WO1997019098A9 - Sequences d'acides nucleiques et d'acides amines relatives au codage de helicobacter pylori a des fins diagnostiques et therapeutiques - Google Patents
Sequences d'acides nucleiques et d'acides amines relatives au codage de helicobacter pylori a des fins diagnostiques et therapeutiquesInfo
- Publication number
- WO1997019098A9 WO1997019098A9 PCT/US1996/018542 US9618542W WO9719098A9 WO 1997019098 A9 WO1997019098 A9 WO 1997019098A9 US 9618542 W US9618542 W US 9618542W WO 9719098 A9 WO9719098 A9 WO 9719098A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pylori
- seq
- nucleic acid
- sequence
- dna
- Prior art date
Links
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Definitions
- He licobacter pylori is a gram-negative, S-shaped, microacrophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren. J.R. and B. Marshall, (1983) Lancet I : 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. Y. 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// 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 //. 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// 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
- the invention features a substantially pure nucleic acid encoding an //. pylori polypeptide having an amino acid sequence of SEQ ID NO:l 16, 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: 1 17, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3.
- the invention features a substantially pure nucleic acid encoding an //. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 18, 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: 1 19, 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 //. 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: 1 1.
- 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 //. 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 //. 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 ac id 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 //. pylori polypeptide having an amino acid sequence of SEQ ID NO: 181 , 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 //. 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, such as 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:21 1, 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 //. 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: l 10.
- 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:l 12.
- 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:l 13.
- 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:l 14.
- 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.
- 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. 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 //. 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 axe 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:l 15-228; 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: 1 15-228.
- 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:l 15-228. The differences, however, are such that: the H. pylori polypeptide exhibits an //. pylori biological activity, e.g., the H.
- the 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: 1 15-228; 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:l 15-228.
- 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%o, 95%, 98%, or 99% homologous to an amino acid sequence contained in SEQ ID NOs: 1 15-228; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NOs: 1 15-228; 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: 1 15- 228.
- the nucleic acid is that of SEQ ID NOs: 1 - 1 14; 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: 1-1 14.
- 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: 1 15-228. The differences, however, are such that: the H. pylori encoded polypeptide exhibits a //. 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:l 15-228; 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: 1-1 14; more preferably to at least 20 consecutive nucleotides contained in SEQ ID NOs: 1-1 14; more preferably to at least 40 consecutive nucleotides contained in SEQ ID NOs: 1-1 14.
- the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences shown in SEQ ID NOs.l 15-228. In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence shown in SEQ ID NOs: 1-1 14 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: 1-1 14, 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.
- the nucleic acid sequences of this invention may be obtained directly from the
- PCR polymerase chain reaction
- Clones carrying the desired sequences described in this invention may be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, 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.
- the nucleic acid will normally comprise approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products.
- the present invention encompasses //. 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 //. 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.
- 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.
- 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: 1-1 14 can be used as a probe to specifically detect //. 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 -1 14 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: 1-1 14 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.
- PCR polymerase chain reaction
- nucleic acid sequences of > 10-15 nucleotides contained in SEQ ID NOs: 1-1 14 have utility in conjunction with suitable enzymes and reagents to create copies of//, 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.
- 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.
- 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// 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: 1-1 14 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: 1 13- 123), and pYES2 (Invitrogen Co ⁇ oration, San Diego, CA).
- Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
- COS cells Gluzman, Y., (1981) Cell 23 : 175- 182
- pCDM 8 are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci.
- Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
- Fusion vectors usually add a number of 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 pRJT5 (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 pETl Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185. Academic Press, San Diego, California (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pETl Id relies on transcription from the T7 gnlO-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 gnl under the transcriptional control of the lacUV 5 promoter.
- T7 gnl coexpressed viral RNA polymerase
- 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 chromatographically isolated from the membrane fraction.
- 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.
- CMC critical micells concentration
- a third property considered when selecting a detergent is the hydrophobicity of the detergent. Typically, membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g. the triton series, would be useful for solubilizing the hydrophobic proteins.
- Another property important to a detergent can be the capability of the detergent to remove the H. 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.
- detergent should be an uncharged detergent.
- Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
- One strategy to maximize recombinant H. pylori peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 1 19-128).
- Another strategy would be to alter the nucleic acid encoding H.
- 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).
- 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 abso ⁇ tion, 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 progress of immunization can be monitored by detection of antibody titers in plasma or serum.
- 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: 1 15-228 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: 1 15-228.
- 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.
- 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.
- 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 ⁇ gtl 1 , ⁇ gtl 8-23, ⁇ ZAP, and ⁇ ORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
- ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
- Antigenic epitopes of a subject H 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.
- the presence of// 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: 1-1 14 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 pharmaceutically acceptable carrier. It is possible to modify the structure of a H pylori protein or peptide for such pu ⁇ oses 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. Another modification is cyclization of the peptide.
- an H pylori protein or peptide can be modified to inco ⁇ orate one or more polymo ⁇ hisms 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
- 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., 4 199 - 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.
- 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).
- specific 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. Biophys. Res. Comm, JJ3L 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. Biophys. Res. Comm, JJ3L 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 (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).
- stimulation proliferation, cytokine secretion
- 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 pe ⁇ etuation 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 MHC molecules, to T cells, in conjunction with the necessary costimulation, has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4.
- the culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines.
- any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are inco ⁇ orated herein by reference.
- a kit for an assay for the production of interferon is also available from Genzyme Co ⁇ oration (Cambridge, MA).
- a common assay for T cell proliferation entails measuring tritiated thymidine inco ⁇ oration.
- the proliferation of T cells can be measured in vitro by determining the amount of ⁇ H-labeled thymidine inco ⁇ orated 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: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- 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.
- 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), 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, inco ⁇ orated 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.
- 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.
- 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 9J_: 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 1 1 : 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
- the vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl ⁇ L-threonyI-D-isoglutamine (thr- MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoy 1-sn- gIycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835 A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion;
- Non-toxic derivatives of cholera toxin including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H pylori bacterial lysates. block polymers or saponins.
- suitable delivery methods include biodegradable microcapsules or immuno- stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue.
- the amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 ⁇ g to 50 ⁇ g, for example 10 ⁇ g to 35 ⁇ g. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage.
- Carrier systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H pylori polypeptide in an insoluble form as fusion proteins.
- Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres.
- Suitable diluents are 0.2 N Na ⁇ C03 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.
- 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.
- a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxi genie 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.
- 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 #y/or/-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.
- V Not-T (not-U) (A or C or G)
- 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 :1 1-15). 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.
- 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) WJ «. Rev. Biochem.
- Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
- the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
- Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989).
- a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
- Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
- Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
- the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
- alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
- Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus inco ⁇ orate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA.
- 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
- fluorescently labeled ligands allows cells to be visually inspected and separated under a fluorescence microscope, or, where the mo ⁇ hology of the cell permits, to be separated by a fluorescence-activated cell sorter.
- a gene library can be expressed as a fusion protein on the surface of a viral particle.
- foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
- coli filamentous phages Ml 3, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle.
- Foreign epitopes can be expressed at the NH2-terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al.
- the pilus Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment.
- Another large surface structure used for peptide display is the bacterial 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) EMBOJ. 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 Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced.
- This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO).
- LacO operator By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
- the associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands.
- a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dyno ⁇ hin B.
- a cohort of peptides was recovered, all related by a consensus sequence corresponding to a six- residue portion of dyno ⁇ hin 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 Lad fusion molecules are confined to the cytoplasm of the host cells.
- the phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles.
- the peptides in the Lacl 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 inco ⁇ oration into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J Med. Chem. 37(9): 1233-1251). These particular biases are not a factor in the Lad display system. The number of small peptides available in recombinant random libraries is enormous. Libraries of 10 ⁇ -10 ⁇ 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.
- 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.
- 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. 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.
- 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.
- /7y/ ⁇ ' -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 polypeptide
- peptidomimetic compounds e.g. diazepine or isoquinoline derivatives
- non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, 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.
- kits for diagnostic pu ⁇ oses 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 uninco ⁇ orated 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 oul-of-frame lacZ gene at the cloning site which becomes in- frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.
- each of the 20 vectors was then transformed into DH5 competent cells (Gibco/BRL, DH5 ⁇ transformation protocol).
- the libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C.
- DNA was purified using the Qiagen Midi-prep kits and Tip-100 columns (Qiagen, Inc.). In this manner, 100 ⁇ g of DNA was obtained per pool. 15 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy with 250-300 base average read-lengths. 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.
- 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.
- Routine assembly of//, pylori sequences was done using the program FALCON (Church, Church et al., Automated DNA Sequenicng and Analysis (J.C. Venter, ed.), Academic Press, 1994). This program has proven to be fast and reliable for most sequences.
- the assembled contigs were displayed using a modified version of GelAssemble, developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic- Acid Res. 12:387-95, 1984) that interacts with REPLICATM. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICATM database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly.
- GCG Genetics Computer Group
- 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.
- 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 Flis-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 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:
- the pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case of H pylori sequence 4821082 (SEQ ID NO: 212) with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
- H pylori sequence 4821082 SEQ ID NO: 212
- Ndel and EcoRI Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994.
- cloning ppiB the pET-28a vector, which encodes a ⁇ is-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with Bam ⁇ I and Xhol restriction endonucleases.
- 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. HMS 174, HB 101 ,
- 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
- Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
- H. pylori sequences 50 nanograms of plasmid DNA isolated as described above 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 (1 16 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
- NTA Ni ⁇ +" 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 OU2go n m, an peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole. Recombinant beta-galactosidase (lacZ) and pept idyl -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 (Ccntriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 91 cm) of Scphacryl 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 Ni ⁇ + -NTA-agarose column were pooled and dialyzed overnight against 1 liter of dialysis buffer (10 mM MOPS, pH 6.5, 50 mM NaCl, 0.1 mM EGTA, 0.02% Brij 35 and 1 mM PMSF). In the morning, a fine white precipitate was removed by centrifugation and the resulting supernatant was loaded onto an 8 ml (8 x 75 mm) MonoS high performance liquid chromatography column (Pharmacia Biotechnology, Inc., Piscataway, NJ, USA) equilibrated in buffer B (10 mM
- 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 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.
- the pellet containing the inclusion bodies was solubilized in buffer B containing 8 M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol, and incubated for 1 hour at room temperature. Insoluble materials were removed by centrifugation at 20,000 x g for 30 min, and the cleared supernatant was loaded onto a 15 ml ( 1.6 x 7.5 cm ) SP-Sepharose column pre-equilibrated in buffer B, 6 M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol. After washing the column with 10 bed volumes, the column was developed with a linear gradient from 0 to 500 mM NaCl.
- 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, IM, 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
- 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 H. pylori
- strain AH244 H. pylori
- 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% C0 2 , 5%0 2 ).
- the animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and 3-5 h after this an oral inoculation of /, 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 //. 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.
- mice were all infected with H. pylori strain AH244 at day 30. Proteins are listed by their Seq ID #'s.
- Protein 179 100 ⁇ g + CT 10 ⁇ g Balb/c 0.3 ml 0, 14, 24, 34 4.
- Protein 181 100 ⁇ g + CT 10 ⁇ g Balb/c 0.3 ml 0, 14, 24, 34
- Mucosal infection The mice were sacrificed by C0 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 co ⁇ us 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
- 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 co ⁇ us and of 4 cm of duodenum were performed in 50% of the mice in order to detect the presence of antibodies in the mucous.
- the antibody titers were determined by regular ELISA technique as for serum antibodies.
- Antibodies in sera All antigens tested given together with CT gave rise to a measurable specific titer in serum. The highest responses were seen with SEQ ID NOs:223, 21 1, 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).
- H. pylori protein LacZ indicating that the effects seen with the Helicobacter pylori antigens were specific. Taken together these data strongly support the use of these H. pylori proteins in a pharmaceutical formulation for the use in humans to treat and/or prevent H. pylori infections.
- 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 mis 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 + TI 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.
- Table 6 Oligonucleotide primers used for PCR amplification of H pylori DNA sequences.
- 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 72°C for 2 min 25 cycles at 94°C for 15 sec, 55°C for 20 sec and 72°C for 2 min Reactions were concluded at 72°C for 8 minutes.
- each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
- Competent bacteria E coli strain TOPI OF' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Briefly, 2 microliters of 0.5 micromolar BME was added to each vial of 50 microliters of competent cells. Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes.
- the cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgS04 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.
- Oligonucleotide primers used for sequencing of H pylori DNA sequences Oligonucleotide primers used for sequencing of H pylori DNA sequences.
- 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.
- 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.
- 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.
- 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.
- Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.
- the sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Madison, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.
- Genomic DNA prepared from the Helicobacter pylori 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
- the PCR is carried out with Perkin Elmer Cctus/GeneAmp PCR System 9600 thermal cyclers.
- each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley a d Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product f 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-l, 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
- 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 MgS0 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 MgS0 4 and 20 mM glucose
- 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 pCTm an (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 lOx T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16°C.
- the ligation products are transformed into XL-1 Blue or DH5- ⁇ E.coli cells by electroporation as described previously.
- cells After recovery in SOC, cells arc 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.
- the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the //. 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.
- the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in //. pylori transformations (see below).
- 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)).
- the orientation of the Kanamycin cassette relative to the H.pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the H.pylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H. pylori.
- 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 //. 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% C0 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 ⁇ m 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% C0 .
- 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% C0 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.
- 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.
- 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: 1 101-1 1 1 1).
- the assay measures the cis-trans isomerization of the Ala- Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S-7388, lot # 84H5805).
- the assay is coupled with ⁇ -chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans.
- test peptide 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 ⁇ M ⁇ -chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction, 10 ⁇ l of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 ⁇ l of reaction mixture at room temperature.
- substrate 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO
- a 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD 60 o n m ⁇ an d resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 ⁇ g/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor.
- the suspension is subjected to 3 cycles of freeze- thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts).
- the lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
- H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
- 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: 1-1 14 (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 inco ⁇ orated 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.
- a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the //. 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. Because peptides such as H.
- a useful H pylori fragment or H. pylori analog is one which exhibits a biological activity in any biological assay for H. pylori activity. Most preferably the fragment or analog possesses 10%), preferably 40%, 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.
- Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln
- Lysine K D-Lys, Arg, D-Arg, homo- Arg, D-homo-Arg, Met, D-Met, Ile, D-lle, Orn, D-Orn
- Tyrosine Y D-Tyr Phe, D-Phe, L-Dopa, His, D-His
- 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.
- 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. TABLE 10
- ORGANISM Helicobacter pylori
- AAATTTAAAA AAGGGGGGAC ATGGTTTTAT GATTTTTAA 519
- ORGANISM Helicobacter pylori
- ORGANISM Helicobacter pylori
- MOLECULE TYPE DNA (genomic)
- ORGANISM Helicobacter pylori
- MOLECULE TYPE DNA (genomic) (ill) HYPOTHETICAL. NO
- ORGANISM Helicobacter pylori
- MOLECULE TYPE DNA (genomic)
- ORGANISM Helicobacter pylori
- ATGTTAAAAA CCCACTTAAG CAGCGCTAGG GGCGTTGTGG TGTTGTCTAA GATTTTACCG 60
- MOLECULE TYPE DNA (genomic) (ill)
- HYPOTHETICAL NO
- ANTI-SENSE NO
- ORIGINAL SOURCE
- ORGANISM Helicobacter pylori
- AAGGCCCACC CTAATGTGTA TGATTTGTTG TTGCAGGTGA TGGAKAACGC CACTTTGAGC 300
- MOLECULE TYPE DNA (genomic)
- ORGANISM Helicobacter pylori
- AATATCGCAG ATATTAAGMR AAACGACTCA GACGGAAGGT TAGTCACAGC RATCAATGCG 840
- GTCACTTCAG AAACCGGTGT GGWAGCTTAT ACGGATCAAA AAGGACGCTT GAATTTGCGC 900
- ORGANISM Helicobacter pylori
- CAAGTTACTC CAATCTCTAT ACAGGATTTT AAAGATATGG CAAGCACTTA TAAGATATTT 300
- ORGANISM Helicobacter pylori
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- ORGANISM Helicobacter pylori
- AAATTTGAAA AACCTGCATA CAAACAACAC AAAATGTCTG AATCCCAATC CATGCAAGAA 240
- MOLECULE TYPE DNA (genomic)
Abstract
L'invention concerne des préparations obtenues par recombinaison ou sensiblement pures de polypeptides de H. pylori. On décrit par ailleurs les acides nucléiques assurant le codage des polypeptides en question, qui sont des constituants utiles dans les compositions de vaccins.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU10554/97A AU1055497A (en) | 1995-11-17 | 1996-11-15 | Nucleic acid and amino acid sequences relating to helicobacter pylori for diagnostics and therapeutics |
US10/335,977 US20040052799A1 (en) | 1996-11-15 | 2002-12-30 | Nucleic acid and amino acid sequences relating to Helicobacter pylori for diagnostics and therapeutics |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US56146995A | 1995-11-17 | 1995-11-17 | |
US08/561,469 | 1995-11-17 |
Publications (2)
Publication Number | Publication Date |
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WO1997019098A1 WO1997019098A1 (fr) | 1997-05-29 |
WO1997019098A9 true WO1997019098A9 (fr) | 1997-11-06 |
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PCT/US1996/018542 WO1997019098A1 (fr) | 1995-11-17 | 1996-11-15 | Sequences d'acides nucleiques et d'acides amines relatives au codage de helicobacter pylori a des fins diagnostiques et therapeutiques |
Country Status (2)
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AU (1) | AU1055497A (fr) |
WO (1) | WO1997019098A1 (fr) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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AU735391B2 (en) * | 1996-11-14 | 2001-07-05 | Human Genome Sciences, Inc. | Helicobacter polypeptides and corresponding polynucleotide molecules |
US6222026B1 (en) | 1998-09-08 | 2001-04-24 | Smithkline Beecham Corporation | Gcp |
WO2000073502A2 (fr) * | 1999-05-31 | 2000-12-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Genes et produits geniques essentiels pour l'identification, le developpement et l'optimisation de principes actifs immunologiques et pharmacologiques pour le traitement d'infections microbiennes |
AUPQ347199A0 (en) * | 1999-10-15 | 1999-11-11 | Csl Limited | Novel polypeptide fragments |
GB0010371D0 (en) * | 2000-04-29 | 2000-06-14 | Astrazeneca Ab | Helicobacter pylori antigens |
US20020107368A1 (en) * | 2000-12-07 | 2002-08-08 | Jing-Hui Tian | Helicobacter proteins, gene sequences and uses thereof |
CN112190703B (zh) * | 2020-10-13 | 2022-11-15 | 宁夏医科大学 | 靶向m细胞的gem颗粒表面展示系统、颗粒疫苗及制备方法与应用 |
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US5403924A (en) * | 1992-10-13 | 1995-04-04 | Vanderbilt University | Taga gene and methods for detecting predisposition to peptic ulceration |
US5434253A (en) * | 1994-03-21 | 1995-07-18 | Vanderbilt University | DNA encoding Helicobacter pylori recombinase |
US5527678A (en) * | 1994-10-21 | 1996-06-18 | Vanderbilt University | CagB and CagC genes of helicobacter pylori and related compositions |
-
1996
- 1996-11-15 AU AU10554/97A patent/AU1055497A/en not_active Abandoned
- 1996-11-15 WO PCT/US1996/018542 patent/WO1997019098A1/fr active Application Filing
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