868, SEQ ID NO: 666, SEQ ID NO: 408, SEQ ID NO: 538, SEQ ID NO: 573, SEQ ID NO: 639, SEQ ID NO: 668, SEQ ID NO: 524, SEQ ID NO: 422, SEQ ID NO: 819, SEQ ID NO: 611, SEQ ID NO: 674, SEQ ID NO: 577, SEQ ID NO: 663, SEQ ID NO: 558, SEQ ID NO: 794, SEQ ID NO: 564, SEQ ID NO: 592, SEQ ID NO: 814, SEQ ID NO: 398, SEQ ID NO: 767, SEQ ID NO: 425, SEQ ID NO: 659, SEQ ID NO: 517, SEQ ID NO: 539, SEQ ID NO: 475, SEQ ID NO: 615, SEQ ID NO: 665, SEQ ID NO: 607, SEQ ID NO: 598, SEQ ID NO: 759, SEQ ID NO: 752, SEQ ID NO: 595, SEQ ID NO: 686, SEQ ID NO: 528, SEQ ID NO: 705, SEQ ID NO: 828, SEQ ID NO: 403, SEQ ID NO: 561, SEQ ID NO: 500, SEQ ID NO: 491, SEQ ID NO: 846, SEQ ID NO: 732, SEQ ID NO: 778, SEQ ID NO: 751, SEQ ID NO: 744, SEQ ID NO: 504, SEQ ID NO: 419, SEQ ID NO: 792, SEQ ID NO: 825, SEQ ID NO: 756, SEQ ID NO: 519, SEQ ID NO: 870, SEQ ID NO: 777, SEQ ID NO: 808, SEQ ID NO: 506, SEQ ID NO: 864, SEQ ID NO: 655, SEQ ID NO: 407, SEQ ID NO: 427, SEQ ID NO: 774, SEQ ID NO: 797, SEQ ID NO: 688, SEQ ID NO: 815, SEQ ID NO: 718, SEQ ID NO: 859, SEQ ID NO: 775, SEQ ID NO: 874, SEQ ID NO: 543, SEQ ID NO: 878, SEQ ID NO: 594, SEQ ID NO: 610, and SEQ ID NO: 600.
Particularly preferred is a purified or isolated H. pylori surface or membrane polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731, SEQ ID NO: 1743, SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO: 1499, SEQ ID NO: 1514, SEQ ID NO: 1641, SEQ ID NO: 1534, SEQ ID NO: 1564, SEQ ID NO: 1673, SEQ ID NO: 1746, SEQ ID NO: 1794, SEQ ID NO: 1843, SEQ ID NO: 1894, SEQ ID NO: 1536, SEQ ID NO: 1544, SEQ ID NO: 1568, SEQ ID NO: 1572, SEQ ID NO: 1582, SEQ ID NO: 1738, SEQ ID NO: 1891, SEQ ID NO: 1660, SEQ ID NO: 1793, SEQ ID NO: 1832, SEQ ID NO: 1841, SEQ ID NO: 1860, SEQ ID NO: 1486, SEQ ID NO: 1465, SEQ ID NO: 1539, SEQ ID NO: 1693, SEQ ID NO: 1629, SEQ ID NO: 1540, SEQ ID NO: 1791, SEQ ID NO: 1525, SEQ ID NO: 1558, SEQ ID NO: 1655, SEQ ID NO: 1517, SEQ ID NO: 875, SEQ ID NO: 676, SEQ ID NO: 801, SEQ ID NO: 860, SEQ ID NO: 747, SEQ ID NO: 529, SEQ ID NO: 387, SEQ ID NO: 391, SEQ ID NO: 515, SEQ ID NO: 625, SEQ ID NO: 568, SEQ ID NO: 454, SEQ ID NO: 800, SEQ ID NO: 499, SEQ ID NO: 779, SEQ ID NO: 648, SEQ ID NO: 643, SEQ ID NO: 537, SEQ ID NO: 511, SEQ ID NO: 616, SEQ ID NO: 829, SEQ ID NO: 546, SEQ ID NO: 780, SEQ ID NO: 553, SEQ ID NO: 549, SEQ ID NO: 410, SEQ ID NO: 608, SEQ ID NO: 513, SEQ ID NO: 656, SEQ ID NO: 845, SEQ ID NO: 563, SEQ ID NO: 570, SEQ ID NO: 805, SEQ ID NO: 851, SEQ ID NO: 423, SEQ ID NO: 810, SEQ ID NO: 604, SEQ ID NO: 636, SEQ ID NO: 757, SEQ ID NO: 412, SEQ ID NO: 816, SEQ ID NO: 771, SEQ ID NO: 418, SEQ ID NO: 662, SEQ ID NO: 512, SEQ ID NO: 679, SEQ ID NO: 605, SEQ ID NO: 421, SEQ ID NO: 552, SEQ ID NO: 576, SEQ ID NO: 571, SEQ ID NO: 725, SEQ ID NO: 565, SEQ ID NO: 717, SEQ ID NO: 536, SEQ ID NO: 647, SEQ ID NO: 501, SEQ ID NO: 838, SEQ ID NO: 428, SEQ ID NO: 494, SEQ ID NO: 479, SEQ ID NO: 711, SEQ ID NO: 722 ,SEQ ID NO: 645, SEQ ID NO: 670, SEQ ID NO: 746, SEQ ID NO: 493, SEQ ID NO: 540, SEQ ID NO: 612, SEQ ID NO: 629, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 433, SEQ ID NO: 385, and SEQ ID NO: 409. In one embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731. SEQ ID NO: 1743, SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 875, SEQ ID NO: 676, SEQ ID NO: 801, SEQ ID NO: 860, SEQ ID NO: 747, SEQ ID NO: 529, SEQ ID NO: 387, SEQ ID NO: 391, SEQ ID NO: 515, SEQ ID NO: 625, SEQ ID NO: 568, SEQ ID NO: 454, SEQ ID NO: 800, SEQ ID NO: 499, SEQ ID NO: 779, and SEQ ID NO: 385.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions selected from the group consisting of SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO:
1499, SEQ ID NO: 1514, SEQ ID NO: 1641, SEQ ID NO: 648, SEQ ID NO: 643, SEQ ID NO: 537, SEQ ID NO: 511, SEQ ID NO: 616, SEQ ID NO: 829, SEQ ID NO: 546, SEQ ID NO: 780, SEQ ID NO: 553, SEQ ID NO: 549, SEQ ID NO: 410, SEQ ID NO: 608, SEQ ID NO: 513, SEQ ID NO: 656, SEQ ID NO: 845, SEQ ID NO: 563, SEQ ID NO: 570, SEQ ID NO: 805, and SEQ ID NO: 851.
In yet another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least three membrane spanning regions selected from the group consisting of SEQ ID NO: 1534, SEQ ID NO: 1564, SEQ ID NO: 1673, SEQ ID NO: 1746, SEQ ID NO: 1794, SEQ ID NO: 1843, SEQ ID NO: 1894, SEQ ID NO: 423, SEQ ID NO: 810, SEQ ID NO: 604, SEQ ID NO: 636, SEQ ID NO: 757, SEQ ID NO: 412, and SEQ ID NO: 816.
In yet a further embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions selected from the group consisting of SEQ ID NO: 1536, SEQ ID NO: 1544, SEQ ID NO: 1568, SEQ ID NO: 1572, SEQ ID NO: 1582, SEQ ID NO:
1738, SEQ ID NO: 1891, SEQ ID NO: 1660, SEQ ID NO: 771, SEQ ID NO: 418, SEQ ID NO: 662, SEQ ID NO: 512, SEQ ID NO: 679, SEQ ID NO: 605, SEQ ID NO: 421, SEQ ID NO: 552, SEQ ID NO: 576, SEQ ID NO: 571.
In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least five membrane spanning regions selected from the group consisting of SEQ ID NO: 1793, SEQ ID NO: 1832, SEQ ID NO: 1841, SEQ ID NO: 1860, SEQ ID NO: 1486, SEQ ID NO: 725, SEQ ID NO: 565, SEQ ID NO: 717, SEQ ID NO: 536, SEQ ID NO: 647, and SEQ ID NO: 409. In another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least six membrane spanning regions selected from the group consisting of SEQ ID NO: 1465, SEQ ID NO: 1539, SEQ ID NO: 1693, SEQ ID NO: 1629, SEQ ID NO: 501, SEQ ID NO: 838, SEQ ID NO: 428, SEQ ID NO: 494, SEQ ID NO: 479, SEQ ID NO: 711. and SEQ ID NO: 722.
In yet another embodiment, the H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions selected from the group consisting of SEQ ID NO: 1540, SEQ ID NO: 1791, SEQ ID NO: 1525, SEQ ID NO: 1558, SEQ ID NO: 1655, SEQ ID NO: 1517, SEQ ID NO: 645, SEQ ID NO: 670, SEQ ID NO: 746, SEQ ID NO: 493, SEQ ID NO: 540, SEQ ID NO: 612, SEQ ID NO: 629, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, and SEQ ID NO: 433.
In another aspect, the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such a member from the above-identified groups of H. pylori polypeptides.
In another aspect, the invention features nucleic acids capable of binding mRNA of H. pylori. Such nucleic acid is capable of acting as antisense nucleic acid to control the translation of mRNA of H. pylori. A further aspect features a nucleic acid which is capable of binding specifically to an H. pylori nucleic acid. These nucleic acids are also referred to herein as complements and have utility as probes and as capture reagents.
In another aspect, the invention features an expression system comprising an open reading frame corresponding to H. pylori nucleic acid. The nucleic acid further comprises a control sequence compatible with an intended host. The expression system is useful for making polypeptides corresponding to H. pylori nucleic acid.
In another aspect, the invention features a cell transformed with the expression system to produce H. pylori polypeptides.
In another aspect, the invention features a method of generating antibodies against H. pylori polypeptides which are capable of binding specifically to H. pylori polypeptides. Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori -specific antigens.
In another aspect, the invention features a method of generating vaccines for immunizing an individual against H. pylori. The method includes: immunizing a subject with an H. pylori polypeptide, e.g., a surface or secreted polypeptide. or active portion thereof, and a pharmaceutically acceptable carrier. Such vaccines have therapeutic and prophylactic utilities.
In another aspect, the invention provides a method for generating a vaccine comprising a modified immunogenic H. pylori polypeptide, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmacologically acceptable carrier. In another aspect, the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori polypeptide. The method includes: contacting the candidate compound with an H. pylori polypeptide and determining if the compound binds or otherwise interacts with an H. pylori polypeptide. Compounds which bind H. pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
In another aspect, the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori nucleic acid, e.g., DNA or RNA. The method includes: contacting the candidate compound with an H. pylori nucleic acid and determining if the compound binds or otherwise interacts with an H. pylori polypeptide. Compounds which bind H. pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
The invention features H. pylori polypeptides, preferably a substantially pure preparation of an H. pylori polypeptide, or a recombinant H. pylori polypeptide. In preferred embodiments: the polypeptide has biological activity; the polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acid residues of the invention contained in the Sequence Listing. In yet another preferred embodiment, the amino acid sequence which differs in sequence identity by about 7% to about 8% from the H pylori amino acid sequences of the invention contained in the Sequence Listing is also encompassed by the invention.
In preferred embodiments: the H. pylori polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.
In a preferred embodiment, the subject H. pylori polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the H. pylori polypeptide exhibits an H. pylori biological activity, e.g., the H. pylori polypeptide retains a biological activity of a naturally occurring H. pylori enzyme. In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
In yet other preferred embodiments, the H. pylori polypeptide is a recombinant fusion protein having a first H. pylori polypeptide portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to H. pylori. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
Polypeptides of the invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
The invention also encompasses an immunogenic component which includes an H. pylori polypeptide in an immunogenic preparation; the immunogenic component being capable of eliciting an immune response specific for the H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogenic component comprises at least one antigenic determinant from a polypeptide of the invention contained in the Sequence Listing.
In another aspect, the invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an H. pylori polypeptide. In preferred embodiments: the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.
In preferred embodiments: the nucleic acid of the invention is that contained in the Sequence Listing; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence of the invention contained in the Sequence Listing.
In a preferred embodiment, the encoded H. pylori polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that: the H. pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.
In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
In preferred embodiments, the subject H. pylori nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the H. pylori gene sequence, e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
In yet a further preferred embodiment, the nucleic acid which encodes an H. pylori polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of the invention contained in the Sequence Listing.
In a preferred embodiment, the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences of the invention contained in the Sequence Listing.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of the invention contained in the Sequence Listing which encodes amino acids of the invention contained in the Sequence Listing.
In another aspect, the invention encompasses: a vector including a nucleic acid which encodes an H. pylori polypeptide or an H. pylori polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant H. pylori polypeptide or H. pylori polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori or H. pylori polypeptide variant, e.g., from the cell or from the cell culture medium.
In another aspect, the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence of the invention contained in the Sequence Listing.
The invention also provides a probe or primer which includes a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of the invention contained in the Sequence Listing, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention further provides nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
The H. pylori strain, from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection(ATCC # 55679) as strain ΗP-J99.
Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference); and, polypeptides specifically bound by antisera to H. pylori polypeptides, especially by antisera to an active site or binding domain of H. pylori polypeptide. The invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as H. pylori polypeptide analogs or variants.
Putative functions have been determined for several of the H. pylori polypeptides of the invention, as shown in Table 1.
Accordingly, uses of the claimed H. pylori polypeptides in these identified functions are also within the scope of the invention.
In addition, the present invention encompasses H. pylori polypeptides characterized as shown in Table 1 below, including: H pylori cell envelope proteins, H. pylori periplasmic/secreted proteins, H. pylori cytoplasmic proteins, and other H. pylori surface and membrane proteins. Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs.
(Polypeptides in the same row of Table 1, i.e., rows 1 and 3, or rows 2 and 4. are related to one another as described in Table 3 below.)
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000038_0002
Figure imgf000039_0002
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000041_0002
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
[In Table 1, "nt" represents nucleotide Seq. ID number and "aa" represents amino
Seq. ID number]
Definitions
A purified or isolated polypeptide or a substantially pure preparation of a polypeptide are used interchangeably herein and, as used herein, mean a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation.
Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 μg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally- occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional H. pylori DNA sequence.
A "contig" as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.
An "open reading frame", also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.
As used herein, a "coding sequence" is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.
A "complement" of a nucleic acid as used herein referes to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
A "gene product" is a protein or structural RNA which is specifically encoded by a gene.
As used herein, the term "probe" refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like.
Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand". Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions. Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions. Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stingency (such as, for example, in a solution of 0.5X SSC, at 65° C) requires that the sequences be essentially completely homologous. Conditions of intermediate stringency (such as, for example, 2X SSC at 65 ° C) and low stringency (such as, for example 2X SSC at 55° C), require correspondingly less overall complementarity between the hybridizing sequences. (1X SSC is 0.15 M NaCl, 0.015 M Na citrate).
The terms peptides, proteins, and polypeptides are used interchangeably herein. As used herein, the term "surface protein" refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
A polypeptide has H. pylori biological activity if it has one, two and preferably more of the following properties: (1) if when expressed in the course of an H. pylori infection, it can promote, or mediate the attachment of H. pylori to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an H pylori protein; (3) or the gene which encodes it can rescue a lethal mutation in an H. pylori gene. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.
A biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the H. pylori polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring H. pylori polypeptides, e.g., one or more of the biological activities described herein. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in CΗO cells. Because peptides such as H. pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H. pylori fragment or H. pylori analog is one which exhibits a biological activity in any biological assay for H. pylori activity. Most preferably the fragment or analog possesses 10%, preferably 40%, more preferably 60%, 70%, 80% or 90% or greater of the activity of H. pylori, in any in vivo or in vitro assay. Analogs can differ from naturally occurring H. pylori polypeptides in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include H. pylori polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the H. pylori polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be made in view of the table below.
Figure imgf000051_0001
Figure imgf000052_0001
Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., β or γ amino acids; and cyclic analogs.
As used herein, the term "fragment", as applied to an H. pylori analog, will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of H. pylori polypeptides can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of H. pylori polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are H. pylori polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
An "immunogenic component" as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal.
An "antigenic component" as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
As used herein, the term "transgene" means a nucleic acid (encoding, e.g., one or more polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the cell's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence. As used herein, the term "transgenic cell" refers to a cell containing a transgene. As used herein, a "transgenic animal" is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by a process of transformation of competent cells or by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
The term "antibody" as used herein is intended to include fragments thereof which are specifically reactive with H. pylori polypeptides.
As used herein, the term "cell-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
Misexpression, as used herein, refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
As used herein, "host cells" and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
As used herein, the term "control sequence" refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.
As used herein, the term "operably linked" refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.
The metabolism of a substance, as used herein, means any aspect of the, expression, function, action, or regulation of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modification, the substance induces in other substances. The metabolism of a substance also includes changes in the distribution of the substance. The metabolism of a substance includes changes the substance induces in the distribution of other substances.
A "sample" as used herein refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; Laboratory Manual 2nd ed. ( 1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985);
Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); the series, Methods in Enzymoloqy (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.) and PCR-A Practical
Approach (McPherson, Quirke, and Taylor, eds., 1991).
I. Isolation of Nucleic Acids of H. pylori and Uses Therefor
H. pylori Genomic Sequence
This invention provides nucleotide sequences of the genome of H. pylori which thus comprises a DNA sequence library of H. pylori genomic DNA. The detailed description that follows provides nucleotide sequences of H. pylori, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are methods of using the disclosed H. pylori sequences in methods including diagnostic and therapeutic applications. Furthermore, the library can be used as a database for identification and comparison of medically important sequences in this and other strains of H. pylori.
To determine the genomic sequence of H. pylori, DNA was isolated from a strain of H. pylori (ATCC # 55679) and mechanically sheared by nebulization to a median size of 2 kb. Following size fractionation by gel electrophoresis, the fragments were blunt-ended, ligated to adapter oligonucleotides, and cloned into each of 20 different pMPX vectors (Rice et al., abstracts of Meeting of Genome Mapping and Sequencing, Cold Spring Harbor, NY, 5/11-5/15, 1994, p. 225) to construct a series of "shotgun" subclone libraries.
DNA sequencing was achieved using multiplex sequencing procedures essentially as disclosed in Church et al., 1988, Science 240: 185; U.S. Patents No. 4,942,124 and 5,149,625). DNA was extracted from pooled cultures and subjected to chemical or enzymatic sequencing. Sequencing reactions were resolved by electrophoresis, and the products were transferred and covalently bound to nylon membranes. Finally, the membranes were sequentially hybridized with a series of labelled oligonucleotides complimentary to "tag" sequences present in the different shotgun cloning vectors. In this manner, a large number of sequences could be obtained from a single set of sequencing reactions. The cloning and sequencing procedures are described in more detail in the Exemplification.
Individual sequence reads obtained in this manner were assembled using the FALCON™ program (Church et al., 1994, Automated DNA Sequencing and Analysis, J.C. Venter, ed., Academic Press) and PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, Jan. 1996, p.157). The average contig length was about 3-4 kb.
A variety of approaches are used to order the contigs so as to obtain a continuous sequence representing the entire H. pylori genome. Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of H. pylori genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined.
The H. pylori sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides. As a result of the analysis of ORFs based on stop-to-stop codon reads, it should be understood that these ORFs may not correspond to the ORF of a naturally-occurring H. pylori polypeptide. These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally-occurring H.
py loriolypeptide. Such start codons within the ORFs provided herein can be identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded H. pylori polypeptide is within the scope of this invention. For example, within the ORFs a codon such as AUG or GUG (encoding methionine or valine) which is part of the initiation signal for protein synthesis can be identified and the ORF modified to correspond to a naturally- occurring H. pylori polypeptide. The predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARK™ (Borodovsky and Mclninch, 1993, Comp. Chem. 17:123).
Other H. pylori Nucleic Acids
The nucleic acids of this invention may be obtained directly from the DNA of the above referenced H. pylori strain by using the polymerase chain reaction (PCR). See "PCR, A Practical Approach" (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products can be checked by conventional sequencing methods. Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
It is also possible to obtain nucleic acids encoding H. pylori polypeptides from a cDNA library in accordance with protocols herein described. A cDNA encoding an H. pylori polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques. Genes encoding H. pylori polypeptides can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. Preferred nucleic acids of the invention are contained in the Sequence Listing.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4.373,071, incorporated by reference herein).
Nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands. antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences. As probes, primers, capture ligands and antisense agents, the nucleic acid normally consists of all or part (approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below.
Probes
A nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect H. pylori. With the sequence information set forth in the present application, sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to H. pylori, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules.
Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques. Individuals skilled in the art will readily recognize that the nucleic acids, for use as probes, can be provided with a label to facilitate detection of a hybridization product.
Nucleic acid isolated and synthesized in accordance with the sequence of the invention contained in the Sequence Listing can also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using appropriate stringency hybridization conditions as described herein.
Capture Ligand
For use as a capture ligand, the nucleic acid selected in the manner described above with respect to probes, can be readily associated with a support. The manner in which nucleic acid is associated with supports is well known. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate H. pylori nucleic acid from the nucleic acid of each other and other organisms. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Helicobacter species from each other and from other organisms. Preferably, the sequence will comprise at least twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques.
Primers
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of H. pylori nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Helicobacter species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of≥ 10-15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of H. pylori nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods.
The copies can be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species. The copies can also be
incorporated into cloning and expression vectors to generate polypeptides corresponding to the nucleic acid synthesized by PCR, as is described in greater detail herein.
Antisense
Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of H. pylori genes. These sequences also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
In one embodiment, nucleic acid or derivatives corresponding to H. pylori nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells. For example, a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA. Preferably, the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA. Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques. Methods for loading antisense nucleic acid in liposomes is known in the art as exemplified by U.S. Patent 4,241,046 issued December 23, 1980 to Papahadjopoulos et al.
II. Expression of H. pylori Nucleic Acids
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides. The nucleic acid of the invention exemplified in the Sequence Listing or fragments of said nucleic acid encoding active portions of H. pylori polypeptides can be cloned into suitable vectors or used to isolate nucleic acid. The isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.
The function of a specific gene or operon can be ascertained by expression in a bacterial strain under conditions where the activity of the gene product(s) specified by the gene or operon in question can be specifically measured. Alternatively, a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to other Helicobacter strains, or other bacterial strains such as E. coli, Norcardia, Corynebacterium, Campylobacter, and Streptomyces species. In some cases the expression host will utilize the natural
Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta- galactosidase promoter for expression in E. coli).
To express a gene product using the natural H. pylori promoter, a procedure such as the following can be used. A restriction fragment containing the gene of interest, together with its associated natural promoter element and regulatory sequences (identified using the DNA sequence data) is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together. The recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
In the case of a gene that requires a different promoter, the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid. This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning.
A suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell. For example, an H. pylori polypeptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CΗO). Other suitable host cells are known to those skilled in the art.
Expression in eucaryotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of a recombinant peptide product. Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Ηerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113- 123), and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39). Generally, COS cells (Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci. USA 84:8573-8577) for transient amplification/expression in mammalian cells, while CHO (dhfr- Chinese Hamster Ovary) cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195) for stable amplification/expression in mammalian cells. Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection. or electroporation.
Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
Expression in procaryotes is most often carried out in E. coli with either fusion or non-fusion inducible expression vectors. Fusion vectors usually add a number of NH2 terminal amino acids to the expressed target gene. These NH2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia,
Piscataway, N J) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein. A preferred reporter group is poly(His), which may be fused to the amino or carboxy terminus of the protein and which renders the recombinant fusion protein easily purifiable by metal chelate chromatography.
Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene
69:301-315) and pET11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pET11d relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides. Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
In the case of membrane bound proteins, these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be
chromatographically isolated from the membrane fraction. Several different criteria are used for choosing a detergent suitable for solubilizing these complexes. For example, one property considered is the ability of the detergent to solubilize the H. pylori protein within the membrane fraction at minimal denaturation of the membrane-associated protein allowing for the activity or functionality of the membrane-associated protein to return upon reconstitution of the protein. Another property considered when selecting the detergent is the critical micelle concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution. A third property considered when selecting a detergent is the hydrophobicity of the detergent. Typically, membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g., the triton series, would be useful for solubilizing the hydrophobic proteins. Another property important to a detergent can be the capability of the detergent to remove the H. pylori protein with minimal protein-protein interaction facilitating further purification. A fifth property of the detergent which should be considered is the charge of the detergent. For example, if it is desired to use ion exchange resins in the purification process then preferably detergent should be an uncharged detergent. Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
One strategy to maximize recombinant H. pylori peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy would be to alter the nucleic acid encoding an H. pylori peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques. The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
III. H. pylori Polypeptides
This invention encompasses isolated H. pylori polypeptides encoded by the disclosed H. pylori genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an H. pylori polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region. This can be acheived, for example, by using the isolated nucleic acid encoding the ORF, or fragments thereof, to prime a polymerase chain reaction with genomic H. pylori DNA as template; this is followed by sequencing the amplified product.
The polypeptides of the invention can be isolated from wild-type or mutant H. pylori cells or from heterologous organisms or cells (including, but not limited to, bacteria, yeast, insect, plant and mammalian cells) into which an H. pylori nucleic acid has been introduced and expressed. In addition, the polypeptides can be part of recombinant fusion proteins.
H. pylori polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein.
Many of the polypeptides of the invention are related to one another. Some of these relationships are described in Table 3 below. Most polypeptides described in Table 3 are over 90% identical to one another as noted in the last two columns; some are between 70% and 90%) identical to one another; and very few share between 60% and 10% identity with each other. The polypeptides represented by the sequence identification numbers in the third column of Table 3 result from translations carried out from stop codon to stop codon in the genomic nucleotide sequence of the invention, while those in the first column result from translations carried out from the first methionine or valine codon following the prior stop codon and proceeding to the final stop codon in the nucleotide sequence. In some cases, the nucleotide sequence encoding the related polypeptides is slightly different, resulting in some differences in amino acid residues of the related polypeptides. In many cases, the related polypeptides differ significantly in length, with one polypeptide containing amino acid residues in addition to those in common between the two polypeptides. In all cases, the relationships described in Table 3 are highly significant, and the nucleotide sequences encoding these related polypeptides are also very similar to one another. For example, the nucleotide probes derived from the coding sequence of the polypeptides in column one can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the polypeptides of column three.
The relationships between the polypeptides shown in Table 3 can be classified in five broad categories as follows. First, for many polypeptides (designated "A" in the last column of the Table 3), the polypeptide denoted in column one is identical to the polypeptide denoted in column three except for an occasional addition of a few putative amino acid residues at the N-terminus which result from the fact that the polypeptides of column three were derived by translating from stop codon to stop codon instead of from a predicted start codon (i.e., Met or Val) to a stop codon as was done for the polypeptides in column one.
Second, for most polypeptides (designated "B" in the last column of the Table 3), the polypeptide of column one is at least 95% identical to the polypeptide of column three except that the polypeptide in column three is longer (at either or both ends) by one or more amino acid residues which do not result from the difference between reading from stop to stop instead of from start to stop.
Third, for some polypeptides (designated "C" in the last column of Table 3), the converse is true, the polypeptide of column one is at least 95% identical to the polypeptide of column three except that the polypeptide in column three is shorter (at either or both ends).
Fourth, for some polypeptides (designated "D" in the last column of Table 3), the polypeptide of column one shares a high level of amino acid identity (i.e., at least 95%) with the polypeptide of column three in the region in which they overlap, but shares little or no identity (i.e., less than 95%) at one or both ends. The level of identity of the
polypeptides in columns one and three in categories "B", "C" and "D" is highly significant. For example, a typical H. pylori gene product will exhibit amino acid sequence identities of between 92%) to 100% among strains of H. pylori isolated from human patients (see Table 10 below).
Finally, a fifth class of polypeptides in column one (designated "E" in the last column of Table 3) are closely related but differ significantly (i.e., less than 95% identical) from the polypeptide of column three. These polypeptides are likely "paralogs," members of related gene families in H. pylori.
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000065_0002
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
IV. Identification of Nucleic Acids Encoding Vaccine Components and Targets for Agents Effective Against H. pylori
The disclosed H. pylori genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids. The invention
encompasses nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H. pylori. Identification of said immunogenic components involved in the determination of the function of the disclosed sequences, which can be achieved using a variety of approaches. Non-limiting examples of these approaches are described briefly below.
Homology to known sequences: Computer-assisted comparison of the disclosed H. pylori sequences with previously reported sequences present in publicly available databases is useful for identifying functional H. pylori nucleic acid and polypeptide sequences. It will be understood that protein-coding sequences, for example, may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80-90%) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc. In addition, many structural features of particular protein classes have been identified and correlate with specific consensus sequences, such as, for example, binding domains for nucleotides, DNA, metal ions, and other small molecules; sites for covalent modifications such as phosphorylation, acylation, and the like; sites of proteimprotein interactions, etc. These consensus sequences may be quite short and thus may represent only a fraction of the entire protein-coding sequence. Identification of such a feature in an H pylori sequence is therefore useful in determining the function of the encoded protein and identifying useful targets of antibacterial drugs.
Of particular relevance to the present invention are structural features that are common to secretory, transmembrane, and surface proteins, including secretion signal peptides and hydrophobic transmembrane domains. H. pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
Identification of essential genes: Nucleic acids that encode proteins essential for growth or viability of H. pylori are preferred drug targets. H. pylori genes can be tested for their biological relevance to the organism by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene "knockout", using techniques known to those skilled in the relevant art. In this manner, essential genes may be identified.
Strain-specific sequences: Because of the evolutionary relationship between different H. pylori strains, it is believed that the presently disclosed H. pylori sequences are useful for identifying, and/or discriminating between, previously known and new H. pylori strains. It is believed that other H. pylori strains will exhibit at least 70% sequence homology with the presently disclosed sequence. Systematic and routine analyses of DNA sequences derived from samples containing H. pylori strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all H. pylori strains. In one embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of H. pylori. Strain- specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more H. pylori strains.
In another embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all H. pylori strains but are not found in other bacterial species. Specific Example: Determination Of Candidate Protein Antigens For Antibody And Vaccine Development
The selection of candidate protein antigens for vaccine development can be derived from the nucleic acids encoding H. pylori polypeptides. First, the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C.
(1985) Biochimica et Biophysica Acta 815, 468-476) for predicting exported and membrane proteins.
Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases. BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database. ORF's with significant homology (e.g. probabilities lower than 1x10-6 that the homology is only due to random chance) to membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to H. pylori genes based on sequence homology to genes cloned in other organisms.
Discriminant analysis (Klein, et al. supra) can be used to examine the ORF amino acid sequences. This algorithm uses the intrinsic information contained in the ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
Infrequently it is not possible to distinguish between multiple possible nucleotides at a given position in the nucleic acid sequence. In those cases the ambiguities are denoted by an extended alphabet as follows:
These are the official IUPAC-IUB single-letter base codes
Figure imgf000075_0001
The amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code. V. Production of Fragments and Analogs of H. pylori Nucleic Acids and Polypeptides
Based on the discovery of the H. pylori gene products of the invention provided in the Sequence Lsiting, one skilled in the art can alter the disclosed structure (of H. pylori genes), e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind H. pylori polypeptides. Such screens are useful for the identification of inhibitors of H. pylori.
Generation of Fragments
Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length. Alteration of Nucleic Acids and Polypeptides: Random Methods
Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein).
(A) PCR Mutagenesis
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15). The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries. (B) Saturation Mutagenesis
Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
(C) Degenerate Oligonucleotides
A library of homologs can also be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815). Alteration of Nucleic Acids and Polypeptides: Methods for Directed Mutagenesis
Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
(A) Alanine Scanning Mutagenesis
Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
(B) Oligonucleotide-Mediated Mutagenesis
Oligonucleotide-mediated mutagenesis is a useful method for preparing
substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second
complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally,
oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
(C) Cassette Mutagenesis
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315[1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide- mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.
(D) Combinatorial Mutagenesis
Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630). In this method, the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences. Other Modifications of H. pylori Nucleic Acids and Polypeptides
It is possible to modify the structure of an H. pylori polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). A modified H. pylori protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.
An H. pylori peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages. In addition, amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.
In order to enhance stability and/or reactivity, an H. pylori polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an H. pylori polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of H. pylori proteins include reduction/alkylation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton N J 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch, of Allergy and Appl. Immunol., 41 : 199 - 215). To facilitate purification and potentially increase solubility of an H. pylori protein or peptide, it is possible to add an amino acid fusion moiety to the peptide backbone. For example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Ηochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325). In addition, to facilitate isolation of peptides free of irrelevant sequences, specific
endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
To potentially aid proper antigen processing of epitopes within an H. pylori polypeptide, canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods. For example, charged amino acid pairs, such as KK or RR, can be introduced between regions withpm a protein or fragment during recombinant construction thereof. The resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes. In addition, such charged amino acid residues can result in an increase in the solubility of the peptide.
Primary Methods for Screening Polypeptides and Analogs
Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to H. pylori polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
(A) Two Hybrid Systems
Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring H. pylori polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an H. pylori protein. (The H. pylori domain is used as the bait protein and the library of variants are expressed as fish fusion proteins.) In an analogous fashion, a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind a H. pylori polypeptide.
(B) Display Libraries
In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologs which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a
fluorescence-activated cell sorter.
A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 1013 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and f1 are most often used in phage display libraries. Either of the phage gIll or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2-terminal end of pIll and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands. e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991 ) Bio/Tech 9, 1369- 1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J.
Bacteriol. 174, 4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).
In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA- binding protein LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the LacI gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six- residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869) This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C- terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pIll and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The LacI fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage.
Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9): 1233-1251). These particular biases are not a factor in the LacI display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries of 107-109 independent clones are routinely prepared. Libraries as large as 101 1 recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available
phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.
In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233-
1251), a molecular DNA library encoding 1012 decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system.
Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ΕLISA, or for binding specificity in a completion phage ΕLISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host. Secondary Screening of Polypeptides and Analogs
The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine for one skilled in the art to obtain analogs and fragments.
Peptide Mimetics of H. pylori Polypeptides
The invention also provides for reduction of the protein binding domains of the subject H. pylori polypeptides to generate mimetics, e.g. peptide or non-peptide agents.
The peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an H. pylori polypeptide binding to a naturally occurring ligand. The critical residues of a subject H. pylori polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate H. pylori -derived peptidomimetics which competitively or noncompetitively inhibit binding of the H. pylori polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31,080A).
For example, scanning mutagenesis can be used to map the amino acid residues of a particular H. pylori polypeptide involved in binding an interacting polypeptide,
peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) can be generated which mimic those residues in binding to an interacting polypeptide, and which therefore can inhibit binding of an H. pylori polypeptide to an interacting polypeptide and thereby interfere with the function of H. pylori polypeptide. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function
(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1 :1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71). VI. Vaccine Formulations for H. pylori Nucleic Acids and Polypeptides
This invention also features vaccine compositions for protection against infection by H. pylori or for treatment of H. pylori infection, a gram-negative spiral microaerophilic bacterium. In one embodiment, the vaccine compositions contain one or more
immunogenic components such as a surface protein from H. pylori, or portion thereof, and a pharmaceutically acceptable carrier. Nucleic acids within the scope of the invention are exemplified by the nucleic acids of the invention contained in the Sequence Listing which encode H. pylori surface proteins. For example, the preferred nucleic acid for a vaccine composition of the invention is isolated from the group of nucleic acids which encode cell envelope proteins as outlined in Table 1. More specifically, the amino acids of SEQ ID NO:812, SEQ ID NO:820, SEQ ID NO:880, SEQ ID NO:658, SEQ ID NO:865, SEQ ID NO: 1729, SEQ ID NO: 1861, or fragments thereof, can be used alone or in combination for the formulation of vaccine compositions of the invention, as well as, their corresponding nucleic acids of SEQ ID NO:977, SEQ ID NO:978, SEQ ID NO:994, SEQ ID NO:215, SEQ ID NO:989, SEQ ID NO:1278, and SEQ ID NO:1410. However, any nucleic acid encoding an immunogenic H. pylori protein, or portion thereof, which is capable of expression in a cell, can be used in the present invention. These vaccines have therapeutic and prophylactic utilities.
One aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains at least one immunogenic fragment of an H. pylori protein and a pharmaceutically acceptable carrier. Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10-20 amino acid residues in length, and more preferably about 12-16 amino acid residues in length.
Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H. pylori protein. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
In one embodiment, immunogenic components are identified by the ability of the peptide to stimulate T cells. Peptides which stimulate T cells, as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate ΗLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen/T cell interaction, and activation of the B cell cascade, leading to the production of antibodies. A T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.
Screening immunogenic components can be accomplished using one or more of several different assays. For example, in vitro, peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture. Presentation of an immunogenic H. pylori peptide in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines. For example, any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incorporated herein by reference. A kit for an assay for the production of interferon is also available from
Genzyme Corporation (Cambridge, MA).
Alternatively, a common assay for T cell proliferation entails measuring tritiated thymidine incorporation. The proliferation of T cells can be measured in vitro by determining the amount of 3H-labeled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
Vaccine compositions of the invention containing immunogenic components (e.g., H. pylori polypeptide or fragment thereof or nucleic acid encoding an H. pylori polypeptide or fragment thereof) preferably include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. For vaccines of the invention containing H. pylori polypeptides, the polypeptide is coadministered with a suitable adjuvant.
It will be apparent to those of skill in the art that the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of antibody administered, whether the protein or DNA is administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or DNA. Vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular
immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 91: 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 11 : 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
The vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl--L-threonyl-D-isoglutamine (thr- MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn- glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835 A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion; and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial lysates, block polymers or saponins.
Other suitable delivery methods include biodegradable microcapsules or immuno- stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue. The amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 μg to 50 μg, for example 10 μg to 35 μg. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.
Carrier systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H. pylori polypeptide in an insoluble form as fusion proteins. Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres. Suitable diluents are 0.2 N NaΗCO3 and/or saline.
Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of H. pylori in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by H. pylori. The vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art. Thus, for adults a suitable dosage will be in the range of 10 μg to 10 g, preferably 10 μg to 100 mg, for example 50 μg to 50 mg. A suitable dosage for adults will also be in the range of 5 μg to 500 mg. Similar dosage ranges will be applicable for children. Those skilled in the art will recognize that the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors. Those skilled in the art will also recognize that appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol. 150:917-921 (1993); Boedecker et al., American
Gastroenterological Assoc. 999:A-222 (1993)). The number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American
Gastroenterological Assoc. 888:A-222 (1993)).
In a preferred embodiment, a vaccine composition of the invention can be based on a killed whole E coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.
It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing H. pylori infection, some are useful only for treating H. pylori infection, and some are useful for both preventing and treating H. pylori infection. In a preferred embodiment, the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H. pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. pylori -caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
VII. Antibodies Reactive With H. pylori Polypeptides
The invention also includes antibodies specifically reactive with the subject H. pylori polypeptide. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Ηarlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the subject H. pylori polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least 95% homologous). In yet a further preferred embodiment of the invention, the anti-H. pylori antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80% percent homologous to a sequence of the invention contained in the Sequence Listing. By "not substantially cross react", it is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no crossreactivity between bacterial and mammalian antigens.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with H. pylori polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. The antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-H. pylori portion.
Both monoclonal and polyclonal antibodies (Ab) directed against H. pylori polypeptides or H. pylori polypeptide variants, and antibody fragments such as Fab' and F(ab')2, can be used to block the action of H. pylori polypeptide and allow the study of the role of a particular H. pylori polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention.
Antibodies which specifically bind H. pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H. pylori antigens. Anti H. pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure. Likewise, the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of an H. pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy.
Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
Another application of anti-H. pylori polypeptide antibodies of the invention is in the immunological screening of cDNA libraries constructed in expression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgt11 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H. pylori polypeptide antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H. pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
VIII. Kits Containing Nucleic Acids. Polypeptides or Antibodies of the Invention
The nucleic acid, polypeptides and antibodies of the invention can be combined with other reagents and articles to form kits. Kits for diagnostic purposes typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels. Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like. Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like. Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like. Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular purpose. Kits further can include instructions for use.
IX. Drug Screening Assays Using H. pylori Polypeptides
By making available purified and recombinant H. pylori polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject H. pylori polypeptides, or of their role in intracellular signaling. Such inhibitors or potentiators may be useful as new therapeutic agents to combat H. pylori infections in humans. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the skilled artisan. In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified H. pylori polypeptide.
Screening assays can be constructed in vitro with a purified H. pylori polypeptide or fragment thereof, such as an H. pylori polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive absorption, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated. A variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the H. pylori polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live H. pylori cells.
EXEMPLIFICATION
I. Cloning and Sequencing of H. pylori DNA
H. pylori chromosomal DNA was isolated according to a basic DNA protocol outlined in Schleif R.F. and Wensink P.C, Practical Methods in Molecular Biology, p.98,
Springer- Verlag, NY., 1981, with minor modifications. Briefly, cells were pelleted, resuspended in TE (10 mM Tris, 1 mM EDTA, pΗ 7.6) and GES lysis buffer (5.1 M guanidium thiocyanate, 0.1 M EDTA, pΗ 8.0, 0.5% N-laurylsarcosine) was added.
Suspension was chilled and ammonium acetate (NΗ4AC) was added to final concentration of 2.0 M. DNA was extracted, first with chloroform, then with phenol-chloroform, and reextracted with chloroform. DNA was precipitated with isopropanol, washed twice with
70%) EtOH. dried and resuspended in TE.
Following isolation whole genomic H. pylori DNA was nebulized (Bodenteich et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994) to a median size of 2000 bp. After nebulization, the DNA was concentrated and separated on a standard 1% agarose gel. Several fractions, corresponding to approximate sizes 900-1300 bp, 1300-1700 bp, 1700-2200 bp, 2200-2700 bp, were excised from the gel and purified by the GeneClean procedure (Bio101, Inc.).
The purified DNA fragments were then blunt-ended using T4 DNA polymerase.
The healed DNA was then ligated to unique BstXI-linker adapters (5'
TCTAGACCACCTGC and 5' GTGGTCTAGA in 100-1000 fold molar excess). These linkers are complimentary to the BstXI-cut pMPX vectors, while the overhang is not self- complimentary. Therefore, the linkers will not concatemerize nor will the cut-vector religate itself easily. The linker-adopted inserts were separated from the unincorporated linkers on a 1% agarose gel and purified using GeneClean. The linker-adopted inserts were then ligated to each of the 20 pMPX vectors to construct a series of "shotgun" subclone libraries. The vectors contain an out-of-frame lacZ gene at the cloning site which becomes in-frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.
All subsequent steps were based on the multiplex DNA sequencing protocols outlined in Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988. Only major modifications to the protocols are highlighted. Briefly, each of the 20 vectors was then transformed into DH5α competent cells (Gibco/BRL, DH5α transformation protocol). The libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C. DNA was purified using the Qiagen Midi-prep kits and Tip- 100 columns
(Qiagen, Inc.). In this manner, 100 μg of DNA was obtained per pool. Fifteen 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy assuming 250- 300 base average read-lengths.
These purified DNA samples were then sequenced using the multiplex DNA sequencing based on chemical degradation methods (Church G.M. and Kieffer-Higgins S., Science 240: 185-188, 1988) or by Sequithrem (Epicenter Technologies) dideoxy sequencing protocols. The sequencing reactions were electrophoresed and transferred onto nylon membranes by direct transfer electrophoresis from 40 cm gels (Richterich P. and Church G.M., Methods in Enzymology 218:187-222, 1993) or by electroblotting (Church, supra). 24 samples were run per gel. 45 successful membranes were produced by chemical sequencing and 8 were produced by dideoxy sequencing. The DNA was covalently bound to the membranes by exposure to ultraviolet light, and hybridized with labeled
oligonucleotides complimentary to tag sequences on the vectors (Church, supra). The membranes were washed to rinse off non-specifically bound probe, and exposed to X-ray film to visualize individual sequence ladders. After autoradiography, the hybridized probe was removed by incubation at 65° C, and the hybridization cycle repeated with another tag sequence until the membrane had been probed 38 times for chemical sequencing
membranes and 10 times for the dideoxy sequencing membranes. Thus, each gel produced a large number of films, each containing new sequencing information. Whenever a new blot was processed, it was initially probed for an internal standard sequence added to each of the pools.
Digital images of the films were generated using a laser-scanning densitometer (Molecular Dynamics, Sunnyvale, CA). The digitized images were processed on computer workstations (VaxStation 4000's) using the program REPLICA™ (Church et al.,
Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994).
Image processing included lane straightening, contrast adjustment to smooth out intensity differences, and resolution enhancement by iterative gaussian deconvolution. The sequences were then automatically picked in REPLICA™ and displayed for interactive proofreading before being stored in a project database. The proofreading was accomplished by a quick visual scan of the film image followed by mouse clicks on the bands of the displayed image to modify the base calls. Many of the sequence errors could be detected and corrected because multiple sequence reads covering the same portion of the genomic DNA provide adequate sequence redundancy for editing. Each sequence automatically received an identification number (corresponding to microtiter plate, probe information, and lane set number). This number serves as a permanent identifier of the sequence so it is always possible to identify the original of any particular sequence without recourse to a specialized database.
Routine assembly of H. pylori sequences was done using the program FALCON (Church, Church et al., Automated DNA Sequenicng and Analysis (J.C. Venter, ed.), Academic Press, 1994). This program has proven to be fast and reliable for most sequences. The assembled contigs were displayed using a modified version of
GelAssemble, developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICA™. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICA™ database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly.
II. Identification, cloning and expression of recombinant H. pylori DNA sequences
To facilitate the cloning, expression and purification of membrane and secreted proteins from H. pylori a powerful gene expression system, the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, was selected. Also, a DNA sequence encoding a peptide tag, the Ηis-Tag, was fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence. The exception to the above was ppiB, a gene cloned for use as a control in the expression studies. In this study, the sequence for H. pylori ppiB contains a DNA sequence encoding a Ηis-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein.
PCR Amplification and cloning of DNA sequences containing ORF's for membrane and secreted proteins from the J99 Strain of Helicobacter pylori.
Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers (Table 4) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an Ncol cloning site at the extreme 5' terminus, except for ΗpSeq. 4821082 (SEQ ID NO: 820) where Ndel was used. These primers were designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native H. pylori DNA sequence. An exception is H. pylori sequence 4821082 (SEQ ID NO: 820) where the initiator methionine is immediately followed by the remainder of the native H. pylori DNA sequence. All reverse primers (specific for the 3' end of any H. pylori ORF) included a EcoRI site at the extreme 5' terminus to permit cloning of each H. pylori sequence into the reading frame of the pET-28b. The pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids (only 19 amino acids in ΗpSeq. 26380318 (SEQ ID NO: 658)and ΗpSeq.14640637 (SEQ ID NO: 447)) including six histidine residues (at the extreme C-terminus), which comprise the Ηis-Tag. An exception to the above, as noted earlier, is the vector construction for the ppiB gene. A synthetic oligonucleotide primer specific for the 5' end of ppiB gene encoded a BamΗI site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encoded a Xhol site at its extreme 5' terminus.
Figure imgf000095_0001
Genomic DNA prepared from the J99 strain of H. pylori (ATCC #55679) was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Sequences 26054702 (SEQ ID NO: 649), 7116626 (SEQ ID NO: 865), 29479681 (SEQ ID NO: 677), 30100332 (SEQ ID NO: 685), 4821082 (SEQ ID NO: 820) and 978477 (SEQ ID NO: 880);
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min
23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes.
Sequence 16225006 (SEQ ID NO: 465);
Denaturation at 94°C for 2 min,
25 cycles at 95°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min
Reaction was concluded at 72°C for 6 minutes.
Sequence 4721061 (SEQ ID NO: 812);
Denaturation at 94°C for 2 min.
2 cycles at 94°C for 15 sec, 36°C for 15 sec and 72°C for 1.5 min
23 cycles at 94°C for 15 sec. 60°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes.
Sequence 26380318 (SEQ ID NO: 658);
Denaturation at 94°C for 2 min.
2 cycles at 94°C for 15 sec. 38°C for 15 sec and 72°C for 1.5 min
23 cycles at 94°C for 15 sec, 62°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes. Sequence 14640637 (SEQ ID NO: 447);
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 33°C for 15 sec and 72°C for 1.5 min
30 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes.
Conditions for amplification of H. pylori ppiB;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 32°C for 15 sec and 72°C for 1.5 min
25 cycles at 94°C for 15 sec, 56°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes
Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, Ncol and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of ΗpSeq. 4821082 (SEQ ID NO: 820), with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA)
Cloning of H. pylori DNA sequences into the pET-28b prokaryotic expression vector.
The pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case of H pylori sequence 4821082 (SEQ ID NO: 820) with Ndel and EcoRI
(Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). In the case of cloning ppiB, the pET-28a vector, which encodes a Ηis-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with BamΗI and Xhol restriction endonucleases.
Following digestion, DNA inserts were cloned (Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB, which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular. John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation reaction was mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 milliliters SOC medium (0.5%) yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
Identification of recombinant pET expression plasmids carrying H. pylori sequences
Individual BL21 clones transformed with recombinant pET-28b-H.pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the expression vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
Isolation and Preparation of plasmid DNA from BL21 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs were picked and incubated in 5 mis of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA). Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacI gene, the lacUN5 promoter and the gene for T7 RΝA polymerase. T7 RΝA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RΝA polymerase transcribes any target plasmid, such as pET-28b, carrying a T7 promoter and a gene of interest. Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 nanograms of plasmid DΝA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Νovagen as part of the pET expression system kit). The lacZ gene (beta-galactosidase) was expressed in the pET-System as described for the H. pylori recombinant constructions. Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 milliliters of cold 10 mM Tris-ΗCl, pΗ 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification.
III. Purification of recombinant proteins from E. coli
Analytical Methods
The concentrations of purified protein preparations were quantified
spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.Η., Rosebrough, N., Fair, A.L. & Randall, R.J. (1951) J. Biol. Chem. 193, pages 265-275, using bovine serum albumin as a standard.
SDS-polyacrylamide gels (12% or 4.0 to 25 % acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
I. Purification of soluble proteins
All steps were carried out at 4°C. Frozen cells were thawed, resuspended in 5 volumes of lysis buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole with 10% glycerol, 0.1 % 2-mercaptoethanol. 200 μg/ ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each of leupeptin, aprotinin, pepstatin, L-1-chloro-3-[4- tosylamido]-7-amino-2-heptanone (TLCK), L-1-chloro-3-[4-tosylamido]-4-phenyl-2- butanone (TPCK), and soybean trypsin inhibitor, and ruptured by several passages through a small volume micro fluidizer (Model M-110S, Microfluidics International Corporation, Newton, MA). The resultant homogenate was made 0.1 % Brij 35, and centrifuged at 100,000 x g for 1 hour to yield a clear supernatant (crude extract).
Following filtration through a 0.8 μm Supor filter (Gelman Sciences, FRG) the crude extract was loaded directly onto a Ni2+- nitrilotriacetate-agarose (NT A) with a 5 milliliter bed volume (Hochuli, E., Dbeli, H., and Schacheer, A. (1987) J. Chromatography 411, 177-184) pre-equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and was eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
Recombinant protein 14640637 (SEQ ID NO: 447) and proteins, beta-galactosidase (lacZ) and peptidyl-prolyl cis-trans isomerase (ppiB)
Fractions containing the recombinant proteins from the Ni2+-NTA-agarose columns were pooled and then concentrated to approximately 5 ml by centrifugal filtration
(Centriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 91 cm) of Sephacryl S-100 HR gel filtration medium equilibrated in Buffer A (10 mM Hepes, pH 7.5, 150 mM NaCl, 0.1 mM EGTA) and run in Buffer A at 18 ml/h. Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled and concentrated by centrifugal filtration.
Recombinant protein 7116626 (SEQ ID NO: 865)
Fractions containing the recombinant protein from the Ni2+ -NTA-agarose column were pooled and dialyzed overnight against 1 liter of dialysis buffer (10 mM MOPS, pH 6.5, 50 mM NaCl, 0.1 mM EGTA, 0.02% Brij 35 and 1 mM PMSF). In the morning, a fine white precipitate was removed by centrifugation and the resulting supernatant was loaded onto an 8 ml (8 x 75 mm) MonoS high performance liquid chromatography column (Pharmacia Biotechnology, Inc., Piscataway, NJ, USA) equilibrated in buffer B (10 mM
MOPS, pH 6.5, 0.1 mM EGTA) containing 50 mM NaCl. The column was washed with 10 bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM). Recombinant protein 7116626 (SEQ ID NO: 865) eluted as a sharp peak at 300 mM NaCl.
2. Purification of insoluble proteins from inclusion bodies
The following steps were carried out at 4°C. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 μg/ ml lysozyme, 5 mM EDTA, ImM PMSF and 0.1 % - mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was made 0.2 % deoxycholate, stirred 10 minutes, then centrifuged at 20,000 x g, for 30 min. The pellets were washed with lysis buffer containing 10 % glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1 % -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol. The resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials..
Recombinant proteins 26054702 (SEQ ID NO: 649), 16225006 (SEQ ID NO: 465), 30100332 (SEQ ID NO: 685), 4721061 (SEQ ID NO: 812)
The following steps were carried out at room temperature. Purified inclusion bodies were dissolved in 20 ml 8.0 M urea in lysis buffer with 1 mM PMSF and 0.1 % 2- mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation. The clear supernatant was filtered, then loaded onto a Ni2+ -NTA agarose column pre-equilibrated in 8.0 M urea in Lysis Buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
Recombinant proteins 29479681 (SEQ ID NO: 677), 978477 (SEQ ID NO: 880), 26380318 (SEQ ID NO: 658)
The pellet containing the inclusion bodies was solubilized in buffer B containing 8
M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol, and incubated for 1 hour at room temperature. Insoluble materials were removed by centrifugation at 20,000 x g for 30 min, and the cleared supernatant was loaded onto a 15 ml ( 1.6 x 7.5 cm ) SP-Sepharose column pre-equilibrated in buffer B, 6 M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol. After washing the column with 10 bed volumes, the column was developed with a linear gradient from 0 to 500 mM NaCl.
Dialysis and concentration of protein samples
Urea was removed slowly from the protein samples by dialysis against Tris- buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M, 4M, 3M, 2M, 1M, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.
After dialysis, samples were concentrated by pressure filtration using Amicon stirred-cells. Protein concentrations were measured using the methods of Perkins (1986
Eur. J. Biochem. 157, 169-180), Bradford ((1976) Anal. Biochem. 72, 248-254) and Lowry ((1951) J. Biol. Chem. 193, pages 265-275).
The recombinant proteins purified by the methods described above are summarized in Table 5 below.
Figure imgf000102_0001
Figure imgf000103_0001
IV. Analysis of H. pylori proteins as Vaccine candidates
To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H. pylori model was used. This model mimics the human H. pylori infection in many respects. The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immunotherapy.
Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding center
(Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
Infection
After a minimum of one week of acclimatization, the animals were infected with a type 2 strain (VacA negative) of H. pylori (strain 244, originally isolated from an ulcer patient). In our hands, this strain has earlier proven to be a good colonizer of the mouse stomach. The bacteria were grown overnight in Brucella broth supplemented with 10 % fetal calf serum, at 37°C in a microaerophilic atmosphere (10% CO2, 5%O2). The animals were given an oral dose of omeprazole (400 μmol/kg) and 3-5 h after this an oral inoculation of H. pylori in broth (approximately 108 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation.
Antigens
Recombinant H. pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa-ΗIS tag for purification reasons and the non-Helicobacter pylori control protein (β-galactosidase from E. coli; LacZ), was constructed in the same way.
All antigens were given in a soluble form, i.e. dissolved in either a ΗΕPΕS buffer or in a buffer containing 0.5% Deoxycholate (DOC).
The antigens are listed in Table 6 below.
Figure imgf000104_0001
Periplastic/Secreted proteins
SEQ ID NO:685 Other cell envelope proteins
SEQ ID NO:820
SEQ ID NO:880
Flagella-associated proteins
SEQ ID NO:658
Control proteins
β-galactosidase (LacZ) Immunizations
Ten animals in each group were immunized 4 times over a 34 day period (day 1, 15, 25 and 35). Purified antigens in solution or suspension were given at a dose of 100 μg/mouse. As an adjuvant, the animals were also given 10 μg/mouse of Cholera toxin (CT) with each immunization. Omeprazole (400 μmol/kg) was given orally to the animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Infected control animals received HEPES buffer + CT or DOC buffer + CT. Animals were sacrificed 2-4 weeks after final immunization. A general outline of the study is shown in Table 7 below.
Figure imgf000105_0001
Analysis of infection
Mucosal infection: The mice were sacrificed by CO2 and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. The mucosa from the antrum and corpus of an area of 25mm2 was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
The urease test was performed essentially as follows. The reagent, Urea Agar Base
Concentrate, was purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61-3).
Urea agar base concentrate was diluted 1:10 with water. 1 ml of if the diluted concentrate was mixed with 100-200 μl of actively growing H. pylori cells. Color change to magenta indicated that cells were urease positive.
The catalase test was performed essentially as follows. The reagent, N,N,N',N'-
Tetramethyl-p-Phenylenediamine, was purchased from Sigma, St. Louis, MO (Catalog #
T3134). A solution of the regent (1% w/v in water) was prepared. H. pylori cells were swabbed onto Whatman filter paper and overlaid with the 1% solution. Color change to dark blue indicated that the cells were catalase positive.
Serum antibodies: From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.
Mucosal antibodies: Gentle scrapings of a defined part of the corpus and of 4 cm of duodenum were performed in 50% of the mice in order to detect the presence of antibodies in the mucous. The antibody titers were determined by regular ELISA technique as for serum antibodies.
Statistical analysis: Wilcoxon-Mann-Whitney sign rank test was used for determination of significant effects of the antigens on Helicobacter pylori colonization.
P<0.05 was considered significant. Because the antrum is the major colonization site for Helicobacter most emphasis was put upon changes in the antral colonization.
Results
Antibodies in sera: All antigens tested given together with CT gave rise to a measurable specific titer in serum. The highest responses were seen with SEQ ID
NOs:865, 812, 658, 447, and 820 (see Figure 1).
Antibodies in mucus: In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with SEQ ID NOs:685, followed by 447, 865, and 658 (see Figure 2). Therapeutic immunization effects:
All control animals (B ALB/c mice) were well colonized with H. pylori (strain AΗ244) in both antrum and corpus of the stomach. Of the antigens tested 3 proteins (SEQ ID NOs: 812, 820, and 447) gave a good and significant reduction and/or eradication of the H. pylori infection. The degree of colonization of the antrum was lower following immunization with SEQ ID NOs: 880, 658, and 865 compared to control. The effect of SEQ ID NOs:465, 677, and 685 did not differ from control. The control protein lacZ, i.e. the non-H. pylori protein, had no eradication effect and in fact had higher Helicobacter colonization compared to the ΗEPES + CT control. All data are shown in Figures 3 and 4 for proteins dissolved in ΗEPES and DOC respectively. Data is shown as geometric mean values. n=8-10 Wilcoxon-Mann-Whitney sign rank test * = p<0.05; x/10 = number of mice showing eradication of H. pylori over the total number of mice examined.
The data presented indicate that all of the H. pylori associated proteins included in this study, when used as oral immunogens in conjunction with the oral adjuvant CT, resulted in stimulation of an immune response as measured by specific serum and mucosal antibodies. A majority of the proteins led to a reduction, and in some cases complete clearance of the colonization of H. pylori in this animal model. It should be noted that the reduction or clearance was due to heterologous protection rather than homologous protection (the polypeptides were based on the H. pylori J99 strain sequence and used in the therapeutic immunization studies against a different (AΗ244) challenge strain), indicating the vaccine potential against a wide variety of H. pylori strains.
The highest colonization in the antrum was seen in animals treated with the non- Helicobacter protein LacZ, indicating that the effects seen with the Helicobacter pylori antigens were specific.
Taken together these data strongly support the use of these H. pylori proteins in a pharmaceutical formulation for the use in humans to treat and/or prevent H. pylori infections.
V. Sequence Variance Analysis of genes in Helicobacter pylori strains
Four genes were cloned and sequenced from several strains of H. pylori to compare the DNA and deduced amino acid sequences. This information was used to determine the sequence variation between the H. pylori strain, J99, and other H. pylori strains isolated from human patients. Preparation of Chromosomal DNA.
Cultures of H. pylori strains (as listed in Table 10) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD600 of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mis of 10 mM Tris-HCl, 0.1 mM EDTA (TE). Lysozyme was added to a final concentration of 1 mg/ml along with, SDS to 1% and RNAse A + T1 to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55 C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1% CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes. At this point, the samples were extracted with chloroform:isoamyl alcohol, extracted with phenol, and extracted again with chloroform:isoamyl alcohol. DNA was precipitated with either EtOH (1.5 x volumes) or isopropanol (0.6 x volumes) at -70°C for 10minutes, washed in 70% EtOH and resuspended in TE.
PCR Amplification and cloning.
Genomic DNA prepared from twelve strains of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (10 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers
(forward and reverse primers, see Table 8) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 0.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc.,
Branchburg, NJ, USA) in a final volume of 20 microliters in duplicate reactions.
Figure imgf000108_0001
The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Sequences (by SEQ ID NO:) 865 and 764;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min
23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes.
Sequence (by SEQ ID NO:) 649 for strains AH5, 5155, 7958, AH24,and J99;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min
25 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min
Reaction was concluded at 72°C for 6 minutes.
Sequences (by SEQ ID NO:) 677 and 649 for strains AH4, AH15, AH61, 5294, 5640, AH18, and Hp244 ;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 20 sec and 72°C for 2 min
25 cycles at 94°C for 15 sec, 55°C for 20 sec and 72°C for 2 min
Reactions were concluded at 72°C for 8 minutes.
Upon completion of thermal cycling reactions, each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
Cloning of H. pylori DNA sequences into the pCR TA cloning vector.
All amplified inserts were cloned into the pCR 2.1 (pCRII in the case of H. pylori sequence 865) vector by the method described in the Original TA cloning kit (Invitrogen, San Diego, CA). Products of the ligation reaction were then used to transform the
TOP10F' (INVaF' in the case of Η. pylori sequence 865) strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria. E coli strain TOP10F' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Briefly, 2 microliters of 0.5 micromolar BME was added to each vial of 50 microliters of competent cells. Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes. The cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of TOPI OF' or INVaF' were then picked and analyzed to evaluate cloned inserts as described below.
Identification of recombinant PCR plasmids carrying H. pylori sequences
Individual TOP10F' or INVaF' clones transformed with recombinant pCR-H.pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the cloning vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
Individual clones of recombinant pCR vectors carrying properly cloned H. pylori ORFs were picked for sequence analysis. Sequence analysis was performed on ABI Sequencers using standard protocols (Perkin Elmer) using vector-specific primers (as found in PCRII or pCR2.1 , Invitrogen, San Diego, CA) and sequencing primers specific to the ORF as listed in Table 9 below.
Figure imgf000111_0001
Results
To establish the PCR error rate in these experiments, five individual clones of SEQ ID NO:649, prepared from five separate PCR reaction mixtures from Η. pylori strain J99, were sequenced over a total length of 897 nucleotides for a cumulative total of 4485 bases of DNA sequence. DNA sequence for the five clones was compared to the DNA sequence of SEQ ID NO:649 obtained previously by a different method, i.e., random shotgun cloning and sequencing. The PCR error rate for the experiments described herein was determined to be 2 base changes out of 4485 bases, which is equivalent to an estimated error rate of less than or equal to 0.04%. DNA sequence analysis was performed on four different open reading frames identified as genes and amplified by PCR methods from a dozen different strains of the bacterium Helicobacter pylori. The deduced amino acid sequences of three of the four open reading frames that were selected for this study showed statistically significant BLAST homology to defined proteins present in other bacterial species. Those ORFs included: SEQ ID NO:649, homologous to the val A & B genes encoding an ABC transporter in F. novicida; SEQ ID NO:865, homologous to lipoprotein e (P4) present in the outer membrane of H. influenzae; SEQ ID NO:677, homologous to fecA, an outer membrane receptor in iron (III) dicitrate transport in E. coli. SEQ ID NO: 764 was identified as an unknown open reading frame, because it showed low homology with sequences in the public databases.
To assess the extent of conservation or variance in the ORFs across various strains of H. pylori, changes in DNA sequence and the deduced protein sequence were compared to the DNA and deduced protein sequences found in the J99 strain of H. pylori (see Table 10 below). Results are presented as percent identity to the J99 strain of H. pylori sequenced by random shotgun cloning. To control for any variations in the J99 sequence each of the four open reading frames were cloned and sequenced again from the J99 bacterial strain and that sequence information was compared to the sequence information that had been collected from inserts cloned by random shotgun sequencing of the J99 strain. The data demonstrate that there is variation in the DNA sequence ranging from as little as 0.12 % difference (SEQ ID NO:764, J99 strain) to approximately 7% change (SEQ ID NO:649, strain AΗ5). The deduced protein sequences show either no variation (SEQ ID NO:764, strains AH18 and AH24) or up to as much as 7.66% amino acid changes (SEQ ID NO:649, Strain AH5).
Figure imgf000113_0001
VI. Experimental Knock-Out Protocol for the Determination of Essential H. pylori Genes as Potential Therapeutic Targets
Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.
The protocol for the deletion of portions of H. pylori genes/ORFs and the insertional mutagenesis of a kanamycin-resistance cassette in order to identify genes which are essential to the cell is modified from previously published methods (Labigne-Roussel et al, 1988, J. Bacteriology 170, pp. 1704-1708; Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573; Reyrat et al., 1995, Proc. Natl. Acad. Sci. 92, pp 8768-8772). The result is a gene "knock-out."
Identification and Cloning of H. pylori Gene Sequences
The sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Plymouth, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame. Genomic DNA prepared from the Helicobacter pylori HpJ99 strain (ATCC 55679) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). For the preparation of genomic DNA from H. pylori, see Example I. PCR amplification is carried out by introducing 10 nanograms of genomic
HpJ99 DNA into a reaction vial containing 10 mM Tris pH 8.3, 50 mM KCl. 2 mM MgCl2, 2 microMolar synthetic oligonucleotide primers (forward=F1 and reverse=R1), 0.2 mM of each deoxynucleotide triphosphate (dATP,dGTP, dCTP, dTTP), and 1.25 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 40 microliters. The PCR is carried out with Perkin Elmer
Cetus/GeneAmp PCR System 9600 thermal cyclers.
Upon completion of thermal cycling reactions, each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction.
Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
PCR products are cloned into the pT7Blue T- Vector (catalog#69820-1, Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
Ligation products are electroporated (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) into electroporation-competent XL-1 Blue or DH5-α E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA). Briefly, 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) at 37°C with shaking for 1 hour. Samples are then spread onto LB (10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/l sodium chloride) plates containing 100 microgram/ml of Ampicillin, 0.3% X-gal, and 100 microgram/ml IPTG. These plates are incubated overnight at 37°C. Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA). To verify that the correct H.pylori DNA inserts had been cloned, these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 H.pylori sequence. Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned. Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage. To minimize errors due to PCR, plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.
The sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other. The pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product. The PCR product is a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, resultes in the deletion of a portion of the ORFs. The PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified.
A Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology 170,
1704-1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid
pCTB8:kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573). The proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA). The fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes. This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides. The "T" overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pΗ 8.3, 50 mM KCl, 2 mM MgCl2, 5 units of DNA Polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP; in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C. The "Kan-T" cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA). The PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter 10x T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16°C.
The ligation products are transformed into XL-1 Blue or DH5-α E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
Several tests by PCR amplification are conducted on these plasmids to verify that the Kanamycin is inserted in the H. pylori gene/ORF, and to determine the orientation of the insertion of the Kanamycin-resistance gene relative to the H. pylori gene/ORF. To verify that the Kanamycin cassette is inserted into the H pylori sequence, the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the H. pylori gene/ORFs. The correct PCR product is the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. To avoid potential polar effects of the kanamycin resistance cassette on H. pylori gene expression, the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in H. pylori
transformations (see below). To determine the orientation of insertion of the kanamycin resistance gene, primers are designed from the ends of the kanamycin resistance gene ("Kan-1" 5'-ATCTTACCTATCACCTCAAAT-3', and "Kan-2" 5'-
AGACAGCAACATCTTTGTGAA-3'). By using each of the cloning primers in conjunction with each of the Kan primers (4 combinations of primers), the orientation of the Kanamycin cassette relative to the H.pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the H.pylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H. pylori. Transformation of Plasmid DNA into H. pylori cells
Two strains of H. pylori are used for transformation: ATCC 55679. the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AH244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach. Cells for transformation are grown at 37°C, 10% CO2, 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated. If grown on plates, cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf microfuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth. An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells. An aliquot (1 to 5 OD600 units/25 microliter) of the resuspended cells is placed onto a prewarmed Sheep-Blood agar plate, and the plate is further incubated at 37°C, 6% CO2, 100% humidity for 4 hours. After this incubation, 10 microliters of plasmid DNA (100 micrograms per microliter) is spotted onto these cells. A positive control (plasmid DNA with the ribonuclease H gene disrupted by kanamycin resistance gene) and a negative control (no plasmid DNA) are done in parallel. The plates are returned to 37°C, 6% CO2 for an additional 4 hours of incubation. Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO2. Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C, 6% CO2, 100% humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep- Blood agar plate containing 25 micrograms/ml Kanamycin.
Three sets of PCR tests are done to verify that the colonies of transformants have arisen from homologous recombination at the proper chromosomal location. The template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1:1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
TEST 1. PCR with cloning primers originally used to amplify the gene/ORF. A positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. A PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.
TEST 2. PCR with F3 (primer designed from sequences upstream of the gene/ORF and not present on the plasmid), and either primer Kan-1 or Kan-2 (primers designed from the ends of the kanamycin resistance gene), depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
TEST 3. PCR with R3 (primer designed from sequences downstream of the gene/ORF and not present on the plasmid) and either primer Kan-1 or Kan-2, depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3). Again, no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
Transformants showing positive results for all three tests above indicate that the gene is not essential for survival in vitro.
A negative result in any of the three above tests for each transformant indicates that the gene had not been disrupted, and that the gene is essential for survival in vitro.
In the event that no colonies result from two independent transformations while the positive control with the disrupted ribonuclease H plasmid DNA produces transformants, the plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3. Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST 2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation VII. High-throughput drug screen assay
Cloning, expression and protein purification
Cloning, transformation, expression and purification of the H. pylori target gene and its protein product,e.g., an H. pylori enzyme, to be used in a high-throughput drug screen assay, is carried out essentially as described in Examples II and III above. Development and application of a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase, is described below as a specific example. Enzymatic Assay
The assay is essentially as described by Fisher (Fischer, G., etal. (1984) Biomed. Biochim. Acta 43:1101-1111). The assay measures the cis-trans isomerization of the Ala- Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S-7388, lot # 84H5805). The assay is coupled with α-chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans. The conversion of the test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second. Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 μM α-chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction, 10 μl of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 μl of reaction mixture at room temperature. Enzymatic assay in crude bacterial extract.
A 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD 600 nm ~ 1) and resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 μg/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor. The suspension is subjected to 3 cycles of freeze- thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts). The lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
Many H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.
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Figure imgf000253_0003
Figure imgf000253_0004
Figure imgf000253_0005
Figure imgf000254_0001
Figure imgf000254_0002
Figure imgf000254_0003
Figure imgf000254_0004
Figure imgf000255_0001
Figure imgf000255_0002
Figure imgf000255_0003
Figure imgf000255_0004
Figure imgf000256_0001
Figure imgf000256_0002
Figure imgf000256_0003
Figure imgf000256_0004
Figure imgf000256_0005
Figure imgf000257_0001
Figure imgf000257_0002
Figure imgf000257_0003
Figure imgf000257_0004
Figure imgf000257_0005
Figure imgf000258_0001
Figure imgf000258_0002
Figure imgf000258_0003
Figure imgf000258_0004
Figure imgf000259_0001
Figure imgf000259_0002
Figure imgf000259_0003
Figure imgf000259_0004
Figure imgf000260_0001
Figure imgf000260_0002
Figure imgf000260_0003
Figure imgf000260_0004
Figure imgf000260_0005
Figure imgf000261_0001
Figure imgf000261_0002
Figure imgf000261_0003
Figure imgf000261_0004
Figure imgf000262_0001
Figure imgf000262_0002
Figure imgf000262_0003
Figure imgf000262_0004
Figure imgf000262_0005
Figure imgf000263_0001
Figure imgf000263_0002
Figure imgf000263_0003
Figure imgf000263_0004
Figure imgf000264_0001
Figure imgf000264_0002
Figure imgf000264_0003
Figure imgf000264_0004
Figure imgf000265_0001
Figure imgf000265_0002
Figure imgf000265_0003
Figure imgf000265_0004
Figure imgf000265_0005
Figure imgf000266_0001
Figure imgf000266_0002
Figure imgf000266_0003
Figure imgf000266_0004
Figure imgf000267_0001
Figure imgf000267_0002
Figure imgf000267_0003
Figure imgf000267_0004
Figure imgf000267_0005
Figure imgf000268_0001
Figure imgf000268_0002
Figure imgf000268_0003
Figure imgf000268_0004
Figure imgf000268_0005
Figure imgf000269_0001
Figure imgf000269_0002
Figure imgf000269_0003
Figure imgf000269_0004
Figure imgf000270_0001
Figure imgf000270_0002
Figure imgf000270_0003
Figure imgf000270_0004
Figure imgf000270_0005
Figure imgf000271_0001
Figure imgf000271_0002
Figure imgf000271_0003
Figure imgf000271_0004
Figure imgf000271_0005
Figure imgf000272_0001
Figure imgf000272_0002
Figure imgf000272_0003
Figure imgf000272_0004
Figure imgf000272_0005
Figure imgf000273_0001
Figure imgf000273_0002
Figure imgf000273_0003
Figure imgf000273_0004
Figure imgf000274_0001
Figure imgf000274_0002
Figure imgf000274_0003
Figure imgf000274_0004
Figure imgf000275_0001
Figure imgf000275_0002
Figure imgf000275_0003
Figure imgf000275_0004
Figure imgf000276_0001
Figure imgf000276_0002
Figure imgf000276_0003
Figure imgf000276_0004
Figure imgf000276_0005
Figure imgf000277_0001
Figure imgf000277_0002
Figure imgf000277_0003
Figure imgf000277_0004
Figure imgf000278_0001
Figure imgf000278_0002
Figure imgf000278_0003
Figure imgf000278_0004
Figure imgf000278_0005
Figure imgf000279_0001
Figure imgf000279_0002
Figure imgf000279_0003
Figure imgf000279_0004
Figure imgf000280_0001
Figure imgf000280_0002
Figure imgf000280_0003
Figure imgf000280_0004
Figure imgf000281_0001
Figure imgf000281_0002
Figure imgf000281_0003
Figure imgf000281_0004
Figure imgf000282_0001
Figure imgf000282_0002
Figure imgf000282_0003
Figure imgf000282_0004
Figure imgf000283_0001
Figure imgf000283_0002
Figure imgf000283_0003
Figure imgf000283_0004
Figure imgf000284_0001
Figure imgf000284_0002
Figure imgf000284_0003
Figure imgf000284_0004
Figure imgf000285_0001
Figure imgf000285_0002
Figure imgf000285_0003
Figure imgf000285_0004
Figure imgf000286_0001
Figure imgf000286_0002
Figure imgf000286_0003
Figure imgf000286_0004
Figure imgf000286_0005
Figure imgf000287_0001
Figure imgf000287_0002
Figure imgf000287_0003
Figure imgf000287_0004
Figure imgf000287_0005
Figure imgf000288_0001
Figure imgf000288_0002
Figure imgf000288_0003
Figure imgf000288_0004
Figure imgf000288_0005
Figure imgf000289_0001
Figure imgf000289_0002
Figure imgf000289_0003
Figure imgf000289_0004
Figure imgf000289_0005
Figure imgf000290_0001
Figure imgf000290_0002
Figure imgf000290_0003
Figure imgf000290_0004
Figure imgf000291_0001
Figure imgf000291_0002
Figure imgf000291_0003
Figure imgf000291_0004
Figure imgf000292_0001
Figure imgf000292_0002
Figure imgf000292_0003
Figure imgf000292_0004
Figure imgf000292_0005
Figure imgf000293_0001
Figure imgf000293_0002
Figure imgf000293_0003
Figure imgf000293_0004
Figure imgf000293_0005
Figure imgf000294_0001
Figure imgf000294_0002
Figure imgf000294_0003
Figure imgf000295_0001
Figure imgf000295_0002
Figure imgf000295_0003
Figure imgf000295_0004
Figure imgf000295_0005
Figure imgf000296_0001
Figure imgf000296_0002
Figure imgf000296_0003
Figure imgf000296_0004
Figure imgf000296_0005
Figure imgf000297_0001
Figure imgf000297_0002
Figure imgf000297_0003
Figure imgf000297_0004
Figure imgf000298_0001
Figure imgf000298_0002
Figure imgf000298_0003
Figure imgf000298_0004
Figure imgf000298_0005
Figure imgf000299_0001
Figure imgf000299_0002
Figure imgf000299_0003
Figure imgf000299_0004
Figure imgf000300_0001
Figure imgf000300_0002
Figure imgf000300_0003
Figure imgf000300_0004
Figure imgf000300_0005
Figure imgf000301_0001
Figure imgf000301_0002
Figure imgf000301_0003
Figure imgf000301_0004
Figure imgf000301_0005
Figure imgf000302_0001
Figure imgf000302_0002
Figure imgf000302_0003
Figure imgf000302_0004
Figure imgf000302_0005
Figure imgf000303_0001
Figure imgf000303_0002
Figure imgf000303_0003
Figure imgf000304_0001
Figure imgf000304_0002
Figure imgf000304_0003
Figure imgf000304_0004
Figure imgf000305_0001
Figure imgf000305_0002
Figure imgf000305_0003
Figure imgf000306_0001
Figure imgf000306_0002
Figure imgf000306_0003
Figure imgf000306_0004
Figure imgf000307_0001
Figure imgf000307_0002
Figure imgf000307_0003
Figure imgf000307_0004
Figure imgf000307_0005
Figure imgf000308_0001
Figure imgf000308_0002
Figure imgf000308_0003
Figure imgf000308_0004
Figure imgf000308_0005
Figure imgf000309_0001
Figure imgf000309_0002
Figure imgf000309_0003
Figure imgf000309_0004
Figure imgf000309_0005
Figure imgf000310_0001
Figure imgf000310_0002
Figure imgf000310_0003
Figure imgf000310_0004
Figure imgf000310_0005
Figure imgf000311_0001
Figure imgf000311_0002
Figure imgf000311_0003
Figure imgf000311_0004
Figure imgf000312_0001
Figure imgf000312_0002
Figure imgf000312_0003
Figure imgf000312_0004
Figure imgf000313_0001
Figure imgf000313_0002
Figure imgf000313_0003
Figure imgf000313_0004
Figure imgf000313_0005
Figure imgf000314_0001
Figure imgf000314_0002
Figure imgf000314_0003
Figure imgf000314_0004
Figure imgf000314_0005
Figure imgf000315_0001
Figure imgf000315_0002
Figure imgf000315_0003
Figure imgf000315_0004
Figure imgf000315_0005
Figure imgf000316_0001
Figure imgf000316_0002
Figure imgf000316_0003
Figure imgf000316_0004
Figure imgf000316_0005
Figure imgf000317_0001
Figure imgf000317_0002
Figure imgf000317_0003
Figure imgf000317_0004
Figure imgf000318_0001
Figure imgf000318_0002
Figure imgf000318_0003
Figure imgf000319_0001
Figure imgf000319_0002
Figure imgf000319_0003
Figure imgf000320_0001
Figure imgf000320_0002
Figure imgf000320_0003
Figure imgf000321_0001
Figure imgf000321_0002
Figure imgf000321_0003
Figure imgf000321_0004
Figure imgf000321_0005
Figure imgf000322_0001
Figure imgf000322_0002
Figure imgf000322_0003
Figure imgf000322_0004
Figure imgf000323_0001
Figure imgf000323_0002
Figure imgf000324_0001
Figure imgf000324_0002
Figure imgf000324_0003
Figure imgf000325_0001
Figure imgf000325_0002
Figure imgf000325_0003
Figure imgf000325_0004
Figure imgf000326_0001
Figure imgf000326_0002
Figure imgf000326_0003
Figure imgf000326_0004
Figure imgf000327_0001
Figure imgf000327_0002
Figure imgf000327_0003
Figure imgf000327_0004
Figure imgf000328_0001
Figure imgf000328_0002
Figure imgf000328_0003
Figure imgf000328_0004
Figure imgf000328_0005
Figure imgf000329_0001
Figure imgf000329_0002
Figure imgf000330_0001
Figure imgf000330_0002
Figure imgf000330_0003
Figure imgf000331_0001
Figure imgf000331_0002
Figure imgf000331_0003
Figure imgf000331_0004
Figure imgf000332_0001
Figure imgf000332_0002
Figure imgf000332_0003
Figure imgf000333_0001
Figure imgf000333_0002
Figure imgf000333_0003
Figure imgf000333_0004
Figure imgf000333_0005
Figure imgf000334_0001
Figure imgf000334_0002
Figure imgf000334_0003
Figure imgf000335_0001
Figure imgf000335_0002
Figure imgf000335_0003
Figure imgf000335_0004
Figure imgf000336_0001
Figure imgf000336_0002
Figure imgf000336_0003
Figure imgf000337_0001
Figure imgf000337_0002
Figure imgf000337_0003
Figure imgf000338_0001
Figure imgf000338_0002
Figure imgf000338_0003
Figure imgf000339_0001
Figure imgf000339_0002
Figure imgf000339_0003
Figure imgf000339_0004
Figure imgf000340_0001
Figure imgf000340_0002
Figure imgf000340_0003
Figure imgf000340_0004
Figure imgf000341_0001
Figure imgf000341_0002
Figure imgf000341_0003
Figure imgf000342_0001
Figure imgf000342_0002
Figure imgf000342_0003
Figure imgf000342_0004
Figure imgf000343_0001
Figure imgf000343_0002
Figure imgf000343_0003
Figure imgf000343_0004
Figure imgf000344_0001
Figure imgf000344_0002
Figure imgf000344_0003
Figure imgf000344_0004
Figure imgf000345_0001
Figure imgf000345_0002
Figure imgf000345_0003
Figure imgf000346_0001
Figure imgf000346_0002
Figure imgf000346_0003
Figure imgf000346_0004
Figure imgf000347_0001
Figure imgf000347_0002
Figure imgf000347_0003
Figure imgf000348_0001
Figure imgf000348_0002
Figure imgf000348_0003
Figure imgf000348_0004
Figure imgf000349_0001
Figure imgf000349_0002
Figure imgf000349_0003
Figure imgf000349_0004
Figure imgf000350_0001
Figure imgf000350_0002
Figure imgf000350_0003
Figure imgf000350_0004
Figure imgf000350_0005
Figure imgf000351_0001
Figure imgf000351_0002
Figure imgf000351_0003
Figure imgf000351_0004
Figure imgf000352_0001
Figure imgf000352_0002
Figure imgf000352_0003
Figure imgf000352_0004
Figure imgf000352_0005
Figure imgf000353_0001
Figure imgf000353_0002
Figure imgf000353_0003
Figure imgf000354_0001
Figure imgf000354_0002
Figure imgf000354_0003
Figure imgf000354_0004
Figure imgf000355_0001
Figure imgf000355_0003
Figure imgf000355_0004
Figure imgf000355_0005
Figure imgf000356_0001
Figure imgf000356_0002
Figure imgf000356_0003
Figure imgf000356_0004
Figure imgf000357_0001
Figure imgf000357_0002
Figure imgf000357_0003
Figure imgf000357_0004
Figure imgf000358_0001
Figure imgf000358_0002
Figure imgf000358_0003
Figure imgf000359_0001
Figure imgf000359_0002
Figure imgf000359_0003
Figure imgf000360_0001
Figure imgf000360_0002
Figure imgf000360_0003
Figure imgf000361_0001
Figure imgf000361_0002
Figure imgf000361_0003
Figure imgf000361_0004
Figure imgf000362_0001
Figure imgf000362_0002
Figure imgf000362_0003
Figure imgf000362_0004
Figure imgf000363_0001
Figure imgf000363_0002
Figure imgf000363_0003
Figure imgf000363_0004
Figure imgf000364_0001
Figure imgf000364_0002
Figure imgf000364_0003
Figure imgf000364_0004
Figure imgf000364_0005
Figure imgf000365_0001
Figure imgf000365_0002
Figure imgf000365_0003
Figure imgf000365_0004
Figure imgf000366_0001
Figure imgf000366_0002
Figure imgf000366_0003
Figure imgf000366_0004
Figure imgf000366_0005
Figure imgf000367_0001
Figure imgf000367_0002
Figure imgf000367_0003
Figure imgf000368_0001
Figure imgf000368_0002
Figure imgf000368_0003
Figure imgf000368_0004
Figure imgf000369_0001
Figure imgf000369_0002
Figure imgf000369_0003
Figure imgf000369_0004
Figure imgf000370_0001
Figure imgf000370_0002
Figure imgf000370_0003
Figure imgf000370_0004
Figure imgf000371_0001
Figure imgf000371_0002
Figure imgf000371_0003
Figure imgf000371_0004
Figure imgf000372_0001
Figure imgf000372_0002
Figure imgf000372_0003
Figure imgf000372_0004
Figure imgf000372_0005
Figure imgf000373_0001
Figure imgf000373_0002
Figure imgf000373_0003
Figure imgf000373_0004
Figure imgf000374_0001
Figure imgf000374_0002
Figure imgf000374_0003
Figure imgf000374_0004
LE 26
Figure imgf000375_0001
Figure imgf000375_0002
Figure imgf000375_0003
Figure imgf000376_0001
Figure imgf000376_0002
Figure imgf000376_0003
Figure imgf000377_0001
Figure imgf000377_0002
Figure imgf000377_0003
Figure imgf000377_0004
Figure imgf000378_0001
Figure imgf000378_0002
Figure imgf000378_0003
Figure imgf000378_0004
Figure imgf000378_0005
Figure imgf000379_0001
Figure imgf000379_0002
Figure imgf000379_0003
Figure imgf000379_0004
Figure imgf000380_0001
Figure imgf000380_0002
Figure imgf000380_0003
Figure imgf000381_0001
Figure imgf000381_0002
Figure imgf000381_0003
Figure imgf000381_0004
Figure imgf000382_0001
Figure imgf000382_0002
Figure imgf000382_0003
Figure imgf000382_0004
Figure imgf000383_0001
Figure imgf000383_0002
Figure imgf000383_0003
Figure imgf000384_0001
Figure imgf000384_0002
Figure imgf000384_0003
Figure imgf000384_0004
Figure imgf000385_0001
Figure imgf000385_0002
Figure imgf000385_0003
Figure imgf000385_0004
Figure imgf000386_0005
Figure imgf000386_0001
Figure imgf000386_0002
Figure imgf000386_0003
Figure imgf000386_0004
Figure imgf000387_0001
Figure imgf000387_0002
Figure imgf000387_0003
Figure imgf000388_0001
Figure imgf000388_0002
Figure imgf000388_0003
Figure imgf000389_0001
Figure imgf000389_0002
Figure imgf000389_0003
Figure imgf000389_0004
Figure imgf000390_0001
Figure imgf000390_0002
Figure imgf000390_0003
Figure imgf000390_0004
Figure imgf000391_0001
Figure imgf000391_0002
Figure imgf000391_0003
Figure imgf000391_0004
Figure imgf000391_0005
Figure imgf000392_0001
Figure imgf000392_0002
Figure imgf000393_0001
Figure imgf000394_0001
Figure imgf000394_0002
Figure imgf000394_0003
Figure imgf000395_0001
Figure imgf000395_0002
Figure imgf000395_0003
Figure imgf000395_0004
Figure imgf000396_0001
Figure imgf000396_0002
Figure imgf000397_0001
Figure imgf000397_0002
Figure imgf000397_0003
Figure imgf000397_0004
Figure imgf000397_0005
Figure imgf000398_0001
Figure imgf000398_0002
Figure imgf000398_0003
Figure imgf000398_0004
Figure imgf000399_0001
Figure imgf000399_0002
Figure imgf000399_0003
Figure imgf000400_0001
Figure imgf000400_0002
Figure imgf000400_0003
Figure imgf000401_0001
Figure imgf000401_0002
Figure imgf000401_0003
Figure imgf000402_0001
Figure imgf000402_0002
Figure imgf000402_0003
Figure imgf000402_0004
Figure imgf000403_0001
Figure imgf000403_0002
Figure imgf000403_0003
Figure imgf000404_0001
Figure imgf000404_0002
Figure imgf000405_0001
Figure imgf000405_0002
Figure imgf000405_0003
Figure imgf000405_0004
Figure imgf000406_0001
Figure imgf000406_0002
Figure imgf000406_0003
Figure imgf000406_0004
Figure imgf000407_0001
Figure imgf000407_0002
Figure imgf000407_0003
Figure imgf000408_0001
Figure imgf000408_0002
Figure imgf000408_0003
Figure imgf000408_0004
Figure imgf000408_0005
Figure imgf000408_0006
Figure imgf000409_0001
Figure imgf000409_0002
Figure imgf000409_0003
Figure imgf000409_0004
Figure imgf000410_0001
Figure imgf000410_0002
Figure imgf000410_0003
Figure imgf000410_0004
Figure imgf000411_0001
Figure imgf000411_0002
Figure imgf000411_0003
Figure imgf000412_0001
Figure imgf000412_0002
Figure imgf000412_0003
Figure imgf000412_0004
Figure imgf000413_0001
Figure imgf000413_0002
Figure imgf000413_0003
Figure imgf000413_0004
Figure imgf000414_0001
Figure imgf000414_0002
Figure imgf000414_0003
Figure imgf000414_0004
Figure imgf000415_0001
Figure imgf000415_0002
Figure imgf000415_0003
Figure imgf000415_0004
Figure imgf000416_0001
Figure imgf000416_0002
Figure imgf000416_0003
Figure imgf000417_0001
Figure imgf000417_0002
Figure imgf000417_0003
Figure imgf000418_0001
Figure imgf000418_0002
Figure imgf000418_0003
Figure imgf000418_0004
Figure imgf000419_0001
Figure imgf000419_0002
Figure imgf000419_0003
Figure imgf000420_0001
Figure imgf000420_0002
Figure imgf000420_0003
Figure imgf000421_0001
Figure imgf000421_0002
Figure imgf000421_0003
Figure imgf000421_0004
Figure imgf000422_0001
Figure imgf000422_0002
Figure imgf000422_0003
Figure imgf000422_0004
Figure imgf000423_0001
Figure imgf000423_0002
Figure imgf000423_0003
Figure imgf000423_0004
Figure imgf000423_0005
Figure imgf000424_0001
Figure imgf000424_0002
Figure imgf000424_0003
Figure imgf000424_0004
Figure imgf000424_0005
Figure imgf000425_0001
Figure imgf000425_0002
Figure imgf000425_0003
Figure imgf000425_0004
Figure imgf000426_0001
Figure imgf000426_0002
Figure imgf000426_0003
Figure imgf000426_0004
Figure imgf000426_0005
Figure imgf000427_0001
Figure imgf000427_0002
Figure imgf000427_0003
Figure imgf000427_0004
Figure imgf000428_0001
Figure imgf000428_0002
Figure imgf000429_0001
Figure imgf000429_0002
Figure imgf000429_0003
Figure imgf000430_0001
Figure imgf000430_0002
Figure imgf000430_0003
Figure imgf000431_0001
Figure imgf000432_0001
Figure imgf000432_0002
Figure imgf000433_0001
Figure imgf000433_0002
Figure imgf000433_0003
Figure imgf000433_0004
Figure imgf000434_0001
Figure imgf000434_0002
Figure imgf000434_0003
Figure imgf000434_0004
Figure imgf000434_0005
Figure imgf000435_0001
Figure imgf000435_0002
Figure imgf000435_0003
Figure imgf000435_0004
Figure imgf000437_0001
Figure imgf000437_0002
Figure imgf000437_0003
Figure imgf000437_0004
Figure imgf000438_0001
Figure imgf000438_0002
Figure imgf000438_0003
Figure imgf000439_0001
Figure imgf000439_0002
Figure imgf000439_0003
Figure imgf000440_0001
Figure imgf000440_0002
Figure imgf000440_0003
Figure imgf000440_0004
Figure imgf000441_0001
Figure imgf000441_0002
Figure imgf000441_0003
Figure imgf000442_0001
Figure imgf000442_0002
Figure imgf000443_0001
Figure imgf000443_0002
Figure imgf000443_0003
Figure imgf000443_0004
Figure imgf000443_0005
Figure imgf000444_0001
Figure imgf000444_0002
Figure imgf000444_0003
Figure imgf000444_0004
Figure imgf000445_0001
Figure imgf000445_0002
Figure imgf000445_0003
Figure imgf000445_0004
Figure imgf000446_0001
Figure imgf000446_0002
Figure imgf000446_0003
Figure imgf000447_0001
Figure imgf000447_0002
Figure imgf000447_0003
Figure imgf000447_0004
Figure imgf000448_0001
Figure imgf000448_0002
Figure imgf000448_0003
Figure imgf000448_0004
Figure imgf000449_0001
Figure imgf000449_0002
Figure imgf000449_0003
Figure imgf000449_0004
Figure imgf000450_0001
Figure imgf000450_0002
Figure imgf000450_0003
Figure imgf000451_0001
Figure imgf000451_0002
Figure imgf000451_0003
Figure imgf000452_0001
Figure imgf000452_0002
Figure imgf000452_0003
Figure imgf000453_0001
Figure imgf000453_0002
Figure imgf000453_0003
Figure imgf000453_0004
Figure imgf000454_0001
Figure imgf000454_0002
Figure imgf000454_0003
Figure imgf000454_0004
Figure imgf000455_0001
Figure imgf000455_0002
Figure imgf000455_0003
Figure imgf000456_0001
Figure imgf000456_0002
Figure imgf000456_0003
Figure imgf000457_0001
Figure imgf000457_0002
Figure imgf000457_0003
Figure imgf000457_0004
Figure imgf000458_0001
Figure imgf000458_0002
Figure imgf000458_0003
Figure imgf000458_0004
Figure imgf000458_0005
Figure imgf000459_0001
Figure imgf000459_0002
Figure imgf000459_0003
Figure imgf000460_0001
Figure imgf000460_0002
Figure imgf000460_0003
Figure imgf000461_0001
Figure imgf000461_0002
Figure imgf000461_0003
Figure imgf000461_0004
Figure imgf000462_0001
Figure imgf000462_0002
Figure imgf000462_0003
Figure imgf000462_0004
Figure imgf000463_0001
Figure imgf000463_0002
Figure imgf000463_0003
Figure imgf000463_0004
Figure imgf000464_0001
Figure imgf000464_0002
Figure imgf000464_0003
Figure imgf000464_0004
Figure imgf000465_0001
Figure imgf000465_0002
Figure imgf000465_0003
Figure imgf000465_0004
Figure imgf000466_0001
Figure imgf000466_0002
Figure imgf000466_0003
Figure imgf000466_0004
Figure imgf000467_0001
Figure imgf000467_0002
Figure imgf000467_0003
Figure imgf000467_0004
Figure imgf000468_0001
Figure imgf000468_0002
Figure imgf000468_0003
Figure imgf000469_0001
Figure imgf000469_0002
Figure imgf000469_0003
Figure imgf000470_0001
Figure imgf000470_0002
Figure imgf000470_0003
Figure imgf000471_0001
Figure imgf000471_0002
Figure imgf000471_0003
Figure imgf000472_0001
Figure imgf000472_0002
Figure imgf000472_0003
Figure imgf000472_0004
Figure imgf000473_0001
Figure imgf000473_0002
Figure imgf000473_0003
Figure imgf000474_0001
Figure imgf000474_0002
Figure imgf000474_0003
Figure imgf000475_0001
Figure imgf000475_0002
Figure imgf000475_0003
Figure imgf000475_0004
Figure imgf000476_0001
Figure imgf000476_0002
Figure imgf000476_0003
Figure imgf000476_0004
Figure imgf000477_0001
Figure imgf000477_0002
Figure imgf000477_0003
Figure imgf000477_0004
Figure imgf000478_0001
Figure imgf000478_0003
Figure imgf000478_0004
Figure imgf000479_0001
Figure imgf000479_0002
Figure imgf000479_0003
Figure imgf000480_0001
Figure imgf000480_0002
Figure imgf000480_0003
Figure imgf000481_0001
Figure imgf000481_0002
Figure imgf000481_0003
Figure imgf000482_0002
Figure imgf000482_0003
Figure imgf000482_0004
Figure imgf000483_0001
Figure imgf000483_0002
Figure imgf000483_0003
Figure imgf000483_0004
Figure imgf000484_0001
Figure imgf000484_0002
Figure imgf000484_0003
Figure imgf000484_0004
Figure imgf000485_0001
Figure imgf000485_0002
Figure imgf000485_0003
Figure imgf000485_0004
Figure imgf000486_0001
Figure imgf000486_0002
Figure imgf000486_0003
Figure imgf000486_0004
Figure imgf000486_0005
Figure imgf000487_0001
Figure imgf000487_0002
Figure imgf000487_0003
Figure imgf000488_0001
Figure imgf000488_0002
Figure imgf000488_0003
Figure imgf000489_0001
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Figure imgf000737_0001
Figure imgf000737_0002
Figure imgf000738_0001
Figure imgf000738_0002
Figure imgf000738_0003
Figure imgf000739_0001
Figure imgf000739_0002
Figure imgf000739_0003
Figure imgf000739_0004
Figure imgf000740_0001
Figure imgf000740_0002
Figure imgf000740_0003
Figure imgf000740_0004
Figure imgf000740_0005
Figure imgf000741_0001
Figure imgf000741_0002
Figure imgf000741_0003
Figure imgf000741_0004
Figure imgf000742_0001
Figure imgf000742_0002
Figure imgf000742_0003
Figure imgf000742_0004
Figure imgf000743_0001
Figure imgf000743_0002
Figure imgf000743_0003
Figure imgf000743_0004
Figure imgf000744_0001
Figure imgf000744_0002
Figure imgf000744_0003
Figure imgf000745_0001
Figure imgf000745_0002
Figure imgf000745_0003
Figure imgf000745_0004
Figure imgf000746_0001
Figure imgf000746_0002
Figure imgf000746_0003
Figure imgf000746_0004
Figure imgf000747_0001
Figure imgf000747_0002
Figure imgf000747_0003
Figure imgf000747_0004
Figure imgf000748_0001
Figure imgf000748_0002
Figure imgf000748_0003
Figure imgf000748_0004
Figure imgf000748_0005
Figure imgf000749_0001
Figure imgf000749_0002
Figure imgf000749_0003
Figure imgf000749_0004
Figure imgf000750_0001
Figure imgf000750_0002
Figure imgf000750_0003
Figure imgf000750_0004
Figure imgf000751_0001
Figure imgf000751_0002
Figure imgf000751_0003
Figure imgf000751_0004
Figure imgf000752_0001
Figure imgf000752_0002
Figure imgf000752_0003
Figure imgf000753_0001
Figure imgf000753_0002
Figure imgf000753_0003
Figure imgf000753_0004
Figure imgf000754_0001
Figure imgf000754_0002
Figure imgf000754_0003
Figure imgf000755_0001
Figure imgf000755_0002
Figure imgf000755_0003
Figure imgf000755_0004
Figure imgf000755_0005
Figure imgf000756_0001
Figure imgf000756_0002
Figure imgf000756_0003
Figure imgf000756_0004
Figure imgf000757_0001
Figure imgf000757_0002
Figure imgf000757_0003
Figure imgf000757_0004
Figure imgf000757_0005
Figure imgf000758_0001
Figure imgf000758_0002
Figure imgf000758_0003
Figure imgf000758_0004
Figure imgf000759_0001
Figure imgf000759_0002
Figure imgf000759_0003
Figure imgf000759_0004
Figure imgf000760_0001
Figure imgf000760_0002
Figure imgf000760_0003
Figure imgf000760_0004
Figure imgf000761_0001
Figure imgf000761_0002
Figure imgf000761_0003
Figure imgf000761_0004
Figure imgf000761_0005
Figure imgf000762_0001
Figure imgf000762_0002
Figure imgf000762_0003
Figure imgf000762_0004
Figure imgf000763_0001
Figure imgf000763_0002
Figure imgf000763_0003
Figure imgf000764_0001
Figure imgf000764_0002
Figure imgf000764_0003
Figure imgf000764_0004
Figure imgf000765_0001
Figure imgf000765_0002
Figure imgf000765_0003
Figure imgf000765_0004
Figure imgf000766_0001
Figure imgf000766_0002
Figure imgf000766_0003
Figure imgf000766_0004
Figure imgf000767_0001
Figure imgf000767_0002
Figure imgf000767_0003
Figure imgf000767_0004
Figure imgf000768_0001
Figure imgf000768_0002
Figure imgf000768_0003
Figure imgf000768_0004
Figure imgf000769_0001
Figure imgf000769_0002
Figure imgf000769_0003
Figure imgf000769_0004
Figure imgf000769_0005
Figure imgf000770_0001
Figure imgf000770_0002
Figure imgf000770_0003
Figure imgf000770_0004
Figure imgf000771_0001
Figure imgf000771_0002
Figure imgf000771_0003
Figure imgf000771_0004
Figure imgf000771_0005
Figure imgf000772_0001
Figure imgf000772_0002
Figure imgf000772_0003
Figure imgf000772_0004
Figure imgf000773_0001
Figure imgf000773_0002
Figure imgf000773_0003
Figure imgf000773_0004
Figure imgf000774_0001
Figure imgf000774_0002
Figure imgf000774_0003
Figure imgf000774_0004
Figure imgf000775_0001
Figure imgf000775_0002
Figure imgf000775_0003
Figure imgf000775_0004
Figure imgf000776_0001
Figure imgf000776_0002
Figure imgf000776_0003
Figure imgf000776_0004
LE 26
Figure imgf000777_0001
Figure imgf000777_0002
Figure imgf000777_0003
Figure imgf000778_0001
Figure imgf000778_0002
Figure imgf000778_0003
Figure imgf000778_0004
Figure imgf000778_0005
Figure imgf000779_0001
Figure imgf000779_0002
Figure imgf000779_0003
Figure imgf000779_0004
Figure imgf000779_0005
Figure imgf000780_0001
Figure imgf000780_0002
Figure imgf000780_0003
Figure imgf000780_0004
Figure imgf000781_0001
Figure imgf000781_0002
Figure imgf000781_0003
Figure imgf000781_0004
Figure imgf000782_0001
Figure imgf000782_0002
Figure imgf000782_0003
Figure imgf000782_0004
Figure imgf000783_0001
Figure imgf000783_0002
Figure imgf000783_0003
Figure imgf000784_0001
Figure imgf000784_0002
Figure imgf000784_0003
Figure imgf000784_0004
Figure imgf000785_0001
Figure imgf000785_0002
Figure imgf000785_0003
Figure imgf000786_0001
Figure imgf000786_0002
Figure imgf000786_0003
Figure imgf000786_0004
Figure imgf000787_0001
Figure imgf000787_0002
Figure imgf000787_0003
Figure imgf000787_0004
Figure imgf000788_0001
Figure imgf000788_0002
Figure imgf000788_0003
Figure imgf000788_0004
Figure imgf000789_0001
Figure imgf000789_0002
Figure imgf000789_0003
Figure imgf000789_0004
Figure imgf000789_0005
Figure imgf000790_0001
Figure imgf000790_0002
Figure imgf000790_0003
Figure imgf000790_0004
Figure imgf000790_0005
Figure imgf000791_0001
Figure imgf000791_0002
Figure imgf000791_0003
Figure imgf000791_0004
Figure imgf000791_0005
Figure imgf000792_0001
Figure imgf000792_0002
Figure imgf000792_0003
Figure imgf000792_0004
Figure imgf000793_0001
Figure imgf000793_0002
Figure imgf000793_0003
Figure imgf000793_0004
Figure imgf000794_0001
Figure imgf000794_0002
Figure imgf000794_0003
Figure imgf000794_0004
Figure imgf000795_0001
Figure imgf000795_0002
Figure imgf000795_0003
Figure imgf000796_0001
Figure imgf000796_0002
Figure imgf000796_0003
Figure imgf000796_0004
Figure imgf000797_0001
Figure imgf000797_0002
Figure imgf000797_0003
Figure imgf000797_0004
Figure imgf000798_0001
Figure imgf000798_0002
Figure imgf000798_0003
Figure imgf000798_0004
Figure imgf000799_0001
Figure imgf000799_0002
Figure imgf000799_0003
Figure imgf000800_0001
Figure imgf000800_0002
Figure imgf000800_0003
Figure imgf000801_0001
Figure imgf000801_0002
Figure imgf000801_0003
Figure imgf000801_0004
Figure imgf000802_0001
Figure imgf000802_0002
Figure imgf000802_0003
Figure imgf000802_0004
Figure imgf000803_0001
Figure imgf000803_0002
Figure imgf000803_0003
Figure imgf000803_0004
Figure imgf000804_0001
Figure imgf000804_0002
Figure imgf000804_0003
Figure imgf000804_0004
Figure imgf000805_0001
Figure imgf000805_0002
Figure imgf000805_0003
Figure imgf000805_0004
Figure imgf000805_0005
Figure imgf000806_0001
Figure imgf000806_0002
Figure imgf000806_0003
Figure imgf000806_0004
Figure imgf000807_0001
Figure imgf000807_0002
Figure imgf000807_0003
Figure imgf000807_0004
Figure imgf000808_0001
Figure imgf000808_0002
Figure imgf000808_0003
Figure imgf000808_0004
Figure imgf000809_0001
Figure imgf000809_0002
Figure imgf000809_0003
Figure imgf000809_0004
Figure imgf000810_0001
Figure imgf000810_0002
Figure imgf000810_0003
Figure imgf000810_0004
Figure imgf000811_0001
Figure imgf000811_0002
Figure imgf000811_0003
Figure imgf000811_0004
Figure imgf000812_0001
Figure imgf000812_0002
Figure imgf000812_0003
Figure imgf000812_0004
Figure imgf000813_0001
Figure imgf000813_0002
Figure imgf000813_0003
Figure imgf000813_0004
Figure imgf000814_0001
Figure imgf000814_0002
Figure imgf000814_0003
Figure imgf000814_0004
Figure imgf000814_0005
Figure imgf000815_0001
Figure imgf000815_0002
Figure imgf000815_0003
Figure imgf000815_0004
Figure imgf000816_0001
Figure imgf000816_0002
Figure imgf000816_0003
Figure imgf000816_0004
Figure imgf000817_0001
Figure imgf000817_0002
Figure imgf000817_0003
Figure imgf000817_0004
Figure imgf000818_0001
Figure imgf000818_0002
Figure imgf000818_0003
Figure imgf000818_0004
Figure imgf000818_0005
Figure imgf000819_0001
Figure imgf000819_0002
Figure imgf000819_0003
Figure imgf000819_0004
Figure imgf000820_0001
Figure imgf000820_0002
Figure imgf000820_0003
Figure imgf000820_0004
Figure imgf000821_0001
Figure imgf000821_0002
Figure imgf000821_0003
Figure imgf000821_0004
Figure imgf000822_0001
Figure imgf000822_0002
Figure imgf000822_0003
Figure imgf000822_0004
Figure imgf000823_0001
Figure imgf000823_0002
Figure imgf000823_0003
Figure imgf000823_0004
Figure imgf000824_0001
Figure imgf000824_0002
Figure imgf000824_0003
Figure imgf000824_0004
Figure imgf000825_0001
Figure imgf000825_0002
Figure imgf000825_0003
Figure imgf000825_0004
Figure imgf000826_0001
Figure imgf000826_0002
Figure imgf000827_0001
Figure imgf000827_0002
Figure imgf000827_0003
Figure imgf000827_0004
Figure imgf000828_0001
Figure imgf000828_0002
Figure imgf000828_0003
Figure imgf000828_0004
Figure imgf000828_0005
Figure imgf000829_0001
Figure imgf000829_0002
Figure imgf000829_0003
Figure imgf000829_0004
Figure imgf000830_0001
Figure imgf000830_0002
Figure imgf000830_0003
Figure imgf000830_0004
Figure imgf000831_0001
Figure imgf000831_0002
Figure imgf000831_0003
Figure imgf000832_0001
Figure imgf000832_0002
Figure imgf000832_0003
Figure imgf000833_0001
Figure imgf000833_0002
Figure imgf000833_0003
Figure imgf000834_0001
Figure imgf000834_0002
Figure imgf000834_0003
Figure imgf000835_0001
Figure imgf000835_0002
Figure imgf000835_0003
Figure imgf000836_0001
Figure imgf000836_0002
Figure imgf000836_0003
Figure imgf000836_0004
Figure imgf000836_0005
Figure imgf000837_0001
Figure imgf000837_0002
Figure imgf000837_0003
Figure imgf000837_0004
Figure imgf000838_0001
Figure imgf000838_0002
Figure imgf000838_0003
Figure imgf000838_0004
Figure imgf000838_0005
Figure imgf000839_0004
Figure imgf000839_0001
Figure imgf000839_0002
Figure imgf000839_0003
Figure imgf000840_0001
Figure imgf000840_0002
Figure imgf000840_0003
Figure imgf000840_0004
Figure imgf000840_0005
Figure imgf000841_0001
Figure imgf000841_0002
Figure imgf000841_0003
Figure imgf000841_0004
Figure imgf000841_0005
Figure imgf000842_0001
Figure imgf000842_0002
Figure imgf000842_0003
Figure imgf000842_0004
Figure imgf000843_0001
Figure imgf000843_0002
Figure imgf000843_0003
Figure imgf000844_0001
Figure imgf000844_0002
Figure imgf000844_0003
Figure imgf000844_0004
Figure imgf000844_0005
Figure imgf000845_0001
Figure imgf000845_0002
Figure imgf000845_0003
Figure imgf000845_0004
Figure imgf000846_0001
Figure imgf000846_0002
Figure imgf000846_0003
Figure imgf000847_0001
Figure imgf000847_0002
Figure imgf000847_0003
Figure imgf000847_0004
Figure imgf000848_0001
Figure imgf000848_0002
Figure imgf000848_0003
Figure imgf000848_0004
Figure imgf000848_0005
Figure imgf000849_0001
Figure imgf000849_0002
Figure imgf000849_0003
Figure imgf000849_0004
Figure imgf000850_0001
Figure imgf000850_0002
Figure imgf000850_0003
Figure imgf000850_0004
Figure imgf000851_0001
Figure imgf000851_0002
Figure imgf000851_0003
Figure imgf000851_0004
Figure imgf000851_0005
Figure imgf000852_0001
Figure imgf000852_0002
Figure imgf000852_0003
Figure imgf000852_0004
Figure imgf000853_0001
Figure imgf000853_0002
Figure imgf000853_0003
Figure imgf000853_0004
Figure imgf000854_0001
Figure imgf000854_0002
Figure imgf000854_0003
Figure imgf000854_0004
Figure imgf000855_0001
Figure imgf000855_0002
Figure imgf000855_0003
Figure imgf000855_0004
Figure imgf000856_0001
Figure imgf000856_0002
Figure imgf000856_0003
Figure imgf000857_0001
Figure imgf000857_0002
Figure imgf000857_0003
Figure imgf000858_0001
Figure imgf000858_0002
Figure imgf000858_0003
Figure imgf000859_0001
Figure imgf000859_0002
Figure imgf000859_0003
Figure imgf000860_0001
Figure imgf000860_0002
Figure imgf000860_0003
Figure imgf000860_0004
Figure imgf000861_0001
Figure imgf000861_0002
Figure imgf000861_0003
Figure imgf000862_0001
Figure imgf000862_0003
Figure imgf000862_0004
Figure imgf000863_0001
Figure imgf000863_0002
Figure imgf000863_0003
Figure imgf000863_0004
Figure imgf000864_0001
Figure imgf000864_0002
Figure imgf000864_0003
Figure imgf000865_0001
Figure imgf000865_0002
Figure imgf000865_0003
Figure imgf000865_0004
Figure imgf000866_0001
Figure imgf000866_0002
Figure imgf000866_0003
Figure imgf000866_0004
Figure imgf000867_0001
Figure imgf000867_0002
Figure imgf000867_0003
Figure imgf000867_0004
Figure imgf000868_0001
Figure imgf000868_0002
Figure imgf000868_0003
Figure imgf000868_0004
Figure imgf000868_0005
Figure imgf000869_0001
Figure imgf000869_0002
Figure imgf000869_0003
Figure imgf000869_0004
Figure imgf000870_0001
Figure imgf000870_0002
Figure imgf000870_0003
Figure imgf000870_0004
Figure imgf000870_0005
Figure imgf000871_0001
Figure imgf000871_0002
Figure imgf000871_0003
Figure imgf000871_0004
Figure imgf000872_0001
Figure imgf000872_0002
Figure imgf000872_0003
Figure imgf000873_0001
Figure imgf000873_0002
Figure imgf000873_0003
Figure imgf000874_0001
Figure imgf000874_0002
Figure imgf000874_0003
Figure imgf000874_0004
Figure imgf000875_0001
Figure imgf000875_0002
Figure imgf000875_0003
Figure imgf000875_0004
Figure imgf000876_0001
Figure imgf000876_0002
Figure imgf000876_0003
Figure imgf000876_0004
Figure imgf000877_0001
Figure imgf000877_0002
Figure imgf000877_0003
Figure imgf000877_0004
Figure imgf000878_0001
Figure imgf000878_0002
Figure imgf000878_0003
Figure imgf000879_0001
Figure imgf000879_0002
Figure imgf000879_0003
Figure imgf000880_0001
Figure imgf000880_0002
Figure imgf000880_0003
Figure imgf000881_0001
Figure imgf000881_0002
Figure imgf000881_0003
Figure imgf000882_0001
Figure imgf000882_0002
Figure imgf000882_0003
Figure imgf000882_0004
Figure imgf000883_0001
Figure imgf000883_0002
Figure imgf000883_0003
Figure imgf000883_0004
Figure imgf000884_0001
Figure imgf000884_0002
Figure imgf000884_0003
Figure imgf000884_0004
Figure imgf000885_0001
Figure imgf000885_0002
Figure imgf000885_0003
Figure imgf000885_0004
Figure imgf000886_0001
Figure imgf000886_0002
Figure imgf000886_0003
Figure imgf000886_0004
Figure imgf000887_0001
Figure imgf000887_0002
Figure imgf000887_0003
Figure imgf000887_0004
Figure imgf000888_0001
Figure imgf000888_0002
Figure imgf000888_0003
Figure imgf000889_0001
Figure imgf000889_0002
Figure imgf000890_0001
Figure imgf000891_0001
Figure imgf000891_0002
Figure imgf000891_0003
Figure imgf000891_0004
Figure imgf000892_0001
Figure imgf000892_0002
Figure imgf000892_0003
Figure imgf000892_0004
Figure imgf000893_0001
Figure imgf000893_0002
Figure imgf000893_0003
Figure imgf000893_0004
Figure imgf000894_0001
Figure imgf000894_0002
Figure imgf000894_0003
Figure imgf000894_0004
Figure imgf000895_0001
Figure imgf000895_0002
Figure imgf000895_0003
Figure imgf000895_0004
Figure imgf000896_0001
Figure imgf000896_0002
Figure imgf000896_0003
Figure imgf000897_0001
Figure imgf000897_0002
Figure imgf000897_0003
Figure imgf000897_0004
Figure imgf000898_0001
Figure imgf000898_0002
Figure imgf000898_0003
Figure imgf000898_0004
Figure imgf000899_0001
Figure imgf000899_0002
Figure imgf000900_0001
Figure imgf000900_0002
Figure imgf000900_0003
Figure imgf000900_0004
Figure imgf000901_0002
Figure imgf000901_0003
Figure imgf000901_0004
Figure imgf000901_0005
Figure imgf000902_0001
Figure imgf000902_0002
Figure imgf000902_0003
Figure imgf000902_0004
Figure imgf000903_0001
Figure imgf000903_0002
Figure imgf000903_0003
Figure imgf000903_0004
Figure imgf000903_0005
Figure imgf000904_0001
Figure imgf000904_0002
Figure imgf000904_0003
Figure imgf000904_0004
Figure imgf000904_0005
Figure imgf000905_0001
Figure imgf000905_0002
Figure imgf000905_0003
Figure imgf000905_0004
Figure imgf000905_0005
Figure imgf000906_0001
Figure imgf000906_0002
Figure imgf000906_0003
Figure imgf000906_0004
Figure imgf000907_0001
Figure imgf000907_0002
Figure imgf000907_0003
Figure imgf000907_0004
Figure imgf000908_0001
Figure imgf000908_0002
Figure imgf000908_0003
Figure imgf000908_0004
Figure imgf000908_0005
Figure imgf000909_0001
Figure imgf000909_0002
Figure imgf000909_0003
Figure imgf000909_0004
Figure imgf000909_0005
Figure imgf000910_0001
Figure imgf000910_0002
Figure imgf000910_0003
Figure imgf000910_0004
Figure imgf000911_0001
Figure imgf000911_0002
Figure imgf000911_0003
Figure imgf000911_0004
Figure imgf000912_0001
Figure imgf000912_0002
Figure imgf000912_0003
Figure imgf000912_0004
Figure imgf000913_0001
Figure imgf000913_0002
Figure imgf000913_0003
Figure imgf000913_0004
Figure imgf000914_0001
Figure imgf000914_0002
Figure imgf000914_0003
Figure imgf000914_0004
Figure imgf000914_0005
Figure imgf000915_0001
Figure imgf000915_0002
Figure imgf000915_0003
Figure imgf000915_0004
Figure imgf000916_0001
Figure imgf000916_0002
Figure imgf000916_0003
Figure imgf000916_0004
Figure imgf000917_0001
Figure imgf000917_0002
Figure imgf000917_0003
Figure imgf000918_0001
Figure imgf000918_0002
Figure imgf000918_0003
Figure imgf000919_0001
Figure imgf000919_0002
Figure imgf000919_0003
Figure imgf000919_0004
Figure imgf000919_0005
Figure imgf000920_0001
Figure imgf000920_0002
Figure imgf000920_0003
Figure imgf000920_0004
Figure imgf000921_0001
Figure imgf000921_0002
Figure imgf000921_0003
Figure imgf000921_0004
Figure imgf000922_0001
Figure imgf000922_0002
Figure imgf000922_0003
Figure imgf000922_0004
Figure imgf000923_0001
Figure imgf000923_0002
Figure imgf000923_0003
Figure imgf000924_0001
Figure imgf000924_0002
Figure imgf000925_0001
Figure imgf000925_0002
Figure imgf000925_0003
Figure imgf000925_0004
Figure imgf000925_0005
Figure imgf000926_0001
Figure imgf000926_0002
Figure imgf000926_0003
Figure imgf000927_0001
Figure imgf000927_0002
Figure imgf000927_0003
Figure imgf000927_0004
Figure imgf000927_0005
Figure imgf000928_0001
Figure imgf000928_0002
Figure imgf000928_0003
Figure imgf000928_0004
Figure imgf000928_0005
Figure imgf000929_0001
Figure imgf000929_0002
Figure imgf000929_0003
Figure imgf000929_0004
Figure imgf000929_0005
Figure imgf000930_0001
Figure imgf000930_0002
Figure imgf000930_0003
Figure imgf000931_0001
Figure imgf000931_0002
Figure imgf000931_0003
Figure imgf000931_0004
Figure imgf000932_0001
Figure imgf000932_0002
Figure imgf000932_0003
Figure imgf000932_0004
Figure imgf000933_0001
Figure imgf000933_0003
Figure imgf000933_0004
Figure imgf000934_0001
Figure imgf000934_0002
Figure imgf000934_0003
Figure imgf000934_0004
Figure imgf000935_0001
Figure imgf000935_0002
Figure imgf000935_0003
Figure imgf000935_0004
Figure imgf000935_0005
Figure imgf000936_0001
Figure imgf000936_0002
Figure imgf000936_0003
Figure imgf000936_0004
Figure imgf000937_0001
Figure imgf000937_0002
Figure imgf000937_0003
Figure imgf000937_0004
Figure imgf000938_0001
Figure imgf000938_0002
Figure imgf000938_0003
Figure imgf000938_0004
Figure imgf000939_0001
Figure imgf000939_0002
Figure imgf000939_0003
Figure imgf000939_0004
Figure imgf000940_0001
Figure imgf000940_0002
Figure imgf000940_0003
Figure imgf000940_0004
Figure imgf000941_0001
Figure imgf000941_0002
Figure imgf000941_0003
Figure imgf000941_0004
Figure imgf000942_0001
Figure imgf000942_0002
Figure imgf000942_0003
Figure imgf000942_0004
Figure imgf000943_0001
Figure imgf000943_0003
Figure imgf000943_0004
Figure imgf000944_0001
Figure imgf000944_0002
Figure imgf000944_0003
Figure imgf000944_0004
Figure imgf000945_0001
Figure imgf000945_0002
Figure imgf000945_0003
Figure imgf000945_0004
Figure imgf000946_0001
Figure imgf000946_0002
Figure imgf000946_0003
Figure imgf000946_0004
Figure imgf000946_0005
Figure imgf000947_0001
Figure imgf000947_0002
Figure imgf000947_0003
Figure imgf000947_0004
Figure imgf000948_0001
Figure imgf000948_0002
Figure imgf000948_0003
Figure imgf000948_0004
Figure imgf000949_0001
Figure imgf000949_0002
Figure imgf000949_0003
Figure imgf000949_0004
Figure imgf000949_0005
Figure imgf000950_0001
Figure imgf000950_0002
Figure imgf000950_0003
Figure imgf000950_0004
Figure imgf000951_0001
Figure imgf000951_0002
Figure imgf000951_0003
Figure imgf000951_0004
Figure imgf000952_0001
Figure imgf000952_0002
Figure imgf000952_0003
Figure imgf000952_0004
Figure imgf000953_0001
Figure imgf000953_0002
Figure imgf000953_0003
Figure imgf000953_0004
Figure imgf000954_0001
Figure imgf000954_0002
Figure imgf000954_0003
Figure imgf000954_0004
Figure imgf000955_0001
Figure imgf000955_0002
Figure imgf000955_0003
Figure imgf000955_0004
Figure imgf000955_0005
Figure imgf000956_0001
Figure imgf000956_0002
Figure imgf000956_0003
Figure imgf000956_0004
Figure imgf000957_0001
Figure imgf000957_0002
Figure imgf000957_0003
Figure imgf000958_0001
Figure imgf000958_0002
Figure imgf000958_0003
Figure imgf000958_0004
Figure imgf000958_0005
Figure imgf000959_0001
Figure imgf000959_0002
Figure imgf000959_0003
Figure imgf000959_0004
Figure imgf000960_0001
Figure imgf000960_0002
Figure imgf000960_0003
Figure imgf000961_0001
Figure imgf000961_0002
Figure imgf000961_0003
Figure imgf000961_0004
Figure imgf000961_0005
Figure imgf000962_0001
Figure imgf000962_0002
Figure imgf000962_0003
Figure imgf000962_0004
Figure imgf000963_0001
Figure imgf000963_0002
Figure imgf000963_0003
Figure imgf000963_0004
Figure imgf000963_0005
Figure imgf000964_0001
Figure imgf000964_0002
Figure imgf000964_0003
Figure imgf000964_0004
Figure imgf000965_0001
Figure imgf000965_0002
Figure imgf000965_0003
Figure imgf000965_0004
Figure imgf000965_0005
Figure imgf000966_0001
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1. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021 , SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 1002, SEQ ID NO: 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1251, SEQ ID NO: 1262, SEQ ID NO: 1149, SEQ ID NO: 1220, SEQ ID NO: 1240, SEQ ID NO: 1164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332, SEQ ID NO: 1345, SEQ ID NO: 1358, SEQ ID NO: 1375, SEQ ID NO: 1417, SEQ ID NO: 1283, SEQ ID NO: 1335, SEQ ID NO: 1368, SEQ ID NO: 1179, SEQ ID NO: 1255, SEQ ID NO: 1258, SEQ ID NO: 1044, SEQ ID NO: 1273, SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ ID NO: 1331, SEQ ID NO: 1432, SEQ ID NO: 1052, SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069, SEQ ID NO: 1005, SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1193, SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1346, SEQ ID NO: 1348, SEQ ID NO: 1350, SEQ ID NO: 1032, SEQ ID NO: 1053, SEQ ID NO: 1081, SEQ ID NO: 1124, SEQ ID NO: 1382, SEQ ID NO: 1437, SEQ ID NO: 1263, SEQ ID NO: 1173, SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1410, SEQ ID NO: 1086, SEQ ID NO: 1322, SEQ ID NO: 1266, SEQ ID NO: 1282, SEQ ID NO: 1271, SEQ ID NO: 1208, SEQ ID NO: 1126, SEQ ID NO: 1270, SEQ ID NO: 1278, SEQ ID NO: 1419, SEQ ID NO: 1125, SEQ ID NO: 1181, SEQ ID NO: 1416, SEQ ID NO: 1096, SEQ ID NO: 1082, SEQ ID NO: 1146, SEQ ID NO: 1145, SEQ ID NO: 1108, SEQ ID NO: 1148, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1424, SEQ ID NO: 1000, SEQ ID NO: 1027, SEQ ID NO: 1175, SEQ ID NO: 1330, SEQ ID NO: 217, SEQ ID NO: 217, SEQ ID NO: 367, SEQ ID NO: 911,SEQ ID NO: 944, SEQ ID NO: 18, SEQ ID NO: 107, SEQ ID NO: 894, SEQ ID NO: 943, SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199, SEQ ID NO: 992, SEQ ID NO: 934, SEQ ID NO: 899, SEQ ID NO: 302, SEQ ID NO: 215, SEQ ID NO: 893, SEQ ID NO: 984, SEQ ID NO: 97,SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 309, SEQ ID NO: 150, SEQ ID NO: 240, SEQ ID NO: 957, SEQ ID NO: 57, SEQ ID NO: 2, SEQ ID NO: 92, SEQ ID NO: 255, SEQ ID NO: 164, SEQ ID NO: 201, SEQ ID NO: 278, SEQ ID NO: 245, SEQ ID NO: 921, SEQ ID NO: 896, SEQ ID NO: 248, SEQ ID NO: 159, SEQ ID NO: 979, SEQ ID NO: 194, SEQ ID NO: 194, SEQ ID NO: 946, SEQ ID NO: 916, SEQ ID NO: 76, SEQ ID NO: 905, SEQ ID NO: 914, SEQ ID NO: 931, SEQ ID NO: 50, SEQ ID NO: 250, SEQ ID NO: 969, SEQ ID NO: 66,SEQ ID NO: 275, SEQ ID NO: 330, SEQ ID NO: 204, SEQ ID NO: 383, SEQ ID NO: 303, SEQ ID NO: 70, SEQ ID NO: 983, SEQ ID NO: 972, SEQ ID NO: 929, SEQ ID NO: 972, SEQ ID NO: 936, SEQ ID NO: 267, SEQ ID NO: 197, SEQ ID NO: 55, SEQ ID NO: 54, SEQ ID NO: 210, SEQ ID NO: 90, SEQ ID NO: 15, SEQ ID NO: 913, SEQ ID NO: 227, SEQ ID NO: 79, SEQ ID NO: 191, SEQ ID NO: 238, SEQ ID NO: 274, SEQ ID NO: 27, SEQ ID NO: 258, SEQ ID NO: 295, SEQ ID NO: 10, SEQ ID NO: 160, SEQ ID NO: 225, SEQ ID NO: 964, SEQ ID NO: 166, SEQ ID NO: 56, SEQ ID NO: 980,SEQ ID NO: 903, SEQ ID NO: 261, SEQ ID NO: 71, SEQ ID NO: 955, SEQ ID NO: 361, SEQ ID NO: 58, SEQ ID NO: 114, SEQ ID NO: 940, SEQ ID NO: 960, SEQ ID NO: 144, SEQ ID NO: 362, SEQ ID NO: 40, SEQ ID NO: 285, SEQ ID NO: 11, SEQ ID NO: 161, SEQ ID NO: 974, SEQ ID NO: 111, SEQ ID NO: 316, SEQ ID NO: 257, SEQ ID NO: 78, SEQ ID NO: 966, SEQ ID NO: 352, SEQ ID NO: 981, SEQ ID NO: 158, SEQ ID NO: 989, SEQ ID NO: 963, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 135, SEQ ID NO: 910, SEQ ID NO: 236, SEQ ID NO: 241, SEQ ID NO: 949, SEQ ID NO: 945, SEQ ID NO: 207, SEQ ID NO: 977, SEQ ID NO: 978, SEQ ID NO: 994, SEQ ID NO: 163, SEQ ID NO: 256, SEQ ID NO: 287, SEQ ID NO: 184, SEQ ID NO: 45, SEQ ID NO: 136, SEQ ID NO: 214, SEQ ID NO: 16, SEQ ID NO: 192, SEQ ID NO: 373, SEQ ID NO: 892, SEQ ID NO: 239, SEQ ID NO: 34, SEQ ID NO: 340, SEQ ID NO: 41, SEQ ID NO: 332, SEQ ID NO: 134, and SEQ ID NO: 330.
2. The purified nucleic acid of claim 1 , wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1020, SEQ ID NO: 1021, SEQ ID NO: 1036, SEQ ID NO: 1050, SEQ ID NO: 1071, SEQ ID NO: 1101, SEQ ID NO: 1135, SEQ ID NO: 1276, SEQ ID NO: 1150, SEQ ID NO: 1187, SEQ ID NO: 1192, SEQ ID NO: 1361, SEQ ID NO: 1379, SEQ ID NO: 1399, SEQ ID NO: 1403, SEQ ID NO: 1400, SEQ ID NO: 1189, SEQ ID NO: 217, SEQ ID NO: 367, SEQ ID NO: 911,SEQ ID NO: 944, SEQ ID NO: 18, SEQ ID NO: 107, SEQ ID NO: 894, SEQ ID NO: 943, SEQ ID NO: 203, SEQ ID NO: 85, SEQ ID NO: 290, SEQ ID NO: 5, SEQ ID NO: 199, SEQ ID NO: 992, SEQ ID NO: 934, SEQ ID NO: 899, SEQ ID NO: 302, and SEQ ID NO: 215.
3. The purified nucleic acid of claim 1, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1002, SEQ ID NO: 1213, SEQ ID NO: 1214, SEQ ID NO: 1215, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1237, SEQ ID NO: 1224, SEQ ID NO: 1251, SEQ ID NO: 1262, SEQ ID NO: 1149, SEQ ID NO: 1220, SEQ ID NO: 1240, SEQ ID NO: 1 164, SEQ ID NO: 1165, SEQ ID NO: 1404, SEQ ID NO: 1 144, SEQ ID NO: 1182, SEQ ID NO: 1157, SEQ ID NO: 1160, SEQ ID NO: 1300, SEQ ID NO: 1321, SEQ ID NO: 1323, SEQ ID NO: 1329, SEQ ID NO: 1332, SEQ ID NO: 1345, SEQ ID NO: 1358, SEQ ID NO: 1375, SEQ ID NO: 1417, SEQ ID NO: 1283, SEQ ID NO: 1335, SEQ ID NO: 1368, SEQ ID NO: 1179, SEQ ID NO: 1255, SEQ ID NO: 1258, SEQ ID NO: 1044, SEQ ID NO: 1273, SEQ ID NO: 893, SEQ ID NO: 984, SEQ ID NO: 97,SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 309, SEQ ID NO: 150, SEQ ID NO: 240, SEQ ID NO: 957, SEQ ID NO: 57, SEQ ID NO: 2, SEQ ID NO: 92, SEQ ID NO: 255, SEQ ID NO: 164, SEQ ID NO: 201, SEQ ID NO: 278, SEQ ID NO: 245, SEQ ID NO: 921, SEQ ID NO: 896, SEQ ID NO: 248, SEQ ID NO: 159, SEQ ID NO: 979, SEQ ID NO: 194, SEQ ID NO: 194, SEQ ID NO: 946, SEQ ID NO: 916, SEQ ID NO: 76, SEQ ID NO: 905, SEQ ID NO: 914, SEQ ID NO: 931, SEQ ID NO: 50, SEQ ID NO: 250, SEQ ID NO: 969, SEQ ID NO: 66,SEQ ID NO: 275, SEQ ID NO: 330, SEQ ID NO: 204, SEQ ID NO: 383, SEQ ID NO: 303, SEQ ID NO: 70, SEQ ID NO: 983, SEQ ID NO: 972, SEQ ID NO: 929, SEQ ID NO: 972, SEQ ID NO: 936, SEQ ID NO: 267, SEQ ID NO: 197, SEQ ID NO: 55, SEQ ID NO: 54, and SEQ ID NO: 210.
4. The purified nucleic acid of claim 1, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1219, SEQ ID NO: 1274, SEQ ID NO: 1210, SEQ ID NO: 1422, SEQ ID NO: 1302, SEQ ID NO: 1308, SEQ ID NO: 1310, SEQ ID NO: 1331, SEQ ID NO: 1432, SEQ ID NO: 1052, SEQ ID NO: 1091, SEQ ID NO: 1421, SEQ ID NO: 1069, SEQ ID NO: 1005, SEQ ID NO: 1007, SEQ ID NO: 1166, SEQ ID NO: 1177, SEQ ID NO: 1193, SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1346, SEQ ID NO: 1348, SEQ ID NO: 1350, SEQ ID NO: 1032, SEQ ID NO: 1053, SEQ ID NO:
1081, SEQ ID NO: 1124, SEQ ID NO: 1382, SEQ ID NO: 1437, SEQ ID NO: 1263, SEQ ID NO: 90, SEQ ID NO: 15, SEQ ID NO: 913, SEQ ID NO: 227, SEQ ID NO: 79, SEQ ID NO: 191, SEQ ID NO: 238, SEQ ID NO: 274, SEQ ID NO: 27, SEQ ID NO: 258, SEQ ID NO: 295, SEQ ID NO: 10, SEQ ID NO: 160, SEQ ID NO: 225, SEQ ID NO: 964, SEQ ID NO: 166, SEQ ID NO: 56, SEQ ID NO: 980,SEQ ID NO: 903, SEQ ID NO: 261, SEQ ID
NO: 71, SEQ ID NO: 955, SEQ ID NO: 361, SEQ ID NO: 58, SEQ ID NO: 114, SEQ ID
NO: 940, SEQ ID NO: 960, SEQ ID NO: 144, SEQ ID NO: 362, SEQ ID NO: 40, SEQ ID
NO: 285, SEQ ID NO: 11, SEQ ID NO: 161, SEQ ID NO: 974, SEQ ID NO: 111, SEQ ID
NO: 316, SEQ ID NO: 257, SEQ ID NO: 78, and SEQ ID NO: 966.
5. The purified nucleic acid of claim 1, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO:
1173, SEQ ID NO: 1405, SEQ IDNO: 1406, SEQ ID NO: 1410, SEQ IDNO: 1086, SEQ ID NO: 1322, SEQ IDNO: 1266, SEQ IDNO: 1282, SEQ ID NO: 1271, SEQ IDNO: 1208, SEQ ID NO: 1126, SEQ ID NO: 1270, SEQ ID NO: 1278, SEQ ID NO: 1419, SEQ ID NO: 1125, SEQ ID NO: 1181, SEQ ID NO: 1416, SEQ ID NO: 1096, SEQ ID NO: 1082, SEQ ID NO: 1146, SEQ ID NO: 1145, SEQ ID NO: 1108, SEQ ID NO: 1148, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1424, SEQ ID NO: 1000, SEQ ID NO: 1027, SEQ ID NO: 1175, SEQ ID NO: 1330, SEQ ID NO: 352, SEQ ID NO: 981, SEQ ID NO: 158, SEQ ID NO: 989, SEQ ID NO: 963, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 135, SEQ ID NO: 910, SEQ ID NO: 236, SEQ ID NO: 241, SEQ ID NO: 949, SEQ ID NO: 945, SEQ ID NO: 207, and SEQ ID NO: 977. 6. A recombinant expression vector comprising the nucleic acid of claim 1 operably linked to a transcription regulatory element.
7. A cell comprising a recombinant expression vector of claim 6. 8. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 7 under conditions that permit expression of the polypeptide.
9. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cytoplasmic polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO: 1229, SEQ ID NO: 1250, SEQ ID NO: 1268, SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ ID NO: 996, SEQ ID NO: 1095, SEQ ID NO: 1156, SEQ ID NO: 1158, SEQ ID NO: 1159, SEQ ID NO: 1277, SEQ ID NO: 1038, SEQ ID NO: 1257, SEQ ID NO: 1357, SEQ ID NO: 1436, SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327, SEQ ID NO: 1412, SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1116, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188, SEQ ID NO: 1198, SEQ ID NO: 1218, SEQ ID NO: 1244, SEQ ID NO: 1306, SEQ ID NO: 1325, SEQ ID NO: 1397, SEQ ID NO: 1398, SEQ ID NO: 1407, SEQ ID NO: 1433, SEQ ID NO: 1216, SEQ ID NO: 1239, SEQ ID NO: 1362, SEQ ID NO: 1017, SEQ ID NO: 1019, SEQ ID NO: 1360, SEQ ID NO: 1423, SEQ ID NO: 1425, SEQ ID NO: 1374, SEQ ID NO: 1028, SEQ ID NO: 1037, SEQ ID NO: 1077, SEQ ID NO: 1 115, SEQ ID NO: 1232, SEQ ID NO: 1241, SEQ ID NO: 1267, SEQ ID NO: 1163, SEQ ID NO: 1068, SEQ ID NO: 1025, SEQ ID NO: 1042, SEQ ID NO: 1046, SEQ ID NO: 1056, SEQ ID NO: 1039, SEQ ID NO: 1072, SEQ ID NO: 1073, SEQ ID NO: 1092, SEQ ID NO: 1100, SEQ ID NO: 1102, SEQ ID NO: 1103, SEQ ID NO: 1104, SEQ ID NO: 1111, SEQ ID NO: 1119, SEQ ID NO: 1136, SEQ ID NO: 1137, SEQ ID NO: 1140, SEQ ID NO: 1142, SEQ ID NO: 1233, SEQ ID NO: 1238, SEQ ID NO: 1243, SEQ ID NO: 1245, SEQ ID NO: 1247, SEQ ID NO: 1249, SEQ ID NO: 1261, SEQ ID NO: 1269, SEQ ID NO: 1279, SEQ ID NO: 1284, SEQ ID NO: 1290, SEQ ID NO: 1297, SEQ ID NO: 1328, SEQ ID NO: 1370, SEQ ID NO: 1372, SEQ ID NO: 1377, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1388, SEQ ID NO: 1401, SEQ ID NO: 1402, SEQ ID NO: 1418, SEQ ID NO: 1420, SEQ ID NO: 1427, SEQ ID NO: 1070, SEQ ID NO: 1151, SEQ ID NO: 1176, SEQ ID NO: 999, SEQ ID NO: 1006, SEQ ID NO: 1012, SEQ ID NO: 1018, SEQ ID NO: 1030, SEQ ID NO: 1033, SEQ ID NO: 1041, SEQ ID NO: 1049, SEQ ID NO: 1054, SEQ ID NO: 1057, SEQ ID NO: 1090, SEQ ID NO: 1097, SEQ ID NO: 1129, SEQ ID NO: 1139, SEQ ID NO: 1143, SEQ ID NO: 1152, SEQ ID NO: 1153, SEQ ID NO: 1155, SEQ ID NO: 1161, SEQ ID NO: 1162, SEQ ID NO: 1169, SEQ ID NO: 1170, SEQ ID NO: 1171, SEQ ID NO: 1180, SEQ ID NO: 1194, SEQ ID NO: 1195, SEQ ID NO: 1199, SEQ ID NO: 1200, SEQ ID NO: 1201, SEQ ID NO: 1202, SEQ ID NO: 1205, SEQ ID NO: 1312, SEQ ID NO: 1336, SEQ ID NO: 1349, SEQ ID NO: 1355, SEQ ID NO: 1359, SEQ ID NO: 1413, SEQ ID NO: 1426, SEQ ID NO: 1430, SEQ ID NO: 882, SEQ ID NO: 382, SEQ ID NO: 130, SEQ ID NO: 230, SEQ ID NO: 269, SEQ ID NO: 312, SEQ ID NO: 211, SEQ ID NO: 959, SEQ ID NO: 938, SEQ ID NO: 110, SEQ ID NO: 244, SEQ ID NO: 328, SEQ ID NO: 235, SEQ ID NO: 315, SEQ ID NO: 296, SEQ ID NO: 976, SEQ ID NO: 321, SEQ ID NO: 43, SEQ ID NO: 281, SEQ ID NO: 326, SEQ ID NO: 272, SEQ ID NO: 344, SEQ ID NO: 139, SEQ ID NO: 30, SEQ ID NO: 220, SEQ ID NO: 364, SEQ ID NO: 369, SEQ ID NO: 372, SEQ ID NO: 991 , SEQ ID NO: 128, SEQ ID NO: 347, SEQ ID NO: 52, SEQ ID NO: 12, SEQ ID NO: 247, SEQ ID NO: 64, SEQ ID NO: 101, SEQ ID NO: 338, SEQ ID NO: 83, SEQ ID NO: 46, SEQ ID NO: 348, SEQ ID NO: 223, SEQ ID NO: 39, SEQ ID NO: 232, SEQ ID NO: 168, SEQ ID NO: 65, SEQ ID NO: 952, SEQ ID NO: 341, SEQ ID NO: 69, SEQ ID NO: 924, SEQ ID NO: 4, SEQ ID NO: 197, SEQ ID NO: 313, SEQ ID NO: 119, SEQ ID NO: 188, SEQ ID NO: 956, SEQ ID NO: 935, SEQ ID NO: 246, SEQ ID NO: 196, SEQ ID NO: 376, SEQ ID NO: 172, SEQ ID NO: 25, SEQ ID NO: 126, SEQ ID NO: 951, SEQ ID NO: 147, SEQ ID NO: 895, SEQ ID NO: 14, SEQ ID NO: 154, SEQ ID NO: 277, SEQ ID NO: 363, SEQ ID NO: 342, SEQ ID NO: 378, SEQ ID NO: 130, SEQ ID NO: 198, SEQ ID NO: 243, SEQ ID NO: 19, SEQ ID NO: 9, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 349, SEQ ID NO: 209, SEQ ID NO: 990, SEQ ID NO: 185, SEQ ID NO: 883, SEQ ID NO: 8, SEQ ID NO: 887, SEQ ID NO: 350, SEQ ID NO: 987, SEQ ID NO: 63, SEQ ID NO: 249, SEQ ID NO: 118, SEQ ID NO: 132, SEQ ID NO: 47, SEQ ID NO: 106, SEQ ID NO: 324, SEQ ID NO: 155, SEQ ID NO: 121, SEQ ID NO: 153, SEQ ID NO: 87, SEQ ID NO: 986, SEQ ID NO: 262, SEQ ID NO: 333, SEQ ID NO: 36, SEQ ID NO: 982, SEQ ID NO: 180, SEQ ID NO: 84, SEQ ID NO: 900, SEQ ID NO: 20, SEQ ID NO: 7, SEQ ID NO: 61, SEQ ID NO: 253, SEQ ID NO: 120, SEQ ID NO: 268, SEQ ID NO: 299, SEQ ID NO: 942, SEQ ID NO: 173, SEQ ID NO: 187, SEQ ID NO: 187, SEQ ID NO: 234, SEQ ID NO: 112, SEQ ID NO: 324, SEQ ID NO: 971, SEQ ID NO: 62, SEQ ID NO: 308, SEQ ID NO: 74, SEQ ID NO: 1, SEQ ID NO: 266, SEQ ID NO: 337, SEQ ID NO: 93, SEQ ID NO: 44, SEQ ID NO: 335, SEQ ID NO: 368, SEQ ID NO: 208, SEQ ID NO: 358, SEQ ID NO: 923, SEQ ID NO: 310, SEQ ID NO: 26, SEQ ID NO: 279, SEQ ID NO: 890, SEQ ID NO: 325, SEQ ID NO: 109, SEQ ID NO: 143, SEQ ID NO: 918, SEQ ID NO: 252, SEQ ID NO: 953, SEQ ID NO: 902, SEQ ID NO: 174, SEQ ID NO: 73, SEQ ID NO: 898, SEQ ID NO: 300, SEQ ID NO: 356, SEQ ID NO: 298, SEQ ID NO: 354, SEQ ID NO: 138, SEQ ID NO: 319, SEQ ID NO: 80, SEQ ID NO: 933, SEQ ID NO: 891, SEQ ID NO: 366, SEQ ID NO: 113, SEQ ID NO: 320, SEQ ID NO: 915, SEQ ID NO: 351, SEQ ID NO: 162, SEQ ID NO: 965, SEQ ID NO: 67, SEQ ID NO: 314, SEQ ID NO: 904, SEQ ID NO: 345, SEQ ID NO: 374, SEQ ID NO: 962, SEQ ID NO: 270, SEQ ID NO: 186, SEQ ID NO: 60, SEQ ID NO: 379, SEQ ID NO: 889, SEQ ID NO: 967, SEQ ID NO: 973, SEQ ID NO: 280, SEQ ID NO: 170, SEQ ID NO: 985, and SEQ ID NO: 932.
10. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1147, SEQ ID NO: 1288, SEQ ID NO: 1324, SEQ ID NO: 1363, SEQ ID NO: 882, SEQ ID NO: 382, SEQ ID NO: 130, and SEQ ID NO: 230. 11. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in amino acid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 997, SEQ ID NO: 1015, SEQ ID NO: 1084, SEQ ID NO: 1094, SEQ ID NO: 1099, SEQ ID NO: 1229, SEQ ID NO: 1250, SEQ ID NO: 1268, SEQ ID NO: 1293, SEQ ID NO: 1339, SEQ ID NO: 1408, SEQ ID NO: 1429, SEQ ID NO: 1434, SEQ ID NO: 1228, SEQ ID NO: 1031, SEQ ID NO: 1034, SEQ ID NO: 1008, SEQ ID NO: 269, SEQ ID NO: 312, SEQ ID NO: 211, SEQ ID NO: 959, SEQ ID NO: 938, SEQ ID NO: 110, SEQ ID NO: 244, SEQ ID NO: 328, SEQ ID NO: 235, SEQ ID NO: 315, SEQ ID NO: 296, SEQ ID NO: 976, SEQ ID NO: 321, SEQ ID NO: 43, SEQ ID NO: 281, SEQ ID NO: 326, and SEQ ID NO: 272.
12. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in nucleotide metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1061, SEQ ID NO: 1064, SEQ ID NO: 1191, SEQ ID NO: 1217, SEQ ID NO: 1365, SEQ ID NO: 1394, SEQ ID NO: 1414, SEQ ID NO: 1415, SEQ ID NO: 1435, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 344, SEQ ID NO: 139, SEQ ID NO: 30, SEQ ID NO: 220, SEQ ID NO: 364, SEQ ID NO: 369, SEQ ID NO: 372,
SEQ ID NO: 991, SEQ ID NO: 128, SEQ ID NO: 347, and SEQ ID NO: 52.
13. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1080, SEQ ID NO: 1128, SEQ ID NO: 1133, SEQ ID NO: 1211, SEQ ID NO: 1252, SEQ ID NO: 1253, SEQ ID NO: 1286, SEQ ID NO: 1289, SEQ ID
NO: 1291, SEQ ID NO: 1303, SEQ ID NO: 1396, SEQ ID NO: 996, SEQ ID NO: 12, SEQ
ID NO: 247, SEQ ID NO: 64, SEQ ID NO: 101, SEQ ID NO: 338, SEQ ID NO: 83, SEQ
ID NO: 46, SEQ ID NO: 348, SEQ ID NO: 223, SEQ ID NO: 39, SEQ ID NO: 232, and
SEQ ID NO: 168.
14. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1095, SEQ ID NO: 1156, SEQ ID NO: 1158, SEQ ID NO: 1159, SEQ ID NO: 1277, SEQ ID NO: 1038, SEQ ID NO: 65, SEQ ID NO: 952, SEQ ID NO: 341, SEQ ID NO: 69, SEQ ID NO: 924, and SEQ ID NO: 4.
15. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1257, SEQ ID NO: 1357, SEQ ID NO: 1436, SEQ ID NO: 197, SEQ ID NO: 313, and SEQ ID NO: 119.
16. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: SEQ ID NO: 1141, SEQ ID NO: 1227, SEQ ID NO: 1327, SEQ ID NO: 1412, SEQ ID NO: 188, SEQ ID NO: 956, SEQ ID NO: 935, SEQ ID NO: 246, SEQ ID NO: 196, and SEQ ID NO: 376.
17. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1003, SEQ ID NO: 1087, SEQ ID NO: 1116, SEQ ID NO: 1130, SEQ ID NO: 1132, SEQ ID NO: 1185, SEQ ID NO: 1188, SEQ ID NO: 1198, SEQ ID NO: 1218, SEQ ID NO: 1244, SEQ ID NO: 1306, SEQ ID NO: 1325, SEQ ID NO: 1397, SEQ ID NO: 1398, SEQ ID NO: 1407, SEQ ID NO: 1433, SEQ ID NO: 172, SEQ ID NO: 25, SEQ ID NO: 126, SEQ ID NO: 951, SEQ ID NO: 147, SEQ ID NO: 895, SEQ ID NO: 14, SEQ ID NO: 154, SEQ ID NO: 277, SEQ ID NO: 363, SEQ ID NO: 342, SEQ ID NO: 378, SEQ ID NO: 130, SEQ ID NO: 198, SEQ ID NO: 243, SEQ ID NO: 19, and SEQ ID NO: 9.
18. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1216, SEQ ID NO: 1239, SEQ ID NO: 1362, SEQ ID NO: 1017, SEQ ID NO: 1019, SEQ ID NO: 1360, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 349, SEQ ID NO: 209, SEQ ID NO: 990, SEQ ID NO: 185, SEQ ID NO: 883, and SEQ ID NO: 8.
19. The purified nucleic acid of claim 9, wherein said H. pylori cytoplasmic polypeptide is an H. pylori chaperone polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of
SEQ ID NO: 1423, SEQ ID NO: 1425, SEQ ID NO: 1374, SEQ ID NO: 887, SEQ ID NO: 350, and SEQ ID NO: 987.
20. A recombinant expression vector comprising the nucleic acid of claim 9 operably linked to a transcription regulatory element.
21. A cell comprising a recombinant expression vector of claim 20.
22. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 21 under conditions that permit expression of the polypeptide.
23. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted or periplasmic polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 1004, SEQ ID NO: 1138, SEQ ID NO: 1067, SEQ ID NO: 1078, SEQ ID NO: 1314, SEQ ID NO: 1319, SEQ ID NO: 1378, SEQ ID NO: 1105, SEQ ID NO: 1114, SEQ ID NO: 1118, SEQ ID NO: 1120, SEQ ID NO: 1123, SEQ ID NO: 1127, SEQ ID NO: 1212, SEQ ID NO: 1223, SEQ ID NO: 1225, SEQ ID NO: 1246, SEQ ID NO: 1248, SEQ ID NO: 1259, SEQ ID NO: 1264, SEQ ID NO: 1265, SEQ ID NO: 1281, SEQ ID NO: 1285, SEQ ID NO: 1294, SEQ ID NO: 1298, SEQ ID NO: 1299, SEQ ID NO: 1315, SEQ ID NO: 1316, SEQ ID NO: 1317, SEQ ID NO: 1318, SEQ ID NO: 1344, SEQ ID NO: 1351, SEQ ID NO: 1353, SEQ ID NO: 1373, SEQ ID NO: 1380, SEQ ID NO: 1387, SEQ ID NO: 1389, SEQ ID NO: 1393, SEQ ID NO: 1411, SEQ ID NO: 1428, SEQ ID NO: 1431, SEQ ID NO: 1439, SEQ ID NO: 1043, SEQ ID NO: 1183, SEQ ID NO: 1184, SEQ ID NO: 1196, SEQ ID NO: 1197, SEQ ID NO: 1203, SEQ ID NO: 995, SEQ ID NO: 998, SEQ ID NO: 1001, SEQ ID NO: 1022, SEQ ID NO: 1023, SEQ ID NO: 1029, SEQ ID NO: 1040, SEQ ID NO: 1051, SEQ ID NO: 1062, SEQ ID NO: 1154, SEQ ID NO: 1320, SEQ ID NO: 1075, SEQ ID NO: 1106, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1221, SEQ ID NO: 1226, SEQ ID NO: 1235, SEQ ID NO: 1301, SEQ ID NO: 1311, SEQ ID NO: 1326, SEQ ID NO: 1341, SEQ ID NO: 1354, SEQ ID NO: 1364, SEQ ID NO: 1366, SEQ ID NO: 1376, SEQ ID NO: 1391, SEQ ID NO: 1395, SEQ ID NO: 1445, SEQ ID NO: 1079, SEQ ID NO: 1186, SEQ ID NO: 1010, SEQ ID NO: 1016, SEQ ID NO: 1172, SEQ ID NO: 1174, SEQ ID NO: 117, SEQ ID NO: 254, SEQ ID NO: 24, SEQ ID NO: 242, SEQ ID NO: 950, SEQ ID NO: 263, SEQ ID NO: 286, SEQ ID NO: 947, SEQ ID NO: 51, SEQ ID NO: 177, SEQ ID NO: 156, SEQ ID NO: 190, SEQ ID NO: 375, SEQ ID NO: 222, SEQ ID NO: 21, SEQ ID NO: 912, SEQ ID NO: 148, SEQ ID NO: 202, SEQ ID NO: 224, SEQ ID NO: 112, SEQ ID NO: 32, SEQ ID NO: 339, SEQ ID NO: 182, SEQ ID NO: 228, SEQ ID NO: 152, SEQ ID NO: 219, SEQ ID NO: 137, SEQ ID NO: 318, SEQ ID NO: 141, SEQ ID NO: 165, SEQ ID NO: 334, SEQ ID NO: 13, SEQ ID NO: 297, SEQ ID NO: 35, SEQ ID NO: 216, SEQ ID NO: 908, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 927, SEQ ID NO: 221, SEQ ID NO: 178, SEQ ID NO: 169, SEQ ID NO: 293, SEQ ID NO: 289, SEQ ID NO: 926, SEQ ID NO: 948, SEQ ID NO: 115, SEQ ID NO: 251, SEQ ID NO: 345, SEQ ID NO: 17, SEQ ID NO: 920, SEQ ID NO: 95, SEQ ID NO: 86, SEQ ID NO: 360, SEQ ID NO: 271, SEQ ID NO: 970, SEQ ID NO: 288, SEQ ID NO: 282, SEQ ID NO: 98, SEQ ID NO: 29, SEQ ID NO: 317, SEQ ID NO: 343, SEQ ID NO: 291, SEQ ID NO: 108, SEQ ID NO: 377, SEQ ID NO: 305, SEQ ID NO: 305, SEQ ID NO: 100, SEQ ID NO: 988, SEQ ID NO: 212,
SEQ ID NO: 884, SEQ ID NO: 37, SEQ ID NO: 968, SEQ ID NO: 975, SEQ ID NO: 237, SEQ ID NO: 335, SEQ ID NO: 260, SEQ ID NO: 370, SEQ ID NO: 91, SEQ ID NO: 276, SEQ ID NO: 311, SEQ ID NO: 173, SEQ ID NO: 102, SEQ ID NO: 304, SEQ ID NO: 380, SEQ ID NO: 127, SEQ ID NO: 993, SEQ ID NO: 925, SEQ ID NO: 181, and SEQ ID NO: 171.
24. A recombinant expression vector comprising the nucleic acid of claim 23 operably linked to a transcription regulatory element. 25. A cell comprising a recombinant expression vector of claim 24.
26. A method for producing an H pylori polypeptide comprising culturing a cell of claim 25 under conditions that permit expression of the polypeptide. 27. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori surface or membrane polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026, SEQ ID NO: 1048, SEQ ID NO: 1063, SEQ ID NO: 1190, SEQ ID NO: 1083, SEQ ID NO: 1113, SEQ ID NO: 1222, SEQ ID NO: 1295, SEQ ID NO: 1343, SEQ ID NO: 1392, SEQ ID NO: 1443, SEQ ID NO: 1085, SEQ ID NO: 1093, SEQ ID NO: 1117, SEQ ID NO: 1121, SEQ ID NO: 1131, SEQ ID NO: 1287, SEQ ID NO: 1440, SEQ ID NO: 1209, SEQ ID NO: 1342, SEQ ID NO: 1381, SEQ ID NO: 1390, SEQ ID NO: 1409, SEQ ID NO: 1035, SEQ ID NO: 1014, SEQ ID NO: 1088, SEQ ID NO: 1242, SEQ ID NO: 1178, SEQ ID NO: 1089, SEQ ID NO: 1340, SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 381, SEQ ID NO: 229, SEQ ID NO: 323, SEQ ID NO: 371, SEQ ID NO: 284, SEQ ID NO: 116, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 907, SEQ ID NO: 193, SEQ ID NO: 145, SEQ ID NO: 59, SEQ ID NO: 322, SEQ ID NO: 94, SEQ ID NO: 306, SEQ ID NO: 939, SEQ ID NO: 205, SEQ ID NO: 123, SEQ ID NO: 906, SEQ ID NO: 928, SEQ ID NO: 346, SEQ ID NO: 129, SEQ ID NO: 307, SEQ ID NO: 133, SEQ ID NO: 131, SEQ ID NO: 886, SEQ ID NO: 179, SEQ ID NO: 104, SEQ ID NO: 213, SEQ ID NO: 359, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO: 327, SEQ ID NO: 365, SEQ ID NO: 33, SEQ ID NO: 331, SEQ ID NO: 175, SEQ ID NO: 200, SEQ ID NO: 292, SEQ ID NO: 23, SEQ ID NO: 336, SEQ ID NO: 301, SEQ ID NO: 28, SEQ ID NO: 941, SEQ ID NO: 103, SEQ ID NO: 231, SEQ ID NO: 176, SEQ ID NO: 31, SEQ ID NO: 917, SEQ ID NO: 151, SEQ ID NO: 922, SEQ ID NO: 265, SEQ ID NO: 142, SEQ ID NO: 259, SEQ ID NO: 122, SEQ ID NO: 206, SEQ ID NO: 96, SEQ ID NO: 353, SEQ ID NO: 38, SEQ ID NO: 89, SEQ ID NO: 77, SEQ ID NO: 954, SEQ ID NO: 264, SEQ ID NO: 937, SEQ ID NO: 226, SEQ ID NO: 283, SEQ ID NO: 88, SEQ ID NO: 125, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 81, SEQ ID NO: 901, SEQ ID NO: 82, SEQ ID NO: 42, SEQ ID NO: 881, and SEQ ID NO: 885.
28. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1060, SEQ ID NO: 1110, SEQ ID NO: 1 112, SEQ ID NO: 1230, SEQ ID NO: 1260, SEQ ID NO: 1280, SEQ ID NO: 1292, SEQ ID NO: 1296, SEQ ID NO: 1307, SEQ ID NO: 1442, SEQ ID NO: 1444, SEQ ID NO: 381, SEQ ID NO: 229, SEQ ID NO: 323, SEQ ID NO: 371, SEQ ID NO: 284, SEQ ID NO: 116, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 907, SEQ ID NO: 193, SEQ ID NO: 145, SEQ ID NO: 59, SEQ ID NO: 322, SEQ ID NO: 94, SEQ ID NO: 306, and SEQ ID NO: 881. 29. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1122, SEQ ID NO: 1254, SEQ ID NO: 1256, SEQ ID NO: 1272, SEQ ID NO: 1275, SEQ ID NO: 1309, SEQ ID NO: 1313, SEQ ID NO: 1347, SEQ ID NO: 1352, SEQ ID NO: 1356, SEQ ID NO: 1438, SEQ ID NO: 1441, SEQ ID NO: 1009, SEQ ID NO: 1026, SEQ ID NO: 1048, SEQ ID NO: 1063, SEQ ID NO: 1190, SEQ ID NO: 939, SEQ ID NO: 205, SEQ ID NO: 123, SEQ ID NO: 906, SEQ ID NO: 928, SEQ ID NO: 346, SEQ ID NO: 129, SEQ ID NO: 307, SEQ ID NO: 133, SEQ ID NO: 131, SEQ ID NO: 886, SEQ ID NO: 179, SEQ ID NO: 104, SEQ ID NO: 213, SEQ ID NO: 359, SEQ ID NO: 140, SEQ ID NO: 146, SEQ ID NO: 327, and SEQ ID NO: 365.
30. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least three membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1083, SEQ ID NO: 1113, SEQ ID NO: 1222, SEQ ID NO: 1295, SEQ ID NO: 1343, SEQ ID NO: 1392, SEQ ID NO: 1443, SEQ ID NO: 33, SEQ ID NO: 331, SEQ ID NO: 175, SEQ ID NO: 200, SEQ ID NO: 292, SEQ ID NO: 23, and SEQ ID NO: 336.
31. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1085, SEQ ID NO: 1093, SEQ ID NO: 1117, SEQ ID NO: 1121, SEQ ID NO: 1131, SEQ ID NO: 1287, SEQ ID NO: 1440, SEQ ID NO: 1209, SEQ ID NO: 301, SEQ ID NO: 28, SEQ ID NO: 941, SEQ ID NO: 103, SEQ ID NO: 231, SEQ ID NO: 176, SEQ ID NO: 31, SEQ ID NO: 917, SEQ ID NO: 151, and SEQ ID NO: 922. 32. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least five membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1342, SEQ ID NO: 1381, SEQ ID NO: 1390, SEQ ID NO: 1409, SEQ ID NO: 1035, SEQ ID NO: 265, SEQ ID NO: 142, SEQ ID NO: 259, SEQ ID NO: 122, SEQ ID NO: 206, and SEQ ID NO: 885. 33. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least six membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1014, SEQ ID NO: 1088, SEQ ID NO: 1242, SEQ ID NO: 1178, SEQ ID NO: 96, SEQ ID NO: 353, SEQ ID NO: 38, SEQ ID NO: 89, SEQ ID NO: 77, SEQ ID NO: 954, and SEQ ID NO: 264.
34. The purified nucleic acid of claim 27, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1089, SEQ ID NO: 1340, SEQ ID NO: 1074, SEQ ID NO: 1107, SEQ ID NO: 1204, SEQ ID NO: 1066, SEQ ID NO: 937, SEQ ID NO: 226, SEQ ID NO: 283, SEQ ID NO: 88, SEQ ID NO: 125, SEQ ID NO: 183, SEQ ID NO: 195, SEQ ID NO: 81, SEQ ID NO: 901, SEQ ID NO: 82, and SEQ ID NO: 42.
35. A recombinant expression vector comprising the nucleic acid of claim 27 operably linked to a transcription regulatory element. 36. A cell comprising a recombinant expression vector of claim 35.
37. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 36 under conditions that permit expression of the polypeptide. 38. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of a nucleic acid of claim 1.
39. A vaccine composition of claim 38, further comprising a pharmaceutically acceptable carrier.
40. A vaccine composition of claim 39, wherein the pharmaceutically acceptable carrier is an adjuvant.
41. A method of treating a subject for H. pylori infection comprising
administering to a subject a vaccine composition of claim 38, such that treatment of H. pylori
infection occurs.
42. A method of claim 41, wherein the treatment is a prophylactic treatment.

Claims

43. A method of claim 41, wherein the treatment is a therapeutic treatment.
44. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of a nucleic acid of claim 9.
45. A vaccine composition of claim 44, further comprising a pharmaceutically acceptable carrier.
46. A vaccine composition of claim 45, wherein the pharmaceutically acceptable carrier is an adjuvant.
47. A method of treating a subject for H. pylori infection comprising
administering to a subject a vaccine composition of claim 44, such that treatment of H. pylori
infection occurs.
48. A method of claim 47, wherein the treatment is a prophylactic treatment.
49. A method of claim 47, wherein the treatment is a therapeutic treatment.
50. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of a nucleic acid of claim 23.
51. A vaccine composition of claim 50, further comprising a pharmaceutically acceptable carrier.
52. A vaccine composition of claim 51, wherein the pharmaceutically acceptable carrier is an adjuvant. 53. A method of treating a subject for H pylori infection comprising
administering to a subject a vaccine composition of claim 50, such that treatment of H. pylori
infection occurs. 54. A method of claim 53, wherein the treatment is a prophylactic treatment.
55. A method of claim 53, wherein the treatment is a therapeutic treatment.
56. A purified H. pylori cell envelope polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812, SEQ ID NO: 1830, SEQ ID NO: 1850, SEQ ID NO: 1854, SEQ ID NO: 1851, SEQ ID NO: 1640, SEQ ID NO: 1453, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1685, SEQ ID NO: 1687, SEQ ID NO: 1688, SEQ ID NO: 1675, SEQ ID NO: 1702, SEQ ID NO: 1713, SEQ ID NO: 1600, SEQ ID NO: 1671, SEQ ID NO: 1691, SEQ ID NO: 1615, SEQ ID NO: 1616, SEQ ID NO: 1855, SEQ ID NO: 1595, SEQ ID NO: 1633, SEQ ID NO: 1608, SEQ ID NO: 1611, SEQ ID NO: 1751, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1780, SEQ ID NO: 1783, SEQ ID NO: 1796, SEQ ID NO: 1809, SEQ ID NO: 1826, SEQ ID NO: 1868, SEQ ID NO: 1734, SEQ ID NO: 1786, SEQ ID NO: 1819, SEQ ID NO: 1630, SEQ ID NO: 1706, SEQ ID NO: 1709, SEQ ID NO: 1495, SEQ ID NO: 1724, SEQ ID NO: 1670, SEQ ID NO: 1725, SEQ ID NO: 1661, SEQ ID NO: 1873, SEQ ID NO: 1753, SEQ ID NO: 1759, SEQ ID NO: 1761, SEQ ID NO: 1782, SEQ ID NO: 1883, SEQ ID NO: 1503, SEQ ID NO: 1542, SEQ ID NO: 1872, SEQ ID NO: 1520, SEQ ID NO: 1456, SEQ ID NO: 1458, SEQ ID NO: 1617, SEQ ID NO: 1628, SEQ ID NO: 1644, SEQ ID NO: 1657, SEQ ID NO: 1658, SEQ ID NO: 1755, SEQ ID NO: 1756, SEQ ID NO: 1797, SEQ ID NO: 1799, SEQ ID NO: 1801, SEQ ID NO: 1483, SEQ ID NO: 1504, SEQ ID NO: 1532, SEQ ID NO: 1575, SEQ ID NO: 1833, SEQ ID NO: 1888, SEQ ID NO: 1714, SEQ ID NO: 1624, SEQ ID NO: 1856, SEQ ID NO: 1857, SEQ ID NO: 1861, SEQ ID NO: 1537, SEQ ID NO: 1773, SEQ ID NO: 1717, SEQ ID NO: 1733, SEQ ID NO: 1722, SEQ ID NO: 1659, SEQ ID NO: 1577, SEQ ID NO: 1721, SEQ ID NO: 1729, SEQ ID NO: 1870, SEQ ID NO: 1576, SEQ ID NO: 1632, SEQ ID NO: 1867, SEQ ID NO: 1547, SEQ ID NO:
1533, SEQ ID NO: 1597, SEQ ID NO: 1596, SEQ ID NO: 1559, SEQ ID NO: 1599, SEQ ID NO: 1788, SEQ ID NO: 1789, SEQ ID NO: 1875, SEQ ID NO: 1451, SEQ ID NO: 1478, SEQ ID NO: 1626, SEQ ID NO: 1781, SEQ ID NO: 660, SEQ ID NO: 660, SEQ ID NO: 855, SEQ ID NO: 534, SEQ ID NO: 675, SEQ ID NO: 404, SEQ ID NO: 518, SEQ ID NO: 464, SEQ ID NO: 672, SEQ ID NO: 640, SEQ ID NO: 490, SEQ ID NO: 755, SEQ ID NO: 389, SEQ ID NO: 635, SEQ ID NO: 877, SEQ ID NO: 637, SEQ ID NO: 477, SEQ ID NO: 772, SEQ ID NO: 658, SEQ ID NO: 463, SEQ ID NO: 852, SEQ ID NO: 503, SEQ ID NO: 411, SEQ ID NO: 441, SEQ ID NO: 782, SEQ ID NO: 575, SEQ ID NO: 691, SEQ ID NO: 724, SEQ ID NO: 452, SEQ ID NO: 386, SEQ ID NO: 497, SEQ ID NO: 712, SEQ ID NO: 591, SEQ ID NO: 638, SEQ ID NO: 740, SEQ ID NO: 697, SEQ ID NO: 569, SEQ ID NO: 470, SEQ ID NO: 700, SEQ ID NO: 586, SEQ ID NO: 823, SEQ ID NO: 627, SEQ ID NO: 627, SEQ ID NO: 684, SEQ ID NO: 551, SEQ ID NO: 478, SEQ ID NO: 508, SEQ ID NO: 545, SEQ ID NO: 628, SEQ ID NO: 443, SEQ ID NO: 702, SEQ ID NO: 776, SEQ ID NO: 461. SEQ ID NO: 737, SEQ ID NO: 809, SEQ ID NO: 642, SEQ ID NO: 879, SEQ ID NO: 773, SEQ ID NO: 468, SEQ ID NO: 842, SEQ ID NO: 788, SEQ ID NO: 624, SEQ ID NO: 788, SEQ ID NO: 644, SEQ ID NO: 727, SEQ ID NO: 631, SEQ ID NO: 450, SEQ ID NO: 448, SEQ ID NO: 653, SEQ ID NO: 495, SEQ ID NO: 400, SEQ ID NO: 541, SEQ ID NO: 673, SEQ ID NO: 482, SEQ ID NO: 622, SEQ ID NO: 689, SEQ ID NO: 736, SEQ ID NO: 417, SEQ ID NO: 716, SEQ ID NO: 762, SEQ ID NO: 395, SEQ ID NO: 587, SEQ ID NO: 669, SEQ ID NO: 758, SEQ ID NO: 593, SEQ ID NO: 451, SEQ ID NO: 827, SEQ ID NO: 502, SEQ ID NO: 719, SEQ ID NO: 469, SEQ ID NO: 715, SEQ ID NO: 847, SEQ ID NO: 453, SEQ ID NO: 527, SEQ ID NO: 652, SEQ ID NO: 745, SEQ ID NO: 567, SEQ ID NO: 848, SEQ ID NO: 430, SEQ ID NO: 748, SEQ ID NO: 396, SEQ ID NO: 588, SEQ ID NO: 795, SEQ ID NO: 523, SEQ ID NO: 791, SEQ ID NO: 714, SEQ ID NO: 481, SEQ ID NO: 765, SEQ ID NO: 837, SEQ ID NO: 833, SEQ ID NO: 585, SEQ ID NO: 865, SEQ ID NO: 764, SEQ ID NO: 440, SEQ ID NO: 465, SEQ ID NO: 555, SEQ ID NO: 526, SEQ ID NO: 687, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 677, SEQ ID NO: 649, SEQ ID NO: 812, SEQ ID NO: 820, SEQ ID NO: 880, SEQ ID NO: 590, SEQ ID NO: 713, SEQ ID NO: 750, SEQ ID NO: 613, SEQ ID NO: 437, SEQ ID NO: 556, SEQ ID NO: 657, SEQ ID NO: 402, SEQ ID NO: 623, SEQ ID NO: 862, SEQ ID NO: 449, SEQ ID NO: 690, SEQ ID NO: 424, SEQ ID NO: 821, SEQ ID NO: 432, SEQ ID NO: 811, SEQ ID NO: 554, and SEQ ID NO: 809.
57. The purified polypeptide of claim 56, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagella-associated polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1471, SEQ ID NO: 1472, SEQ ID NO: 1487, SEQ ID NO: 1501, SEQ ID NO: 1522, SEQ ID NO: 1552, SEQ ID NO: 1586, SEQ ID NO: 1727, SEQ ID NO: 1601, SEQ ID NO: 1638, SEQ ID NO: 1643, SEQ ID NO: 1812, SEQ ID NO: 1830, SEQ ID NO: 1850, SEQ ID NO: 1854, SEQ ID NO: 1851, SEQ ID NO: 1640, SEQ ID NO: 660, SEQ ID NO: 660, SEQ ID NO: 855, SEQ ID NO: 534, SEQ ID NO: 675, SEQ ID NO: 404, SEQ ID NO: 518, SEQ ID NO: 464, SEQ ID NO: 672, SEQ ID NO: 640, SEQ ID NO: 490, SEQ ID NO: 755, SEQ ID NO: 389, SEQ ID NO: 635, SEQ ID NO: 877, SEQ ID NO: 637, SEQ ID NO: 477, SEQ ID NO: 772, and SEQ ID NO: 658.
58. The purified polypeptide of claim 56, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1453, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1685, SEQ ID NO: 1687, SEQ ID NO: 1688, SEQ ID NO: 1675, SEQ ID NO: 1702, SEQ ID NO: 1713, SEQ ID NO: 1600, SEQ ID NO: 1671, SEQ ID NO: 1691, SEQ ID NO: 1615, SEQ ID NO: 1616, SEQ ID NO: 1855, SEQ ID NO: 1595, SEQ ID NO: 1633, SEQ ID NO: 1608, SEQ ID NO: 161 1. SEQ ID NO: 1751, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1780, SEQ ID NO: 1783, SEQ ID NO: 1796, SEQ ID NO: 1809, SEQ ID NO: 1826, SEQ ID NO: 1868, SEQ ID NO: 1734, SEQ ID NO: 1786, SEQ ID NO: 1819, SEQ ID NO: 1630, SEQ ID NO: 1706, SEQ ID NO: 1709, SEQ ID NO: 1495, SEQ ID NO: 1724, SEQ ID NO: 463, SEQ ID NO: 852, SEQ ID NO: 503, SEQ ID NO: 411, SEQ ID NO: 441, SEQ ID NO: 782, SEQ ID NO: 575, SEQ ID NO: 691, SEQ ID NO: 724, SEQ ID NO: 452, SEQ ID NO: 386, SEQ ID NO: 497, SEQ ID NO: 712, SEQ ID NO: 591, SEQ ID NO: 638, SEQ ID NO: 740, SEQ ID NO: 697, SEQ ID NO: 569, SEQ ID NO: 470, SEQ ID NO: 700, SEQ ID NO: 586, SEQ ID NO: 823, SEQ ID NO: 627, SEQ ID NO: 627, SEQ ID NO: 684, SEQ ID NO: 551, SEQ ID NO: 478, SEQ ID NO: 508, SEQ ID NO: 545, SEQ ID NO: 628, SEQ ID NO: 443, SEQ ID NO: 702, SEQ ID NO: 776, SEQ ID NO: 461, SEQ ID NO: 737, SEQ ID NO: 809, SEQ ID NO: 642, SEQ ID NO: 879, SEQ ID NO: 773, SEQ ID NO: 468, SEQ ID NO: 842, SEQ ID NO: 788, SEQ ID NO: 624, SEQ ID NO: 788, SEQ ID NO: 644, SEQ ID NO: 727, SEQ ID NO: 631, SEQ ID NO: 450, SEQ ID NO: 448, and SEQ ID NO: 653.
59. The purified polypeptide of claim 56, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transporter polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1670, SEQ ID NO: 1725, SEQ ID NO: 1661, SEQ ID NO: 1873, SEQ ID NO: 1753, SEQ ID NO: 1759, SEQ ID NO:
1761, SEQ ID NO: 1782, SEQ ID NO: 1883, SEQ ID NO: 1503, SEQ ID NO: 1542, SEQ ID NO: 1872, SEQ ID NO: 1520, SEQ ID NO: 1456, SEQ ID NO: 1458, SEQ ID NO: 1617, SEQ ID NO: 1628, SEQ ID NO: 1644, SEQ ID NO: 1657, SEQ ID NO: 1658, SEQ ID NO: 1755, SEQ ID NO: 1756, SEQ ID NO: 1797, SEQ ID NO: 1799, SEQ ID NO: 1801, SEQ ID NO: 1483, SEQ ID NO: 1504, SEQ ID NO: 1532, SEQ ID NO: 1575, SEQ ID NO: 1833, SEQ ID NO: 1888, SEQ ID NO: 1714, SEQ ID NO: 495, SEQ ID NO: 400, SEQ ID NO: 541, SEQ ID NO: 673, SEQ ID NO: 482, SEQ ID NO: 622, SEQ ID NO: 689, SEQ ID NO: 736, SEQ ID NO: 417, SEQ ID NO: 716, SEQ ID NO: 762, SEQ ID NO: 395, SEQ ID NO: 587, SEQ ID NO: 669, SEQ ID NO: 758, SEQ ID NO: 593, SEQ ID NO: 451, SEQ ID NO: 827, SEQ ID NO: 502, SEQ ID NO: 719, SEQ ID NO: 469, SEQ ID NO: 715, SEQ ID NO: 847, SEQ ID NO: 453, SEQ ID NO: 527, SEQ ID NO: 652, SEQ ID NO: 745, SEQ ID NO: 567, SEQ ID NO: 848, SEQ ID NO: 430, SEQ ID NO: 748, SEQ ID NO: 396, SEQ ID NO: 588, SEQ ID NO: 795, SEQ ID NO: 523, SEQ ID NO: 791, SEQ ID NO: 714, SEQ ID NO: 481, and SEQ ID NO: 765.
60. The purified polypeptide of claim 56, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1624, SEQ ID NO: 1856, SEQ ID NO: 1857, SEQ ID NO: 1861, SEQ ID NO: 1537, SEQ ID NO: 1773, SEQ ID NO: 1717, SEQ ID NO: 1733, SEQ ID NO: 1722, SEQ ID NO: 1659, SEQ ID NO: 1577, SEQ ID NO: 1721, SEQ ID NO: 1729, SEQ ID NO: 1870, SEQ ID NO: 1576, SEQ ID NO: 1632, SEQ ID NO: 1867, SEQ ID NO: 1547, SEQ ID NO: 1533, SEQ ID NO: 1597, SEQ ID NO: 1596, SEQ ID NO: 1559, SEQ ID NO: 1599, SEQ ID NO: 1788, SEQ ID NO: 1789, SEQ ID NO: 1875, SEQ ID NO: 1451, SEQ ID NO: 1478, SEQ ID NO: 1626, SEQ ID NO: 1781, SEQ ID NO: 837, SEQ ID NO: 833, SEQ ID NO: 585, SEQ ID NO: 865, SEQ ID NO: 764, SEQ ID NO: 440, SEQ ID NO: 465, SEQ ID NO: 555, SEQ ID NO: 526, SEQ ID NO: 687, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 677, SEQ ID NO: 649, and SEQ ID NO: 812.
61. A purified H. pylori cytoplasmic polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485, SEQ ID NO: 1459, SEQ ID NO: 1512, SEQ ID NO: 1515, SEQ ID NO: 1642, SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866, SEQ ID NO: 1886, SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1737, SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1728, SEQ ID NO: 1489, SEQ ID NO: 1708, SEQ ID NO: 1808, SEQ ID NO: 1887, SEQ ID NO: 1498, SEQ ID NO: 1506, SEQ ID NO: 1592, SEQ ID NO: 1678, SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636, SEQ ID NO: 1639, SEQ ID NO: 1649, SEQ ID NO: 1669, SEQ ID NO: 1695, SEQ ID NO: 1757, SEQ ID NO: 1776, SEQ ID NO: 1848, SEQ ID NO: 1849, SEQ ID NO: 1858, SEQ ID NO: 1884, SEQ ID NO: 1667, SEQ ID NO: 1690, SEQ ID NO: 1813, SEQ ID NO: 1468, SEQ ID NO: 1470, SEQ ID NO: 1811, SEQ ID NO: 1874, SEQ ID NO: 1876, SEQ ID NO: 1825, SEQ ID NO: 1479, SEQ ID NO: 1488, SEQ ID NO: 1528, SEQ ID NO: 1566, SEQ ID NO: 1683, SEQ ID NO: 1692, SEQ ID NO: 1718, SEQ ID NO: 1614, SEQ ID NO: 1519, SEQ ID NO: 1476, SEQ ID NO: 1493, SEQ ID NO: 1497, SEQ ID NO: 1507, SEQ ID NO: 1490, SEQ ID NO: 1523, SEQ ID NO: 1524, SEQ ID NO: 1543, SEQ ID NO: 1551, SEQ ID NO: 1553, SEQ ID NO: 1554, SEQ ID NO: 1555, SEQ ID NO: 1562, SEQ ID NO: 1570, SEQ ID NO: 1587, SEQ ID NO: 1588, SEQ ID NO: 1591, SEQ ID NO: 1593, SEQ ID NO: 1684, SEQ ID NO: 1689, SEQ ID NO: 1694, SEQ ID NO: 1696, SEQ ID NO: 1698, SEQ ID NO: 1700, SEQ ID NO: 1712, SEQ ID NO: 1720, SEQ ID NO: 1730, SEQ ID NO: 1735, SEQ ID NO: 1741, SEQ ID NO: 1748, SEQ ID NO: 1779, SEQ ID NO: 1821, SEQ ID NO: 1823, SEQ ID NO: 1828, SEQ ID NO: 1834, SEQ ID NO: 1835, SEQ ID NO: 1836, SEQ ID NO: 1839, SEQ ID NO: 1852, SEQ ID NO: 1853, SEQ ID NO: 1869, SEQ ID NO: 1871, SEQ ID NO: 1878, SEQ ID NO: 1521, SEQ ID NO: 1602, SEQ ID NO: 1627, SEQ ID NO: 1450, SEQ ID NO: 1457, SEQ ID NO: 1463, SEQ ID NO: 1469, SEQ ID NO: 1481, SEQ ID NO: 1484, SEQ ID NO: 1492, SEQ ID NO: 1500, SEQ ID NO: 1505, SEQ ID NO: 1508, SEQ ID NO: 1541, SEQ ID NO: 1548, SEQ ID NO: 1580, SEQ ID NO: 1590, SEQ ID NO: 1594, SEQ ID NO: 1603, SEQ ID NO: 1604, SEQ ID NO: 1606, SEQ ID NO: 1612, SEQ ID NO: 1613, SEQ ID NO: 1620, SEQ ID NO: 1621, SEQ ID NO: 1622, SEQ ID NO: 1631, SEQ ID NO: 1645, SEQ ID NO: 1646, SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, SEQ ID NO: 1653, SEQ ID NO: 1656, SEQ ID NO: 1763, SEQ ID NO: 1787, SEQ ID NO: 1800, SEQ ID NO: 1806, SEQ ID NO: 1810, SEQ ID NO: 1864, SEQ ID NO: 1877, SEQ ID NO: 1881, SEQ ID NO: 390, SEQ ID NO: 876, SEQ ID NO: 547, SEQ ID NO: 678, SEQ ID NO: 729, SEQ ID NO: 786, SEQ ID NO: 654, SEQ ID NO: 734, SEQ ID NO: 646, SEQ ID NO: 522, SEQ ID NO: 696, SEQ ID NO: 807, SEQ ID NO: 683, SEQ ID NO: 790, SEQ ID NO: 763, SEQ ID NO: 806, SEQ ID NO: 799, SEQ ID NO: 434, SEQ ID NO: 743, SEQ ID NO: 804, SEQ ID NO: 733, SEQ ID NO: 826, SEQ ID NO: 562, SEQ ID NO: 420, SEQ ID NO: 664, SEQ ID NO: 850, SEQ ID NO: 857, SEQ ID NO: 861, SEQ ID NO: 872, SEQ ID NO: 544, SEQ ID NO: 830, SEQ ID NO: 446, SEQ ID NO: 397, SEQ ID NO: 699, SEQ ID NO: 459, SEQ ID NO: 509, SEQ ID NO: 818, SEQ ID NO: 488, SEQ ID NO: 438, SEQ ID NO: 831, SEQ ID NO: 667, SEQ ID NO: 429, SEQ ID NO: 680, SEQ ID NO: 597, SEQ ID NO: 460, SEQ ID NO: 709, SEQ ID NO: 822, SEQ ID NO: 466, SEQ ID NO: 584, SEQ ID NO: 388, SEQ ID NO: 631, SEQ ID NO: 787, SEQ ID NO: 532, SEQ ID NO: 619, SEQ ID NO: 723, SEQ ID NO: 641, SEQ ID NO: 698, SEQ ID NO: 630, SEQ ID NO: 869, SEQ ID NO: 601, SEQ ID NO: 415, SEQ ID NO: 542, SEQ ID NO: 704, SEQ ID NO: 572, SEQ ID NO: 467, SEQ ID NO: 399, SEQ ID NO: 579, SEQ ID NO: 739, SEQ ID NO: 849, SEQ ID NO: 824, SEQ ID NO: 871, SEQ ID NO: 547, SEQ ID NO: 633, SEQ ID NO: 695, SEQ ID NO: 405, SEQ ID NO: 394, SEQ ID NO: 761, SEQ ID NO: 574, SEQ ID NO: 596, SEQ ID NO: 832, SEQ ID NO: 651, SEQ ID NO: 867, SEQ ID NO: 614, SEQ ID NO: 401, SEQ ID NO: 393, SEQ ID NO: 413, SEQ ID NO: 835, SEQ ID NO: 863, SEQ ID NO: 458, SEQ ID NO: 701, SEQ ID NO: 531, SEQ ID NO: 550, SEQ ID NO: 439, SEQ ID NO: 516, SEQ ID NO: 802, SEQ ID NO: 581, SEQ ID NO: 535, SEQ ID NO: 578, SEQ ID NO: 492, SEQ ID NO: 858, SEQ ID NO: 720, SEQ ID NO: 813, SEQ ID NO: 426, SEQ ID NO: 834, SEQ ID NO: 609, SEQ ID NO: 489, SEQ ID NO: 480, SEQ ID NO: 406, SEQ ID NO: 392, SEQ ID NO: 456, SEQ ID NO: 707, SEQ ID NO: 533, SEQ ID NO: 728, SEQ ID NO: 769, SEQ ID NO: 671, SEQ ID NO: 602, SEQ ID NO: 618, SEQ ID NO: 618, SEQ ID NO: 682, SEQ ID NO: 524, SEQ ID NO: 802, SEQ ID NO: 785, SEQ ID NO: 457, SEQ ID NO: 781, SEQ ID NO: 473, SEQ ID NO: 384, SEQ ID NO: 726, SEQ ID NO: 817, SEQ ID NO: 498, SEQ ID NO: 436, SEQ ID NO: 815, SEQ ID NO: 856, SEQ ID NO: 650, SEQ ID NO: 844, SEQ ID NO: 580, SEQ ID NO: 783, SEQ ID NO: 416, SEQ ID NO: 741, SEQ ID NO: 442, SEQ ID NO: 803, SEQ ID NO: 520, SEQ ID NO: 566, SEQ ID NO: 557, SEQ ID NO: 706, SEQ ID NO: 710, SEQ ID NO: 487, SEQ ID NO: 603, SEQ ID NO: 472, SEQ ID NO: 476, SEQ ID NO: 770, SEQ ID NO: 841, SEQ ID NO: 768, SEQ ID NO: 839, SEQ ID NO: 560, SEQ ID NO: 796, SEQ ID NO: 483, SEQ ID NO: 634, SEQ ID NO: 445, SEQ ID NO: 853, SEQ ID NO: 525, SEQ ID NO: 798, SEQ ID NO: 549, SEQ ID NO: 836, SEQ ID NO: 589, SEQ ID NO: 760, SEQ ID NO: 462, SEQ ID NO: 789, SEQ ID NO: 507, SEQ ID NO: 828, SEQ ID NO: 866, SEQ ID NO: 754, SEQ ID NO: 730, SEQ ID NO: 617, SEQ ID NO: 455, SEQ ID NO: 873, SEQ ID NO: 435, SEQ ID NO: 766, SEQ ID NO: 793, SEQ ID NO: 742, SEQ ID NO: 599, SEQ ID NO: 854, and SEQ ID NO: 632.
62. The purified polypeptide of claim 61 , wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in energy conversion selected from the group consisting of SEQ ID NO: 1598, SEQ ID NO: 1739, SEQ ID NO: 1775, SEQ ID NO: 1814, SEQ ID NO: 390, SEQ ID NO: 876, SEQ ID NO: 547, SEQ ID NO: 678. 63. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in amino acid metabolism selected from the group consisting of SEQ ID NO: 1448, SEQ ID NO: 1466, SEQ ID NO: 1535, SEQ ID NO: 1545, SEQ ID NO: 1550, SEQ ID NO: 1680, SEQ ID NO: 1701, SEQ ID NO: 1719, SEQ ID NO: 1744, SEQ ID NO: 1790, SEQ ID NO: 1859, SEQ ID NO: 1880, SEQ ID NO: 1885, SEQ ID NO: 1679, SEQ ID NO: 1482, SEQ ID NO: 1485, SEQ ID NO: 1459, SEQ ID NO: 729, SEQ ID NO: 786, SEQ ID NO: 654, SEQ ID NO: 734, SEQ ID NO: 646, SEQ ID NO: 522, SEQ ID NO: 696, SEQ ID NO: 807, SEQ ID NO: 683, SEQ ID NO: 790, SEQ ID NO: 763, SEQ ID NO: 806, SEQ ID NO: 799, SEQ ID NO: 434, SEQ ID NO: 743, SEQ ID NO: 804, SEQ ID NO: 733.
64. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide or a fragment thereof involved in nucleotide metabolism selected from the group consisting of SEQ ID NO: 1512, SEQ ID NO: 1515, SEQ ID NO: 1642, SEQ ID NO: 1668, SEQ ID NO: 1816, SEQ ID NO: 1845, SEQ ID NO: 1865, SEQ ID NO: 1866, SEQ ID NO: 1886, SEQ ID NO: 1509, SEQ ID NO: 1510, SEQ ID NO: 826, SEQ ID NO: 562, SEQ ID NO: 420, SEQ ID NO: 664, SEQ ID NO: 850, SEQ ID NO: 857, SEQ ID NO: 861, SEQ ID NO: 872, SEQ ID NO: 544, SEQ ID NO: 830, and SEQ ID NO: 446.
65. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in carbohydrate metabolism selected from the group consisting of SEQ ID NO: 1531, SEQ ID NO: 1579, SEQ ID NO: 1584, SEQ ID NO: 1662, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1737, SEQ ID NO: 1740, SEQ ID NO: 1742, SEQ ID NO: 1754, SEQ ID NO: 1847, SEQ ID NO: 1447, SEQ ID NO: 397, SEQ ID NO: 699, SEQ ID NO: 459, SEQ ID NO: 509, SEQ ID NO: 818, SEQ ID NO: 488, SEQ ID NO: 438, SEQ ID NO: 831, SEQ ID NO: 667, SEQ ID NO: 429, SEQ ID NO: 680, and SEQ ID NO: 597. 66. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in cofactor metabolism selected from the group consisting of SEQ ID NO: 1546, SEQ ID NO: 1607, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1728, SEQ ID NO: 1489, SEQ ID NO: 460, SEQ ID NO: 709, SEQ ID NO: 822, SEQ ID NO: 466, SEQ ID NO: 584, and SEQ ID NO: 388.
67. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in lipid metabolism selected from the group consisting of SEQ ID NO: 1708, SEQ ID NO: 1808, SEQ ID NO: 1887, SEQ ID NO: 631, SEQ ID NO: 787, and SEQ ID NO:
532.
68. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in mRNA translation and ribosome biogenesis selected from the group consisting of SEQ ID NO: 1498, SEQ ID NO: 1506, SEQ ID NO: 1592, SEQ ID NO: 1678, SEQ ID NO: 1778, SEQ ID NO: 1863, SEQ ID NO: 619, SEQ ID NO: 723, SEQ ID NO: 641, SEQ ID NO: 698, SEQ ID NO: 630, and SEQ ID NO: 869. 69. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in genome replication, transcription, recombination and repair selected from the group consisting of SEQ ID NO: 1454, SEQ ID NO: 1538, SEQ ID NO: 1567, SEQ ID NO: 1581, SEQ ID NO: 1583, SEQ ID NO: 1636, SEQ ID NO: 1639, SEQ ID NO: 1649, SEQ ID NO: 1669, SEQ ID NO: 1695, SEQ ID NO: 1757, SEQ ID NO: 1776, SEQ ID NO: 1848, SEQ ID NO: 1849, SEQ ID NO: 1858, SEQ ID NO: 1884, SEQ ID NO: 601, SEQ ID NO: 415, SEQ ID NO: 542, SEQ ID NO: 704, SEQ ID NO: 572, SEQ ID NO: 467, SEQ ID NO: 399, SEQ ID NO: 579, SEQ ID NO: 739, SEQ ID NO: 849, SEQ ID NO: 824, SEQ ID NO: 871, SEQ ID NO: 547, SEQ ID NO: 633, SEQ ID NO: 695, SEQ ID NO: 405, SEQ ID NO: 394, and SEQ ID NO: 761.
70. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof involved in outer membrane or cell wall biosynthesis selected from the group consisting of SEQ ID NO: 1667, SEQ ID NO: 1690, SEQ ID NO: 1813, SEQ ID NO: 1468, SEQ ID NO: 1470, SEQ ID NO: 1811, SEQ ID NO: 574, SEQ ID NO: 596, SEQ ID NO: 832, SEQ ID NO: 651, SEQ ID NO: 867, SEQ ID NO: 614, SEQ ID NO: 401, and SEQ ID NO: 393.
71. The purified polypeptide of claim 61, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori chaperone polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 1874, SEQ ID NO: 1876, SEQ ID NO: 1825, SEQ ID NO: 413, SEQ ID NO: 835, and SEQ ID NO: 863.
72. A purified H. pylori secreted or periplasmic polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO:
1455, SEQ ID NO: 1589, SEQ ID NO: 1518, SEQ ID NO: 1529, SEQ ID NO: 1765, SEQ ID NO: 1770, SEQ ID NO: 1829, SEQ ID NO: 1556, SEQ ID NO: 1565, SEQ ID NO: 1569, SEQ ID NO: 1571, SEQ ID NO: 1574, SEQ ID NO: 1578, SEQ ID NO: 1663, SEQ ID NO: 1674, SEQ ID NO: 1676, SEQ ID NO: 1697, SEQ ID NO: 1699, SEQ ID NO: 1710, SEQ ID NO: 1715, SEQ ID NO: 1716, SEQ ID NO: 1732, SEQ ID NO: 1736, SEQ ID NO: 1745, SEQ ID NO: 1749, SEQ ID NO: 1750, SEQ ID NO: 1766, SEQ ID NO: 1767, SEQ ID NO: 1768, SEQ ID NO: 1769, SEQ ID NO: 1795, SEQ ID NO: 1802, SEQ ID NO: 1804, SEQ ID NO: 1824, SEQ ID NO: 1831, SEQ ID NO: 1838, SEQ ID NO: 1840, SEQ ID NO: 1844, SEQ ID NO: 1862, SEQ ID NO: 1879, SEQ ID NO: 1882, SEQ ID NO: 1890, SEQ ID NO: 1494, SEQ ID NO: 1634, SEQ ID NO: 1635, SEQ ID NO: 1647, SEQ ID NO: 1648, SEQ ID NO: 1654, SEQ ID NO: 1446, SEQ ID NO: 1449, SEQ ID NO: 1452, SEQ ID NO: 1473, SEQ ID NO: 1474, SEQ ID NO: 1480, SEQ ID NO: 1491, SEQ ID NO: 1502, SEQ ID NO: 1513, SEQ ID NO: 1605, SEQ ID NO: 1771, SEQ ID NO: 1526, SEQ ID NO: 1557, SEQ ID NO: 1560, SEQ ID NO: 1585, SEQ ID NO: 1672, SEQ ID NO: 1677, SEQ ID NO: 1686, SEQ ID NO: 1752, SEQ ID NO: 1762, SEQ ID NO: 1777, SEQ ID NO: 1792, SEQ ID NO: 1805, SEQ ID NO: 1815, SEQ ID NO: 1817, SEQ ID NO: 1827, SEQ ID NO: 1842, SEQ ID NO: 1846, SEQ ID NO: 1896, SEQ ID NO: 1530, SEQ ID NO: 1637, SEQ ID NO: 1461, SEQ ID NO: 1467, SEQ ID NO: 1623, SEQ ID NO: 1625, SEQ ID NO: 530, SEQ ID NO: 708, SEQ ID NO: 414, SEQ ID NO: 694, SEQ ID NO: 703, SEQ ID NO: 721, SEQ ID NO: 749, SEQ ID NO: 685, SEQ ID NO: 444, SEQ ID NO: 606, SEQ ID NO: 582, SEQ ID NO: 621, SEQ ID NO: 868, SEQ ID NO: 666, SEQ ID NO: 408, SEQ ID NO: 538, SEQ ID NO: 573, SEQ ID NO: 639, SEQ ID NO: 668, SEQ ID NO: 524, SEQ ID NO: 422, SEQ ID NO: 819, SEQ ID NO: 611, SEQ ID NO: 674, SEQ ID NO: 577, SEQ ID NO: 663, SEQ ID NO: 558, SEQ ID NO: 794, SEQ ID NO: 564, SEQ ID NO: 592, SEQ ID NO: 814, SEQ ID NO: 398, SEQ ID NO: 767, SEQ ID NO: 425, SEQ ID NO: 659, SEQ ID NO: 517, SEQ ID NO: 539, SEQ ID NO: 475, SEQ ID NO: 615, SEQ ID NO: 665, SEQ ID NO: 607, SEQ ID NO: 598, SEQ ID NO: 759, SEQ ID NO: 752, SEQ ID NO: 595, SEQ ID NO: 686, SEQ ID NO: 528, SEQ ID NO: 705, SEQ ID NO: 828, SEQ ID NO: 403, SEQ ID NO: 561, SEQ ID NO: 500, SEQ ID NO: 491, SEQ ID NO: 846, SEQ ID NO: 732, SEQ ID NO: 778, SEQ ID NO: 751, SEQ ID NO: 744, SEQ ID NO: 504, SEQ ID NO: 419, SEQ ID NO: 792, SEQ ID NO: 825, SEQ ID NO: 756, SEQ ID NO: 519, SEQ ID NO: 870, SEQ ID NO: 777, SEQ ID NO: 808, SEQ ID NO: 506, SEQ ID NO: 864, SEQ ID NO: 655, SEQ ID NO: 407, SEQ ID NO: 427, SEQ ID NO: 774, SEQ ID NO: 797, SEQ ID NO: 688, SEQ ID NO: 815, SEQ ID NO: 718, SEQ ID NO: 859, SEQ ID NO: 775, SEQ ID NO: 874, SEQ ID NO: 543, SEQ ID NO: 878, SEQ ID NO: 594, SEQ ID NO: 610, and SEQ ID NO: 600. 73. A purified H. pylori surface or membrane polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731, SEQ ID NO: 1743, SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO: 1499, SEQ ID NO: 1514, SEQ ID NO: 1641, SEQ ID NO: 1534, SEQ ID NO: 1564, SEQ ID NO: 1673, SEQ ID NO: 1746, SEQ ID NO: 1794, SEQ ID NO: 1843, SEQ ID NO: 1894, SEQ ID NO: 1536, SEQ ID NO: 1544, SEQ ID NO: 1568, SEQ ID NO: 1572, SEQ ID NO: 1582, SEQ ID NO: 1738, SEQ ID NO:
1891, SEQ ID NO: 1660, SEQ ID NO: 1793, SEQ ID NO: 1832, SEQ ID NO: 1841, SEQ ID NO: 1860, SEQ ID NO: 1486, SEQ ID NO: 1465, SEQ ID NO: 1539, SEQ ID NO: 1693, SEQ ID NO: 1629, SEQ ID NO: 1540, SEQ ID NO: 1791, SEQ ID NO: 1525, SEQ ID NO: 1558, SEQ ID NO: 1655, SEQ ID NO: 1517, SEQ ID NO: 875, SEQ ID NO: 676, SEQ ID NO: 801, SEQ ID NO: 860, SEQ ID NO: 747, SEQ ID NO: 529, SEQ ID NO: 387, SEQ ID NO: 391, SEQ ID NO: 515, SEQ ID NO: 625, SEQ ID NO: 568, SEQ ID NO: 454, SEQ ID NO: 800, SEQ ID NO: 499, SEQ ID NO: 779, SEQ ID NO: 648, SEQ ID NO: 643, SEQ ID NO: 537, SEQ ID NO: 511, SEQ ID NO: 616, SEQ ID NO: 829, SEQ ID NO: 546, SEQ ID NO: 780, SEQ ID NO: 553, SEQ ID NO: 549, SEQ ID NO: 410, SEQ ID NO: 608, SEQ ID NO: 513, SEQ ID NO: 656, SEQ ID NO: 845, SEQ ID NO: 563, SEQ ID NO: 570, SEQ ID NO: 805, SEQ ID NO: 851, SEQ ID NO: 423, SEQ ID NO: 810, SEQ ID NO: 604, SEQ ID NO: 636, SEQ ID NO: 757, SEQ ID NO: 412, SEQ ID NO: 816, SEQ ID NO: 771, SEQ ID NO: 418, SEQ ID NO: 662, SEQ ID NO: 512, SEQ ID NO: 679, SEQ ID NO: 605, SEQ ID NO: 421, SEQ ID NO: 552, SEQ ID NO: 576, SEQ ID NO: 571, SEQ ID NO: 725, SEQ ID NO: 565, SEQ ID NO: 717, SEQ ID NO: 536, SEQ ID NO: 647, SEQ ID NO: 501, SEQ ID NO: 838, SEQ ID NO: 428, SEQ ID NO: 494, SEQ ID NO: 479, SEQ ID NO: 711, SEQ ID NO: 722 ,SEQ ID NO: 645, SEQ ID NO: 670, SEQ ID NO: 746, SEQ ID NO: 493, SEQ ID NO: 540, SEQ ID NO: 612, SEQ ID NO: 629, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 433, SEQ ID NO: 385, and SEQ ID NO: 409.
74. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least one membrane spanning region selected from the group consisting of SEQ ID NO: 1511, SEQ ID NO: 1561, SEQ ID NO: 1563, SEQ ID NO: 1681, SEQ ID NO: 1711, SEQ ID NO: 1731, SEQ ID NO: 1743, SEQ ID NO: 1747, SEQ ID NO: 1758, SEQ ID NO: 1893, SEQ ID NO: 1895, SEQ ID NO: 875, SEQ ID NO: 676, SEQ ID NO: 801, SEQ ID NO: 860, SEQ ID NO: 747, SEQ ID NO: 529, SEQ ID NO: 387, SEQ ID NO: 391, SEQ ID NO: 515, SEQ ID NO: 625, SEQ ID NO: 568, SEQ ID NO: 454, SEQ ID NO: 800, SEQ ID NO: 499, SEQ ID NO: 779, and SEQ ID NO: 385.
75. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least two membrane spanning regions selected from the group consisting of SEQ ID NO: 1573, SEQ ID NO: 1705, SEQ ID NO: 1707, SEQ ID NO: 1723, SEQ ID NO: 1726, SEQ ID NO: 1760, SEQ ID NO: 1764, SEQ ID NO: 1798, SEQ ID NO: 1803, SEQ ID NO: 1807, SEQ ID NO: 1889, SEQ ID NO: 1892, SEQ ID NO: 1460, SEQ ID NO: 1477, SEQ ID NO: 1499, SEQ ID NO: 1514, SEQ ID NO: 1641, SEQ ID NO: 648, SEQ ID NO: 643, SEQ ID NO: 537, SEQ ID NO: 511, SEQ ID NO: 616, SEQ ID NO: 829, SEQ ID NO: 546, SEQ ID NO: 780, SEQ ID NO: 553, SEQ ID NO: 549, SEQ ID NO: 410, SEQ ID NO: 608, SEQ ID NO: 513, SEQ ID NO: 656, SEQ ID NO: 845, SEQ ID NO: 563, SEQ ID NO: 570, SEQ ID NO: 805, and SEQ ID NO: 851. 76. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least three membrane spanning regions selected from the group consisting of SEQ ID NO: 1534, SEQ ID NO: 1564, SEQ ID NO: 1673, SEQ ID NO: 1746, SEQ ID NO: 1794, SEQ ID NO: 1843, SEQ ID NO: 1894, SEQ ID NO: 423, SEQ ID NO: 810, SEQ ID NO: 604, SEQ ID NO: 636, SEQ ID NO: 757, SEQ ID NO: 412, and SEQ ID NO: 816.
77. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least four membrane spanning regions selected from the group consisting of SEQ ID NO: 1536, SEQ ID NO: 1544, SEQ ID NO: 1568, SEQ ID NO: 1572, SEQ ID
NO: 1582, SEQ ID NO: 1738, SEQ ID NO: 1891, SEQ ID NO: 1660, SEQ ID NO: 771,
SEQ ID NO: 418, SEQ ID NO: 662, SEQ ID NO: 512, SEQ ID NO: 679, SEQ ID NO:
605, SEQ ID NO: 421, SEQ ID NO: 552, SEQ ID NO: 576, and SEQ ID NO: 571.
78. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least five membrane spanning regions selected from the group consisting of SEQ ID NO: 1793, SEQ ID NO: 1832, SEQ ID NO: 1841, SEQ ID NO: 1860, SEQ ID NO: 1486, SEQ ID NO: 725, SEQ ID NO: 565, SEQ ID NO: 717, SEQ ID NO: 536, SEQ ID NO: 647, SEQ ID NO: 409.
79. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least six membrane spanning regions selected from the group consisting of SEQ ID NO: 1465, SEQ ID NO: 1539, SEQ ID NO: 1693, SEQ ID NO: 1629, SEQ ID NO: 501, SEQ ID NO: 838, SEQ ID NO: 428, SEQ ID NO: 494, SEQ ID NO: 479, SEQ ID NO: 711, and SEQ ID NO: 722. 80. The purified polypeptide of claim 73, wherein said H. pylori surface or membrane polypeptide or a fragment thereof is an H. pylori polypeptide or a fragment thereof having at least seven membrane spanning regions selected from the group consisting of SEQ ID NO: 1540, SEQ ID NO: 1791, SEQ ID NO: 1525, SEQ ID NO: 1558, SEQ ID NO: 1655, SEQ ID NO: 1517, SEQ ID NO: 645, SEQ ID NO: 670, SEQ ID NO: 746, SEQ ID NO: 493, SEQ ID NO: 540, SEQ ID NO: 612, SEQ ID NO: 629, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, and SEQ ID NO: 433.
81. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an H. pylori polypeptide of claim 56.
82. A vaccine composition of claim 81, further comprising a pharmaceutically acceptable carrier.
83. A vaccine composition of claim 82, wherein the pharmaceutically acceptable carrier is an adjuvant.
84. A method of treating a subject for H. pylori infection comprising
administering to a subject a vaccine composition of claim 81, such that treatment of H. pylori
infection occurs.
85. A method of claim 84, wherein the treatment is a prophylactic treatment.
86. A method of claim 84, wherein the treatment is a therapeutic treatment.
87. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an H. pylon polypeptide of claim 61.
88. A vaccine composition of claim 87, further comprising a pharmaceutically acceptable carrier.
89. A vaccine composition of claim 88, wherein the pharmaceutically acceptable carrier is an adjuvant. 90. A method of treating a subject for H. pylori infection comprising
administering to a subject a vaccine composition of claim 87, such that treatment of H. pylori
infection occurs. 91. A method of claim 90, wherein the treatment is a prophylactic treatment.
92. A method of claim 90, wherein the treatment is a therapeutic treatment.
93. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an H. pylon polypeptide of claim 72.
94. A vaccine composition of claim 93, further comprising a pharmaceutically acceptable carrier. 95. A vaccine composition of claim 94, wherein the pharmaceutically acceptable carrier is an adjuvant.
96. A method of treating a subject for H. pylori infection comprising
administering to a subject a vaccine composition of claim 93, such that treatment of H. pylori
infection occurs.
97. A method of claim 96, wherein the treatment is a prophylactic treatment.
98. A method of claim 96, wherein the treatment is a therapeutic treatment.
99. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an H. pylon polypeptide of claim 73.
100. A vaccine composition of claim 99, further comprising a pharmaceutically acceptable carrier.
101. A vaccine composition of claim 100, wherein the pharmaceutically acceptable carrier is an adjuvant.
102. A method of treating a subject for H. pylori infection comprising administering to a subject a vaccine composition of claim 99, such that treatment of H. pylori
infection occurs.
103. A method of claim 102, wherein the treatment is a prophylactic treatment.
104. A method of claim 102, wherein the treatment is a therapeutic treatment.
105. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising:
(a) contacting a sample with a nucleic acid of claim 1 under conditions in which a hybrid can form between the probe and a Helicobacter nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a Helicobacter nucleic acid in the sample.
106. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising:
(a) contacting a sample with a nucleic acid of claim 9 under conditions in which a hybrid can form between the probe and a Helicobacter nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a Helicobacter nucleic acid in the sample.
107. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising: (a) contacting a sample with a nucleic acid of claim 23 under conditions in which a hybrid can form between the probe and a Helicobacter nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a Helicobacter nucleic acid in the sample.
108. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising:
(a) contacting a sample with a nucleic acid of claim 27 under conditions in which a hybrid can form between the probe and a Helicobacter nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a Helicobacter nucleic acid in the sample.
PCT/US1996/009122 1995-06-07 1996-06-06 Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics WO1996040893A1 (en)

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EE9700373A EE9700373A (en) 1995-06-07 1996-06-06 Nucleic acid and amino acid sequences associated with Helicobacter pylori for diagnosis and treatment
AU63278/96A AU710880C (en) 1995-06-07 1996-06-06 Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics
NZ311874A NZ311874A (en) 1995-06-07 1996-06-06 Recombinant and/or pure preparations of H. pylori polypeptides and nucleic acids which are useful for diagnostics and vaccines
JP9501519A JPH11504220A (en) 1995-06-07 1996-06-06 Helicobacter pylori related nucleic acid and amino acid sequences for diagnostic and therapeutic agents
SK1651-97A SK165197A3 (en) 1995-06-07 1996-06-06 Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics
BR9609430A BR9609430A (en) 1995-06-07 1996-06-06 µ nucleic acid recombinant expression cell vector polypeptide vaccine composition and processes for producing a h pylori polypeptide to treat an individual for h pylori infection and to detect the presence of acid nuclei of heliocobacter in a sample
EP96922393A EP0842270A4 (en) 1995-06-07 1996-06-06 Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics
IS4630A IS4630A (en) 1995-06-07 1997-12-05 Nucleic and amino acid sequences for Helicobacter Pylori for diagnostic and therapeutic purposes
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