CA2201353A1 - The taga gene and methods for detecting predisposition to peptic ulceration and gastric carcinoma - Google Patents

Abstract

The present invention provides an isolated nucleic acid encoding an approximately 120-128 kilodalton antigen of Helicobacter pylori, or an antigenic fragment thereof, wherein the antigen is associated with peptic ulceration. The present invention also provides methods of detecting the presence of a Helicobacter pylori strain possessing the 120-128 kilodalton antigen in a subject, comprising the steps of contacting an antibody-containing sample from the subject with a detectable amount of the tagA
antigen or antigenic polypeptide of the present invention and detecting the binding of the antigen or fragment and the antibody. The detection of a strain expressing the TagA antigen is an indication of predisposition to peptic ulceration and gastric carcinoma. A mutant H. pylori not expressing a functional TagA antigen is also provided.

Description

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~ WO96/10639 22 Q 1 3 5 ~ PCT~S9~,l2669 THE tagA GENE AND hhl~vS FOR DETECTING
PREDISPOSITION TO PEPTIC ULCERATION AND GASTRIC ~CTNOMA

~AC~ROUND OF T~ INv~ ON

Helicobacter pylori is now recognized as an important pathogen of humans in that the chronic gastritis it causes is a risk factor for the development of peptic ulcer disease. There is also increasing evidence that persistent infection with Helicobacter pylori is a risk factor for the development of gastric adenocarcinoma (1,2), especially of the distal stomach (3-5). The evidence comes mainly from epidemiologic investigations (6-9), including nested case-control studies (4,5,10), and molecular and pathological studies support its biological plausibility (11). However, although essentially all infected persons develop gastritis, clinical consequences of H. pylori infection are recognized in only a minority of persons. Also, while H. pylori infection is highly prevalent in patients with gastric cancer, most H. pylori-infected persons never develop these neoplasms (12).
Thus, it is important to identify other factors that more precisely determine risk among H. pylori-infected persons.

H. pylori strains are highly diverse (13-15) at a genetic level, but most phenotypic characteristics are well-conserved. Furthermore, individuals can be infected with more than one strain (4,15). Thus, it is important to isolate particular characteristics of H. pylori strains that might affect risk of gastric cancer development.

WO96/10639 ~ ~ Q ~ 3 ~ 3 rcT~sgs/1~66g Two exceptions to the phenotypic homogeneity are currently recognized. First, about 50~-60~ of H. pylori strains produce a vacuolating cytotoxin in vi tro (20,39), and toxin production is associated with peptic ulceration (40). Second, there is heterogeneity in whether an antigenic protein migrating at approximately 120-128 kilodalton (kDa) on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis tSDS-PAGE] is produced (20). More recent measurements of this protein (referred to interchangably as TagA or CagA) have yielded molecular weights as high as 140 kDa. Although toxic activity is mediated by an 87 kDa protein (23,41), toxin production itself is associated with the presence of the antigenic 120-128 kDa protein (20). Previous studies have found that about 60-80~ of H. pylori isolates express the 120-128 kDa protein (20,42). Notably, presence of antibodies to the 120-128 kDa protein in either serum or mucosal secretions is associated with the presence of peptic ulceration (18,20).
Until now, little was known about the association between toxin production, ulcers or gastric carcinoma and the 120-128 kDa antigen. This is due to the previous inability to further characterize the 120-128 kDa antigen after its initial visualization.

In previous studies, the 120-128 kDa antigen was visualized by Western blotting, but virtually no other characterization was performed (20). In contrast to the ease with which this antigen has been visualized by Western blotting, the 120-128 kDa band has not been easily ~ WO96110639 22 Q 7 3 ~ ~ PCT~S95/12669 visualized by other methods such as silver staining (Figure 2 in Cover et al., 1990 (20)). The explanation for this phenomenon is that this antigen is present only in minute quantities, relative to other H. pylori proteins. Recently, Gerstenecker et al. (43) have reported the isolation of an approximately 120 kDa protein from H. pylori which reacts with positive human control serum. However, virtually no characterization (such as N-terminal sequencing) of this antigen has been performed.
Despite the difficulty of purification, the present invention provides the cloning and sequence of the gene and deduced amino acid sequence encoding the 120-128 kDa protein. This data was obtained using alternate methodology that did not require purification of the 120-128 kDa antigen. The invention also provides diagnostic, therapeutic, and prophylactic compositions and methods.

Immunoblot studies suggest that persons infected with tagA+ strains have higher degrees of gastric inflammation and epithelial cell damage than do persons from whom tagA~ strains have been isolated (18). Persons infected with tagA+ H. pylori strains have enhanced expression of IL-1~, IL-l~, and IL-8 in gastric biopsies compared to uninfected persons, and patients infected with tagA- strains (19). Both intensity of inflammation and epithelial damage may be involved in the pathogenesis of gastric cancer (1). Thus, there is a need to ~m; ne the importance of tagA expressing H. pylori strains in this context.

2 ~ ~ 1 3 ~ 3 PcT~S95/12669 The invention meets these needs by providing a new serologic assay based on a recombinant fragment of tagA, and a method of determining predisposition to gastric cancer.
. ST ~ ~Y QF THE ~Nv~Nl~ON

The present invention provides an isolated nucleic acid encoding an approximately 120-128 kilodalton antigen of Helicobacter pylori, or an antigenic fragment thereof, wherein the antigen is associated with peptic ulceration and gastric carcinoma. The present invention also provides methods of detecting the presence of a Helicobacter pylori strain possessing the 120-128 kilodalton antigen in a subject, comprising the steps of contacting an antibody-containing sample from the sub~ect with a detectable amount of the TagA antigen or fragment thereof of the present invention and detecting the reaction of the fragment and the antibody.
~RIEF DESCRIPTIQN QF THE FIGU~

Figure 1 shows physical maps of plasmids pMC1, pMC2, and pMC3. The large arrow beneath pMC3 represents the location of the tagA gene and the direction of transcription as determined by deletion mutations and immunoblotting. The small arrows represent the strand and extent of DNA sequenced from exonuclease III-derived fragments. Restriction endonuclease cleavage sites: B, 30 BglII; Ba, Ba~I; E, EcoRI; H, HindIII; N, NdeI; S, SacI.

WO96110639 2 ~ ~ 1 3 ~ 3 PCT~S95112669 Figure 2 shows a restriction map of pMC3:km used in construction of H. pylori mutant. The km cassette from pILL600 was ligated into the NdeI site of pMC3 to create pMC3:km. The arrows represent open reading frames including the truncated 2577 bp tagA open reading frame.
Restriction sites are E, EcoRI; B, BglII; H, HindIII; Ba, BamHI; N, NdeI; S, SacI; Sm, SmaI. pMC4.represents the 2.9 kb EcoRI to SacI fragment of pMC3.

Figure 3 shows physical maps of plasmids pMC3, pYB2 and pUT2. The large arrow beneath pUT2 represents the location of the tagA gene and the direction of transcription as determined by deletion mutations and ;mmllnoblotting. Restriction endonuclease cleavage sites:
B, BglII; Ba, BamHI; E, EcoRI; EV, EcoRV; H, HindIII; N, NdeI; S, SacI, X; XbaI.

Figure 4 shows physical mpas of the DNA
fragments from tagA which were used to make overlapping gene fusions. The arrows represent the direction of transcription. The size of each product is indicated.
The clear boxed areas represent those areas of the gene that possess repeated nucleotide regions and which have been determined to express immunogenic epitopes of this protein.

DETATT-T~ DES~IPTION OF THE lNv~NllON

Nucleic Acids The present invention provides an isolated nucleic acid encoding an approximately 120-128 kDa antigen WO96/10639 2 a Q 7 3 5 ~ PCT~S95/12669 or fragment of H. pylori, associated with peptic ulceration (TagA). By "isolated" is meant sufficiently separated from other nucleic acids, proteins and other cellular components found in the naturally occurring organism to be useful in a clinical diagnostic or other scientific protocol. The nucleic acid encoding the 120-128 kDa antigen can be the native tagA gene, which is specific for strains of H. pylori expressing the 120-128 kDa antigen. By ~'specific~ is meant an isolated sequence which does not hybridize to a significant degree above background with nucleic acids from H. pylori strains that do not express the antigen.

An example of such an isolated nucleic acid is an open reading frame of 3543 base pairs comprising nucleotides 1072 through 4614 contained in a 4821 base pair insert (SEQ ID N0:3). A cell line containing a plasmid having the full length tagA gene is deposited with the American Type Culture Collection (1230 Parklawn Drive, Rockville MD 20852) under ATCC Accession No. 69273. This isolated H. pylori-specific nucleic acid can be used to detect H. pylori expressing the 120-128 kDa antigen in methods such as polymerase chain reaction (PCR), ligase chain reaction and hybridization as further described below. The nucleic acid or fragments thereof can be in a suitable expression vector and host, and utilized to produce the full length TagA protein as further described hereln .

An example of a nucleic acid encoding an antigenic fragment of the 120-128 kDa antigen is a ~ WO96/10639 ~2 ~ ~ 3 ~ 3 PCT~S95/12669 truncated open reading frame of 2577 base pairs comprising nucleotides 1072 through 3648 contained in a 3648 base pair insert (SEQ ID NO:1). This specific nucleic acid can be used to detect H. pylori possessing the 120-128 kDa antigen in methods such as polymerase chain reaction, ligase chain reaction and hybridization. Alternatively, the 3648 base pair sequence can be in a vector in a suitable host and utilized to produce an antigenic fragment of TagA.
Another example of a nucleic acid encoding an antigenic fragment of the 120-128 kDa antigen is a recombinant fragment (orv220) of the tagA gene comprising nucleotides 1921 through 3648 of SEQ ID Nos:1 and 3 (Example 4). This specific nucleic acid can be used to detect H. pylori strains expressing the 120-128 kDa antigen in methods such as polymerase chain reaction, ligase chain reaction and hybridization. Alternatively, the nucleic acid can be in a vector in a suitable host and utilized to produce an antigenic fragment of TagA.

Nucleic acids encoding other antigenic fragments of TagA can be obtained by the skilled artisan using routine methods and a routine amount of experimentation following the procedures described herein for obtaining the exemplary nucleic acids. Other methods for obtaining nucleic acid fragments of a known isolated nucleic acid, such as restriction digests, can be applied to the larger coding sequence to obtain nucleic acids encoding other fragments of the antigen. The nucleic acids so obtained WO9~/10639 PCT~S95/12669 2~U~53 can be routinely screened for antigenicity, specificity, etc., according to the examples herein.

Changes in the nucleotide sequence of the exemplary nucleic acids encoding TagA or antigenic fragments thereof are also contemplated as long as the antigenicity of the polypeptide encoded by the nucleic acids is maintained. Likewise, nucleic acids used as primers or probes can have substitutions so long as enough complementary bases exist for selective or specific hybridization (44).

Another example of the nucleic acid encoding the TagA antigen is an alternative coding sequence for the antigen, obtained based on the degeneracy of the genetic code. Having provided one amino acid sequence of the TagA
antigen (SEQ ID NO:3), the skilled artisan can determine the nucleotide sequence that encodes the antigen or a fragment of the antigen, and can generate the nucleic acid using the existing techniques.

An isolated nucleic acid that selectively hybridizes with the nucleic acid comprising nucleotides 1072 through 4614 contained in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO:3 under polymerase chain reaction conditions is also contemplated.
The term "selectively" as used herein refers to a nucleic acid that will hybridize under PCR conditions with the reference nucleic acid at a level distinguishable from background or random hybridization, but may also hybridize with selected other nucleic acids at a level clearly ~ WO96/10639 ~ 3 5 3 PCT~S95/12669 distinguishable over background or random hybridization.
The selectively hybridizing nucleic acid can be complementary to the above nucleic acid. The degree of sequence identity and length of the hybridizing sequences can be selected based on the intended use of the particular sequence. If used as primers, the invention provides compositions including at least two nucleic acids which selectively hybridize with different regions so as to amplify a desired region. Exemplary PCR conditions are provided herein, are well known in the art, and are taught by the providers of PCR reagents and apparatus.

An isolated nucleic acid that specifically hybridizes with the nucleic acid comprising nucleotides 1072 through 4614 contained in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO:3 under the stringency conditions of 68C for 16 hours in buffer containing 6X SSC, 0.5~ sodium dodecyl sulfate, 5X
Denhardt's solution, with washing at 60C in 0.5X SSC is also contemplated (Example 1, southern hybridization).
Other examples of hybridization conditions are also provided. Specific hybridization conditions exclude nucleic acids that hybridize with nucleic acids other than the reference nucleic acid. Thus, the hybridizing nucleic acid can be complementary to a segment of or all of the above nucleic acid, or it can have sufficient complementarity with the segment to which it hybridizes to prevent it from binding to other naturally occurring nucleic acids. The sequences to which the nucleic acid will hybridize can be selected based on the nucleotide sequence and the utility of the particular sequence. For WO96/10639 ~ 3 ~ ~ PCT~S95/12669 example, the specifically hybridizing nucleic acids can be used as probes for detecting the presence of an organism that has the nucleic acid to which it hybridizes.
Alternatively, the hybridizing nucleic acids can encode the fragments of TagA provided herein.

Also provided is an isolated nucleic acid that hybridizes with the nucleic acid encoding the H. pylori TagA under stringent conditions and has at least 70~
complementarity with the segment and strand of the nucleic acid of SEQ ID NO:3 to which it hybridizes. The hybridizing nucleic acids of the invention can have at least 70~, 80~, 85~, 90~, 95~, 97~, 98~ and 99~
complementarity with the segment and strand of the exemplary sequence to which it hybridizes. The nucleic acids can range from at least 18 to 4000 nucleotides in length. Thus, the nucleic acid can encode TagA from another tagA+ strain, or can be used as a probe or primer for detecting the presence of tagA+ H pylori.
The invention provides examples of these nucleic acids of H. pylori, so that the degree of complementarity required to distinguish specifically hybridizing from nonspecifically hybridizing nucleic acids under specific conditions can be clearly determined for each nucleic acid. The nucleic acids can be double stranded or single stranded depending on the intended use (e.g. as a coding sequence or a primer/probe, respectively). It should also be clear that a specifically hybridizing nucleic acid will not hybridize with nucleic acids encoding unrelated proteins.

~ WO96/10639 ~ 2~ ~ ~ 3 5 3 PCT~Sg5/12669 One skilled in the art can readily obtain the nucleic acids of the present invention using routine methods to synthesize a full gene as well as shorter nucleotide fragments. For example, techniques for obtaining nucleic acids such as those provided in the Sequence Listing are specifically provided in the application. Furthermore, additional methods are provided in the art that can be utilized without significant modification. Ferretti et al. (75) and Wosnick et al.
(76) show routine methods to synthesize a gene of known sequence. More specifically, Ferretti et al. teach the synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides. The synthesized gene was faithful to the known sequence (first sentence, page 603), demonstrating the reliability of this method of gene synthesis. Additionally, Wosnick et al. teach the synthesis of a maize glutathione-transferase (GST) gene using an efficient, one-step annealing/ligation protocol.
This technique also produced a complete synthetic gene with 100~ fidelity, which demonstrates the routine nature of this protocol.

Antigen Purified antigenic polypeptides encoded by the nucleic acids of the present invention are also contemplated. As used herein, "purified" means the antigen is sufficiently free of cont~m;n~nts or cell components with which the antigen normally occurs to distinguish the antigen from the cont~m;n~nts or components. Thus, the purified antigenic polypeptide is WO96/10639 ~ ~ ~ 7 3 ~ ~ PCT~S95112669 sufficiently separated from cont~m;n~nts, so that it can be used in a clinical diagnostic or other laboratory protocol. The purified approximately 120-128 kDa full-length antigen and antigenic fragments of the present invention are also referred to herein as "the antigen,"
'~the TagA antigen" or "the cagA antigen."

Specifically, an approximately 130 kDa full length TagA antigenic polypeptide (SEQ ID NO:4) is encoded by an open reading frame of 3543 base pairs within the 4821 base pair cloned insert, consisting essentially of the amino acids encoded by nucleotides 1072 through 4614 contained in the nucleotide sequence defined in the Sequence Listing as SEQ ID NO:3.
An approximately 96 kDa antigenic fragment of TagA is encoded by an open reading frame of 2577 base pairs within the 3648 base pair cloned insert, consisting essentially of the amino acids encoded by nucleotides 1072 through 3648 contained in the nucleotide sequence defined in the Sequence Listing as SEQ ID NO:l.

Another antigenic fragment of TagA is encoded by an open reading frame (orv220), consisting essentially of the amino acids encoded by nucleotides 1921 through 3648 contained in the nucleotide sequence defined in the Sequence Listing as SEQ ID NO:l.

An antigenic fragment of the antigen can be selected by applying the routine technique of epitope mapping to the TagA protein to determine the regions of 96/10639 ~ PCT~S95/12669 the protein that contain epitopes reactive with serum antibodies or capable of eliciting an immune response in an animal. Once the epitope is selected, an antigenic polypeptide containing the epitope can be synthesized S directly, or produced recombinantly by cloning nucleic acids encoding the polypeptide in an expression system, according to the standard methods. Alternatively, an antigenic fragment of the antigen can be isolated from the whole antigen or a larger fragment by chemical or mechanical disruption. The purified fragments thus obtained can be tested to determine their antigenicity and specificity by the methods taught herein. An antigenic fragment is defined as an amino acid sequence of at least about 5 consecutive amino acids derived from the antigen amino acid sequence that is reactive with (binds) an antibody.

Once the amino acid sequence of the antigenic polypeptide is provided, antigenic polypeptides can be designed that correspond to amino acid sequences of the native antigen, but with modifications in the form of substitutions, inclusions or deletions of particular amino acid residues in the derived sequences. The modifications can include attaching the antigen to sequences designed to provide for some additional property, such as solubility.
The modifications can include other amino acids that provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase bio-longevity, alter enzymatic activity, or alter interactions with gastric acidity. In any case, the peptide must possess a bioactive property, such as WO96/10639 ~ 2 Q 7 3 ~ ~ PCT~S95/12669 antigenicity, immunogenicity, speciflcity etc. The polypeptides so designed can be tested for antigenicity, immunogenicity and specificity by the methods used and described herein. These polypeptides can then be synthesized, using standard peptide synthesis techniques.
Thus, synthesis or purification of an extremely large number of polypeptides derived from the exemplary sequence of the TagA antigen is possible.

10 Dete~^m;n;n~J Tmml~nogenicity The purified antigenic polypeptides can be tested to determine their immunogenicity and specificity.
Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to an animal and the immunological response ~e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human or a guinea pig, the condition of the subject, the size of the subject, etc. Thereafter an animal so inoculated with the antigen can be exposed to the bacterium to determine the vaccine effect of the specific antigenic fragment. The speciflcity of the fragment can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other closely related bacteria.

Vectors and Host~
A vector comprising the nucleic acids of the present invention is also provided. The vectors of the invention can be in a host suitable for expressing the -WO96/10639 ~2 ~ ~ 3 ~ 3 PCT~S95/12669 polypeptides encoded by the nucleic acid. Specific example of vectors and hosts are provided below in the Examples.

There are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseu~o~on~.~ species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, the carboxy-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used.
There are several advantages to yeast expression systems.
First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing.
Second, post-translational glycosylation is efficiently WO96/10639 ~ 3 ~ ~ PCT~S95/12669 carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MF~-l gene) is routinely used to direct protein secretion from yeast (45). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
The antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or ~-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.

Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of antigen in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal. The wog6/1063s 22 0 1 3 53 Pcrlusssn2669 vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
The antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector. Presence of the vector DNA in transformed cells can be confirmed by Southern analysis and production of an RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.

Alternative vectors for the expression of antigen in m~mm~l ian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, WO96/10639 2 a Q ~ ~ 5 3 PCT~S95/1266s hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted DNAs in m~mm~l ian cells (such as COS7).

The DNA sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of, an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells .
transformed with the desired DNA sequences (see, e.g., U.S. Patent 4,704,362).

Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.

~09611~639 ~ 2 0 1 ~ ~ 3 PCT~S95/1~669 Purified Antibodies A purified monoclonal antibody that specifically binds the TagA antigen or antigenic fragment is also provided. The antibody can specifically bind a unique epitope of the antigen it can also bind epitopes of other organisms. The term "bind" means the well understood antigen/antibody binding as well as other nonrandom association with an antigen. "Specifically bind" as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, the TagA antigen. Antibodies can be made as described in the Examples (see also, Harlow and Lane (46)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Polyclonal antibodies can be purified directly, or spleen cells from the animal can be fused with an immortal cell line and screened for monoclonal antibody secretion. Thus, nonhuman polyclonal antibodies that specifically bind the antigen are within the scope of the present invention.

A ligand that specifically binds the antigen is also contemplated. The ligand can be a fragment of an antibody or a smaller molecule designed to bind an epitope of the TagA antigen. The antibody or ligand can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the composition of the present invention are those listed below in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers.

WO96/10639 ~ 3 5 3 PCT~S95/12669 Antigen Bound to Substrate A purified TagA antigen bound to a substrate is also provided. The antigen can also be bound to a purified antibody or ligand. The antibody can be a monoclonal antibody obtained by standard methods and as described herein.

Serological Detection (Diagnosis) Methods Detecting Antibody with Antigen The present invention provides a method of detecting the presence of a H. pylori strain possessing the 120-128 kDa antigen in a subject, comprising the steps of contacting an antibody-containing sample from the subject with a detectable amount of the TagA or TagA
antigenic fragment of the present invention and detecting the binding reaction of the TagA or fragment and the antibody produced by the subject, the binding indicating the presence of the toxic H. pylori strain or previous infection with the toxic H. pylori strain. There are numerous routine immunological assays that can be used in the present detection and predisposition methods.
Examples are provided below.

Detecting Antigen with Antibody/Ligand One example of the method of detecting H. pylori possessing the antigen is performed by contacting a fluid or tissue sample from the subject with an amount of a purified antibody specifically reactive with the antigen, and detecting the binding of the antibody with the antigen. It is contemplated that the antigen will be on WO96110639 ~ 5 3 PCT~S95/12669 intact cells containing the antigen, or will be fragments of the antigen. As contemplated herein, the antibody includes any ligand which binds the antigen, for example, an intact antibody, a fragment of an antibody or another reagent that has reactivity with the antigen. The fluid sample of this method can comprise any body fluid which would contain the antigen or a cell containing the antigen, such as blood, plasma, serum, saliva and urine.
Other possible examples of body fluids include sputum, mucus, gastric juice and the like.

ELISA
Immunoassays such as immunofluorescence assays (IFA), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection of the antigen. An ELISA method effective for the detection of the antigen can, for example, be as follows: (l) bind the antibody to a substrate; (2) contact the bound antibody with a fluid or tissue sample containing the antigen; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect antibody as well as antigen.

Competitive Inhibition Assay Another immunologic technique that can be useful in the detection of H. pylori expressing tagA or previous H. pylori infection utilizes monoclonal antibodies (MAbs) WO96/10639 22 ~ ~ 3 5 3 PCT~S95/12669 for detection of antibodies specifically reactive with TagA antigen. Briefly, sera or other body fluids from the subject is reacted with the antigen bound to a substrate (e.g. an ELISA 96-well plate). Excess sera is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted antigen-serum antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control (no patient serum antibody). The degree of monoclonal antibody inhibition is a very specific test for a particular variety or strain since it is based on monoclonal antibody binding specificity. MAbs can also be used for detection directly in cells by IFA.
Micro-Agglutination Assay A micro-agglutination test can also be used to detect the presence of the TagA-possessing H. pyl ori strain in a subject. Briefly, latex beads (or red blood cells) are coated with the antigen and mixed with a sample from the subject, such that antibodies in the tissue or body fluids that are specifically reactive with the antigen crosslink with the antigen, causing agglutination.
The agglutinated antigen-antibody complexes form a precipitate, visible with the naked eye or by spectrophotometer. In a modification of the above test, antibodies specifically reactive with the antigen can be ` bound to the beads and antigen in the tissue or body fluid thereby dete¢ted.

WO96/10639 ~ Z U ~ 3 ~ 3 PCT~S95/12669 Sandwich Assay/Flow Cytometry/Immunoprecipitation In addition, as in a typical sandwich assay, the antibody can be bound to a substrate and reacted with the antigen. Thereafter, a secondary labeled antibody is S bound to epitopes not recognized by the first antibody and the secondary antibody is detected. Since the present invention provides TagA antigen for the detection of toxic H. pylori or previous H. pylori infection other serological methods such as flow cytometry and immunoprecipitation can also be used as detection methods.

In the diagnostic methods taught herein, the antigen can be bound to a substrate and contacted by a fluid sample such as serum, urine, saliva or gastric juice. This sample can be taken directly from the patient or in a partially purified form. In this manner, antibodies specific for the antigen (the primary antibody) will specifically react with the bound antigen.
Thereafter, a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody. Generally, the secondary antibody or other ligand which is reactive, either specifically with a different epitope of the antigen or nonspecifically with the ligand or reacted antibody, will be selected for its ability to react with multiple sites on the primary antibody. Thus, for example, several molecules of the secondary antibody can react with each primary antibody, making the primary antibody more detectable.

WO96/10639 ~ 3 5 ~ PCT~S9S/12669 Detectable Moieties The detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like.
Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy), alkaline phosphatase (for biochemical detection by color change) and radioisotopes (for radiography). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (46).

Detecting Disease or Predispo~ition to Disease Peptic Ulceration Because the purified TagA antigen provided herein is associated with peptic ulceration, the present invention also provides a method of determining predisposition to peptic ulceration in a subject. The method can be accomplished according to the methods set forth above for the detection of H. pyl ori expressing the TagA antigen or for the detection of antibodies specific to the TagA antigen or for the detection of specific antibodies to the TagA antigen. The presence of the TagA
antigen or TagA specific antibodies indicates a predisposition of the subject to peptic ulceration. The methods described below for detecting nucleic acids specific for tagA+ strains can also be used.

W096llU639 ~ ~ n 1 3 5 ~ rCT~S95/12669 Gastric Carcinoma Because the purified TagA protein provided herein is associated with gastric cancer, the present invention also provides a method of determining predisposition to gastric carcinoma in a subject. The method can be accomplished according to the methods set forth above for the detection of tagA+ H. pylori strains or for the detection of antibodies specific to the TagA
antigen or for the detection of specific antibodies to the TagA antigen. The presence of the TagA antigen or TagA
specific antibodies indicates a predisposition of the subject to gastric carcinoma. Example 4 provides supporting human data and an example of a specific protocol for practicing this method. The methods described below for detecting nucleic acids specific for tagA+ strains can also be used.

Treatment Methods Methods of treating peptic ulcers in a subject using the compositions of the present invention are provided. For example, in one such method an amount of ligand (e.g., antibody or antibody fragment) specifically reactive with the approximately 120-128 kDa antigen of H.
pylori sufficient to bind the antigen in the subject and improve the subject's clinical condition is administered to the subject. Such improvement results from the ligand interfering with the antigen's normal function in inducing inflammation and cellular damage. The ligand can be a purified monoclonal antibody specifically reactive with the antigen, a purified polyclonal antibody derived from a nonhuman animal, or other reagent having specific WO96/10639 PCT~S95/12669 2~ n 13 ~3 reactivity with the antigen. Additionally, cytotoxic moieties can be conjugated to the ligand/antibody by standard methods. Examples of cytotoxic moieties include ricin A chain, diphtheria toxin and radioactive isotopes.
Another method of treating peptic ulcers in a subject comprises administering to the subject an amount of a ligand/antagonist for a receptor for the 120-128 kDa antigen of H. pylori sufficient to react with the receptor and prevent the binding of the 120-128 kDa antigen to the receptor. An antagonist for the receptor is thus contemplated. The result is an improvement in the subject's clinical condition. Alternatively, the treatment method can include administering to the subject an amount of an analogue of a TagA receptor to result in competitive binding of the TagA antigen, thus inhibiting binding of the TagA antigen to its wild type receptor.
The receptor is localized on cells present in the gastroduodenal mucosa, such as epithelial cells, inflammatory cells, or endothelial cells.

Because the expression of TagA is shown to be associated with gastric carcinoma, the above treatment methods are applicable to the treatment or prevention of gastric carcinoma.

Mutant Organism The present invention also provides a mutant H.
pylori in which the tagA gene product has been rendered nonfunctional. The mutant can either not express tagA or express a non-functioning TagA antigen. In one example, WO96/1~639 ~ I 0 1 3 53 PCT~S95/l2669 the mutant H. pylori strain is obtained by making a substitution mutation in the coding sequence for the TagA
antigen as described in Example 2. Since the present invention provides the nucleic acid encoding the antigen, other methods of mutating the coding sequence of the antigen can be used to obtain other mutant strains as contemplated herein. An example of the mutant H. pylori strain of the present invention is designated 84-183:M22 and is deposited with the American Type Culture Collection (1230 Parklawn Drive, Rockville, MD 20852) under ATCC
Accession Number 55359.

Additional isogenic mutants can be prepared, for example, by inserting a nucleic acid in the tagA gene or deleting a portion of the tagA gene so as to render the gene non-functional or produced in such low amounts that the organism is non-infectious. Furthermore, by providing the nucleotide sequence for the nucleic acid encoding the antigen, the present invention permits the making of specific point mutations having the desired effect. The deletion, insertion or substitution mutations can be made in the gene sequence in either the regulatory or coding region to prevent transcription or to render the transcribed product nonfunctional.
One such approach to the construction of a deletion or insertion mutant is via the Donnenberg method (47). A deletion in tagA is created by deleting a 0.2 kb BamH1-NdeI fragment and religating the tagA clone. This mutant is cloned into suicide vector pILL570. The sacB
gene of Bacillus subtilis is also cloned into the suicide WO96/10639 PCT~S95/12669 22~1353 vector to provide a conditionally lethal phenotype. This construct is transformed into H. pylori by electroporation, and transformants selected by spectinomycin resistance. The merodiploid strain which contains the suicide vector and the mutated version of the tagA gene are exposed to sucrose to directly select for organisms that have undergone a second recombination, resulting in the loss of the vector. These and other well known methods of making mutations can be applied to the nucleic acids provided herein to obtain other desired mutations.

Non-isogenic mutants are also within the scope of the invention. For example, a live attenuated H.
pylori that is also a tagA- mutant according to the present invention, is provided. A tagA-recA- mutant strain is constructed, for example, by insertion mutation of both the tagA and recA genes, according to the methods taught herein and taught in U.S. serial no. 08/215,928 for recA.
A tagA-vacA- mutant strain is constructed, for example, by insertion mutation of both the tagA and vacA genes, according to the methods taught herein for tagA and in U.S. Application Serial No. 08/215,928, which describes the generation of a vacA mutant. A recA-tagA-vacA~ mutant strain is constructed, for example, by insertion mutation of the recA, tagA and vacA genes, according to the methods taught herein for recA and vacA, and taught in U.S.
serial no. 08/215,928. Any of the well known methods of mutating a gene can be used in the present invention to generate H. pylori mutant strains. The strains can be tested as provided for immunogenicity.

WO96/10639 ~2 ~ ~ 3 5 ~ PCT~S95/12669 Vaccine~
The antigen, antigenic fragment or mutant H.
pylori of this invention can be used in the construction of a vaccine comprising an immunogenic amount of the antigen or mutant H. pylori and a pharmaceutically acceptable carrier. The vaccine can be the entire antigen, the antigen on an intact H. pylori, E. coli or other strain. The vaccine can then be used in a method of preventing peptic ulceration or other complications of H.
pylori infection (including atrophic gastritis and malignant neoplasms of the stomach).

Immunogenic amounts of the antigen can be determined using standard procedures. Briefly, various concentrations of a putative specific immunoreactive epitope are prepared, administered to an animal and the immunological response (e.g., the production of antibodies) of an animal to each concentration is determined.
The pharmaceutically acceptable carrier in the vaccine of the instant invention can comprise saline or other suitable carriers (48). An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the subject (48).
Methods of administration can be by oral or sublingual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.

W096/10639 ~2 0 1 3 5 3 PCT~S95/12669 It can be appreciated from the above that the vaccine can be used as a prophylactic or a therapeutic modality. Thus, the invention provides methods of preventing or treating H. pylori infection and the associated diseases by administering the vaccine to a subject.

Nucleic Acid Detection (Diagnosis) Methods The presence of the TagA antigen and H. pyl ori possessing the TagA antigen can also be determined by detecting the presence of a nucleic acid specific for the antigen. The specificity of these sequences for the antigen can be determined by conducting a computerized comparison with known sequences, catalogued in GenBank, a computerized database, using the computer programs Word Search or FASTA of the Genetics Computer Group (Madison, WI), which search the catalogued nucleotide sequences for similarities to the gene in question.

The nucleic acid specific for the antigen can be detected utilizing a nucleic acid amplification technique, such as polymerase chain reaction or ligase chain reaction. Alternatively, the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism. For example, the present invention provides a method of detecting the presence of H. pylori, possessing the TagA antigen, comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site. In addition, PCR primers which hybridize only with nucleic acids specific for the antigen can be WO96/10639 ~ PCT~S9S/12669 utilized. The presence of amplification indicates the presence of the antigen. In another embodiment a restriction fragment of a DNA sample can be sequenced directly using, for example, Sanger ddNTp sequencing or 7-deaza-2'-deoxyguanosine 5'-triphosphate and Taq polymerase and compared to the known unique sequence to detect H.
pylori. In a further embodiment, the present invention provides a method of detecting the presence of tagA-containing H. pylori by selective amplification by the methods described above. In yet another embodiment H.
pylori can be detected by directly hybridizing the unique sequence with a tagA specific nucleic acid probe.
Furthermore, the nucleotide sequence could be amplified prior to hybridization by the methods described above.
Once specific variable sequences are shown to be associated with peptic ulceration, the methods to detect these sequences are standard in the art. Detection of point mutations or variable sequences using direct probing involves the use of oligonucleotide probes which may be prepared, for example, synthetically or by nick translation. The probes may be suitably labeled using, for example, a radio label, enzyme label, fluorescent label, biotin-avidin label and the like for subsequent visualization in the example of Southern blot hybridization procedure. The labeled probe is reacted with a bound sample DNA, e.g., to a nitrocellulose sheet under conditions such that only fully complementary sequences hybridize. The areas that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction.

W096/10639 ~ ~ n 1 3 5 ~ PCT~S95/12669 The areas of the filter that exhibit such labeling may then be visualized, for example, by autoradiography. The labeled probe is reacted with a DNA sample bound to, for example, nitrocellulose under conditions such that only fully complementary sequences will hybridize. The stringency of hybridization is usually 5C below the Ti (the irreversible melting temperature of the hybrid formed between the probe and its target sequence) for the given chain length. For 2Omers the recommended hybridization temperature is about 58C. The washing temperatures are unique to the sequence under investigation and need to be optimized for each variant.

Alternative probing techniques, such as ligase chain reaction (LCR), involve the use of mismatch probes, i.e., probes which are fully complementary with the target except at the point of the mutation. The target sequence is then allowed to hybridize both with oligonucleotides which are fully complementary and have oligonucleotides containing a mismatch, under conditions which will distinguish between the two. By manipulating the reaction conditions, it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present there is significantly reduced hybridization.
The polymerase chain reaction (PCR) is a technique that amplifies specific DNA sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealing and extension carried out with polymerase, e.g., a heat stable enzyme Taq polymerase, leads to exponential increases in the concentration of , WO96/10639 ~ 3 PCT~S95/12669 desired DNA sequences. Given a knowledge of the nucleotide sequence of a mutation, synthetic oligonucleotides can be prepared which are complementary to sequences which flank the DNA of interest. Each 5 oligonucleotide is complementary to one of the two strands. The DNA can be denatured at high temperatures ~e.g., 95C) and then reannealed in the presence of a large molar excess of oligonucleotides. The oligonucleotides, oriented with their 3' ends pointing 10 towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates. The end product is then denatured again for another cycle. After this three-15 step cycle has been repeated several times, amplification of a DNA segment by more than one million-fold can be achieved. The resulting DNA may then be directly sequenced in order to locate any genetic alteration.
Alternatively, it may be possible to prepare 20 oligonucleotides that will only bind to altered DNA, so that PCR will only result in multiplication of the DNA if a mutation is present. Following PCR, direct visualization or allele-specific oligonucleotide hybridization may be used to detect disease associated 25 with a point mutation. Alternatively, an adaptation of PCR called amplification of specific alleles (PASA) can be employed; this uses differential amplification for rapid and reliable distinction between alleles that differ at a v single base pair. Other techniques, such as 3SR, which 30 utilize RNA polymerase to achieve high copy number, can also be used where appropriate.

WO96/10639 a ~ o l 3 ~ 3 PCT~S9S/12669 In yet another method, PCR may be followed by restriction endonuclease digestion with subsequent analysis of the resultant products. Nucleotide substitutions can result in the gain or loss of specific restriction endonuclease site. The gain or loss of a restriction endonuclease recognition site facilitates the detection of the disease associated mutation using restriction fragment length polymorphism (RFLP) analysis or by detection of the presence or absence of a polymorphic restriction endonuclease site in a PCR product that spans the sequence of interest.

For RFLP analysis, DNA is obtained, for example from the blood, gastric specimen, saliva, dental plaque, other bodily fluids or stool of the subject suspected of containing tagA-possessing H. pylori, or H. pylori isolated from subject, and from a subject infected with nontoxic H. pylori, is digested with a restriction endonuclease, and subsequently separated on the basis of size by agarose gel electrophoresis. The Southern blot technique can then be used to detect, by hybridization with labeled probes, the products of endonuclease digestion. The patterns obtained from the Southern blot can then be compared. Using such an approach, tagA DNA is detected by determining the number of bands detected and comparing this number to the DNA from H. pylori strains that are not associated with severe disease. Restriction endonucleases can also be utilized effectively to detect mutations in the tagA gene.

WO96/10639 ~ ~ fi ~ 3 ~ ~ PCT~S95/12669 Similar creation of additional restriction sites by nucleotide substitutions at the disclosed mutation sites can be readily calculated by reference to the genetic code and a list of nucleotide sequences recognized by restriction endonucleases.
.

Single strand conformational analysis (SSCA) offers a relatively quick method of detecting sequence changes which may be appropriate in at least some instances.

In general, primers for PCR and LCR are usually about 20 bp in length and the preferable range is from 15-25 bp. Better amplification is obtained when both primers are the same length and with roughly the same nucleotide composition. PCR conditions can include denaturation of strands usually takes place at 94C and extension from the primers is usually at 72C. The annealing temperature varies according to the sequence under investigation.
Examples of reaction times are: 20 mins denaturing; 35 cycles of 2 min, 1 min, 1 min for annealing, extension and denaturation; and finally a 5 min extension step.

PCR amplification of specific alleles (PASA) is a rapid method of detecting single-base mutations or polymorphisms. PASA (also known as allele specific amplification) involves amplification with two oligonucleotide primers such that one is allele-specific.
The desired allele is efficiently amplified, while the other allele(s) is poorly amplified because it mismatches with a base at or near the 3' end of the allele-specific WO96110639 22 Q 1 3 ~ 3 PCT~S95/12669 ~

primer. Thus, PASA or the related method of PAMSA may be used to specifically amplify the mutation sequences of the invention. Where such amplification is done on H. pylori isolates or samples obtained from an individual, it can serve as a method of detecting the presence of the mutations.

As mentioned above, a method known as ligase chain reaction (LCR) can be used to successfully detect a single-base substitution. LCR probes may be combined or multiplexed for simultaneously screening for multiple different mutations. Thus, LCR can be particularly useful where, as here, multiple mutations are predictive of the same disease.
Antigen-Detecting Rit The present invention provides a kit for the diagnosis of infection by strains of H. pylori possessing the TagA antigen. Particularly, the kit can detect the presence of TagA antigen specifically reactive with an antibody or an immunoreactive fragment thereof. The kit can include an antibody bound to a substrate, a secondary antibody reactive with the antigen and a reagent for detecting binding of the secondary antibody to the antigen. Such a kit can be an ELISA kit and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above. The diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.

WO96/10639 PCT~S95/1266g

3 ~ 3 Antibody-Detecting Rit The diagnostic kit of the present invention can be used to detect the presence of a primary antibody specifically reactive with TagA or an antigenic fragment thereof. The kit can include the antigen bound to a substrate, a secondary antibody reactive with the antibody specifically reactive with the TagA antigen and a reagent for detecting binding of the secondary antibody to the primary antibody. Such a kit can be an ELISA kit and can comprise the substrate, antigen, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above. The diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.

Nucleic Acid Detection (Diagnostic) Kits Once the nucleotide sequence of the TagA antigen is determined, the diagnostic kit of the present invention can alternatively be constructed to detect nucleotide sequences specific for the antigen comprising the standard kit components such as the substrate and reagents for the detection of nucleic acids. Because H. pylori infection can be diagnosed by detecting nucleic acids specific for the antigen in gastric or duodenal tissue and body fluids such as gastric juice, urine, stool, and saliva, it will be apparent to an artisan that a kit can be constructed that utilizes the nucleic acid detection methods, such as ~ specific nucleic acid probes, primers or restriction fragment length polymorphisms in analyses. It is contemplated that the diagnostic kits will further WO96/10639 22 ~ PCT~S95/12669 comprise a positive and negative control test.

The particular reagents and other components included in the diagnostic kits of the present invention can be selected from those available in the art in accord with the specific diagnostic method practiced in the kit.
Such kits can be used to detect the antigen in tissue and fluid samples from a subject.

The following examples are intended to illustrate, but not limit, the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed.

Cloning and Expression of TagA Antigen Bacterial strains and growth conditionæ.
H. pylori strain 84-183 (ATCC 53726) was used to clone the gene for the TagA antigen. Thirty-two clinical H. pylori isolates from humans, including strains that had been previously shown to possess the antigen, were used to assess conservation of the gene and correlation with cytotoxin production (Table 1). Stock cultures were maintained at -70C in Brucella broth (BBL Microbiology Systems, Cockeysville, MD) supplemented with 15~ glycerol.
H. pylori strains were cultured in Brucella broth supplemented with 5~ fetal bovine serum in a microaerobic atmosphere (generated by CampyPak-Plus (BBL) at 37C for WO96/10639 22 Q ~ 3 ~ 3 PCT~S95/12669 48 hours. For transformation and protein expression, E.
coli strains XL1-Blue (Stratagene, La Jolla, CA), HB101 (ATCC 33694), and DH5~ (Stratagene, La Jolla, CA) were cultured in Luria-Bertoli (LB) medium with shaking at 37~C. The final concentrations of ampicillin when added to media was 100 ~g/ml.

Chemicals and enzymes.
Isopropyl-~-D-thiogalactopyranoside (IPTG) was purchased from Sigma Chemical Co. (St. Louis, MO) and used at 57 ~g/ml, and 5-bromo-4-chloro-3-indolyl-$-D-galactoside (X-GAL; final concentration 40 ~g/ml) was from Boehringer-Mannheim (Indianapolis, IN). Restriction enzymes, T4 DNA ligase, E. coli DNA polymerase large (Klenow) fragment and Sequenase~ were from Promega and United States Biochemicals (Cleveland, OH). [~_32p] dATP
(650 Ci/mmol) was from ICN Radiochemicals (Irvine, CA).

Genetic techniques and nucleotide sequence analysis.
To obtain chromosomal DNA from H. pylori 84-183, the strain was cultured for 48 h in Brucella broth containing 5~ fetal bovine serum, the cells pelleted, and resuspended in 100 mM Tris-HC1 (pH 7.2) containing 100mM
NaCl. Cells were lysed using 1~ SDS in 100 mM Tris-HC1 (pH 8.8). After chloroform-phenol extractions, the chromosomal DNA was precipitated with 100~ ethanol.
Plasmids were isolated by the rapid alkaline extraction procedure of Birnboim and Doly (49) and purification was ~ completed by precipitation in the presence of 800 mM NaCl and 6.5~ polyethylene glycol. All other standard molecular genetic techniques, including sequential ordered WO96/10639 ` 2 ~ 3 PCT~S9~/12669 deletions, were performed as described (50). The nucleotide sequence was determined unambiguously on both strands using double-stranded DNA templates and the dideoxy chain termination procedure as described previously (51). Oligonucleotide primers were synthesized by the Vanderbilt University DNA Core Facility using a Milligen 7500 DNA synthesizer, using the manufacturer's protocol. Nucleotide sequences were compiled and analyzed with the aid of the DNA-Star program (DNA Star, Inc., Madison, WI); putative promoter and Shine-Dalgarno sequences were identified by comparison with consensus sequences (52).

Construction of a genomic library from N. pylori.
Strain 84-183 chromosomal DNA was sheared by sonication and the resulting fragments were electrophoresed on a 0.7~ low melting temperature agarose gel. Fragments in the 2-10 kb size range were excised, treated with T4 DNA polymerase to produce blunt ends, and ligated to phosphorylated EcoRI octamer linkers (New England Biolabs, Beverly, MA). The DNA was digested with EcoRI and ligated to the EcoRI arms of the ~ZapII vector, according to the manufacturer's protocol. The ligation mixtures were added to the Gigapack IIa packaging mix (Stratagene) and titered on XL1-blue cells (lambda ZapII) or Y1088 (lambda gtll) cells. The amplified phage libraries were screened with adsorbed sera from an H.
pylori-infected person or by plaque hybridization.

, WO96/10639 2~ n ~ 3 5 3 PCT~S95/12669 Cloning of H. pylori-specific gene~.
Serum from an H. pylori-infected person that strongly recognizes the 120-128 kDa antigen was adsorbed with H. pylori strain 86-313, which does not produce the 120-128 kDa band, and with E. coli cells to reduce the likelihood of nonspecific reactivity and then used to screen a bank of genes from the amplified AZapII phage library (53). The bank contained approximately 4x104 insertions. The amplified phage library was screened by allowing approximately 105 plaques to grow on XL1 Blue cells for 2.5 h at 42C, overlaying with a nitrocellulose filter previously impregnated with 10 mM IPTG, and incubating for 2 h at 37C. The filters were then screened with the adsorbed serum to detect 9 reactive clones. Positive plaques were then plaque purified, and lysates were prepared from these infected E. coli cells.
The lysates were immunoblotted with the adsorbed serum and clones expressing recombinant proteins were saved. By immunoblotting with the adsorbed human serum, each of the XLl-Blue lysates showed a strongly immunoreactive band migrating at either approximately 75, 85, or 96 kDa, corresponding to plasmids pMCl, pMC2, or pMC3, respectively.

From~the three representative clones, the pBluescript plasmids containing the cloned DNA inserts were excised by co-infèction with helper phage, as detailed (54), and fresh XLl-Blue cells transformed. After plasmid purification, restriction enzyme cleavage maps were generated and the plasmids used for further characterization. In a parallel study, four clones were WO96/10639 22 ~ 53 pcT~s9sll266s isolated from a Agtll library of H. pylori 84-183 DNA by the same methodology, and the DNA insert from one of four positive clones was amplified by polymerase chain reaction (PCR) using primers based on the known flanking Agtll sequences. Recombinant phage DNA from four positive plaques was purified, and each contained a 0.6 kb insert.
Immunoblot analysis of lysates from two clones (AYBl and AYB2) showed similar sized 130 kDa bands that reacted with the adsorbed human antiserum. To determine whether the 130 kDa protein was synthesized by a recombinant phage as a fusion protein, cell lysate prepared from AYBl was subjected to immunoblot analysis using ~-galactosidase specific antiserum. The cross-reactivity shown indicates that the recombinant clone AYBl contains a fusion of the Agtll ~-galactosidase large (116 kDa) fragment and an H.
pylori open reading frame. We cloned the AYBl insert into pUCl9, but the recombinant (pYBl) did not express any protein.

Gel electrophoresis and immunoblot analysis.
Lysates from E. coli carrying recombinant lambda gtll, AZapII or pBluescript were analyzed by SDS-PAGE and immunoblotting with adsorbed human serum. Discontinuous sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (P~GE) was performed as described previously (55) by using a 4.5~ stacking gel and a 7.0 separating gel. Samples containing 3 ~g of protein were applied to each gel lane. After electrophoresis, gels were fixed and proteins were resolved by the modified silver stain method of Oakley et al. (56). Concentrated ~ WO96/10639 ~2 ~ 1 3 5 3 PCT~S95/12669 culture supernatants containing protein were diluted in sample buffer and were layered onto the surface of a polyacrylamide gel in a Mini-PROTEAN II slab cell (Bio-Rad Laboratories, Richmond, Calif.). Following electrophoresis, proteins were transferred to nitrocellulose paper by electro blotting for 1 h at 1 amp.
After nonspecific binding was blocked, the nitrocellulose paper was incubated at room temperature for 1 h with 1:100 dilutions of serum samples. Alkaline-phosphatase conjugates of goat anti-human IgG, (Tago, Inc., Burlingame, Calif.), in a dilution of 1:2,000 were used as the second antibody.

Southern hybridization.
H. pylori or C. jejuni chromosomal DNA was digested with either HindIII or EcoRI and BamHI and the resulting fragments were electrophoresed on a 0.7~ agarose gel in 0.04 M Tris-acetate -2 mM EDTA buffer (pH 8.2).
All hybridization conditions and procedures were exactly as described (50). Probes were radiolabeled by primer extension using random hexamers (57). Hybridization was carried out at 68C for 16h in buffer containing 6X SSC
(lX SSC is 0.15M NaCl, 0.015M sodium citrate), 0.5~ sodium dodecyl sulfate (SDS), 5X Denhardt's solution (lX
Denhardt's solution is 0.02~ Ficoll, 0.02~
polyvinylpyrrolidone, 0.02~ bovine serum albumin), and 100 ~g/ml salmon sperm DNA. The blots were washed at 60C in 0.5X SSC and exposed to XAR-2 X-ray film (Eastman Kodak, Rochester, NY).

WO96/10639 2~ ~ 1 3 5 3 PCr/US95/12669 ~

Colony hybridization.
H. pylori strains were grown on trypticase soy blood agar plates (BBL) and replica copies of these colonies were transferred to nitrocellulose filters. Each 5 filter was placed on 3 mm Whatman paper saturated with 0.2 M NaOH/1.5M NaCl. After 3 min the filter was transferred to 3 mm Whatman paper saturated with 0.4M Tris-Cl (pH
7.6)/2X SSC for 3 min, and then to 2X SSC for 3 min. The colony blot filters were dried in a vacuum oven for 90 min 10 at 80C and hybridized with radiolabeled pMC3 as described (50).

Cytotoxin production.
H. pylori broth culture supernatants were 15 concentrated 30-fold by ultrafiltration, passaged through a 0.2 ~M filter, and incubated with HeLa cells. Briefly, H. pylori strains were cultured at 37C in brucella broth (BBL, Microbiology Systems, Cockeysville, MD.) containing 5~ defined fetal bovine serum (Hyclone, Logan, Utah), 20 supplemented with 10 mM ammonium chloride to potentiate cytotoxin activity. Broth cultures were incubated in a microaerobic atmosphere on a gyratory shaker at 100 rpm for 72 h. Cultures were centrifuged at 3,000 x g for 15 min, and the cell-free supernatants were stored at -70C.
25 After thawing, supernatants were concentrated 30-fold by using a 30-kDa ultrafiltration membrane, and retentates were sterilized by passage through a 0.22-~m-pore-size filter. These concentrated culture supernatants (CCSs) were incubated with HeLa cells (obtained from Allison 30 O'Brien, Uniformed Services University of the Health Sciences, Bethesda, Md.) in twofold dilutions from 1:10 to WO96/10639 ~2 n ~ 3 ~3 PCT~sg5/1266s 1:320 as described previously (39), except that toxicity assays were performed in a total volume of 100 ~1 in 96-well microtiter plates (Falcon; Becton Dickinson and Co., Lincoln Park, N.J.).
Vacuolation of HeLa cells was quantitated using a neutral red uptake assay. Briefly, a stock solution of 0.5~ purified grade neutral red (Sigma Chemical Co., St.
Louis, Mo.) was prepared in 0.9~ saline and filtered with Whatman no. 1 filter pater. Staining solutions were prepared before each experiment by diluting the stock solution 1:10 in Eagle medium containing 10~ fetal bovine serum. After incubation with test samples for 24 h the medium overlaying HeLa cells was removed and replaced with 100 ~l of staining solution per well for 4 min. The cells were washed twice with 150 ~l of 0.9~ saline per well, and the neutral red was extracted from cells by the addition of 100 ~l of acidified alcohol per well (58). The optical density (OD) at 540 nm of wells was determined by using an MR700 enzyme-linked immunosorbent assay reader (Dynatech, Alexandria, VA.). All assays were performed in triplicate. In all experiments, the mean OD of wells containing cells incubated with medium alone was less than 0.130 (mean, 0.101 + 0.007); this background OD was subtracted from the OD of experimental wells to yield a net OD. Of the 32 H. pylori strains tested, 15 produced the vacuolating cytotoxin, as determined in this assay (Table 3).
.

WO96/10639 PCT~S95/12669 2a Q 13 ~3 Mapping the pBluescript inserts.
After digestion with EcoRI, plasmids pMCl, pMC2, and pMC3 were found to contain DNA inserts of approximately 2.5, 2.7, and 3.6 kb, respectively.
Analysis of restriction endonuclease treatment of the recombinant plasmids identified a conserved l.2 kb HindIII-digestion fragment in all three (Figure l). As such, further studies concentrated on pMC3, which contained the largest insert. Analysis of deletion mutations produced by exonuclease III digestion, identified the orientation and approximate location of the open reading frame (ORF) (Figure l, large arrow).

Sequence analysis of pMC3 and pYBl.
To determine the sequence of the 3.6 kb~insert in pMC3, a series of nested ordered deletions of the plasmid using exonuclease III (Figure l) was performed, as described (50). In total, the sequence for the entire pMC3 insert representing 3648 bp was determined on both strands (SEQ ID NO:l). The nucleotides are numbered on the right of each line. The nucleotides encoding the glycine at residue number 859 of SEQ ID NO:l are an artifact of the cloning process and are not a part of the tagA gene. SEQ ID NO:2 provides the deduced amino acid sequence of the nucleic acid shown in SEQ ID NO:l.

A long open reading frame commencing at nucleotide 1072 continues to the termination of the insert. Two other open reading frames in the opposite orientation begin at 645 bp and 264 bp. The deduced amino acids are shown beneath the nucleotides. Potential WO96/10639 ~2 Q ~ 3 5 3 PcrluS95/12669 ribosomal binding-sites (Shine-Delgarno sequence; SD), and putative promoter elements (-35 and -l0 sequences) are indicated. Only a single ORF exceeding 300 bases was found in any of the six possible reading frames. This ORF
5 encodes a TagA antigen of 859 amino acids, yielding a predicted protein with a molecular weight of 96,022 (SEQ ID NO: 2). The direction of transcription deduced from this ORF is also in agreement with that determined previously by the use of the deletion mutants. However, l0 there is no translation termination signal, indicating that the ORF in pMC3 is truncated. The truncated fragment is rich in basic amino acids (Table 2) and the predicted isoelectric point is 8Ø A potential ribosomal binding site (AGGAG) ends 6 bp upstream of the ORF. The sequence 15 112 bp upstream of the translational start site exhibits the promoter sequence TATAGT (SEQ ID NO: l) which resembles the Pribnow consensus promoter sequence TATNATN
(Hawley and McClure). This putative -l0 region, which is similar to a sigma-70 promoter, is associated with a -35 20 region, ATGCCA, which shares 4 of 6 bases with the corresponding consensus sequence, TTGACA (52). The deduced amino acid composition of the truncated polypeptide is shown in Table 2.

Two smaller ORFs, each proceeding in the opposite direction, also were identified (SEQ ID NO: l).
The first, encoding a polypeptide of 79 amino acids, commences at bp 645 and is not proceeded by an obvious Shine Delgarno or putative promoter sequence. The second ORF commences at bp 264 and encodes 88 amino acids before the end of the insert. This truncated ORF is preceded by WO96/10639 ~2 ~ 1 ~ 9 3 PCT~S95/12669 a Shine Dalgarno sequence, and the sequence TTTGAT 90 bp upstream of the translational start site resembles the -10 consensus promoter site, followed by the sequence TTGTCA, which shares 5 of 6 bases with the -35 consensus sequence (52).

The 0.6 kb insert in pYB1 was sequenced using both forward and reverse primers of the known Agtll flanking sequences along with additional primers based on experimentally-derived insert sequences. The first 464 bases of the 620 bp pYBl sequence overlapped with the end of pMC3, but the ORF still continued.

Serologic recognition of the truncated recombinant TagA
antigen.
In addition to the index case, sera from H.
pylori-infected persons that recognize the TagA antigen from H. pylori strain 84-183 recognize the recombinant polypeptide. For this analysis, we studied serum from 6 persons not infected with H. pylori, and from 14 infected persons (7 did and 7 did not recognize the 120-128 kDa antigen from strain 84-183). Using lysates of E. coli XL1-Blue transformed with pMC3 and immunoblotting, there is clear recognition of the 96 kDa antigen by human serum IgG. In total, 4 of 7 sera that recognize the native 120-128 kDa band also strongly recognize the recombinant protein versus none of the 13 sera tested that do not recognize the 120-128 kDa band (p=0.007, Fisher's exact test, 2-tailed). If weak reactions to the pMC3 band are considered, then all 7 sera that recognize the 120-128 kDa band, and 3 of 13 of the non-recognizing sera react to the ~ WO96/10639 22 0 1 3 ~ 3 PCT~S95112669 recombinant protein (p=0.003, Fisher's exact test, 2-tailed). Thus, the recombinant protein produced by pMC3 can be used for serologic assays to detect antibodies to the H. pylori 120-128 kDa antigen.

Con~ervation of the tagA gene.
To determine whether other H. pylori strains possess the tagA gene or homologous sequences, 32 strains were studied by colony hybridization using pMC3 as a probe (Table 3). A positive signal was obtained from 19 (59.3~) of these strains. SDS-PAGE and immunoblotting of whole cells of these strains indicated that 19 (59.3~) of the 32 strains expressed a band at 120-128 kDa. The immllnohlot and colony hybridization findings correlated completely;
all 19 H. pylori strains expressing the protein possessed a gene homolog, in comparison to none of the 13 strains not expressing the protein (p<0.001, Fisher's exact test, one-tailed). In addition, all 15 strains producing the vacuolating cytotoxin showed both pMC3 hybridization and presence of the 120-128 kDa band (Table 3).

To gain information on the restriction fragment polymorphism of the tagA gene and whether there are multiple homologous genes in each bacterial genome, genomic DNA from 4 H. pylori strains was prepared and Southern hybridization performed using pMC3 as the probe.
Two strains expressing the 120-128 kDa protein and with positive colony hybridization now showed strong hybridization to a HindIII restriction fragment migrating at approximately 1.2 kb, and weaker bands at 3.0 and 3.3 kb. For a third strain that showed the phenotype and had WO96/10639 22 ~ 1 3 ~ 3 PCT~S9~/12669 ~

a positive colony hybridization, the probe hybridized strongly in the Southern analysis to a band of about 1.1 kb; no weaker bands were seen. A band migrating at less than 0.5 kb that hybridized weakly with the probe was present in all three strains. An H. pylori strain that expressed no 120-128 kDa protein and that had a negative colony hybridization, as well as a C. jejuni strain used as a control, showed lack of hybridization in the Southern analysis. Hybridization of pMC3 to chromosomal DNA from strains 84-183 and 60190 digested with EcoRI and BamHI
also showed polymorphism, confirming the heterogeneity observed with the other restriction enzyme. These studies indicate that although homologs of TagA exist in other H.
pylori strains, there is heterogeneity in either intragenic or flanking sequences.

The present example provides a cloned fragment of H. pylori genomic DNA that includes the majority of a gene that encodes an important H. pylori antigen. The evidence that pMC3 contains the gene encoding the TagA
antigen may be summarized as follows: (i) neither the protein nor the gene are present in all H. pylori strains;
(ii) only strains expressing the 120-128 kDa protein hybridize with pMC3 and strains that do not express the protein do not hybridize; (iii) sera from H. pylori-infected persons that recognize the 120-128 kDa antigen recognize the product of recombinant tagA significantly more frequently than do control sera.

The partial sequences of tagA and the two other ORFs have no identity with the N-terminus or 3 internal W096tlO639 2~ n ~ 3 ~3 PCT~S95/12669 sequences from the 87 kDa cytotoxin. This finding is consistent with earlier observations that the 120-128 kDa and 87 kDa proteins are antigenically unrelated (41).
Comparison of the truncated deduced gene product revealed little direct homology with known proteins.

The tagA gene or homologous genes are present in approximately 60~ of the H. pylori isolates studied but absent from the others. As indicated by the Southern analysis, there is evidence for restriction fragment polymorphism even when only a small number of strains are examined. Absence of a homolog correlated exactly with lack of expression of an antigenic band at 120-128 kDa.
Thus, the phenotype lacking this band is not due to deficiencies related to transcription or expression but rather to the absence of the implicated gene.

The presence of genomic DNA containing at least the truncated tagA gene is highly associated with cytotoxin production. A minority of strains that possess the tagA gene do not produce detectable levels of cytotoxin. This phenomenon may reflect suboptimal sensitivity in the cell culture assay to detect toxin, or may indicate that factors other than the TagA antigen are associated with toxin activity.

As shown by the immunoblot studies, the pMC3 products are excellent diagnostic reagents for detection of human serum antibodies to the TagA antigen. Use of this recombinant protein can readily supply sufficient antigen to aid in development of immunoassays to determine WO96/10639 22 0 1 3 5 ~ PCT~S95112669 which persons are infected with H. pylori strains producing the native 120-128 kDa protein, and heterologous antibodies raised against the pMC3 gene product can be used to determine which strains produce the TagA antigen.
Knowledge of the DNA sequence of pMC3 permits the construction of oligonucleotides for use as hybridization probes or for primers for PCR. Such techniques are also used for rapid detection of infection due to a strain with the implicated genotype. Creation of deletion mutants enables elucidation of the role of this gene product and provides both therapeutic reagents and vaccine candidates.
Such diagnostic methods and mutants are detailed herein.

~ WO96/10639 22 C 1 3 5 3 ~ 2~9 Table 1 Nelicobacter pylori strains used in thiæ study Expression of Expression of Strain Isolation120-128 kDavacuolating designation localeantigenacytotoxin activityb Tx30a Texas 84-183 Texas + +
60190 England + +
87-29 Colorado + +
86-313 Colorado 87-199 Colorado + +
86-385 Colorado 87-33 Colorado + +
87-81 Colorado + +
87-91 Colorado + +
87-90 Colorado 87-226 Colorado +
87-225 Colorado 87-230 Colorado 87-75 Colorado 87-203 Colorado 87-6 Colorado +
86-338 Colorado 86-63 New York +
86-86 New York + +
86-332 Minnesota + +
92-18 Tennessee + +
92-19 Tennessee + +
92-20 Tennessee 92-21 Tennessee + +
92-22 Tennessee +

92-23 Tennessee SUBSTITUTE SHEET (RULE 26) 2 2 ~ 1 3 ~ 3 ~ 2669 ~

WO96/10639 ~ 3~/12669 Expression of Expression of Strain Isolation 120-128 kDa vacuolating designation locale antigena cytotoxin activityb 92-24 Tennessee 92-25 Tennessee + +
92-26 Tennessee + +
92-27 Tennessee + +

92-28 Tennessee a Recognition of 120-128 kDa band in cell lysates by human serum as detected by immunoblot (20).
b Production of vacuolating cytotoxin as detected in HeLa cell culture (59).

SUBSTITUTE SHEET (RULE 26) I2~nl3s3 WO96/10639 PCT~S95/12669 Table 2 Amino acid composition of truncated 859 amino acid TagA
polypeptide as deduced from pMC3 Percent of Number of 859 amino ~m; no acid residues ac Ala 60 7.0 Cys 2 0.2 Asp 62 7.2 Asn 82 9.5 Glu 59 6.9 Gln 48 5.6 Phe 44 5.l Gly 54 6.3 His 12 l.4 Ile 50 5.8 Lys lOl ll.8 Leu 67 7.8 Met 12 . l.4 Pro 24 2.8 Arg 22 2.6 Ser 63 7.3 Thr 30 3.5 Val 47 5.5 Trp 4 0.4 Tyr 16 l.9 ~ WO96/10639 2~ ~ ~ 3 5 3 PCT~S95/12669 Table 3 Correlation between presence of 120-128 kDa band by immunoblot, hybridization with pMC3, and cytotoxin production by 32 N. pylori isolate~ from hll~~

Strain characterist;cs Presence of Hybridization Cytotoxin 120-128 kDa of pMC3 to production band on H. pylori in cell Number ;mmllnohlota colonyb culture of assayC strains + + + 15 _ _ - 13 + _ 4 + O
+ -- O
+ _ -- O
+ _ + O
+ + O

a Recognition of 120-128 kDa band in cell lysates by human serum as detected by immunoblot (20).
b Hybridization of pMC3 to lysed H. pylori cells in colony blot (50).
c Production of vacuolating cytotoxin as detected in HeLa cell culture (59).

22 013 ~3 WO96/10639 PCT~S9Sl12669 Construction and characterization of a tagA-negative strain of Helicobacter pylori Bacterial strains, vectors and growth conditions.
H. pylori strain 84-183 (ATCC 53726) used in this study was from the culture collection of the Vanderbilt University Campylobacter/Helicobacter Laboratory and was chosen because it has been extensively characterized. Stock cultures were maintained at -70C in Brucella broth (BBL Microbiology Systems, Cockeysville, MD) supplemented with 15~ glycerol. H. pylori strains were grown in Brucella broth supplemented with 5~ fetal bovine serum or on blood agar plates supplemented with nalidixic acid (50 mg/liter), vancomycin (10 mg/liter), polymyxin B
(5000 U/liter), and trimethoprim (5 mg/liter) under microaerobic conditions at 37C for 48 hours. E. coli strain DH5~ (Stratagene, La Jolla, CA) used for transformation, was grown in LB medium. As described above, pMC3 contains the truncated tagA gene on a 3.5 kb insert in pBluescript. Plasmid pILL600 (60) was used as a source of a C. coli kanamycin (km) resistance gene.
Chemicals and enzymes.
Final concentrations of ampicillin (100 ~g/ml) and kanamycin (50 ~g/ml) were used whenever necessary.
Restriction enzymes, T4 DNA ligase, E. coli DNA polymerase large (Klenow) fragment were from Promega and United States Biochemicals (Cleveland, OH). ~-32P-dATP (650 Ci/mmol) was from ICN Radiochemicals (Irvine, CA).

~ W096tlO639 22 0 1 3 5 3 PCT~S95/12669 Genetic techniqueQ.
Chromosomal DNA was prepared as described above.
Plasmids were isolated by the procedure of Birnboim and Doly (49). All other standard molecular genetic techniques were performed as described (50). DNA
fragments used as probes for hybridization experiments were gel-purified.

Introduction of ~m cassette into H. pylori strain 84-183.
An E. coli kanamycin-resistance gene was inserted into the unique NdeI site of pMC3 to create pMC3:km (Figure 2). This construct was introduced directly into H. pylori strain 84-183 by electroporation.
Briefly, H. pylori cells grown on blood agar plates for 48 h were harvested, washed three times in electroporation buffer (15~ glycerol/5~ sucrose) and suspended in 200 ~l of the buffer. Plasmid DNA from pMC3:km was isolated by a rapid (mini-prep) alkaline-lysis method of Birnboim and Doly and was added to the cells and incubated for 5 min on ice. The cells and DNA were transferred to 0.2 cm electroporation cuvette in a Gene-pulsar apparatus ~Bio-Rad), and high voltage pulses (25F, 2.5 kv and 200Q) were delivered as described previously (61). Following electroporation, the cells were suspended in 400 ~l of LB
media and spread on blood agar plates. The plates were incubated at 37C under microaerobic conditions for 24 h, then cells were harvested, plated on blood agar plates containing 50 ~g/ml of kanamycin, and incubated microaerobically for 48 h.

22~353 WO96/10639 PCT~S95112669 The cloning vector used was unable to replicate in H. pylori and selection on kanamycin-containing media yielded kanamycin-resistant recombinants. From approximately l0l H. pylori cfu, 3000 transformants (10-7) were obtained when 500 ng of plasmid DNA was used.

Colony hybridization.
Fifty kanamycin-resistant transformants obtained by electroporation were grown on blood agar plates and replica copies of these colonies were transferred to nitrocellulose filters. Each filter was placed on 3 mM
Whatman paper saturated with 0.2 M NaOH/l.5 M NaCl. After 3 min the filter was transferred to 3 mM Whatman paper, saturated with 0.4 M Tris-HCl (pH 7.6)/2 X SSC for 3 min, and then to 2 X SSC for 3 min. The colony blot filters were dried in a vacuum oven for 90 min at 80C and hybridized with radiolabeled pBluescript or the km-resistance gene, as described above. The colony blots were washed at 60C in 0.5X SSC and exposed to XAR-2 X-Ray film (Eastman Kodak, Rochester, NY).

Gel electrophoresis and ;mml~nohlot analy~is.
Lysates of E. coli carrying pBluescript, pMC3 or pMC3:km or of H. pylori cells were prepared and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE~.
Immunoblotting of whole cell extracts derived from wild-type and mutants l, 21 and 22 was performed as detailed above using a 1:300 dilution of adsorbed human sera, and a l:2000 dilution of goat anti-human immunoglobulin alkaline phosphatase conjugate as the secondary antibody, as described above. These studies showed that isogenic ~ WO96/10639 2t 0 1 3 5 ~ PCT~S95/12669 mutant strains 1, 21, and 22 have no antigenic tagA gene product.

Southern hybridizations.
Southern hybridization of wild-type H. pyl ori strain 84-183 and kanamycin-resistant transformants M21 and M22 were performed. H. pyl ori chromosomal DNA was digested with HindIII or BamHI and SacI and the resulting fragments were electrophoresed on a 0.7~ agarose gel and transferred to nylon membrane. Probes were gel-purified DNA fragments derived from pMC4 or pILL600 and were radiolabeled by primer extension using random hexameric oligonucleotides as described above. The DNA was then transferred to a nylon membrane and hybridized with 32p_ labeled pMC4 or the 1.3 kb km cassette under conditions of high stringency. Hybridizations were performed in a solution of 6X SSC, 0.5~ SDS, 5X Denhardt's solution and 100 ~g/ml salmon sperm DNA and the blots were washed for 30 min. at 60C in 0.5X SSC/0.1~ SDS.
Genotypic characterization of the transformants.
To provide genetic evidence that the tagA gene is disrupted in the transformant strains, DNA isolated from wild-type strain 84-183 and H. pylori mutants 21 and 22 was digested with the restriction endonuclease HindIII
or BamHI and SacI. After separation of the digested DNA
on an agarose gel the DNA was transferred to a nylon membrane and hybridized to pMC4 which is a tagA probe.
This probe hybridized to approximately 20 and 1.0 kb BamHI-SacI fragments in the wild-type strain, whereas the 1.O kb BamHI-SacI fragment is lost and a new 2.3 kb WO96/10639 ~2 ~ ~ 3 ~ 3 PCT~S95/12669 hybridizing fragment was observed in both mutant strains without disruption of the other bands. Similarly, a 1.2 kb HindIII fragment was lost and a 2.2 kb fragment gained in both mutants because of the kanamycin resistance gene insertion. The kanamycin gene probe hybridized only with the 2.3 kb BamHI - SacI and 2.2 kb HindIII fragment in mutants 21 and 22 strains, which indicate that replacement had occurred in the tagA gene. Thus, the tagA gene in strain 84-183 had been mutagenized by insertion of the km gene.

Cytotoxin production.
Cytotoxin production was assayed as described above and the results shown in Table 4. The results indicate that neither the intact TagA antigen nor the intact tagA gene is required for vacuolation.

W096/10639 22 0 1 3 5 3 PCT~S95/12669 Table 4 Cytotoxin production by wild-type H. pylori strains and tagA~ mutants Supernatant Optical dilution dens;tya 84-183 M1 M22 87-203b 1:50.21 iO.04 0.26 i0-040.23 iO.05 iO.02 1:100.16 iO.02 0.20 i0.030.13 iO.02 0.01 +0.01 1:200.13 iO.Ol 0.15 i0.020.10 iO.02 0.01 iO.O1 1:400.09 iO-02 0.06 iO.Ol0.06 iO.02 0.01 i 1:800.03 iO-O1 0.04 i 0.02 iO.O1 0.01 i 1:160-0.01 ~0.02 -0.01 iO.O1-0.01 +0.02 -0.02 +0.02 a Net optical density as measured in neutral red assay of cytotoxin-induced vacuolation, as described above (59).
b Strain 87-203 is a strain known not to produce cytotoxin.

Full Length tagA Gene and Gene Product Cloning and sequencing of the full length gene.
To isolate the full-length gene, we next used the 0.6 kb fragment of pYB1 as a probe to screen the AZapII library of H. pylori 84-183. Five positive plaques were purified and the pBluescript plasmids containing the WO96110639 2 ~ 3 PCT~S95/12669 cloned DNA inserts were excised by co-infection with the helper phage. Each of the five positive clones contained DNA inserts of 2 to 3 kb (data not shown). The clone designated pYB2, which contains a 2.7 kb insert, was chosen for further study and a restriction map generated (Figure 3). A series of nested deletions starting at either end of the 2.7 kb insert of pYB2 was performed using exonuclease III. Using overlapping deletion clones of pYB2, we determined 1969 bp sequence in both strands.
As expected, the first 785 bases of this sequence (SEQ ID
NO:3, beginning with nucleotide 2864) overlapped with the end of pMC3. Translation of the complete nucleotide sequence generated from pMC3, pYB1 and pYB2 in all possible reading frames revealed a single open reading frame of 3,543 nucleotides initiated by an ATG codon at position 1072 and terminated by a TAA codon at position

4,615. The sequence encodes a protein of 1181 amino acid residues (SEQ ID NO:4) and the calculated molecular weight of the deduced polypeptide is 131,517 daltons. A sequence that could form a potential stem-loop structure in the mRNA and which could serve as a transcription termination site (~G= -14.4 Kcal) extends from nucleotides 4642 to 4674 (SEQ ID NO:3).

Homologies of the TagA polypeptide with other proteins.
Search of Swiss.Prot version 21, and NBRF-PIR
protein data banks showed no striking homologies with the full length TagA antigen (SEQ ID NO:4). However, among the homologies with the highest scores were chloroplast H+-transporting ATP synthases (62,63), and a sodium channelprotein (74) with 16.8~ and 17.3~ identity, and 50~ and ~WO96/10639 ~2 ~ ~ 3 5 3 PCT~S95/12669 42.6~ conserved amino acids in the region between residues 1-482 and 123-1182, respectively. No significant homologies were observed when the amino acid sequences of the other two ORFs contained in pMC3 were compared with the protein data bases.

The content of basic amino acids [141 lysines, (11.9~) and 117 asparagines (9.9~)] TagA was unusually high and was consistent with the predicted isoelectric point of the peptide (8.89). A hydropathicity plot indicated that the deduced protein is predominantly hydrophilic. An interesting feature of the primary structure of this protein is the presence of structures of homopolymeric amino acid sequence, most notably polyasparagine (SEQ ID NO:4, Position 3705). In searches comparing this asparagine-rich region with various protein sequence data bases, there was strong homology with sequences from yeast (64,65,66,67,68,69,70) and Plasmodium (71) nucleotide-binding proteins. Polyasparagine is also found in the DNA-binding regulatory product of the lac9 gene of Kluyveromyces var.lactis (72) and potassium transport protein (TRK1) of Saccharomyces cerevisiae ( 73) Con~truction of the full length tagA gene.
To construct the full length tagA gene, we utilized the unique SacI restriction site located in both pMC3 and pYB2 (Figure 3). First, the 3.6 kb tagA fragment of pMC3 was cloned into a pUC19 vector under the control of the lacZ prometer, to generate pUT1. Next, the 2.6 kb SacI fragment from pYB2 was cloned into sacI-digested pUT1. A clone with the correct orientation was selected, WO96/10639 2~ ~ ~ 3 5 3 PCT~S95112669 which was named pUT2. An identical clone (pEM3), but present in the pGEM3z vector, has been deposited with the ATCC in compliance with the requirements of the Budapest Treaty under Accession No. 69273. E. coli cells containing pUT2 or pEM3 expressed the immunoreactive H.
pylori 128 kDa protein.

Detection of human serologic responses to the recombinant TagA protein by Western blotting.
To determine whether human sera reacted with the full-length recombinant TagA protein, lysate from pEM3-containing cells was electrophoresed on a 7~ acrylamide gel, and electroblotted onto nitrocellulose paper. Sera from lO H. pylori infected hl]m~nq and lO uninfected humans were diluted l:lO0 and tested for reactivity with the recombinant protein. Sera from 7 H. pylori infected persons recognized the TagA protein, compared to sera from l of lO uninfected persons (p=O.Ol, one-tailed Fisher's exact test). Thus, the recombinant full-length protein was a useful antigen for assessing human responses to H.
pylori.

H. pylori strains possessing tagA associated with an increased risk of developing adenocarcinoma of the stomach This example describes a serologic assay to determine whether Helicobacter pylori infection is due to a tagA+ strain. This serum IgG assay, utilizing orv220, a recombinant tagA fragment was found to have a sensitivity ~ WO96/10639 22 0 1 3 5 3 PCT~S95112669 of 94.4~ and specificity of 92.5~ when used in a clinically-defined population.

The present example also shows that H. pylori infection is associated with gastric cancer risk. Serum specimens obtained from a cohort of Japanese-American men in Hawaii was used in a study of 103 H. pylori-infected men who eventually (13 years after the serum specimens were collected) developed gastric cancer, and 103 matched H. pylori-infected control specimens from the men who did not develop cancer within the same period of time. Serum antibodies to TagA were associated with a l.9-fold (0.9-4.0, p=0.08) increased risk of developing gastric cancer during the observation period. Among the 75 men with intestinal type cancer of the distal stomach, the odds ratio (OR) was 2.3 (1.0-5.2, p=0.056). Young age (~72 years) and late stage at tumor diagnosis each were significantly associated with TagA-positivity.

The following results indicate that presence of serum antibodies to the product of a recombinant tagA
accurately determines the tagA status of an infecting H.
pylori strain, and indicates that infection with a tagA+
strain is associated with an enhanced risk of development of gastric cancer, especially intestinal type affecting the distal stomach.

Selection of patients for validation study.
To determine the utility of the TagA serologic assay, sera from 181 persons whose H. pylori status had been previously defined (9,22-24) were studied.

WO96/10639 ~2 ~ ~ 3 5 3 PCT~S95/12669 Uninfected persons (n=115) were those who underwent endoscopy and had biopsies that did not reveal H. pylori infection by rapid urease test or by histologic e~m;n~tion; all patients also had negative serology in a standardized enzyme-linked immunosorbent assay (ELISA) for serum IgG directed to H. pylori (25). The 115 uninfected patients were arbitrarily divided into a reference group (n=35) and a test group (n=80). Infected persons were those from whom H. pylori was obtained in culture on biopsy; these 66 patients included those with duodenal ulceration (n=14), gastric ulceration (n=6), gastritis alone (n=36), or other diagnoses (n=10). For each patient, a single isolate was evaluated to determine tagA
status by colony hybridization with a gene-specific probe, as previously described (6,24). On the basis of the hybridization assay, 36 patients were defined as being infected by a tagA+ strain, and 30 patients by a tagA-strain. All sera had been stored at -20C until used.

Selection of patients for cancer study.
All the patients in this study were part of the Japan-Hawaii Study cohort, as previously described.
During the 21-year period from 1968-1989, 109 cases of pathologically-confirmed gastric carcinoma had been identified, and previous serologic testing indicated that 103 of these patients had been infected with H. pylori at the time of their original serum submission in the 1960's

(5). Of the 103 matched controls for these cases, 83 had been H. pylori-infected. For each of the rem~;ning 20 H.
pylori-infected cases, 3 further controls were identified according to the previous criteria (5). Serology to ~WO96/10639 22 0 1 ~ ~ 3 PCT~S95112669 determine whether H. pylori infection was present was done on coded samples, and from the positives, one H. pylori-infected control was randomly selected for each of the 20 unmatched cases. In total, the study consisted of 103 H.
pylori-infected men who developed gastric cancer, and their 103 matched controls, who also were H. pylori-infected but did not develop gastric cancer during the study period.

Preparation of the recombinant TagA antigen.
A 1.7kb Bam~l fragment containing bp 1921-3648 of tagA cloned in pMC3 (16) was subcloned into the BamHI
site of pET15b (Novagen, Madison WI), downstream of the T7 promoter. This plasmid (pORV220) was used to transform E.
coli host strain BL21, a ADE3 lysogen containing the T7 RNA polymerase gene under control of the lacW5 promoter (26). Addition of IPTG to a growing culture of the lysogen induces T7 polymerase to transcribe the target DNA
on the recombinant plasmid. This vector allows transcriptionally-regulated expression of a fusion protein consisting of the TagA fragment with an N-terminal histidine-tag. The fusion protein consists of 600 residues (24 vector + 576 insert), and has a predicted molecular mass of 66.4 kDa. The protein was purified from lysates of induced broth cultures using a nickel-chelating resin, and eluted with imidazole, as described (27).

Serologic methods.
The presence of serum IgG antibodies to H.
pylori was determined by ELISA with the Pyloristat kit (Biowhittaker, Walkersville, MD) as previously described WO96/10639 2 ~ PCT~S95/12669 (5). For the TagA ELISAs, optimal concentrations of antigen, patient serum, and anti-human IgG conjugate were determined by checkerboard titrations. For orv220, the optimal antigen concentration was 5~g/ml and l00~l aliquots were loaded into wells in a 96-well microtiter plate. The optimal dilution of human serum was l:l00, and horseradish peroxidase-conjugated goat-anti-human IgG was used at a dilution of l:4000. Other details of the serologic methods were exactly as described in similar assays (23,25).

Statistical analysis.
The t-test for paired samples was used for the comparison of means, and McNemar's test was used for the comparison of the distribution of various characteristics between patients and control subjects. Odds ratios (OR) for stomach cancer, based on the results of the TagA
assay, were derived from conditional logistic regression methods (29). Tests for linear trend in the logit of risk were derived from conditional logistic regression models through the use of grouped TagA test results (coded by quartile as l, 2, 3, or 4). All models of conditional logistic regression were fitted by using iterative maximum likelihood methods and a special application of the proportional hazards regression model (30). The estimate of the attributable risk of gastric carcinoma related to a tagA+ strain was based on the method of Walter (3l). A
receiver operator characteristic (ROC) curve was constructed to summarize the sensitivity and specificity estimates (32).

WO96/10639 ~2 ~ 1 3 5 3 PCT~S95/12669 Diagnostic accuracy of TagA serology using orv220.
Previous studies of TagA serology used a recombinant antigen that was purified from E. coli cell lysates (21). Because purification of this antigen was tedious and prolonged, overlapping tagA gene fragments were expressed as fusion proteins in an alternate prokaryotic expression system. Preliminary studies indicated that orv220 expressed the best of several of the candidate antigens based on the TagA fragments studied thus far. The truncated protein was compared with the antigen purified from E. coli strains transformed with pEM3, which encodes the entire tagA ORF. By linear regression analysis, serum IgG results for 41 persons (19 infected and 22 uninfected), correlated closely between assays using the 2 different antigens (r=0.96, p~0.001).
Using the same threshold of the mean value for uninfected persons + 2 SD, the orv220 antigen gave a negative result for 21 (95~) of the 22 uninfected persons; and the exception was weakly positive. For the 19 H. pylori-infected persons, the results with orv220 were exactly thesame as for the pEM3 antigen (12 positive and 7 negative).
Thus, serologic reactivity with the 66.4 kDa TagA fragment encoded by orv220 was nearly identical with that detected with a larger TagA fragment.
To establish the assay, sera from 36 persons known not to be infected with H. pylori were used for reference, and after multiple runs, thresholds based on mean values plus intervals of standard deviation were established. Concurrently, sera were tested from a second group of 80 uninfected persons, 36 persons known to be W096/10639 ~ 3 ~ 3 PCT~S95/12669 infected with a tagA+ strain, and 30 persons from whom the only H. pylori isolate obtained was tagA~. Not surprisingly, optical density ratios for the uninfected persons were nearly identical to those for the reference groups. In contrast, the values for the persons infected with tagA+ strains were significantly higher (Students' T-test, p~O.OO1, one-tailed). Among the 30 sera obtained from persons from whom the only isolate was a tagA~ strain, a bimodal distribution was observed. For 27 of the sera, the values were similar to those in the two groups of uninfected persons, but for three sera the values were much higher and similar to those for patients infected with tagA+ strains.

To establish a threshold for use in diagnostic assays, the accuracy of several optical density ratio cut-offs were examined (Table 5). Overall, the highest accuracy was obtained when the mean value for the 35 uninfected (reference) patients + 3 intervals of standard deviation was used, with sensitivity of 94.4% and specificity of 92.5%. This type of threshold was used in all future studies. On the basis of receiver operator characteristic (ROC) curves, there was high level discrimination of tagA status using orv220.
Assessment of stability of antibody levels.
To determine whether serum antibodies to the TagA product persist over the course of chronic H. pylori infection, paired serum specimens from 36 epidemiologists who were part of a cohort previously studied for clinical and epidemiological features associated with H. pylori ~ WO96/10639 22 0 ~ 3 5 ~ PCT~S95/12669 infection ~28) were evaluated. On average, the specimens were obtained 7.59 years apart, and values in the standard - H. pyl ori ELISA (28) were compared to values in the TagA
ELISA.

.
Among 36 participants, none had seroconverted from positive to negative antibody status or vice versa.
Mean optical density scores produced by the first and second sera were highly similar (0.217 vs 0.249; p=0.3, paired t-test; Table 6).

Association of tagA Positivity With Gastric Cancer.
Having developed an assay to detect persons infected with H. pylori strains possessing TagA using TagA
or antigenic fragments thereof, the invention shows that infection with a tagA~ strain is associated with the risk of developing gastric cancer. From an earlier study of 109 Japanese-American patients, who developed gastric cancer over a 21-year observation period (5), 103 were found to be H. pylori-infected based on serum specimens obtained from them a mean of 13 years prior to cancer diagnosis. Each of these cases was matched to a control from the same cohort, of the same age, and who also was H.
pylori-infected; the characteristics of the 103 H. pylori infected men who developed gastric cancer and their age-matched controls are shown in Table 7. The two groups of men were similar with respect to demographic characteristics and laboratory values.

For this population, presence of serum antibodies to TagA (i.e., infection with a tagA positive WO96/10639 ~ 3 PCT~S95/12669 H. pyl ori strain) was associated with increased risk of cancer development (OR=1.9, p=0.08) (Table 8). An analysis confined to the 101 cases of cancer of the distal stomach and their controls showed highly similar values, as expected (OR=1.8, p=0.11). When the cases with distal cancers were stratified by the histologic type of tumor, the strongest association was with the intestinal type (OR=2.3, p=0.056) but not the diffuse type (OR=1.0, p=1.0); 3 men had an indeterminate histological pattern.
Positivity in the TagA ELISA was not the result of seroconversion during the interval between when serum was obtained and diagnosis of cancer, nor was the magnitude of the TagA antibody response a factor in the risk developing cancer. In the population of H. pylori-infected persons, the presence of tagA was associated with an attributable risk of 28~ (95~ C.I.=0-57~) for gastric cancer. There was no association between levels of the IgG antibodies to TagA and levels of IgG to the conserved H.
pyl ori antigens.
The TagA antibody analysis was stratified by the patients' age at which gastric cancer was diagnosed. TagA
seropositivity was associated with a 3.0-fold (95~
C.I.=1.0-9.3; p=0.057) increase in gastric cancer risk for 52 men diagnosed under the age of 72. In contrast, for 51 men who were 272 years at time of diagnosis, the association was not significant (OR=1.3, 95~ C.I.=0.5-3.5). TagA seropositivity was associated with risk of developing an advanced stage tumor (3 or 4), OR=2.6, 95 C.I.= 1.1-6.2; p=0.03, but not an earlier (1 or 2) stage tumor (OR=1.0). The risk of developing gastric cancer W096tlO639 22 01 3 5 ~ PCT~S95/12669 .

associated with TagA seropositivity was not increased when subjects were stratified according to birth order or sibship size.

Although some of the above OR values are not statistically significant using the conservative 2-tailed analysis of significance, use of 1-tailed analysis indicates that the associations with all cancers and intestinal type cancers reach statistical significance (p=0.04 and 0.028 respectively). Furthermore, undetected differences in risk between the two groups, lack of perfect accuracy of the assay, and variation in patient-to-patient response to this infection could help account for lack of significance.
Multiple infection.
Simultaneous gastric infection with two H.
pylori strains has been reported with frequencies of 10 to 13~ (14,15), and simultaneous infection with three different strains also has been observed (33). In the initial validation study, only a single colony from each patient had been picked, so there was no opportunity to examine for multiple infection. However, that 3 of 30 (10~) persons from whom a tagA- strain was the only isolate had high-level serologic responses to TagA suggests that these persons were co-infected with a tagA+ strain. Since only the conjunction of simultaneous i~fection with a tagA-index strain and a second tagA+ strain and not vice versa, could show dichotomous results between colony testing and serologic assay, the data suggest that the frequency of multiple infection may be substantially higher than the -WO96/10639 ~ 5 ~ PCT~S95/12669 previously reported frequency, which was based on a small number of biopsies (4,15).

The present Example further supports the utility of non-invasive serologic assays for H. pylori infections, because they are shown to be global assays that in essence sample the entire stomach. In contrast, biopsy-based techniques only sample a minuscule fraction (~ ) of the gastric mucosa. Because TagA is an immunodominant antigen (17), serologic assays potentially have the power to detect infections with tagA+ organisms even if numbers are low in relation to tagA~ isolates.

The associations with the subset of more aggressive tumors (younger age, higher stage when diagnosed), and the consistency of the data with the underlying hypothesis, show that the effect is real as further supported by a number of realities of TagA
biology. First, infection with tagA~ strains is associated with enhanced epithelial cell injury (18,19), injury to surface gastric epithelial cells promoting or possibly initiating the oncogenesis. Second, infection with cagA+
strains is associated with higher degrees of gastric inflammation (8,19), and with enhanced expression of pro-inflammatory cytokines such as IL-1 and IL-8 (19,35).
These may contribute to epithelial injury. Third, most tagA+ strains also express vacuolating cytotoxin activity (16,17). Expression of cytotoxin as assessed by serum neutralizing antibodies, may be associated with gastric cancer (36). Finally, intestinal type gastric cancer is WO96/10639 22 Q 1 3 5 3 PCT~S95/12669 more highly associated with increased inflammation and atrophic gastritis than is the diffuse type (37,38).

It has been shown that high titered antibody responses to conserved H. pylori antigens were associated with gastric cancer risk (5,8). One interpretation of this phenomenon is that high antibody levels are markers for the degree of inflammation, active inflammation being considered a precursor of oncogenic events (1). The lack of strict correlation of gastric cancer and anti-TagA
antibody levels may reflect overmatching, since only persons infected with H. pylori were selected, all of whom have some degree of inflammation. In any event, the significance of these findings must be tempered by the observation that TagA-positivity was present in 78~ of the H. pylori-infected controls who did not develop cancer.
Thus, infection with a tagA+ strain is neither necessary nor sufficient for oncogenesis but is shown to be a factor involved in this process.
The presence of tagA in a strain may be a marker for adjacent genes or for a particular phenotype that itself is relevant to inflammation or to the oncogenic process. Nevertheless, this Example shows that particular H. pylori strains are associated with differential risk of gastric cancer.

Summary.
Among persons known to be infected with tagA+
strains, there was a broad range in serologic reactivity, but without substantial overlap with the reactivity of WO96/10639 ~ 3 PCT~S95/12669 uninfected persons. The stability of the results over years in the absence of antimicrobial therapy further indicates the utility of the present TagA assay.

Having provided an accurate assay for detecting infection with a tagA+ strain of H. pylori, the invention also permitted the determination that infection with a tagA+ strain is a risk factor for the development of gastric cancer. Infection with a tagA+ strain nearly doubled the risk of developing gastric cancer over the ensuing 21 years, compared with infection with tagA~
strain, and the effect was more marked for persons who developed intestinal type neoplasms.

Having provided a purified TagA and antigenic fragments of TagA, and shown that infection with a TagA
expressing strain indicates predisposition to gastric carcinoma, further uses of the present discoveries are enabled.

W096/10639 ~2 Q ~ ~ ~ 3 PCT~S9S/12669 Table 5 Diagnostic value of TagA ELISA using orv220 Group Number of Number (~) exceeding threshold*
subjects lSD 2SD 3SD 4SD
Uninfected 80 12(15~) 9(11.3~ 6(7.5~) 3(3.8~) Infected, 30 9(30~) 6(20~) 5(16.7~) 4(13.3~) tagA- strain Infected, 36 36(100~) 3597.2~) 3494.4~) 31(86.1~) tagA+ strain * Threshold defined as mean plus stated intervals of standard deviation (SD) of optical density ratio (ODR) values for reference group of 35 persons known not to be infected by H.
pyl ori.

Table 6 Stability of serum antibodies to N. pylori antigens in 36 subjects Serum IgG response to indicated antigen Serum specimen Conserved H. pylori TagAt antigens**
Initial 3260 0.217iO.30 Follow-up* 2913 0.249+0.35 * Follow-up specimen obtained an average of 7.59+1.0 years after initial specimen.
** Antibody level indicated by reciprocal geometric mean titer to pool of sonicates of 5 H. pylori strains.
Antibody level indicated by Mean + SD optical density ratio.

WO96/10639 ~ ~ n ~ 3 53 PCT~S95/12669 Table 7 Characteristics of H. pylori-infected patients with gastric cancer and control subjects at the time the serum specimen was obt~; ne~

Characteristic Patients Controls P-value (n=103) (n=103) Mean age at 58.8 58.7 0.18 ex~m;n~tion (year) Born in United 83 83 0.83 States (~) Married (~) 93 95 0.72 Alcohol use (~) 65 73 0.27 Mean body-mass index 23.5 24.0 0.33 Mean diastolic blood 81.6 82.1 0.74 pressure (mm Hg) Mean serum cholesterol 5.7 5.6 0.74 (mmol/liter) Mean serum glucose 9.3 9.2 0.92 (mmol/liter) W096/10639 '~ ~ Q 1 3 5 ~ PCT~S95112669 Table 8 Odds Ratios for the Association between Infection with a tagA' N.
pylori Strain and Gastric Cancer Gastric Matched-pair Statusa Odds 95~
Cancer (Patients/Controls) Total Ratio Confidence Type Intervalb + / / + _ / _ All6921 ll 2 103 l.9 (0.9 - 4.0) Distal 68 20 ll 2 lOl l.8 (0.9 - 3.8) Intesti- 48 18 8 l 75 2.3 (l.O - 5.2) nal Diffuse 18 2 2 l 23 l.O (O.l - 7.l) a +/+, both patient and matched control show serologic response to TagA.
+/-, patient but not matched control show serologic resonse to TagA.
-/+, control but not patient show serologic response to TagA.
-/-, neither patient nor control show serologic response to TagA.
b two-tailed analysis WO96/10639 ~ 2 ~ ~ ~ 5 ~ PCT~S95/12669 Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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(1) GENERAL INFORMATION:
(i) APPLICANT: COVER, TIMOTHY L.
BLASER, MARTIN J.
HARRY KLEANTHOUS
TUMMURU, MURALI K.R.

(ii) TITLE OF lNv~NllON: THE tagA GENE AND METHODS FOR DETECTING
PREDISPOSITION TO PEPTIC ULCERATION AND GASTRIC CARCINOMA
(iii) NUMBER OF ~QU~N~S: 4 (iv) CORRESP~N~N~ ~nD~-~S:
(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.
(B) STREET: 127 Peachtree Street, Suite 1200 (C) CITY: Atlanta (D) STATE: Georgia (E) C'~UN1~Y: USA
(F) ZIP: 30303 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SPRATT, GWENDOLYN D.
(B) REGISTRATION NUMBER: 36,016 (C) REFERENCE/DOCKET NUMBER: 2200.030 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 404/688-0770 (B) TELEFAX: 404/688-9880 (2) INFORMATION FOR SEQ ID NO:1:
~yu~N~ CHARACTERISTICS:
(A) LENGTH: 3648 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO96/10639 2~ ~ ~ 3 5 3 PCI'IUS95/12669 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1072..3648 (xi) S~yu~:~ DESCRIPTION: SEQ ID NO:1:

ATGGGCTGCG CGTAACGAAA AACAGTCGCT TGAC~L~Lll TGATGTCATC AGAGATTTTC 60 ATTAAAGGAT TGGCTGCTAT CGCTAGCCCT AAAGTTACTA CAAw ll~lL GTAGCTGTCA 240 ATC:AAAAAA AGTATTTTTA TACTATTCAT ACAAGCGCTA CTTTATAATT TAAATCAAAA 360 CCGACGCTTT l~L-L-L~AcAA CTGATATAAT TTAGGAACAA TAAACCTACT TGTCCCAACC 420 ALL1L1~L1"L CTCAAGTCAT CGTAGAATTG TAGATCTTTA GGAL-LLlGA TGTATTTTTT 480 AALC~L~L~A GGTTGAAACC TAAAAACAAG CAGAAACAAA CCCAAGCTGA TCAGAGTGAG 540 AATAAAGCTC CATTTTAAGC AACTCCATAA ACCACTAAAG AAA~LlLLLL TGAGACTCTC 600 TTTGAAAATC L~LC~lATTG ALLL~LLLLC CALLLL~Lll CCCATGCGGA TCACAAACGC 660 GCTTCATGCA CTCACCTTGC TCCTAACCAT L-l~Lc~AAcc ATCTTTAGCG TTGCATTTGA 780 lLl-~lL~AAA AAGGCTCATT TCTTAGTTTC TTTTATTCTT AAAATTTTTC CATTCTAGCA 840 AALLLLl~LL AATTGTGGGT AAAAATGTGA A1C~LLC~:IA GCTTTTAGAC GCTTGCAACG 900 ATCGGACTTT TTTCAATATT AATGAAAAAA TGCCA~ATAT TCTAAATATT GTGGTATAGT 960 ATACCAATA AGATACCGAT AGGTATGA~A CTAGGTATAG AAGGAGAAAC A ATG ACT 1077 Met Thr Asn Glu Thr Ile Asp Gln Gln Pro Gln Thr Glu Ala Ala Phe Asn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val Asp Asn W 096/10639 22 n 1 3 ~ 3 PCTrUS95112669 . ~

Ala Val Ala Ser Tyr Asp Pro Asp Gln Lys Pro Ile Val Asp Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Gly Ile Ser Gln Leu Arg Glu GAA TAC TCC AAT A~A GCG ATC AAA AAT CCT ACC AAA AAG AAT CAG TAT 1317 Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn Gln Tyr TTT TCA GAC TTT ATC AAT AAG AGC AAT GAT TTA ATC AAC A~A GAC AAT 1365 Phe Ser Asp Phe Ile Asn Lys Ser Asn Asp Leu Ile Asn Lys Asp Asn Leu Ile Val Val Glu Ser Ser Thr Lys Ser Phe Gln Lys Phe Gly Asp Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn Asp Pro Ser Lys Ile Asn Thr Arg Cys Ile Arg Asn Phe Met Glu His Thr Ile CAA CCC CCT ATC CCT GAT GAC AAA GAA AAA GCA GAG TTT TTG A~A TCT 1557 Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu Lys Ser GCC AAA CAA TCT TTT GCA GGA ATC ATC ATA GGG AAT CA~ ATC CGA ACG 1605 Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile Arg Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu Arg Gln Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu Asp Ile 195 . 200 205 210 TTT TTA TCA TTT ATA TTT GAC AAA AAA CAA TCT TCT GAT GTC A~A GAA 1749 Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys Glu Ala Ile Asn Gln Glu Pro Leu Pro His Val Gln Pro Asp Ile Ala Thr W 096/10639 ~2 g 1 ~ ~ 3 PCTrUS95/12669 Ser Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp Leu Leu GAT GAA AGG GGT AAT TTT TCT A~A TTC ACT CTT GGC GAT ATG GAA ATG 1893 Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met Glu Met Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe Asn Gln Leu Leu Ile His Asn Asn Thr Leu Ser Ser Val Leu Met Gly Ser CAT GAT GGC ATA GAA CCT GAA A~A GTT TCA TTA TTG TAT GCG GGC AAT 2037 His Asp Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala Gly Asn GGT GGT TTT GGA GCC AAG CAC GAT TGG AAC GCC ACC GTT GGT TAT A~A 2085 Gly Gly Phe Gly Ala Lys His Asp Trp Asn Ala Thr Val Gly Tyr Lys GAC CAA CAA GGT AAC AAT GTG GCT ACA ATA ATT AAT GTG CAT ATG A~A 2133 Asp Gln Gln Gly Asn Asn Val Ala Thr Ile Ile Asn Val His Met Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn CCT AGT TTT TAT CTC TAC A~A GAA GAC CAA CTC ACA GGC TCA CAA CGA 2229 Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln Arg GCA TTG AGT CAA GAA GAG ATC CAA AAC A~A ATA GAT TTC ATG GAA TTT 2277 Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys Ile Asp Phe Met Glu Phe CTT GCA CAA AAC AAT GCT AAA TTA GAC AGC TTG AGC GAG A~A GAG A~A 2325 Leu Ala Gln Asn Asn Ala Lys Leu Asp Ser Leu Ser Glu Lys Glu Lys GAA A~A TTC CGA AAT GAG ATT AAG GAT TTC CAA A~A GAC TCT AAG CCT 2373 Glu Lys Phe Arg Asn Glu Ile Lys Asp Phe Gln Lys Asp Ser Lys Pro TAT TTA GAC GCC CTA GGG AAT GAT CGT ATT GCT TTT GTT TCT A~A A~A 2421 Tyr Leu Asp Ala Leu Gly Asn Asp Arg Ile Ala Phe Val Ser Lys Lys GAC CCA A~A CAT TCA GCT TTA ATT ACT GAG TTT AAT AAG GGG GAT TTG 2469 Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Asn Lys Gly Asp Leu W O 96/10639 ~2 Q 1 3 ~ 3 PCTtUS95tl2669 AGC TAC ACT CTC AAA GTT ATG GGA A~A AAG CAG ATA AAG GCT TTA GAT 2517 Ser Tyr Thr Leu Lys Val Met Gly Lys Lys Gln Ile Lys Ala Leu Asp AGG GAG A~A AAT GTC ACT CTT CAA GGT AAC CTA A~A CAT GAT GGC GTG 2565 Arg Glu Lys Asn Val Thr Leu Gln Gly Asn Leu Lys His Asp Gly Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser Lys Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu Glu Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn Asn Leu Ala Ile Thr Ser Val Val Arg Arg Asp Leu Glu Asp hys Leu Ile Ala AAA GGA TTG TCC CCA CAA GAA GCT AAT AAG CTT GTC A~A GAT TTT TTG 2805 Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys Leu Val Lys Asp Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys Ala GTA GCT GAA GCT A~A AAC ACA GGC AAC TAT GAC GAG GTG AAA CGA GCT 2901 Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg Ala CAG AAA GAT CTT GAA A~A TCT CTA AAG AAA CGA GAG CAT TTG GAG A~A 2949 Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu His Leu Glu Lys W 096/10639 ~ 3 PCTrUS95/12669 Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn Lys Met Glu Ala Lys Ala Gln Ala Asn Ser Gln Lys Asp Glu Ile Phe Ala Leu Ile Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala Ile Ala Tyr Ala Gln Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glu Asn Ile AAC AAG GAT TTG AAA GAC TTT AGT AAA TCT TTT GAT GGA TTC AAA A~T 3189 Asn Lys Asp Leu Lys Asp Phe Ser Lys Ser Phe Asp Gly Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Gly Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Val Glu Asn Leu Asn Ala Ala Leu Asn Glu Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Gln Glu Asn Ser Ile Lys Asp Val Ile Ile Asn Gln hys Ile Thr Asp Lys Val Asp Glu Leu Asn Gln Ala Val Ser Val Ala Lys Ile Ala Cys Asp Phe Ser Gly Val Glu Gln Ala Leu Ala Aæp Leu Lys Asn Phe Ser Lys Glu Gln Leu Ala Gln Gln Ala Gln Lys Asn Glu Ser Phe Asn Val Gly Lys Ser Glu Ile Tyr Gln Ser Val Lys Asn Gly Val Asn Gly Thr Leu Val Gly Asn -W 096tlO639 22 0 1 3 5 ~ PCTAJS95/12669 Gly Leu Ser Gly Ile Glu Ala Thr Gly (2) INFORMATION FOR SEQ ID NO:2:
(i) ~yUhN~ CHARACTERISTICS:
tA) LENGTH: 859 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Thr Asn Glu Thr Ile Asp Gln Gln Pro Gln Thr Glu Ala Ala Phe sn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val Asp Asn Ala Val Ala Ser Tyr Asp Pro Asp Gln Lys Pro Ile Val Asp Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Gly Ile Ser Gln Leu Arg Glu Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn ln Tyr Phe Ser Asp Phe Ile Asn Lys Ser Asn Asp Leu Ile Asn Lys sp Asn Leu Ile Val Val Glu Ser Ser Thr Lys Ser Phe Gln Lys Phe Gly Asp Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn Asp Pro Ser Lys Ile Asn Thr Arg Cys Ile Arg Asn Phe Met Glu His Thr Ile Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu ys Ser Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile rg Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu rg Gln Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu W O96/10639 2 2 0 1 3 9 3 PCTnUS95/12669 Asp Ile Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys Glu Ala Ile Asn Gln Glu Pro Leu Pro His Val Gln Pro Asp Ile la Thr Ser Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp eu Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met lu Met Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe Asn Gln Leu Leu Ile His Asn Asn Thr Leu Ser Ser Val Leu Met Gly Ser His Asp Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala ly Asn Gly Gly Phe Gly Ala Lys His Asp Trp Asn Ala Thr Val Gly yr Lys Asp Gln Gln Gly Asn Asn Val Ala Thr Ile Ile Asn Val His et Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln Arg Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys Ile Asp Phe Met lu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Ser Leu Ser Glu Lys lu Lys Glu Lys Phe Arg Asn Glu Ile Lys Asp Phe Gln Lys Asp Ser ys Pro Tyr Leu Asp Ala Leu Gly Asn Asp Arg Ile Ala Phe Val Ser Lys Lys Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Asn Lys Gly Asp Leu Ser Tyr Thr Leu Lys Val Met Gly Lys Lys Gln Ile Lys Ala Leu Asp Arg Glu Lys Asn Val Thr Leu Gln Gly Asn Leu Lys His Asp =
WO96110639 22 0 1 3 5 ~ PCT/US95112669 Gly Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser Lys Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu Glu Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn Asn Leu Ala Ile Thr Ser Val Val Arg Arg Asp Leu Glu Asp Lys Leu le Ala Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys Leu Val Lys Asp he Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg Ala Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu His Leu Glu Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn ys Met Glu Ala Lys Ala Gln Ala Asn Ser Gln Lys Asp Glu Ile Phe la Leu Ile Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala Ile Ala Tyr Ala Gln Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glu Asn Ile Asn Lys Asp Leu Lys Asp Phe Ser Lys Ser Phe Asp Gly Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys la Leu Lys Gly Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile er Lys Val Glu Asn Leu Asn Ala Ala Leu Asn Glu Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Gln Glu Asn Ser Ile Lys Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Glu Leu Asn Gln Ala Val Ser Val Ala Lys Ile Ala Cys Asp Phe W 096/10639 22 0 1 3 5 3 PCTrUS95/~2669 Ser Gly Val Glu Gln Ala Leu Ala Asp Leu Lys Asn Phe Ser Lys Glu Gln Leu Ala Gln Gln Ala Gln Lys Asn Glu Ser Phe Asn Val Gly Lys Ser Glu Ile Tyr Gln Ser Val Lys Asn Gly Val Asn Gly Thr Leu Val Gly Asn Gly Leu Ser Gly Ile Glu Ala Thr Gly (2) INFORMATION FOR SEQ ID NO:3:
( i ) ~QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 4821 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~.~S: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1072..4614 (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:3:
ATGGGCTGCG CGTAACGA~A AACAGTCGCT TGAC~L~llL TGATGTCATC AGAGATTTTC 60 CA~ATATCCG CTATACCTTT GACTCCTAGA GCGCAACCAC CTACGATCGC TAGAACAGAA 120 ATGATCTGAA CCACCA~AGT TTTAGTCTCA GTAATGCCTG ATGCAGGACT GTCGA~AGCC 180 ATTA~AGGAT TGGCTGCTAT CGCTAGCCCT A~AGTTACTA CAA~lLl~ll GTAGCTGTCA 240 GTGATTCTTG TAAAAAATTT CATGCGTTTC CTTTCA~ATT GA~ATCAATC GTTTGAGTAT 300 ATCA~AAAAA AGTATTTTTA TACTATTCAT ACAAGCGCTA CTTTATAATT TA~ATCA~AA 360 CCGACGCTTT L~llL~ACAA CTGATATAAT TTAGGAACAA TA~ACCTACT TGTCCCAACC 420 AL11LL~LL1 CTCAAGTCAT CGTAGAATTG TAGATCTTTA GGAL~llL~A TGTATTTTTT 480 AAlC~l~L~A GGTTGA~ACC TA~AAACAAG CAGA~ACA~A CCCAAGCTGA TCAGAGTGAG 540 AATA~AGCTC CATTTTAAGC AACTCCATAA ACCACTAAAG A~A~LLLLLL TGAGACTCTC 600 TTTGA~AATC TGTCCTATTG A~LLL~1LL1C CAlLLL~LLL CCCATGCGGA TCACAAACGC 660 . ~ L ~ L ~ 3 1 1 - ~Jt~ , : (J 1 1 ~ CJ~ 'L J ~? ~ .3~3~ # ~
/B.~ ' t W0~110C39 ~! g O 1 3 ~ 3 PCT/l~s9~12669 g7 TTA2~ACAA ATAC~5'AC~A TA~TA~aT~ GGCAt~ACAZL AACCZL~ rAGAAC~ 720 G~ ATGCA CTC~CC1~GC q:CGTAPCCAT ~ L~ A~CC AT~1~CG TrGCp.~TGA 7~D
~-L1'~:LL~AA A~ CA~r TC~TAGTTTC m'rATTCIrT A~AAL~LLL~ C~5TCTAGCA ~40 A~LL~-~LIG~L AA1L~ A~aAI~G~rG;A A~ A G~.-LlAGAC G~TTaCA~; 9 ATCGGA~rTT TTTCA~TATT hAl~Aa~ TGCr'~A~ T TC~ Arr GTGGTAT~GT 360 G~TAACCTTC ~AG.7`'Y~GP. ATTG/:~ATACS QAA~ At3~i ~5~ A6CW;~ L~ 1020 GPT~C'AATZ~ AG~$~CCGAT AGGTAT~A CTAGGT~TAG ~G~AAAC A ATG AC'r 1077 Met Thr ~C G~A ACr A~ C CAA CAA CC2~ C~A ACC ~AA GCG GCT TTr AAC CCG 11'~5 A~n ':lu Thr Ile ~sp Gln Gl~ Pro Gln ~ Glu ~a ~;la Phe ~g7~ Pro 5 lO ~
C~G C~ 5'TT ATC AAT A~T CTT CAA GTA ~ T rrT CTT A~A GTT GAT AAC 1173 Gln Gln Phe ~le As~ Asn ~eu Gln ~al Ala Phe Leu Lys Val A~p A~

GCT GTC GCT TCA ~AC ~AT ~:CT GAT ~A AAA CU~ ~TC GTT G:AT ~AG ~AC 1221 Ala Val Al~ Ser q~yr A~p E~-o ~9p Gln ~y~ ~ra Ile V~l ~p Lys Asn 35 ~0 5 50 GA~ AGG ~T AAC AGG Cl~ GC'r rrT 5AG GG~L ATC TC/~ C~ TTA AGG ~-AA 12 ~ 3 A~p Arg A6p ~sn Arg C1~ Ala Phe Glu Gly Ile Ser Gln ~eu Arg Glu 55 6~ 65 GAP~ TAC TCC AAT ~A GCG ATC A~A ~Al' CC~ ~CC A~ AA¢ A~T ~AG ~A~ 17 Glu ~yr 5er Asn ~ys Ala Ile Lys ~9~ Pro Shr ~y~ Ly A~;n Gl~
70 75 ~0 TCA G~C TTT ~rc AP~T AA~ A~C ~T GAT 5'TA ATC ~AC A~A GAC AAT 13 65 Phe Ser Asp Phe Ile A~n Ly~ Se~ Asn ~sp Leu Ile A~3~ hy~ A~p ~s~

CTC ~ 3TC GTG G~ TCT TCC ACA A~LOE AGC 1~ CAG ~A~ m' GGG G~ 1413 ~eu Ile V~l Val Gl~ Ser Ser Thr Lys Ser Phe ~:ln Lys Phe Gly ~p 100 105 llo C~G CGT I'AC Ct~A A5~ ACA AGT TGG GTG TCC CAT CAP. A~C GAT C~:G 1461 G1A ~Y Tyr Arg 3:le Phe Thr Ber Trp ~ral Ser Hi~ Glr~ Asn Asp Pro 115 12~ 125 130 TCT ~AA ATC ~C ACC OE:A TGC ATC CGA Al~T 'rTT AT~ GAA CAT ~CC ATA 150 Ser Ly~ Ile AS~ ~r Ar~ Cys Ile Arg Asn Phe Met Glu ~is ~r I1 1~5 1~ 145 C~ CCC CCT ~TC CCT ~T GAC ~A GA~ ~ GCA GAG rTT TTG AAA ~ 557 Gln ~ro i?ro Il~ Pro ~sp A~p Lys~ GlU ~y~ ~a G1U Phe T~U Lys Ser 150 ~55 16~

h~ s~E~;

TOTRL P . 04 22~13S3 W 096tlO639 PCTtUS9~/12669 GCC A~A CAA TCT TTT GCA GGA ATC ATC ATA GGG AAT CAA ATC CGA ACG 1605 Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile Arg Thr GAT CAA A~A TTC ATG GGC GTG TTT GAT GAA TCC TTG A~A GAA AGG CAA 1653 Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu Arg Gln GAA GCA GAA A~A AAT GGA GGG CCT ACT GGT GGG GAT TGG TTG GAT ATT 1701 Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu Asp Ile TTT TTA TCA TTT ATA TTT GAC A~A A~A CAA TCT TCT GAT GTC A~A GAA 1749 Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys Glu Ala Ile Asn Gln Glu Pro Leu Pro His Val Gln Pro Asp Ile Ala Thr Ser Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp Leu Leu GAT GAA AGG GGT AAT TTT TCT A~A TTC ACT CTT GGC GAT ATG GAA ATG 1893 Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met Glu Met TTA GAT GTT GAG GGC GTC GCC GAC ATG GAT CCC AAT TAC A~G TTC AAT 1941 Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe Asn Gln ~eu Leu Ile His Asn Asn Thr Leu Ser Ser Val Leu Met Gly Ser CAT GAT GGC ATA GAA CCT GAA A~A GTT TCA TTA TTG TAT GCG GGC AAT 2037 His Asp Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala Gly Asn GGT GGT TTT GGA GCC AAG CAC GAT TGG AAC GCC ACC GTT GGT TAT A~A 2085 Gly Gly Phe Gly Ala Lys His Asp Trp Asn Ala Thr Val Gly Tyr Lys GAC CAA CAA GGT AAC AAT GTG GCT ACA ATA ATT AAT GTG CAT ATG A~A 2133 Asp Gln Gln Gly Asn Asn Val Ala Thr Ile Ile Asn Val His Met Lys AAC GGC AGT GGC TTA GTC ATA GCA GGT GGT GAG A~A GGG ATT AAC AAC 2181 Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn CCT AGT TTT TAT CTC TAC A~A GAA GAC CAA CTC ACA GGC TCA CAA CGA 2229 W 096110639 2~ ~ ~ 3 ~ 3 PCT~US95/12669 .

Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln Arg Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys Ile Asp Phe Met Glu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Ser Leu Ser Glu Lys Glu Lys Glu Lys Phe Arg Asn Glu Ile Lys Asp Phe Gln Lys Asp Ser Lys Pro Tyr Leu Asp Ala Leu Gly Asn Asp Arg Ile Ala Phe Val Ser Lys Lys Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Asn Lys Gly Asp Leu Ser Tyr Thr Leu Lys Val Met Gly Lys Lys Gln Ile Lys Ala Leu Asp Arg Glu Lys Asn Val Thr Leu Gln Gly Asn Leu Lys His Asp Gly Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser Lys Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu Glu Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn Asn Leu Ala Ile Thr Ser Val Val Arg Arg Asp Leu Glu Asp Lys Leu Ile Ala Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys Leu Val Lys Asp Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys Ala W 096/10639 22 ~ 1 3 ~ 3 PCTnUS9S/12669 ~

GTA GCT GAA GCT A~A AAC ACA GGC AAC TAT GAC GAG GTG A~A CGA GCT 2901 Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg Ala CAG A~A GAT CTT GAA A~A TCT CTA AAG A~A CGA GAG CAT TTG GAG AAG 2949 Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu His Leu Glu Lys GAT GTA GCG A~A AAT TTG GAG AGC A~A AGC GGC AAC A~A AAT A~A ATG 2997 Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn Lys Met GAA GCA A~A GCT CAA GCT AAC AGC CAA A~A GAT GAG ATT TTT GCG TTG 3045 Glu Ala Lys Ala Gln Ala Asn Ser Gln Lys Asp Glu Ile Phe Ala Leu ATC AAT A~A GAG GCT AAT AGA GAC GCA AGA GCA ATC GCT TAC GCT CAA 3093 Ile Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala Ile Ala Tyr Ala Gln AAT CTT A~A GGC ATC A~A AGG GAA TTG TCT GAT A~A CTT GAA AAT ATC 3141 Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glu Asn Ile AAC AAG GAT TTG A~A GAC TTT AGT A~A TCT TTT GAT GGA TTC A~A AAT 3189 Asn Lys Asp Leu Lys Asp Phe Ser Lys Ser Phe Asp Gly Phe Lys Asn GGC A~A AAT AAG GAT TTC AGC AAG GCA GAA GAA ACG CTA A~A GCC CTT 3237 Gly Lys Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu A~A GGC TCG GTG A~A GAT TTA GGT ATC AAT CCG GAA TGG ATT TCA A~ 3285 Lys Gly Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys GTT GAA AAC CTT AAT GCA GCT TTG AAT GAA TTC A~A AAT GGC A~A AAT 3333 Val Glu Asn Leu Asn Ala Ala Leu Asn Glu Phe Lys Asn Gly Lys Asn AAG GAT TTC AGC AAG GTA ACG CAA GCA A~A AGC GAC CAA GAA AAT TCC 3381 Lys Asp Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Gln Glu Asn Ser ATT A~A GAT GTG ATC ATC AAT CAA AAG ATA ACG GAT A~A GTT GAT GAA 3429 Ile Lys Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Glu CTC AAT CAA GCG GTA TCA GTG GCT A~A ATA GCG TGC GAT TTC AGT GGG 3477 Leu Asn Gln Ala Val Ser Val Ala Lys Ile Ala Cys Asp Phe Ser Gly WO96/10639 ~2 ~ 3 PCT/US9!j112669 Val Glu Gln Ala Leu Ala Asp Leu Lys Asn Phe Ser Lys Glu Gln Leu Ala Gln Gln Ala Gln Lys Asn Glu Ser Phe Asn Val Gly Lys Ser Glu Ile Tyr Gln Ser Val Lys Asn Gly Val Asn Gly Thr Leu Val Gly Asn Gly Leu Ser Gly Ile Glu Ala Thr Ala Leu Ala Lys Asn Phe Ser Asp Ile Lys Lys Glu Leu Asn Glu Lys Phe Lys Asn Phe Asn Asn Asn Asn Asn Asn Gly Leu Lys Asn Gly Gly Glu Pro Ile Tyr Ala Gln Val Asn Lys Lys Lys Thr Gly Gln Val Ala Ser Pro Glu Glu Pro Ile Tyr Ala Gln Val Ala Lys Lys Val Thr Lys Lys Ile Asp Gln Leu Asn Gln Ala Ala Thr Ser Gly Phe Gly Gly Val Gly Gln Ala Gly Phe Pro Leu Lys Arg His Asp Lys Val Glu Asp Leu Ser Lys Val Gly Arg Ser Val Ser Pro Glu Pro Ile Tyr Ala Thr Ile Asp Asp Leu Gly Gly Ser Phe Pro Leu Lys Arg His Asp Lys Val Asp Asp Leu Ser Lys Val Gly Leu Ser Arg Asn Gln Glu Leu Thr Gln Lys Ile Asp Asn Leu Ser Gln Ala Val W O 96/10639 2~ ~ 1 3 5 ~ PCTrUS95112669 Ser Glu Ala Lys Ala Gly Phe Phe Gly Asn Leu Glu Gln Thr Ile Asp AAG CTC A~A GAT TTT ACA A~A AAC AAT CCT GTG AAT CTA TGG GCT GAA 4197 Lys Leu Lys Asp Phe Thr Lys Asn Asn Pro Val Asn Leu Trp Ala Glu 1030 1035 i040 AGC GCA A~A A~A GTG CCT GCT AGT TTG T Q GCG A~A CTA GAC AAT TAC 4245 Ser Ala Lys Lys Val Pro Ala Ser Leu Ser Ala Lys Leu Asp Asn Tyr Ala Thr Asn Ser His Thr Arg Ile Asn Ser Asn Ile Gln Asn Gly Ala ATC AAT GAA A~A GCG ACC GGC ACT GAA CGG CAA A~A AAC CCT GAG TGG 4341 Ile Asn Glu Lys Ala Thr Gly Thr Glu Arg Gln Lys Asn Pro Glu Trp CTC A~A CTC GTG AAT GAT AAG ATC GTT GCG CAT AAT GTG GGA AGC GTT 4389 Leu Lys Leu Val Asn Asp Lys Ile Val Ala His Asn Val Gly Ser Val Pro Leu Ser Glu Tyr Asp Asn Ile Gly Phe Ser Gln Lys Asn Met Lys Asp Tyr Ser Asp Ser Phe Lys Phe Ser Thr Lys Leu Asn Asn Ala Val A~A GAC ATT AAG TCT GGC TTT ACG CAA TTT TTA GCC AAT GCA TTT TCT 4533 Lys Asp Ile Lys Ser Gly Phe Thr Gln Phe Leu Ala Asn Ala Phe Ser ACA GGA TAT TAC TCC ATG GCG AGA GAA AAT GCG GAG CAT GGA ATC A~A 4581 Thr Gly Tyr Tyr Ser Met Ala Arg Glu Asn Ala Glu His Gly Ile Lys AAT GCT AAT ACA A~A GGT GGT TTC CAA A~A TCT TAAAGGATTA AGGAACAC Q 4634 Asn Ala Asn Thr Lys Gly Gly Phe Gln Lys Ser A~AACG Q~A AACCACCTTG CTA~AAGCAA GGG~ ll AACTTA~AAT ATCCCGACAG 4694 A QCTAACGA AAGGCTTTGT TCTTTA~AGT CTG QTAGAT ATTTCCTACC CCAaAAAGAC 4754 TTAACCCTTT GCTTA~AATT A~ATTTGATT GTGCTAGTGG ~LlC~lGCTT TATAGTGCGG 4814 (2) INFORMATION FOR SEQ ID NO:4:

W O96/10639 PCT~US95/12669 ~2 0 ~ ~ ~ 3 (i) S~u~N~ CHARACTERISTICS:
(A) LENGTH: 1181 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear lii) MOLECULE TYPE: protein (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:4:
Met Thr Asn Glu Thr Ile Asp Gln Gln Pro Gln Thr Glu Ala Ala Phe sn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val Asp Asn Ala Val Ala Ser Tyr Asp Pro Asp Gln Lys Pro Ile Val Asp Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Gly Ile Ser Gln Leu Arg Glu Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn ln Tyr Phe Ser Asp Phe Ile Asn Lys Ser Asn Asp Leu Ile Asn Lys sp Asn Leu Ile Val Val Glu Ser Ser Thr Lys Ser Phe Gln Lys Phe Gly Asp Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn Asp Pro Ser Lys Ile Asn Thr Arg Cys Ile Arg Asn Phe Met Glu His Thr Ile Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu ys Ser Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile rg Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu Arg Gln Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu Asp Ile Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys Glu Ala Ile Asn Gln Glu Pro Leu Pro His Val Gln Pro Asp Ile W 096/10639 22 ~ 1 3 5 3 PCTnUS95/12669 ~

Ala Thr Ser Thr Thr His Ile Gln Gly Leu Pro Pro Glu Ser Arg Asp eu Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met lu Met Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe Asn Gln Leu Leu Ile His Asn Asn Thr Leu Ser Ser Val Leu Met Gly Ser His Asp Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala ly Asn Gly Gly Phe Gly Ala Lys His Asp Trp Asn Ala Thr Val Gly yr Lys Asp Gln Gln Gly Asn Asn Val Ala Thr Ile Ile Asn Val His et Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln Arg Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys Ile Asp Phe Met lu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Ser Leu Ser Glu Lys lu Lys Glu Lys Phe Arg Asn Glu Ile Lys Asp Phe Gln Lys Asp Ser ys Pro Tyr Leu Asp Ala Leu Gly Asn Asp Arg Ile Ala Phe Val Ser Lys Lys Asp Pro Lys His Ser Ala Leu Ile Thr Glu Phe Asn Lys Gly Asp Leu Ser Tyr Thr Leu Lys Val Met Gly Lys Lys Gln Ile Lys Ala eu Asp Arg Glu Lys Asn Val Thr Leu Gln Gly Asn Leu Lys His Asp ly Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser ys Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu lu Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn -~ W 096/10639 22 0 1 ~ 5 3 PCT~US95/12669 Asn Leu Ala Ile Thr Ser Val Val Arg Arg Asp Leu Glu Asp Lys Leu Ile Ala Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys Leu Val Lys Asp Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg Ala Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu His Leu Glu Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn Lys Met Glu Ala Lys Ala Gln Ala Asn Ser Gln Lys Asp Glu Ile Phe Ala Leu Ile Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala Ile Ala Tyr Ala Gln Asn Leu Lys Gly Ile Lys Arg Glu Leu Ser Asp Lys Leu Glù

Asn Ile Asn Lys Asp Leu Lys Asp Phe Ser Lys Ser Phe Asp Gly Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Gly Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Val Glu Asn Leu Asn Ala Ala Leu Asn Glu Phe Lys Asn Gly Lys Asn Lys Asp Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Gln Glu Asn Ser Ile Lys Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Glu Leu Asn Gln Ala Val Ser Val Ala Lys Ile Ala Cys Asp Phe Ser Gly Val Glu Gln Ala Leu Ala Asp Leu Lys Asn Phe Ser Lys Glu Gln Leu Ala Gln Gln Ala Gln Lys Asn Glu Ser Phe Asn Val Gly Lys ~ ~ Q ~ 3 5 3 -Ser Glu Ile Tyr Gln Ser Val Lys Asn Gly Val Asn Gly Thr Leu Val Gly Asn Gly Leu Ser Gly Ile Glu Ala Thr Ala Leu Ala Lys Asn Phe Ser Asp Ile Lys Lys Glu Leu Asn Glu Lys Phe Lys Asn Phe Asn Asn Asn Asn Asn Asn Gly Leu Lys Asn Gly Gly Glu Pro Ile Tyr Ala Gln Val Asn Lys Lys Lys Thr Gly Gln Val Ala Ser Pro Glu Glu Pro Ile Tyr Ala Gln Val Ala Lys Lys Val Thr Lys Lys Ile Asp Gln Leu Asn Gln Ala Ala Thr Ser Gly Phe Gly Gly Val Gly Gln Ala Gly Phe Pro Leu Lys Arg His Asp Lys Val Glu Asp Leu Ser Lys Val Gly Arg Ser Val Ser Pro Glu Pro Ile Tyr Ala Thr Ile Asp Asp Leu Gly Gly Ser Phe Pro Leu Lys Arg His Asp Lys Val Asp Asp Leu Ser Lys Val Gly Leu Ser Arg Asn Gln Glu Leu Thr Gln Lys Ile Asp Asn Leu Ser Gln Ala Val Ser Glu Ala Lys Ala Gly Phe Phe Gly Asn Leu Glu Gln Thr Ile Asp Lys Leu Lys Asp Phe Thr Lys Asn Asn Pro Val Asn Leu Trp Ala Glu Ser Ala Lys Lys Val Pro Ala Ser Leu Ser Ala Lys Leu Asp Asn Tyr Ala Thr Asn Ser His Thr Arg Ile Asn Ser Asn Ile Gln Asn Gly Ala Ile Asn Glu Lys Ala Thr Gly Thr Glu Arg Gln Lys Asn Pro Glu Trp Leu Lys Leu Val Asn Asp Lys Ile Val Ala His Asn Val Gly Ser Val Pro Leu Ser Glu Tyr Asp Asn Ile Gly Phe Ser Gln Lys Asn ~ 3 5 3 Met Lys Asp Tyr Ser Asp Ser Phe Lys Phe Ser Thr Lys Leu Asn Asn Ala Val Lys Asp Ile Lys Ser Gly Phe Thr Gln Phe Leu Ala Asn Ala Phe Ser Thr Gly Tyr Tyr Ser Met Ala Arg Glu Asn Ala Glu His Gly 1155 ~1160 1165 Ile Lys Asn Ala Asn Thr Lys Gly Gly Phe Gln Lys Ser

Claims (26)

What is claimed is:

1. An isolated nucleic acid encoding an approximately 120-128 kilodalton antigen of Helicobacter pylori or an antigenic fragment thereof, wherein the antigen is associated with peptic ulceration.

2. A vector comprising the nucleic acid of claim 1.

3. The vector of claim 2 in a host suitable for expressing the polypeptide encoded by the nucleic acid.

4. The nucleic acid of claim 1, comprising nucleotides 1072 through 4614 contained in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO:3.

5. The nucleic acid of claim 1, comprising nucleotides 1072 through 3648 contained in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO:1.

6. The nucleic acid of claim 1, comprising nucleotides 1921 through 3648 contained in the nucleotide sequence.
defined in the Sequencing Listing as SEQ ID NO:1.

7. An isolated nucleic acid that selectively hybridizes with the nucleic acid of claim 4 under polymerase chain reaction conditions.

8. An isolated nucleic acid that hybridizes with the nucleic acid of claim 4 under the stringency conditions of 68°C for 16 hours in buffer containing 6X SSC, 0.5% sodium dodecyl sulfate, 5X Denhardt's solution, with washing at 60°C in 0.5X SSC.

9. A purified antigenic polypeptide encoded by the nucleic acid of claim 1.

10. The antigenic polypeptide of claim 9, wherein the polypeptide consists essentially of the amino acids encoded by nucleotides 1072 through 4614 contained in the nucleotide sequence defined in the Sequence Listing as SEQ
ID NO:3.

11. The antigenic polypeptide of claim 9, wherein the polypeptide consists essentially of the amino acids encoded by nucleotides 1072 through 3648 contained in the nucleotide sequence defined in the Sequence Listing as SEQ
ID NO:1.

12. The antigenic polypeptide of claim 9, wherein the polypeptide consists essentially of the amino acids encoded by nucleotides 1921 through 3648 contained in the nucleotide sequence defined in the Sequence Listing as SEQ
ID NO:1.

13. A method of detecting the presence of a Helicobacter pylori strain possessing the 120-128 kilodalton antigen in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 10;

b. detecting the binding of the polypeptide and the antibody, the reaction indicating the presence of the Helicobacter pylori strain.

14. A method of detecting the presence of a Helicobacter pylori strain possessing the 120-128 kilodalton antigen in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 11;

b. detecting the binding of the polypeptide and the antibody, the binding indicating the presence of the Helicobacter pylori strain.

15. A method of detecting the presence of a Helicobacter pylori strain possessing the 120-128 kilodalton antigen in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 12;

b. detecting the binding of the polypeptide and the antibody, the binding indicating the presence of the Helicobacter pylori strain.

16. A method of determining predisposition to peptic ulceration in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 10;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to peptic ulceration.

17. A method of determining predisposition to peptic ulceration in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 11;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to peptic ulceration.

18. A method of determining predisposition to peptic ulceration in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 12;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to peptic ulceration.

19. A method of determining predisposition to gastric carcinoma in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 10;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to gastric carcinoma.

20. A method of determining predisposition to gastric carcinoma in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 11;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to gastric carcinoma.

21. A method of determining predisposition to gastric carcinoma in a subject, comprising the steps of:

a. contacting an antibody-containing sample from the subject with a detectable amount of the polypeptide of claim 12;

b. detecting the binding of the polypeptide and the antibody, the binding indicating a predisposition of the subject to gastric carcinoma.

22. A method of detecting the presence of the Helicobacter pylori associated with peptic ulceration in a subject, comprising detecting the presence of the nucleic acid of claim 1.

23. The polypeptide of claim 9 in a pharmaceutically acceptable carrier.

24. A mutant Helicobacter pylori in which the product of the nucleic acid of Claim 1 has been rendered nonfunctional.

25. The Helicobacter pylori strain of claim 24, wherein the Helicobacter pylori is deposited with the American Type Culture Collection under ATCC Accession Number 55359.

26. The mutant Helicobacter pylori of claim 24 in a pharmaceutically acceptable carrier.