WO2005023851A1

WO2005023851A1 - Plasminogen/plasmin binding polypeptides and nucleic acids therefore

Info

Publication number: WO2005023851A1
Application number: PCT/SE2004/001272
Authority: WO
Inventors: Göran Kronvall; Klas Jönsson
Original assignee: Karolinska Innovations Ab
Priority date: 2003-09-05
Filing date: 2004-09-06
Publication date: 2005-03-17

Abstract

The invention provides Helicobacter pylori polynucleotides corresponding to the genes HP0508 and HP0863, respectively. The polynucleotides are responsible for coding of two new proteins with plasminogen/plasmin binding properties. The corresponding polypeptides and polynucleotides that encode these polypeptides, are useful in medical methods, such as in for preventing or treating Helicobacter pylori infection. They can also potentially be used as antigens in tests to detect antibodies in individuals exposed to Helicobacter pylori.

Description

Plasminogen/plasmin binding polypeptides and nucleic acids therefore

FIELD OF THE INVENTION

The present invention relates to polypeptides derived from Helicobacter pylori and their corresponding nucleic acids, as well as to the use thereof within the medical field.

BACKGROUND OF THE INVENTION

Helicobacter pylori is a microaerophilic, gram-negative bacterium that colonizes the mucosa of the human stomach. One of the most common pathogens of humans, this microbe is associated with chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach. Some well-characterized virulence factors are known to aid in its pathogenesis by altering the microbe's environment: lowering pH (urease), collagenase, affecting host cells (vacA), or disrupting tight junctions (cagA). All have enzymatic activity that can alter their environment to the bacteriums' own advantage. Secreted or surface-localized enzymes such as urease are powerful tools for the microbe to alter its environment and proteases and toxins are classically virulence factors. The ability of H. pylori to bind the proenzyme plasminogen and therefore harness protease activity may be crucial to its unique persistence in the human stomach.

Recently, plasminogen (Pig) binding activity was described in H pylori ( Ringner, M., Valkonen, K. H. & Wadstrom, T. (1994) FEMS Immunol. Med. Microbiol. 9, 29-34; Pantzar, M., Ljungh, A. & Wadstrom, T. (1998) Infect. Immun. 66, 4976-4980; Yarzabal, A. (2000) Braz. J. Biol. Res. 33, 1015-1021).

In a recent patent application (WO 02/077022) surface proteins of H. pylori have been identified with binding properties for polysulphated molecules. The application claims purification procedures for the identification of the peptides as well as diagnostic and therapeutic use. The five peptide sequences are all different from the plasminogen binding protein genes (HP0508 and HP0863) according to the present invention.

Pig binding is common in host-bacteria interaction and plays a major role in the virulence of the pathogen. Among several, Pig binding has been reported for group A, C, and G streptococci, Neisseria meningitidis, Salmonella enterica, Haemophilus influenzae, and Borrelia burgdorferi, and is believed to facilitate tissue penetration after the surface-bound Pig is converted to plasmin, a broad-specificity protease, by a Pig activator. In a clear example of host exploitation, both Pig and activator (with the exceptions of Yersinia pestis and Streptococcus), are supplied by the host. For Borrelia burgdorferi and Yersinia pestis, Pig binding has been demonstrated to be important for virulence in vivo. Pig binding is also utilized by the influenza A virus to enter host cells and the disease-associated prion protein PrP(Sc) (transmissible spongiform encephalopathy) has been reported to bind Pig, leading to enhanced Pig activation by tPA, although the physiological significance of this binding is unclear.

Pig is an enzymatically inactive 91 -kDa single chain plasma and extracellular glycoprotein. It is the main component of the fibrinolytic system that during wound healing is activated by proteolytic cleavage into the active form plasmin, a serine protease, which binds to and cleaves fibrin, the main component of blood clots. Plasmin consists of an A and B chain which are connected by disulfide bridges. The A chain consists of loop structures (kringles) which have lysine binding capacity and enables the protein to bind to fibrin. Following binding to fibrin, the B chain, which contains the proteolytically active domain, degrades fibrin to fibrin fragments. Plasmin can also degrade several extracellular matrix molecules accumulated in tissues and blood where the plasmin activity is controlled by specific inactivators such as α₂-antiplasmin and -microglobulirι and the plasminogen activator inhibitors PAI-1, PAI-2, which regulates the Pig activators tPA and uPA.

Although Pig binding proteins of MW 42 kDa and 57-58.9 kDa had previously been shown to exist (Pantzar, Ljungh et al.; Yarzabal, vide supra) until now no DNA or amino acid sequences had been reported for H pylori specific Pig binding protein (Plg-bp) genes or gene products.

SUMMARY OF THE INVENTION

According to a first aspect the present invention provides an isolated nucleic acid molecule encoding an amino acid sequence selected from SEQ ID NO:l and SEQ ID NO: 2 of the appended sequence listing.

In one embodiment of this aspect said nucleic acid is derived from any of the genes HP0508 or HP0863.

In another embodiment said nucleic acid is derived from a sequence SEQ ID NO:3 of the appended sequence listing.

According to a second aspect the present invention provides a polypeptide encoded by a nucleic acid according to the invention. In one embodiment the polypeptide has an amino acid sequence selected from SEQ ED NO:l and SEQ ID NO:2.

According to a further aspect the present invention provides an antibody to the polypeptide according to the invention.

According to still a further aspect the invention provides a pharmaceutical composition comprising at least one nucleic acid, polypeptide or antibody according to the invention together with a physiologically acceptable diluent or carrier.

hi one embodiment this pharmaceutical composition is for use as a vaccine.

According to still another aspect the invention provides a method of preventing or treating Helicobacter infection in a mammal by administering to said mammal a prophylactically or therapeutically effective amount of a pharmaceutical composition according to the invention.

According to another aspect, the invention provides primers for, or probes that hybridize with, a nucleotide sequence of a nucleic acid according to the invention for use in a diagnostic method for the purpose of detecting Helicobacter pylori.

According to still a further aspect, the invention provides a diagnostic kit for assessing the presence of Helicobacter pylori in a mammal or in a biological or environmental sample, comprising a primer or probe, a polypeptide or an antibody according to the invention

According to still another aspect the invention provides a method of detecting Helicobacter pylori in a mammal or in a biological or environmental sample by:

- bringing a primer or probe, a polypeptide or an antibody into contact with a biological sample from said mammal or with said biological or environmental sample; and

- detecting any product of binding of said primer or probe, said polypeptide or said antibody to any components derived from Helicobacter pylori present in any of said samples.

Further aspects of the invention are as defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Protein sequence alignment of C-termini of genes corresponding to HP0508 and

HP0863 from strains 26695, J99 and CCUG 17874. The sequences from phage display clones #18 and #21 are shown. Numbers indicate residue number on protein. Protein sequences (Gene HP0508 and HP0863) from GeneMark.

FIGURE 2. Attachment of Plg-bps to Pig. Various concentrations of recombinant Plg-bps containing His tag fusions were incubated with Pig bound to microtiter wells. Attachment was detected by incubation with alkaline phosphatase conjugated anti-His antibody followed by p-nitrophenyl phosphate colorimetric development. Absorbance readings were taken at 405 nm in triplicate and results are representative of at least three independent experiments. Standard deviations are shown.

FIGURE 3. Specificity of Plg-bps attachment to Pig. Recombinant Plg-bps containing His tag fusions were preincubated with Pig or fetuin before being allowed to attach to Pig bound to microtiter wells. Absorbance readings were taken at 405 nm in triplicate and results are representative of at least three independent experiments. Standard deviations are shown.

FIGURE 4. Inhibition of PgbA (A) and PgbB (B) attachment to Pig with lysine and EACA. Absorbance readings were taken at 405 nm in triplicate and results are representative of at least three independent experiments. Standard deviations are shown.

FIGURE 5. Expression of Plg-bps by H pylori strains of diverse geographical origins. Western blot of H. pylori whole cell lysates using antibody 114, which recognizes both Plg- bps. Lanes: (1) Poland 43; (2) Turkey 33; (3) Iran 23; (4) Sweden 5; (5) Somalia 3; (6) CCUG 17874; (7) 26695; (8) AH244; (9) E. coli BL21 Star. Kaleidascope prestained (Bio- Rad) molecular weight markers (kDa) are indicated (sizes run larger than actual).

FIGURE 6. Surface localization of C-termini of Plg-bps PgbA and PgbB. Western blot and of proteinase K-treated intact H. pylori CCUG17874. Treated and untreated whole cell lysates were probed with antibody 114, which recognizes the C-termini of both Plg-bps. Benchmark prestained (Invitrogen) molecular weight markers (kDa) are indicated.

FIGURE 7. Recombinant Plg-bps inhibit attachment of Pig to whole H. pylori. Digoxigenin- labeled Pig was preincubated with purified, recombinant Plg-bps before being allowed to attach to H pylori strain CCUG 17874 immobilized onto microtiter wells. Attached Pig was detected by incubating with alkaline phosphatase conjugated anti-digoxigenin antibodies and colorimetric development in the presence of p-nitrophenyl phosphate. Absorbance readings were taken at 405 nm in triplicate and results are representative of at least three independent experiments. Standard deviations are shown. FIGURE 8. Pig bound to Plg-bps can be activated to plasmin. Pig was allowed to attach to Plg-bps immobilized on microtiter wells. Wells with no Pig added were used as controls. The Pig bound to Plg-bp was incubated with and without tPA. Activation to plasmin was assessed by development in the presence of a chromogenic plasmin substrate, V-0882. Absorbance readings were taken at 405 nm in triplicate and results are representative of at least three independent experiments. Standard deviations are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated nucleic acid corresponding to the genes HP0508 and HP0863 of Helicobacter pylori.

Herein below, these genes will be referred to as pgbA and pgbB, respectively, and the plasminogen/plasmin binding polypeptide, or protein, encoded by HP0508 (i.e. pgbA) will be referred to as PgbA, whereas the plasminogen/plasmin binding polypeptide, or protein, encoded by HP0863 (i.e. pgbB) will be referred to as PgbB.

The term "isolated", in respect of the inventive nucleic acid, is to be construed as referring to a nucleic acid which is essentially separated from other genes that naturally occur in H. pylori. Also, for the purpose of the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated by the context. Thus, for example, reference to "an antibody" includes multiple copies of the antibody and can also include more than one particular species of the antibody.

The nucleic acid of the present invention can include the positive and/or negative RNA strand, the sense and/or antisense DNA strand, as well as any combinations thereof. The nucleic acid can also be modified, e.g. by containing methylated bases.

The nucleic acid can comprise the coding sequence for the inventive polypeptides, or the coding sequence with the gene's upstream and downstream regulatory sequences, or any combination thereof. The nucleic acid can, for example, comprise a DNA and include its own promoter, or another promoter may be operatively linked to the nucleic acid such that the coding sequence of the polypeptide is expressed. Alternatively, the nucleic acid can comprise an RNA which on translation provides the polypeptide of the invention.

The nucleic acid encoding the polypeptide according to the invention may be obtained by any number of techniques well known to one skilled in the art. As an example, a recombinant nucleic acid molecule may be synthesized. Oligonucleotide synthesis procedures are well known to the art and oligonucleotides coding for a particular protein are readily obtainable through automated DNA synthesis. One strand of a double-stranded nucleic acid molecule can be synthesized and hybridized to its complementary strand. The oligonucleotides may be designed so as to obtain a corresponding double-stranded molecule having either internal restriction sites or appropriate overhangs at the termini for cloning into an appropriate vector.

The present invention also provides a purified polypeptide of a sequence shown as SEQ ED NO:l or SEQ ED NO:2, any functionally equivalent variant thereof, and a purified polypeptide encoded by a nucleic acid according to the invention. The polypeptide can be used e.g. as a vaccine component and as a reagent for identifying antibodies raised in a host infected by H. pylori.

PgbA and PgbB, identified as described herein below, have molecular weights of 63 kDa and 70 kDa apparent and 50 and 60 kDa calculated molecular weights, respectively. The differences between calculated and apparent molecular weights are likely due to posttranslational modifications. As mentioned herein above, is intended that the invention also encompasses functionally equivalent variants as well as fragments of the inventive polypeptides and these can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the functionality, e.g. plasminogen/plasmin binding activity, antibody binding capacity or immunogenicitiy, of the peptide is not significantly impaired compared to the native proteins. Functional or active regions of the plasminogen binding protein may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are well known to a person skilled in the art.

For the purpose of the present specification "purified polypeptide" means that the protein or fragment is sufficiently free from contaminants or cell components with which the protein normally occurs to be useful for the intended purpose, e.g. a serological assay.

The inventive polypeptides can be synthesized e.g. by recombinant DNA technology, such as is well known to the person skilled in the art, and also can be chemically synthesized using currently available laboratory equipment and standard chemical reactions for protein synthesis.

The present invention further provides a pharmaceutical composition comprising at least one immunogenic polypeptide derived from PgbA or PgbB. In an advantageous embodiment the pharmaceutical composition comprises polypeptides derived from both PgbA and PgbB. Suitably, the immunogenic polypeptide will comprise at least five, and more preferably at least ten, contiguous amino acid residues. An immunologically effective amount of the inventive polypeptide or a fragment thereof can be used to inoculate a host organism such that the host generates an active immune response thereto which can later provide protection to the host from infection by H pylori.

By use of the term "immunologically effective amount", it is meant that the administration of that amount to a mammalian host, either in a single dose or as part of a series, is effective for treatment or prevention of Helicobacter infection. The amount varies depending upon e.g. the health and physical condition of the subject to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Examples of afflictions and disorders that may advantageously be treated by administration of the pharmaceutical compositions of the invention are chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach.

In avaccine composition of the invention the amount of polypeptide administered will depend on the subject to be treated, the condition of the subject, the size of the subject, etc., but will be at least an immunogenic amount. The polypeptide of the present invention may be the sole active immunogen in the vaccine composition. Alternatively the vaccine composition may include other active immunogens, including other Helicobacter antigens such as e.g. urease, lipopolysaccharide and catalase, as well as immunologically active antigens against other pathogenic species.

In another embodiment the polypeptide of the invention or an immunogenic fragment thereof may be delivered to the mammal to be treated using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the polypeptide. A number of bacteria that colonise the gastrointestinal tract have been developed as vaccine vectors, e.g. Salmonella, Shigella, Yersinia and Escherichia.

The present invention also provides a purified antibody which selectively binds with the polypeptide of SEQ ID NO:l or SEQ ID NO:2. The antibody (either polyclonal or monoclonal) can be raised to the plasminogen binding protein of the invention or an immunogenic fragment thereof. Methods of preparing antibodies are well known to the person skilled in the art.The antibody can be used in techniques or procedures such as diagnostics, treatment or vaccination.

Immunization against H. pylori may further be achieved by a "naked" DNA vaccine approach, wherein DNA constructs containing promoter sequences upstream of the sequences coding for the plasminogen binding polypeptides can be injected into muscle tissue or administered via the mucosa and result in expression of antigens that induce a protective immune response.

The pharmaceutically acceptable diluent, carrier or adjuvant in the pharmaceutical composition of the present invention, such as a vaccine, can be selected by standard criteria. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained. The diluent, carrier or adjuvant will of course depend on the selected way of administration, which can be e.g.oral, sublingual mucosal, inhaled, absorbed, or by injection.

As an example, an oral vaccine composition according to the invention may comprise a mucosal adjuvant, such as cholera toxin, non-toxic derivatives of cholera toxin, chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. Further, other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; and antibiotics such as streptomycin.

Other adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in the prophylactic or therapeutic pharmaceutical composition of this invention. The formulation of such prophylactic or therapeutic compositions is well known to persons skilled in the field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Also, the compositions may include additional active ingredients. For details on pharmaceutical formulations reference may be made to Remington: "The Science and Practice of Pharmacy," 20th Edn., AR Gennaro, Editor, Mack Publishing Co., Easton, PA (2003) (ISBN 0-7817-5025-3).

The present invention also provides a method of detecting the presence of H. pylori in a mammal or in a biological or environmental sample, e.g. comprising the steps of contacting a sample suspected of containing H. pylori, or derived from a mammal suspected of being infected with H pylori, with nucleic acid primers capable of hybridizing to a nucleic acid according to the invention, amplifying the nucleic acid and detecting the presence of an amplification product, the presence of the amplification product indicating the pprreesseennce of H. pylori in the sample or in the mammal.

Amplification methods are well known in the art and encompass e.g. the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the self-sustained sequence replication (3SR) system, the transcription-based amplification system (TAS), and the RNA replication system based on Q.beta. replicase.

The amplified nucleic acid can be detected in any suitable detection assay, such as by use of radio-labelled primers, or by use of primers containing other detectable moieties, such as biotin. The amplified nucleic acid can also be stained and visualized, such as with ethidium bromide staining.

The present invention also provides a method of detecting the presence of H. pylori in a mammal by contacting an antibody-containing sample from the mammal with a polypeptide according to the invention, and detecting the binding of the antibody with the polypeptide, the binding indicating the presence of H. pylori in the mammal.

Furthermore, the present invention provides a method of detecting the presence of H. pylori in a mammal, comprising the steps of contacting a sample from the mammal with an antibody according to the invention and detecting the binding of the antibody with an antigen, the binding indicating the presence of H pylori in the mammal.

Immunodiagnostic methods that can be used to detect as well as quantitate antigen or antibody are well known to the person skilled in the art. As an example, the detection and quantitation of the antigen or antibody may be performed by use of immunoassays such as immunofluorescence assays (IF A), enzyme linked immunosorbent assays (ELISA) and immunoblotting.

In an exemplary ELISA method for detecting the antigen, the antibody is bound to a substrate; the bound antibody is brought into contact with a biological sample containing the antigen; the antigen-antibody complex is brought into with a second antibody that is bound to a detectable moiety such as alkaline phosphatase; the whole complex is brought into contact with the substrate for the enzyme; finally a colour reagent is added and the colour change is observed. As is well known to the person skilled in the art the ELISA method is also adaptable to detection of antibody. According to one aspect the present invention provides a method of preventing or treating Helicobacter pylori infection in a mammal by administering to the subject a prophylactically or therapeutically effective amount of a vaccine comprising an immunogenic polypeptide according to the invention either alone or with a pharmaceutically acceptable carrier.

Another method of preventing or treating aH. pylori infection in a mammal may comprise administering to the mammal a prophylactically or therapeutically effective amount of an antibody to the polypeptide of the invention either alone or with a pharmaceutically acceptable carrier.

EXAMPLES

Identification of Pig binding activity byphage display and database search of the identified gene/protein

Previous studies had shown that H. pylori CCUG 17874 can bind Pig, an activity attributed to 42, 57 (Pantzar, Ljungh et al. vide supra), and 58.9 kDa (Yarzabal vide supra) water- extractable surface proteins, however no gene(s) were identified. In order to clone gene(s) responsible for H. pylori Pig binding, we constructed a phage display library containing sonicated chromosomal DNA fragments. The library was panned against immobilized Pig, which revealed two clones, designated #18 and #21, which exhibited binding activity. Sequencing of both clones revealed overlapping regions and by searching the H pylori 26695 sequence database (www.tigr.org), the combined #18 and #21 sequences which we designated "comb" (Fig. 1), were shown to have 95% identity on the DNA level to the gene PGBA. Surprisingly, a subsequent database search of the amino acid sequence of PgbA revealed 100% identity of the C-terminal 54 amino acids, a lysine-rich domain, with those of PgbB in strain 26695 (Fig. 1). Plg-bps are characteristically lysine-rich and binding is mediated by these lysine residues, therefore this finding suggested that the protein encoded by HP0863 may also bind Pig. The molecular weights of these proteins based on their sequences were 50 and 60 kDa so it is unclear whether they are the same as those previously reported.

Recombinant Plg-bp H pylori CCUG 17874 PgbA and PgbB bind Pig To test the proteins for Pig binding activity, both PgbA and HP0863 were cloned without their signal sequences, expressed as His tag fusions, and affinity purified by Ni-chelate chromatography. The purified recombinant proteins, PgbA and PgbB, appeared as single bands at approximately 55 kDa and 65 kDa, respectively, when analyzed by SDS-PAGE (data not shown). To assay binding to Pig, Pig was coated onto microtiter wells and the remaining available sites were blocked with bovine serum albumin (BSA) before allowing purified recombinant Plg-bps to attach. Attachment was detected by incubating with an alkaline phosphatase conjugated antibody against the His-tag and colorimetric visualization. Both proteins exhibited concentration-dependent Pig-attachment (Fig. 2). In this assay, PgbA attaches to Pig approximately 20% better than PgbB, although both reach saturation at 0.25 μM (Fig. 2). Henceforth we will refer to PgbA and PgbB proteins as Plg-bps.

To determine whether the binding of Pig to Plg-bps is specific, we inhibited the interaction using Pig and fetuin and showed that Pig was able to disrupt the interaction, whereas fetuin could not (Fig. 3). With Pig as an inhibitor, attachment could be decreased by more than 80% for both Plg-bps, whereas with fetuin, attachment of PgbB was decreased by only 20% and not at all for PgbA (Fig. 3). These results demonstrate that the interactions are specific and not dependent on sialic acid.

Pig is known to have lysine binding domains and other Plg-bps characteristically exhibit lysine-dependent binding via interaction with these domains. In previous studies, it was shown that such binding can be inhibited by lysine and the lysine analog epsilon- aminocaproic acid (EACA). Since the amino acid sequences encoded by pgbA and HP0863 are highly lysine-rich in their carboxyl terminal ends, we investigated the potential lysine binding dependence. We added lysine or EACA with Plg-bp to compete for binding to Pig- coated microtiter wells in the same assay as described above. Indeed, the binding could be inhibited by lysine and EACA with EACA being a slightly more effective for inhibitor of Pig binding to both proteins (Fig. 4). It is possible that this discrepancy is due to the different mechanisms by which lysine and EACA inhibit Pig binding. Lysine competitively inhibits by binding to the Pig lysine-binding sites whereas EACA alters the conformation of Pig such that these sites are not available for binding. Because Pig is immobilized in this assay, some sites may not be exposed.

Detection of Pig binding proteins in purified proteins, laboratory strains and clinical isolates using antibodies raised against recombinant PgbA.

In a Western blot, purified recombinant PgbA and PgbB were detected (data not shown) with a polyclonal antibody raised against a recombinant protein of the sequence designated "comb", which is shared by both Plg-bps (Fig. 1). In a Western blot analysis of whole cell lysates of laboratory strains and clinical isolates, two proteins were detected roughly corresponding to the sizes of PgbA and PgbB (Fig. 5). Prebleed sera did not recognize any proteins (data not shown). The proteins were detected in clinical isolates from diverse geographical regions, Poland, Turkey, Iran, Sweden, Somalia, USA, as well as common laboratory strains CCUG 17874, 26695 (Fig. 5), G27, ATCC 43504, SSI (data not shown). In total, we have tested 11 H pylori strains, all of which express Plg-bps under laboratory culture conditions. These data demonstrate that PgbA and PgbB are widely expressed proteins, suggesting they may have an important function. Attempts to knock out pgbA were not successful, leading to the speculation that one or both genes are essential.

PgbA and PgbB on H pylori are surface localized

Previous studies demonstrated that whole H. pylori CCUG 17874 organisms can bind radiolabeled Pig (Pantzar, Ljungh et al. vide supra), suggesting that the binding moieties are located on the surface of the organism. To determine whether the Plg-bps identified in this study are surface-localized, we treated whole H. pylori with proteinase K to degrade surface- exposed protein domains. Treated and untreated cells were analyzed by Western blot (Fig. 6) using anti-sera raised against the overlapping domain, "comb", of the Plg-bps (Fig. 1). The antisera showed reduced reactivity against proteinase K-treated H. pylori (Fig. 6), indicating that this antigen is exposed on the surface and therefore was degraded during the treatment.

PgbA and PgbB are responsible for the majority of Pig binding activity Because presumably many proteins are surface-exposed on H. pylori, we wanted to determine whether the PgbA and PgbB proteins are in fact responsible for most or all of the organism's Pig binding activity. To answer this question, we determined the degree to which the recombinant proteins could inhibit binding of intact H pylori to Pig. Digoxigenin-labeled Pig was preincubated with various concentrations of purified PgbA, PgbB, a combination of the two, Pig, or fetuin. The preincubated mixture was allowed to attach to intact H. pylori CCUG 17874 coated on microtiter wells. Pig attachment was detected by incubating with alkaline phosphatase conjugated anti-digoxigenin antibodies followed by colorimetric development. Recombinant Plg-bps were able to inhibit Pig attachment to whole H pylori whereas fetuin could not (Fig. 7). At 0.2 μM, both Plg-bps individually inhibited Pig attachment by -78%. Interestingly, combining both proteins resulted in slightly greater inhibition at any concentration compared to either Plg-bp alone, suggesting that they work synergistically. Perhaps they form a complex resulting in higher affinity cooperative binding. Although it is possible that the recombinant proteins were sterically hindering binding by other Plg-bps, these data suggest that PgbA and PgbB do in fact mediate the majority of Pig binding observed with whole organisms.

Pig bound to Plg-bps can be converted into enzymatically active plasmin Because plasmin can cleave extracellular matrix proteins, bacterial binding to Pig has been hypothesized to aid the organism by enabling tissue penetration after the surface-bound Pig is converted to plasmin by a host-derived Pig activator. We asked whether Pig bound to PgbA and PgbB could subsequently be converted to plasmin without inhibiting its protease function, thus enabling the organism to become surface-coated with a broad-specificity protease. To address this question, we used a chromogenic assay to measure proteolytic activity after Pig bound to Plg-bps was incubated with a Pig activator. Microtiter plates were coated with recombinant Plg-bps, additional available sites were blocked with BSA, and Pig was allowed to bind. After washing, tissue plasminogen activator (tPA) was allowed to activate the bound Pig. Addition of the chromogenic substrate, V-0882 (Sigma), allowed us to measure plasmin activity by reading absorbance at 405 nm. We demonstrated that Pig bound to either Plg-bps was capable of being converted to functionally active plasmin (Fig. 8). No substrate cleavage was detected when either tPA or Pig was omitted from the assay or when wells were coated with BSA instead of Plg-bps (Fig. 8). These results show that binding of Plg-bps to Pig does not inhibit the activation to plasmin and that Plg-bps alone cannot activate Pig.

Materials and Methods

Bacterial strains, culture media and DNA extraction

H. pylori CCUG 17874 and H. pylori 26695 (CCUG 41936) were obtained from the Culture

Collection at the University of Gothenburg and grown on blood-agar plates (Columbia blood- agar base, (Acumedia, Svenska Labfas, Ljusne, Sweden) 43 g, DL-tryptophan 0.1 g, deionized water 1 1, and horse blood (citrate) 60 ml in a humidified microaerobic environment

(nitrogen 85% v/v, carbon dioxide 10% v/v and oxygen 5% v/v) at 36° C for 3 days.

Chromosomal DNA was extracted and purified as described previously (Monstein, Jonsson et al. Scand J Gastroenterol. 2002 Jan;37(l):l 12-9).

Oligonucleotides and PCR amplification

Primers used in this study were obtained from Cybergene (Cybergene AB, Novum, Huddinge, Sweden). Sense and antisense primers for sequencing and amplification of phage display clones are shown in Table 1. Sense and antisense primers for amplification, sequencing and cloning full length H. pylori CCUG 17874 Plg-bp PgbA and PgbB and "comb" are shown in Table 1.

TABLE 1.

Oligonucleotides used in phage display, PCR amplification and DNA sequence analysis.

PCR amplifications to create near full-length Plg-bp H pylori CCUG 17874 and HP0863 amplicons were performed using AmpliTaq Gold (PE- Applied Biosystems, Stockholm, Sweden) in a final reaction volume of 25 μl, 2.5 mM MgCl₂ and 25 pmol of each primer. The following cycle program was used: initial denaturation step at 94° C for 8 min, followed by 25 cycles of denaturation at 94°C for 30 sec, annealing at 55° C for 30 sec, extension at 72° C for 30 sec, followed by a final extension step at 72° C for 7 min. Subsequently, the integrity of the PCR amplicons was analyzed by agarose gel electrophoresis. Products were sequenced on an ABI-PRISM 310 genetic analyzer (PE-applied Biosystems). Appropriate H pylori DNA sequences were retrieved from the NCBI-GenBank. Sequence alignment was performed using the Sequencher 3.1.1 software program (Gene Codes Corp., Ann Arbor, Michigan, USA). The accession number corresponding to the near full-length H pylori CCUG 17874 putative Pig binding protein gene (corresponding to H pylori 26695 gene pgbA) is given in the acknowledgments section.

Construction and biopanning of the Ml 3 phage library

Chromosomal DNA was fragmented by sonication at maximum power at 4°C for 10 seconds during 3-5 minutes using a microprobe sonicator (Soniprep 150 MSE, Crawley, West Sussex, UK). Samples were taken at different times and DNA fragmentation was analyzed by agarose gel electrophoresis. DNA fragments in the range of 100-2000 base-paires were selected to construct a phage display library.

In brief, fragmented chromosomal DNA was ligated into the phagemide pG8SAET and subsequently transformed into E. coli TGI followed by infection with the helper - phage R408, thereby enabling the expression of potential in frame fusion proteins with the Ml 3 phage major outer protein VIII.

The biopanning procedure was performed. In brief, 200 μl of the phage library was added to plasminogen coated (20 μg each well) microtiter plates (MaxiSorp, Nunc, Copenhagen, Denmark) and incubated for 4 hours at room temperature. Subsequently, the wells were washed using PBS (phosphate buffered saline pH 7.5) supplemented with 0.05 % Tween 20. Bound phage particles were eluted from the wells using a low pH (50 M Na-citrate, 140 mM NaCl, pH 5.0, followed by neutralization using a 2M Tris-HCl pH 8.0 buffer at a final concentration of 250 mM. This eluate was than used in a second round of biopanning for the enrichment of binding clones. After a second round of biopanning, the phagemid clones were analyzed by PCR amplification and DNA sequence analysis using primers as indicated in Table 1. DNA sequence and alignment analysis

Appropriate amounts (2-5 μl) of purified plasmid, phagemide (Wizard plasmid purification kit, Promega, Scandinavien Diagnostic Services Falkenberg, Sweden) or PCR amplicons (JETquick PCR purification spin kit, Saven, Malmδ, Sweden) were sequenced using an ABI- PRISM BigDye terminator cycle sequencing kit V2.0 (PE- Applied Biosystems).

Expression ofH. pylori Plg-bps as His-tag fusions

H. pylori CCUG 17874 PCR amplicon corresponding to H pylori 26695 gene pgbA and H pylori 26695 gene HP0863 PCR amplicon (both amplicons were near full-length genes but without signal sequences) were cloned into a directional cloning, histidine-tag fusion protein expression vector (pET-lOOD, Invitrogen) and initially transformed into E. coli TOP 10 cells (Invitrogen). Subsequently, positive colonies were picked and inserts were analyzed by PCR amplification and DNA sequence analysis using pET-lOOD vector primers and H. pylori specific primers (for details see Table 1). Plasmid DNA from appropriate clones were transformed into E. coli BL21 star cells ( vitrogen) and subsequently expressed in the presence of EPTG at a final concentration of 0.5 mM following the instruction manual (Invitrogen). Finally, cells were harvested by centrifugation at 3000 x g (4°C for 15 min) in a Sorvall RC5C centrifuge. Fusion proteins were isolated and purified from cell pellets using a Bug-buster purification procedure (Novagen, VWR International AB, Stockholm, Sweden) and affinity purified using Ni-chelate columns (Novagen) in the presence of 6 M urea. The integrity of the purified His-tag fusion proteins was analyzed by SDS-PAGE.

ELISA Pig attachment assays

One μg of human plasminogen (Pig) in 100 μl phosphate buffered saline (PBS) was coated onto microtiter wells (Nunc) for 1 hr at room temperature or overnight at 4°C. The remaining sites were blocked by incubating with 200 μl of 1% BSA in PBS for 1 hr at room-temperature or over night at 4°C, followed by incubation in 200 μl PBS, 0.02% sodium azide for 5 min. and stored dry at 4°C for 2-3 weeks. Plg-bp, expressed N-terminal His-tag fusion proteins attachment to coated wells was assayed by incubating the indicated concentrations of Plg-bp (Fig. 2) in 100 μl of 0.1% BSA, PBS (PBSB) for 1 hr, followed by 3 x 5 min washing steps with 200 μl PBSB. Next, attached Plg-bp was visualized by an alkaline phosphatase conjugated N-terminal recognizing anti-his-tag antibody (Invitrogen) for 1 hr, and developing in 1 M diethanolamine, 1 mM MgCl2 with 1 mg/ml p-nitrophenyl phosphate at 37° C for 30 min.

Inhibition of attachment of Plg-bp by fetuin or Pig was assayed by pre-incubation of various concentrations of fetuin or Pig (Fig. 3) with 0.5 μM of Pig binding proteins in PBSB for 1 hr at room-temperature before allowing the Plg-bp to attach to the Pig-coated microtiter wells. Inhibition of Pig binding was assayed using a mixture of either D-aminocaproic acid (EACA) or lysine (Fig. 4) with the indicated amount of Plg-bp which was added to the Pig-coated microwells.

Antibodies

Polyclonal antisera (114) was raised in rabbits against recombinant H. pylori Plg-bp "comb" CCUG 17874 (BioDesign International USA). See Fig. 1 for the amino acid sequence of "comb." Antisera was adsorbed against soluble proteins from E. coli BL21 Star, the strain from which the protein was purified for immunization. Cells were sonicated and proteins in the supernatant were coupled to CNBr 4B Sepharose (Pharmacia) according to the manufacturer's instructions. Fifteen μl of antisera was diluted 1:1000 in TBS, passed over a 1 ml column, and stored at 4° C until further use for up to 4 months.

Western blots

Purified recombinant Plg-bps and whole cell lysates of H pylori were electrophoretically separated on a premade NuPAGE 10% Bis-Tris gel (Invitrogen) in MOPS buffer or a 10% Tris-HCl Ready Gel (BioRad) and electroblotted onto a PVDF membrane using a BioRad Mini-PROTEAN II cell, following the instruction manual (BioRad Laboratories, Sundbyberg, Sweden). The membranes were incubated in blocking buffer (5% nonfat dried milk powder in PBS) for 1 hr or overnight, washed three times with Tris Buffered Saline (TBS: 20mM Tris- HCl pH 7.5, 140 mM NaCl) and probed with polyclonal antibody 114 diluted 1:1000 in TBS, 0.05% Tween 20. After three washes in TBST for 5 min. each, the membranes were incubated for 1 hr with alkaline phosphatase conjugated goat anti-rabbit antibody (SIGMA- Aldrich Sweden AB, Stockholm, Sweden) diluted 1:30,000 in TBST, followed by three washes in TBST at room temperature. Finally, the blot was developed with Sigma FastTab NBT/BCEP substrate (Sigma).

Surface localization ofH pylori Plg-bps

H. pylori in PBS, 5 mM MgCl were treated with 10 mg/ml proteinase K at 37° C for 40 min..

Cells were centrifuged (3000xg), washed with PBS, 5 mM MgCl twice and resuspended in the same volume. Cells were subjected to Western blot analysis and probed with antibody

114.

Inhibition of Pig binding to H. pylori

Whole H. pylori were washed in TBS, adjusted to an OD of 1, and 200 μl was coated onto microtiter plates for 1 hr at RT or overnight at 4° C. Remaining sites on the wells were blocked with 1% BSA in PBS for 1 hr at RT or overnight at 4° C. Pig was labeled with digoxigenin (Roche) according to the manufacturer's instructions, preincubated with inhibitors Plg-bps or fetuin and added to the wells in PBS, 0.1% BSA (PBSB) for 1 hr. After washing with PBSB, the wells were incubated for 1 hr with alkaline phosphatase conjugated anti-digoxigenin (Roche) in PBSB at 1:3000. After washing, the wells were incubated with 1 mg/ml phosphatase substrate in diethanolamine buffer and absorbance was read at 405 nm after 30 min at 37° C.

Plasmin chromogenic assay

Microtiter wells were coated with 100 μl of 10 μg/ml Plg-bps in PBS for 1 hr at RT. 200 μl of 1% BSA in PBS was used to block remaining sites overnight at 4° C. All subsequent washes and incubations were done in PBS with 1% BSA. Pig at 10 μg/ml was allowed to attach for 1 hr, the wells were washed, and 0.32 μg/ml tissue Pig activator (Sigma) was incubated for 1 hr at RT. After washing, the wells were incubated with a chromogenic plasmin substrate at 0.5 mg/ml at 37° C for 30 min. Plasmin cleavage was detected by reading absorbance at 405 nm.

SEQUENCE LISTING

<110> Karolinska Innovations AB

<120> Plasminogen/plasmin binding polypeptides and nucleic acids therefore

<130> P06422PC00

<150> US 60/500,253 <151> 2003-09-05

<160> 3

<170> Patentln version 3.3

<210> 1

<211> 452

<212> PRT

<213> Helicobacter pylori

<400> 1 Met Leu Arg Leu Leu He Gly Leu Leu Leu Met Ser Phe He Ser Leu 1 5 10 15

Gin Ser Ala Ser Tip Gin Glu Pro Leu Arg Val Ser He Glu Phe Val 20 25 30

Asp Leu Pro Lys Lys He He Arg Phe Pro Ala His Asp Leu Gin Val 35 40 45

Gly Glu Phe Gly Phe Val Val Thr Lys Leu Ser Asp Tyr Glu He Val 50 55 60

Asn Ser Glu Val Val He He Ala Val Glu Asn Gly Val Ala Thr Ala 65 70 75 80

Lys Phe Arg Ala Phe Glu Ser Met Lys Gin Arg His Leu Pro Thr Pro 85 90 95

Arg Met Val Ala Arg Lys Gly Asp Leu Val Tyr Phe Arg Gin Phe Asn 100 105 110

Asn Gin Ala Phe Leu He Ala Pro Asn Asp Glu Leu Tyr Glu Gin He 115 120 125

Arg Ala Thr Asn Thr Asp He Asn Phe He Ser Ser Asp Leu Leu Val 130 135 140

Thr Phe Leu Asn Gly Phe Asp Pro Lys He Ala Asn Leu Arg Lys Ala 145 150 155 160

Cys Asn Val Tyr Ser Val Gly Val He Tyr He Val Thr Thr Asn Thr 165 170 175

Leu Asn He Leu Ser Cys Glu Ser Phe Glu He Leu Glu Lys Arg Glu 180 185 190

Leu Asp Thr Ser Gly Val Thr Lys Thr Ser Thr Pro Phe Phe Ser Arg 195 200 205

Val Glu Gly He Asp Ala Gly Thr Leu Gly Lys Leu Phe Ser Gly Ser 210 215 220

Gin Ser Lys Asn Tyr Phe Ala Tyr Tyr Asp Ala Leu Val Lys Lys Glu 225 230 235 240

Lys Arg Lys Glu Val Arg He Lys Lys Arg Glu Glu Lys He Asp Ser 245 250 255

Arg Glu He Lys Arg Glu He Lys Gin Glu Ala He Lys Glu Pro Lys 260 265 270

Lys Ala Asn Gin Gly Thr Gin Asn Ala Pro Thr Leu Glu Glu Lys Asn 275 280 285

Tyr Gin Lys Ala Glu Arg Lys Leu Asp Ala Lys Glu Glu Arg Arg Tyr 290 295 300 Leu Arg Asp Glu Arg Lys Lys Ala Lys Ala Thr Lys Lys Ala Met Glu 305 310 315 320

Phe Glu Glu Arg Glu Lys Glu His Asp Glu Arg Asp Glu Gin Glu Thr 325 330 335

Glu Gly Arg Arg Lys Ala Leu Glu Met Asp Lys Gly Asp Lys Lys Glu 340 345 350

Glu Arg Val Lys Pro Lys Glu Asn Glu Arg Glu He Lys Gin Glu Ala 355 360 365

He Lys Glu Pro Ser Asp Gly Asn Asn Ala Thr Gin Gin Gly Glu Lys 370 375 380

Gin Asn Ala Pro Lys Glu Asn Asn Ala Gin Lys Glu Glu Asn Lys Pro 385 390 395 400

Asn Ser Lys Glu Glu Lys Arg Arg Leu Lys Glu Glu Lys Lys Lys Ala 405 410 415

Lys Ala Glu Gin Arg Ala Arg Glu Phe Glu Gin Arg Ala Arg Glu His 420 425 430

Gin Glu Arg Asp Glu Lys Glu Leu Glu Glu Arg Arg Lys Ala Leu Glu 435 440 445

Ala Gly Lys Lys 450 <210> 2 <211> 298 <212> PRT <213> Helicobacter pylori

<400> 2

Met Phe Cys Phe Glu Asn Leu Asn He Gin Asn Asp He Lys Ser Lys 1 5 10 15

Ser Phe Gly Gly He Val Lys Ser He Ser Met Asn Asp Leu Gin Gin 20 25 30

He Thr He Pro He Pro Pro Leu Glu He Gin Gin Glu He Val Lys 35 40 45

He Leu Asp Ala Phe Thr Glu Leu Asn Thr Glu Leu Asn Thr Glu Leu 50 55 60

Lys Ala Arg Lys Lys Gin Tyr Glu Tyr Tyr Gin Asn Met Leu Leu Asp 65 70 75 80

Phe Asn Asp He Asn Gin Asn His Lys Asp Ala Lys He Lys Thr Tyr 85 90 95

Pro Lys Arg Leu Lys Thr Leu Leu His Thr Leu Ala Pro Lys Gly Val 100 105 110

Glu Phe Arg Lys Leu Gly Glu Val Cys Glu Ser Thr Asn Lys Lys Thr 115 120 125

Leu Lys He Ser Glu Val Ser Glu Val Lys Asn Lys Gly Met Tyr Pro 130 135 140

Val He Asn Ser Gly Arg Asp Leu Tyr Gly Tyr Tyr His Asp Phe Asn 145 150 155 160

Asn Asp Gly Glu Asn He Thr He Ala Ser Arg Gly Glu Tyr Ala Gly 165 170 175

Phe He Asn Tyr Phe Asn Glu Lys Phe Phe Ala Gly Gly Leu Cys Tyr 180 185 190

Pro Tyr Lys Val Lys Asp Thr Asn Glu Leu Leu Thr Lys Phe Leu Tyr 195 200 205

Phe Tyr Leu Lys Thr Asn Glu He Gin He Met Glu Asn Leu Val Phe 210 215 220

Arg Gly Ser He Pro Ala Leu Asn Lys Ala Asp He Glu Thr Leu Thr 225 230 235 240

He Pro He Pro Pro Leu Glu He Gin Gin Glu He Val Lys He Leu 245 250 255

Asp Gin Phe Ser Ala Leu Thr Thr Asp Leu Leu Ala Gly He Pro Ala 260 265 270 Glu He Lys Ala Arg Lys Lys Gin Tyr Glu Tyr Tyr Arg Glu Lys Leu 275 280 285

Leu Thr Phe Lys Pro Leu Gin Asn Lys Glu 290 295

<210> 3

<211> 1344

<212> DNA

<213> Helicobacter pylori

<400> 3 atgttaaggc ttttgatagg acttctttta atgagtttta taagcttgca atcagcctct 60

tggcaagaac ccttaagagt gagtatagaa tttgtggatt tgcctaaaaa aatcattcgt 120

tttccggctc atgatttgca agtgggggag tttggttttg tcgttactaa actttcagat 180

tatgaaatcg ttaattctga agtggtcatt atcgccgttg aaaatggtgt cgcaacggct 240

aaattcagag cgtttgagtc tatgaaacaa agccatttac ccactccaag aatggtcgct 300

agaaaggggg atttggtcta ttttaggcaa ttcaacaacc aagcgttttt aatcgctcct 360

aatgatgaac tctatgagca aatcagggcg actaacaccg atattaattt tagctctgat 420

ttgttggtta cttttttgaa tgggtttgac ccaaaaatcg ctaatttaag aaaagcgtgc 480

aacgtttata gcgtgggggt gatttatatt gtaaccacca acacgctcaa tattttaagc 540

tgtgagagtt ttgaaatttt agaaaaaaga gagttggata caagcggcgt tattaaaact 600

tctacgccgt ttttttctag ggttgagggc attgatgcag gcacgctagg gaaacttttt 660

tcaggcagtc agtctaaaaa ttacttcgct tactatgacg ctttagtgaa aaaagaaaaa 720 cgcaaagaag taaggattaa aaagagggaa ggaaagattg attctagaga aattaaacga 780

gaaatcaagc aagaggccat taaagagcct aaaaaagcca atcaaggcac agaaaacgct 840

cctactttag aagagaaaaa ctaccaaaaa gcagagcgca aacttgatgc taaagaagaa 900

aggcgctatt taagagatga aaggaaaaaa gccaaagcca ccaaaaaggc tatggaattt 960

gaagaaagag aaaaagagca tgatgaaagg gacgaacaag agactgaagg aagaagaaaa 1020

gctttagaaa tggataaagg caatgaaaaa gtcaatgcca aagaaaatga gcgagaaatc 1080

aagcaagaag ccattaaaga gccagataat ggaaataacg ccacccaaca aggcgaaaaa 1140

caaaacgctc ctaaagagaa caacgcttct aaagaagaga aaaaaccaaa ttctaaagaa 1200

gaaaaacgcc gcttgaaaga agaaaagaaa aaagccaaag ccgaacaaag agcgagagaa 1260

tttgaacaaa gagcgagaga gcatcaagaa agagatgaaa aagagcttga agaaagaaga 1320

aaagctttag aaatgaataa gaag 1344

Claims

1. An isolated nucleic acid molecule encoding an amino acid sequence selected from SEQ ID NO:l and SEQ ID NO:2.

2. An isolated nucleic acid molecule according to claim 1 derived from any of the genes HP0508 (pgbA) or HP086β (pgbB) of Helicobacter pylori.

3. An isolated nucleic acid molecule according to claim 1 or claim 2 having the sequence SEQ ID NO:3.

4. A polypeptide encoded by a nucleic acid according to any of the claims 1-3.

5. A polypeptide according to claim 4 having an amino acid sequence selected from SEQ ID NO: l and SEQ ID NO:2.

6. An antibody to the polypeptide according to claim 4 or claim 5.

7. A pharmaceutical composition comprising a nucleic acid according to any of claims 1-3 together with a physiologically acceptable diluent or carrier.

8. A pharmaceutical composition comprising at least one polypeptide according to claim 4 or 5 together with a physiologically acceptable diluent or carrier.

9. A pharmaceutical composition comprising an antibody according to claim 6 together with a physiologically acceptable diluent or carrier.

10. A pharmaceutical composition according to any of the claims 7-9 for use as a vaccine.

11. A method of preventing or treating Helicobacter infection in a mammal by administering to said mammal a prophylactically or therapeutically effective amount of a pharmaceutical composition according to any of the claims 7-9.

12. Primers for, or probes that hybridize with, a nucleotide sequence of a nucleic acid according to any of claims 1 -3 for use in a diagnostic method for the purpose of detecting Helicobacter pylori.

13. A diagnostic kit for assessing the presence of Helicobacter pylori in a mammal or in a biological or environmental sample, comprising a primer or probe according to claim 12, a polypeptide according to claim 4 or claim 5 or an antibody according to claim 6.

14. A method of detecting Helicobacter pylori in a mammal or in a biological or environmental sample by:

- bringing a primer or probe according to claim 12 into contact with a biological sample from said mammal or with said biological or environmental sample; and

- detecting any product of binding of said primer or probe to any nucleic acid present in the sample.

15. A method of detecting Helicobacter pylori in a mammal or in a biological sample by:

- bringing a polypeptide according to claim 4 or 5 into contact with a biological sample from said mammal or with said biological sample; and

- detecting any product of binding of said polypeptide to any antibody present in the sample.

16. A method of detecting Helicobacter pylori in a mammal or in a biological or environmental sample by:

- bringing an antibody according to claim 6 into contact with a biological sample from said mammal or with said biological or environmental sample; and

- detecting any product of binding of said antibody to any polypeptide or protein present in any of said samples.