WO1997019098A1 - Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics - Google Patents

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

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Publication number
WO1997019098A1
WO1997019098A1 PCT/US1996/018542 US9618542W WO9719098A1 WO 1997019098 A1 WO1997019098 A1 WO 1997019098A1 US 9618542 W US9618542 W US 9618542W WO 9719098 A1 WO9719098 A1 WO 9719098A1
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WO
WIPO (PCT)
Prior art keywords
pylori
nucleic acid
seq
polypeptide
dna
Prior art date
Application number
PCT/US1996/018542
Other languages
French (fr)
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WO1997019098A9 (en
Inventor
Douglas H. Smith
Original Assignee
Astra Aktiebolag
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Filing date
Publication date
Application filed by Astra Aktiebolag filed Critical Astra Aktiebolag
Priority to AU10554/97A priority Critical patent/AU1055497A/en
Publication of WO1997019098A1 publication Critical patent/WO1997019098A1/en
Publication of WO1997019098A9 publication Critical patent/WO1997019098A9/en
Priority to US10/335,977 priority patent/US20040052799A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • H. pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, ( 1983) Lancet 1 : 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83-88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H.
  • H. pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma.
  • Boser M. J. (1993) Trends Microbiol. 1 : 255-260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age.
  • H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
  • urease an enzyme that may play a role in neutralizing gastric acid pH (Eaton et al., (1991) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee
  • This invention relates to novel genes, e.g., genes encoding bacterial surface proteins, from the organism Helicobacter 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 surface proteins or parts thereof nucleic acids capable of binding mRNA from H. pylori surface proteins to block protein translation, and methods for producing H. pylori surface proteins or parts thereof using peptide synthesis and recombinant DNA techniques.
  • This invention also includes antibodies and nucleic acids sequences useful as probes to detect H. pylori infection.
  • vaccine compositions and methods for the protection against infection by H. pylori are described.
  • Figure 1 is a table which contains information from homology searches performed on the sequences of this invention using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package.
  • Figure 2 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.
  • Figure 3 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.
  • Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
  • Figure 5 is a bar graph that depicts therapeutic immunization of H., pylori infected mice with specific antigens dissolved in buffer containing DOC.
  • the invention features a substantially pure nucleic acid encoding an
  • H. pylori polypeptide having an amino acid sequence of SEQ ID NO:115 such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1.
  • the invention features a substantially pure nucleic acid encoding an H.. pylori polypeptide having an amino acid sequence of SEQ ID NO:116, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:117, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:118. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:4.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:119, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:5.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:120, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:6.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:121, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:7.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:122, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:8.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:123, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:9.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:124. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:10.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:125, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:11.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:126, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:12.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:127, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:13.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:128, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:14.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:129, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:15.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:130, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:16.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:131 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:17.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:132, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:18.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:133, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:19.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:134, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:20.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:135, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:21.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:136, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:22.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:137, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:23.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:138, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:24.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:139, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:25.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:140, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:26.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:141, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:27.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:142, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:28.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:143, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:29.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:144, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:30.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:145, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:31.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:146, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:32.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:147, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:33.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:148, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:34.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:149, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:35.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:150, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:36.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:151, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:37.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:152, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:38.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:153, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:39.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:154, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:40.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:155, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:41.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:156, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:42.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:157, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:43.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 158, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:44.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:159, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:45.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:160, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:46.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:161, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:47.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:162, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:48.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:163, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:49.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:164, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:50.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:165, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 51.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:166, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:52.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:167, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:53.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:168, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:54.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:169, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:55.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:170, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:56.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:171, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:57.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:172, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:58.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:173, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:59.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:174, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:60.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:175, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:61.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:176, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:62.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:177, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:63.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:178, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:64.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:179, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:65.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:180, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:66.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:182, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:67.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:182, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:68.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:183, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:69.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:184, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:70.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:185, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:71.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:186, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:72.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:187, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:73.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:188, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:74.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:189, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:75.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:190, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:76.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:191, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:77.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:192, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:78.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:193.
  • a nucleic acid comprising a nucleotide sequence of SEQ ID NO:79.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:194, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:80.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:195, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:81.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:196, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:82.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:197, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:83.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:198, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:84.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:199, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:85.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:200, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:86.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:201 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:87.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:202, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:88.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:203, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:89.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:204, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:90.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:205, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:91.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:206, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:92.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:207, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:93.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:208, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:94.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:209, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:95.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:210, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:96.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:211 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:97.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:212, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:98.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:213, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:99.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:214, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:100.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:215, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:101.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:216, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:102.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:217, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:103.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:218, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:104.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:219, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:105.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:220, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:106.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:221, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:107.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:222, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:108.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:223, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:109.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:224, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:110.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:225, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:111.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:226, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:112.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:227, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:113.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:228. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:114.
  • the invention comprises nucleic acids capable of binding mRNA of H. pylori.
  • nucleic acid is capable of acting as anti-sense 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. Such nucleic acid has 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 make H. pylori polypeptides.
  • the invention features a method of generating antibodies against H. pylori proteins which are capable of binding specifically to H. pylori proteins.
  • Such antibody has utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori-specific antigens.
  • the invention features a method of generating vaccines for immunizing an individual against H. pylori.
  • the method includes: immunizing a subject with an H. pylori protein, e.g., a surface protein, or portion thereof, and a pharmaceutically acceptable carrier.
  • H. pylori protein e.g., a surface protein, or portion thereof
  • a pharmaceutically acceptable carrier e.g., 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 protein, e.g., a surface protein, or 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%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence contained in SEQ ID NOs:115-228; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NOs:115-228; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids 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 acids contained in SEQ ID NOs:115-228.
  • the H. pylori polypeptide is encoded by a nucleic acid in SEQ ID NOs: 1 -114, or by a nucleic acid having at least 60%. 70%, 80%, 90%, 95%. 98%, or 99% homology with a nucleic acid of SEQ ID NOs: 1-114.
  • the subject H. pylori polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues from a sequence in SEQ ID NOs:115-228.
  • the differences 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 SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
  • 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 includes an immunogen which includes an H. pylori polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
  • the immunogen comprises an antigenic determinant from a protein contained in SEQ ID NOs:115-228.
  • 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%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence contained in SEQ ID NOs:115-228; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
  • the nucleic acid is that of SEQ ID NOs:1-114; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence contained in SEQ ID NOs:1-114.
  • the encoded H. pylori polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NOs:115-228.
  • the differences are such that: the H. pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.
  • the encoded polypeptide includes all or a fragment of an amino acid sequence contained in SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
  • 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 12 consecutive nucleotides contained in SEQ ID NOs:1-114; more preferably to at least 20 consecutive nucleotides contained in SEQ ID NOs:1-114; more preferably to at least 40 consecutive nucleotides contained in SEQ ID NOs:1-114.
  • the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences shown in SEQ ID NOs:115-228.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence shown in SEQ ID NOs:1-114 which encodes amino acids shown in SEQ ID NOs:115-228.
  • the invention includes: a vector including a nucleic acid which encodes an H. pylori-like polypeptide e g , an H. pylori polypeptide; a host cell transfected with the vector; and a method of producing a recombinant H. pylori-like polypeptide. e.g., an H. pylori polypeptide; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori-like polypeptide, e.g., an H. pylori polypeptide, 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 SEQ ID NOs: 1-114.
  • 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 SEQ ID NOs:1-114, 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 involves nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
  • the H. pylori strain from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection ATCC # 55679) as strain ⁇ P-J99.
  • nucleic acid sequences of this invention may be obtained directly from the
  • PCR polymerase chain reaction
  • Nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands, antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences.
  • the nucleic acid will normally comprise approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products.
  • H. pylori polypeptides characterized as shown in Table 1 below, including: H. pylori outer membrane proteins. H. pylori periplasmic/secreted proteins, and other H. pylori surface proteins. Members of these groups were identified by BLAST homology searches.
  • the H. pylori polypeptides identified in Table 1 are representative members of the groups identified above and are in no way limiting. Additional members of the groups can be identified within the H. pylori polypeptides disclosed herein by the methods known to those skilled in the art.
  • 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: 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.
  • 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.
  • a substantially pure nucleic acid e.g., a substantially pure DNA
  • 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.
  • Homologous refers to the sequence similarity or sequence identity between two polypeptide molecules 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.
  • peptides, proteins, and polypeptides are used interchangeably herein.
  • surface protein refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
  • transgene means a nucleic acid sequence (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.
  • 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.
  • a polypeptide has H. pylori biological activity if it has one, two, three, 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.
  • Misexpression refers to a non- wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
  • host cells and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA. and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
  • control sequence refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site 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.
  • ORF an "open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a peptide. This region may represent a portion of a coding sequence or a total sequence.
  • a "coding sequence” is a nucleic acid sequence which is transcribed into messenger RNA and/or translated into a peptide 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, or recombinant nucleic acid sequences.
  • 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.
  • Capture ligand a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand".
  • Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
  • a nucleic acid isolated or synthesized in accordance with SEQ ID NOs:1-114 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 acid sequences 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 acid sequences 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 SEQ ID NOs:1-114 may also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using relaxed stringency hybridization conditions, as will be obvious to anybody skilled in the art.
  • 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 SEQ ID NOs:1-114 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 SEQ ID NOs:1-114 may 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 acid sequences in other Helicobacter species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of ⁇ 10-15 nucleotides contained in SEQ ID NOs:1-114 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.
  • PCR polymerase chain reaction
  • 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.
  • 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
  • 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 may also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
  • Nucleic acid or derivatives corresponding to H. pylori nucleic acid sequences 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. 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.
  • 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 could 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).
  • the expression host will utilize the natural Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta-galactosidase promoter for expression in E. coli).
  • a promoter sequence derived from the expressing organism e.g., an E. coli beta-galactosidase promoter for expression in E. coli.
  • a 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 the following components: an origin of replication that functions in the host organism, and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together.
  • the recombinant plasmid is introduced into the host organism by electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
  • the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid.
  • This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning.
  • Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate proteins and peptides.
  • the nucleic acid exemplified in SEQ ID NOs:1-114 or fragments of said nucleic acid sequences encoding immunogenic portions of H. pylori proteins (SEQ ID NO: 115-228) 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 host cell may be any procaryotic or eucaryotic cell.
  • an H. pylori peptide may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CHO).
  • bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CHO).
  • baculovirus baculovirus
  • yeast or mammalian cells
  • Chinese hamster ovary cell CHO
  • Other suitable host cells are known to those skilled in the art.
  • yeast S. cerivisae examples include pYepSecl (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) Virolosv 170:31-39).
  • COS cells Gluzman, Y., (1981 ) Cell 23: 175-182
  • pCDM 8 are used in conjunction with such vectors as pCDM 8 (Aruffo. A. and Seed, B., (1987) Proc. Natl. Acad. Sci. USA 84:8573-8577) for transient
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation.
  • Fusion vectors usually add a number of NH 2 terminal amino acids to the expressed target gene. These NH 2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1 ) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein.
  • Such enzymes include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia,
  • Piscataway, 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
  • target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc
  • expression of target genes inserted into pET11d relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gn1 under the transcriptional control of the lacUV 5 promoter.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori peptide can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the peptide may be secreted and isolated from a mixture of cells and medium containing the peptide.
  • the peptide 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.
  • Peptides 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 peptides. Additionally, in many situations, peptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
  • membrane bound proteins these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be
  • a detergent suitable for solubilizing these complex 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.
  • 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.
  • 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).
  • 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 may be constructed in vitro with a purified H. pylori enzyme such that the action of the enzyme produces an easily 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 may be tested in the assay to identify those which inhibit or potentiate the activity of the H. pylori enzyme. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same enzymatic activity in whole, live H. pylori cells.
  • the invention also includes antibodies specifically reactive with the subject H. pylori-like 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 subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide shown in SEQ ID NOs:115-228 or a closely related human or non-human mammalian homolog (e.g. 90% percent homologous, more preferably at least 95 percent homologous).
  • the anti-H. pylori antibodies do not substantially cross react (i.e.
  • a protein which is: e.g., less than 80% percent homologous to a sequence shown in SEQ ID NOs:115-228.
  • a protein which is: e.g., less than 80% percent homologous to a sequence shown in SEQ ID NOs:115-228.
  • 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 SEQ ID NOs:115-228.
  • 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 present 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 present invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention.
  • Antibodies which specifically bind H. pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H. pylori antigens.
  • Anti-H. pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure.
  • the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
  • the level of an H. pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy.
  • Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections.
  • the present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
  • anti-H. pylori polypeptide antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gt11 , ⁇ gt18-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gt11 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H.
  • H. pylori polypeptide antibodies Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H. pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
  • the present invention also includes vaccine compositions for protection against infection by H. pylori or for treatment of H. pylori infection, a gram-negative spiral microaerophilic bacterium.
  • the vaccine compositions contain immunogenic surface proteins 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 SEQ ID NOs:1-114 and which encode H. pylori surface proteins shown in SEQ ID NOs:115-228.
  • 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.
  • vaccines can have therapeutic and prophylactic utilities.
  • Another aspect of the present invention provides vaccine compositions for protection against infection by H. pylori or for treatment of H. pylori infection, which contain a modified immunogenic H. pylori protein or portion thereof, and a
  • H. pylori protein or peptide 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.
  • H. pylori peptide Another example of modification of an H. pylori peptide is 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.
  • an H. pylori protein or peptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation.
  • 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.
  • an H. pylori protein 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.
  • PEG polyethylene glycol
  • pylori proteins include reduction/alkylation (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) Bio/Technology, 6: 1321 - 1325).
  • immobilized metal ion affinity chromatography Hochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325.
  • endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
  • canonical protease sensitive sites can be engineered between regions, each comprising at least one T cell 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 T cell epitopes.
  • such charged amino acid residues can result in an increase in the solubility of the peptide.
  • Site-directed mutagenesis of a nucleic acid encoding an H. pylori protein can be used to modify the structure of the peptide by methods known in the art. Such methods may, among others, include polymerase chain reaction (PCR) with oligonucleotide primers bearing one or more mutations ( ⁇ o et al., (1989) Gene, 77: 51 - 59) or total synthesis of mutated genes ( ⁇ ostomsky, Z. et al., ( 1989) Biochem. Biophvs. Res. Comm, 161 : 1056 - 1063).
  • PCR polymerase chain reaction
  • oligonucleotide primers bearing one or more mutations ⁇ o et al., (1989) Gene, 77: 51 - 59
  • total synthesis of mutated genes ⁇ ostomsky, Z. et al., ( 1989) Biochem. Biophvs. Res. Comm, 161 : 1056 - 1063).
  • Another aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains an immunogenic fragment of an H. pylori protein or portion thereof, 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 peptides of the invention can be obtained, for example, by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H. pylori protein.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • an H. pylori protein may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced
  • immunogenic H. pylori fragments can be identified by the ability of the peptide to stimulate T cells.
  • Peptides which stimulate T cells as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope.
  • T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate ⁇ LA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope.
  • 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.
  • IgE is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events at the level of the T helper cell, by the nature of the lymphokines secreted.
  • 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. Amino acid sequences which mimic those of the T cell epitopes and which modify the allergic response to protein allergens 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.
  • 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.
  • 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 M ⁇ C molecules in a T cell culture.
  • Presentation of an immunogenic H. pylori peptide in association with appropriate M ⁇ C 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 ⁇ -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.
  • an expression vector containing a nucleic acid encoding all or a portion of a H pylori protein, operably linked to at least one regulatory sequence can be used.
  • Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
  • Regulatory sequences are art-recognized and include promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel, Gene Expression Technology
  • the expression vector includes nucleic acid. preferably a DNA, encoding a modified H. pylori protein or immunogenic fragment having all or a portion of the amino acid sequence .
  • Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.
  • Host cells suitable for transfection and recombinant production of H. pylori proteins of the invention include any procaryotic or eucaryotic cell.
  • an H. pylori protein or peptide may be expressed in bacterial cells such as E coli, insect cells
  • baculovirus baculovirus
  • yeast yeast
  • mammalian cells such as Chinese hamster ovary cells (CHO).
  • suitable host cells can be found in Goeddel, (1990) supra or known to those skilled in the art.
  • H. pylori proteins and fragments of the invention can also be chemically
  • 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).
  • compositions of the present invention containing DNA encoding immunogenic protein from H. pylori, or containing modified protein or fragments contain both the DNA or protein and 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.
  • the protein or peptide must be coadministered with a suitable adjuvant.
  • Vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular
  • Oral immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 91 : 9866-9870.
  • Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
  • Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 11 : 637-642.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl--L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, referred to a MTP-PE);
  • an adjuvant including, but not limited to aluminum hydroxide; N-acetyl-muramyl--L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-mur
  • 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.
  • Suitable delivery methods include biodegradable microcapsules or immuno-stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g.; bluetongur.
  • 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 with 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 dependant 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 immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888:A-222 (1993)).
  • a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.
  • the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H. pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. pylori-caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
  • ORF's open reading frames
  • ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
  • amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code.
  • novel gene products e.g. bacterial surface gene products, from the organism H. pylori.
  • novel gene products e.g. bacterial surface gene products
  • H. pylori bacterial surface gene products
  • 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). This is a very powerful and relatively rapid method of introducing random mutations.
  • the DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase. e.g., by using a dGTP/dATP ratio of five and adding Mn 2+ to the PCR reaction.
  • the pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
  • Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242).
  • This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand.
  • the mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements. 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.
  • 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 d locations or domains for mutagenesis, Cunningham and Wells (Science 244: 1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
  • Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
  • Oligonucleotide-mediated mutagenesis is a useful method for preparing
  • the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad Sci USA, 75: 5765[1978]).
  • the starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA to be mutated are identified.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are compatible 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.
  • 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 homologs which retain ligand-binding activity.
  • the use of fluorescently labeled ligands allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a
  • a gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gIII or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle.
  • Foreign epitopes can be expressed at the NH 2 -terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
  • E. coli the outer membrane protein, LamB
  • LamB the outer membrane protein
  • Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals.
  • Other cell surface proteins e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al.
  • Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment.
  • Another large surface structure used for peptide display is the bacterial motile organ, the flagellum.
  • Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083).
  • Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane 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. Capturing the peptide captures the particle and the DNA within.
  • An alternative scheme uses the DNA- binding protein LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the LacI gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a LacI-peptide fusion protein is produced.
  • This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO).
  • LacO operator By installing two copies of LacO on the expression plasmid, the LacI-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
  • the associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands.
  • a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B.
  • a cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869)
  • peptides-on-plasmids differs in two important ways from the phage display methods.
  • the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini.
  • Both of the filamentous phage coat proteins, pill and pVIII are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains.
  • the phage-displayed peptides are presented right at the amino terminus of the fusion protein.
  • a second difference is the set of biological biases affecting the population of peptides actually present in the libraries.
  • the LacI fusion molecules are confined to the cytoplasm of the host cells.
  • the phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles.
  • the peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.
  • the phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage.
  • phage/phagemid or plasmid libraries Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.
  • RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening.
  • the polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector.
  • This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification.
  • a DNA sequencing vector for peptide identification.
  • the high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists.
  • the type of a secondary screen used will depend on the desired activity that needs to be tested.
  • an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
  • the invention also provides for reduction of the protein binding domains of the subject H. pylori-like family polypeptides, e.g., an H. pylori polypeptide, to generate mimetics, e.g. peptide or non-peptide agents.
  • the peptide mimetics are able to disrupt binding of a H. pylori 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 noncompetatively inhibit binding of the H. pylori polypeptide with an interacting polypeptide
  • an interacting polypeptide see, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" 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
  • peptidomimetic compounds e.g. diazepine or isoquinoline derivatives
  • peptidomimetic compounds 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.
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:
  • kits for diagnostic purposes typically comprise the nucleic acid, peptides 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.
  • labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like.
  • 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.
  • TE 10 mM Tris, 1 mM EDTA, p ⁇ 7.6
  • GES lysis buffer 5.1 M guanidium thiocyanate, 0.1 M EDTA, p ⁇ 8.0, 0.5% N-laurylsarcosine
  • the purified DNA fragments were then blunt-ended using T4 DNA polymerase.
  • the healed DNA was then ligated to unique BstXI-linker adapters 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-adapted 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
  • sequencing protocols 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
  • 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..
  • 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.
  • 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.
  • All forward primers (specific for the 5' end of the sequence) were designed to include an NcoI cloning site at the extreme 5' terminus, except for H. pylori sequence 4821082 (SEQ ID NO: 212) where NdeI was used. These primers were designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native H. pylori DNA sequence. An exception is H. pylori sequence 4821082 (SEQ ID NO: 212) where the initiator methionine is immediately followed by the remainder of the native H. pylori DNA sequence. All reverse primers (specific for the 3' end of any H.
  • pylori ORF included a EcoRI site at the extreme 5' terminus to permit cloning of each H. pylori sequence into the reading frame of the pET-28b.
  • the pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids 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.
  • Genomic DNA prepared from the J99 strain of H. pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • genomic DNA 50 nanograms was introduced into a reaction vial containing 2 mM MgCl 2 , 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H.
  • pylori ORF 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 microliters.
  • the following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
  • the pET-28b vector was prepared for cloning by digestion with NcoI and EcoRI, or in the case of H. pylori sequence 4821082 (SEQ ID NO: 212) with NdeI and EcoRI
  • cloning ppiB the pET-28a vector, which encodes a ⁇ is-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with Bam ⁇ I and XhoI restriction endonucleases.
  • DNA inserts were cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB. which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
  • Competent bacteria E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc.. F. Ausubel et al., eds., 1994).
  • the pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101.
  • 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 a T7 promoter and a gene of interest.
  • IPTG isopropyl-B-D-thiogalactoside
  • 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 was 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) was expressed in the pET-System as described for the H. pylori recombinant constructions.
  • Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate.
  • bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
  • SDS-polyacrylamide gels (12% or 4.0 to 25 % acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
  • Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
  • NTA Ni 2+- nitrilotriacetate-agarose
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and was eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD 280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole. Recombinant beta-galactosidase (lacZ) and peptidyl-prolyl cis-trans isomerase (ppiB)
  • Recombinant protein 7116626 (SEQ ID NO: 223)
  • the resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials.
  • Recombinant proteins 30100332 (SEQ ID NO: 181), 4721061 (SEQ ID NO: 211)
  • the pellet containing the inclusion bodies was solubilized in buffer B containing 8 M urea, 1 mM PMSF and 0 1 % 2-mercaptoethanol, and incubated for 1 hour at room temperature. Insoluble materials were removed by centrifugation at 20,000 x g for 30 min, and the cleared supernatant was loaded onto a 15 ml ( 1.6 ⁇ 7.5 cm ) SP-Sepharose column pre-equilibrated in buffer B, 6 M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol. After washing the column with 10 bed volumes, the column was developed with a linear gradient from 0 to 500 mM NaCl.
  • Urea was removed slowly from the protein samples by dialysis against Tris-buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M, 4M, 3M, 2M, 1 M, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.
  • TBS Tris-buffered saline
  • DOC deoxycholate
  • H. pylori To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H. pylori model was used. This model mimics the human H. pylori infection in many respects The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immunotherapy
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding center (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 strain A ⁇ 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% CO 2 , 5%O 2 )
  • the animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and 3-5 h after this an oral inoculation of H. pylori in broth (approximately 10 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation.
  • Recombinant H. pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa- ⁇ IS tag for purification reasons and the non-Helicobacter pylori control protein ( ⁇ -galactosidase from E. coli; LacZ), was constructed in the same way.
  • the antigens are listed in Table 4 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 ⁇ EPES 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 5 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 was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
  • the urease test was performed essentially as follows.
  • the catalase test was performed essentially as follows.
  • a solution of the reagent (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 mucus In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with SEQ ID NOs:181, followed by 223 (see Figure 3).
  • H. pylori Three 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.
  • H. pylori strains (as listed in Table 8) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD 600 of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mls of 10 mM Tris- ⁇ Cl, 0.1 mM EDTA (TE).
  • BLBB 1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum
  • Lysozyme was added to a final concentration of 1 mg/ml along with SDS to 1% and RNAse A + T1 to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55°C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1 % CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes.
  • Genomic DNA prepared from twelve strains oi 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 6) 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.
  • each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
  • Competent bacteria E coli strain TOP10F' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994). Briefly, 2 microliters of 0.5 micromolar BME was added to each vial of 50 microliters of competent cells. Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes.
  • the cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of TOP10F' or INVaF' were then picked and analyzed to evaluate cloned inserts as described below. Identification of recombinant PCR plasmids carrying H. pylori sequences
  • 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.
  • SEQ ID NO: 199 was identified as an unknown open reading frame, because it showed low homology with sequences in the public databases.
  • Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis.
  • the sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Madison, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.
  • Genomic DNA prepared from the Helicobacter pylori HP-J99 strain (ATCC 55679) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons. Inc., F. Ausubel et al., editors, 1994).
  • PCR polymerase chain reaction
  • each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction.
  • Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
  • PCR products are cloned into the pT7Blue T-Vector (catalog#69820-1 , Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • the ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
  • 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose) at 37°C with shaking for 1 hour.
  • SOC medium 0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose
  • LB 10 g/l bacto tryptone, 5 g/l bacto yeast extract, 10 g/l sodium chloride
  • LB 10 g/l bacto tryptone, 5 g/l bacto yeast extract, 10 g/l sodium chloride
  • Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
  • these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 H. pylori sequence.
  • Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned.
  • Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage.
  • plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.
  • the sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other.
  • the pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product.
  • the PCR product is a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, results in the deletion of a portion of the ORFs.
  • the PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified.
  • a Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology 170, 1704-1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • the Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid
  • pCTB8:kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573).
  • the proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA).
  • the fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes.
  • This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides.
  • the "T” overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris p ⁇ 8.3, 50 mM KCl, 2 mM MgCl 2 , 5 units of DNA Polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP, in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C.
  • the "Kan-T” cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA).
  • the PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter 10x T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16oC.
  • the ligation products are transformed into XL-1 Blue or DH5- ⁇ E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
  • H. pylori Two strains of H. pylori are used for transformation: HP-J99 (ATCC 55679), the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AH244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach.
  • Cells for transformation are grown at 37°C, 10% CO 2 . 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated.
  • cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf micro fuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells.
  • Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO 2 .
  • Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C. 6% CO 2 , 100% humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.
  • the template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1 :1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
  • TEST 1 PCR with cloning primers originally used to amplify the gene/ORF.
  • a positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette.
  • a PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.
  • PCR with F3 primer designed from sequences upstream of the gene/ORF and not present on the plasmid
  • primer Kan-1 or Kan-2 primers designed from the ends of the kanamycin resistance gene
  • Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
  • PCR with R3 primer designed from sequences downstream of the gene/ORF and not present on the plasmid
  • primer Kan-1 or Kan-2 depending on whether the plasmid DNA used was of "A" or "B” orientation.
  • Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3).
  • no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
  • plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H. pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3.
  • TEST 2 and TEST 3 Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST 2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation
  • H. pylori target gene and its protein product e.g., an H. pylori enzyme
  • H. pylori enzyme e.g., a H. pylori enzyme
  • a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase is described below as a specific example.
  • the assay is essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Acta 43:1101-1111).
  • the assay measures the cis-trans isomerization of the Ala- Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S-7388, lot # 84H5805).
  • the assay is coupled with ⁇ -chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans.
  • test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer.
  • the data are collected every second with an average scanning of time of 0.5 second Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 ⁇ M ⁇ -chymotrypsin (type 1-5 from bovine Pancreas. Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction.
  • a 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD 600 nm ⁇ 1 ) and resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 ⁇ g/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor.
  • the suspension is subjected to 3 cycles of freeze-thaw ( 15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts).
  • the lysate is centrifuged (12,000 g ⁇ 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
  • H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
  • 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 SEQ ID NOs:1-114 (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, or analogs of H. pylori polypeptides
  • a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the H. pylori polypeptides shown in SEQ ID NOs:115-228, or of other naturally occurring H. pylori polypeptides. e.g., one or more of the biological activities described above.
  • fragments which exist in vivo e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in C ⁇ O cells.
  • 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%. or at least 90% 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
  • 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 abolish the biological activity of the H. pylori polypeptide.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.
  • analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs.
  • fragment as applied to a 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.
  • H. pylori polypeptide-encoding DNA can be introduced into an expression vector, the vector introduced into a cell suitable for expression of the desired protein, and the peptide recovered and purified, by prior art methods.
  • Antibodies to the peptides an proteins can be made by immunizing an animal, e.g.. a rabbit or mouse, and recovering anti-H. pylori polypeptide antibodies by prior art methods.

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 vaccine compositions.

Description

NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO HELICOBACTER
PYLORI FOR DIAGNOSTICS AND THERAPEUTICS
Background of the Invention
Helicobacter pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, ( 1983) Lancet 1 : 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83-88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H. pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. (Blaser M. J., (1993) Trends Microbiol. 1 : 255-260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age. (Taylor, D.N. and M. J. Blaser, (1991 ) Epidemiol. Rev 13: 42-50). H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
The bacterial factors necessary for colonization of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of the putative virulence factors include the following: urease, an enzyme that may play a role in neutralizing gastric acid pH (Eaton et al., (1991) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee
(1991) Microb. Ecol. Hlth. Dis. 4: 121-134; Labigne et al., (1991) J. Bacterial. 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. Bakl. 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 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 bacterial surface proteins, from the organism Helicobacter 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 surface proteins or parts thereof, nucleic acids capable of binding mRNA from H. pylori surface proteins to block protein translation, and methods for producing H. pylori surface proteins or parts thereof using peptide synthesis and recombinant DNA techniques. This invention also includes antibodies and nucleic acids sequences useful as probes to detect H. pylori infection. In addition, vaccine compositions and methods for the protection against infection by H. pylori are described.
Brief Description of the Drawings
Figure 1 is a table which contains information from homology searches performed on the sequences of this invention using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package.
Figure 2 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.
Figure 3 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.
Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
Figure 5 is a bar graph that depicts therapeutic immunization of H., pylori infected mice with specific antigens dissolved in buffer containing DOC.
Detailed Description of the Invention
In one aspect, the invention features a substantially pure nucleic acid encoding an
H. pylori polypeptide having an amino acid sequence of SEQ ID NO:115, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1. In another aspect, the invention features a substantially pure nucleic acid encoding an H.. pylori polypeptide having an amino acid sequence of SEQ ID NO:116, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:117, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:118. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:4.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:119, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:5.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:120, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:6.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:121, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:7.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:122, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:8.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:123, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:9.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:124. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:10.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:125, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:11.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:126, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:12.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:127, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:13. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:128, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:14.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:129, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:15.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:130, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:16.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:131 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:17.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:132, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:18.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:133, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:19.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:134, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:20.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:135, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:21.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:136, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:22.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:137, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:23.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:138, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:24.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:139, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:25. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:140, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:26.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:141, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:27.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:142, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:28.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:143, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:29.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:144, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:30.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:145, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:31.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:146, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:32.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:147, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:33.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:148, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:34.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:149, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:35.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:150, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:36.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:151, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:37. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:152, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:38.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:153, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:39.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:154, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:40.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:155, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:41.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:156, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:42.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:157, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:43.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 158, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:44.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:159, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:45.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:160, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:46.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:161, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:47.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:162, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:48.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:163, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:49. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:164, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:50.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:165, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 51.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:166, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:52.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:167, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:53.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:168, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:54.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:169, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:55.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:170, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:56.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:171, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:57.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:172, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:58.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:173, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:59.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:174, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:60.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:175, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:61. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:176, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:62.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:177, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:63.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:178, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:64.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:179, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:65.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:180, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:66.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:182, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:67.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:182, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:68.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:183, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:69.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:184, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:70.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:185, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:71.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:186, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:72.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:187, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:73. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:188, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:74.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:189, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:75.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:190, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:76.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:191, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:77.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:192, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:78.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:193. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:79.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:194, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:80.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:195, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:81.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:196, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:82.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:197, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:83.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:198, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:84.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:199, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:85. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:200, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:86.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:201 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:87.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:202, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:88.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:203, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:89.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:204, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:90.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:205, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:91.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:206, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:92.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:207, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:93.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:208, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:94.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:209, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:95.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:210, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:96.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:211 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:97. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:212, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:98.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:213, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:99.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:214, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:100.
In another aspect the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:215, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:101.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:216, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:102.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:217, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:103.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:218, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:104.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:219, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:105.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:220, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:106.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:221, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:107.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:222, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:108.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:223, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:109. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:224, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:110.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:225, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:111.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:226, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:112.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:227, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:113.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO:228. such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:114.
In another aspect, the invention comprises nucleic acids capable of binding mRNA of H. pylori. Such nucleic acid is capable of acting as anti-sense 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. Such nucleic acid has 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 make H. pylori polypeptides.
In another aspect, the invention features a method of generating antibodies against H. pylori proteins which are capable of binding specifically to H. pylori proteins. Such antibody has utility as reagents for immunoassays to evaluate the abundance and distribution of H. pylori-specific antigens.
In another aspect, the invention features a method of generating vaccines for immunizing an individual against H. pylori. The method includes: immunizing a subject with an H. pylori protein, e.g., a surface protein, or 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 protein, e.g., a surface protein, or 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%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence contained in SEQ ID NOs:115-228; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NOs:115-228; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids 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 acids contained in SEQ ID NOs:115-228.
In preferred embodiments: the H. pylori polypeptide is encoded by a nucleic acid in SEQ ID NOs: 1 -114, or by a nucleic acid having at least 60%. 70%, 80%, 90%, 95%. 98%, or 99% homology with a nucleic acid of SEQ ID NOs: 1-114.
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 SEQ ID NOs:115-228. 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 SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
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 includes an immunogen which includes an H. pylori polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant from a protein contained in SEQ ID NOs:115-228.
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%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence contained in SEQ ID NOs:115-228; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
In preferred embodiments: the nucleic acid is that of SEQ ID NOs:1-114; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence contained in SEQ ID NOs:1-114.
In a preferred embodiment, the encoded H. pylori polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NOs:115-228. 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 SEQ ID NOs:115-228; 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 SEQ ID NOs:115-228.
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 12 consecutive nucleotides contained in SEQ ID NOs:1-114; more preferably to at least 20 consecutive nucleotides contained in SEQ ID NOs:1-114; more preferably to at least 40 consecutive nucleotides contained in SEQ ID NOs:1-114.
In a preferred embodiment, the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences shown in SEQ ID NOs:115-228.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence shown in SEQ ID NOs:1-114 which encodes amino acids shown in SEQ ID NOs:115-228.
In another aspect, the invention includes: a vector including a nucleic acid which encodes an H. pylori-like polypeptide e g , an H. pylori polypeptide; a host cell transfected with the vector; and a method of producing a recombinant H. pylori-like polypeptide. e.g., an H. pylori polypeptide; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori-like polypeptide, e.g., an H. pylori polypeptide, 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 SEQ ID NOs: 1-114.
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 SEQ ID NOs:1-114, 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 involves nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
The H. pylori strain, from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection ATCC # 55679) as strain ΗP-J99.
The nucleic acid sequences 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, eg, Sambrook et al.. Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
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 will normally comprise approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products.
Putatiive functions have been determined for several of the H. pylori polypeptides of the invention, as shown in Figure 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 outer membrane proteins. H. pylori periplasmic/secreted proteins, and other H. pylori surface proteins. Members of these groups were identified by BLAST homology searches. The H. pylori polypeptides identified in Table 1 are representative members of the groups identified above and are in no way limiting. Additional members of the groups can be identified within the H. pylori polypeptides disclosed herein by the methods known to those skilled in the art.
Figure imgf000018_0001
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.
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 substantially pure nucleic acid, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: 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 sequence 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.
Homologous refers to the sequence similarity or sequence identity between two polypeptide molecules 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.
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.
As used herein, the term "transgene" means a nucleic acid sequence (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.
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.
A polypeptide has H. pylori biological activity if it has one, two, three, 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.
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.
An "open reading frame", also referred to herein as ORF, is a region of nucleic acid which encodes a peptide. This region may represent a portion of a coding sequence or a total sequence.
As used herein, a "coding sequence" is a nucleic acid sequence which is transcribed into messenger RNA and/or translated into a peptide 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, or recombinant nucleic acid sequences.
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.
The experimental manipulation of such conditions has been well described in the literature including such books as Molecular Cloning: A Laboratory Manual. Sambrook, J., Fritsch, E.F., Maniatis, T., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2nd ed. (1989).
The practice of the present 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). Probes
A nucleic acid isolated or synthesized in accordance with SEQ ID NOs:1-114 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 acid sequences 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 acid sequences, 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 SEQ ID NOs:1-114 may also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using relaxed stringency hybridization conditions, as will be obvious to anybody skilled in the art.
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 SEQ ID NOs:1-114 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 SEQ ID NOs:1-114 may 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 acid sequences in other Helicobacter species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of≥ 10-15 nucleotides contained in SEQ ID NOs:1-114 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 will be described in greater detail below. 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 may also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
Nucleic acid or derivatives corresponding to H. pylori nucleic acid sequences 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.
Expressing H. pylori Genes
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 could 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 is 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 the following components: an origin of replication that functions in the host organism, and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together. The recombinant plasmid is introduced into the host organism by 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. Expressed Genes in Therapeutics
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate proteins and peptides. The nucleic acid exemplified in SEQ ID NOs:1-114 or fragments of said nucleic acid sequences encoding immunogenic portions of H. pylori proteins (SEQ ID NO: 115-228) 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 host cell may be any procaryotic or eucaryotic cell. For example, an H. pylori peptide may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CHO). 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 pYepSecl (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) Virolosv 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), EMBOJ. 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 pET11d relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl). 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 peptide can be cultured under appropriate conditions to allow expression of the peptide to occur. The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the peptide 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. Peptides 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 peptides. Additionally, in many situations, peptides 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) 119-128). 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.20:2111-2118). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071 , incorporated by reference herein).
Drug Screening Assays
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 may be constructed in vitro with a purified H. pylori enzyme such that the action of the enzyme produces an easily 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 may be tested in the assay to identify those which inhibit or potentiate the activity of the H. pylori enzyme. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same enzymatic activity in whole, live H. pylori cells.
Antibodies
The invention also includes antibodies specifically reactive with the subject H. pylori-like 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 SEQ ID NOs:115-228 or a closely related human or non-human mammalian homolog (e.g. 90% percent homologous, more preferably at least 95 percent homologous). In yet a further preferred embodiment of the present invention, the anti-H. pylori antibodies do not substantially cross react (i.e. react specifically) with a protein which is: e.g., less than 80% percent homologous to a sequence shown in SEQ ID NOs:115-228. 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 SEQ ID NOs:115-228. 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 present 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 present invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention.
Antibodies which specifically bind H. pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H. pylori antigens. Anti-H. pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure. Likewise, the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of an H. pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy.
Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
Another application of anti-H. pylori polypeptide antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as λgt11 , λgt18-23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgt11 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H. pylori polypeptide antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H. pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
Vaccines
The present invention also includes 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 immunogenic surface proteins 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 SEQ ID NOs:1-114 and which encode H. pylori surface proteins shown in SEQ ID NOs:115-228. 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 can have therapeutic and prophylactic utilities. Another aspect of the present invention provides vaccine compositions for protection against infection by H. pylori or for treatment of H. pylori infection, which contain a modified immunogenic H. pylori protein or portion thereof, and a
pharmaceutically acceptable carrier. It is possible to modify the structure of a H. pylori protein or peptide 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.
Another example of modification of an H. pylori peptide is 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 protein or peptide 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 protein can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of H. pylori proteins include reduction/alkylation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton 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 T cell epitopes within an H. pylori protein of the invention, canonical protease sensitive sites can be engineered between regions, each comprising at least one T cell 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 T cell epitopes. In addition, such charged amino acid residues can result in an increase in the solubility of the peptide.
Site-directed mutagenesis of a nucleic acid encoding an H. pylori protein can be used to modify the structure of the peptide by methods known in the art. Such methods may, among others, include polymerase chain reaction (PCR) with oligonucleotide primers bearing one or more mutations (Ηo et al., (1989) Gene, 77: 51 - 59) or total synthesis of mutated genes (Ηostomsky, Z. et al., ( 1989) Biochem. Biophvs. Res. Comm, 161 : 1056 - 1063). To enhance recombinant protein expression, the aforementioned methods can be applied to change the codons present in the cDNA sequence of the invention to those preferentially utilized by the host cell in which the recombinant protein is being expressed (Wada et al., supra). An extensive discussion of mutagenesis protocols is provided in the "Production of Fragments and Analogs" section herein.
Another aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains an immunogenic fragment of an H. pylori protein or portion thereof, 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 peptides of the invention can be obtained, for example, by screening peptides 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. For example, an H. pylori protein may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced
(recombinantly or by chemical synthesis) and tested to identify those peptides having the ability to induce a T cell response, such as stimulation (proliferation, cytokine secretion). An extensive discussion of peptide analogs and fragments is provided in the "Production of Fragments and Analogs" section herein.
In one embodiment, immunogenic H. pylori fragments can be identified by the ability of the peptide to stimulate T cells. Peptides which stimulate T cells, as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate ΗLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen/T cell interaction, and activation of the B cell cascade, leading to the production of antibodies. One isotype of these antibodies, IgE, is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events at the level of the T helper cell, by the nature of the lymphokines secreted. 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. Amino acid sequences which mimic those of the T cell epitopes and which modify the allergic response to protein allergens 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 peptides for those which are immunogenic 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 MΗC molecules in a T cell culture. Presentation of an immunogenic H. pylori peptide in association with appropriate MΗC 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 3Η-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.
To produce modified proteins or immunogenic fragments by recombinant DNA techniques, an expression vector containing a nucleic acid encoding all or a portion of a H pylori protein, operably linked to at least one regulatory sequence can be used. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and include promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel, Gene Expression Technology
Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. In one embodiment of the present invention, the expression vector includes nucleic acid. preferably a DNA, encoding a modified H. pylori protein or immunogenic fragment having all or a portion of the amino acid sequence . Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.
Host cells suitable for transfection and recombinant production of H. pylori proteins of the invention include any procaryotic or eucaryotic cell. For example, an H. pylori protein or peptide may be expressed in bacterial cells such as E coli, insect cells
(baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO). Other suitable host cells can be found in Goeddel, (1990) supra or known to those skilled in the art.
H. pylori proteins and fragments of the invention can also be chemically
synthesized, using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. 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).
Vaccine compositions of the present invention containing DNA encoding immunogenic protein from H. pylori, or containing modified protein or fragments, contain both the DNA or protein and 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 modified H. pylori protein or immunogenic protein fragments, the protein or peptide must be coadministered with a suitable adjuvant.
It will be apparent to those of skill in the art that the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of antibody administered, whether the protein or DNA is
administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or DNA.
Vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular
immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 91 : 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 11 : 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
The vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl--L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 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), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g.; bluetongur. 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 with 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 dependant upon the patient's body weight, disease, the route of administration, and other factors. Those skilled in the art will also recognize that appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol. 150:917-921 (1993); Boedecker et al., American
Gastroenterological Assoc. 999:A-222 (1993)). The number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888:A-222 (1993)).
In a preferred embodiment, a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.
It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing H. pylori infection, some are useful only for treating H. pylori infection, and some are useful for both preventing and treating H. pylori infection. In a preferred embodiment, the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H. pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. pylori-caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
Determination of candidate protein antigens for antibody and vaccine development
The selection of candidate protein antigens for vaccine development were derived from the nucleotide sequence. First, all possible open reading frames (ORF's) greater than 50 nucleotides in all six reading frames were identified and translated into amino acid sequences. Second, the identified ORF's were analyzed for homology to other known exported or membrane proteins and the ORF's were also analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985)
Biochimica et Biophysica Acta 815, 468-476) for predicting exported and membrane proteins.
Homology searches were performed using the BLAST algorithm contained in the
Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases. BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database. ORF's with significant homology (e.g. probabilities better than 1×10 (ee-6)) to membrane or exported proteins represent likely protein antigens for vaccine development. Possible functions are provided to some of the H. pylori genes as indicated in Figure 560 based on sequence homology to genes cloned in other organisms.
Discriminant analysis (Klein, et al. supra) was used to examine the ORF amino acid sequences using our own software. This algorithm uses the intrinsic information contained in the ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
Infrequently it is not possible to distinguish between multiple possible nucleotides at a given position in the nucleic acid sequence. In those cases the ambiguities are denoted by an extended alphabet as follows:
These are the official IUPAC-IUB single-letter base codes
Figure imgf000038_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.
Production of Fragments and Analogs
The inventor has discovered novel gene products, e.g. bacterial surface gene products, from the organism H. pylori. Once an example of this core structure has been provided 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 prior art methods 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 analogues 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.
Production of Altered DNA and Peptide Sequences: Random Methods
Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein).
PCR Mutagenesis
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). This is a very powerful and relatively rapid method of introducing random mutations. 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.
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. Degenerate Oligonucleotides
A library of homologs can also be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA ( 1983) Tetrahedron 39:3 ; Itakura et al. (1981 ) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409. 5,198,346, and 5,096,815).
Production of Altered DNA and Peptide Sequences: 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.
Alanine Scanning Mutagenesis
Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred d 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.
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]).
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 compatible 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
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.
Primary High-Through-Put Methods for Screening Libraries of Peptide Fragments or Homologs
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.
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.
Display Libraries
In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18: 136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologs which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a
fluorescence-activated cell sorter.
A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can he applied to affinity matrices at concentrations well over 10* J phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gIII 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 motile organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane 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 LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the LacI gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a LacI-peptide fusion protein is produced. This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the LacI-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869)
This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The LacI fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage.
Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9): 1233- 1251). These particular biases are not a factor in the LacI display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries of 107-109 independent clones are routinely prepared. Libraries as large as 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 Screens
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
The invention also provides for reduction of the protein binding domains of the subject H. pylori-like family polypeptides, e.g., an H. pylori polypeptide, to generate mimetics, e.g. peptide or non-peptide agents. The peptide mimetics are able to disrupt binding of a H. pylori 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 noncompetatively inhibit binding of the H. pylori polypeptide with an interacting polypeptide (see, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,762A and EP- B31 ,080A). For example, scanning mutagenesis can be used to map the amino acid residues of a particular H. pylori polypeptide involved in binding an interacting
polypeptide, peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) can be generated which mimic those residues in binding to an interacting polypeptide, and which therefore can inhibit binding of an H. pylori polypeptide to an interacting polypeptide and thereby interfere with the function of H. pylori polypeptide. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:
Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology. G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene
pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in
Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. ( 1986) J Chem Soc Perkin Trans 1 :1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71). Kits
The nucleic acid, peptides and antibodies of the present invention can be combined with other reagents and articles to form kits. Kits for diagnostic purposes typically comprise the nucleic acid, peptides 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.
Exemplification
1. 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 (Biol 01 , Inc.).
The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The healed DNA was then ligated to unique BstXI-linker adapters 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-adapted 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
Gel Assemble, developed by the Genetics Computer Group (GCG) (Devereux et al.. Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICA™. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICA™ database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly. II. Identification, cloning and expression of recombinant H. pylori DNA sequences
To facilitate the cloning, expression and purification of membrane and secreted proteins from H. pylori a powerful gene expression system, the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, was selected. Also, a DNA sequence encoding a peptide tag, the Ηis-Tag, was fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence. The exception to the above was ppiB, a gene cloned for use as a control in the expression studies. In this study, the sequence for H. pylori ppiB contains a DNA sequence encoding a Ηis-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein. PCR Amplification and cloning of DNA sequences containing ORF s for membrane and secreted proteins from the J99 Strain of Helicobacter pylori.
Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers (Table 2) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life
Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an NcoI cloning site at the extreme 5' terminus, except for H. pylori sequence 4821082 (SEQ ID NO: 212) where NdeI was used. These primers were designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native H. pylori DNA sequence. An exception is H. pylori sequence 4821082 (SEQ ID NO: 212) where the initiator methionine is immediately followed by the remainder of the native H. pylori DNA sequence. All reverse primers (specific for the 3' end of any H. pylori ORF) included a EcoRI site at the extreme 5' terminus to permit cloning of each H. pylori sequence into the reading frame of the pET-28b. The pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids including six histidine residues (at the extreme C-terminus), which comprise the Ηis-Tag. An exception to the above, as noted earlier, is the vector construction for the ppiB gene. A synthetic oligonucleotide primer specific for the 5' end of ppiB gene encoded a BamΗI site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encoded a XhoI site at its extreme 5' terminus.
Figure imgf000050_0001
Figure imgf000051_0001
Genomic DNA prepared from the J99 strain of H. pylori (ATCC #55679) was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Sequences 7116626 (SEQ ID NO: 223), 29479681 (SEQ ID NO: 179), 30100332 (SEQ ID NO: 181), 4821082 (SEQ ID NO: 212) and 978477 (SEQ ID NO: 228);
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 4721061 (SEQ ID NO: 211);
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 36°C for 15 sec and 72°C for 1.5 min
23 cycles at 94°C for 15 sec, 60°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes.
Conditions for amplification of H. pylori ppiB;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 32°C for 15 sec and 72°C for 1.5 min
25 cycles at 94°C for 15 sec, 56°C for 15 sec and 72°C for 1.5 min
Reactions were concluded at 72°C for 6 minutes Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD. USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, NcoI and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of H. pylon sequence 4821082 (SEQ ID NO: 212), with NdeI and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples were then subjected to electrophoresis on 1.0 % NuSeive (FMC
BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).
Cloning ofH. pylori DNA sequences into the pET-28b prokaryotic expression vector.
The pET-28b vector was prepared for cloning by digestion with NcoI and EcoRI, or in the case of H. pylori sequence 4821082 (SEQ ID NO: 212) with NdeI and EcoRI
(Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). In the case of cloning ppiB, the pET-28a vector, which encodes a Ηis-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with BamΗI and XhoI restriction endonucleases.
Following digestion, DNA inserts were cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB. which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc.. F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation reaction was mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below. Identification of recombinant pET expression plasmids carrying H. pylori sequences
Individual BL21 clones transformed with recombinant pET-28b-H.pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the expression vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
Isolation and Preparation of plasmid DNA from BL21 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H.. pylori ORFs were picked and incubated in 5 mls of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA. USA).
Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101.
JM109, DH5, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the 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 a T7 promoter and a gene of interest. Strains used include: BL21 (DE3) (Studier, F.W., Rosenberg. A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 nanograms of plasmid DNA isolated as described above was 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) was expressed in the pET-System as described for the H. pylori recombinant constructions. Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by
centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification. III. Purification of recombinant proteins from E. coli
Analytical Methods
The concentrations of purified protein preparations were quantified
spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.II., 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) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
1. Purification of soluble proteins
All steps were carried out at 4°C. Frozen cells were thawed, resuspended in 5 volumes of lysis buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole with 10% glycerol, 0.1 % 2-mercaptoethanol, 200 μg/ ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each of leupeptin, aprotinin, pepstatin, L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone (TLCK), L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK), and soybean trypsin inhibitor, and ruptured by several passages through a small volume microfluidizer (Model M-110S, Microfluidics International Corporation, Newton, MA). The resultant homogenate was made 0.1 % Brij 35, and centrifuged at 100,000 x g for 1 hour to yield a clear supernatant (crude extract).
Following filtration through a 0.8 μm Supor filter (Gelman Sciences, FRG) the crude extract was loaded directly onto a Ni2+- nitrilotriacetate-agarose (NTA) with a 5 milliliter bed volume (Hochuli, E., Dbeli, H., and Schacheer, A. (1987) J. Chromatography 411, 177-184) pre-equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and was eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole. Recombinant beta-galactosidase (lacZ) and peptidyl-prolyl cis-trans isomerase (ppiB)
Fractions containing the recombinant proteins from the Ni2+-NTA-agarose columns were pooled and then concentrated to approximately 5 ml by centrifugal filtration
(Centriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 9] cm) of Sephacryl S-100 HR gel filtration medium equilibrated in Buffer A (10 mM Hepes, pH 7.5, 150 mM NaCl, 0.1 mM EGTA) and run in Buffer A at 18 ml/h. Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled and concentrated by centrifugal filtration.
Recombinant protein 7116626 (SEQ ID NO: 223)
Fractions containing the recombinant protein from the Ni2+ -NTA-agarose column were pooled and dialyzed overnight against 1 liter of dialysis buffer (10 mM MOPS, pH 6.5, 50 mM NaCl, 0.1 mM EGTA, 0.02% Brij 35 and 1 mM PMSF). In the morning, a fine white precipitate was removed by centrifugation and the resulting supernatant was loaded onto an 8 ml (8 × 75 mm) MonoS high performance liquid chromatography column (Pharmacia Biotechnology, Inc., Piscataway, NJ, USA) equilibrated in buffer B (10 mM
MOPS, pH 6.5, 0.1 mM EGTA) containing 50 mM NaCl. The column was washed with 10 bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM). Recombinant protein 7116626 (SEQ ID NO: 223) eluted as a sharp peak at 300 mM NaCl.
2. Purification of insoluble proteins from inclusion bodies
The following steps were carried out at 4°C. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 μg/ ml lysozyme, 5 mM EDTA, ImM PMSF and 0.1 % - mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was made 0.2 % deoxycholate, stirred 10 minutes, then centrifuged at 20,000 x g, for 30 min. The pellets were washed with lysis buffer containing 10 % glycerol, 10 mM EDTA, 1% Triton X-100. 1 mM PMSF and 0.1% -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol. The resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials..
Recombinant proteins 30100332 (SEQ ID NO: 181), 4721061 (SEQ ID NO: 211)
The following steps were carried out at room temperature. Purified inclusion bodies were dissolved in 20 ml 8.0 M urea in lysis buffer with 1 mM PMSF and 0.1 % 2- mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation. The clear supernatant was filtered, then loaded onto a Ni2+ -NTA agarose column pre-equilibrated in 8.0 M urea in Lysis Buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF. 0.1 % 2 -mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD280 nm, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
Recombinant proteins 29479681 (SEQ ID NO: 179), 978477 (SEQ ID NO: 228)
The pellet containing the inclusion bodies was solubilized in buffer B containing 8 M urea, 1 mM PMSF and 0 1 % 2-mercaptoethanol, and incubated for 1 hour at room temperature. Insoluble materials were removed by centrifugation at 20,000 x g for 30 min, and the cleared supernatant was loaded onto a 15 ml ( 1.6 × 7.5 cm ) SP-Sepharose column pre-equilibrated in buffer B, 6 M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol. After washing the column with 10 bed volumes, the column was developed with a linear gradient from 0 to 500 mM NaCl.
Dialysis and concentration of protein samples
Urea was removed slowly from the protein samples by dialysis against Tris-buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M, 4M, 3M, 2M, 1 M, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.
After dialysis, samples were concentrated by pressure filtration using Amicon stirred-cells. Protein concentrations were measured using the methods of Perkins (1986 Eur. J. Biochem. 157, 169-180), Bradford ((1976) Anal. Biochem. 72, 248-254) and Lowry ((1951) J. Biol. Chem. 193, pages 265-275).
The recombinant proteins purified by the methods described above are summarized in Table 3 below.
Figure imgf000056_0001
Figure imgf000057_0001
IV. Analysis of H. pylori proteins as Vaccine candidates
To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H. pylori model was used. This model mimics the human H. pylori infection in many respects The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immunotherapy
Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding center (Denmark). They were kept in ordinary makrolon cages with free supply of water and food The animals were 4-6 weeks old at arrival.
Infection
After a minimum of one week of acclimatization, the animals were infected with a type 2 strain (VacA negative) of H. pylori (strain AΗ244, 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 ΗEPES buffer or in a buffer containing 0.5%> Deoxycholate (DOC).
The antigens are listed in Table 4 below.
Figure imgf000058_0001
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 ΗEPES 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 5 below.
Figure imgf000059_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 25mm was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
The urease test was performed essentially as follows. The reagent, Urea Agar Base
Concentrate, was purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61 -3). Urea agar base concentrate was diluted 1:10 with water. 1 ml of 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 reagent (1% w/v in water) was prepared. H. pylori cells were swabbed onto Whatman filter paper and overlaid with the 1% solution. Color change to dark blue indicated that the cells were catalase positive.
Serum antibodies: From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.
Mucosal antibodies: Gentle scrapings of a defined part of the corpus and of 4 cm of duodenum were performed in 50% of the mice in order to detect the presence of antibodies in the mucous. The antibody titers were determined by regular ELISA technique as for serum antibodies.
Statistical analysis: Wilcoxon-Mann- Whitney sign rank test was used for determination of significant effects of the antigens on Helicobacter pylori colonization. P<0.05 was considered significant. Because the antrum is the major colonization site for Helicobacter most emphasis was put upon changes in the antral colonization.
Results
Antibodies in sera: All antigens tested given together with CT gave rise to a measurable specific titer in serum The highest responses were seen with SEQ ID
NOs:223, 211, and 212 (see Figure 2).
Antibodies in mucus: In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with SEQ ID NOs:181, followed by 223 (see Figure 3).
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 2 proteins (SEQ ID NOs: 211 and 212) gave a good and significant reduction and/or eradication of the H. pylori infection. The degree of colonization of the antrum was lower following
immunization with SEQ ID NOs: 228 and 223 compared to control. The effect of SEQ ID NOs:179, and 181 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 4 and 5 for proteins dissolved in ΗEPES and DOC respectively. Data is shown as geometric mean values. n=8-10 Wilcoxon-Mann- Whitney sign rank test * = p<0.05; x/10 = number of mice showing eradication of H. pylori over the total number of mice examined.
The data presented indicate that all of the H. pylori associated proteins included in this study, when used as oral immunogens in conjunction with the oral adjuvant CT, resulted in stimulation of an immune response as measured by specific serum and mucosal antibodies. A majority of the proteins led to a reduction, and in some cases complete clearance of the colonization of H. pylori in this animal model. It should be noted that the reduction or clearance was due to heterologous protection rather than homologous protection (the polypeptides were based on the H. pylori J99 strain sequence and used in the therapeutic immunization studies against a different (AΗ244) challenge strain), indicating the vaccine potential against a wide variety of H. pylori strains.
The highest colonization in the antrum was seen in animals treated with the non- Helicobacter protein LacZ, indicating that the effects seen with the Llelicobacter pylori antigens were specific. Taken together these data strongly support the use of these H. pylori proteins in a pharmaceutical formulation for the use in humans to treat and/or prevent H. pylori infections.
V. Sequence Variance Analysis of genes in Helicobacter pylori strains
Three genes were cloned and sequenced from several strains of H. pylori to compare the DNA and deduced amino acid sequences. This information was used to determine the sequence variation between the H. pylori strain, J99, and other H. pylori strains isolated from human patients. Preparation of Chromosomal DNA.
Cultures of H. pylori strains (as listed in Table 8) 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 mls 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 EtOΗ (1.5 x volumes) or isopropanol (0.6 x volumes) at -70°C for 10 minutes, washed in 70% EtOΗ and resuspended in TE.
PCR Amplification and cloning.
Genomic DNA prepared from twelve strains oi 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 6) 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 imgf000062_0001
The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Sequences (by SEQ ID NO:) 223 and 199;
Denaturation at 94ºC for 2 min,
2 cycles at 94ºC for 15 sec, 30°C for 15 sec and 72ºC 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.
Sequences (by SEQ ID NO:) 179;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 20 sec and 72ºC for 2 min
25 cycles at 94°C for 15 sec, 55°C for 20 sec and 72°C for 2 min
Reactions were concluded at 72°C for 8 minutes.
Upon completion of thermal cycling reactions, each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
Cloning of H. pylori DNA sequences into the pCR TA cloning vector.
All amplified inserts were cloned into the pCR 2.1 (pCRII in the case of H. pylori sequence 223) 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 TOPI 0F' (INVaF' in the case of H. pylori sequence 223) strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain TOP10F' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994). Briefly, 2 microliters of 0.5 micromolar BME was added to each vial of 50 microliters of competent cells. Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes. The cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of 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 TOPI 0F' 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 7 below.
Figure imgf000063_0001
Figure imgf000064_0001
Results
The PCR error rate for the experiments described herein was determined to be 2 base changes out of 4485 bases, which is equivalent to an estimated error rate of less than or equal to 0.04%
DNA sequence analysis was performed on four different open reading frames identified as genes and amplified by PCR methods from a dozen different strains of the bacterium Helicobacter pylori. The deduced amino acid sequences of three of the four open reading frames that were selected for this study showed statistically significant BLAST homology to defined proteins present in other bacterial species. Those ORFs included: SEQ ID NO:223, homologous to lipoprotein e (P4) present in the outer membrane of H. influenzae; SEQ ID NO:179, homologous to fecA, an outer membrane receptor in iron (III) dicitrate transport in E. coli. SEQ ID NO: 199 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 8 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.
Figure imgf000065_0001
VI. Experimental Knock-Out Protocol for the Determination of Essential H. pylori Genes as Potential Therapeutic Targets
Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis.
transcription, translation, regulation and colonization/virulence.
The protocol for the deletion of portions of H. pylori genes/ORFs and the insertional mutagenesis of a kanamycin-resistance cassette in order to identify genes which are essential to the cell is modified from previously published methods (Labigne-Roussel et al., 1988, J. Bacteriology 170, pp. 1704-1708; Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573; Reyrat et al., 1995, Proc. Natl. Acad. Sci. 92, pp 8768-8772). The result is a gene "knock-out." Identification and Cloning of H. pylori Gene Sequences
The sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Plymouth, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame. Genomic DNA prepared from the Helicobacter pylori HP-J99 strain (ATCC 55679) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons. Inc., F. Ausubel et al., editors, 1994). For the preparation of genomic DNA from H. pylori, see Example I. PCR amplification is carried out by introducing 10 nanograms of genomic HP-J99 DNA into a reaction vial containing 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2, 2 microMolar synthetic oligonucleotide primers (forward=F 1 and reverse=R1 ). 0.2 mM of each deoxynucleotide triphosphate (dATP,dGTP, dCTP, dTTP), and 1.25 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg. NJ, USA) in a final volume of 40 microliters The PCR is carried out with Perkin Elmer
Cetus/GeneAmp PCR System 9600 thermal cyclers.
Upon completion of thermal cycling reactions, each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction.
Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
PCR products are cloned into the pT7Blue T-Vector (catalog#69820-1 , Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
Ligation products are electroporated (Current Protocols in Molecular Biology, John
Wiley and Sons. Inc., F. Ausubel et al., editors, 1994) into electroporation-competent XL- 3 Blue or DH5-α E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA). Briefly, 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) at 37°C with shaking for 1 hour. Samples are then spread onto LB (10 g/l bacto tryptone, 5 g/l bacto yeast extract, 10 g/l sodium chloride) plates containing 100 microgram/ml of Ampicillin, 0.3% X-gal, and 100 microgram/ml IPTG. These plates are incubated overnight at 37°C. Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA). To verify that the correct Hpylori DNA inserts had been cloned, these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 H. pylori sequence. Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned. Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage. To minimize errors due to PCR, plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.
The sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other. The pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product. The PCR product is a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, results in the deletion of a portion of the ORFs. The PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified.
A Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology 170, 1704-1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid
pCTB8:kan (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573). The proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA). The fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes. This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides. The "T" overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pΗ 8.3, 50 mM KCl, 2 mM MgCl2, 5 units of DNA Polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP, in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C. The "Kan-T" cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA). The PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter 10x T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16ºC.
The ligation products are transformed into XL-1 Blue or DH5-α E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
Several tests by PCR amplification are conducted on these plasmids to verify that the Kanamycin is inserted in the H. pylori gene/ORF, and to determine the orientation of the insertion of the Kanamycin-resistance gene relative to the H. pylori gene/ORF. To verify that the Kanamycin cassette is inserted into the H. pylori sequence, the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the H. pylori gene/ORFs. The correct PCR product is the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. To avoid potential polar effects of the kanamycin resistance cassette on H. pylori gene expression, the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in H. pylori
transformations (see below). To determine the orientation of insertion of the kanamycin resistance gene, primers are designed from the ends of the kanamycin resistance gene ("Kan-1 " 5'-ATCTTACCTATCACCTCAAAT-3' (SEQ ID NO:267), and "Kan-2" 5'-AGACAGCAACATCTTTGTGAA-3' (SEQ ID NO:268)). By using each of the cloning primers in conjunction with each of the Kan primers (4 combinations of primers), the orientation of the Kanamycin cassette relative to the H. pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the Hpylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H. pylori. Transformation of Plasmid DNA into H. pylori cells
Two strains of H. pylori are used for transformation: HP-J99 (ATCC 55679), the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AH244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach. Cells for transformation are grown at 37°C, 10% CO2. 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated. If grown on plates, cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf micro fuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells. An aliquot (1 to 5 OD600 units/25 microliter) of the resuspended cells is placed onto a prewarmed Sheep-Blood agar plate, and the plate is further incubated at 37°C, 6% CO2, 100%o humidity for 4 hours. After this incubation, 10 microliters of plasmid DNA (100 micrograms per microliter) is spotted onto these cells. A positive control (plasmid DNA with the ribonuclease H gene disrupted by kanamycin resistance gene) and a negative control (no plasmid DNA) are done in parallel. The plates are returned to 37°C, 6% CO2 for an additional 4 hours of incubation. Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO2. Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C. 6% CO2, 100% humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.
Three sets of PCR tests are done to verify that the colonies of transformants have arisen from homologous recombination at the proper chromosomal location. The template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1 :1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
TEST 1. PCR with cloning primers originally used to amplify the gene/ORF. A positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. A PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location. TEST 2. PCR with F3 (primer designed from sequences upstream of the gene/ORF and not present on the plasmid), and either primer Kan-1 or Kan-2 (primers designed from the ends of the kanamycin resistance gene), depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
TEST 3. PCR with R3 (primer designed from sequences downstream of the gene/ORF and not present on the plasmid) and either primer Kan-1 or Kan-2 depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3). Again, no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
Transformants showing positive results for all three tests above indicate that the gene is not essential for survival in vitro.
A negative result in any of the three above tests for each transformant indicates that the gene had not been disrupted, and that the gene is essential for survival in vitro.
In the event that no colonies result from two independent transformations while the positive control with the disrupted ribonuclease H plasmid DNA produces transformants, the plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H. pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3. Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST 2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation
VII. High-throughput drug screen assay
Cloning, expression and protein purification
Cloning, transformation, expression and purification of the H. pylori target gene and its protein product,e.g., an H. pylori enzyme, to be used in a high-throughput drug screen assay, is carried out essentially as described in Examples II and III above. Development and application of a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase, is described below as a specific example. Enzymatic Assay
The assay is essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Acta 43:1101-1111). The assay measures the cis-trans isomerization of the Ala- Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S-7388, lot # 84H5805). The assay is coupled with α-chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans. The conversion of the test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 μM α-chymotrypsin (type 1-5 from bovine Pancreas. Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction. 10 μl of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 μl of reaction mixture at room temperature. Enzymatic assay in crude bacterial extract.
A 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD 600 nm ~ 1 ) and resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 μg/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor. The suspension is subjected to 3 cycles of freeze-thaw ( 15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts). The lysate is centrifuged (12,000 g × 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
Many H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
Other Embodiments
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 SEQ ID NOs:1-114 (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, or analogs of H. pylori polypeptides A biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the H. pylori polypeptides shown in SEQ ID NOs:115-228, or of other naturally occurring H. pylori polypeptides. e.g., one or more of the biological activities described above. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in CΗO cells. Because peptides such as H. pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H. pylori fragment or H. pylori analog is one which exhibits a biological activity in any biological assay for H. pylori activity. Most preferably the fragment or analog possesses 10%), preferably 40%. or at least 90% 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 abolish the biological activity of the H. pylori polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.
Figure imgf000072_0001
Figure imgf000073_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 a 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.
In order to obtain an H. pylori polypeptide, H. pylori polypeptide-encoding DNA can be introduced into an expression vector, the vector introduced into a cell suitable for expression of the desired protein, and the peptide recovered and purified, by prior art methods. Antibodies to the peptides an proteins can be made by immunizing an animal, e.g.. a rabbit or mouse, and recovering anti-H. pylori polypeptide antibodies by prior art methods.
The nucleic acids and corresponding polypeptides of the invention were disclosed previously in the corresponding US application, U.S.S.N. 08/561 ,469, filed November 17, 1995 (Attorney Docket No.: GTN-001CP). The correlation between sequence
identification numbers in the above-identified parent applications and sequence
identification numbers provided herein is outlined in Table 10 below.
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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.
Other embodiments are within the following claims.
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Claims

1. A substantially pure nucleic acid encoding an H. pylori polypeptide, said nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-114.
2. A substantially pure nucleic acid from naturally occurring H. pylori which hybridizes under stringent conditions to a nucleic acid which encodes an H. pylori polypeptide, said nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1-114.
3. A method of evaluating a compound for the ability to bind an H. pylori nucleic acid comprising: contacting said compound with an H. pylori nucleic acid selected from the group consisting of SEQ ID NO:1-114 and determining if said compound binds said H. pylori nucleic acid.
4. The method of claim 3, wherein said compound is an activator of the bacterial life cycle.
5. The method of claim 3, wherein said compound is an inhibitor of the bacterial life cycle.
6. The method of claim 3, wherein said method is performed in vitro.
7. The method of claim 3, wherein said method is performed in vivo.
8. A method of generating a vaccine for immunizing a subject against H. pylori infection comprising: immunizing said subject with a nucleic acid 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:1-114, and a therapeutically acceptable carrier.
9. A method of detecting the presence of a Helicobacter species in a sample comprising:
contacting said sample with a nucleic acid encoding an H. pylori polypeptide, said nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1-114;
hybridizing said sample to said nucleic acid; said hybridization being indicative of the presence of said Helicobacter species in said sample.
10. The method of claim 9, wherein said Helicobacter species is H. pylori.
11. The method of claim 9, wherein said nucleic acid is 20 or more nucleotides in length.
12. A method of inhibiting expression of a gene from a Helicobacter species comprising: administering to said species an H. pylori antisense nucleic acid selected from the group consisting of SEQ ID NO:1-114.
13. The method of claim 12, wherein said Helicobacter species is H. pylori.
14. The method of claim 12, wherein said antisense nucleic acid is administered in a carrier.
15. The method of claim 12, wherein said carrier is a liposome or a bacteriophage.
16. The method of claim 12, wherein said antisense nucleic acid is 20 or more nucleotides in length.
17. The method of claim 12. wherein said antisense nucleic acid is capable of binding to Helicobacter nucleic acid or mRNA.
18. A method of generating a vaccine for immunizing a subject against H. pylori infection comprising: immunizing said subject with a nucleic acid encoding an H. pylori polypeptide or a fragment thereof, said polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:115-228, and a therapeutically acceptable carrier.
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WO2000073502A2 (en) * 1999-05-31 2000-12-07 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Essential genes and gene products for identifying, developing and optimising immunological and pharmacological active ingredients for the treatment of microbial infections
US6222026B1 (en) 1998-09-08 2001-04-24 Smithkline Beecham Corporation Gcp
WO2001083533A1 (en) * 2000-04-29 2001-11-08 Astrazeneca Ab Helicobacter pylori antigens: fusion of hpaa and hpa44
EP1409683A2 (en) * 2000-12-07 2004-04-21 Antex Biologics, Inc. i HELICOBACTER /i PROTEINS, NUCLEIC ACIDS AND USES THEREOF
US7786260B1 (en) * 1999-10-15 2010-08-31 Csl Limited Polypeptide fragments comprising c terminal portion of helicobacter catalase
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
WO1998021225A1 (en) * 1996-11-14 1998-05-22 Merieux Oravax Helicobacter polypeptides and corresponding polynucleotide molecules
US6222026B1 (en) 1998-09-08 2001-04-24 Smithkline Beecham Corporation Gcp
WO2000073502A2 (en) * 1999-05-31 2000-12-07 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Essential genes and gene products for identifying, developing and optimising immunological and pharmacological active ingredients for the treatment of microbial infections
WO2000073502A3 (en) * 1999-05-31 2002-10-03 Max Planck Gesellschaft Essential genes and gene products for identifying, developing and optimising immunological and pharmacological active ingredients for the treatment of microbial infections
US7786260B1 (en) * 1999-10-15 2010-08-31 Csl Limited Polypeptide fragments comprising c terminal portion of helicobacter catalase
WO2001083533A1 (en) * 2000-04-29 2001-11-08 Astrazeneca Ab Helicobacter pylori antigens: fusion of hpaa and hpa44
EP1409683A2 (en) * 2000-12-07 2004-04-21 Antex Biologics, Inc. i HELICOBACTER /i PROTEINS, NUCLEIC ACIDS AND USES THEREOF
EP1409683A4 (en) * 2000-12-07 2005-03-23 Antex Biolog Inc i HELICOBACTER /i PROTEINS, NUCLEIC ACIDS AND USES THEREOF
CN112190703A (en) * 2020-10-13 2021-01-08 宁夏医科大学 GEM particle surface display system of targeting M cells, particle vaccine, preparation method and application

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