WO1999021959A2

WO1999021959A2 - Helicobacter pylori vaccine formulations

Info

Publication number: WO1999021959A2
Application number: PCT/US1998/022883
Authority: WO
Inventors: Ronald W. Ellis; Brian M. Noonan; Richard A. Alm; Douglas Smith; Braydon C. Guild
Original assignee: Genome Therapeutics Corporation
Priority date: 1997-10-28
Filing date: 1998-10-28
Publication date: 1999-05-06
Also published as: AU1698699A

Abstract

H. pylori vaccine formulations are described. H. pylori vaccine formulations which include, as a pharmaceutically acceptable carrier, live attenuated bacterial vectors are also described. The H. pylori vaccine formulations are useful for prevention or treatment of an H. pylori infection.

Description

HELICOBΛCTER PYLORI VACCINE FORMULATIONS

Background of the Invention

Helicobacter pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet 1 : 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83-88). H pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H. pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. (Blaser M. J., (1993) Trends Microbiol. J_: 255- 260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age. (Taylor, D.N. and M. J. Blaser, (1991) Epidemiol. Rev 1_3: 42-50). H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).

The bacterial factors necessary for colonization of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of the putative virulence factors include the following: urease, an enzyme that may play a role in neutralizing gastric acid pH (Eaton et al., (1991) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee (1991) Microb. Ecol. Hlth. Dis. 4: 121-134; Labigne et al., (1991) J. Bacteriol. 173: 1920-1931); the bacterial flagellar proteins responsible for motility across the mucous layer. (Hazell et al., (1986) J. Inf. Dis. 153: 658-663; Leying et al., (1992) Mol. Microbiol. 6: 2863-2874; and Haas et al., (1993) Mol. Microbiol. 8: 753-760); Vac A, a bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells (Schmitt, W. and R. Haas, (1994) Molecular Microbiol. 12(2}: 307-319); and several gastric tissue-specific adhesins. (Boren et al., (1993) Science 262: 1892- 1895; Evans et al., (1993) J. Bacteriol. 1_75: 674-683; and Falk et al., (1993) Proc. Natl. Acad. Sci. USA 90: 2035-203).

Numerous therapeutic agents are currently available that eradicate H. pylori infections in vitro. (Huesca et. al, (1993) Zbl. Bakt. 280: 244-252; Hopkins, R. J. and J. G. Morris, supra). However, many of these treatments are suboptimally effective in vivo because of bacterial resistance, altered drug distribution, patient non-compliance or poor drug availabilty. (Hopkins, R. J. and j. G. Morris, supra). Treatment with antibiotics combined with bismuth are part of the standard regime used to treat H. pylori infection. (Malfertheiner, P. and J. E. Dominguez-Munoz (1993) Clinical Therapeutics j_5 Supp. B: 37-48). Recently, combinations of a proton pump inhibitors and a single antibiotic have been shown to ameliorate duodenal ulcer disease. (Malfertheiner, P. and J. E. Dominguez-Munoz supra). However, methods employing antibiotic agents can have the problem of the emergence of bacterial strains which are resistant to these agents. (Hopkins, R. J. and J. G. Morris, supra). These limitations demonstrate that new more effective methods are needed to combat H. pylori infections in vivo. In particular, the design of new vaccines that may prevent infection by this bacterium is highly desirable.

Summary of the Invention This invention relates to H. pylori vaccine formulations, and uses thereof. For example, the vaccine formulations of the present invention can be used for the protection against or treatment of H. pylori infection. Preferred H. pylori vaccine formulations include, as a pharmaceutically acceptable carrier, a bacterial vector. Preferably, the bacterial vector is attenuated. Reference is made to the Sequence Listings of the applications mentioned above (see "Related Applications") which are incorporated herein by reference. The Sequence Listings include amino acid and nucleotide sequences which relate to H. pylori polypeptides. The disclosure of the present application expressly includes the use of at least one of these polypeptides or fragments thereof in the vaccine formulation of the present invention. Hence, for example, the bacterial vector used in the formulation can comprise a nucleotide sequence encoding an H. pylori polypeptide or a fragment thereof, wherein the nucleotide sequence given is given in a Sequence listing of one of the incorporated applications.

Detailed Description of the Drawings Figure 1 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.

Figure 2 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.

Figure 3 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.

Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in buffer containing DOC.

Detailed Description of the Invention In one aspect, the invention features a vaccine formulation for prophylactic or therapeutic treatment of an H. pylori infection, which includes an effective amount of at least one isolated H. pylori polypeptide or a fragment thereof in a pharmaceutically acceptable carrier. Preferred vaccine formulations include an effective amount of at lest one isolated H pylori polypeptide or a fragment thereof in a bacterial vector. Preferably, the bacterial vector includes a nucleotide sequence encoding the H. pylori polypeptide or fragment thereof. Preferred bacterial vectors for use in vaccine formulations of the invention include a species of Salmonella, Vibrio cholerae and Shigella.

Another embodiment of the invention features vaccine formulations which include two or more H pylori polypeptides or fragments thereof. In one embodiment, the two or more of the H pylori polypeptides or fragments thereof, are contained and expressed by the same bacterial vector. For example, the bacterial vector can include two or more nucleotide sequences encoding two or more H. pylori polypeptides or fragments thereof. Alternatively, two or more H pylori polypeptides or fragments thereof are included in and expressed by two or more of the same or different bacterial vectors. For example, two bacterial vectors (e.g., two different Salmonella vectors or a Salmonella and a V. cholerae vector) can each contain the same or different H. pylori polypeptides or fragments thereof (e.g., one bacterial vector includes a nucleotide sequence encoding an H. pylori polypeptide or a fragment thereof and a second bacterial vector includes a nucleotide sequence encoding the same or different H. pylori polypeptide or a fragment thereof). When the H pylori polypeptides or fragments thereof are expressed in two or more different bacterial vectors, the vectors can be combined to form a vaccine formulation of the invention.

In a preferred embodiment, the vaccine formulation further includes an adjuvant. In one embodiment, the bacterial vector includes both a nucleotide sequence encoding an H pylori polypeptide or a fragment thereof and a nucleotide sequence encoding an adjuvant. In another embodiment, one bacterial vector contains a nucleotide sequence encoding an H pylori polypeptide or a fragment thereof and a second bacterial vector contains a nucleotide sequence encoding an adjuvant (e.g., a nucleotide sequence encoding a cytokine). When the H. pylori polypeptide or a fragment thereof and the adjuvant are expressed by a different bacterial vectors, the vectors can be combined to from a vaccine formulation of the invention.

Another embodiment of the invention features vaccine formulations in which an H. pylori polypeptide or a fragment thereof, other than urease, is contained in a Salmonella vector.

In one embodiment, the invention features vaccine formulations in which an H. pylori polypeptide or a fragment thereof is contained in and expressed by a species of Salmonella (e.g., S. typhi or S. typhimurium). Preferably, the Salmonella vector is attenuated by a mutation in at least one of the genes selected from the group consisting of cya, crp, and cdt. In a preferred embodiment, the Salmonella vector is attenuated by a mutation in the cya gene. In another embodiment, the Salmonella vector is attenuated by a mutation in the crp gene. In yet another embodiment, the Salmonella vector is attenuated by a mutation in the cdt gene. Another embodiment of the invention features vaccine formulations in which an

H pylori polypeptide or a fragment thereof is contained in and expressed by a species of Vibrio cholerae. Yet another embodiment of the invention features vaccine formulations in which an H pylori polypeptide or a fragment thereof is contained in and expressed by a species of Shigella. In a preferred embodiment, the vaccine formulation includes a bacterial vector which is attenuated.

In a preferred embodiment, the vaccine formulation includes an H. pylori outer membrane polypeptide or a fragment thereof. Preferred H pylori outer membrane polypeptides or fragments thereof are selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63 SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.

In another embodiment, the vaccine formulation includes an H. pylori outer membrane polypeptide or a fragment thereof which is encoded by a nucleic acid selected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, or a complement thereof. In another embodiment, the vaccine formulation includes an H pylori outer membrane polypeptide having a terminal phenylalanine residue or a fragment thereof.

In another embodiment, the vaccine formulation includes an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from a group consisting of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103.

In another embodiment, the vaccine formulation includes an H pylori polypeptide having a terminal phenylalanine residue or a fragment thereof which is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a complement thereof.

In another embodiment, the vaccine formulation includes an H. pylori outer membrane polypeptide having a C-terminal tyrosine cluster or a fragment thereof.

In another embodiment, the vaccine formulation includes an H. pylori polypeptide having a C-terminal tyrosine clusterter or a fragment thereof selected from a group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 11 1.

In another embodiment, the vaccine formulation includes an H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof which is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 28. SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, or a complement thereof.

In another embodiment, the vaccine formulation includes an H. pylori outer membrane polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof. In another embodiment, the vaccine formulation includes an H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103.

In another embodiment, the vaccine formulation includes an H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof which is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a complement thereof.

In another aspect, the invention features a vaccine formulation for prophylactic or therapeutic treatment of an H. pylori infection. The formulation includes an effective amount of at least one isolated H pylori polypeptide or a fragment thereof in a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier includes a bacterial vector (e.g., a Salmonella vector) which includes a nucleotide sequence encoding an H pylori polypeptide or a fragment thereof, provided that the H pylori polypeptide is not urease.

In yet another aspect, the invention features a method for treatment or prophylaxis of an H pylori infection in a subject. The method includes administering to a subject a vaccine formulation of the invention, such that treatment or prophylaxis of H. pylori infection occurs.

In another aspect, the invention features a method of producing an H. pylori vaccine formulation. The method includes introducing at least one isolated H. pylori polypeptide or a fragment thereof into a pharmaceutically acceptable carrier to thereby form a vaccine formulation, wherein the pharmaceutically acceptable carrier is a bacterial vector which includes a nucleotide sequence encoding the H pylori polypeptide or fragment thereof. Preferably, the bacterial vector is selected from the group consisting of a species of Salmonella, Vibrio cholerae and Shigella. In another embodiment, the method further includes the steps of isolating the H pylori polypeptide or fragment thereof. For example, the method can include the steps of providing a recombinant H pylori polypeptide or a fragment thereof in which a cell is cultured under conditions that permit expression of an H. pylori polypeptide or a fragment thereof and the H. pylori polypeptide or a fragment thereof is isolated from the cell. In another aspect, the invention features a method of generating vaccines for immunizing an individual against H. pylori. The vaccination method includes: immunizing a subject with at least one H. pylori polypeptide according to the present invention, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmaceutically acceptable carrier. Such vaccines have therapeutic and/or prophylactic utilities.

In another aspect, the invention provides a method for generating a vaccine comprising a modified immunogenic H. pylori polypeptide, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmacologically acceptable carrier. The invention further features vaccine formulations which include H. pylori polypeptides, preferably a substantially pure preparation of an H. pylori polypeptide, or a recombinant H. pylori polypeptide. In preferred embodiments: the polypeptide has biological activity; the polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%), 95%), 98%o, or 99% identical or homologous to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65%) sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acid residues of the invention contained in the Sequence Listing. In yet another preferred embodiment, the amino acid sequence which differs in sequence identity by about 7% to about 8%> from the H. pylori amino acid sequences of the invention contained in the Sequence Listing is also encompassed by the invention.

In preferred embodiments: the H pylori polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.

In a preferred embodiment, the subject H. pylori polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the H pylori polypeptide exhibits an H pylori biological activity, e.g., the H pylori polypeptide retains a biological activity of a naturally occurring H. pylori polypeptide.

In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5 ' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.

In yet other preferred embodiments, the H pylori polypeptide is a recombinant fusion protein having a first H pylori polypeptide portion and a second polypeptide portion, e.g., a second -polypeptide portion having an amino acid sequence unrelated to H. pylori. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.

Polypeptides for use in the vaccine formulations of invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.

The invention also encompasses vaccine formulations which include an immunogenic component which includes at least one H pylori polypeptide in an immunogenic preparation; the immunogenic component being capable of eliciting an immune response specific for the H pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogenic component comprises at least one antigenic determinant from a polypeptide of the invention contained in the Sequence Listing.

In another aspect, the vaccine formulations of the invention include a substantially pure nucleic acid having a nucleotide sequence which encodes an H. pylori polypeptide. In preferred embodiments: the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%), 95%), 98%), or 99%) homologous to an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the

Sequence Listing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.

In preferred embodiments: the nucleic acid for use in the invention is that contained in the Sequence Listing; the nucleic acid is at least 60%>, 70%, 80%, 90%>, 95%>, 98%, or 99% homologous with a nucleic acid sequence of the invention contained in the Sequence Listing.

In a preferred embodiment, the encoded H. pylori polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that: the H pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.

In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.

In preferred embodiments, the subject H. pylori nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the H. pylori gene sequence, e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell. In yet a further preferred embodiment, the nucleic acid which encodes an H. pylori polypeptide for use in the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of the invention contained in the

Sequence Listing. In a preferred embodiment, the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences of the invention contained in the

Sequence Listing. In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of the invention contained in the Sequence Listing which encodes amino acids of the invention contained in the Sequence Listing.

In another aspect, the vaccine formulation of the invention features a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence of the invention contained in the Sequence Listing.

The vaccine formulation of the invention further encompasses isolated H. pylori polypeptides which are encoded by a nucleic acids which hybridizes under stringent hybridization conditions to a nucleic acid contained in the Sequence Listing. The invention further encompasses the use of 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; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) as strain ΗP-J99.

Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6 and 6.4.1-6.4.10, hereby incorporated by reference); and, polypeptides specifically bound by antisera to H. pylori polypeptides, especially by antisera to an active site or binding domain of H. pylori polypeptide. The invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as H. pylori polypeptide analogs or variants.

Putative functions have been determined for several of the H. pylori polypeptides of the invention, as shown in Table 1.

In addition, the present invention encompasses the use of H. pylori polypeptides characterized as shown in Table 1 below, including: H. pylori cell envelope proteins, e.g., outer membrane cell envelope proteins. Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs. Polypeptides related by significant homology to the polypeptides of Table 1 are also considered to be classified in the manner of the homologs shown in Table 1. TABLE 1

[In Table 1 , "nt" represents nucleotide Seq. ID number and "aa" represents amino acid Seq. ID number]

Definitions The terms "purified polypeptide" and "isolated polypeptide" and "a substantially pure preparation of a polypeptide" are used interchangeably herein and, as used herein, mean a polypeptide that has been substantially, and preferably completely, separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 μg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide. Furthermore, the terms "purified polypeptide" and "isolated polypeptide" and "a substantially pure preparation of a polypeptide," as used herein, refer to both a polypeptide obtained from nature or produced by recombinant DNA techniques as described herein.

For example, an "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the H pylori protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of H pylori protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of H. pylori protein having less than about 30% (by dry weight) of non-H. pylori protein (also referred to herein as a "contaminating protein"), more preferably less than about 20%) of non-H. pylori protein, still more preferably less than about 10%> of non-H. pylori protein, and most preferably less than about 5%> non-H. pylori protein. When the H. pylori protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%), more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of H. pylori protein in which the protein is separated from chemical precusors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of H. pylori protein having less than about 30% (by dry weight) of chemical precursors or non-H. pylori chemicals, more preferably less than about 20% chemical precursors or non-H. pylori chemicals, still more preferably less than about 10% chemical precursors or non-H. pylori chemicals, and most preferably less than about 5% chemical precursors or non-H. pylori chemicals. A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50%) of the subject cells.

A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional H. pylori DNA sequence.

A "contig" as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.

An "open reading frame", also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.

As used herein, a "coding sequence" is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.

A "complement" of a nucleic acid as used herein referes to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence. A "gene product" is a protein or structural RNA which is specifically encoded by a gene.

As used herein, the term "probe" refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like. Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand". Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.

Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%) homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50%> homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions. Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions. Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stingency (such as, for example, in a solution of 0.5X SSC, at 65° C) requires that the sequences be essentially completely homologous. Conditions of intermediate stringency (such as, for example, 2X SSC at 65 ° C) and low stringency (such as, for example 2X SSC at 55° C), require correspondingly less overall complementarity between the hybridizing sequences. (IX SSC is 0.15 M NaCl, 0.015 M Na citrate). A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. The terms peptides, proteins, and polypeptides are used interchangeably herein. -

As used herein, the term "surface protein" refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.

A polypeptide has H pylori biological activity if it has one, two and preferably more of the following properties: ( 1 ) if when expressed in the course of an H pylori infection, it can promote, or mediate the attachment of H pylori to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an H pylori protein; (3) the gene which encodes it can rescue a lethal mutation in an H pylori gene; (4) or it is immunogenic in a subject. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.

A biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the H. pylori polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring H pylori polypeptides, e.g., one or more of the biological activities described herein. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in CΗO cells. Because peptides such as H pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H pylori fragment or H. pylori analog is one which exhibits a biological activity in any biological assay for H. pylori activity. Most preferably the fragment or analog possesses 10%, preferably 40%, more preferably 60%, 70%, 80%) or 90%) or greater of the activity of H pylori, in any in vivo or in vitro assay. Analogs can differ from naturally occurring H. pylori polypeptides in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include H pylori polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the H. pylori polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be made in view of the table below.

TABLE 2

CONSERVATIVE AMINO ACID REPLACEMENTS

Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids; and cyclic analogs.

As used herein, the term "fragment", as applied to an H. pylori analog, will - ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of H pylori polypeptides can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of H. pylori polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are H pylori polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.

An "immunogenic component" as used herein is a moiety, such as an H pylori polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal alone or in combination with an adjuvant. An "antigenic component" as used herein is a moiety, such as an H pylori polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.

As used herein, the term "Gram negative bacteria" refers to cocci, nonenteric rods, enteric rods, and spirilla. The genera of gram negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,

Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agribacterium, Azotobacter, Spirrila, Serratia, Rhizobium, Chlamydia, Rickettsia, Trepanema, and Fusobacterium. As used herein, the term "attenuated live bacteria", refers to an avirulent microorganism which has the ability to colonize and replicate in an infected individual, but which does not cause disease symptoms usually associated with virulent strains of the same species of microorganism. Avirulent does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is treated. The microbe may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent. Avirulent strains are incapable of inducing a full suite of symptoms of the disease that is normally associated with its virulent pathogenic counterpart. Avirulent strains of microorganisms may be derived from virulent strains by mutation. The preffered attenuted live bacteria of the invention is selcted from the group consiting of Salmonella, V. cholerae and Shigella. Example of an attenuated Salmonella is a S. typhi or S. typhimurium strain which has a mutation, e.g., a deletion, in at least one of the genes selected from the group consisting of cya, crp and cdt.

As used herein, the term "H. pylori cell envelope polypeptide", refers to both H pylori inner and outer membrane proteins.

As used herein, the term "H. pylori outer membrane polypeptide", refers to a polypeptide which has an N-terminal signal sequence and which is expressed on the outer membrane by H. pylori. As used herein, a "signal sequence" refers to a peptide containing about 20 amino acids which occurs at the extreme N-terminal end of secretory and integral membrane proteins and which contains large numbers of hydrophobic amino acid residues. Such a "signal sequence", also referred to in the art as a "signal peptide", serves to direct a protein containing such a sequence to a lipid bilayer. An outer membrane polypeptide as used herein does not include proteins which are found on the H. pylori surface due to cell death or lysis. Thus, proteins such as urease which is found on the surface of H. pylori due to altruistic lysis are not encompassed in the term outer membrane polypeptide. H pylori outer membrane polypeptides have been further classified into subgroups, e.g., outer membrane proteins having a terminal phenylalanine residue and a C-terminal tyrosine cluster, outer membrane proteins having a terminal phenylalanine residue, and outer membrane proteins having a C-terminal tyrosine cluster, as set forth in Table 1 herein and in Table 1 of each of the following applications the contents of which, including Table 1, are incorporated herein by reference: USSN 08/660,742, filed June 6, 1996, USSN 08/736,791, filed Ocotber 25, 1996, USSN 08/739,150, filed October 28, 1996, USSN 08/759,739, filed December 6, 1996, USSN 08/761,184, filed December 6, 1996, USSN 08/821,931, filed March 21, 1997, USSN 08/625,811, filed March 29, 1996, USSN 08/738,859, filed October 28, 1996, USSN 08/761,318, filed December 6, 1996, USSN 08/824,132, filed March 27, 1997, USSN 08/758,731, filed April 2, 1996, USSN 08/736,905, filed October 25, 1996, USSN 08/769,224, filed December 6, 1996, USSN 08/759,625, filed December 5, 1996, USSN 08/823,745, filed March 25, 1997, USSN 08/761,066, filed December 5, 1996, USSN 08/981,928, filed July 14, 1997, USSN 08/892,020, filed July 14, 1997, USSN 08/959,131, filed Ocotber 28, 1997, and USSN , filed on even date herewith, entitled "Nucleic Acid and Amino Acid

Sequences Relating to Helicobacter Pylori for Diagnostics and Therapeutics", (Attorney Docket No.: GTN-018). As used herein, the term "transgene" means a nucleic acid (encoding, e.g., one or more polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the cell's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence. As used herein, the term "transgenic cell" refers to a cell containing a transgene.

As used herein, a "transgenic animal" is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by a process of transformation of competent cells or by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

The term "antibody" as used herein is intended to include fragments thereof which are specifically reactive with H pylori polypeptides. As used herein, the term "cell-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.

Misexpression, as used herein, refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post- transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

As used herein, "host cells" and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.

As used herein, the term "control sequence" refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.

As used herein, the term "operably linked" refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.

The metabolism of a substance, as used herein, means any aspect of the, expression, function, action, or regulation of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance. The metabolism of a substance includes modifications, e.g., covalent or non- covalent modification, the substance induces in other substances. The metabolism of a substance also includes changes in the distribution of the substance. The metabolism of a substance includes changes the substance induces in the distribution of other substances.

A "sample" as used herein refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.

The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); the series, Methods in Enzymoloqy (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and

Grossman, eds.) and PCR-A Practical Approach (McPherson, Quirke, and Taylor, eds.,

1991).

I. Isolation of Nucleic Acids of//, pylori and Uses Therefor

H. pylori Genomic Sequence This invention provides nucleotide sequences of the genome of H. pylori which thus comprises a DNA sequence library of H. pylori genomic DNA. The detailed description that follows provides nucleotide sequences of H. pylori, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are methods of using the disclosed H pylori sequences in methods including diagnostic and therapeutic applications. Furthermore, the library can be used as a database for identification and comparison of medically important sequences in this and other strains of H. pylori.

To determine the genomic sequence of H. pylori, DNA was isolated from a strain of H pylori (ATCC # 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) and mechanically sheared by nebulization to a median size of 2 kb. Following size fractionation by gel electrophoresis, the fragments were blunt-ended, ligated to adapter oligonucleotides, and cloned into each of 20 different pMPX vectors (Rice et al., abstracts of Meeting of Genome Mapping and Sequencing, Cold Spring Harbor, NY, 5/11-5/15, 1994, p. 225) to construct a series of "shotgun" subclone libraries.

DNA sequencing was achieved using multiplex sequencing procedures essentially as disclosed in Church et al., 1988, Science 240:185; U.S. Patents No. 4,942,124 and 5,149,625). DNA was extracted from pooled cultures and subjected to chemical or enzymatic sequencing. Sequencing reactions were resolved by electrophoresis, and the products were transferred and covalently bound to nylon membranes. Finally, the membranes were sequentially hybridized with a series of labelled oligonucleotides complimentary to "tag" sequences present in the different shotgun cloning vectors. In this manner, a large number of sequences could be obtained from a single set of sequencing reactions. The cloning and sequencing procedures are described in more detail in the Exemplification.

Individual sequence reads obtained in this manner were assembled using the FALCON™ program (Church et al. , 1994, Automated DNA Sequencing and Analysis, J.C. Venter, ed., Academic Press) and PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, Jan. 1996, p.157). The average contig length was about 3-4 kb.

A variety of approaches are used to order the contigs so as to obtain a continuous sequence representing the entire H. pylori genome. Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of H. pylori genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined.

The H. pylori sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides. As a result of the analysis of ORFs based on stop-to-stop codon reads, it should be understood that these ORFs may not correspond to the ORF of a naturally-occurring H. pylori polypeptide. These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally- occurring H. pylori polypeptide. Such start codons within the ORFs provided herein can be identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded H. pylori polypeptide is within the scope of this invention. For example, within the ORFs a codon such as AUG or GUG (encoding methionine or valine) which is part of the initiation signal for protein synthesis can be identified and the ORF modified to correspond to a naturally-occurring H. pylori polypeptide. The predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARK™ (Borodovsky and Mclninch, 1993, Comp. Chem. 17:123). Other H. pylori Nucleic Acids

The nucleic acids of this invention may be obtained directly from the DNA of the above referenced H. pylori strain by using the polymerase chain reaction (PCR). See "PCR, A Practical Approach" (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products can be checked by conventional sequencing methods. Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY). It is also possible to obtain nucleic acids encoding H. pylori polypeptides from a cDNA library in accordance with protocols herein described. A cDNA encoding an H. pylori polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques. Genes encoding H. pylori polypeptides can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. Preferred nucleic acids of the invention are contained in the Sequence Listing. The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et ak U.S. Patent No. 4,598,049; Caruthers et a U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

Nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands, antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences. As probes, primers, capture ligands and antisense agents, the nucleic acid normally consists of all or part

(approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below. Probes A nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect H. pylori. With the sequence information set forth in the present application, sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to H. pylori, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques. Individuals skilled in the art will readily recognize that the nucleic acids, for use as probes, can be provided with a label to facilitate detection of a hybridization product. Nucleic acid isolated and synthesized in accordance with the sequence of the invention contained in the Sequence Listing can also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using appropriate stringency hybridization conditions as described herein. Capture Ligand For use as a capture ligand, the nucleic acid selected in the manner described above with respect to probes, can be readily associated with a support. The manner in which nucleic acid is associated with supports is well known. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate H. pylori nucleic acid from the nucleic acid of each other and other organisms. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Helicobacter species from each other and from other organisms. Preferably, the sequence will comprise at least twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques. Primers

Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of H pylori nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Helicobacter species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of > 10-15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of H pylori nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods. The copies can be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species. The copies can also be incorporated into cloning and expression vectors to generate polypeptides corresponding to the nucleic acid synthesized by PCR, as is described in greater detail herein.

Antisense

Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of H pylori genes. These sequences also have utility as antisense agents to prevent expression of genes of other Helicobacter species.

In one embodiment, nucleic acid or derivatives corresponding to H. pylori nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells. For example, a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA. Preferably, the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA. Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques. Methods for loading antisense nucleic acid in liposomes is known in the art as exemplified by U.S. Patent 4,241,046 issued December 23, 1980 to Papahadjopoulos et al. II. Expression of H. pylori Nucleic Acids Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides. The nucleic acid of the invention exemplified in the Sequence Listing or fragments of said nucleic acid encoding active portions of H pylori polypeptides can be cloned into suitable vectors or used to isolate nucleic acid. The isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.

The function of a specific gene or operon can be ascertained by expression in a bacterial strain under conditions where the activity of the gene product(s) specified by the gene or operon in question can be specifically measured. Alternatively, a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to other Helicobacter strains, or other bacterial strains such as E. coli, Norcardia, Corynebacterium, Campylobacter, and Streptomyces species. In some cases the expression host will utilize the natural Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta-galactosidase promoter for expression in E. coli). To express a gene product using the natural H pylori promoter, a procedure such as the following can be used. A restriction fragment containing the gene of interest, together with its associated natural promoter element and regulatory sequences (identified using the DNA sequence data) is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together. The recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.

In the case of a gene that requires a different promoter, the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid. This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning. A suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell. For example, an H pylori polypeptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CΗO). Other suitable host cells are known to those skilled in the art.

Expression in eucaryotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of a recombinant peptide product. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Ηerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:3 \ -39). Generally, COS cells (Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci. USA 84^*8573-8577) for transient amplification/expression in mammalian cells, while CΗO (dhfr Chinese Hamster Ovary) cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195) for stable amplification/expression in mammalian cells. Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

Expression in procaryotes is most often carried out in E. coli with either fusion or non-fusion inducible expression vectors. Fusion vectors usually add a number of NH2 terminal amino acids to the expressed target gene. These NH2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein. A preferred reporter group is poly(His), which may be fused to the amino or carboxy terminus of the protein and which renders the recombinant fusion protein easily purifiable by metal chelate chromatography.

Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pETl ld (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 λ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.

For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides.

Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.

In the case of membrane bound proteins, these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be chromatographically isolated from the membrane fraction. Several different criteria are used for choosing a detergent suitable for solubilizing these complexes. For example, one property considered is the ability of the detergent to solubilize the H. pylori protein within the membrane fraction at minimal denaturation of the membrane- associated protein allowing for the activity or functionality of the membrane-associated protein to return upon reconstitution of the protein. Another property considered when selecting the detergent is the critical micelle concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution. A third property considered when selecting a detergent is the hydrophobicity of the detergent. Typically, membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g., the triton series, would be useful for solubilizing the hydrophobic proteins. Another property important to a detergent can be the capability of the detergent to remove the H pylori protein with minimal protein-protein interaction facilitating further purification. A fifth property of the detergent which should be considered is the charge of the detergent. For example, if it is desired to use ion exchange resins in the purification process then preferably detergent should be an uncharged detergent. Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.

One strategy to maximize recombinant H. pylori peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy would be to alter the nucleic acid encoding an H. pylori peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein). III. H. pylori Polypeptides

This invention encompasses isolated H. pylori polypeptides encoded by the disclosed H pylori genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an H pylori polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region. This can be acheived, for example, by using the isolated nucleic acid encoding the ORF, or fragments thereof, to prime a polymerase chain reaction with genomic H. pylori DNA as template; this is followed by sequencing the amplified product.

The polypeptides of the invention can be isolated from wild-type or mutant H. pylori cells or from heterologous organisms or cells (including, but not limited to, bacteria, yeast, insect, plant and mammalian cells) into which an H pylori nucleic acid has been introduced and expressed. In addition, the polypeptides can be part of recombinant fusion proteins.

H pylori polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein. IV. Identification of Nucleic Acids Encoding Vaccine Components and Targets for Agents Effective Against H. pylori

The disclosed H. pylori genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids. The invention encompasses nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H. pylori. Identification of said immunogenic components involved in the determination of the function of the disclosed sequences can be achieved using a variety of approaches. Non-limiting examples of these approaches are described briefly below.

Homology to known sequences: Computer-assisted comparison of the disclosed H. pylori sequences with previously reported sequences present in publicly available databases is useful for identifying functional H. pylori nucleic acid and polypeptide sequences. It will be understood that protein-coding sequences, for example, may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80-90%) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc. In addition, many structural features of particular protein classes have been identified and correlate with specific consensus sequences, such as, for example, binding domains for nucleotides, DNA, metal ions, and other small molecules; sites for covalent modifications such as phosphorylation, acylation, and the like; sites of protein :protein interactions, etc. These consensus sequences may be quite short and thus may represent only a fraction of the entire protein-coding sequence. Identification of such a feature in an H. pylori sequence is therefore useful in determining the function of the encoded protein and identifying useful targets of antibacterial drugs. Of particular relevance to the present invention are structural features that are common to secretory, transmembrane, and surface proteins, including secretion signal peptides and hydrophobic transmembrane domains. H. pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines. Identification of essential genes: Nucleic acids that encode proteins essential for growth or viability of H. pylori are preferred drug targets. H. pylori genes can be tested for their biological relevance to the organism by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene "knockout", using techniques known to those skilled in the relevant art. In this manner, essential genes may be identified. Strain-specific sequences: Because of the evolutionary relationship between different H. pylori strains, it is believed that the presently disclosed H. pylori sequences are useful for identifying, and/or discriminating between, previously known and new H. pylori strains. It is believed that other H. pylori strains will exhibit at least 70% sequence homology with the presently disclosed sequence. Systematic and routine analyses of DNA sequences derived from samples containing H. pylori strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all H. pylori strains. In one embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of H. pylori. Strain-specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more H pylori strains. In another embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all H. pylori strains but are not found in other bacterial species.

Specific Example: Determination Of Candidate Protein Antigens For Antibody And Vaccine Development The selection of candidate protein antigens for vaccine development can be derived from the nucleic acids encoding H pylori polypeptides. First, the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985) Biochimica et Biophysica Acta 815, 468-476) for predicting exported and membrane proteins.

Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases. BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database. ORF's with significant homology (e.g. probabilities lower than lxlO"⁶ that the homology is only due to random chance) to membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to H. pylori genes based on sequence homology to genes cloned in other organisms.

Discriminant analysis (Klein, et al. supra) can be used to examine the ORF amino acid sequences. This algorithm uses the intrinsic information contained in the ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.

Surface exposed outer membrane proteins are likely to represent the best antigens to provide a protective immune response against H. pylori. Among the algorithms that can be used to aid in prediction of these outer membrane proteins include the presence of an amphipathic beta-sheet region at their C-terminus. This region which has been detected in a large number of outer membrane proteins in Gram negative bacteria is often characterized by hydrophobic residues (Phe or Tyr) clustered at alternating positions from the C-terminus. Importantly, these sequences have not been detected at the C-termini of periplasmic proteins, thus allowing preliminary distinction between these classes of proteins based on primary sequence data. This phenomenon has been reported previously by Stray ve et al. (J. Mol. Biol. 218:141-148, 1991).

Additional amino acid sequence motifs are found in many outer membrane proteins of H pylori. Five or six distinct blocks of similar amino acid residues are found in these motifs including the distinctive hydrophobic residues (Phe or Tyr) frequently found at positions near the C-terminus of outer membrane proteins. The presence of several shared motifs clearly establishes the similarity between members of this group of proteins.

One skilled in the art would know that these shared sequence motifs are highly significant and establish a similarity among this group of proteins.

Infrequently it is not possible to distinguish between multiple possible nucleotides at a given position in the nucleic acid sequence. In those cases the ambiguities are denoted by an extended alphabet as follows:

These are the official IUPAC-IUB single-letter base codes

Code Base Description

G Guanine

A Adenine

T Thymine

C Cytosine

R Purine (A or G)

Y Pyrimidine (C or T or U)

M Amino (A or C)

K Ketone (G or T)

S Strong interaction (C or G) w Weak interaction (A or T)

Η Not-G (A or C or T)

B Not-A (C or G or T)

V Not-T (not-U) (A or C or G)

D Not-C (A or G or T)

N Any (A or C or G or T) The amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code. V. Production of Fragments and Analogs of H. pylori Nucleic Acids and Polypeptides

Based on the discovery of the H pylori gene products of the invention provided in the Sequence Lsiting, one skilled in the art can alter the disclosed structure (of H pylori genes), e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind H pylori polypeptides. Such screens are useful for the identification of inhibitors of H pylori. Generation of Fragments

Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

Alteration of Nucleic Acids and Polypeptides: Random Methods

Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein). (A) PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1 :11- 15). The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.

(B) Saturation Mutagenesis Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.

(C) Degenerate Oligonucleotides A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429- 2433; Devlin et al. (1990) Science 249: 404-406; Cwiria et al. (1990) PNAS 87: 6378- 6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815). Alteration of Nucleic Acids and Polypeptides: Methods for Directed Mutagenesis

Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

(A) Alanine Scanning Mutagenesis Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.

(B) Oligonucleotide-Mediated Mutagenesis Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]). (C) Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315[1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.

(D) Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630). In this method, the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences. Other Modifications of , pylori Nucleic Acids and Polypeptides

It is possible to modify the structure of an H. pylori polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). A modified H pylori protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.

An H. pylori peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages. In addition, amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.

In order to enhance stability and/or reactivity, an H. pylori polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an H. pylori polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of H pylori proteins include reduction/alkylation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch, of Allergy and Appl. Immunol, 41 : 199 - 215).

To facilitate purification and potentially increase solubility of an H. pylori protein or peptide, it is possible to add an amino acid fusion moiety to the peptide backbone. For example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325). In addition, to facilitate isolation of peptides free of irrelevant sequences, specific endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide. To potentially aid proper antigen processing of epitopes within an H. pylori polypeptide, canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods. For example, charged amino acid pairs, such as KK or RR, can be introduced between regions within a protein or fragment during recombinant construction thereof. The resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes. In addition, such charged amino acid residues can result in an increase in the solubility of the peptide. Primary Methods for Screening Polypeptides and Analogs Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to H pylori polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.

(A) Two Hybrid Systems

Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring H. pylori polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an H. pylori protein. (The H. pylori domain is used as the bait protein and the library of variants are expressed as fish fusion proteins.) In an analogous fashion, a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind a H. pylori polypeptide.

(B) Display Libraries

In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371 ; and Goward et al. (1992) 7755 18:136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologs which retain ligand- binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 10 3 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2- terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).

A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 and Klauser et al. (1990) EMBOJ. 9, 1991- 1999).

In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface.

Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3 '-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869)

This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage- displayed peptides are presented right at the amino terminus of the fusion protein. (Cwiria, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The Lad fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9): 1233-1251). These particular biases are not a factor in the Lad display system.

The number of small peptides available in recombinant random libraries is enormous. Libraries of 10 '-lθ" independent clones are routinely prepared. Libraries as large as 10^ recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251), a molecular DNA library encoding 10^2 decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ELISA, or for binding specificity in a completion phage ELISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host. Secondary Screening of Polypeptides and Analogs

The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above. Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine for one skilled in the art to obtain analogs and fragments. Peptide Mimetics of H. pylori Polypeptides

The invention also provides for reduction of the protein binding domains of the subject H. pylori polypeptides to generate mimetics, e.g. peptide or non-peptide agents. The peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an H. pylori polypeptide binding to a naturally occurring ligand. The critical residues of a subject H. pylori polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate H. pylori-devived peptidomimetics which competitively or noncompetitively inhibit binding of the H. pylori polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31 ,080A).

For example, scanning mutagenesis can be used to map the amino acid residues of a particular H. pylori polypeptide involved in binding an interacting polypeptide, peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) can be generated which mimic those residues in binding to an interacting polypeptide, and which therefore can inhibit binding of an H. pylori polypeptide to an interacting polypeptide and thereby interfere with the function of H pylori polypeptide. For instance, non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,

Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto- methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1 :1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:4l9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71). VI. Vaccine Formulations for H. pylori Nucleic Acids and Polypeptides This invention also features vaccine compositions or formulations (used interchangeably herein) for protection against infection by H. pylori or for treatment of H. pylori infection. As used herein, the term "treatment of H pylori infection" refers to therapeutic treatment of an existing or established H. pylori infection. The terms "protection against H pylori infection" or "prophylactic treatment" refer to the use of H. pylori vaccine formulations for reducing the risk of or preventing an infection in a subject at risk of H. pylori infection. In one embodiment, the vaccine compositions contain one or more immunogenic components, such as a surface protein, from H. pylori, or portion thereof, and a pharmaceutically acceptable carrier. For example, in one embodiment, the vaccine formulations of the invention contain at least one or combination of H pylori polypeptides or fragments thereof, from same or different H. pylori antigens. Nucleic acids and H. pylori polypeptides for use in the vaccine formulations of the invention include the nucleic acids and polypeptides set forth in the Sequence Listing, preferably those H. pylori nucleic acids that encode surface proteins and surface proteins or fragments thereof. For example, a preferred nucleic acid and H pylori polypeptide for use in a vaccine composition of the invention is selected from the group of nucleic acids which encode cell envelope proteins and H pylori cell envelope proteins as set forth in Table 1. However, any nucleic acid encoding an immunogenic H. pylori protein and H. pylori polypetide, or portion thereof, can be used in the present invention. These vaccines have therapeutic and/or prophylactic utilities.

One aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains at least one immunogenic fragment of an H. pylori protein and a pharmaceutically acceptable carrier. Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10-20 amino acid residues in length, and more preferably about 12-16 amino acid residues in length.

Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H. pylori protein. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.

In one embodiment, immunogenic components are identified by the ability of the peptide to stimulate T cells. Peptides which stimulate T cells, as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen/T cell interaction, and activation of the B cell cascade, leading to the production of antibodies. A T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.

In another embodiment, immunogenic components of the invention are identified through genomic vaccination. The basic protocol is based on the idea that expression libraries consisting of all or parts of a pathogen genome, e.g., an H pylori genome, can confer protection when used to genetically immunize a host. This expression library immunization (ELI) is analogous to expression cloning and involves reducing a genomic expression library of a pathogen, e.g., H pylori, into plasmids that can act as genetic vaccines. The plasmids can also be designed to encode genetic adjuvants which can dramatically stimulate the humoral response. These genetic adjuvants can be introduced at remote sites and act as well extracelluraly as intracellularly.

This is a new approach to vaccine production that has many of the advantages of live/attenuated pathogens but no risk of infection. An expression library of pathogen DNA is used to immunize a host thereby producing the effects of antigen presentation of a live vaccine without the risk. For example, in the present invention, random fragments from the H. pylori genome or from cosmid or plasmid clones, as well as PCR products from genes identified by genomic sequencing, can be used to immunize a host. The feasibility of this approach has been demonstrated with Mycoplasma pulmonis (Barry et al., Nature 377:632-635, 1995), where even partial expression libraries of Mycoplasma pulmonis, a natural pathogen in rodents, provided protection against challenge from the pathogen.

ELI is a technique that allows for production of a non-infectious multipartite vaccine, even when little is known about pathogen's biology, because ELI uses the immune system to screen candidate genes. Once isolated, these genes can be used as genetic vaccines or for development of recombinant protein vaccines. Thus, ELI allows for production of vaccines in a systematic, largely mechanized fashion.

Screening immunogenic components can be accomplished using one or more of several different assays. For example, in vitro, peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MΗC molecules in a T cell culture. Presentation of an immunogenic H. pylori peptide in association with appropriate MΗC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines. For example, any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incorporated herein by reference. A kit for an assay for the production of interferon is also available from Genzyme Corporation (Cambridge, MA).

Alternatively, a common assay for T cell proliferation entails measuring tritiated thymidine incorporation. The proliferation of T cells can be measured in vitro by determining the amount of -1H-labeled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.

Vaccine compositions or formulations of the invention containing one or more immunogenic components (e.g., H. pylori polypeptide or fragment thereof or nucleic acid encoding an H. pylori polypeptide or fragment thereof) preferably include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. 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 H. pylori nucleic acid or polypeptide. For vaccine formulations of the invention containing H. pylori polypeptides, the polypeptide is preferably administered with a suitable adjuvant and/or a delivery system described herein.

It will be apparent to those of skill in the art that the therapeutically effective amount of DNA or protein of this invention will depend, røter alia, upon the administration schedule, the unit dose of an H pylori nucleic acid or polypeptide administered, whether the protein or nucleic acid 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 nucleic acid.

Vaccine formulations are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 91_: 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori. Czinn et. al. (1993) Vaccine 11 : 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. In one embodiment, the vaccine formulation includes, as a pharmaceutically acceptable carrier, an adjuvant. Examples of the suitable adjuvants for use in the vaccine formulations of the invention include, but are not limited, to aluminum hydroxide; N-acetyl-muramyl— L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3- hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion; and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the H. pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial lysates, block polymers or saponins. As used herein, the term adjuvant is also intended to encompass immunomodulators, e.g., cytokines. Such adjuvants can be coadministered with the H. pylori polypeptide or fragment thereof in the vaccine formulation of the invention. Alternatively, a nucleic acid encoding an adjuvant (e.g., a nucleotide sequence encoding a cytokine) can be expressed as described herein in a delivery system which can further include an H. pylori polypeptide or a fragment thereof. Accordingly, in one embodiment, the vaccine formulation includes a delivery system, such as a bacterial vector, which contains and expresses a nucleotide sequence encoding an H. pylori polypeptide or a fragment thereof and a nucleotide sequence encoding an adjuvant. Alternatively, the vaccine formulation can include two or more of the same or different delivery systems, such as two or more of the same or different bacterial vectors, one of which contains and expresses an H. pylori polypeptide or a fragment thereof and another of which contains and expresses an adjuvant. A nucleic acid encoding an adjuvant can be introduced into a delivery system, such as a bacterial vector, e.g., a species of Salmonella, V. cholera or Shigella, using recombinant techniques known in the art. For example, the nucleic acid encoding the adjuvant can be introduced into a same or a different bacterial vector from the one expressing the H pylori polypeptide or fragment thereof. If the adjuvant is expressed from a different bacterial vector, the vectors can be combined to produce a vaccine formulation of the invention. In another embodiment, the vaccine formulation includes, as a pharmaceutically acceptable carrier, a delivery system. Suitable delivery systems for use in the vaccine formulations of the invention 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. A number of such live recombinant viral vaccine systems are known, such as the Vaccinia virus system (US Patent 4,603,112; Brochier et al., (1991) Nature 354: 520). Examples of attenuated live bacterial systems include, but are not limited to, attenuated species of Salmonella , Vibrio Cholerae and Shigella live vectors. In another embodiment of the invention, the vaccine formulation includes both a delivery system and an adjuvant.

Delivery systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H pylori polypeptide in an insoluble form as fusion proteins. Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres. Suitable diluents are 0.2 N NaΗCO3 and/or saline.

Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of H. pylori in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by H. pylori. The vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art. Thus, for adults a suitable dosage will be in the range of 10 μg to 10 g, preferably 10 μg to 100 mg, for example 50 μg to 50 mg. A suitable dosage for adults will also be in the range of 5 μg to 500 mg. Similar dosage ranges will be applicable for children.

The amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 μg to 50 μg, for example 10 μg to 35 μg. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.

Those skilled in the art will recognize that the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors. Those skilled in the art will also recognize that appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol. 150:917-921 (1993)); Boedecker et al., American Gastroenterological Assoc. 999:A-222 (1993)). The number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888: A-222 (1993)).

In a preferred embodiment, a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.

It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing H. pylori infection, some are useful only for treating H. pylori infection, and some are useful for both preventing and treating H. pylori infection. In a preferred embodiment, the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H. pylori. It should be understood that amelioration of any of the symptoms of H pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. τ /or/^'-caused disease, or an increase in the production of antibodies in the serum or mucous of patients. Live Attenuated Bacterial Vector Systems

As an illustrative example, "new generation" Salmonella mutants have been identified, which are attenuated and immunogenic in both human and animal models. Of interest are strains harboring mutations in genes whose products are involved in key biosynthetic pathways (aro mutants), regulatory genes (cya-crp and phoP-phoQ) and genes encoding stress proteins (htrA) and combinations of these mutations (Ηoiseth et al. (1981) Nature 291 : 238-240; Dougan et al. (1988) J Infect Dis 158: 1329-1335; Miller et al. (1989) Mol Gen Gennet 215: 312-316; Roberts et al. (1993) Biologicals 2k 4-5; Curtiss et al. (1987) Infect Immun 55: 3035-3043; Galan et al. (1989) Microb Pathog 6: 433-443; Pulkkiner et al. (1991) J Bacteriol 173: 86-93; Johnson et al. (1991) Mol Microbiol 5: 401-407; Chatfield et al. (1992) Microb Pathog 12: 145-151).

Two classes of Salmonella (e.g., S. typhi or S. typhimurium) mutants that have been extensively employed as vectors in animal models are the auxotrophic strains (aro mutants), harboring defined mutations in genes encoding enzymes in the prechorismate pathway, and strains having mutations in the adenylate cyclase and the cAMP genes (cya, crp mutants). Strains harboring mutations in aro A., aroC, and aroD and combinations thereof have been demonstrated to be effective vaccines in several animal species (Dougan et al. (1988) J. Infect Dis L58: 1389-1335; Miller et al. (1989) Mol Gen Genet 215: 312-316; Roberts et al. (1993) Biologicals 2±: 4-5; Jones et al. (1990) Vaccine 9: 29-36; Cooper et al. (1992) Vaccine Η): 247-254). The cya crp Salmonella mutants have also been shown to orally vaccinate against wild-type infection. These two genes affect the expression of a large number of genes, particularly those involved in carbohydrate and amino acid metabolism.

The expression of a heterologous antigen in Salmonella can be accomplished using techniques known in the art for expression of heterologous DNA in Escherichia coli, given the similarities between these organisms. Moreover, many of the conventional expression vectors used in E. coli also perform well in Salmonella and well developed technologies, such as electroporation, can be used to transfer recombinant DNA constructs into Salmonella (see, also, e.g., US 5,387,744; US 5,294,441 ; US 5,389,368; EP 0315682; WO 91/06317; and WO 92/08486, which are incorporated herein by reference).

Like Salmonella, Vibrio cholerae has many features that make it useful as a delivery vehicle for H. pylori polypeptides or fragments thereof. For example, V. cholerae has the advantage of being noncommensal and noninvasive and is capable of inducing mucosal and systemic humoral immunity (Levine et al. (1981) J Infect Dis 143: 818-820). V. cholerae attaches selectively to intestinal M cells, resulting in direct antigenic presentation to underlying lymphoid tissue, thereby invoking a strong mucosal immune response (Owen et al. ( 1986) J infect Dis 153: 1108-1118; Svennerholm et al (1982) Lancet \ : 305-308; Czerkinsky et al. (1991) Infect Immun 59: 996-1001). In addition, V. cholerae colonizes human intestinal surfaces for 7-14 days and thus allows for sustained antigenic presentation after a single oral administration. Furthermore, based on the knowledge of cholera pathogenesis, live oral attenuated V. cholerae vaccines have been developed and shown to be safe and effective. For example, attenuated strains of V. cholerae have been developed as vaccines by deletion of internal fragments of a cholera toxin gene ctxA (CVD103-ΗgR (Levine (1988) Lancet 2: 467- 470); 0395-N1 (Mekalanos et al. (1983) Nature 306: 551-557)). More recently, several different El Tor Ol and 0139 Bengal strains have been developed as cholera vaccine strains by deletion of the entire bacteriophage-encoded cholera toxin (CTX) genetic element and flanking attRSl sites, a 17-bp "end-repeat" (ER) sequence that share 17 out of 18 bp with the chromosomal insertion site of the CTX genetic element on the V. cholerae chromosome (Taylor et al. (1994) J Infect Dis 170: 1518-1523; Waldor et al. (1994) J Infect Dis 170: 278-283; Coster et al. (1995) Lancet 345: 949-952).

It is generally desirable to employ a vector strain that expresses heterologous antigens having the ability to insert into the host chromosome, and genetic systems have been developed in V. cholerae for introducing stable, chromosomal insertions of heterologous antigens by in vivo marker exchange systems. V. cholerae has the further advantage of having a large genomic capacity (approximately 4 megabase pairs) that will accommodate a large amount of heterologous DNA. In addition, several active promoters have been identified in V. cholerae, including a number that are active in vivo, facilitating the expression of heterologous antigens after inoculation. In fact, V. cholerae has been shown to be capable of secreting heterologous antigens fused to B subunit of cholera toxin (CtxB) and to the homologous B subunit of Escherichia coli heat-labile enterotoxin (LTB) (Backstrom et al. (1994) Gene 149: 21 1-217; Schodel et al. (1991) Gene 99: 255-259). In addition, to assess the immunological response to heterologous antigens delivered by V. cholerae vectors, there are several animal models available which are predictive of efficacy in humans (see, also, e.g., US 5,628,994; US 5,399,494; WO 95/10300; EP 0581329 and EP 0119031, which are incorporated herein by reference).

Like Salmonella and Vibrio cholerae, Shigella (e.g., S.flexneri or S. dysenteriae) has many features that make it useful as a delivery vehicle for H pylori polypeptides or fragments thereof. For example, Shigella is a useful biological delivery system due to its ability to invade cells, escape from the phagosome, and enter into the cytoplasm of eucaryotic cells. Based on the knowledge of Shigella pathogenesis, live oral attenuated Shigella vaccines have been developed and shown to be safe and effective. For example, attenuated strains of Shigella have been developed as vaccines by deleting the gene encoding aspartate b-semialdehyde dehydrogenase (ASD), an essential enzyme required for synthesizing the bacterial cell wall constituent diaminopimelic acid (DAP). This mutant strain retains the ability to enter mammalian cells, but once inside the cell, but is unable to replicate due to the absence of DAP, which is unavailable for scavenge from mammalian cells. As a result, the bacteria dies, releasing its contents including intact DNA and antigens already present in the bacteria. For example, the Shigella flexneri 2a strain 2457T has a mutation which results from the integration of a deleted E. coli asd gene containing a 553 bp deletion from position 439 to 991 of the structural gene into its chromosome. A kanamycin resistance cassette containing the complete Tn5 kanamycin gene was cloned between the flanking sequences of the mutant asd gene (see, e.g., WO 97/08955, which is incorporated herein by reference).

Preferred Shigella strains have an asd mutation to thereby provide an attenuated Shigella vector. The strain does not need to be virulent, but preferably should have the ability to enter or be taken up by the target cell. The asd mutation will facilitate the destruction of the bacteria once the bacteria is inside the cell. In addition, any gene other than asd can be mutated to have the same effect on the bacteria; namely retain the ability to enter the cell and die once inside the cell or be attenuated to such an extent that clinical symptoms in the host organism are acceptable. Examples of such genes include, but are not limited to, thy A, genes for LPS production, htrA and btr-5, and dut. Methods of Making Attenuated Live Bacterial Vectors

The following techniques apply to attenuation of any bacterial vector, e.g., a species of Salmonella, Vibrio cholerae or Shigella. Salmonella is provided as an illustrative example. Certain mutations can render a microbe avirulent without substantially affecting its immunogenicity. Accordingly, in one embodiment, this invention relates to H pylori microbial vaccines in which the microbe carries a mutation in at least one adenylate cycclase or cAMP genes. Preferably, the microbe has the deletion (open triangle or delta) mutations Δcya and/or Δcrp eliminating the ability to synthesize adenylate cyclase (ATP pyrophoshate lyase (cyclizing) EC 4.6.1.1) and the cyclic AMP receptor protein (CRP), respectively.

Cyclic-3'5'-AMP (cAMP) and the cyclic AMP receptor protein are necessary for the transcription of a large number of genes and operons concerned with the transport and breakdown of a large number of catabolites. Evidence has been provided that demonstrates that systems used for transporting fuel/carbon sources are all under positive control by cAMP, as are several amino acid permeases. In addition to its very important role in catabolism, the cAMP concentration in cells also influences lysogenization by temperate phages, synthesis of fimbriae, synthesis of flagella and synthesis of at least one outer membrane protein. Although cAMP is present in mammalian cells, the concentrations present in macrophages and other cells in which Salmonella can invade and multiply are below the concentration of 0.1 to 1.0 mM cAMP necessary to allow Δcya mutants to exhibit a wild-type phenotype in vitro. Furthermore, the inclusion of the Δcrp mutation would essentially abolish any benefit that could accrue from uptake of c AMP in vitro or in vivo by such Δcya mutants. Introduction of the mutations into cya and crp of S. typhi can be accomplished by use of transposons, to transfer the mutations from other Salmonella strains into S. typhi. Transposons can be added to a bacterial chromosome at many locations. The characteristics of transposon insertion and deletion have been reviewed in Kleckner et al. (1977), J. Mol. Biol. 116:125. For example, the transposon TnlO, which confers resistance to tetracycline (and sensitivity to fusaric acid) can be used to create Δcya and Δcrp mutations in a variety of bacterial species, including, for example, E. coli and S. typhimurium. Methods for the creation and deletion of these mutants in S. typhimurium are described in EPO Pub. No. 315,682, hereby incorporated by reference. Utilizing TnlO, these mutations can be transposed into various isolates of S. typhi, preferably those which are highly pathogenic. Once rendered avirulent by the introduction of the Δcya and/or Δcrp mutations, the microbes can serve as the immunogenic component of a vaccine to induce immunity against the microbe.

In another embodiment of the invention, the S. typhi which are cya mutants and/or crp mutants are further mutated, preferably by a deletion, in a gene adjacent to the crp gene which governs virulence of Salmonella. Mutation in this gene, the cdt gene, diminishes the ability of the bacteria to effectively colonize deep tissues, e.g., the spleen. When a plasmid having the crp⁺ gene is placed in a strain with the Δ(crp-cdt), it retains its avirulence and immunogenicity thus having a phenotype similar to cya and crp mutants. Mutants with the Δ(crp-cdt) mutation containing a crp⁺ gene on a plasmid retain the normal ability to colonize the intestinal tract and GALT, but have a diminished ability to colonize deeper tissues. Mutations in cdt in S. typhi can be either created directly, or can be introduced via transposition from S. typhimurium strains. In addition, the cdt mutation can be created in other strains of Salmonella using techniques known in the art, and phenotypic selection using the characteristics described herein; alternatively, the Δcdt mutation can be transposed from the S. typhimurium into other strains of Salmonella using techniques of transposon mutagenesis which are known in the art.

Furthermore, recombinant DNA techniques can be employed to develop vaccines that do not have any material capable of cross-reacting with vertebrate host antigens or capable to eliciting an autoimmune state.

Methods of preparing organisms, particularly Salmonella, which can function as carrier bacteria are discussed in WO 89/03427 (published Apr. 20, 1989), incorporated herein by reference. Generally, the Salmonella are treated to cause a mutation in a chromosomal gene which encodes an enzyme that is essential for cell survival, wherein this enzyme catalyzes a step in the biosynthesis of an essential cell wall structural component. An extrachromosomal genetic element, for example, a recombinant vector, is introduced into the mutant cell. This genetic element contains a first recombinant gene which encodes an enzyme which is functional replacement for the native enzyme, but the first recombinant gene cannot replace the defective chromosomal gene. The first recombinant gene is structurally linked to a second recombinant gene encoding a desired product, which is to be expressed in the carrier microorganism. Loss of the first recombinant gene causes the cells to lyse when the cells are in an environment where a product due to the expression of the first recombinant gene is absent. A number of genes which encode enzymes essential for cell survival, which catalyze a step in the biosynthesis of an essential cell wall structural component, are known in the art, for e.g., aspartate semialdehyde dehydrogenase (Asd), which is encoded by the asd gene. A method for introducing a deletion mutation in the asd gene of S. typhi utilizing transposon mutagenesis is known in the art. Described in the Examples is the construction of a genetic element which carries the functional replacement for the asd gene, linked to a gene encoding an H pylori antigen which is to be expressed in the avirulent carrier S. typhi.

The creation of cya and/or crp mutants of S. typhi, including those which also contain mutations in cdt, and/or the mutations in carrier microorganisms can also be accomplished using other techniques known in the art. These techniques include for example, standard techniques of mutagenesis and/or the use of recombinant DNA techniques. The desired mutants are then selected on the basis of phenotypic characteristics, some of which are shown in Table 3.

TABLE 3

^Ωphage sensitivity was assayed by soft agar overlay technique or by transduction. S- sensitive; R = resistant.

"Motility determined by stabbing a loopful of a standing overnight culture into media containing 1.0% casein, 0.5% NaC-2, 0.5% Difco agar, 50 μg/mg triphenyltetrazolium chloride indicator agar; incubation at 37° C, and motility recorded at 24 and 48 h. ^cMean Generation Time (min) = determined in Luria broth with aeration (150 rpm New

Brunswick platform shaker) at 37° C.

"minimal inhibitory concentrations (μg/ml) of antibiotics were determined by streaking standing overnight cultures of each strain onto agar containing defined concentration of antibiotics.

VII. Antibodies Reactive With H. pylori Polypeptides

The invention also includes antibodies specifically reactive with the subject H. pylori polypeptide. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by

Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide.

Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the subject H. pylori polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least 95% homologous). In yet a further preferred embodiment of the invention, the anti-H. pylori antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80% percent homologous to a sequence of the invention contained in the Sequence Listing. By "not substantially cross react", it is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no crossreactivity between bacterial and mammalian antigens.

The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with H. pylori polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. The antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-H. pylori portion.

Both monoclonal and polyclonal antibodies (Ab) directed against H. pylori polypeptides or H. pylori polypeptide variants, and antibody fragments such as Fab' and F(ab")2, can be used to block the action of H pylori polypeptide and allow the study of the role of a particular H. pylori polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention. Antibodies which specifically bind H pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H pylori antigens. Anti H. pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure. Likewise, the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of an H pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy. Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens. Another application of anti-H. pylori polypeptide antibodies of the invention is - in the immunological screening of cDNA libraries constructed in expression vectors such as λgtl 1, λgtl8-23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgtl 1 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject H pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H. pylori polypeptide antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.

VIII. Kits Containing Nucleic Acids, Polypeptides or Antibodies of the Invention

The nucleic acid, polypeptides and antibodies of the invention can be combined with other reagents and articles to form kits. Kits for diagnostic purposes typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels. Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like. Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like. Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like. Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular purpose. Kits further can include instructions for use.

IX. Drug Screening Assays Using H. pylori Polypeptides

By making available purified and recombinant H. pylori polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject H. pylori polypeptides, or of their role in intracellular signaling. Such inhibitors or potentiators may be useful as new therapeutic agents to combat H. pylori infections in humans. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the skilled artisan.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free- systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified H. pylori polypeptide.

Screening assays can be constructed in vitro with a purified H pylori polypeptide or fragment thereof, such as an H pylori polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive absorption, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated. A variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the H pylori polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live H pylori cells.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.

EXEMPLIFICATION I. Cloning and Sequencing of H. pylori DNA H. pylori chromosomal DNA was isolated according to a basic DNA protocol outlined in Schleif R.F. and Wensink P.C., Practical Methods in Molecular Biology, p.98, Springer- Verlag, NY., 1981, with minor modifications. Briefly, cells were pelleted, resuspended in TE (10 mM Tris, 1 mM EDTA, pH 7.6) and GES lysis buffer (5.1 M guanidium thiocyanate, 0.1 M EDTA, pH 8.0, 0.5% N-laurylsarcosine) was added. Suspension was chilled and ammonium acetate (NH₄Ac) was added to final concentration of 2.0 M. DNA was extracted, first with chloroform, then with phenol- chloroform, and reextracted with chloroform. DNA was precipitated with isopropanol, - washed twice with 70%> EtOH, dried and resuspended in TE.

Following isolation whole genomic H. pylori DNA was nebulized (Bodenteich et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994) to a median size of 2000 bp. After nebulization, the DNA was concentrated and separated on a standard 1% agarose gel. Several fractions, corresponding to approximate sizes 900-1300 bp, 1300-1700 bp, 1700-2200 bp, 2200-2700 bp, were excised from the gel and purified by the GeneClean procedure (BiolOl, Inc.).

The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The healed DNA was then ligated to unique BstXI-linker adapters in 100-1000 fold molar excess. These linkers are complimentary to the BstXI-cut pMPX vectors, while the overhang is not self-complimentary. Therefore, the linkers will not concatemerize nor will the cut- vector religate itself easily. The linker-adopted inserts were separated from the unincorporated linkers on a 1% agarose gel and purified using GeneClean. The linker-adopted inserts were then ligated to each of the 20 pMPX vectors to construct a series of "shotgun" subclone libraries. The vectors contain an out-of-frame lacZ gene at the cloning site which becomes in-frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.

All subsequent steps were based on the multiplex DNA sequencing protocols outlined in Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988. Only major modifications to the protocols are highlighted. Briefly, each of the 20 vectors was then transformed into DH5α competent cells (Gibco/BRL, DH5α transformation protocol). The libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C. DNA was purified using the Qiagen Midi-prep kits and Tip-100 columns (Qiagen, Inc.). In this manner, 100 μg of DNA was obtained per pool. Fifteen 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy assuming 250-300 base average read-lengths. These purified DNA samples were then sequenced using the multiplex DNA sequencing based on chemical degradation methods (Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988) or by Sequithrem (Epicenter Technologies) dideoxy sequencing protocols. The sequencing reactions were electrophoresed and transferred onto nylon membranes by direct transfer electrophoresis from 40 cm gels (Richterich P. and Church G.M., Methods in Enzymology 218:187-222, 1993) or by electroblotting (Church, supra). 24 samples were run per gel. 45 successful membranes were produced by chemical sequencing and 8 were produced by dideoxy sequencing. The DNA was covalently bound to the membranes by exposure to ultraviolet light, and hybridized with labeled oligonucleotides complimentary to tag sequences on the vectors (Church, supra). The membranes were washed to rinse off non-specifically bound probe, and exposed to X-ray film to visualize individual sequence ladders. After autoradiography, the hybridized probe was removed by incubation at 65° C, and the hybridization cycle repeated with another tag sequence until the membrane had been probed 38 times for chemical sequencing membranes and 10 times for the dideoxy sequencing membranes. Thus, each gel produced a large number of films, each containing new sequencing information. Whenever a new blot was processed, it was initially probed for an internal standard sequence added to each of the pools.

Digital images of the films were generated using a laser-scanning densitometer (Molecular Dynamics, Sunnyvale, CA). The digitized images were processed on computer workstations (VaxStation 4000's) using the program REPLICA™ (Church et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994). Image processing included lane straightening, contrast adjustment to smooth out intensity differences, and resolution enhancement by iterative gaussian deconvolution. The sequences were then automatically picked in REPLICA™ and displayed for interactive proofreading before being stored in a project database. The proofreading was accomplished by a quick visual scan of the film image followed by mouse clicks on the bands of the displayed image to modify the base calls. Many of the sequence errors could be detected and corrected because multiple sequence reads covering the same portion of the genomic DNA provide adequate sequence redundancy for editing. Each sequence automatically received an identification number (corresponding to microtiter plate, probe information, and lane set number). This number serves as a permanent identifier of the sequence so it is always possible to identify the original of any particular sequence without recourse to a specialized database.

Routine assembly of H pylori sequences was done using the program FALCON (Church, Church et al., Automated DNA Sequenicng and Analysis (J.C. Venter, ed.), Academic Press, 1994). This program has proven to be fast and reliable for most sequences. The assembled contigs were displayed using a modified version of

GelAssemble, developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICA™. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICA™ database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly. II. Identification, cloning and expression of recombinant H. pylori DNA sequences To facilitate the cloning, expression and purification of membrane and secreted proteins from H pylori a powerful gene expression system, the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, was selected. Also, a DNA sequence encoding a peptide tag, the Ηis-Tag, was fused to the 3' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence. The exception to the above was ppiB, a gene cloned for use as a control in the expression studies. In this study, the sequence for H pylori ppiB contains a DNA sequence encoding a Ηis-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein.

PCR Amplification and cloning of DNA sequences containing ORF's for membrane and secreted proteins from the J99 Strain of Helicobacter pylori.

Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers (Table 4) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an Ncol cloning site at the extreme 5' terminus, except for ΗpSeq. 4821082 where Ndel was used. These primers were designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native H. pylori DNA sequence. An exception is H pylori sequence 4821082 where the initiator methionine is immediately followed by the remainder of the native H. pylori DNA sequence. All reverse primers (specific for the 3' end of any H. pylori ORF) included a EcoRI site at the extreme 5' terminus to permit cloning of each H. pylori sequence into the reading frame of the pET-28b. The pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids (only 19 amino acids in ΗpSeq. 26380318 and ΗpSeq.14640637) including six histidine residues (at the extreme C-terminus), which comprise the Ηis-Tag. An exception to the above, as noted earlier, is the vector construction for the ppiB gene. A synthetic oligonucleotide primer specific for the 5' end of ppiB gene encoded a BamHI site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encoded a Xhol site at its extreme 5' terminus.

TABLE 4 Oligonucleotide primers used for PCR amplification of H. pylori DNA sequences

Genomic DNA prepared from the J99 strain of H pylori (ATCC #55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler: Protein 26054702, Protein 7116626, Protein 29479681, Protein 30100332, and Protein 4821082; Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes. Protein 16225006;

Denaturation at 94°C for 2 min,

25 cycles at 95°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reaction was concluded at 72°C for 6 minutes. Protein 4721061;

Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 36°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 60°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes. Protein 26380318;

Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 38°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 62°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes. Protein 14640637;

Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 33°C for 15 sec and 72°C for 1.5 min 30 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes. Conditions for amplification of H. pylori ppiB; Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 32°C for 15 sec and 72°C for 1.5 min 25 cycles at 94°C for 15 sec, 56°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, Ncol and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of HpSeq. 4821082 (SEQ ID NO: 1309), with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples were then subjected to electrophoresis on 1.0 %> NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).

Cloning ofH. pylori DNA sequences into the pET-28b prokaryotic expression vector. The pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case ofH pylori protein 4821082 with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). In the case of cloning ppiB, the pET-28a vector, which encodes a Ηis-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with BamΗI and Xhol restriction endonucleases.

Following digestion, DNA inserts were cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB, which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below. Transformation of competent bacteria with recombinant plasmids

Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H pylori sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation reaction was mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 milliliters SOC medium (0.5%> yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below. Identification of recombinant pET expression plasmids carrying H. pylori sequences Individual BL21 clones transformed with recombinant pET-28b-Η.pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the expression vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Isolation and Preparation of plasmid DNA from BL21 transformants

Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs were picked and incubated in 5 mis of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA). Expression of recombinant H. pylori sequences in E. coli

The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying a T7 promoter and a gene of interest. Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89). To express recombinant H. pylori sequences, 50 nanograms of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). The lacZ gene (beta-galactosidase) was expressed in the pET-System as described for the H. pylori recombinant constructions. Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions. After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification. III. Purification of recombinant proteins from E. coli Analytical Methods

The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough, N., Fair, A.L. & Randall, R.J. (1951) J. Biol. Chem. 193, pages 265-275, using bovine serum albumin as a standard.

SDS-polyacrylamide gels (12% or 4.0 to 25 % acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.

Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).

/. Purification of soluble proteins All steps were carried out at 4°C. Frozen cells were thawed, resuspended in 5 volumes of lysis buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole with 10% glycerol, 0.1 % 2-mercaptoethanol, 200 μg/ ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each of leupeptin, aprotinin, pepstatin, L-l-chloro-3-[4- tosylamido]-7-amino-2-heptanone (TLCK), L-l -chloro-3-[4-tosylamido]-4-phenyl-2- butanone (TPCK), and soybean trypsin inhibitor, and ruptured by several passages through a small volume microfluidizer (Model M-l 10S, Microfluidics International Corporation, Newton, MA). The resultant homogenate was made 0.1 %> Brij 35, and centrifuged at 100,000 x g for 1 hour to yield a clear supernatant (crude extract).

Following filtration through a 0.8 μm Supor filter (Gelman Sciences, FRG) the crude extract was loaded directly onto a Ni^⁺- nitrilotriacetate-agarose (NT A) with a 5 milliliter bed volume (Hochuli, E., Dbeli, H., and Schacheer, A. (1987) J. Chromatography 411, 177-184) pre-equilibrated in lysis buffer containing 10 % glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 % glycerol, 0.1 % Brij 35, and was eluted with sequential steps of lysis buffer containing 10 % glycerol, 0.05 % Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD2go lun, and peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole. Recombinant protein 14640637 and proteins, beta-galactosidase (lacZ) and peptidyl- prolyl cis-trans isomerase (ppiB)

Fractions containing the recombinant proteins from the Ni2⁺-NTA-agarose columns were pooled and then concentrated to approximately 5 ml by centrifugal filtration (Centriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 91 cm) of Sephacryl S-100 HR gel filtration medium equilibrated in Buffer A (10 mM Hepes, pH 7.5, 150 mM NaCl, 0.1 mM EGTA) and run in Buffer A at 18 ml/h.

Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled and concentrated by centrifugal filtration.

Recombinant protein 7116626 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 MOPS, pH 6.5, 0.1 mM EGTA) containing 50 mM NaCl. The column was washed with 10 bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM). Recombinant protein 7116626 eluted as a sharp peak at 300 mM NaCl. 2. Purification of insoluble proteins from inclusion bodies

The following steps were carried out at 4°C. Cell pellets were resuspended in lysis buffer with 10% glycerol 200 μg/ ml lysozyme, 5 mM EDTA, 1 mM PMSF and 0.1 % -mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was made 0.2 % deoxycholate, stirred 10 minutes, then centrifuged at 20,000 x g, for 30 min. The pellets were washed with lysis buffer containing 10 % glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2- mercaptoethanol. The resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials.. Recombinant proteins 26054702, 16225006, 30100332, 4721061

The following steps were carried out at room temperature. Purified inclusion bodies were dissolved in 20 ml 8.0 M urea in lysis buffer with 1 mM PMSF and 0.1 % 2-mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation. The clear supernatant was filtered, then loaded onto a Ni^⁺ -NTA agarose column pre-equilibrated in 8.0 M urea in Lysis Buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF, 0.1 % 2- mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD28O ^raXi-_> ^an^ P^ea^ fractions were analyzed by SDS- PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole. Recombinant proteins 29479681, 26380318

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. - 70

Dialysis and concentration of protein samples

Urea was removed slowly from the protein samples by dialysis against Tris- buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M, 4M, 3M, 2M, IM, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.

After dialysis, samples were concentrated by pressure filtration using Amicon stirred-cells. Protein concentrations were measured using the methods of Perkins (1986 Eur. J. Biochem. 157, 169-180), Bradford ((1976) Anal. Biochem. 72, 248-254) and

10 Lowry ((1951) J. Biol. Chem. 193, pages 265-275).

The recombinant proteins purified by the methods described above are summarized in Table 5 below.

TABLE 5

71

IV. Analysis of H. pylori proteins as Vaccine candidates

To analyze H. pylori proteins for use in the vaccine formulations of the invention, several H. pylori proteins were expressed, characterized immunologically and 5 tested in animal efficacy studies as outlined below. Specifically, the immunomodulatory effects ofH pylori proteins were investigated in a mouse///, pylori model which mimics the human H. pylori infection in humans. In these studies, the effect of oral immunization of selected H. pylori polypeptides in H. pylori infected mice was determined. Those skilled in the art will recognize that in the examples outlined below,

10 the use of cholera toxin (CT) was only illustrative and is not essential for achieving oral immunization. Identification, cloning and expression of recombinant Helicobacter pylori sequences.

To facilitate the cloning, expression and purification of membrane and/or secreted proteins from H. pylori, the pET gene expression system (Novagen), for cloning

15 and expression of recombinant proteins in Escherichia coli was selected. Further, for proteins that have a signal sequence at their amino-terminal end, a DNA sequence encoding a peptide tag (His-tag) was fused to the 5' end of the H. pylori DNA sequences of interest in order to facilitate purification of the recombinant protein products. PCR amplification and cloning of DNA sequences containing ORFs for membrane

20 and secreted proteins from the J99 strain of Helicobacter pylori

The sequences selected (from the list of the DNA sequences of the invention) for cloning from H. pylori strain J99 were prepared for amplification cloning by the polymerase chain reaction (PCR). All of the selected sequences encode for outer membrane H. pylori proteins, with vac9 (SEQ ID NO: 60), vaclO (SEQ ID NO: 61),

25 vac22 (SEQ ID NO: 71) and vac41 (SEQ ID NO: 77) sequences all sharing a terminal phenylalanine residue. Likewise, the vac32 (SEQ ID NO: 95), vac36 (SEQ ID NO: 96) and vac37 (SEQ ID NO: 97) sequences all share a terminal phenylalanine residue and a tyrosine cluster at the C-terminus. Synthetic oligonucleotide primers for each ORF of interest (Table 6) specific for the predicted mature 5' end of the ORF and downstream (3') of the predicted translational termination codon were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' terminus of the region of ORF of interest) were designed to include a -3-w.III restriction site followed by a Ndel restriction site. These primers were designed to permit the initiation of protein translation at a methionine residue encoding within the Ndel restriction site sequence (in the case of producing a non His-tagged recombinant protein) or to fuse in frame with the DNA sequence encoding the His-tag (for producing His-tagged recombinant protein), followed by the coding sequence for the remainder of the native H. pylori DNA. All reverse oligonucleotide primers (specific for downstream (3') of the predicted translational termination codon of the ORF) were designed to include an EcoRI restriction site at the 5' terminus. This combination of primers would enable each ORF of interest to be cloned into pΕT28b (to produce a His-tagged recombinant protein) or pET30a (to produce a non His-tagged or native recombinant protein). The pET28b vector provides sequence encoding an additional 20 amino- terminal amino acids (plus the methionine in the Ndel restriction site) including a stretch of six histidine residues which makes up the His-tag.

Genomic DNA prepared from H pylori strain J99 (ATCC 55679) was used as the source of template DNA for the PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing a specific H. pylori ORF, genomic DNA (50 nanograms) was introduced into a reaction tube containing 200 nanograms of both the forward and reverse synthetic oligonucleotide primer specific for the ORF of interest, and 45 microliters of PCR SuperMix purchased (GibcoBRL Life Technologies,

Gaithersburg, MD, USA) in a total of 50 microliters. The PCR SuperMix is supplied in 1.1X concentrations and contains 22mM Tris-HCl (pH 8.4), 55mM KCl, 1.65 mM MgCl2, 220 micromolar of each dATP, dCTP, dGTP and dTTP, 22 units recombinant Taq polymerase/ml and stabilizers. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/Gene Amp PCR System thermal cycler. Table 6: Oligonucleotide primers

Sequences for Vac32, Vac9 and Vac22

Denaturation at 94°C for 30 sec

35 cycles at 94°C for 15 sec, 55°C for 15 sec, and 72°C for 1.5 min Reactions were concluded at 72°C for 8 minutes

Sequences for VaclO and Vac41

Denaturation at 94°C for 30 sec

35 cycles at 94°C for 15 sec, 55°C for 15 sec, and 72°C for 2.5 min

Reactions were concluded at 72°C for 8 minutes Sequences for Vac36 and Vac37

Denaturation at

2 cycles at 94°C for 15 sec, 30°C for 15 sec, and 72°C for 1.5 min

23 cycles at 94°C for 15 sec, 55°C for 15 sec, and 72°C for 1.5 min

Reactions were concluded at 72°C for 6 minutes Upon completion of the thermal cycling reactions, each sample of amplified

DNA was subjected to electrophoresis on 1.0% agarose gels. The DNA was visualized by exposure to ethidium bromide and long wave UV irradiation, and cut out in gel slices. DNA was purified using the Wizard PCR Preps Kit (Promega Corp., Madison,

WI, USA), and then subjected to digestion with BamHI and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). The digested PCR amplicon was then re-electrophoresed and purified as before. Ligation ofH. pylori DNA sequences into cloning vectors

The pOK12 vector (J. Vieira and J. Messing, Gene 100:189-194, 1991) was prepared for cloning for digestion with BamHI and EcoRI in the case of Vac9, 10, 22, 31 and 32, whereas the pSU21 vector (B. Bartolome et al., Gene 102:75-78, 1991) was prepared for cloning by digestion with BamHI and EcoRI in the case of Vac 41 (Current . Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). The vectors were subjected to electrophoresis on 1.0% agarose gels and purified using the Wizard PCR Preps kit (Promega Corp., Madison, WI, USA). Following ligation of the purified, digested vector and the purified, digested amplified H pylori ORF, the products of the ligation reaction were transformed into E. coli JM109 competent cells according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Individual bacterial colonies were screened for those containing the correct recombinant plasmids by incubating in LB broth overnight (plus 25ug/ml kanamycin sulfate for the pOK12 based plasmids or 25ug/ml chloramphenicol for the pSU21 based plasmids) followed by plasmid DNA preparation using the Magic Minipreps system (Promega Corp., Madison, WI, USA), and then analyzed by restriction digestion (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Cloning ofH. pylori DNA sequences into thepET28b and pET30a prokary otic expression vectors

Both the pΕT28b and pET30a expression vectors were prepared for cloning by digestion with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994). The H. pylori DNA sequences were removed from pOK12 (Vac9, 10,23,31 and 32) or pSU21 (Vac41) plasmid backbones by digestion with Nt/el and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994). The pΕT28b, pET30a and H. pylori DNA sequences were all electrophoresed on a 1% agarose gel and purified using the Wizard PCR Preps kit (Promega Corp., Madison WI, USA). Following ligation of the purified, digested expression vector and the purified, digest H pylori DNA sequences, the products of the ligation reaction were transformed into E. coli JM109 competent cells (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994). Individual bacterial colonies were screened for those containing the correct recombinant plasmids by preparing plasmid DNA as described above followed by analysis by restriction digestion profiles and DNA sequencing (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994). These recombinant plasmids were then used to transform specific E. coli expression strains. Transformation of competent bacteria with recombinant expression plasmids

Competent bacterial strains (BL21(DE3), BL21(DE3)pLyS, HMS174(DE3) and HMS174(DE3)pLysS were prepared and transformed with the recombinant pET28b 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., eds., 1994). These expression host strains contain a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivate that carries the lad gene, the lacUVS promoter and the gene for T7 RNA polymerase. T7 RNA polymerase expression is induced by the addition of isopropyl-β-D thiogalactoside (IPTG), and the T7 RNA polymerase then transcribes any target plasmid, such as pET28b, that carries a T7 promoter sequence and a gene of interest. Expression of recombinant H. pylori sequences in E. coli

Transformants were collected from LB agar plates containing 25ug/ml kanamycin sulfate (ensures maintenance of the pET28b-based recombinant plasmids) and used to inoculate LB broth containing 25ug/ml kanamycin sulfate and grown to an optical density at 600nm of 0.5 to 1.0 OD units, at which point ImM IPTG was added to the culture for one to three hours to induce gene expression of the H. pylori recombinant DNA constructions. After induction of gene expression with IPTG, bacteria were pelleted by centrifugation and resuspended in SDS-PAGE solubilization buffer and subjected to SDS-PAGE (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994). Proteins were visualized by staining with Coomassie Brilliant Blue or detected by western immunoblotting using the specific anti-His tag monoclonal antibody (Clontech, Palo Alto, CA, USA) using standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). The host strain that provided the highest level of recombinant protein production was then chosen for use in a large-scale induction in order to purify the recombinant protein. All of the following proteins listed were expressed recombinantly and the strain giving the highest level of expression listed: BL21(DE3) (vac31, vac26, vac37); BL21(DE3) pLysS (vac 9, 32); HMS174(DE3) (vac 10, 11). Purification of recombinant proteins and generation of specific antiserum

Large scale cultures were inoculated and grown as above, and induced with ImM IPTG for 3 hours. After induction, bacteria were pelleted by centrifugation in a Sorvall centrifuge at 3500 x g for 15 min at 4°C. All of the expressed recombinant proteins were present in the insoluble inclusion body fraction. Inclusion bodies were purified according to standard protocols (Antibodies, Cold Spring Harbor Laboratory Press, E. Harlow and D. Lane, eds., 1988). The recombinant protein produced by vac32 was solubilized in 8M urea and partially purified by nickel chromatography (REF here). Denatured recombinant proteins were purified by electrophoresis on SDS-PAGE gels, and after visualization with Coomassie Brilliant Blue, the protein was excised from the gel and the gel slices homogenized. This material was used to raise specific polyclonal antibodies in mice or rabbits according to standard protocols (Antibodies, Cold Spring Harbor Laboratory Press, E. Harlow and D. Lane, eds., 1988). Immunological characterization of recombinant proteins

In all cases where antibody was attempted to be raised, high titre antisera was generated, confirming the immunogenicity of the recombinant proteins. Further, these specific antisera were used to analyze whether the protein encoded by the cloned gene was expressed in H pylori. Western immunoblot analysis using standard protocols (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al, eds., 1994) confirmed that the H pylori strain J99 did express proteins of the expected molecular weight that reacted with the vaclO, vac32, vac31, vac36 antiserum. The specific antiserum was also used to determine the level of antigenic conservation between a large number ofH. pylori isolates that had been obtained from distinct geographical sites around the world, and from all types of clinical manifestations, including gastritis, duodenal ulcer, gastric ulcer and gastric cancer. It was found that every strain produced a protein that reacted specifically with each antiserum. Further, H. pylori cells from strains J99, 17874, AΗ244 and SSI were fractionated into different cellular compartments (Doig and Trust 1994 Infect. Immun. 62:4526-4533: O'Toole et al. 1995 J. Bacteriol. 177:6049-6057). The specific antiserum was used to probe these fractions by western immunoblot to identify in which fraction the protein was localized. In all cases, the immunoreactive protein was present in the outer membrane as had been predicted by the sequence features and motif searches described herein.

Demonstration of protein efficacy as a vaccine Purification of vac36 (SEQ ID NO: 96) for efficacy studies All the following steps were carried out at 4°C. Cell pellets were resuspended in

5 volumes per gram of cell of lysis buffer (50mM Sodium Phosphate pH 8.0, 0.5 M NaCl, 5mM Imidazole) with lOmM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 % β-mercaptoethanol, and ruptured by several passages through a small volume microfluidizer (Model M-l 10S, Microfluidics International Corporation, Newton, MA). The resulting homogenate was made 0.2% sodium deoxycholate (DOC), stirred 20 minutes, then centrifuged (10,000 g x 30 min). The pellets were washed twice with Lysis Buffer containing lOmM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% β -mercaptoethanol, then with lysis buffer containing IM urea, ImM PMSF and 0.1 % β- mercaptoethanol. The resulting white pellet is composed primarily of inclusion bodies, free of unbroken cells and membranous materials.

The inclusion bodies were dissolved in 20 ml 6M guanidine-HC 1 in lysis buffer with 1 mM PMSF and 0.1 % β-mercaptoethanol, and incubated on ice for 1 hour. Materials that did not dissolve were removed by centrifugation (100,000 g x 30 min.) The clear supernatant was filtered through a 0.8 μm Supor filter (Gelman Sciences, FRG) and then load directly onto a 10 ml Ni^⁺ - NTA agarose column (Hochuli et al. 1987) pre-equilibrated in 6M guanidine-HCl in Lysis Buffer containing 1 mM PMSF and 0.1% β-Mercaptoethanol. The column was washed with 20ml (2 bed volumes) of Lysis Buffer containing 6M guanidine-HCl, ImM PMSF and 0.1 %β-mercaptoethanol, then guanidine-HCl was removed slowly with a 100 ml linear gradient (from 6M to 0 M Guanidine-HCl) of lysis buffer containing 0.5% Brij 35, 1 mM PMSF, 0.1% β- mercaptochanol. Next, the column was developed with a 25 ml linear gradient of increasing imidazole (5 to 500 mM) in Lysis buffer containing 0.5%> Brij 35, 1 mM

PMSF and 0.1% β-mercaptoethanol. The recombinant proteins elute as a peak centered at 1 OOmM imidazole.

Fractions containing the recombinant proteins were pooled and then concentrated to approximately 8 ml by centrifugal filtration (Centriprep-10, Amicon, MA), and loaded directly onto a 350-ml column (2.2 X 91 cm) of Sephacyl S-100 HR gel filtration medium equilibrated in Buffer A (50mM Sodium Phosphate, pH 8.0, 500 mM NaCl, 0.1 mM EGTA, 1 mM PMSF, 0.1%β-mercaptoethanol, 0.5% Brij 35) and ran in Buffer A at 30 ml/h. Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled, concentrated to 1.5 to 2 mg/ml and dialysed overnight against 10 mM Potassium

Phosphate pH 7.5, 150 mM NaCl, 0.1 mM EGTA and 0.5% Brij 35. The concentration of protein in the dialysate was quantified, then aliquoted prior to freezing at - 20°C. Mouse model of Heliocobacter pylori infection

A murine model ofH. pylori infection was produced by infection of C57BL/6 mice with with H. pylori Sydney strain SSI and was used to assess the efficacy of recombinant H pylori vac36. This mouse-adapted H. pylori strain is cagA+ vacA+, shows colonization levels in C57BL/6 mice equivalent to those observed in humans, forms adhesion pedestals, colonizes for at least 8 months, and elicits a chronic-active gastritis and mucosal atrophy (Lee et al, Gastroenterology, 112:1386-1397, 1997). Dose-response studies have shown 100% infection rates of inbred C57BL/6 and Balb/C mice at 8 weeks post-challenge with a single inoculation of 10" organisms. Animals

Female SPF BALB/c mice were purchased from Bomholt Breeding center (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival. Infection

After a minimum of one week of acclimatization, the animals were infected with a type 2 strain (Vac A negative) ofH. pylori (strain 244, originally isolated from an ulcer patient). In our hands, this strain has earlier proven to be a good colonizer of the mouse stomach. The bacteria were grown overnight in Brucella broth supplemented with 10 % fetal calf serum, at 37°C in a microaerophilic atmosphere (10% CO₂, 5%O₂). The animals were given an oral dose of omeprazole (400 μmol/kg) and 3-5 h after this an oral inoculation ofH pylori in broth (approximately 10 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation. Antigens Recombinant H. pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa-ΗIS tag for purification reasons and the non-Helicobacter pylori control protein (b-galactosidase from E. coli; LacZ), was constructed in the same way.

All antigens were given in a soluble form, i.e. dissolved in either a ΗEPES buffer or in a buffer containing 0.5% Deoxycholate (DOC). The antigens are listed in Table 7 below.

Table 7

Helicobacter pylori proteins Outer membrane Proteins

Protein 7116626 Protein 4721061

Protein 16225006

Protein 29479681

Protein 14640637

Periplasmic/Secreted Proteins Protein 30100332

Other cell envelope proteins

Protein 4821082 Flagella-associated proteins

Protein 26380318 Control proteins β-galactosidase (LacZ) Immunizations

Ten animals in each group were immunized 4 times over a 34 day period (day 1 , 15, 25 and 35). Purified antigens in solution or suspension were given at a dose of 100 mg/mouse. As an adjuvant, the animals were also given 10 μg/mouse of Cholera toxin (CT) with each immunization. Omeprazole (400 mmol/kg) was given orally to the animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Infected control animals received HEPES buffer + CT or DOC buffer + CT. Animals were sacrificed 2-4 weeks after final immunization. A general outline of the study is shown in Table 8 below.

Table 8

Study outline, therapeutic immunization:

Mice were all infected with H. pylori strain Ah244 at day 30.

Analysis of infection

Mucosal infection: The mice were sacrificed by CO₂ and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. The mucosa from the antrum and corpus of an area of 25mm was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity ofH. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.

The urease test was performed essentially as follows. The reagent, Urea Agar Base Concentrate, was purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61-3). Urea agar base concentrate was diluted 1 :10 with water. 1 ml of if the diluted concentrate was mixed with 100-200 ml of actively growing H pylori cells. Color change to magenta indicated that cells were urease positive.

The catalase test was performed essentially as follows. The reagent, N,N,N',N'- Tetramethyl-p-Phenylenediamine, was purchased from Sigma, St. Louis, MO (Catalog # T3134). A solution of the regent (1% w/v in water) was prepared. H pylori cells were swabbed onto Whatman filter paper and overlaid with the 1 % solution. Color change to dark blue indicated that the cells were catalase positive.

Serum antibodies: From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.

Mucosal antibodies: Gentle scrapings of a defined part of the corpus and of 4 cm of duodenum were performed in 50% of the mice in order to detect the presence of antibodies in the mucous. The antibody titers were determined by regular ELISA technique as for serum antibodies.

Statistical analysis: Wilcoxon-Mann- Whitney sign rank test was used for determination of significant effects of the antigens on Helicobacter pylori colonization. PO.05 was considered significant. Because the antrum is the major colonization site for Helicobacter most emphasis was put upon changes in the antral colonization. Assessment of gastric H. pylori infection

The presence ofH pylori organisms in gastric tissue was determined by culture of gastric tissue and by a quantitative urease assay. In the latter method, a longitudinal segment of antrum, representing approximately ^lA of the total antral region was placed in 1 ml of urea broth. After 4 hr, the extent of color change resulting from urea hydrolysis and increased pΗ was quantiated by spectrophotometric measurement of A550 (Fox et al, Immunol. 88:400-406, 1996). The assay sensitivity is ~ 10^ H pylori organisms. A positive (H # /or/-infected) gastric tissue was defined as that sample showing 2 standard deviations above the mean A550 value derived from a group of unchallenged uninfected age-matched control mice. Assessment of local immune response to immunization in gastric tissue

Longitudinal sections of gastric tissues from the esophageal to the duodenal junction were embedded in OCT embedding compound, frozen in liquid nitrogen, and cryosections immunostained with monoclonal antibodies recognizing CD4⁺ or CD8⁺T cells or with antisera against mouse IgA for identification of IgA containing (IgACC) plasma cells (Pappo et al, Infect. Immun. 63:1246-1252, 1995). The degree of local gastric immune response was expressed quantitatively as the number of CD4⁺' CD8⁺ or

IgACC cells per mm^ of gastric region examined.

Results

Antibodies in sera: All antigens tested given together with CT gave rise to a measurable specific titer in serum. The highest responses were seen with Protein

7116626, Protein 4721061, Protein 26380318, Protein 14640637 and Protein 4821082 (see Figure 1). Antibodies in mucus: In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with Protein 30100332, followed by Protein 14640637, and Protein 26380318 (see Figure 2). Therapeutic immunization effects: All control animals (BALB/c mice) were well colonized with H. pylori (strain

AΗ244) in both antrum and corpus of the stomach. Of the antigens tested 3 proteins (Protein 4721061, Protein 4821082, and Protein 14640637) gave a good and significant reduction and/or eradication of the H. pylori infection. The degree of colonization of the antrum was lower following immunization with Protein 7116626 and Protein 26380318 compared to control. The effect of Proteins 16225006, 29479681, and 30100332 did not differ from control. The control protein lacZ, i.e. the non-H pylori protein, had no eradication effect and in fact had higher Helicobacter colonization compared to the ΗEPES + CT control. All data are shown in Figures 3 and 4 for proteins dissolved in ΗEPES and DOC respectively. Data is shown as geometric mean values. n=8-10 Wilcoxon-Mann- Whitney sign rank test * = p<0.05; x 10 = number of mice showing eradication ofH pylori over the total number of mice examined. Protective activity of purified recombinant / . pylori vac36 (SEQ ID NO: 96) antigen

The ability of purified recombinant vac36 antigen derived from H pylori to interfere with the establishment of an H. pylori infection was examined in mice. Groups (n=10) of 6-8 week-old female C57BL/6 mice were immunized orally 4 times at weekly intervals as follows: 1) 100 μg of recombinant vac36 antigen and 10 μg cholera toxin (CT) adjuvant, 2) 1 mg H pylori lysate antigens and 10 μg CT, and 3) 0.2 M bicarbonate buffer and 10 wg CT adjuvant. The mice were challenged 2 weeks later on 3 consecutive days by oral administration of 10^ H. pylori organisms. The experiment was terminated 2 weeks post-challenge, and the H pylori infection level assessed by bacterial colony counts and by quantitative urease assays.

Oral immunization with vac36 antigen interfered with the establishment ofH. pylori infection upon challenge with live H pylori organisms. Mice immunized with purified recombinant vac36 antigen exhibited a significantly lower level of colonization by H. pylori, as assessed by gastric urease activity and bacterial count assays (Table 9). Oral immunization with vac36 antigen also resulted in the generation of a local protective gastric immune response. Greater numbers of CD4⁺T cells and of IgACC were recruited in the gastric tissues of vac36-immunized mice when compared with unimmunized H. pylori-infected mice (Table 10). Table 9

Recombinant vac36 antigen protects mice from challenge with H. pylori

a Urease activity is expressed as mean A55Q± SEM of duplicate antral samples from n=10 mice/group. b by Wilcoxon Rank Sum Test compared with mice immunized with CT adjuvant alone c The level ofH pylori in gastric tissue was assessed by bacterial counts, and shown as mean colony forming units±SEM

Table 10 vac36-immunized mice generate a local gastric immune response upon challenge with H. pylori

^a Mean number of cells/mm^ of gastric region ± SEM

* p<0.05 by Wilcoxon Rank Sum Test when compared with unimmunized H. pylori infected mice The data presented indicate that all of the H. pylori associated proteins included in this study, when used as oral immunogens in conjunction with the oral adjuvant CT, resulted in stimulation of an immune response as measured by specific serum and mucosal antibodies. A majority of the proteins led to a reduction, and in some cases complete clearance of the colonization ofH pylori in this animal model. It should be noted that the reduction or clearance was due to heterologous protection rather than homologous protection (the polypeptides were based on the H pylori J99 strain sequence and used in the therapeutic immunization studies against a different (AΗ244) challenge strain, indicating the vaccine potential against a wide variety ofH. pylori strains.

The highest colonization in the antrum was seen in animals treated with the non- Helicobacter protein LacZ, indicating that the effects seen with the Helicobacter pylori antigens were specific.

Taken together these data strongly support the use of these H. pylori proteins in a pharmaceutical formulation for the use in humans to treat and/or prevent H pylori infections. V. Sequence Variance Analysis of genes in Helicobacter pylori strains

Four genes were cloned and sequenced from several strains ofH pylori to compare the DNA and deduced amino acid sequences. This information was used to determine the sequence variation between the H. pylori strain, J99, and other H. pylori strains isolated from human patients. Preparation of Chromosomal DNA.

Cultures ofH pylori strains (as listed in Table 13) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD₆₀₀ of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mis of 10 mM Tris- ΗC1, 0.1 mM EDTA (TE). Lysozyme was added to a final concentration of 1 mg/ml along with, SDS to 1% and RNAse A + Tl to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55 C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1% CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes. At this point, the samples were extracted with chloroform: isoamyl alcohol, extracted with phenol, and extracted again with chloroform:isoamyl alcohol. DNA was precipitated with either

EtOΗ (1.5 x volumes) or isopropanol (0.6 x volumes) at -70°C for lOminutes, washed in 70%) EtOΗ and resuspended in TE. PCR Amplification and cloning.

Genomic DNA prepared from twelve strains of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). To amplify a DNA sequence containing an H. pylori ORF, genomic DNA (10 nanograms) was introduced into a reaction vial containing 2 mM MgCl₂, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers, see Table 11 ) 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 11

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:

Protein 7116626 and Protein vac38 (SEQ ID NO: 98); Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes. Protein 26054702 for strains AH5, 5155, 7958, AH24,and J99; Denaturation at 94°C for 2 min, 2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min

25 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min

Reaction was concluded at 72°C for 6 minutes.

Protein 26054702 and Protein 294796813 for strains AH4, AH15, AH61, 5294, 5640, AH18, and Hp244 ;

Denaturation at 94°C for 2 min,

2 cycles at 94°C for 15 sec, 30°C for 20 sec and 72°C for 2 min

25 cycles at 94°C for 15 sec, 55°C for 20 sec and 72°C for 2 min

Reactions were concluded at 72°C for 8 minutes. Upon completion of thermal cycling reactions, each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.

Cloning ofH. pylori DNA sequences into the pCR TA cloning vector.

All amplified inserts were cloned into the pCR 2.1 vector by the method described in the Original TA cloning kit (Invitrogen, San Diego, CA). Products of the ligation reaction were then used to transform the TOPI OF' (INVaF' in the case ofH pylori sequence 350) strain of E. coli as described below.

Transformation of competent bacteria with recombinant plasmids

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 BMΕ was added to each vial of 50 microliters of competent cells.

Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes. The cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM

MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of TOPI OF' or INVaF' were then picked and analyzed to evaluate cloned inserts as described below.

Identification of recombinant PCR plasmids carrying H. pylori sequences

Individual TOPI OF' or INVaF' clones transformed with recombinant pCR-

H.pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H pylori sequences in the cloning vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).

Individual clones of recombinant pCR vectors carrying properly cloned H pylori ORFs were picked for sequence analysis. Sequence analysis was performed on ABI Sequencers using standard protocols (Perkin Elmer) using vector-specific primers (as found in PCRII or pCR2.1, Invitrogen, San Diego, CA) and sequencing primers specific to the ORF as listed in Table 12 below.

Table 12 li onucleotide rimers used for se uencin ofH. lori DNA se uences.

Results

To establish the PCR error rate in these experiments, five individual clones of Protein 26054702, prepared from five separate PCR reaction mixtures from H. pylori strain J99, were sequenced over a total length of 897 nucleotides for a cumulative total of 4485 bases of DNA sequence. DNA sequence for the five clones was compared to a DNA sequence obtained previously by a different method, i.e., random shotgun cloning and sequencing. The PCR error rate for the experiments described herein was determined to be 2 base changes out of 4485 bases, which is equivalent to an estimated error rate of less than or equal to 0.04%).

DNA sequence analysis was performed on four different open reading frames identified as genes and amplified by PCR methods from a dozen different strains of the bacterium Helicobacter pylori. The deduced amino acid sequences of three of the four open reading frames that were selected for this study showed statistically significant BLAST homology to defined proteins present in other bacterial species. Those ORFs included: Protein 26054702, homologous to the val A & B genes encoding an ABC transporter in F. novicida; Protein 7116626, homologous to lipoprotein e (P4) present in the outer membrane ofH. influenzae; Protein 29479681, homologous to fecA, an outer pylori (see Table 13 below). Results are presented as percent identity to the J99 strain of H pylori sequenced by random shotgun cloning. To control for any variations in the J99 sequence each of the four open reading frames were cloned and sequenced again from the J99 bacterial strain and that sequence information was compared to the sequence information that had been collected from inserts cloned by random shotgun sequencing of the J99 strain. The data demonstrate that there is variation in the DNA sequence ranging from as little as 0.12 % difference (Protein 38 (SEQ ID NO: 98), J99 strain) to approximately 7% change (Protein 26054702, strain AΗ5). The deduced protein sequences show either no variation ( Protein 38 (SEQ ID NO: 98), strains AH18 and AH24) or up to as much as 7.66% amino acid changes (Protein 26054702, Strain AH5).

Table 13

Multiple Strain DNA Sequence analysis of H. pylori Vaccine Candidates

J99 Protien #: 26054702 26054702 71 16626 71 16626 29479681 29479681 vac38 vac38

Length of Region 248 a.a. 746 nt. 232 a.a. 696 nt. 182 a.a. 548 nt. 273 a.a. 819 nt.

Sequenced:

Strain Tested

AA Nuc. AA Nuc. AA Nuc. AA identity Nuc. identity identity identity identity identity identity Identity

J99 100.00% 100.00% 100.00% 100.00% 100.00% 100.00% 99.63% 99.88%

AH244 95.16% 95.04% n.d. n.d. 99.09% 96.71% 98.90% 96.45%

AH4 95.97% 95.98% 97.84% 95.83% n.d. n.d. 97.80% 95.73%

AH5 92.34% 93.03% 98.28% 96.12% 98.91% 96.90% 98.53% 95.73%

AH15 95.16% 94.91% 97.41% 95.98% 99.82% 97.99% 99.63% 96.09%

AH61 n.d. n.d. 97.84% 95.98% 99.27% 97.44% n.d. n.d.

5155 n.d. n.d. n.d. n.d. 99.45% 97.08% 98.53% 95.60%

5294 94.35% 94.37% 98.28% 95.40% 99.64% 97.26% 97.07% 95.48%

7958 94.35% 94.10% 97.84% 95.40% n.d. n.d. 99.63% 96.46%

5640 95.16% 94.37% 97.41% 95.69% 99.09% 97.63% 98.53% 95.48%

AH18 n.d. n.d. 98.71% 95.69% 99.64% 97.44% 100.00% 95.97%

AH24 94.75% 95.04% 97.84% 95.40% 99.27% 96.71% 100.00% 96.46% n.d. = not done

VI. Experimental Knock-Out Protocol for the Determination of Essential H. pylori Genes as Potential Therapeutic Targets

Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.

The protocol for the deletion of portions ofH pylori genes/ORFs and the insertional mutagenesis of a kanamycin-resistance cassette in order to identify genes which are essential to the cell is modified from previously published methods (Labigne- Roussel et al., 1988, J. Bacteriology 170, pp. 1704-1708; Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573; Reyrat et al., 1995, Proc. Natl. Acad. Sci. 92, pp 8768-8772). The result is a gene "knock-out." Identification and Cloning ofH. pylori Gene Sequences

The sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Plymouth, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.

Genomic DNA prepared from the Helicobacter pylori ΗpJ99 strain (ATCC 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). For the preparation of genomic DNA from H pylori, see Example I. PCR amplification is carried out by introducing 10 nanograms of genomic HpJ99 DNA into a reaction vial containing 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl₂, 2 microMolar synthetic oligonucleotide primers (forward=Fl and reverse=Rl), 0.2 mM of each deoxynucleotide triphosphate (dATP,dGTP, dCTP, dTTP), and 1.25 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 40 microliters. The PCR is carried out with Perkin Elmer Cetus/GeneAmp PCR System 9600 thermal cyclers.

Upon completion of thermal cycling reactions, each sample of amplified DNA is visualized on a 2% TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction. Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). PCR products are cloned into the pT7Blue T-Vector (catalog#69820-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 Wiley and Sons, Inc., F. Ausubel et al., editors, 1994) into electroporation- competent XL-1 Blue or DH5-a E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA). Briefly, 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) at 37°C with shaking for 1 hour. Samples are then spread onto LB (10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/1 sodium chloride) plates containing 100 microgram/ml of Ampicillin, 0.3% X-gal, and 100 microgram/ml IPTG. These plates are incubated overnight at 37°C. Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).

To verify that the correct H.pylori DNA inserts had been cloned, these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 H.pylori sequence. Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned. Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage. To minimize errors due to PCR, plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.

The sequences of the genes/ORFs are again used to design a second pair of primers which flank the region ofH pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other. The pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product. The PCR product is a linear piece of DNA with H pylori DNA • at each end and the pT7Blue vector backbone between them which, in essence, resultes in the deletion of a portion of the ORFs. The PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified. A Kanamycin-resistance cassette (Labigne-Roussel et al., 1988 J. Bacteriology

170, 1704-1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid pCTB8:£α« (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573). The proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA). The fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes. This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides. The "T" overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pΗ 8.3, 50 mM KCl, 2 mM MgCl₂, 5 units of DNA Polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP, in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C. The "Kan-T" cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA). The PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter 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 DΗ5-a E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram/ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).

Several tests by PCR amplification are conducted on these plasmids to verify that the Kanamycin is inserted in the H. pylori gene/ORF, and to determine the orientation of the insertion of the Kanamycin-resistance gene relative to the H pylori gene/ORF. To verify that the Kanamycin cassette is inserted into the H pylori sequence, the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the H. pylori gene/ORFs. The correct PCR product is the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. To avoid potential polar effects of the kanamycin resistance cassette on H pylori gene expression, the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in H pylori transformations (see below). To determine the orientation of insertion of the kanamycin resistance gene, primers are designed from the ends of the kanamycin resistance gene ("Kan-1" 5'-ATCTTACCTATCACCTCAAAT-3' (SEQ ID NO: 179)), and "Kan-2" 5'-AGACAGCAACATCTTTGTGAA-3' (SEQ ID NO: 180)). By using each of the cloning primers in conjunction with each of the Kan primers (4 combinations of primers), the orientation of the Kanamycin cassette relative to the H.pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the H.pylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H. pylori. Transformation of Plasmid DNA into H. pylori cells Two strains ofH. pylori are used for transformation: ATCC 55679, the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AΗ244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach. Cells for transformation are grown at 37°C, 10% CO , 100% humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated. If grown on plates, cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf microfuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth. An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells.. An aliquot (1 to 5 OD₆₀₀ units/25 microliter) of the resuspended cells is placed onto a prewarmed Sheep-Blood agar plate, and the plate is further incubated at 37°C, 6% CO₂, 100%) humidity for 4 hours. After this incubation, 10 microliters of plasmid DNA (100 micrograms per microliter) is spotted onto these cells. A positive control (plasmid DNA with the ribonuclease H gene disrupted by kanamycin resistance gene) and a negative control (no plasmid DNA) are done in parallel. The plates are returned to 37°C, 6% CO₂ for an additional 4 hours of incubation. Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6% CO₂. Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C, 6% CO₂, 100%) humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.

Three sets of PCR tests are done to verify that the colonies of transformants have arisen from homologous recombination at the proper chromosomal location. The template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1:1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.

TEST 1. PCR with cloning primers originally used to amplify the gene/ORF. A positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. A PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.

TEST 2. PCR with F3 (primer designed from sequences upstream of the gene/ORF and not present on the plasmid), and either primer Kan-1 or Kan-2 (primers designed from the ends of the kanamycin resistance gene), depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out. TEST 3. PCR with R3 (primer designed from sequences downstream of the gene/ORF and not present on the plasmid) and either primer Kan-1 or Kan-2, depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3). Again, no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.

Transformants showing positive results for all three tests above indicate that the gene is not essential for survival in vitro. A negative result in any of the three above tests for each transformant indicates that the gene had not been disrupted, and that the gene is essential for survival in vitro. In the event that no colonies result from two independent transformations while the positive control with the disrupted ribonuclease H plasmid DNA produces transformants, the plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3. Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST 2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation. VII. High-throughput drug screen assay Cloning, expression and protein purification

Cloning, transformation, expression and purification of the H pylori target gene and its protein product,e.g., an H. pylori enzyme, to be used in a high-throughput drug screen assay, is carried out essentially as described in Examples II and III above. Development and application of a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase, is described below as a specific example. Enzymatic Assay

The assay is essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Acta 43:1101-1111). The assay measures the cis-trans isomerization of the Ala-Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S- 7388, lot # 84H5805). The assay is coupled with α-chymotrypsin, where the ability of . the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans. The conversion of the test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second. Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 μM α-chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction, 10 μl of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 μl of reaction mixture at room temperature. Enzymatic assay in crude bacterial extract. A 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD ₆₀o _n ~ 1) ¹¹⁰¹ resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 μg/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor. The suspension is subjected to 3 cycles of freeze-thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts). The lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.

Many H pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays. VIII. H. pylori Attenuated Salmonella Vaccine

A Salmonella strain, such as S. typhi or S. typhimurium is attenuated by making a deletion mutation in at least one of the following genes: cya, crp and cdt using conventional mutagenesis techniques. The resulting knockout is then recombined into a Salmonella genome to create a new avirulent strain of Salmonella. The avirulent strain of Salmonella preferably further lacks the asd gene, which encodes aspartate semialdehyde dehydrogenase, essential for survival (Curtiss et al., (1988) Avirulent Salmonella expressing virulence antigens from other pathogens for use as orally administered vaccines. In, Virulence Mechanisms of Bacterial Pathogens (J.A. Roth, ed.), ASM Press, Washington DC; Curtiss et al. (1994) Recombinant Salmonella vectors in vaccine development. In, Recombinat Vectors in Vaccine Development (F. Brown, ed.) Dev. Biol. Stand. Basel, Karger. Vol 82, pp.23-33). The H. pylori gene of interest is placed on the pBR322 or pUC-based plasmid which has a promoter active in Salmonella and a copy of the asd gene which was deleted from the Salmonella strain discussed above. The plasmid may have an antibiotic selection marker (e.g., Amp^R or Tet^R) for construction in E. coli. This pBR pr pUC plasmid is then used to transform the attenuated Salmonella strain. Transformants are grown in culture and are then introduced into mice to confirm that the Salmonella strain is attenuated. Virulent strains of Salmonella are lethal at certain dosages in mice (3 X 10⁵ bacteria = oral LD₅₀; < 50 bacteria = IP LD₅₀).

Expression of the H. pylori protein of interest by the attenuated Salmonella strain is tested by immunoblotting with an antibody raised against that specific protein. Such antibodies are produced by recombinantly expressing the H pylori gene of interest in E. coli, purifying the H pylori protein, and using the purified H pylori protein to raise antibodies in mice as described herein.

Immunization Protocol Recombinant attenuated Salmonella strain expressing the H pylori protein of interest is given orally to C57 black mice in one or two weekly doses. Ten animals in each group are immunized with a single dose (10⁷-10⁸ bacteria).

The animals are then challenged 2-3 weeks later with 3 x 10⁸ cfu of the wild type Helicobacter strain, SSI . Attenuated Salmonella strain that lacks the H. pylori gene of interest is used as a negative control. H pylori whole-cell lysate mixed with cholera toxin is administered in four weekly doses and is used as a positive control for immunization experiments (cholera toxin at 10 μg/mouse; lysate at 100 μg/mouse).

Analysis of infection

Mucosal infection: The immunized mice are sacrificed by CO2 and cervical dislocation. The abdomen is opened and the stomach removed. After cutting the stomach along the greater curvature, it is rinsed in saline. The mucosa from the antrum and corpus of an area of 22mm^ is scraped separately with a surgical scalpel. The mucosa scraping is suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates are incubated under microaerophilic conditions for 3-5 days and the number of colonies is counted. The identity ofH. pylori is ascertained by urease test and by direct microscopy or Gram staining.

The urease test is performed essentially as follows. The reagent, Urea Agar Base Concentrate, is purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61-3). Urea agar base concentrate is diluted 1 :10 with water. 1 ml of the diluted concentrate is mixed with 100-200 ml of actively growing H pylori cells. Color change to magenta indicated that cells are urease positive. Therapeutic Immunization

Ten C57 black mice in each group are challenged with a single dose of 3 x 10^ cfu of the H pylori strain, SSI . Two weeks later the mice are immunized with one or two (weekly) doses of a recombinant Salmonella strain carrying the H. pylori gene of interest or with four weekly doses ofH. pylori lysate with cholera toxin (positive control). Cholera toxin by itself is given as a negative control (10 μg/ml).

Two weeks after immunization, the mice are sacrificed and the same H pylori colony counts and urease tests are performed as outlined above.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

1. A vaccine formulation for prophylactic or therapeutic treatment of an H pylori infection comprising an effective amount of at least one isolated H pylori polypeptide or a fragment thereof in a pharmaceutically acceptable carrier, wherein said . pharmaceutically acceptable carrier comprises a bacterial vector comprising a nucleotide sequence encoding the H. pylori polypeptide or fragment thereof, said bacterial vector selected from the group consisting of a species of Salmonella, Vibrio cholerae and Shigella.

2. The vaccine formulation of claim 1, wherein said bacterial vector is a species of Salmonella.

3. The vaccine formulation of claim 1, wherein said bacterial vector is a species of Vibrio cholerae.

4. The vaccine formulation of claim 1, wherein said bacterial vector is a species of Shigella.

5. The vaccine formulation of claim 1, wherein said bacterial vector is attenuated.

6. The vaccine formulation of claim 1, wherein said H pylori polypeptide is an outer membrane polypeptide.

7. The vaccine formulation of claim 6, wherein said H. pylori outer membrane polypeptide or a fragment thereof is selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.

8. The vaccine formulation of claim 6, wherein said H pylori outer membrane polypeptide or a fragment thereof is encoded by a nucleic acid selected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, or a complement thereof.

9. The vaccine formulation of claim 6, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an is an H pylori polypeptide having a terminal phenylalanine residue or a fragment thereof.

10. The vaccine formulation of claim 9, wherein said H pylori polypeptide having a terminal phenylalanine residue or a fragment thereof is selected from a group consisting of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103.

11. The vaccine formulation of claim 9, wherein said H pylori polypeptide having a terminal phenylalanine residue or a fragment thereof is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID Γûá NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a complement thereof.

12. The vaccine formulation of claim 6, wherein said H pylori outer membrane polypeptide or a fragment thereof is an is an H pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof.

13. The vaccine formulation of claim 12, wherein said H pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof is selected from a group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111.

14. The vaccine formulation of claim 12, wherein said H. pylori polypeptide having a C-terminal tyrosine cluster or a fragment thereof is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, or a complement thereof.

15. The vaccine formulation of any of claims 9, 10, 11, 12, 13 or 14, wherein said H pylori outer membrane polypeptide or a fragment thereof is an is an H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof.

16. The vaccine formulation of claim 15, wherein said H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof is selected from the group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103.

17. The vaccine formulation of claim 16, wherein said H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a complement thereof.

18. The vaccine formulation of claim 2, wherein said Salmonella is attenuated by a mutation in at least one of the genes selected from the group consisting of cya, crp, and cdt.

19. The vaccine formulation of claim 18, wherein said Salmonella is attenuated by a mutation in the cya gene.

20. The vaccine formulation of claim 18, wherein said Salmonella is attenuated by a mutation in the crp gene.

21. The vaccine formulation of claim 18, wherein said Salmonella is attenuated by a mutation in the cdt gene.

22. The vaccine formulation of claim 1 , wherein said pharmaceutically acceptable carrier further comprises an adjuvant.

23. The vaccine formulation of claim 22, wherein the bacterial vector further comprises a nucleic acid encoding an adjuvant.

24. A vaccine formulation for prophylactic or therapeutic treatment of an H. pylori infection comprising an effective amount of at least one isolated H pylori polypeptide or a fragment thereof in a pharmaceutically acceptable carrier, wherein said pharmaceutically acceptable carrier comprises a Salmonella vector comprising a nucleotide sequence encoding an H pylori polypeptide or a fragment thereof, provided that said H. pylori polypeptide is not urease.

25. The vaccine formulation of claim 24, wherein said H. pylori polypeptide or a fragment thereof is an H pylori outer membrane protein or a fragment thereof.

26. The vaccine formulation of claim 24, wherein said pharmaceutically acceptable carrier further comprises an adjuvant.

27. The vaccine formulation of claim 26, wherein said Salmonella vector further comprises a nucleic acid encoding an adjuvant.

28. A vaccine formulation for prophylactic or therapeutic treatment of an H pylori infection comprising an effective amount of at least one isolated H pylori outer membrane polypeptide or a fragment thereof in a Salmonella vector, wherein said Salmonella vector comprises a nucleotide sequence encoding an H pylori polypeptide or a fragment thereof, said vector having a mutation in at least one of the genes selected from the group consisting of cya, crp, and cdt.

29. The vaccine formulation of claim 1, wherein said H. pylori polypeptide or a fragment thereof is not urease.

30. A method for treatment or prophylaxis of an H. pylori infection in a subject comprising administering to a subject a vaccine formulation of any of claims 1, 24 or 28, such that treatment or prophylaxis of an H. pylori infection occurs.

31. A method of producing an H pylori vaccine formulation comprising introducing at least one isolated H pylori polypeptide or a fragment thereof into a pharmaceutically acceptable carrier to thereby form a vaccine formulation, wherein said pharmaceutically acceptable carrier comprises a bacterial vector comprising a nucleotide sequence encoding the H pylori polypeptide or fragment thereof, said bacterial vector selected from the group consisting of a species of Salmonella, Vibrio cholerae and Shigella.