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WO1997037044A1 - Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof - Google Patents

Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof Download PDF

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Publication number
WO1997037044A1
WO1997037044A1 PCT/US1997/005223 US9705223W WO9737044A1 WO 1997037044 A1 WO1997037044 A1 WO 1997037044A1 US 9705223 W US9705223 W US 9705223W WO 9737044 A1 WO9737044 A1 WO 9737044A1
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WIPO (PCT)
Prior art keywords
seq
pylori
polypeptide
fragment
nucleic acid
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Ceased
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PCT/US1997/005223
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French (fr)
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WO1997037044A9 (en
Inventor
Douglas Smith
Richard A. Alm
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AstraZeneca AB
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Astra AB
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Filing date
Publication date
Priority to EE9800334A priority Critical patent/EE9800334A/en
Priority to HU0100267A priority patent/HUP0100267A3/en
Priority to JP9529649A priority patent/JP2000501621A/en
Priority to EP97917731A priority patent/EP0901530A1/en
Priority to IL12580897A priority patent/IL125808A0/en
Priority to BR9708456A priority patent/BR9708456A/en
Priority to SK1305-98A priority patent/SK130598A3/en
Priority to NZ332565A priority patent/NZ332565A/en
Application filed by Astra AB filed Critical Astra AB
Priority to AU25984/97A priority patent/AU726892B2/en
Publication of WO1997037044A1 publication Critical patent/WO1997037044A1/en
Priority to IS4831A priority patent/IS4831A/en
Priority to NO984517A priority patent/NO984517L/en
Anticipated expiration legal-status Critical
Publication of WO1997037044A9 publication Critical patent/WO1997037044A9/en
Ceased legal-status Critical Current

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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
  • 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 (Eaton et al., (1991) Infect. Immunol. 59: 2470-2475;
  • 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.
  • 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 against 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.
  • Figure 5 is a graph depicting the activity of recombinant PPIase.
  • Figure 6 is a graph depicting PPIase activity in an H. pylori extract.
  • Figure 7 is a graph depicting a decrease of glutamate racemase activity with L- Serine-O-Sulfate.
  • Figure 8 depicts the amino acid sequence alignment in a portion of the sequence of 12 H. pylori proteins (depicted in the single letter amino acid code and designated by their amino acid Sequence ID Numbers; shown N-terminal to C-terminal, left to right).
  • Figure 9 depicts the N-terminal portion of nine H. pylori proteins (depicted in the single letter amino acid code and designated by their amino acid Sequence ID Numbers; shown N-terminal to C-terminal, left to right).
  • the invention features a recombinant or substantially pure preparation of H. pylori polypeptide of SEQ ID NO: 492.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 492, such nucleic acid is contained in SEQ ID NO: 1.
  • the H. pylori polypeptide sequences described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides 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: 492 through SEQ ID NO: 541.
  • 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: 492 through SEQ ID NO: 541 , 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: 542 through SEQ ID NO: 591.
  • 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: 542 through SEQ ID NO: 591, 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: 592 through SEQ ID NO: 641.
  • 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: 592 through SEQ ID NO: 641 , such nucleic acids are contained in SEQ ID NO: 101 through SEQ ID NO: 150.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H .
  • 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: 642 through SEQ ID NO: 691, 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: 692 through SEQ ID NO: 741.
  • 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: 692 through SEQ ID NO: 741, such nucleic acids are contained in SEQ ID NO: 201 through SEQ ID NO: 250.
  • 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: 742 through SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765 through SEQ ID NO: 791.
  • 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: 742 through SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765 through SEQ ID NO: 791 , such nucleic acids are contained in SEQ ID NO: 251 through SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, and SEQ ID NO: 274 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: 792 through SEQ ID NO: 818 and SEQ ID NO: 820 through SEQ ID NO: 841.
  • 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: 792 through SEQ ID NO: 818 and SEQ ID NO: 820 through SEQ ID NO: 841, such nucleic acids are contained in SEQ ID NO: 301 through SEQ ID NO: 327 and SEQ ID NO: 329 throgh SEQ ID NO: 350.
  • 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: 842 through SEQ ID NO: 846 and SEQ ID NO: 848 through SEQ ID NO: 891.
  • 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: 842 through SEQ ID NO: 846 and SEQ ID NO: 848 through SEQ ID NO: 891, such nucleic acids are contained in SEQ ID NO: 351 through SEQ ID NO: 364 and SEQ ID NO: 366 through SEQ ID NO: 400.
  • 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: 892 through SEQ ID NO: 896 and SEQ ID NO: 898 through SEQ ID NO: 941.
  • 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: 892 through SEQ ID NO: 896 and SEQ ID NO: 898 through SEQ ID NO: 941, such nucleic acids are contained in SEQ ID NO: 401 through SEQ ID NO: 405 and SEQ ID NO: 407 through SEQ ID NO: 450.
  • 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: 942 through SEQ ID NO: 963 and SEQ ID NO: 966 through SEQ ID NO: 982.
  • 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: 942 through SEQ ID NO: 963 and SEQ ID NO: 966 through SEQ ID NO: 982, such nucleic acids are contained in SEQ ID NO: 451 through SEQ ID NO: 472 and SEQ ID NO: 475 through SEQ ID NO: 491.
  • 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: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041 through SEQ ID NO: 1087 and SEQ ID NO: 1090.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide selected from the group consisting of H.
  • SEQ ID NO: 1037 SEQ ID NO: 1038, SEQ ID NO: 1041 through SEQ ID NO: 1087 and SEQ ID NO: 1090, such nucleic acids are contained in SEQ ID NO: 983, SEQ ID NO: 984, SEQ ID NO: 987 through SEQ ID NO: 1033 and SEQ ID NO: 1036.
  • 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: 1296 through SEQ ID NO: 1298.
  • 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: 1296 through SEQ ID NO: 1298, such nucleic acids are contained in SEQ ID NO: 1293 through SEQ ID NO: 1295.
  • the invention features a recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of H. pylori polypeptides 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 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: 494.
  • this invention encompasses a recombinant or substantially pure preparation of an H. pylori polypeptide of SEQ ID NO: 494.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 494.
  • 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: 542-SEQ ID NO: 591) or nucleic acids (e.g., SEQ ID NO: 51 -SEQ ID NO: 100), as well as any subgroups from within the above- identified groups.
  • 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: 692 through SEQ ID NO: 741 can be divided into one or more subgroups as follows: SEQ ID NO: 692-SEQ ID NO: 680; SEQ ID NO: 681 -SEQ ID NO: 710; SEQ ID NO: 711-SEQ ID NO: 730; SEQ ID NO: 731-SEQ ID NO: 741 ; 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO:
  • SEQ ID NO: 1294 SEQ ID NO: 1295, SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, SEQ ID NO: 1031 , SEQ ID NO: 254, SEQ ID NO: 352, SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381, SEQ ID NO: 389, SEQ ID NO: 1010, SEQ ID NO: 1012, SEQ ID NO: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421, SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 41 1 , SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO:
  • 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1 , SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391 , SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398
  • SEQ ID NO: 352 SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381, SEQ ID NO: 389, SEQ ID NO: 1010, and SEQ ID NO: 1012.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321 , SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391 , SEQ ID NO: 392, SEQ ID NO: 3
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294, and SEQ ID NO: 1295.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, and SEQ ID NO: 1031.
  • 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: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421 , SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 411, SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO: 290, SEQ ID NO: 417, SEQ ID NO: 430, SEQ ID NO: 992, and SEQ ID NO: 1025.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 354.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421, and SEQ ID NO: 1022.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 463.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion or adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 281 and SEQ ID NO: 988.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 407 and SEQ ID NO: 1017.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 477.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transport polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 414.
  • 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: 470, SEQ ID NO: 1033, SEQ ID NO: 357, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 1030, SEQ ID NO: 345, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 455, SEQ ID NO: 1003, SEQ ID NO: 351, SEQ ID NO: 416, SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, SEQ ID NO: 1293, SEQ ID NO: 318, SEQ ID NO: 322, SEQ ID NO:
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 470 and SEQ ID NO: 1033.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 357 and SEQ ID NO: 457.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 461 and SEQ ID NO: 1030.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 345, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 455, and SEQ ID NO: 1003.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in lipid metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 351 and SEQ ID NO: 416.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, and SEQ ID NO: 1293.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 355, SEQ ID NO: 1006, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 301
  • SEQ ID NO: 442. SEQ ID NO: 451. SEQ ID NO: 471, SEQ ID NO: 478, SEQ ID NO: 488. SEQ ID NO: 987, SEQ ID NO: 990, SEQ ID NO: 991 , SEQ ID NO: 993, SEQ ID NO: 1001 , SEQ ID NO: 1002, SEQ ID NO: 1007, SEQ ID NO: 1013, SEQ ID NO: 1016, SEQ ID NO: 1018, SEQ ID NO: 1021 , and SEQ ID NO: 1026.
  • the H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 355 and SEQ ID NO: 1006.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori cellular polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 256, SEQ ID NO: 267, SEQ ID NO: 282, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 332, SEQ ID NO: 333.
  • SEQ ID NO: 336 SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341 , SEQ ID NO: 344, SEQ ID NO: 369, SEQ ID NO: 376, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 423, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481 , SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 489, SEQ ID NO: 984, SEQ ID NO: 994, SEQ ID NO: 1024, and SEQ ID NO: 1036.
  • H. pylori cell envelope polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 746, SEQ ID
  • 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: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 7
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757.
  • SEQ ID NO: 768 SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901 , SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063, SEQ ID NO: 1077, SEQ ID NO: 1297. and SEQ ID NO: 1298.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, and SEQ ID NO: 1085.
  • 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: 845, SEQ ID NO: 863, SEQ ID NO: 891 , SEQ ID NO: 912, SEQ ID NO: 1076, SEQ ID NO: 954, SEQ ID NO: 772, SEQ ID NO: 1042, SEQ ID NO: 902, SEQ ID NO: 898, SEQ ID NO: 1071, SEQ ID NO: 781, SEQ ID NO: 908, SEQ ID NO: 921, SEQ ID NO: 1046, and SEQ ID NO: 1079.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 845.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 863, SEQ ID NO: 891. SEQ ID NO: 912, and SEQ ID NO: 1076.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 954.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in secretion or adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 772 and SEQ ID NO: 1042.
  • the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof selected from the group consisting of SEQ ID NO: 898 and SEQ ID NO: 1071.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 968.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transport polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 905.
  • H. pylori cytoplasmic polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 961, SEQ ID NO: 1087, SEQ ID NO: 848, SEQ ID NO: 948, SEQ ID NO: 952, SEQ ID NO: 1084, SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, SEQ ID NO: 1057, SEQ ID NO: 842, SEQ ID NO: 907, SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841, SEQ ID NO: 910, SEQ ID NO: 951, SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, SEQ ID NO: 1296, SEQ ID NO: 809, SEQ ID NO: 813, SEQ ID NO:
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 961 and SEQ ID NO: 1087.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 848 and SEQ ID NO: 948.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 952 and SEQ ID NO: 1084.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, and SEQ ID NO: 1057.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in lipid metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 842, SEQ ID NO: 907.
  • the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof selected from the group consisting of SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841 , SEQ ID NO: 910, SEQ ID NO: 951 , SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, and SEQ ID NO: 1296.
  • H. pylori secreted polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 846, SEQ ID NO: 1060, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 751 , SEQ ID NO: 752, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 770, SEQ ID NO: 774, SEQ ID NO: 775, SEQ ID NO: 778, SEQ ID NO: 779, SEQ ID NO: 780, SEQ ID NO: 782, SEQ ID NO: 786, SEQ ID NO: 787, SEQ ID NO: 788, SEQ ID NO: 789, SEQ ID NO: 791, SEQ ID NO: 7
  • the H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 846 and SEQ ID NO: 1060. Particularly preferred is a purified or isolated H.
  • polypeptide selected from the group consisting of SEQ ID NO: 747, SEQ ID NO: 758, SEQ ID NO: 773, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801, SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 835, SEQ ID NO: 860, SEQ ID NO: 867, SEQ ID NO: 873, SEQ ID NO: 877, SEQ ID NO: 914, SEQ ID NO: 922, SEQ ID NO: 926, SEQ ID NO: 927, SEQ ID NO: 949, SEQ ID NO: 953, SEQ ID NO:
  • 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 antibody has utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori-specif ⁇ c 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 polypeptides 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% homologous to an amino acid sequence contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence 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 a polypeptide contained in the Sequence Listing.
  • the H. pylori polypeptide is encoded by a nucleic acid 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 shown 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 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 contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence 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 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%
  • the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence 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 contained in the Sequence Listing.
  • the nucleic acid is that as shown in the Sequence
  • nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%
  • 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 in the Sequence
  • 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 contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence 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 a nucleic acid contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of a nucleic acid contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of a nucleic acid contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of a nucleic acid contained in the Sequence Listing.
  • the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences shown in the Sequence Listing.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence shown in the Sequence Listing which encodes amino acids shown 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 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 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) as strain HP-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 as shown 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 secreted proteins, H. pylori cytoplasmic proteins and H. pylori cellular proteins.
  • H. pylori cell envelope proteins H. pylori secreted proteins
  • H. pylori cytoplasmic proteins H. pylori cellular proteins.
  • Polypeptides related by significant homology to the polypeptides of Table 1 are also considered to be classified in the manner of the homolog shown in Table 1.
  • a purified preparation or a substantially pure preparation of a polypeptide means 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:
  • polypeptide 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 substantially pure nucleic acid e.g., a substantially pure DNA
  • 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.
  • 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
  • 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 for 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.
  • 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
  • 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
  • Conditions of intermediate stringency such as, for example, 2X SSC at 65 ° C
  • low stringency such as, for example 2X SSC at 55° C
  • 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 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 shown 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 CHO cells. Because peptides such as H.
  • 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 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 are outlined in Table 2 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.
  • 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 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
  • 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 and terminators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers.
  • 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.
  • 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 was then determined.
  • PCR polymerase chain reaction
  • ORFs open reading frames
  • ORFs open reading frames
  • ORFs of at least 180 nucleotides were predicted.
  • 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. p yloripolypeptide.
  • 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 ORF modified to correspond to a naturally-occurring H. pylori polypeptide 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 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, amplified products can be checked by conventional sequencing methods.
  • PCR polymerase chain reaction
  • Clones carrying the desired sequences described in this invention may 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 cell line. Double stranded cDNAs can then be prepared from the total mRNA.
  • the cDNAs can be inserted into a suitable plasmid or viral (e.g..
  • 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 are shown 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.
  • 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.
  • probes, primers, capture ligands and antisense agents the nucleic acid normally consists of all or part
  • a nucleic acid isolated or synthesized in accordance with the nucleotide sequences set forth 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 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 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 shown 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 acids of ⁇ 10-15 nucleotides 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
  • 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 acids 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 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.
  • 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 and other bacterial strains such as E. coli, Norcardia,
  • 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.
  • telomere 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 intro- Jerusalem 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
  • 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 Herskowitz, (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).
  • 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).
  • COS 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 include the pAc series (Smith
  • pCDM 8 Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci. USA 84:8573-8577
  • CHO dhfr Chinese Hamster Ovary
  • 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.
  • 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, 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, NJ) 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 pETl Id 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
  • 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.
  • 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 chromatographically isolated from the membrane fraction.
  • a detergent suitable for solubilizing these complex 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 micells concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution.
  • CMC critical micells concentration
  • 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.
  • 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) 1 19-128).
  • Another strategy would be to alter the nucleic acid encoding 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. 20:2111-21 18).
  • 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).
  • This invention encompasses isolated H. pylori polypeptides encoded by the disclosed H. pylori genomic sequences, including the polypeptides contained in the Sequence Listing.
  • Polypeptides of the invention are preferably at least 5 amino acid residues in length.
  • 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 isolatedand identified based on an ORF that endoes 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.
  • 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 Tables 3-6 below. All of the polypeptide lengths in Table 3 are from stop codon to stop codon in the nucleotide sequence of H. pylori. As is known in the art, the actual polypeptide lengths are usually shorter than the stop-to-stop codon lengths because a start codon for an initiator charged tRNA usually appears a few nucleotides downstream from the prior stop codon and within a few nucleotides following a ribosome binding site (also known as a "Shine-Delgarno sequence"). Since most of the ribosome binding sites in H.
  • polypeptide sequences of S ⁇ Q ID NOs:492-743 of this invention represent the stop- to-stop codon lengths of the open reading frames of S ⁇ Q ID NOs: 1-252. All other polypeptide sequences of this invention represent the predicted start to stop protein lengths from the nucleotide sequences.
  • One skilled in the art can recognize start sites in the stop-to-stop open reading frames of the nucleotide sequences presented herein.
  • polypeptides shown in Table 3 can be described as follows. First, all of the polypeptides of Table 3 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
  • the stop-to-stop lengths are different for some of the homologous pairs of polypeptides.
  • the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility.
  • some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
  • nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another.
  • nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
  • polypeptide lengths in Table 4 below are measured from stop codon to stop codon in the nucleotide sequence of H. pylori.
  • polypeptides shown in Table 4 can be described as follows. First, all of the polypeptides of Table 4 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
  • the stop-to-stop lengths are different for some of the homologous pairs of polypeptides.
  • the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility.
  • some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
  • nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another.
  • nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
  • polypeptide lengths in Table5 below are measured from start codon to stop codon in the nucleotide sequence of H. pylori.
  • polypeptides shown in Table 5 can be described as follows. First, all of the polypeptides of Table 5 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
  • start-to-stop lengths are different for some of the homologous pairs of polypeptides.
  • the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility.
  • some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
  • nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another.
  • nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
  • polypeptide lengths in Table 6 below are measured from stop codon to stop codon in the nucleotide sequence of H. pylori.
  • polypeptides shown in Table 6 can be described as follows. First, all of the polypeptides of Table 6 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other. Second, the stop-to-stop lengths are different for the homologous pairs of polypeptides. In some cases, the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved uti lity .
  • nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another.
  • nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
  • 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 the identification of nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H. pylori. An important aspect of this identification is to determine the function of the disclosed sequences, which can be achieved using a variety of approaches. Non-limiting examples of these methods are described briefly below.
  • H. pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
  • 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.
  • S train-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, although whether or not this is correct is not essential to the invention.
  • 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.
  • 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. (1985) Biochimica et Biophysica Ada 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 5371 1) 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 better than 1x10 (ee-6)
  • Possible functions can be provided to H. pylon 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.
  • outer membrane proteins are likely to represent the best antigens to provide a protective immune response against H. pylori.
  • algorithms that can be used to aid in prediction of these outer membrane proteins include the presence of an amphipathic beta-sheet region at their C-terminus. This region which has been detected in a large number of outer membrane proteins in Gram negative bacteria is often characterized by hydrophobic residues (Phe or Tyr) approximately at positions 1, 3, 5, 7 and 9 from the C-terminus (e.g., see Figure 8, block F). Importantly, these sequences have not been detected at the C-termini of periplasmic proteins, thus allowing preliminary distinction between these classes of proteins based on primary sequence data. This phenomenon has been reported previously by Struyve et al. (J. Mol. Biol. 218:141-148, 1991).
  • FIG. 8 Also illustrated in Figure 8 are additional amino acid sequence motifs found in many outer membrane proteins of H. pylori.
  • the amino acid sequence alignment in Figure 8 depicts portions of the sequence of 12 H. pylori proteins (depicted in the single letter amino acid code) labeled with their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right.
  • Six distinct blocks (labeled A through F) of similar amino acid residues are found including the distinctive hydrophobic residues (Phe or Tyr; F or Y according to the single letter code for amino acid residues) frequently found at positions near the C-terminus of outer membrane proteins.
  • the presence of several shared motifs clearly establishes the similarity between members of this group of proteins.
  • FIG. 9 depicts the N- terminal portion of nine H. pylori proteins (designated by their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right).
  • 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 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 discovery 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.
  • 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
  • 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.
  • 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 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
  • oligonucleotide 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. 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.
  • 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.
  • 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.
  • 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/alky lation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 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).
  • H. pylori protein or peptide it is possible to add an amino acid fusion moiety to the peptide backbone.
  • an amino acid fusion moiety for example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988)
  • 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
  • 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, can be used to detect homolog 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.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages Ml 3, fd., and fl 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).
  • 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).
  • the pilus 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 may peptides 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 protease IgA of Neisseria (Hansson et al. (1992) J Bacteriol. 174, 4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991- 1999).
  • 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.
  • An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89: 1865-1869).
  • This system uses a plasmid containing the Lad 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 Lad 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.
  • LacO LacO operator
  • 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.
  • 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 Lad 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 Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.
  • 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 Lad display system.
  • 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.
  • 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.
  • This invention also features vaccine compositions for protection against infection by H pylori or for treatment of H pylori infection, a gram-negative spiral
  • 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.
  • immunogenic components such as a surface protein from H pylori, or portion thereof
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier.
  • Nucleic acids within the scope of the invention are exemplified by the nucleic acids shown in the Sequence Listing which encode H. pylori surface proteins.
  • 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
  • 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 HLA 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.
  • immunogenic components of the invention are identified through genomic vaccination.
  • the basic protocol is based on the idea that expression libraries consisting of all or parts of a pathogen genome, e.g., an H. pylori genome, can confer protection when used to genetically immunize a host.
  • This expression library immunization (ELI) is analogous to expression cloning and involves reducing a genomic expression library of a pathogen, e.g., H. pylori, into plasmids that can act as genetic vaccines.
  • the plasmids can also be designed to encode genetic adjuvants which can dramatically stimulate the humoral response. These genetic adjuvants can be introduced at remote sites and act as well extracelluraly as intracellularly.
  • An expression library of pathogen DNA is used to immunize a host thereby producing the effects of antigen presentation of a live vaccine without the risk.
  • random fragments from the H. pylori genome or from cosmid or plasmid clones, as well as PCR products from genes identified by genomic sequencing can be used to immunize a host.
  • the feasibility of this approach has been demonstrated with Mycoplasma pulmonis (Barry et al.. Nature 377:632-635, 1995), where even partial expression libraries of Mycoplasma pulmonis, a natural pathogen in rodents, provided protection against challenge from the pathogen.
  • ELI is a technique that allows for production of a non-infectious multipartite vaccine, even when little is known about pathogen's biology, because ELI uses the immune system to screen candidate genes. Once isolated, these genes can be used as genetic vaccines or for development of recombinant protein vaccines. Thus. ELI allows for production of vaccines in a systematic, largely mechanized fashion. 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.
  • 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).
  • 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.
  • 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.
  • 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 1 1637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl- sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, 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%
  • an adjuvant including, but not limited to aluminum hydrox
  • squalene/Tween 80 emulsion squalene/Tween 80 emulsion
  • cholera toxin 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.
  • Suitable delivery methods include biodegradable microcapsules or immuno-stimulating complexes (ISCOMs) 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 NaHCO3 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 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
  • compositions of the invention are usefuls 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.
  • 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.
  • 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 Harlow 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 shown 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 shown 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 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 mi croinj ection 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
  • 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 ⁇ gtl 1, ⁇ gtl8-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gtl 1 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 are examples of a subject H.
  • 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
  • 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.
  • 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.
  • 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, Springer-Verlag, NY., 1981, with minor modifications. Briefly, cells were pelleted, resuspended in TE (10 mM Tris, 1 mM EDTA, p ⁇ 7.6) and GES lysis buffer (5.1 M guanidium thiocyanate, 0.1 M EDTA, p ⁇ 8.0, 0.5% N-laurylsarcosine) was added. Suspension was chilled and ammonium acetate (N ⁇ 4 AC) 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.
  • the purified DNA fragments were then blunt-ended using T4 DNA polymerase.
  • the healed DNA was then ligated to unique BstXI-linker adopters 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.
  • 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 (Qiagen, Inc.). In this manner, 100 ⁇ g of DNA was obtained per pool. 15 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy with 250-300 base average read-lengths.
  • 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., Automated DNA Sequenicng 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 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.
  • the error rate of the REPLICATM base calling software was 2-5% with most errors occurring near the end of a sequence read. 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 a number correspond to (microtiter plate and probe information) and lane set number (corresponding to microtiter plate columns). 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 polypeptides of the invention can be performed essentially as outlined below.
  • a gene expression system such as the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, is selected.
  • a DNA sequence encoding a peptide tag, the ⁇ is-Tag is fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products.
  • the 3' end is 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.
  • 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.
  • Nucleic acids chosen for example, from the nucleic acids set forth in the Sequence Listing) for cloning from the J99 strain of H. pylori are prepared for amplification cloning by polymerase chain reaction (PCR).
  • Synthetic oligonucleotide primers specific for the 5' and 3' ends of open reading frames (ORFs) are designed and purchased from GibcoBRL Life Technologies (Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) are designed to include an Ncol cloning site at the extreme 5' terminus.
  • primers are 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.
  • All reverse primers include 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 including six histidine residues (at the extreme C-terminus), which comprise the ⁇ is-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 encodes a Bam ⁇ I site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encodes a Xhol site at its extreme 5' terminus.
  • Genomic DNA prepared from the J99 strain of H pylori is used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular
  • genomic DNA 50 nanograms is 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.
  • each sample of amplified DNA is washed and purified using the Qiaquick Spin PCR purification kit (Qiagen,
  • the pET-28b vector is prepared for cloning by digestion with endonucleases, e.g., Ncol and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • endonucleases e.g., Ncol and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • endonucleases e.g., Ncol and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • the pET-28a vector which encodes a His-Tag that can be fused to the 5' end of an inserted gene, is used and the cloning site prepared for cloning with the ppiB gene by digestion with B
  • Competent bacteria E coli strain BL21 or E. coli strain BL21(DE3), are 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).
  • 1 microliter of ligation reaction is mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples are incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples are then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight.
  • SOC medium 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose
  • Transformed colonies of BL21 are then picked and analyzed to evaluate cloned inserts as described below.
  • the pET vector can be propagated in any E. coli K-12 strain e.g. ⁇ MS174, 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 lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
  • T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its 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 DNA isolated as described above is used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit).
  • the lacZ gene (beta-galactosidase) is expressed in the pET-System as described for the H. pylori recombinant constructions.
  • Transformed cells are cultured in SOC medium for 1 hour, and the culture is then plated on LB plates containing 25 micrograms/ml kanamycin sulfate.
  • bacterial colonies are 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 are pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets are resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells are then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets are weighed and frozen at -80°C until ready for protein purification.
  • the concentrations of purified protein preparations are quantified
  • SDS-polyacrylamide gels (12% or 4.0 to 25 % acrylamide gradient gels) are purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
  • Molecular weight markers include rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (1 16 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).
  • Frozen cells are 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 % -mercaptoethanol, 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 microfluidizer (Model M-l 10S, Microfluidics International Corporation, Newton, MA).
  • lysis buffer 20 mM Tri
  • NTA Ni 2+- nitrolotriacetate-agarose
  • the column is washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and are eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and: either 20, 100, 200, or 500 mM imidazole.
  • Fractions are monitored by absorbance at OD 280 nm, and Peak fractions are analyzed by SDS-PAGE.
  • the pellets are 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 % -mercaptoethanol.
  • the resulting white pellet is composed primarily of inclusion bodies, free of unbroken cells and membranous materials.
  • Urea is 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 %
  • DOC deoxycholate
  • Purification of outer membranes form H pylon can be performed by essentially follwing the protocol outlined below.
  • H pylori strains J99 (ATCC# 55679) and Ah244 are grown on chocolate blood agar containing 5% (vol/vol) horse blood, at 37(C in an atmosphere containing 10%
  • Bacteria were harvested by suspension in 20 mM Tris, p ⁇ 7.5. The cells are collected by centrifugation at 12,000 Xg, for 20 min at 4(C and washed 3 times with 20 mM Tris, p ⁇ 7.5. Cells are suspended in 20 mM Tris, p ⁇ 7.5 and broken by sonication on ice (eight bursts of 30 s at 60 watts with 60 s pauses between bursts). DNase (0.1 mg) and RNase (0.5 mg) are added to the cell suspension, and the mixture is incubated for 30 minutes at room temperature. The cell suspension is centrifuged at 12,000 Xg for 20 min, at 4(C.
  • the supernatant was retained and centrifuged again. Total membranes are collected from the supernatant by centrifugation at 40,000 Xg for 30 minute, at 4°C. The pellet are washed twice in 20 mM Tris, pFI 7.5. The protein content is assayed using the Bradford protein assay, with bovine serum albumin (BSA) as a standard. The suspension is then adjusted to 1 mg protein /ml. The solubilization of the membranes is realized by adding N-lauryl-sarcosine to this suspension in a ratio of 6 mg of N-lauryl-sarcosine per mg of protein. The suspension is incubated for 30 minutes at room temperature in presence of N-lauryl-sarcosinc.
  • BSA bovine serum albumin
  • Outer membrane antigens can be identified using a protocol outlined below. Proteins are separated on sodium dodecyl sulfate polyacrylamide gels (SDS- PAGE) according to the method described by Laemmli, U.K. (1970) Nature (London) Volume 227, 680-685. Samples are prepared by suspension in standard treatment buffer and heated at 100°C for 10 min. Approximately 1 -5 mg of protein is loaded per well on 8X10 cm minigels (0.75 mm). The separated proteins are then transferred to PVDF membranes as described below.
  • SDS- PAGE sodium dodecyl sulfate polyacrylamide gels
  • Electroblotting of separated proteins to PVDF membranes is performed in a Bio Rad Mini-Trans Blot Electrophoretic Transfer cell.
  • the PVDF membrane Immobilon- p SQ is employed. Electroblotting is carried out for 60 min at 50V using CAPS transfer buffer (10mM 3-[Cyclohexylamino]-1-propanesulfonic acid, 10% methanol). The membrane is stained with 0.2% Ponceau S and destained with Milli Q quality water.
  • Antigens within the preparation are then identified using western
  • Alkaline phosphatase conjugated anti-mouse Ig from goat is then added in 10 mM Tris-HCl-0.9% NaCl-0.5% Tween 20-0.5% BSA, pH 7.5 and incubated for lh at room temperature. After this incubation, the membrane is washed three times in TTBS. The reactive bands are revealed using 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad) as the Alkaline phosphatase substrate and Nitro Blue Tetrazolium (Bio-Rad) as the color development reagent.
  • Bio-Rad 5-bromo-4-chloro-3-indolyl phosphate
  • Bio-Rad Nitro Blue Tetrazolium
  • proteins that are identified as immunoreactive are cut from a fresh unreacted immobilon membranes and microsequenced at the Worcester Foundation microsequencing facility. Membranes from which the protein bands are cut are then subjected to western immunoblot as described above to confirm that the appropriate band had been excised.
  • mice 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.
  • 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 8 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 9 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 ofH. 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 ⁇ 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.
  • 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.
  • 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 Proteins 3, 4, 9, 1 , and 7 (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 Protein 6, followed by 1 , 3, and 9 (see Figure 2).
  • 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
  • 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.
  • 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 (forward and reverse primers, see Table 10) 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.
  • 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.
  • Therapeutic targets were 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 were identified from the H pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs.
  • All synthetic oligonucleotide primers (Table 13) were designed with the aid of the OLIGO program (National Biosciences. Inc.. Madison, MN 55447, USA), and were purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA).
  • Specific primers Fl and RI ) were chosen which flanked most or all of the ORF, depending on its size. If the ORF was smaller than 800 to 1000 base pairs, flanking primers were chosen outside of the open reading frame.
  • Genomic DNA prepared from the Helicobacter pylori HpJ99 strain was 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
  • the PCR was carried out with Perkin Elmer Cetus/GeneAmp PCR System 9600 thermal cyclers. The thermal cycling conditions used to obtain amplified DNA products for each knock-out target are shown in Table 14.
  • each sample of amplified DNA was 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 was then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
  • PCR products were 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 was 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 was allowed to proceed for 16 hours at 16°C.
  • Ligation products were 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). Briefly, 1 microliter of ligation reaction was mixed with 40 microliters of
  • the sequences of the genes/ORFs were again used to design a second pair of primers (F2 and R2) which flanked the region ofH pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but were oriented away from each other.
  • the pool of circular plasmid DNAs of the previously isolated clones were used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers was away from each other, the portion of the ORF between the primer would not be included in the resultant PCR product.
  • the PCR product was a linear piece of DNA with H.
  • a Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology 170, 1704- 1708) was ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F.
  • the Kanamycin cassette containing a Campylobacter kanamycin resistance gene was obtained by carrying out an EcoRI digestion of the recombinant plasmid pCTBS.kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566- 10573). The proper fragment ( 1.4 kb) was isolated on a 1 % TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA).
  • the fragment was 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 was then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides.
  • the OTO overhang was then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pH 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 was purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA).
  • the PCR product of the deletion primers was 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 reaction and incubating for 16 hours at 16°C.
  • the ligation products were transformed into XL-1 Blue or DH5- ⁇ E.coli cells by electroporation as described previously. After recovery in SOC, cells were plated onto LB plates containing 100 microgram/ml Ampicilhn and grown overnight at 37°C. These plates were then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies had both the Ampicilhn resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies were picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA was isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
  • the orientation of the Kanamycin cassette relative to the H. pylori sequence was determined. Positive clones were classified as either in the "A" orientation (the same direction of transcription was present for both the H pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the Hpylori gene was opposite to that of the Kanamycin resistance gene). Clones which shared the same orientation (A or B) were pooled for subsequent experiments and independently transformed into H pylori.
  • H pylori Two strains ofH pylori were used for transformation: ATCC 55679. the clinical isolate which provided the DNA from which H pylori sequence database was obtained, and A ⁇ 244, an isolate which had been passaged in, and had the ability to colonize the mouse stomach.
  • Cells for transformation were grown at 37°C, 10% CO 2 , 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells were grown to exponential phase, and examined microscopically to determine that the cells were "healthy" (actively moving cells) and not contaminated.
  • cells were 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 were 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 was taken to determine the optical density at 600 nm, in order to calculate the concentration of cells.
  • the template for PCR was obtained by a rapid boiling DNA preparation method. An aliquot of the colony (stab of the colony with a toothpick) was 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) was added and vortexed. The mixture was microfuged for 5 minutes and the supernatant was used as DNA template for PCR with combinations of the following primers to verify
  • TEST 1 PCR with F1 and R1 primers (cloning primers originally used to amplify the gene/ORF).
  • F1 and R1 primers 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 was proof that the gene had not been knocked out and that the transformant was 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
  • primer Kan-1 or Kan-2 primers designed from the ends of the kanamycin resistance gene
  • a positive result of homologous recombination at the correct chromosomal location of the sequences of the gene/ORFs upstream from the kanamycin resistance gene should show a single PCR product, the expected size to be from the location of F3 to the insertion site of kanamycin resistance gene. No PCR product or PCR product(s) of incorrect size(s) would prove that the plasmid had not been 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
  • primer Kan-1 or Kan-2 primer used was of "A" or "B” orientation.
  • a positive result of homologous recombination at the correct chromosomal location downstream from the kanamycin resistance gene would show a single PCR product, the expected size to be 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) would prove that the plasmid had not been integrated at the correct site and that the gene had not been knocked out.
  • ORF encoded a protein with PPiase activity
  • the gene was isolated by polymerase chain reaction (PCR) amplification cloning, overexpressed in E. coli, and the protein purified to homogeneity.
  • PCR polymerase chain reaction
  • a polyhistidine tag was added to the N- terminus of this ORF.
  • a simple assay using PPIase to evaluate protein folding function was developed for future use as a high-throughput drug screen.
  • PPIases are divided into three unrelated families: the cyclophilins, the FK506-binding (FKBPs) and the parvulins.
  • FKBPs FK506-binding
  • parvulins PPIase mutants have been reported from yeast and fruit fly, attempts to isolate disruption mutants in
  • Escherichia coli were unsuccessful (Shieh, B. ⁇ ., et.al. (1989) Nature 338:67-70). This suggests that this activity is essential for viability in bacteria.
  • H. pylori a powerful gene expression system, the p ⁇ T System, for cloning and expression of recombinant ppi in ⁇ . coli.
  • the sequence for H. pylori ppi contains a DNA sequence encoding a His-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.
  • a synthetic oligonucleotide primer (5'-TTATGGATCCAAACCAATTAAAA CT-3' (S ⁇ Q ID NO: 1287)) specific for the 5' end of ppi gene encoded a BamHI site at its extreme 5' terminus and a primer (5'-TATCTCGAGTTATAGAGAAGGGC-3' (S ⁇ Q ID NO: 1288)) specific for the 3' end of the ppi gene encoded a Xhol site at its extreme 5' terminus.
  • Genomic DNA prepared from the J99 strain 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.
  • 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:
  • the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
  • the amplified DNA sample was subjected to digestion with the restriction endonucleases, BamHI and Xhol (New England BioLabs, Beverly, MA, USA) (Current Protocols in Molecular Biology, Ibid).
  • the assay for PPIase was essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Acta 43 : 1101 - 11 1 1).
  • 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 were collected every second with an average scanning of time of 0.5 second. Assays were 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 was 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 leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor.
  • Ther suspension was 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 was centrifuged (12,000 g x 30 minutes) and the supernatant was assayed for PPiase activity.
  • PPI from H. pylori was expressed in E. coli using the pET-28b expression vector from Novagen (cat # 69868-1).
  • the expressed recombinant protein was isolated from the soluble fraction of bacterial cells that had been disrupted by cavitation in a
  • Microfluidics Cell disruption chamber The expression levels of recombinant PPI produced 100 mg of protein.
  • the recombinant protein could be purified to homogeneity by Ni 2+ chelate chromatography and gel filtration. On sodium dodecyl sulfate polyacrylamide gels, the recombinant protein migrates as a single band at 21 kDa, in accordance with the predicted molecular weight of 20,975 deduced from the gene sequence.
  • the PPIase activity was assayed using the chromogenic tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
  • An initial velocity of 4.9 ⁇ mole/min/mg protein was measured with the purified enzyme (Figure 5). This corresponds to a k cat of 1.6 sec -1 which is similar to the one obtained for the E. coli PPIase (Liu, J. and Walsh, CT. (1990) Proc.Natl. Acad. Sci. USA 87:4028-4032) and the one from porcine kidney (Fischer, G. (1989) Nature 337:476-478).
  • the recombinant protein has a high catalytic efficiency of 2.06 X 10 9 M -1 s -1 when the assay is measured at 25°C. These values are one to two orders of magnitude higher than that observed for other characterized PPIases. However, in those studies, the ppiase assay was conducted at 10 C, which may account for the discrepency.
  • the calalytic efficiency is very close to the 1 X 10 8 to 1 X 10 9 M -1 s -1 upper diffusinal limit for "kinetically perfect" enzymes (Albery, W.J. and Knowles, J.R. (1976) Biochemistry 15:5631 -5640) and suggests that by at least one measure, the H.
  • PPIase is a highly effective catalyst in the cis-trans isomerisation of the Ala-Pro bond in the oligopeptide substrate.
  • the presence of PPIase was also determined in an H pylori extract. As with the assay for the recombinant protein, PPIase activity was detected, and was dependent on the concentration of extract added ( Figure 6).
  • the Helicobacter pylori genome contains an open reading frame (ORF) of 255 amino acids that was found to have homology to the Staphylococcus haemolyticus glutamate racemase gene (dga) (NCBI Accession number U 12405) and to the E. coli murl gene which encodes glutamate racemase activity in that organism.
  • ORF open reading frame
  • the gene was isolated by polymerase chain reaction (PCR) amplification cloning, overexpressed in E. coli, and the protein purified to apparent homogeneity.
  • PCR polymerase chain reaction
  • a simple assay for glutamate racemase activity resulting in the isomerization of D-glutamic acid to L- glutamic acid was developed to facilitate purification and for future use as a high- throughput drug screen.
  • H pylori The ORF in H pylori has been found by gene disruption studies to be essential for viability ofH. pylori cells in laboratory culture (see Example VII above). Therefore, inhibition of the enzymatic activity would be expected to be lethal for the organism, and such inhibitors may have utility in antimicrobial therapy of human infectious diseases.
  • a 765 base pair DNA sequence encoding the murl gene of H. pylori was isolated by polymerase chain reaction (PCR) amplification cloning.
  • a synthetic oligonucleotide primer (5'-AAATAGTCATATGAAAATAGGCGTTTTTG -3' (SEQ ID NO: 1289)) encoding an Ndel restriction site and the 5' terminus of the murl gene and a primer (5'-AGAATTCTATTACAATTTGAGCCATTCT -3' (SEQ ID NO: 1290)) encoding an EcoRI restriction site and the 3' end of the murl gene were used to amplify the murl gene of H. pylori using genomic DNA prepared from the J99 strain of H. pylori as the template DNA for the PCR amplification reactions (Current Protocols in Molecular
  • genomic DNA 25 nanograms was introduced into each of two reaction vials containing 1.0 micromole of each synthetic oligonucleotide primer, 2.0 mM MgCl 2 . 0.2 mM of each deoxynucleotide triphosphate (dATP, dGTP, dCTP & dTTP), and 1.25 units of heat stable DNA polymerases (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 50 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for the murl gene using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
  • the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
  • the amplified DNA sample was subjected to digestion with the restriction endonucleases, Ndel and EcoRI (New England Biolabs, Beverly, MA USA) (Current Protocols in Molecular Biology, Ibid).
  • the DNA samples from each of two reaction mixtures were pooled and subjected to electrophoresis on a 1.0% SeaPlaque (FMC BioProducts, Rockland, ME, USA) agarose gel. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation.
  • Amplified DNA encoding the H pylori murl gene was isolated from agarose gel slices and purified using the Bio 101
  • the pET-23b vector can be propagated in any E. coli K-12 strain, e.g., ⁇ MS174, 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 RNA polymerase.
  • hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacl gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
  • T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest.
  • IPTG isopropyl-B-D-thiogalactoside
  • Strains used in our laboratory include: BL21(DE3) (Studier, F.W.,
  • the pET-23b vector (Novagen, Inc., Madison, WI, USA) was prepared for cloning by digestion with Ndel and EcoRI (Current Protocols in Molecular Biology, Ibid). Following digestion, the amplified, agarose gel-purified DNA fragment carrying the murl gene was cloned (Current Protocols in Molecular Biology, Ibid) into the previously digested pET-23b expression vector. Products of the ligation reaction were then used to transform the BL21(DE3) strain of E. coli.
  • Competent bacteria E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET23-murI expression plasmid carrying the cloned H pylori sequence according to standard methods (Current Protocols in Molecular, Ibid). Briefly, 1 microliter of ligation reaction was mixed with 50 microliters of
  • Colonies carrying pEY-23-murI vectors were picked and incubated in 5 mis of LB broth plus 100 microgram/ml ampicillin overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).
  • NCBI The 2210 bp DNA sequence encoding the groE operon of E. coli
  • a synthetic oligonucleotide primer (5'-GCGAATTCGATCAG AATTTTTTTTCT-3' (SEQ ID NO: 1291)) encoding an EcoRI restriction site and the 5' terminus of the groE operon containing the endogenous promoter region of the groE operon and a primer (5'-ATAAGTACTTGTGAATCTTATACTAG -3' (SEQ ID NO: 1291)) encoding an EcoRI restriction site and the 5' terminus of the groE operon containing the endogenous promoter region of the groE operon and a primer (5'-ATAAGTACTTGTGAATCTTATACTAG -3' (SEQ ID NO: 1291)) encoding an EcoRI restriction site and the 5' terminus of the groE operon containing the endogenous promoter region of the groE operon and a primer (5'-ATAAGTACTTGTGAATCTTATACTAG -3' (SEQ ID NO: 1291)) encoding an EcoRI restriction site
  • oligonucleotide primer 1.5 mM MgCl 2 , 0.2 mM each deoxynucleotide triphosphate (dATP, dGTP, dCTP & dTTP) and 2.6 units heat stable DNA polymerases (Expanded High Fidelity PCR System, Boehringer Mannheim, Indianapolis, Indiana) in a final volume of 50 microliters.
  • the following thermal cycling conditions were used to obtain amplified DNA products for the groE operon using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
  • the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA sample was subjected to digestion with the restriction endonucleases. EcoRI and Scal (New England Biolabs. Beverly, MA USA) (Current Protocols in Molecular Biology, Ibid). The DNAs from each of two reaction mixtures were pooled and subjected to electrophoresis in a 1.0% SeaPlaque (FMC BioProducts, Rockland, ME, USA) agarose gel. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarsoe gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista. CA, USA).
  • a DNA fragment, EcoRI to Seal, containing the E. coli groE operon was cloned into the corresponding sites of the pACYC184 expression vector (New England Biolabs, Beverly, MA, USA) to make pACYC 184-groE.
  • the BL21 (DE3) strain of E. coli was transformed with pACYC-groE.
  • a tetracycline-resistant transformant overexpressing proteins of M r ⁇ 14,000 (Gro ⁇ S) and M r ⁇ 60,000 (Gro ⁇ L) was isolated.
  • Competent bacteria derived from a clone of strain BL21(D ⁇ 3) carrying the pACYC-groE plasmid were transformed with 50 nanograms of pET23-murI plasmid
  • a bacterial clone of BL21(D ⁇ 3) carrying both the pACYC-graE expression plasmid and the p ⁇ T-23-murI plasmid was cultured in LB broth supplemented with 1.0 mM D,L-glutamic acid and 100 microgram/ml ampicillin and 10 micrograms/ml tetracycline at 30°C until an optical density at 600 nM of 0.5 to 1.0 O.D. units was reached, at which point, isopropyl-beta-D-thiogalactoside (IPTG) was added to the culture at a final concentration of 1.0 mM. Cells were cultured overnight to induce gene expression of the H. pylori recombinant DNA constructions .
  • IPTG isopropyl-beta-D-thiogalactoside
  • EGTA 10% glycerol, 10 mM D,L-glutamic acid, 0.1 % ⁇ -mercaptoethanol, 200 ⁇ g/ ml lysozyme, 1 mM PMSF, and 10 ug/ml each of leupeptin, aprotinin, pepstatin.
  • the resultant homogenate was diluted with 1 volume of buffer A (10 mM Tris-HCl pH 7.0, 0.1 mM EGTA, 10 % glycerol, 1 mM DL- Glutamic acid, 1 mM PMSF, 0.1% beta-mercaptoethanol), made 0.1 % Brij-35, and centrifuged (100,000 x g, 1 h) to yield a clear supernatant (crude extract).
  • buffer A 10 mM Tris-HCl pH 7.0, 0.1 mM EGTA, 10 % glycerol, 1 mM DL- Glutamic acid, 1 mM PMSF, 0.1% beta-mercaptoethanol
  • the extract was loaded directly onto a 20 ml Q-Sepharose column pre-equilibrated in buffer A containing 100 mM NaCl and 0.02 % Brij -35.
  • the column was washed with 100 ml (5 bed volumes) of Buffer A containing 100 mM NaCl and 0.02 % Brij-35, then developed with a 100-ml linear gradient of increasing NaCl (from 100 to 500 mM) in Buffer A.
  • a band of M r 28,000 corresponding to glutamate racemase, the product of the recombinant H pylori murl gene, eluted at a gradient concentration of approximately 200-280 mM NaCl.
  • Individual column fractions were then characterized for glutamate racemase activity (see below for description of assay) and the protein profile of the fractions were analyzed on 12 % acrylamide SDS-PAGE gels.
  • Glutamate racemase eluted as a sharp peak at 310 mM NaCl.
  • Fractions containing a glutamate racemase activity were pooled, concentrated by dialysis against storage buffer [50% glycerol, 10 mM 3- (N-morpholino-propanesulfonic acid (MOPS) pH 7.0, 150 mM NaCl, 0.1 mM EGTA, 0.02 % Brij-35, 1 mM dithiothreitol (DTT)], and stored at -20°C. Assays for glutamate racemase activity.
  • MOPS N-morpholino-propanesulfonic acid
  • the measurement of the activity of glutamate racemase is linked to an OD change in the visible range in a series of coupled reactions to the activities of L-glutamate dehydrogenase (reduction of NAD to NAD ⁇ ) and diaphorase (reduction of the dye p-iodonitrotetrazolium violet, INT).
  • Initial rates were determined by following the increase in absorbance at 500 nm in a reaction volume of 200 ⁇ l containing 50 mM Tris- ⁇ Cl, p ⁇ 7.8, 4% v/v glycerol, 10 mM NAD, 2 mM INT, 60 Units/ml L-glutamate dehydrogenase, 5 Units/ml diaphorase, and varying concentrations of either substrate (from 0.063 mM to 250 mM D-glutamic acid) or purified enzyme (from 1 ⁇ g to 50 ⁇ g).
  • the conversion of D-glutamic acid to L-glutamic acid is coupled to the conversion of L-glutamic acid and NAD + by L-glutamate dehydrogenase to 2- oxoglutarate, ammonia.
  • the production of NADH is measured as an increase of absorbance at 340 nm (the reduction of NAD + to NADH) at 37°C.
  • the standard assay mixture (adapted from Choi, S-Y meticulous Esaki, N., Yoshimura, T., and Soda, K., 1991.
  • Protein Expression and Purification 2, 90-93) contained 10 mM Tris-HCl, pH 7.5, 5 mM NAD+, 5 Units/ml L-glutamate dehydrogenase, varying concentrations of the substrate D-Glutamic Acid (0.063 mM to 250 mM), and the purified recombinant H. pylori enzyme glutamate racemase (1 ⁇ g to 50 ⁇ g).
  • the reaction was started by the addition of either the substrate D-glutamic acid or the recombinant glutamate racemase after a preincubation at 37°C for 5 minutes with all of the other assay ingredients.
  • the change in absorbance at 340 nm was measured in a Spectra MAX 250. Initial velocities were derived from the initial slopes.
  • the coupled reactions can be summarized as shown below:
  • the H pylori glutamate racemase was overexpressed in E. coli and purified. In the presence of the E. coli chaperones GroES and GroEL, the glutamate racemase was expressed as a soluble protein. About 20 mg of soluble Murl was produced per liter of culture as judged by intensity of the protein band after SDS-PAGE. No band corresponding to the molecular weight of murl protein was seen in control gel lanes containing extracts from cells transformed with the pET vector lacking a murl insert. Addition of 1 mM DL-glutamic acid during cultivation of the expressing cells increased the apparent expression level by about five-fold.
  • Murl was purified by cation exchange chromatography and gel filtration. Upon SDS-PAGE analysis, the purified protein migrated as a single polypeptide species with an apparent mass 29 kDa which is consistent with the predicted mass of 28.858. 3) Kinetic properties of recombinant H. pylroi murl enzyme
  • the H pylori glutamate racemase was tested for inactivation with a sucuide inhibitor, L-serine-O sulfate, which is known to inhibit murl from E. coli.
  • the enzyme was incubated in the presence of 20 mM L-serine-O sulfate, and at different times interval, aliquots were removed to determine residual activity.
  • the initial velocity of purified recombinant H. pylroi murl protein was determined in the single enzyme coupled asssay following incubation with the inhibitor L-serine-O-sulfate (LSOS) at 20 mM for the times indicated on the x-axis.
  • the control was incubated in an identical manner but without LSOS. As shown in Figure 7, the H pylori glutamate racemase can be readily inactivated by the inhibitor.

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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 in diagnostic and vaccine compositions.

Description

NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO HELICOBACTER PYLORI AND VACCINE COMPOSITIONS THEREOF
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 against 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.
Figure 5 is a graph depicting the activity of recombinant PPIase. Figure 6 is a graph depicting PPIase activity in an H. pylori extract.
Figure 7 is a graph depicting a decrease of glutamate racemase activity with L- Serine-O-Sulfate.
Figure 8 depicts the amino acid sequence alignment in a portion of the sequence of 12 H. pylori proteins (depicted in the single letter amino acid code and designated by their amino acid Sequence ID Numbers; shown N-terminal to C-terminal, left to right).
Figure 9 depicts the N-terminal portion of nine H. pylori proteins (depicted in the single letter amino acid code and designated by their amino acid Sequence ID Numbers; shown N-terminal to C-terminal, left to right).
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: 492. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 492, such nucleic acid is contained in SEQ ID NO: 1. The H. pylori polypeptide sequences described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides 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: 492 through SEQ ID NO: 541. 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: 492 through SEQ ID NO: 541 , 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: 542 through SEQ ID NO: 591. 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: 542 through SEQ ID NO: 591, 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: 592 through SEQ ID NO: 641. 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: 592 through SEQ ID NO: 641 , 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: 642 through SEQ ID NO: 691. 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: 642 through SEQ ID NO: 691, 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: 692 through SEQ ID NO: 741. 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: 692 through SEQ ID NO: 741, 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: 742 through SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765 through SEQ ID NO: 791. 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: 742 through SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765 through SEQ ID NO: 791 , such nucleic acids are contained in SEQ ID NO: 251 through SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, and SEQ ID NO: 274 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: 792 through SEQ ID NO: 818 and SEQ ID NO: 820 through SEQ ID NO: 841. 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: 792 through SEQ ID NO: 818 and SEQ ID NO: 820 through SEQ ID NO: 841, such nucleic acids are contained in SEQ ID NO: 301 through SEQ ID NO: 327 and SEQ ID NO: 329 throgh 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: 842 through SEQ ID NO: 846 and SEQ ID NO: 848 through SEQ ID NO: 891. 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: 842 through SEQ ID NO: 846 and SEQ ID NO: 848 through SEQ ID NO: 891, such nucleic acids are contained in SEQ ID NO: 351 through SEQ ID NO: 364 and SEQ ID NO: 366 through SEQ ID NO: 400.
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: 892 through SEQ ID NO: 896 and SEQ ID NO: 898 through SEQ ID NO: 941. 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: 892 through SEQ ID NO: 896 and SEQ ID NO: 898 through SEQ ID NO: 941, such nucleic acids are contained in SEQ ID NO: 401 through SEQ ID NO: 405 and SEQ ID NO: 407 through SEQ ID NO: 450.
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: 942 through SEQ ID NO: 963 and SEQ ID NO: 966 through SEQ ID NO: 982. 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: 942 through SEQ ID NO: 963 and SEQ ID NO: 966 through SEQ ID NO: 982, such nucleic acids are contained in SEQ ID NO: 451 through SEQ ID NO: 472 and SEQ ID NO: 475 through SEQ ID NO: 491.
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: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041 through SEQ ID NO: 1087 and SEQ ID NO: 1090. 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: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041 through SEQ ID NO: 1087 and SEQ ID NO: 1090, such nucleic acids are contained in SEQ ID NO: 983, SEQ ID NO: 984, SEQ ID NO: 987 through SEQ ID NO: 1033 and SEQ ID NO: 1036.
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: 1296 through SEQ ID NO: 1298. 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: 1296 through SEQ ID NO: 1298, such nucleic acids are contained in SEQ ID NO: 1293 through SEQ ID NO: 1295.
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 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 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: 494. Therefore, this invention encompasses a recombinant or substantially pure preparation of an H. pylori polypeptide of SEQ ID NO: 494. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 494.
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: 542-SEQ ID NO: 591) or nucleic acids (e.g., SEQ ID NO: 51 -SEQ ID NO: 100), 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: 692 through SEQ ID NO: 741 can be divided into one or more subgroups as follows: SEQ ID NO: 692-SEQ ID NO: 680; SEQ ID NO: 681 -SEQ ID NO: 710; SEQ ID NO: 711-SEQ ID NO: 730; SEQ ID NO: 731-SEQ ID NO: 741 ; or any combinations thereof.
Particularly preferred 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441 , SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 1011, SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, SEQ ID NO: 1032, SEQ ID NO: 259, SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023. SEQ ID NO: 1294, SEQ ID NO: 1295, SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, SEQ ID NO: 1031 , SEQ ID NO: 254, SEQ ID NO: 352, SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381, SEQ ID NO: 389, SEQ ID NO: 1010, SEQ ID NO: 1012, SEQ ID NO: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421, SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 41 1 , SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO: 290, SEQ ID NO: 417, SEQ ID NO: 430, SEQ ID NO: 992, SEQ ID NO: 1025, SEQ ID NO: 477, SEQ ID NO: 414, SEQ ID NO: 253, SEQ ID NO: 293, SEQ ID NO: 334, SEQ ID NO: 343, SEQ ID NO: 418, SEQ ID NO: 424, and SEQ ID NO: 443.
In another 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1 , SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391 , SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 101 1, SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, SEQ ID NO: 1032, SEQ ID NO: 259, SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294, SEQ ID NO: 1295, SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, SEQ ID NO: 1031, SEQ ID NO:
254, SEQ ID NO: 352, SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381, SEQ ID NO: 389, SEQ ID NO: 1010, and SEQ ID NO: 1012.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321 , SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391 , SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 101 1, SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, and SEQ ID NO: 1032.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294, and SEQ ID NO: 1295.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, and SEQ ID NO: 1031.
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: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421 , SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 411, SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO: 290, SEQ ID NO: 417, SEQ ID NO: 430, SEQ ID NO: 992, and SEQ ID NO: 1025.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 354.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421, and SEQ ID NO: 1022. In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 463.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion or adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 281 and SEQ ID NO: 988.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 407 and SEQ ID NO: 1017.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 477.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transport polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 414.
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: 470, SEQ ID NO: 1033, SEQ ID NO: 357, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 1030, SEQ ID NO: 345, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 455, SEQ ID NO: 1003, SEQ ID NO: 351, SEQ ID NO: 416, SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, SEQ ID NO: 1293, SEQ ID NO: 318, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 330, SEQ ID NO: 347, SEQ ID NO: 440, SEQ ID NO: 446, SEQ ID NO: 464, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 995, SEQ ID NO: 997, SEQ ID NO: 1005, and SEQ ID NO: 1028.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 470 and SEQ ID NO: 1033.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 357 and SEQ ID NO: 457. In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 461 and SEQ ID NO: 1030.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 345, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 455, and SEQ ID NO: 1003.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in lipid metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 351 and SEQ ID NO: 416.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, and SEQ ID NO: 1293.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 355, SEQ ID NO: 1006, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 323, SEQ ID NO: 338, SEQ ID NO: 342, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 356, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362. SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 373, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 401. SEQ ID NO: 403, SEQ ID NO: 405, SEQ ID NO: 408, SEQ ID NO: 420, SEQ ID NO: 426, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 432, SEQ ID NO: 439. SEQ ID NO: 442. SEQ ID NO: 451. SEQ ID NO: 471, SEQ ID NO: 478, SEQ ID NO: 488. SEQ ID NO: 987, SEQ ID NO: 990, SEQ ID NO: 991 , SEQ ID NO: 993, SEQ ID NO: 1001 , SEQ ID NO: 1002, SEQ ID NO: 1007, SEQ ID NO: 1013, SEQ ID NO: 1016, SEQ ID NO: 1018, SEQ ID NO: 1021 , and SEQ ID NO: 1026.
In another embodiment, the H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 355 and SEQ ID NO: 1006.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cellular polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 256, SEQ ID NO: 267, SEQ ID NO: 282, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 332, SEQ ID NO: 333. SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341 , SEQ ID NO: 344, SEQ ID NO: 369, SEQ ID NO: 376, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 423, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481 , SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 489, SEQ ID NO: 984, SEQ ID NO: 994, SEQ ID NO: 1024, and SEQ ID NO: 1036.
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: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940. SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, SEQ ID NO: 1086, SEQ ID NO: 750, SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063, SEQ ID NO: 1077, SEQ ID NO: 1297, SEQ ID NO: 1298, SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, SEQ ID NO: 1085, SEQ ID NO: 745, SEQ ID NO: 843, SEQ ID NO: 906. SEQ ID NO: 1073, SEQ ID NO: 872, SEQ ID NO: 880, SEQ ID NO: 1064. SEQ ID NO: 1066, SEQ ID NO: 845, SEQ ID NO: 863, SEQ ID NO: 891, SEQ ID NO: 912, SEQ ID NO: 1076, SEQ ID NO: 954, SEQ ID NO: 772, SEQ ID NO: 1042, SEQ ID NO: 902, SEQ ID NO: 898, SEQ ID NO: 1071 , SEQ ID NO: 781 , SEQ ID NO: 908, SEQ ID NO: 921, SEQ ID NO: 1046, SEQ ID NO: 1079, SEQ ID NO: 968, SEQ ID NO: 905, SEQ ID NO: 744, SEQ ID NO: 784, SEQ ID NO: 825, SEQ ID NO: 834, SEQ ID NO: 909, SEQ ID NO: 915, and SEQ ID NO: 934.
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: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901 , SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, SEQ ID NO: 1086, SEQ ID NO: 750, SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063, SEQ ID NO: 1077, SEQ ID NO: 1297, SEQ ID NO: 1298, SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081 , SEQ ID NO: 1085, SEQ ID NO: 745, SEQ ID NO: 843, SEQ ID NO: 906, SEQ ID NO: 1073, SEQ ID NO: 872, SEQ ID NO: 880, SEQ ID NO: 1064, and SEQ ID NO: 1066.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757. SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901 , SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932. SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, and SEQ ID NO: 1086.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063, SEQ ID NO: 1077, SEQ ID NO: 1297. and SEQ ID NO: 1298.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, and SEQ ID NO: 1085.
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: 845, SEQ ID NO: 863, SEQ ID NO: 891 , SEQ ID NO: 912, SEQ ID NO: 1076, SEQ ID NO: 954, SEQ ID NO: 772, SEQ ID NO: 1042, SEQ ID NO: 902, SEQ ID NO: 898, SEQ ID NO: 1071, SEQ ID NO: 781, SEQ ID NO: 908, SEQ ID NO: 921, SEQ ID NO: 1046, and SEQ ID NO: 1079.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 845.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 863, SEQ ID NO: 891. SEQ ID NO: 912, and SEQ ID NO: 1076.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 954. In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in secretion or adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 772 and SEQ ID NO: 1042.
In another embodiment, the H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof selected from the group consisting of SEQ ID NO: 898 and SEQ ID NO: 1071.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 968.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transport polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 905.
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: 961, SEQ ID NO: 1087, SEQ ID NO: 848, SEQ ID NO: 948, SEQ ID NO: 952, SEQ ID NO: 1084, SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, SEQ ID NO: 1057, SEQ ID NO: 842, SEQ ID NO: 907, SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841, SEQ ID NO: 910, SEQ ID NO: 951, SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, SEQ ID NO: 1296, SEQ ID NO: 809, SEQ ID NO: 813, SEQ ID NO: 815, SEQ ID NO: 821, SEQ ID NO: 838, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 955, SEQ ID NO: 981, SEQ ID NO: 982, SEQ ID NO: 1049, SEQ ID NO: 1051, SEQ ID NO: 1059, and SEQ ID NO: 1082.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 961 and SEQ ID NO: 1087.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 848 and SEQ ID NO: 948.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 952 and SEQ ID NO: 1084.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, and SEQ ID NO: 1057.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in lipid metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 842, SEQ ID NO: 907.
In another embodiment, the H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof selected from the group consisting of SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841 , SEQ ID NO: 910, SEQ ID NO: 951 , SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, and SEQ ID NO: 1296.
Particularly preferred is a purified or isolated H. pylori secreted polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 846, SEQ ID NO: 1060, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 751 , SEQ ID NO: 752, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 770, SEQ ID NO: 774, SEQ ID NO: 775, SEQ ID NO: 778, SEQ ID NO: 779, SEQ ID NO: 780, SEQ ID NO: 782, SEQ ID NO: 786, SEQ ID NO: 787, SEQ ID NO: 788, SEQ ID NO: 789, SEQ ID NO: 791, SEQ ID NO: 792, SEQ ID NO: 793, SEQ ID NO: 794, SEQ ID NO: 795, SEQ ID NO: 796, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 814, SEQ ID NO: 829, SEQ ID NO: 833, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 847, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851 , SEQ ID NO: 852, SEQ ID NO: 853, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 864, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 870, SEQ ID NO: 871, SEQ ID NO: 879, SEQ ID NO: 881, SEQ ID NO: 885, SEQ ID NO: 886, SEQ ID NO: 887, SEQ ID NO: 892, SEQ ID NO: 894, SEQ ID NO: 896, SEQ ID NO: 899, SEQ ID NO: 911, SEQ ID NO: 917, SEQ ID NO: 919, SEQ ID NO: 920, SEQ ID NO: 923, SEQ ID NO: 930, SEQ ID NO: 933, SEQ ID NO: 942, SEQ ID NO: 962, SEQ ID NO: 969, SEQ ID NO: 979, SEQ ID NO: 1041, SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: 1056, SEQ ID NO: 1061, SEQ ID NO: 1067, SEQ ID NO: 1070, SEQ ID NO: 1072, SEQ ID NO: 1075, and SEQ ID NO: 1080.
In another embodiment, the H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 846 and SEQ ID NO: 1060. Particularly preferred is a purified or isolated H. pylori cellular polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 747, SEQ ID NO: 758, SEQ ID NO: 773, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801, SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 835, SEQ ID NO: 860, SEQ ID NO: 867, SEQ ID NO: 873, SEQ ID NO: 877, SEQ ID NO: 914, SEQ ID NO: 922, SEQ ID NO: 926, SEQ ID NO: 927, SEQ ID NO: 949, SEQ ID NO: 953, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 970, SEQ ID NO: 971, SEQ ID NO: 972, SEQ ID NO: 973, SEQ ID NO: 974, SEQ ID NO: 975, SEQ ID NO: 976, SEQ ID NO: 977, SEQ ID NO: 978, SEQ ID NO: 980, SEQ ID NO: 1038, SEQ ID NO: 1048, SEQ ID NO: 1078, and SEQ ID NO: 1090.
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 antibody has utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori-specifιc 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 polypeptides 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% homologous to an amino acid sequence contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence 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 a polypeptide contained in the Sequence Listing.
In preferred embodiments: the H. pylori polypeptide is encoded by a nucleic acid 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 shown 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 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 contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence 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 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 contained in the Sequence Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence 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 contained in the Sequence Listing.
In preferred embodiments: the nucleic acid is that as shown in the Sequence
Listing; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%
homologous with a nucleic acid sequence 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 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 contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence 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 a nucleic acid contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of a nucleic acid contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of a nucleic acid contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of a nucleic acid 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 shown in the Sequence Listing.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence shown in the Sequence Listing which encodes amino acids shown 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 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 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) as strain HP-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 as shown 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 secreted proteins, H. pylori cytoplasmic proteins and H. pylori cellular proteins. Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs. Polypeptides related by significant homology to the polypeptides of Table 1 (as reflected by FASTA compar- isons of the amino acid sequences and indicated in many cases in Tables 3-6 below) are also considered to be classified in the manner of the homolog shown in Table 1.
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
[In Table 1, "nt" represents nucleotide Seq. ID number and "aa" represents amino acid Seq. ID number] Definitions
A purified preparation or a substantially pure preparation of a polypeptide, as used herein, means 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 substantially pure nucleic acid, e.g., a substantially pure DNA, 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.
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 for 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. (IX 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 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 shown 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 CHO 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 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 are outlined in Table 2 below.
Figure imgf000032_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 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 and terminators; 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 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, 5/1 1-5/15, 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). A resulting assembly of contigs, each representing a continuous stretch of DNA or DNA sequence was obtained. The average contig length was about 3 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 was then determined. The H. pylori sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides. ORFs of at least 180 nucleotides (based on stop-to-stop codon reads) were predicted. 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. p yloripolypeptide. 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, amplified products can be checked by conventional sequencing methods. Clones carrying the desired sequences described in this invention may 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 be 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 cell line. 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 are shown 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 shown in the Sequence Listing. These uses are described in further detail below.
Probes
A nucleic acid isolated or synthesized in accordance with the nucleotide sequences set forth 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 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 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 shown 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 acids of≥ 10-15 nucleotides 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 acids 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, and other bacterial strains such as E. coli, Norcardia,
Corynebacterium, 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 intro- duced 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 Herskowitz, (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, NJ) 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 pETl Id 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 gn1 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 complex. 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 micells 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) 1 19-128). Another strategy would be to alter the nucleic acid encoding 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. 20:2111-21 18). 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 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 isolatedand identified based on an ORF that endoes 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 Tables 3-6 below. All of the polypeptide lengths in Table 3 are from stop codon to stop codon in the nucleotide sequence of H. pylori. As is known in the art, the actual polypeptide lengths are usually shorter than the stop-to-stop codon lengths because a start codon for an initiator charged tRNA usually appears a few nucleotides downstream from the prior stop codon and within a few nucleotides following a ribosome binding site (also known as a "Shine-Delgarno sequence"). Since most of the ribosome binding sites in H. pylori have many of the same general features of those known in E. coli, one skilled in the art can predict the actual start codon with good reliability from the stop-to-stop nucleotide sequence of an open reading frame. The polypeptide sequences of SΕQ ID NOs:492-743 of this invention represent the stop- to-stop codon lengths of the open reading frames of SΕQ ID NOs: 1-252. All other polypeptide sequences of this invention represent the predicted start to stop protein lengths from the nucleotide sequences. One skilled in the art can recognize start sites in the stop-to-stop open reading frames of the nucleotide sequences presented herein. In addition, one skilled in the art will occasionally detect alternative start sites, some of which may be utilized in vivo by the cellular machinery. The number of these alternative start sites is sufficiently small that one skilled in the art can readily test them in a recombinant expression systems known in the art to determine which ones provide authentic functional protein products.
The relationship between the polypeptides shown in Table 3 can be described as follows. First, all of the polypeptides of Table 3 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
Second, the stop-to-stop lengths are different for some of the homologous pairs of polypeptides. In some cases, the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility. Third, some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
In all cases, the homology relationships described in Table 3 are highly significant. For example, a typical H pylori gene product will exhibit amino acid sequence identities of between 92% and 100% among different strains of H. pylori selected from human patients. The nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another. For example, nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Additional relationships between polypeptides of the invention are described in Table 4 below. All of the polypeptide lengths in Table 4 below are measured from stop codon to stop codon in the nucleotide sequence of H. pylori.
The relationship between the polypeptides shown in Table 4 can be described as follows. First, all of the polypeptides of Table 4 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
Second, the stop-to-stop lengths are different for some of the homologous pairs of polypeptides. In some cases, the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility. Third, some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
In all cases, the homology relationships described in Table 4 are highly significant. For example, a typical H. pylori gene product will exhibit amino acid sequence identities of between 92% and 100% among different strains of H. pylori selected from human patients. The nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another. For example, nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Additional relationships between polypeptides of the invention are described in Table 5 below. All of the polypeptide lengths in Table5 below are measured from start codon to stop codon in the nucleotide sequence of H. pylori.
The relationship between the polypeptides shown in Table 5 can be described as follows. First, all of the polypeptides of Table 5 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other.
Second, the start-to-stop lengths are different for some of the homologous pairs of polypeptides. In some cases, the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved utility. Third, some polypeptides in the second column are homologous to two shorter polypeptides in the fifth column.
In all cases, the homology relationships described in Table 5 are highly significant. For example, a typical H. pylori gene product will exhibit amino acid sequence identities of between 92% and 100% among different strains of H. pylori selected from human patients. The nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another. For example, nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
Figure imgf000054_0001
Additional relationships between polypeptides of the invention are described in Table 6 below. All of the polypeptide lengths in Table 6 below are measured from stop codon to stop codon in the nucleotide sequence of H. pylori.
The relationship between the polypeptides shown in Table 6 can be described as follows. First, all of the polypeptides of Table 6 are at least 90% identical with each other over most of their lengths, and most are over 95% identical with each other. Second, the stop-to-stop lengths are different for the homologous pairs of polypeptides. In some cases, the shorter polypeptide contains the relevant portion of the protein exhibiting utility in this invention; in some cases, the longer polypeptide may exhibit improved uti lity .
In all cases, the homology relationships described in Table 6 are highly significant. For example, a typical H. pylori gene product will exhibit amino acid sequence identities of between 92% and 100% among different strains of H. pylori selected from human patients. The nucleotide sequences encoding the related polypeptides of this invention are also very similar to one another. For example, nucleotide probes derived from the coding sequence of a polypeptide of this invention can be used in PCR or hybridization experiments to identify clones carrying the nucleotide sequence encoding the homologous related polypeptide.
Figure imgf000056_0001
Figure imgf000057_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 the identification of nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H. pylori. An important aspect of this identification is to determine the function of the disclosed sequences, which can be achieved using a variety of approaches. Non-limiting examples of these methods 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 a useful tool 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 is strongly suggestive 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 protein.protein 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 potentially 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. S train-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, although whether or not this is correct is not essential to the invention. 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 Ada 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 5371 1) 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 better than 1x10 (ee-6)) to membrane or exported proteins represent likely protein antigens for vaccine development. Possible functions can be provided to H. pylon 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.
Surface exposed outer membrane proteins are likely to represent the best antigens to provide a protective immune response against H. pylori. Among the algorithms that can be used to aid in prediction of these outer membrane proteins include the presence of an amphipathic beta-sheet region at their C-terminus. This region which has been detected in a large number of outer membrane proteins in Gram negative bacteria is often characterized by hydrophobic residues (Phe or Tyr) approximately at positions 1, 3, 5, 7 and 9 from the C-terminus (e.g., see Figure 8, block F). Importantly, these sequences have not been detected at the C-termini of periplasmic proteins, thus allowing preliminary distinction between these classes of proteins based on primary sequence data. This phenomenon has been reported previously by Struyve et al. (J. Mol. Biol. 218:141-148, 1991).
Also illustrated in Figure 8 are additional amino acid sequence motifs found in many outer membrane proteins of H. pylori. The amino acid sequence alignment in Figure 8 depicts portions of the sequence of 12 H. pylori proteins (depicted in the single letter amino acid code) labeled with their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right. Six distinct blocks (labeled A through F) of similar amino acid residues are found including the distinctive hydrophobic residues (Phe or Tyr; F or Y according to the single letter code for amino acid residues) frequently found at positions near the C-terminus of outer membrane proteins. The presence of several shared motifs clearly establishes the similarity between members of this group of proteins.
In addition, outer membrane proteins isolated from H. pylori frequently share a motif near the mature N-terminus (i.e., after processing to remove the secretion signal) as illustrated in the blocked amino acid residues in Figure 9. Figure 9 depicts the N- terminal portion of nine H. pylori proteins (designated by their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right).
One skilled in the art would know that these shared sequence motifs are highly significant and establish a similarity among this group of proteins.
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:
Figure imgf000061_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 provided in the Sequence
Listing, 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 discovery 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. 1 1 :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/alky lation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 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 (Hochuli, 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 within 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 homolog 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 10^ 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 Ml 3, fd., and fl 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 may peptides 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 protease IgA 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 Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89: 1865-1869). This system uses a plasmid containing the Lad 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 Lad 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 Lad 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 Lad 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 Lad display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries oi 107-1 09 independent clones are routinely prepared. Libraries as large as 10^ 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 ELISA, or for binding specificity in a completion phage ELISA (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 to perform 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 ct 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
Commun126:419; 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 shown in the Sequence Listing which encode H. pylori surface proteins. 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 HLA 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.
In another embodiment, immunogenic components of the invention are identified through genomic vaccination. The basic protocol is based on the idea that expression libraries consisting of all or parts of a pathogen genome, e.g., an H. pylori genome, can confer protection when used to genetically immunize a host. This expression library immunization (ELI) is analogous to expression cloning and involves reducing a genomic expression library of a pathogen, e.g., H. pylori, into plasmids that can act as genetic vaccines. The plasmids can also be designed to encode genetic adjuvants which can dramatically stimulate the humoral response. These genetic adjuvants can be introduced at remote sites and act as well extracelluraly as intracellularly.
This is a new approach to vaccine production that has many of the advantages of live/attenuated pathogens but no risk of infection. An expression library of pathogen DNA is used to immunize a host thereby producing the effects of antigen presentation of a live vaccine without the risk. For example, in the present invention, random fragments from the H. pylori genome or from cosmid or plasmid clones, as well as PCR products from genes identified by genomic sequencing, can be used to immunize a host. The feasibility of this approach has been demonstrated with Mycoplasma pulmonis (Barry et al.. Nature 377:632-635, 1995), where even partial expression libraries of Mycoplasma pulmonis, a natural pathogen in rodents, provided protection against challenge from the pathogen.
ELI is a technique that allows for production of a non-infectious multipartite vaccine, even when little is known about pathogen's biology, because ELI uses the immune system to screen candidate genes. Once isolated, these genes can be used as genetic vaccines or for development of recombinant protein vaccines. Thus. ELI allows for production of vaccines in a systematic, largely mechanized fashion. 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 1 1 : 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 1 1637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl- sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, 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) 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 NaHCO3 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)).
It will be apparent to those skilled in the art that some of the vaccine
compositions of the invention are usefuls 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.
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 Harlow 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 shown 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 shown 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 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 mi croinj ection 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 λgtl 1, λgtl8-23, 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, λgtl 1 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.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.
Other Embodiments
Many of the nucleic acids and corresponding polypeptides of the invention were disclosed previously in the parent applications, U.S.S.N. 08/761 ,318, filed December 6, 1996 (Attorney Docket No.: GTN-009CP2), U.S.S.N. 08/736,905, filed October 25,
1996 (Attorney Docket No.: GTN-010CP) and U.S.S.N. 08/738,859, filed October 28,
1996 (Attorney Docket No.: GTN-009CP), which are a continuation-in-part of U.S.S.N.
08/625,81 1, filed March 29, 1996 (Attorney Docket No.: GTN-009), and U.S.S.N.
08/758,731 , filed April 2, 1996 (Attorney Docket No.: GTN-010). The correlation between sequence identification numbers in the above-identified parent applications and sequence identification numbers provided herein is outlined in Table 7 below.
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
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 (Bio 101 , Inc.).
The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The healed DNA was then ligated to unique BstXI-linker adopters 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. 15 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy with 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 Enzemology 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 has 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 Sequenicng 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. For typical sequences derived by chemical sequencing, the error rate of the REPLICA™ base calling software was 2-5% with most errors occurring near the end of a sequence read. 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 a number correspond to (microtiter plate and probe information) and lane set number (corresponding to microtiter plate columns). 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 H. pylori Nucleic Acids
Expression and purification of the H. pylori polypeptides of the invention can be performed essentially as outlined below.
To facilitate the cloning, expression and purification of membrane and secreted proteins from H pylori, a gene expression system, such as the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, is selected. Also, a DNA sequence encoding a peptide tag, the Ηis-Tag, is fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3' end is selected for fusion in order to avoid alteration of any 5' terminal signal sequence. The exception to the above is 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 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 Nucleic Acids Containing ORF's for
Membrane and Secreted Polypeptides from H. pylori
Nucleic acids chosen (for example, from the nucleic acids set forth in the Sequence Listing) for cloning from the J99 strain of H. pylori are prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers specific for the 5' and 3' ends of open reading frames (ORFs) are designed and purchased from GibcoBRL Life Technologies (Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) are designed to include an Ncol cloning site at the extreme 5' terminus. These primers are 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. All reverse primers (specific for the 3' end of any H. pylori ORF) include 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 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 encodes a BamΗI site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encodes a Xhol site at its extreme 5' terminus.
Genomic DNA prepared from the J99 strain of H pylori is 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) is 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.
Upon completion of thermal cycling reactions, each sample of amplified DNA is washed and purified using the Qiaquick Spin PCR purification kit (Qiagen,
Gaithersburg, MD, USA). All amplified DNA samples are subjected to digestion with the restriction endonucleases, e.g., Ncol and EcoRI (New England BioLabs, Beverly, MA. USA)(Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples are then subjected to electrophoresis on 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA is visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel is purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, C A, USA)
Cloning ofH. pylori Nucleic Acids Into an Expression Vector
The pET-28b vector is prepared for cloning by digestion with endonucleases, e.g., Ncol 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 His-Tag that can be fused to the 5' end of an inserted gene, is used and the cloning site prepared for cloning with the ppiB gene by digestion with BamHI and Xhol restriction endonucleases.
Following digestion, DNA inserts are 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 is cloned into the pET-28a expression vector. Products of the ligation reaction are 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), are 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 is mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples are incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples are then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight.
Transformed colonies of BL21 are then picked and analyzed to evaluate cloned inserts as described below.
Identification Of Recombinant Expression Vectors With H. Pylori Nucleic Acids Individual BL21 clones transformed with recombinant pET-28b-H.pylori ORFs are 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 verifies 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 Nucleic Acids From Transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs are picked and incubated in 5 mis of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA is 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. ΗMS174, 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 lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its 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 DNA isolated as described above is used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). The lacZ gene (beta-galactosidase) is expressed in the pET-System as described for the H. pylori recombinant constructions. Transformed cells are cultured in SOC medium for 1 hour, and the culture is then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies are 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 are pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets are resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells are then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets are 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 are quantified
spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins. S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations are also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough, N., Farr, 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) are purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
Molecular weight markers include rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (1 16 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).
1. Purification of soluble proteins
All steps are carried out at 4°C. Frozen cells are 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 % -mercaptoethanol, 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 microfluidizer (Model M-l 10S, Microfluidics International Corporation, Newton, MA). The resultant homogenate is 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 (m Supor filter (Gelman Sciences, FRG) the crude extract is loaded directly onto a Ni2+- nitrolotriacetate-agarose (NTA) with a 5 milliliter bed volume (Hochuli, E., Dbeli, H., and Schacheer, A. (1987) J.
Chromatography 41 1, 177-184) pre-equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column is washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and are eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and: either 20, 100, 200, or 500 mM imidazole. Fractions are monitored by absorbance at OD280 nm, and Peak fractions are analyzed by SDS-PAGE.
2. Purification of insoluble proteins from inclusion bodies
The following steps are carried out at 4°C. Cell pellets are resuspended in lysis buffer with 10% glycerol 200 (g/ ml lysozyme, 5 mM EDTA, ImM PMSF and 0.1 % - mercaptoethanol. After passage through the cell disrupter, the resulting homogenate is made 0.2 % deoxycholate, stirred 10 minutes, then centrifuged at 20,000 x g, for 30 min. The pellets are 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 % -mercaptoethanol. The resulting white pellet is composed primarily of inclusion bodies, free of unbroken cells and membranous materials.
Dialysis and concentration of protein samples
Urea is 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 as follows; 6M, 4M, 3M, 2M, 1M, 0.5 M and finally TBS without any urea. Each dialysis step is conducted for a minimum of 4 hours at room temperature.
After dialysis, samples are concentrated by pressure filtration using Amicon stirred-cells. Protein concentrations are 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). IV. Assessment Of The Antigenicity Of Outer Membrane Localized Antigens Of H. pylori
Purification of outer membranes form H pylon can be performed by essentially follwing the protocol outlined below.
H pylori strains J99 (ATCC# 55679) and Ah244 are grown on chocolate blood agar containing 5% (vol/vol) horse blood, at 37(C in an atmosphere containing 10%
CO2 for 48 h. Bacteria were harvested by suspension in 20 mM Tris, pΗ 7.5. The cells are collected by centrifugation at 12,000 Xg, for 20 min at 4(C and washed 3 times with 20 mM Tris, pΗ 7.5. Cells are suspended in 20 mM Tris, pΗ 7.5 and broken by sonication on ice (eight bursts of 30 s at 60 watts with 60 s pauses between bursts). DNase (0.1 mg) and RNase (0.5 mg) are added to the cell suspension, and the mixture is incubated for 30 minutes at room temperature. The cell suspension is centrifuged at 12,000 Xg for 20 min, at 4(C. The supernatant was retained and centrifuged again. Total membranes are collected from the supernatant by centrifugation at 40,000 Xg for 30 minute, at 4°C. The pellet are washed twice in 20 mM Tris, pFI 7.5. The protein content is assayed using the Bradford protein assay, with bovine serum albumin (BSA) as a standard. The suspension is then adjusted to 1 mg protein /ml. The solubilization of the membranes is realized by adding N-lauryl-sarcosine to this suspension in a ratio of 6 mg of N-lauryl-sarcosine per mg of protein. The suspension is incubated for 30 minutes at room temperature in presence of N-lauryl-sarcosinc. Outer membranes are collected by centrifugation at 40,000 Xg for 30 minutes at 4°C. The pellet is washed 3 times with Milli Q quality water, aliquoted and stored at -20°C until use. Identification Of Outer Membrane Antigens ofH. pylori
Outer membrane antigens can be identified using a protocol outlined below. Proteins are separated on sodium dodecyl sulfate polyacrylamide gels (SDS- PAGE) according to the method described by Laemmli, U.K. (1970) Nature (London) Volume 227, 680-685. Samples are prepared by suspension in standard treatment buffer and heated at 100°C for 10 min. Approximately 1 -5 mg of protein is loaded per well on 8X10 cm minigels (0.75 mm). The separated proteins are then transferred to PVDF membranes as described below.
Electroblotting of separated proteins to PVDF membranes is performed in a Bio Rad Mini-Trans Blot Electrophoretic Transfer cell. The PVDF membrane Immobilon- pSQ is employed. Electroblotting is carried out for 60 min at 50V using CAPS transfer buffer (10mM 3-[Cyclohexylamino]-1-propanesulfonic acid, 10% methanol). The membrane is stained with 0.2% Ponceau S and destained with Milli Q quality water.
Antigens within the preparation are then identified using western
immunoblotting. After electroblotting, non specific binding sites of the PVDF membrane are blocked with 5% non fat dry milk in 10 mM Tris-HCl-0.9% NaCl, pH 7.5. The membrane is incubated with a appropriate dilution of normal mouse serum in 10 mM Tris-HCl-0.9% NaCl-0.5% Tween 20-0.5% BSA, pH 7.5, for 2 h at 37(C and then washed three times with 10 mM Tris-HCl-0.9% NaCl-0.5% Tween 20, pH 7.5 (TTBS). Alkaline phosphatase conjugated anti-mouse Ig, from goat is then added in 10 mM Tris-HCl-0.9% NaCl-0.5% Tween 20-0.5% BSA, pH 7.5 and incubated for lh at room temperature. After this incubation, the membrane is washed three times in TTBS. The reactive bands are revealed using 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad) as the Alkaline phosphatase substrate and Nitro Blue Tetrazolium (Bio-Rad) as the color development reagent.
For amino acid microsequencing, proteins that are identified as immunoreactive are cut from a fresh unreacted immobilon membranes and microsequenced at the Worcester Foundation microsequencing facility. Membranes from which the protein bands are cut are then subjected to western immunoblot as described above to confirm that the appropriate band had been excised.
V. Analysis Of H. Pylori Proteins As Vaccine Candidates
To investigate the immunomodulatory effect ofH. 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 (Vac A negative) ofH 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 10 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 8 below.
Figure imgf000099_0001
Figure imgf000100_0002
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 9 below.
Figure imgf000100_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 ofH. 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 Proteins 3, 4, 9, 1 , and 7 (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 Protein 6, followed by 1 , 3, and 9 (see Figure 2).
Therapeutic immunization effects:
All control animals (BALB/c mice) were well colonized with H. pylori (strain AΗ244) in both antrum and corpus of the stomach. Of the antigens tested 3 proteins (Proteins 4, 7, and 1) 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 Proteins 8, 9, and 3 compared to control. The effect of Proteins 5, 2, and 6 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 ofH 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.
VI. 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 ofH pylori strains (as listed in Table 12) 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- ΗCl, 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 10) 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 imgf000103_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 for Proeins 12 and 14;
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 for Protem 11 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 for Protein 11 and Protein 13 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 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 H pylori sequence 350) 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 MgC12, 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 ovemight. Transformed colonies of TOP10F' 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 1 1 below.
Figure imgf000105_0001
Figure imgf000106_0001
Results
To establish the PCR error rate in these experiments, five individual clones of Protein 1 1, prepared from five separate PCR reaction mixtures from H 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 Protein 1 1 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: Protein 11 , homologous to the vai A & B genes encoding an ABC transporter in F. novicida; Protein 12, homologous to lipoprotein e (P4) present in the outer membrane of Η. influenzae; Protein 13, homologous to fecA, an outer membrane receptor in iron (III) dicitrate transport in E. coli. Protein 14 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 12 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 (Protein 14, J99 strain) to approximately 7% change (Protein 1 1 , strain AH5) The deduced protein sequences show either no variation (Protein 14, strains AH 18 and AH24) or up to as much as 7.66% amino acid changes (Protein 1 1, Strain AH5).
Figure imgf000107_0001
VII. Experimental Knock-Out Protocol for the Determination of Essential H. pylori
Genes as Potential Therapeutic Targets
Therapeutic targets were 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 was 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).
Identification and Cloning ofH pylori Gene Sequences
The sequences of the genes or ORFs (open reading frames) selected as knock-out targets were identified from the H pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers (Table 13) were designed with the aid of the OLIGO program (National Biosciences. Inc.. Plymouth, MN 55447, USA), and were purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). Specific primers (Fl and RI ) were chosen which flanked most or all of the ORF, depending on its size. If the ORF was smaller than 800 to 1000 base pairs, flanking primers were chosen outside of the open reading frame.
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Genomic DNA prepared from the Helicobacter pylori HpJ99 strain (ATCC 55679) was 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 was 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 was carried out with Perkin Elmer Cetus/GeneAmp PCR System 9600 thermal cyclers. The thermal cycling conditions used to obtain amplified DNA products for each knock-out target are shown in Table 14.
Figure imgf000112_0001
Figure imgf000113_0001
Upon completion of thermal cycling reactions, each sample of amplified DNA was 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 was then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
PCR products were 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 was 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 was allowed to proceed for 16 hours at 16°C.
Ligation products were 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 was 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 were 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 were then spread onto LB (10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/1 sodium chloride) plates containing 100 microgram/ml of Ampicilhn, 0.3% X-gal, and 100 microgram/ml IPTG. These plates were incubated overnight at 37°C. Ampicillin-resistant colonies with white color were selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicilhn, and plasmid DNA was 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 DN As were used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers (F1 and R1) 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 were
confirmation that the correct inserts had been cloned. Two to six such verified clones were 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 were pooled, and used in subsequent cloning steps.
The sequences of the genes/ORFs were again used to design a second pair of primers (F2 and R2) which flanked the region ofH pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but were oriented away from each other. The pool of circular plasmid DNAs of the previously isolated clones were used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers was away from each other, the portion of the ORF between the primer would not be included in the resultant PCR product. The PCR product was a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, resulted in the deletion of a portion of the ORFs. The PCR product was visualized on a 1 % TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size had been amplified.
A Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology 170, 1704- 1708) was 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 was obtained by carrying out an EcoRI digestion of the recombinant plasmid pCTBS.kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566- 10573). The proper fragment ( 1.4 kb) was isolated on a 1 % TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA). The fragment was 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 was then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides. The OTO overhang was then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pH 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 was purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA). The PCR product of the deletion primers (F2 and R2) was 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 reaction and incubating for 16 hours at 16°C.
The ligation products were transformed into XL-1 Blue or DH5-α E.coli cells by electroporation as described previously. After recovery in SOC, cells were plated onto LB plates containing 100 microgram/ml Ampicilhn and grown overnight at 37°C. These plates were then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies had both the Ampicilhn resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies were picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA was isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
Several tests by PCR amplification were conducted on these plasmids to verify that the Kanamycin was 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 was inserted into the H. pylori sequence, the plasmid DNAs were used as templates for PCR amplification with the set of primers (F1 and R1) originally used to clone the H pylori gene/ORFs. The correct PCR product was 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 was determined and both orientations were eventually used in H. pylori transformations (see below). To determine the orientation of insertion of the kanamycin resistance gene, primers were designed from the ends of the kanamycin resistance gene ("Kan-1 " 5'-
ATCTTACCTATCACCTCAAAT-3' (SEQ ID NO: 1285), and "Kan-2" 5'- AGACAGCAACATCTTTGTGAA-3' (SEQ ID NO: 1286)). By using each of the cloning primers (F1 and R1) in conjunction with each of the Kan primers (4
combinations of primers), the orientation of the Kanamycin cassette relative to the H. pylori sequence was determined. Positive clones were classified as either in the "A" orientation (the same direction of transcription was present for both the H pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the Hpylori gene was opposite to that of the Kanamycin resistance gene). Clones which shared the same orientation (A or B) were pooled for subsequent experiments and independently transformed into H pylori.
Transformation of Plasmid DNA into H. pylori cells
Two strains ofH pylori were used for transformation: ATCC 55679. the clinical isolate which provided the DNA from which H pylori sequence database was obtained, and AΗ244, an isolate which had been passaged in, and had the ability to colonize the mouse stomach. Cells for transformation were grown at 37°C, 10% CO2, 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells were grown to exponential phase, and examined microscopically to determine that the cells were "healthy" (actively moving cells) and not contaminated. If grown on plates, cells were 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 were 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 was 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 was placed onto a prewarmed Sheep-Blood agar plate, and the plate was further incubated at 37°C, 6% CO2, 100% humidity for 4 hours. After this incubation, 10 microliters of plasmid DNA (100 micrograms per microliter), was 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) were done in parallel. The plates were returned to 37°C, 6% CO2 for an additional 4 hours of incubation. Cells were then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO2. Cells were 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 appeared, they were picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.
Three sets of PCR (three tests) were done to verify that the colonies of transformants have arose from homologous recombination at the proper chromosomal location. The template for PCR (DNA from the colony) was obtained by a rapid boiling DNA preparation method. An aliquot of the colony (stab of the colony with a toothpick) was 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) was added and vortexed. The mixture was microfuged for 5 minutes and the supernatant was 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 F1 and R1 primers (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 was proof that the gene had not been knocked out and that the transformant was 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 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. A positive result of homologous recombination at the correct chromosomal location of the sequences of the gene/ORFs upstream from the kanamycin resistance gene should show a single PCR product, the expected size to be from the location of F3 to the insertion site of kanamycin resistance gene. No PCR product or PCR product(s) of incorrect size(s) would prove that the plasmid had not been 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 either primer Kan-1 or Kan-2, depending on whether the plasmid DNA used was of "A" or "B" orientation. A positive result of homologous recombination at the correct chromosomal location downstream from the kanamycin resistance gene would show a single PCR product, the expected size to be 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) would prove that the plasmid had not been integrated at the correct site and that the gene had not been knocked out.
Genes that are not essential for survival in vitro normally resulted in many transformants as observed for the positive control of ribonuclease H gene. Any transformants showing positive results for all three tests above would result in the conclusion that the gene was not essential for survival in vitro.
Genes that are essential for survival in vitro normally showed very few transformants. All transformants would be screened. A negative result of any of the three above tests for each transformant would lead to the conclusion that the gene had not been disrupted, and that the gene was essential for survival in vitro.
In the event that no colonies resulted from two independent transformations while the positive control with the disrupted ribonuclease H plasmid DNA produced transformants, the plasmid DNA was further analyzed by PCR on DNA from
transformant populations prior to plating for colony formation, to verify that it can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA was incubated according to the transformation protocol described above. DNA was extracted from the H. pylori cells immediately after incubation with the plasmid DNA and the DNA was used as templates for the above TEST 2 and TEST 3. Positive results of 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
Genes used in these experiments have been found to be essential, non-essential, or are still in progress, as indicated in Table 15.
Figure imgf000119_0001
VIII. Cloning, purification and characterization of the gene encoding the peptidyl- propyl cis-trans isomerase of H pylori
The Helicobacter pylori genome contains an open reading frame (ORF) of 170 amino acids was found to have homology with the Synechococcus sp. (strain PCC 7942).ppi gene (NCBI Accession number P29820). Therefore, to evaluate whether this
ORF encoded a protein with PPiase activity, the gene was isolated by polymerase chain reaction (PCR) amplification cloning, overexpressed in E. coli, and the protein purified to homogeneity. To facilitate purification, a polyhistidine tag was added to the N- terminus of this ORF. A simple assay using PPIase to evaluate protein folding function was developed for future use as a high-throughput drug screen.
Currently, the class of PPIases is divided into three unrelated families: the cyclophilins, the FK506-binding (FKBPs) and the parvulins. Although PPIase mutants have been reported from yeast and fruit fly, attempts to isolate disruption mutants in
Escherichia coli were unsuccessful (Shieh, B.Η., et.al. (1989) Nature 338:67-70). This suggests that this activity is essential for viability in bacteria.
Cloning, expression and protein purification
To facilitate the cloning, expression and purification of ppi from H. pylori, a powerful gene expression system, the pΕT System, for cloning and expression of recombinant ppi in Ε. coli. In this study, the sequence for H. pylori ppi contains a DNA sequence encoding a His-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.
A synthetic oligonucleotide primer (5'-TTATGGATCCAAACCAATTAAAA CT-3' (SΕQ ID NO: 1287)) specific for the 5' end of ppi gene encoded a BamHI site at its extreme 5' terminus and a primer (5'-TATCTCGAGTTATAGAGAAGGGC-3' (SΕQ ID NO: 1288)) specific for the 3' end of the ppi gene encoded a Xhol site at its extreme 5' terminus. Genomic DNA prepared from the J99 strain 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 the H. pylori ppi gene, 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:
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, the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA sample was subjected to digestion with the restriction endonucleases, BamHI and Xhol (New England BioLabs, Beverly, MA, USA) (Current Protocols in Molecular Biology, Ibid). The DNA as subjected to electrophoresis on a 1.0 % NuSeive (FMC BioProducts, Rockland, ME USA) agarose gel. 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, transformation, expression and purification of the PPI gene was carried out essentially as described in Examples II and III above.
Assay for PPiase activity
The assay for PPIase was essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Acta 43 : 1101 - 11 1 1). 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 were collected every second with an average scanning of time of 0.5 second. Assays were carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 μ M 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) was added to 390 μl of reaction mixture at room temperature. PPiase assay in crude bacterial extract.
A 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth was 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. Ther suspension was 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 was centrifuged (12,000 g x 30 minutes) and the supernatant was assayed for PPiase activity. Results
PPI from H. pylori was expressed in E. coli using the pET-28b expression vector from Novagen (cat # 69868-1). The expressed recombinant protein was isolated from the soluble fraction of bacterial cells that had been disrupted by cavitation in a
Microfluidics Cell disruption chamber. The expression levels of recombinant PPI produced 100 mg of protein. The recombinant protein could be purified to homogeneity by Ni2+ chelate chromatography and gel filtration. On sodium dodecyl sulfate polyacrylamide gels, the recombinant protein migrates as a single band at 21 kDa, in accordance with the predicted molecular weight of 20,975 deduced from the gene sequence.
The PPIase activity was assayed using the chromogenic tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. An initial velocity of 4.9 μmole/min/mg protein was measured with the purified enzyme (Figure 5). This corresponds to a kcat of 1.6 sec -1 which is similar to the one obtained for the E. coli PPIase (Liu, J. and Walsh, CT. (1990) Proc.Natl. Acad. Sci. USA 87:4028-4032) and the one from porcine kidney (Fischer, G. (1989) Nature 337:476-478).
The recombinant protein has a high catalytic efficiency of 2.06 X 109 M-1 s-1 when the assay is measured at 25°C. These values are one to two orders of magnitude higher than that observed for other characterized PPIases. However, in those studies, the ppiase assay was conducted at 10 C, which may account for the discrepency. The calalytic efficiency is very close to the 1 X 108 to 1 X 10 9 M-1 s-1 upper diffusinal limit for "kinetically perfect" enzymes (Albery, W.J. and Knowles, J.R. (1976) Biochemistry 15:5631 -5640) and suggests that by at least one measure, the H. pylori PPIase is a highly effective catalyst in the cis-trans isomerisation of the Ala-Pro bond in the oligopeptide substrate. The presence of PPIase was also determined in an H pylori extract. As with the assay for the recombinant protein, PPIase activity was detected, and was dependent on the concentration of extract added (Figure 6).
These results show that PPIase activity can be measured on either H. pylori extracts or on the recombinant protein in E. coli. The high catalytic efficiency also demonstrates that H. pylori enzymes, such as PPIase, 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. IX. Cloning, purification, and characterization of the gene encoding the glutamate racemase of H pylori.
The Helicobacter pylori genome contains an open reading frame (ORF) of 255 amino acids that was found to have homology to the Staphylococcus haemolyticus glutamate racemase gene (dga) (NCBI Accession number U 12405) and to the E. coli murl gene which encodes glutamate racemase activity in that organism. To evaluate whether this H pylori ORF encodes a protein with glutamate racemase activity, the gene was isolated by polymerase chain reaction (PCR) amplification cloning, overexpressed in E. coli, and the protein purified to apparent homogeneity. A simple assay for glutamate racemase activity resulting in the isomerization of D-glutamic acid to L- glutamic acid was developed to facilitate purification and for future use as a high- throughput drug screen.
The ORF in H pylori has been found by gene disruption studies to be essential for viability ofH. pylori cells in laboratory culture (see Example VII above). Therefore, inhibition of the enzymatic activity would be expected to be lethal for the organism, and such inhibitors may have utility in antimicrobial therapy of human infectious diseases.
Cloning of H. pylori murl gene encoding glutamate racemase
A 765 base pair DNA sequence encoding the murl gene of H. pylori was isolated by polymerase chain reaction (PCR) amplification cloning. A synthetic oligonucleotide primer (5'-AAATAGTCATATGAAAATAGGCGTTTTTG -3' (SEQ ID NO: 1289)) encoding an Ndel restriction site and the 5' terminus of the murl gene and a primer (5'-AGAATTCTATTACAATTTGAGCCATTCT -3' (SEQ ID NO: 1290)) encoding an EcoRI restriction site and the 3' end of the murl gene were used to amplify the murl gene of H. pylori using genomic DNA prepared from the J99 strain of H. pylori as the template DNA for the 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 the murl gene, genomic DNA (25 nanograms) was introduced into each of two reaction vials containing 1.0 micromole of each synthetic oligonucleotide primer, 2.0 mM MgCl2. 0.2 mM of each deoxynucleotide triphosphate (dATP, dGTP, dCTP & dTTP), and 1.25 units of heat stable DNA polymerases (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 50 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for the murl gene using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Conditions for amplification of H. pylori murl;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 30 sec and 72°C for 15 sec
23 cycles at 94°C for 15 sec, 53°C for 30 sec and 72°C for 15 sec
Reactions were concluded at 72°C for 20 minutes
Upon completion of thermal cycling reactions, the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA sample was subjected to digestion with the restriction endonucleases, Ndel and EcoRI (New England Biolabs, Beverly, MA USA) (Current Protocols in Molecular Biology, Ibid). The DNA samples from each of two reaction mixtures were pooled and subjected to electrophoresis on a 1.0% SeaPlaque (FMC BioProducts, Rockland, ME, USA) agarose gel. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. Amplified DNA encoding the H pylori murl gene was isolated from agarose gel slices and purified using the Bio 101
GeneClean Kit protocol (Bio 101 Vista, CA, USA).
Cloning ofH pylori DNA sequences into the pET-23 prokaryotic expression
vector.
The pET-23b vector can be propagated in any E. coli K-12 strain, e.g., ΗMS174, HB101, JM109, DH5α, 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 lacl gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest. Strains used in our laboratory include: BL21(DE3) (Studier, F.W.,
Rosenberg, A.H.. Dunn, J.J.. and Dubendorff, J.W. (1990) Meth. Enzymol. 185. 60-89).
The pET-23b vector (Novagen, Inc., Madison, WI, USA) was prepared for cloning by digestion with Ndel and EcoRI (Current Protocols in Molecular Biology, Ibid). Following digestion, the amplified, agarose gel-purified DNA fragment carrying the murl gene was cloned (Current Protocols in Molecular Biology, Ibid) into the previously digested pET-23b expression vector. Products of the ligation reaction were then used to transform the BL21(DE3) strain of E. coli.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET23-murI expression plasmid carrying the cloned H pylori sequence according to standard methods (Current Protocols in Molecular, Ibid). 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 100 microgram/ml ampicillin 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-23-murI 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, Ibid).
Isolation and Preparation of plasmid DNA from BL21 transformants
Colonies carrying pEY-23-murI vectors were picked and incubated in 5 mis of LB broth plus 100 microgram/ml ampicillin overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).
Cloning and expression of the E. coli groE operon
It has been demonstrated that coexpression of the E. coli murl gene with the genes in the E. coli groE operon reduces the formation of insoluble inclusion bodies containing recombinant glutamate racemase (Ashiuchi, M., Yoshimura, T., Kitamura, T., Kawata, Y., Nagai, J., Gorlatov, S., Esaki, N. and Soda, K., 1995, J. Biochem. 1 17, 495-498). The groE operon encodes two proteins, GroES (97 amino acids) and GroEL (548 amino acids), which are molecular chaperones. Molecular chaperones cooperate to assist the folding of new polypeptide chains (F. Ulrich Hartl , 1996, Nature London 381 , pp. 571-580).
The 2210 bp DNA sequence encoding the groE operon of E. coli (NCBI
Accession number X07850) was isolated by polymerase chain reaction (PCR) amplification cloning. A synthetic oligonucleotide primer (5'-GCGAATTCGATCAG AATTTTTTTTCT-3' (SEQ ID NO: 1291)) encoding an EcoRI restriction site and the 5' terminus of the groE operon containing the endogenous promoter region of the groE operon and a primer (5'-ATAAGTACTTGTGAATCTTATACTAG -3' (SEQ ID
NO: 1292)) encoding a Seal restriction site and the 3' end of the groEL gene contained in the groE operon were used to amplify the groE operon of E. coli using genomic DNA prepared from E. coli strain MG1655 as the template DNA for the PCR amplification reactions (Current Protocols in Molecular Biology, Ibid). To amplify a DNA sequence containing the E. coli groE operon, genomic DNA (12.5 nanograms) was introduced into each of two reaction vials containing 0.5 micromoles of each synthetic
oligonucleotide primer, 1.5 mM MgCl2, 0.2 mM each deoxynucleotide triphosphate (dATP, dGTP, dCTP & dTTP) and 2.6 units heat stable DNA polymerases (Expanded High Fidelity PCR System, Boehringer Mannheim, Indianapolis, Indiana) in a final volume of 50 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for the groE operon using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Conditions for amplification and cloning of the E. coli groE operon;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 30 sec and 72°C for 2 min
23 cycles at 94°C for 15 sec, 55°C for 30 sec and 72°C for 2 min
Reactions were concluded at 72°C for 8 minutes
Upon completion of thermal cycling reactions, the amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA sample was subjected to digestion with the restriction endonucleases. EcoRI and Scal (New England Biolabs. Beverly, MA USA) (Current Protocols in Molecular Biology, Ibid). The DNAs from each of two reaction mixtures were pooled and subjected to electrophoresis in a 1.0% SeaPlaque (FMC BioProducts, Rockland, ME, USA) agarose gel. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarsoe gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista. CA, USA).
A DNA fragment, EcoRI to Seal, containing the E. coli groE operon was cloned into the corresponding sites of the pACYC184 expression vector (New England Biolabs, Beverly, MA, USA) to make pACYC 184-groE. The BL21 (DE3) strain of E. coli was transformed with pACYC-groE. A tetracycline-resistant transformant overexpressing proteins of Mr ~ 14,000 (GroΕS) and Mr ~ 60,000 (GroΕL) was isolated.
Transformation of E. coli strain BL21(DE3) carrying the pACYC-groE plasmid ofE. coli.
Competent bacteria derived from a clone of strain BL21(DΕ3) carrying the pACYC-groE plasmid were transformed with 50 nanograms of pET23-murI plasmid
DNA, isolated as described above (Current Protocols in Molecular Biology, Ibid). A clone of BL21(DΕ3) carrying both the pACYC-groE expression plasmid and the pΕT- 23-murI plasmid was isolated and used for expression of recombinant glutamate racemase as described below.
Expression of recombinant H pylori murl
A bacterial clone of BL21(DΕ3) carrying both the pACYC-graE expression plasmid and the pΕT-23-murI plasmid was cultured in LB broth supplemented with 1.0 mM D,L-glutamic acid and 100 microgram/ml ampicillin and 10 micrograms/ml tetracycline at 30°C until an optical density at 600 nM of 0.5 to 1.0 O.D. units was reached, at which point, isopropyl-beta-D-thiogalactoside (IPTG) was added to the culture at a final concentration of 1.0 mM. Cells were cultured overnight 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 3000 x g for 20 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. Pellets were weighed (average wet weight = 6 grams/liter) and processed to purify recombinant protein as described below.
Purification of soluble glutamate racemase
All steps were carried out at 4°C. Cells were suspended in 4 volumes of lysis buffer (50 mM Potassium phosphate, pΗ 7.0, 100 mM NaCl, 2 mM EDTA. 2 mM
EGTA, 10% glycerol, 10 mM D,L-glutamic acid, 0.1 % β-mercaptoethanol, 200 μg/ ml lysozyme, 1 mM 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 three passages through a small volume microfluidizer (Model M-110S, Micro fluidics
International Corporation, Newton, MA). The resultant homogenate was diluted with 1 volume of buffer A (10 mM Tris-HCl pH 7.0, 0.1 mM EGTA, 10 % glycerol, 1 mM DL- Glutamic acid, 1 mM PMSF, 0.1% beta-mercaptoethanol), made 0.1 % Brij-35, and centrifuged (100,000 x g, 1 h) to yield a clear supernatant (crude extract).
After filtation through a 0.80-um filter, the extract was loaded directly onto a 20 ml Q-Sepharose column pre-equilibrated in buffer A containing 100 mM NaCl and 0.02 % Brij -35. The column was washed with 100 ml (5 bed volumes) of Buffer A containing 100 mM NaCl and 0.02 % Brij-35, then developed with a 100-ml linear gradient of increasing NaCl (from 100 to 500 mM) in Buffer A. A band of Mr = 28,000 corresponding to glutamate racemase, the product of the recombinant H pylori murl gene, eluted at a gradient concentration of approximately 200-280 mM NaCl. Individual column fractions were then characterized for glutamate racemase activity (see below for description of assay) and the protein profile of the fractions were analyzed on 12 % acrylamide SDS-PAGE gels.
Fractions containing glutamate racemase were pooled, brought to 70% saturation with solid (NH4)2SO4, stirred for 20 min, and then centrifuged at 27,000 x g for 20 min. The resulting pellet was resuspended in lysis buffer to a final volume of 8 ml and loaded directly onto a 350-ml column (2.2 x 92 cm) of Sephacryl S-100HR gel filtration medium equilibrated in buffer B (10 mM Hepes pH 7.5, 150 mM NaCl, 0.1 mM EGTA, 10% glycerol, 1 mM D,L-glutamatic acid, 0.1 mM PMSF, 0.1 % beta-mercaptoethanol) and run at 30 ml/h. Fractions found to contain a glutamate racemase activity were pooled, and 0.5 volume of buffer C (10 mM Tris pH 7.5, 0.1 mM EGTA, 10% glycerol, 1 mM D,L-glutamic acid, 0.1 mM PMSF, 0.1 % B -mercaptoethanol) was added (to reduce the NaCl concentration to 100 mM), and loaded onto a MonoQ 10/10 high- pressure liquid chromatography column equilibrated in buffer C containing 100 mM NaCl. The column was washed with 5 bed volumes of this buffer and developed with a 40 ml linear gradient of increasing NaCl (from 100 to 500). Glutamate racemase eluted as a sharp peak at 310 mM NaCl. Fractions containing a glutamate racemase activity were pooled, concentrated by dialysis against storage buffer [50% glycerol, 10 mM 3- (N-morpholino-propanesulfonic acid (MOPS) pH 7.0, 150 mM NaCl, 0.1 mM EGTA, 0.02 % Brij-35, 1 mM dithiothreitol (DTT)], and stored at -20°C. Assays for glutamate racemase activity.
Conversion of D-glutamate to L-glutamate (two enzyme coupled assay)
The activity of glutamate racemase, interconversion of the enantiomers of glutamic acid, was measured using D-glutamic acid as substrate. The method of Gallo and Knowles (Gallo, K.A. and Knowles, J.R., 1993, Biochmistry 32, 3981-3990) that was used to measure the glutamate racemase activity of Lactobacillus fermenti was adapted for the measurement of glutamate racemase activity of the H pylori murl gene product isolated as a recombinant protein from E. coli. In this assay, the measurement of the activity of glutamate racemase is linked to an OD change in the visible range in a series of coupled reactions to the activities of L-glutamate dehydrogenase (reduction of NAD to NADΗ) and diaphorase (reduction of the dye p-iodonitrotetrazolium violet, INT). Initial rates were determined by following the increase in absorbance at 500 nm in a reaction volume of 200 μl containing 50 mM Tris-ΗCl, pΗ 7.8, 4% v/v glycerol, 10 mM NAD, 2 mM INT, 60 Units/ml L-glutamate dehydrogenase, 5 Units/ml diaphorase, and varying concentrations of either substrate (from 0.063 mM to 250 mM D-glutamic acid) or purified enzyme (from 1μg to 50 μg). After a preincubation of all reagents except either the substrate (D-glutamic acid) or the enzyme (murl gene product) for a period of 5 minutes, reactions were initiated by adding the missing ingredient (i.e., the enzyme or the subtrate, as required), and the increase in optical density at 500 nm was measured in a Microplate Spectophotometer System (Molecular Devices, Spectra MAX 250). Measurements were followed for 20 minutes, and initial velocities were derived by calculating the maximum slope for the absorbance increases. The coupled reaction can be summarized as shown below:
1) D-glutamate → L-glutamate
glutamate racemase
2) L-glutamate + Η2O + NAD+ → 2-oxoglutarate + NH3 + NADH
L-glutamate dehydrogenase
3) NADH + INT → NAD+ + formazan (color)
diaphorase Conversion of D-glutamate to L-glutamate (single enzyme coupled assay)
In this assay, the conversion of D-glutamic acid to L-glutamic acid is coupled to the conversion of L-glutamic acid and NAD+ by L-glutamate dehydrogenase to 2- oxoglutarate, ammonia. The production of NADH is measured as an increase of absorbance at 340 nm (the reduction of NAD+ to NADH) at 37°C. The standard assay mixture (adapted from Choi, S-Y„ Esaki, N., Yoshimura, T., and Soda, K., 1991.
Protein Expression and Purification 2, 90-93) contained 10 mM Tris-HCl, pH 7.5, 5 mM NAD+, 5 Units/ml L-glutamate dehydrogenase, varying concentrations of the substrate D-Glutamic Acid (0.063 mM to 250 mM), and the purified recombinant H. pylori enzyme glutamate racemase (1 μg to 50 μg). The reaction was started by the addition of either the substrate D-glutamic acid or the recombinant glutamate racemase after a preincubation at 37°C for 5 minutes with all of the other assay ingredients. The change in absorbance at 340 nm was measured in a Spectra MAX 250. Initial velocities were derived from the initial slopes. The coupled reactions can be summarized as shown below:
1 ) D-glutamate → L-glutamate
glutamate racemase
2) L-glutamate + H2O + NAD+ → 2-oxoglutarate + NH3 + NADH
L-glutamate dehydrogenase
Results
1) Expression of the H. pylori murl gene in E. coli cells
To examine its biochemical properties, the H pylori glutamate racemase was overexpressed in E. coli and purified. In the presence of the E. coli chaperones GroES and GroEL, the glutamate racemase was expressed as a soluble protein. About 20 mg of soluble Murl was produced per liter of culture as judged by intensity of the protein band after SDS-PAGE. No band corresponding to the molecular weight of murl protein was seen in control gel lanes containing extracts from cells transformed with the pET vector lacking a murl insert. Addition of 1 mM DL-glutamic acid during cultivation of the expressing cells increased the apparent expression level by about five-fold.
2) Purification of recombinant H. pylori murl protein
Murl was purified by cation exchange chromatography and gel filtration. Upon SDS-PAGE analysis, the purified protein migrated as a single polypeptide species with an apparent mass 29 kDa which is consistent with the predicted mass of 28.858. 3) Kinetic properties of recombinant H. pylroi murl enzyme
Kinetic constant for recombinant glutamate racemase were estimated by assaying its activity at various concentrations of protein and D-glutamic acid as described above. Purified recombinant H pylori glutamate racemase exhibits a Vmax of ~ 300
nmoles/min/mg protein (kcat = 8.6 min -1 ) and a Km of ~100 μM for D-glutamate. Although the Vmax value is lower than that observed for highly purified glutamate racemase from some other bacterial species, its Km for D-glutamic acid is higher than that observed for the enzyme from most other species, resulting in a catalytic efficiency (kcat/Km) which is typical of purified preparation from E. coli and P. Pentococcus.
4) Characterization of Murl: Inhibition by L-serine-O sulfate
The H pylori glutamate racemase was tested for inactivation with a sucuide inhibitor, L-serine-O sulfate, which is known to inhibit murl from E. coli. The enzyme was incubated in the presence of 20 mM L-serine-O sulfate, and at different times interval, aliquots were removed to determine residual activity. The initial velocity of purified recombinant H. pylroi murl protein was determined in the single enzyme coupled asssay following incubation with the inhibitor L-serine-O-sulfate (LSOS) at 20 mM for the times indicated on the x-axis. The control was incubated in an identical manner but without LSOS. As shown in Figure 7, the H pylori glutamate racemase can be readily inactivated by the inhibitor.
Future application of the glutamate racemase activity in high throughput drug screening assays.
The assays for measurement of H. pylori glutamate racemase activity described above have been carried out in 96-well plates in which multiple reactions were conducted simultaneously. Measurements of activity in a multi-well format are readily amenable to scale-up to permit rapid analysis of numerous compounds for inhibition of the glutamate racemase activity. Compunds which inhibit the activity of glutamate racemase may have application as novel antibiotics and may be suitable for the treatment and eradication of bacterial (e.g., H. pylori) infections in humans. Known inhibitors of glutamate racemase, such as L-serine-O-sulfate, can be used to calibrate high throughput screens of new compound libraries to facilitate identification of new compounds with properties suitable for in vivo human therapeutics. 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 imgf001111_0001

Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori polypeptide selected from the group consisting of SEQ ID NO: 492-SEQ ID NO: 759, SEQ ID NO: 761 , SEQ ID NO: 763, SEQ ID NO: 765-SEQ ID NO: 818, SEQ ID NO: 820-SEQ ID NO: 846, SEQ ID NO: 848-SEQ ID NO: 896, SEQ ID NO: 898- SEQ ID NO: 963, SEQ ID NO: 966-SEQ ID NO: 982, SEQ ID NO: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041-SEQ ID NO: 1087, SEQ ID NO.1090 and SEQ ID NO: 1296- SEQ ID NO: 1298.
2. A recombinant expression vector comprising the nucleic acid of claim 1 operably linked to a transcription regulatory element.
3. A cell comprising a recombinant expression vector of claim 2.
4. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 3 under conditions that permit expression of the polypeptide.
5. A probe comprising a nucleotide sequence consisting of at least 8 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274-SEQ ID NO: 327, SEQ ID NO: 329-SEQ ID NO: 364, SEQ ID NO: 366-SEQ ID NO: 405, SEQ ID NO: 407-SEQ ID NO: 472, SEQ ID NO: 475-SEQ ID NO: 491 , SEQ ID NO: 983, SEQ ID NO: 984, SEQ ID NO: 987-SEQ ID NO: 1033, SEQ ID NO: 1036 and SEQ ID NO: 1293-SEQ ID NO: 1295 or the complement thereof.
6. An isolated nucleic acid comprising a nucleotide sequence of at least 8 nucleotides in length, wherein the sequence is hybridizable to a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274-SEQ ID NO: 327, SEQ ID NO: 329-SEQ ID NO: 364, SEQ ID NO: 366-SEQ ID NO: 405, SEQ ID NO: 407-SEQ ID NO: 472, SEQ ID NO: 475-SEQ ID NO: 491, SEQ ID NO: 983, SEQ ID NO: 984, SEQ ID NO: 987-SEQ ID NO: 1033, SEQ ID NO: 1036 and SEQ ID NO: 1293-SEQ ID NO: 1295 or the complement thereof.
7. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori polypeptide or a fragment thereof, said nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274-SEQ ID NO: 327, SEQ ID NO: 329-SEQ ID NO: 364, SEQ ID NO: 366-SEQ ID NO: 405, SEQ ID NO: 407-SEQ ID NO: 472, SEQ ID NO: 475-SEQ ID NO: 491, SEQ ID NO: 983, SEQ ID NO: 984, SEQ ID NO: 987-SEQ ID NO: 1033, SEQ ID NO: 1036 and SEQ ID NO: 1293-SEQ ID NO: 1295.
8. A vaccine composition of claim 7, further comprising a pharmaceutically acceptable carrier.
9. A vaccine composition of claim 8, wherein the pharmaceutically acceptable carrier is an adjuvant.
10. A method of treating a subject for H. pylori infection comprising administering to a subject a vaccine composition of claim 7, such that treatment ofH pylori infection occurs.
11. A method of claim 10, wherein the treatment is a prophylactic treatment.
12. A method of claim 10, wherein the treatment is a therapeutic treatment.
13. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising:
(a) contacting a sample with a probe of claim 5 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.
14. A recombinant or substantially pure preparation of an H. pylori polypeptide selected from the group consisting of SEQ ID NO: 492-SEQ ID NO: 759, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 765-SEQ ID NO: 818, SEQ ID NO: 820-SEQ ID NO: 846, SEQ ID NO: 848-SEQ ID NO: 896, SEQ ID NO: 898-SEQ ID NO: 963, SEQ ID NO: 966-SEQ ID NO: 982, SEQ ID NO: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041 -SEQ ID NO: 1087, SEQ ID NO: 1090 and SEQ ID NO: 1296-SEQ ID NO: 1298.
15. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of a purified H pylori polypeptide or a fragment thereof, wherein said H pylori polypeptide is selected from the group consisting of SEQ ID NO: 492-SEQ ID NO: 759, SEQ ID NO: 761 , SEQ ID NO: 763, SEQ ID NO: 765-SEQ ID NO: 818, SEQ ID NO: 820-SEQ ID NO: 846, SEQ ID NO: 848-SEQ ID NO: 896, SEQ ID NO: 898-SEQ ID NO: 963, SEQ ID NO: 966-SEQ ID NO: 982, SEQ ID NO: 1037, SEQ ID NO: 1038, SEQ ID NO: 1041-SEQ ID NO: 1087, SEQ ID NO:1090 and SEQ ID NO: 1296-SEQ ID NO: 1298.
16. A vaccine composition of claim 15, further comprising a
pharmaceutically acceptable carrier.
17. A vaccine composition of claim 16, wherein the pharmaceutically acceptable carrier is an adjuvant.
18. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 15, such that treatment of H pylori infection occurs.
19. A method of claim 18, wherein the treatment is a prophylactic treatment.
20. A method of claim 18, wherein the treatment is a therapeutic treatment.
21. 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384. SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 101 1 , SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, SEQ ID NO: 1032, SEQ ID NO: 259, SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294, SEQ ID NO: 1295, SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437. SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, SEQ ID NO: 1031, SEQ ID NO: 254, SEQ ID NO: 352, SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381, SEQ ID NO: 389, SEQ ID NO: 1010, SEQ ID NO: 1012, SEQ ID NO: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421, SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 41 1, SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO: 290, SEQ ID NO: 417, SEQ ID NO: 430, SEQ ID NO: 992, SEQ ID NO: 1025, SEQ ID NO: 477, SEQ ID NO: 414, SEQ ID NO: 253, SEQ ID NO: 293, SEQ ID NO: 334, SEQ ID NO: 343, SEQ ID NO: 418, SEQ ID NO: 424, and SEQ ID NO: 443.
22. The purified nucleic acid of claim 21 , 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: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 311 , SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466. SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 1011, SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, SEQ ID NO: 1032, SEQ ID NO: 259, SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294. SEQ ID NO: 1295, SEQ ID NO: 319. SEQ ID NO: 325, SEQ ID NO: 425. SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, SEQ ID NO: 1031, SEQ ID NO: 254, SEQ ID NO: 352, SEQ ID NO: 415, SEQ ID NO: 1019, SEQ ID NO: 381 , SEQ ID NO: 389, SEQ ID NO: 1010, and SEQ ID NO: 1012.
23. he purified nucleic acid of claim 22, wherein said H pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 255, SEQ ID NO: 263, SEQ ID NO: 266, SEQ ID NO: 277, SEQ ID NO: 280, SEQ ID NO: 285, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 299, SEQ ID NO: 31 1, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331 , SEQ ID NO: 353, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 368, SEQ ID NO: 375, SEQ ID NO: 384, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 404, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 427, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 441 , SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 983, SEQ ID NO: 989, SEQ ID NO: 1008, SEQ ID NO: 101 1, SEQ ID NO: 1014, SEQ ID NO: 1015, SEQ ID NO: 1029, and SEQ ID NO: 1032.
24. The purified nucleic acid of claim 22, wherein said H pylori outer membrane polypeptide or a fragment thereof is an H pylori polypeptide having a C- terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 286, SEQ ID NO: 326, SEQ ID NO: 374, SEQ ID NO: 399, SEQ ID NO: 422, SEQ ID NO: 454, SEQ ID NO: 465, SEQ ID NO: 998, SEQ ID NO: 1009, SEQ ID NO: 1023, SEQ ID NO: 1294, and SEQ ID NO: 1295.
25. The purified nucleic acid of claim 22, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 319, SEQ ID NO: 325, SEQ ID NO: 425, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 467, SEQ ID NO: 996, SEQ ID NO: 1027, and SEQ ID NO: 1031.
26. The purified nucleic acid of claim 21 , wherein said II. 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: 354, SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421. SEQ ID NO: 1022, SEQ ID NO: 463, SEQ ID NO: 281, SEQ ID NO: 988, SEQ ID NO: 41 1 , SEQ ID NO: 407, SEQ ID NO: 1017, SEQ ID NO: 290, SEQ ID NO: 417, SEQ ID NO: 430, SEQ ID NO: 992, and SEQ ID NO: 1025.
27. The purified nucleic acid of claim 26, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 354.
28. The purified nucleic acid of claim 26, wherein said H. pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 372, SEQ ID NO: 400, SEQ ID NO: 421 , and SEQ ID NO: 1022.
29. The purified nucleic acid of claim 26, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 463.
30. The purified nucleic acid of claim 26, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in secretion and adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 281 and SEQ ID NO: 988.
31. The purified nucleic acid of claim 26, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 407 and SEQ ID NO: 1017.
32. The purified nucleic acid of claim 21 , wherein said H pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 477.
33. The purified nucleic acid of claim 21, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori transport polypeptide or a fragment thereof encoded by the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 414.
34. A recombinant expression vector comprising the nucleic acid of claim 21 operably linked to a transcription regulatory element.
35. A cell comprising a recombinant expression vector of claim 34.
36. A method for producing an H pylori polypeptide comprising culturing a cell of claim 35 under conditions that permit expression of the polypeptide.
37. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 355, SEQ ID NO: 1006, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 261 , SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 323, SEQ ID NO: 338, SEQ ID NO: 342, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 356, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 373, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 401, SEQ ID NO: 403, SEQ ID NO: 405, SEQ ID NO: 408, SEQ ID NO: 420, SEQ ID NO: 426, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 432, SEQ ID NO: 439, SEQ ID NO: 442, SEQ ID NO: 451 , SEQ ID NO: 471 , SEQ ID NO: 478, SEQ ID NO: 488, SEQ ID NO: 987, SEQ ID NO: 990, SEQ ID NO: 991 , SEQ ID NO: 993, SEQ ID NO: 1001 , SEQ ID NO: 1002, SEQ ID NO: 1007, SEQ ID NO: 1013, SEQ ID NO: 1016, SEQ ID NO: 1018, SEQ ID NO: 1021 , and SEQ ID NO: 1026.
38. The purified nucleic acid of claim 37, wherein said H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 355 and SEQ ID NO: 1006.
39. A recombinant expression vector comprising the nucleic acid of claim 38 operably linked to a transcription regulatory element.
40. A cell comprising a recombinant expression vector of claim 39.
41. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 40 under conditions that permit expression of the polypeptide.
42. 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: 470, SEQ ID NO: 1033, SEQ ID NO: 357, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 1030, SEQ ID NO: 345, SEQ ID NO: 383,
SEQ ID NO: 387, SEQ ID NO: 455, SEQ ID NO: 1003, SEQ ID NO: 351, SEQ ID NO: 416, SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, SEQ ID NO: 1293, SEQ ID NO: 318, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 330, SEQ ID NO: 347, SEQ ID NO: 440, SEQ ID NO: 446, SEQ ID NO: 464, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 995, SEQ ID NO: 997, SEQ ID NO: 1005, SEQ ID NO: 1028.
43. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 470 and SEQ ID NO: 1033.
44. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 357 and SEQ ID NO: 457.
45. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 461 and SEQ ID NO: 1030.
46. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in cofactor metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 345, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 455, and SEQ ID NO: 1003.
47. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in lipid metabolism or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 351 and SEQ ID NO: 416.
48. The purified nucleic acid of claim 42, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 278, SEQ ID NO: 335, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 419, SEQ ID NO: 460, SEQ ID NO: 472, SEQ ID NO: 1000, SEQ ID NO: 1004, SEQ ID NO: 1020, and SEQ ID NO: 1293.
49. A recombinant expression vector comprising the nucleic acid of claim 42 operably linked to a transcription regulatory element.
50. A cell comprising a recombinant expression vector of claim 49.
51. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 50 under conditions that permit expression of the polypeptide.
52. An isolated nucleic acid comprising a nucleotide sequence encoding an
H pylori cellular polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 256, SEQ ID NO: 267, SEQ ID NO: 282. SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309. SEQ ID NO: 310, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 344, SEQ ID NO: 369, SEQ ID NO: 376, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 423, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481 , SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 489, SEQ ID NO: 984, SEQ ID NO: 994, SEQ ID NO: 1024, and SEQ ID NO: 1036.
53. A recombinant expression vector comprising the nucleic acid of claim 52 operably linked to a transcription regulatory element.
54. A cell comprising a recombinant expression vector of claim 53.
55. A method for producing an H pylori polypeptide comprising culturing a cell of claim 54 under conditions that permit expression of the polypeptide.
56. A vaccine composition for prevention or treatment of an H pylori infection comprising an effective amount of a nucleic acid of claim 21.
57. A vaccine composition of claim 56, further comprising a
pharmaceutically acceptable carrier.
58. A vaccine composition of claim 57, wherein the pharmaceutically acceptable carrier is an adjuvant.
59. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 56, such that treatment of H. pylori infection occurs.
60. A method of claim 59, wherein the treatment is a prophylactic treatment.
61. A method of claim 59, wherein the treatment is a therapeutic treatment.
62. A vaccine composition for prevention or treatment of an H pylori infection comprising an effective amount of a nucleic acid of claim 37.
63. A vaccine composition of claim 62, further comprising a
pharmaceutically acceptable carrier.
64. A vaccine composition of claim 63, wherein the pharmaceutically acceptable carrier is an adjuvant.
65. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 62, such that treatment of H. pylori infection occurs.
66. A method of claim 65, wherein the treatment is a prophylactic treatment.
67. A method of claim 65, wherein the treatment is a therapeutic treatment.
68. A vaccine composition for prevention or treatment of an H pylori infection comprising an effective amount of a nucleic acid of claim 42.
69. A vaccine composition of claim 68, further comprising a
pharmaceutically acceptable carrier.
70. A vaccine composition of claim 69, wherein the pharmaceutically acceptable carrier is an adjuvant.
71. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 68, such that treatment of H. pylori infection occurs.
72. A method of claim 71 , wherein the treatment is a prophylactic treatment.
73. A method of claim 71 , wherein the treatment is a therapeutic treatment.
74. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of a nucleic acid of claim 52.
75. A vaccine composition of claim 74, further comprising a
pharmaceutically acceptable carrier.
76. A vaccine composition of claim 75, wherein the pharmaceutically acceptable carrier is an adjuvant.
77. A method of treating a subject for H. pylori infection comprising administering to a subject a vaccine composition of claim 74, such that treatment ofH. pylori infection occurs.
78. A method of claim 77, wherein the treatment is a prophylactic treatment.
79. A method of claim 77, wherein the treatment is a therapeutic treatment.
80. A purified H. pylori cell envelope polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941 , SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, SEQ ID NO: 1086, SEQ ID NO: 750, SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063. SEQ ID NO: 1077, SEQ ID NO: 1297, SEQ ID NO: 1298, SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938. SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, SEQ ID NO: 1085, SEQ ID NO: 745, SEQ ID NO: 843, SEQ ID NO: 906, SEQ ID NO: 1073, SEQ ID NO: 872, SEQ ID NO: 880, SEQ ID NO: 1064, SEQ ID NO: 1066, SEQ ID NO:
845, SEQ ID NO: 863, SEQ ID NO: 891 , SEQ ID NO: 912, SEQ ID NO: 1076, SEQ ID NO: 954, SEQ ID NO: 772, SEQ ID NO: 1042, SEQ ID NO: 902, SEQ ID NO: 898, SEQ ID NO: 1071 , SEQ ID NO: 781 , SEQ ID NO: 908, SEQ ID NO: 921. SEQ ID NO: 1046, SEQ ID NO: 1079, SEQ ID NO: 968, SEQ ID NO: 905, SEQ ID NO: 744, SEQ ID NO: 784, SEQ ID NO: 825, SEQ ID NO: 834, SEQ ID NO: 909, SEQ ID NO: 915, and SEQ ID NO: 934.
81. The purified polypeptide of claim 80, 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: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, SEQ ID NO: 1086, SEQ ID NO: 750, SEQ ID NO: 777, SEQ ID NO: 817, SEQ ID NO: 865, SEQ ID NO: 890, SEQ ID NO: 913, SEQ ID NO: 945, SEQ ID NO: 956, SEQ ID NO: 1052, SEQ ID NO: 1063, SEQ ID NO: 1077, SEQ ID NO: 1297, SEQ ID NO: 1298, SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, SEQ ID NO: 1085, SEQ ID NO: 745, SEQ ID NO: 843, SEQ ID NO: 906, SEQ ID NO: 1073, SEQ ID NO: 872, SEQ ID NO: 880, SEQ ID NO: 1064, and SEQ ID NO: 1066.
82. The purified polypeptide of claim 81 , wherein said H pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, and SEQ ID NO: 1086.
83. The purified polypeptide of claim 81 , wherein said H pylori outer membrane polypeptide or a fragment thereof is an H pylori polypeptide a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 746, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 776, SEQ ID NO: 783, SEQ ID NO: 785, SEQ ID NO: 790, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 882, SEQ ID NO: 844, SEQ ID NO: 855, SEQ ID NO: 857, SEQ ID NO: 859, SEQ ID NO: 866, SEQ ID NO: 875, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 893, SEQ ID NO: 895, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 903, SEQ ID NO: 918, SEQ ID NO: 924, SEQ ID NO: 925, SEQ ID NO: 932, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 957, SEQ ID NO: 959, SEQ ID NO: 960, SEQ ID NO: 1037, SEQ ID NO: 1043, SEQ ID NO: 1062, SEQ ID NO: 1065, SEQ ID NO: 1068, SEQ ID NO: 1069, SEQ ID NO: 1083, and SEQ ID NO: 1086.
84. The purified polypeptide of claim 81, wherein said H pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 810, SEQ ID NO: 816, SEQ ID NO: 916, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 958, SEQ ID NO: 1050, SEQ ID NO: 1081, and SEQ ID NO: 1085.
85. The purified polypeptide of claim 80, 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: 845, SEQ ID NO: 863, SEQ ID NO: 891, SEQ ID NO: 912, SEQ ID NO: 1076, SEQ ID NO: 954, SEQ IDNO: 772, SEQ IDNO: 1042, SEQ ID NO: 902, SEQ ID NO: 898, SEQ ID NO: 1071 , SEQ ID NO: 781 , SEQ ID NO: 908, SEQ ID NO: 921 , SEQ ID NO: 1046, SEQ ID NO: 1079.
86. The purified polypeptide of claim 85, wherein said II. pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in outer membrane and cell wall synthesis or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 845.
87. The purified polypeptide of claim 85, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 863, SEQ ID NO: 891 , SEQ ID NO: 912, and SEQ ID NO: 1076.
88. The purified polypeptide of claim 85, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in cofactor metabolism or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 954.
89. The purified polypeptide of claim 85, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H pylori polypeptide involved in secretion and adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 772 and SEQ ID NO: 1042.
90. The purified polypeptide of claim 85, wherein said H pylori inner membrane polypeptide or a fragment thereof is an H. pylori polypeptide involved in transport or a fragment thereof selected from the group consisting of SEQ ID NO: 898 and SEQ ID NO: 1071.
91. The purified polypeptide of claim 80, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori flagellar polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 968.
92. The purified polypeptide of claim 80, wherein said H pylori cell envelope polypeptide or a fragment thereof is an H pylori transport polypeptide or a fragment thereof comprising an amino acid sequence of SEQ ID NO: 905.
93. A purified H. pylori cellular polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 747, SEQ ID NO: 758, SEQ ID NO: 773, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801 , SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 830, SEQ ID NO: 831 , SEQ ID NO: 832, SEQ ID NO: 835, SEQ ID NO: 860, SEQ ID NO: 867, SEQ ID NO: 873, SEQ ID NO: 877, SEQ ID NO: 914, SEQ ID NO: 922, SEQ ID NO: 926, SEQ ID NO: 927, SEQ ID NO: 949, SEQ ID NO: 953, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 970, SEQ ID NO: 971, SEQ ID NO: 972, SEQ ID NO: 973, SEQ ID NO: 974, SEQ ID NO: 975, SEQ ID NO: 976, SEQ ID NO: 977, SEQ ID NO: 978, SEQ ID NO: 980, SEQ ID NO: 1038, SEQ ID NO: 1048, SEQ ID NO: 1078, and SEQ ID NO: 1090.
94. A purified H pylori secreted polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 846, SEQ ID NO: 1060, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 759, SEQ ID NO: 761 , SEQ ID NO: 763, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 770, SEQ ID NO: 774, SEQ ID NO: 775, SEQ ID NO: 778, SEQ ID NO: 779, SEQ ID NO: 780, SEQ ID NO: 782, SEQ ID NO: 786, SEQ ID NO: 787, SEQ ID NO: 788, SEQ ID NO: 789, SEQ ID NO: 791 , SEQ ID NO: 792, SEQ ID NO: 793, SEQ ID NO: 794, SEQ ID NO: 795, SEQ ID NO: 796, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 814, SEQ ID NO: 829, SEQ ID NO: 833, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851 , SEQ ID NO: 852, SEQ ID NO: 853, SEQ ID NO: 854, SEQ ID NO: 858, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 864, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 870, SEQ ID NO: 871 , SEQ ID NO: 879, SEQ ID NO: 881 , SEQ ID NO: 885, SEQ ID NO: 886, SEQ ID NO: 887, SEQ ID NO: 892, SEQ ID NO: 894, SEQ ID NO: 896, SEQ ID NO: 899, SEQ ID NO: 911, SEQ ID NO: 917, SEQ ID NO: 919, SEQ ID NO: 920, SEQ ID NO: 923, SEQ ID NO: 930, SEQ ID NO: 933, SEQ ID NO: 942, SEQ ID NO: 962, SEQ ID NO: 969, SEQ ID NO: 979, SEQ ID NO: 1041, SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1047, SEQ ID NO: 1055, SEQ ID NO: 1056, SEQ ID NO: 1061, SEQ ID NO: 1067, SEQ ID NO: 1070, SEQ ID NO: 1072, SEQ ID NO: 1075, and SEQ ID NO: 1080.
95. The purified polypeptide of claim 94, wherein said H. pylori secreted polypeptide or a fragment thereof is an H. pylori polypeptide involved in secretion and adhesion or a fragment thereof selected from the group consisting of SEQ ID NO: 846 and SEQ ID NO: 1060.
96. A purified H pylori cytoplasmic polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 961 , SEQ ID NO: 1087, SEQ ID NO: 848, SEQ ID NO: 948, SEQ ID NO: 952, SEQ ID NO: 1084, SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, SEQ ID NO: 1057, SEQ ID NO: 842, SEQ ID NO: 907, SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841, SEQ ID NO: 910, SEQ ID NO: 951, SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, SEQ ID NO: 1296, SEQ ID NO: 809, SEQ ID NO: 813, SEQ ID NO: 815, SEQ ID NO: 821, SEQ ID NO: 838, SEQ ID NO: 931 , SEQ ID NO: 937, SEQ ID NO: 955, SEQ ID NO: 981 , SEQ ID NO: 982, SEQ ID NO: 1049, SEQ ID NO: 1051 , SEQ ID NO: 1059, and SEQ ID NO: 1082.
97. The purified polypeptide of claim 96, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H. pylori polypeptide involved in energy conversion or a fragment thereof selected from the group consisting of SEQ ID NO: 961 and SEQ ID NO: 1087.
98. The purified polypeptide of claim 96, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in amino acid metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 848 and SEQ ID NO: 948.
99. The purified polypeptide of claim 96, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in nucleotide metabolism and transport or a fragment thereof selected from the group consisting of SEQ ID NO: 952 and SEQ ID NO: 1084.
100. The purified polypeptide of claim 96, wherein said H. pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in cofactor metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 836, SEQ ID NO: 874, SEQ ID NO: 878, SEQ ID NO: 946, and SEQ ID NO: 1057.
101. The purified polypeptide of claim 96, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in lipid metabolism or a fragment thereof selected from the group consisting of SEQ ID NO: 842 and SEQ ID NO: 907.
102. The purified polypeptide of claim 96, wherein said H pylori cytoplasmic polypeptide or a fragment thereof is an H pylori polypeptide involved in genome replication, transcription, recombination and repair or a fragment thereof selected from the group consisting of SEQ ID NO: 769, SEQ ID NO: 826, SEQ ID NO: 837, SEQ ID NO: 841 , SEQ ID NO: 910, SEQ ID NO: 951 , SEQ ID NO: 963, SEQ ID NO: 1054, SEQ ID NO: 1058, SEQ ID NO: 1074, and SEQ ID NO: 1296.
103. A vaccine composition for prevention or treatment of an H pylori infection comprising an effective amount of an H. pylori polypeptide of claim 80.
104. A vaccine composition of claim 103, further comprising a
pharmaceutically acceptable carrier.
105. A vaccine composition of claim 104, wherein the pharmaceutically acceptable carrier is an adjuvant.
106. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 103, such that treatment ofH pylori infection occurs.
107. A method of claim 106, wherein the treatment is a prophylactic treatment.
108. A method of claim 106, wherein the treatment is a therapeutic treatment.
109. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amount of an H pylori polypeptide of claim 93.
110. A vaccine composition of claim 109, further comprising a
pharmaceutically acceptable carrier.
11 1. A vaccine composition of claim 110, wherein the pharmaceutically acceptable carrier is an adjuvant.
1 12. A method of treating a subject for H. pylori infection comprising administering to a subject a vaccine composition of claim 109, such that treatment ofH. pylori infection occurs.
113. A method of claim 112, wherein the treatment is a prophylactic treatment.
1 14. A method of claim 1 12, wherein the treatment is a therapeutic treatment.
115. A vaccine composition for prevention or treatment of an H pylori infection comprising an effective amount of an H. pylori polypeptide of claim 94.
116. A vaccine composition of claim 1 15, further comprising a
pharmaceutically acceptable carrier.
117. A vaccine composition of claim 1 16, wherein the pharmaceutically acceptable carrier is an adjuvant.
118. A method of treating a subject for H. pylori infection comprising administering to a subject a vaccine composition of claim 1 15, such that treatment of H. pylori infection occurs.
1 19. A method of claim 1 18, wherein the treatment is a prophylactic treatment.
120. A method of claim 1 18, wherein the treatment is a therapeutic treatment.
121. A vaccine composition for prevention or treatment of an H. pylori infection comprising an effective amoimt of an H pylori polypeptide of claim 96.
122. A vaccine composition of claim 121, further comprising a
pharmaceutically acceptable carrier.
123. A vaccine composition of claim 122, wherein the pharmaceutically acceptable carrier is an adjuvant.
124. A method of treating a subject for H pylori infection comprising administering to a subject a vaccine composition of claim 121 , such that treatment of H. pylori infection occurs.
125. A method of claim 124, wherein the treatment is a prophylactic treatment.
126. A method of claim 124, wherein the treatment is a therapeutic treatment.
127. 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 21 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.
128. 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 37 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.
129. 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 42 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.
130. 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 52 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/US1997/005223 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof Ceased WO1997037044A1 (en)

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SK1305-98A SK130598A3 (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
JP9529649A JP2000501621A (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences related to Helicobacter pylori and vaccine compositions thereof
EP97917731A EP0901530A1 (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
IL12580897A IL125808A0 (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
BR9708456A BR9708456A (en) 1996-03-29 1997-03-27 Sequence of nucleic acid amino acids referring to helicobacter pylori and vaccine compositions with the same
NZ332565A NZ332565A (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
AU25984/97A AU726892B2 (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
EE9800334A EE9800334A (en) 1996-03-29 1997-03-27 Nucleic and amino acid sequences associated with Helicobacter pylor and vaccine compositions derived therefrom
HU0100267A HUP0100267A3 (en) 1996-03-29 1997-03-27 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
IS4831A IS4831A (en) 1996-03-29 1998-08-21 Nucleic and amino acid sequences related to helicobacter pylori and their vaccines
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US73690596A 1996-10-25 1996-10-25
US08/736,905 1996-10-25
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WO2000022135A1 (en) * 1998-10-15 2000-04-20 Chiron Behring Gmbh & Co. Helicobacter pylori vaccine
WO2000029432A1 (en) * 1998-11-17 2000-05-25 Provalis Uk Limited Heliobacter pylori antigen
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WO2002040516A3 (en) * 2000-11-15 2002-08-08 Ludwig Deml Helicobacter cysteine rich protein a (hcpa) and uses thereof
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CN115991745A (en) * 2022-07-19 2023-04-21 四川大学华西医院 A Helicobacter pylori recombinant antigen protein TatB and its preparation method and application

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ID18542A (en) 1998-04-16
NZ332565A (en) 2000-03-27
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IL125808A0 (en) 1999-04-11
AU2598497A (en) 1997-10-22
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IS4831A (en) 1998-08-21
EE9800334A (en) 1999-04-15
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BR9708456A (en) 1999-08-03
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