WO2000046242A2 - 19 KILODALTON PROTEIN OF $i(HELICOBACTER PYLORI) - Google Patents

Abstract

The present invention relates to a 19,000 Dalton protein produced by Helicobacter pylori and the use of this protein and nucleic acid sequences encoding this protein as a vaccine against infection from Helicobacter bacterium, such as Helicobacter pylori.

Description

19 Kilodalton Protein of Helicobacter Pylori

Field of the Invention The present invention relates to a protein of about 19,000 daltons, which protein is produced by Helicobacter pylori and relates to the use of this protein and nucleic acid sequences encoding the protein in a vaccine against infection caused by a Helicobacter bacterium, such as Helicobacter pylori.

Background of the Invention

Helicobacter pylori (H. pylori) is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen infection. [Warren, J.R. et al., Lancet, 1: 1273-1275, (1983) ; and Marshall et al., Microbios Lett, 25: 83-88, (1984)]. H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. [Rathbone et al., Gut, 27: 635-641,

(1986)]. Additional evidence has developed for an etiological role of H. pylori in non- ulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. [Blaser, M J., Trends Microbiol, 1: 255-260, (1993)]. H pylori colonizes the human gastric mucosa, establishing an infection that usually persists for many years. About 30-50% of the human population appear to be chronically infected. [Rainer, Η. et al,

Biologicals, 25: 175-177, (1997)]. The current recommended treatment for chronic H pylori infection is multiple antibiotic treatment combined with a proton pump inhibitor, or with bismuth salts. However, this treatment may not fully resolve the infection and resistance to antibiotics can occur. H. pylori infection in humans induces a strong local and systemic immune response; however, the immune response if often unable to clear the infection. Accordingly, work has led to developing potential H pylori vaccines. Narious antigens, proteins and genes have been reported in this area. [See Bolin et al, PCT Application No. 96/38475, filed June 3, 1996; Doidge et al., PCT Application No. 96/33220, filed April 19, 1996; Clancy et al., PCT Application No. 96/25430, filed February 15, 1996; Chan et al., PCT Application No. 96/12965, filed October 19, 1995; Byrne et al., PCT Application No. 96/01273, filed July 3, 1995; Alemohammad M.M., in U.S. Patent 5,262,156, filed August 12, 1991; Allan et al., PCT Application No. 97/03359, filed June 28, 1996; and Dettmar et al., German Patent Application 195235554 Al based on Great Britain Application No. 9413074.] As an alternative to potential vaccines and diagnostic currently provided in the art referenced above, novel methods of curing or preventing H. pylori infection through the use of novel H. pylori proteins, as well as genes encoding such proteins are described herein. The protein is selected for use as a vaccine candidate based on the ability of the protein, or polypeptide thereof, to confer protection to mice challenged with a live organism.

Summary of the Invention

This invention relates to an isolated and purified H. pylori protein of about 19 kDa and nucleic acid sequences encoding therefor. The mature processed form of this protein has the following amino-terminal amino acid sequence: MLSKDIIKLLNEQNN (SEQ ID NO.3). The isolated and purified H. pylori protein has an apparent molecular weight of about 19 kDa as measured on a 10 % to 20% gradient SDS polyacrylamide gel. More specifically, this protein has a molecular weight of about 19.3 kDa as measured by mass spectrometry. The protein and nucleic acid sequences of the present invention have diagnostic and therapeutic utility for H. pylori and other Helicobacter species. They can be used to detect the presence of H. pylori and other Helicobacter species in a sample, and to screen compounds for the ability to interfere with the H. pylori life cycle or to inhibit H. pylori infection. The protein and portions thereof can be used in vaccine compositions. In additional embodiments, this invention includes embodiments relating to isolated nucleic acid sequences corresponding to the entire coding sequences of the 19 kDa H. pylori proteins or portions thereof, nucleic acids capable of binding mRNA from H. pylori surface proteins and methods for producing H. pylori surface proteins or portions thereof using peptide synthesis and recombinant techniques. Preferred embodiments of this invention are directed to vaccine compositions based on agents prepared from the proteins and nucleic acids of this invention and methods for treatment and prevention of H. pylori infections employing such compositions. Brief Description of the Drawings

Figure 1: This Figure depicts the purity of the native 19 kDa protein, as analyzed by use SDS-PAGE as described in example 3.

Figure 2: This Figure depicts mouse protection data from the H. pylori SSI experimental challenge following intragastric vaccination of mice with an isolated and purified 19 kDa protein of H. pylori, as described in the Example 5. The asterisk in the Figure represents a statistically significant reduction in colonization compared to the respective KLΗ control.

Figure 3: This Figure is gel depicting the results of example 6, which examined the conservation of the 19 kDa protein in five Η. pylori strains (PBCC 1105, PBCC 1107, LET 13, ATCC 43579 and SSI, which are represented in lanes 2, 3, 4, 5 and 7 respectively). Both lanes 1 and 6 contain a 1Kb ladder of DNA markers, and lane 8 contains no DNA as a PCR control.

Detailed Description of the Invention

One aspect of the present invention provides an isolated, substantially purified H. pylori protein having a molecular weight of about 19 kDa. As used herein polypeptide means the amino acid sequences and/or fragments thereof which comprise the 19 kDa protein of H. pylori, and, specifically the amino acid sequences and/or fragments thereof which comprise the 19 kDa protein of H. pylori strain ATCC 43579; having the N-terminal amino acid sequence of SEQ ID No. 3; wherein the mature processed form of such protein has an N-terminal amino acid sequence consisting essentially of SEQ. ID NO.3. Preferably, the polypeptides of this invention have antigenic properties, such as being reactive with H. pylori antibodies. In alternative embodiments the 19 kDa H. pylori protein has the amino acid sequence depicted in SEQ ID NO. 2. Antigens for use as vaccines can be based on the above-described isolated polypeptides sequences, or allelic or other variants thereof, which are biological equivalents. Suitable biological equivalents have about 70 to about 80%, and most preferably at least about 90%, similarity to one of the amino acid sequences referred to above, or to a portion thereof, provided the equivalent is capable of eliciting substantially the same antigenic properties as the isolated and purified 19 kDa protein of H. pylori strain ATCC 43579; having the N-terminal amino acid sequence of SEQ ID No. 3. wherein the mature processed form of such protein has an N-terminal amino acid sequence consisting essentially of SEQ. ID NO.3.

The biological equivalents are obtained by generating variants and modifications to the isolated polypeptides of this invention. These variants and modifications to the isolated polypeptides are obtained by altering the amino acid sequences by insertion, deletion or substitution of one or more amino acids. The polypeptides are then selected for use as an antigen and/or vaccine candidate based on the following criteria: (i) the antigen is conserved among H. pylori clinical isolates, and (ii) the antigen is able to confer protection to vaccinated mice from challenge with a live organism. Preferably, the antigen is located on the bacterial surface. The amino acid sequence is modified, for example by substitution in order to create a polypeptide having substantially the same or improved qualities. A preferred means of introducing alterations comprises making predetermined mutations of the nucleic acid sequence of the polypeptide by site-directed mutagenesis.

The amino acid changes are achieved by changing the codons of the nucleic acid sequence. It is known that such modified polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve antigenic or immunogenic activity (see, e.g. Kyte and Doolittle, 1982, Ηopp, US Patent 4,554,101, each incorporated herein by reference). For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide which result in increased activity or enhanced immune response. Alternatively, amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide-molecule conjugates which retain sufficient antigenic properties of the starting polypeptide to be useful for other purposes. For example, a selected polypeptide of the present invention may be bound to a solid support in order to have particular advantages for diagnostic applications.

One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, as discussed by Kyte and Doolittle (1982), wherein it was found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated is intended for use in immunological embodiments. See, for example, U.S. Patent 4,554,101, which states that the greatest local average hydrophilicity of a "protein," as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid.

In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to introduce substitutions of amino acids where these values are +2, with +1 being particularly preferred, and those within + 0.5 being the most preferred substitutions.

Preferable characteristics of the polypeptides of this invention include one or more of the following: (a) being a membrane protein or being a protein directly associated with a membrane; (b) capable of being separated as a protein using an SDS acrylamide (10%) gel; and (c) reducing the colonization of H. pylori in mice upon delivery thereto. The polypeptides based on the 19kDa protein of H. pylori for this invention are particularly useful in vaccine compositions since the compositions are capable of significantly reducing or preventing colonization of H. pylori, as verified in animal models, such as mice. Preferably, compositions containing such polypeptides can reduce colonization by at least about 50 percent when compared against a non- vaccinated control model. More preferably, the reduction in colonization is at least about 60 or 70 percent, or even at least about 80 percent. Comparisons for colonization reduction can be conducted by vaccination (for example by oral vaccination) with the polypeptides admixed with cholera toxin.

Accordingly, further embodiments of this invention relate to a vaccine composition comprising (i) at least one isolated polypeptide as disclosed above and (ii) at least one of a pharmaceutically acceptable buffer or diluent, adjuvant or carrier. The vaccine composition may comprise a carrier, which in turn may be conjugated to said polypeptide. In additional embodiments, the vaccine composition may further comprise an adjuvant. Preferably, these compositions have therapeutic and prophylactic applications as vaccines in preventing and/or ameliorating H. pylori infection. In such applications, an immunologically effective amount of at least one polypeptide of this invention is employed in such amount to cause a substantial reduction in colonization of the H. pylori, as described above.

The formulation of such vaccine compositions is well known to persons skilled in this field. Naccine compositions of the invention containing antigenic components (e.g., H. pylori polypeptide or fragment thereof or nucleic acid encoding an H. pylori polypeptide or fragment thereof) preferably include a pharmaceutically acceptable carrier. 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. The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions of the present invention is contemplated.

Such vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. and by Sedegah et al. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H. pylori (See Czinn et al.). Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

The vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; aluminum phosphate; Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA); MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Hamilton, MT), IL-12 (Genetics Institute, Cambridge, MA); N-acetyl-muramyl~L-theronyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( l'-2'- dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy~ethylamine (CGP 19835 A, referred to a MTP-PE); and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial lysates, block polymers or saponins.

Preferably also, the isolated polypeptides of this invention are used in a vaccine composition for oral administration which includes a mucosal adjuvant and used for the treatment or prevention of H. pylori infection in a human host.

The mucosal adjuvant can be cholera toxin; however, preferably, mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. For specific cholera toxins which may be particularly useful in preparing vaccine compositions of this invention, see the mutant cholera toxin E29Η as disclosed in U.S. Provisional Application No. 60/102,430 which is hereby incorporated herein. These may be added to, or conjugated with, the H. pylori polypeptides of this invention. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin (LT). Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine. Stimulon™ , QS-21, MPL™, and IL-12, which described above, may also be used.

The vaccine compositions of this invention may be delivered in the form of ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells. Alternatively, micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors. The Helicobacter isolated polypeptides of this invention may also be incorporated into oily emulsions.

The isolated polypeptides of the present invention may be administered as the sole active immunogen in a vaccine composition. Alternatively, however, the vaccine, composition may include other active immunogens, including other Helicobacter antigens such as urease, lipopolysaccharide, Hsp60, NacA, CagA or catalase, as well as immunologically active antigens against other pathogenic species.

One of the important aspects of this invention relates to a method of inducing immune responses in a mammal comprising the step of providing to said mammal a vaccine composition of this invention. The vaccine composition is a composition which is antigenic in the treated animal or human such that the immunologically effective amount of the polypeptide(s) contained in such composition brings about the desired response against H pylori infection. Preferred embodiments relate to a method for the treatment, including amelioration, or prevention of Helicobacter infection in a human comprising administering to a human an immunologically effective amount of the antigenic composition. Immunologically effective amount, as used herein, means the administration of that amount to a mammalian host (preferably human), either in a single dose or as part of a series of doses, sufficient to at least cause the immune system of the individual treated to generate a response that reduces the clinical impact of the bacterial infection. This may range from a minimal decrease in bacterial burden to prevention of the infection. Ideally, the treated individual will not exhibit the more serious clinical manifestations of the Helicobacter infection. The dosage amount can vary depending upon specific conditions of the individual. This amount can be determined in routine trials by means known to those skilled in the art.

Another specific aspect of the present invention relates to using a vaccine vector expressing an isolated Helicobacter polypeptide, or an immunogenic fragment thereof. Accordingly, a further aspect this invention provides a method of inducing an immune response in a mammal, which comprises providing to a mammal a vaccine vector expressing at least one, or a mixture of isolated Helicobacter polypeptides, or an immunogenic fragment thereof. The isolated polypeptide of the present invention can be delivered to the mammal 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 an antigenic polypeptide or immunogenic fragment as a foreign polypeptide. Particularly, bacteria that colonize the gastrointestinal tract, such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discussed by Holmgren et al. (1992) and McGhee et al. (1992).

An additional embodiment of the present invention relates to a method of inducing an immune response in a human comprising administering to said human an amount of a DNA molecule encoding an isolated polypeptide of this invention, optionally with a transfection-facilitating agent, where said polypeptide, when expressed, retains immunogenicity and, when incorporated into an antigenic composition or vaccine and administered to a human, provides protection without inducing enhanced disease upon subsequent infection of the human with Helicobacter pathogen, such as H pylori. Transfection-facilitating agents are known in the art and include bupivicaine, and other local anesthetics (for examples see U.S. Patent 5,739,118) and cationic polyamines (as published in International Patent Application WO 96/10038), which are hereby incorporated by reference.

The present invention also relates to an antibody, which may either be a monoclonal or polyclonal antibody, specific for antigenic polypeptides as described above. Such antibodies may be produced by methods which are well known to those skilled in the art. The antibodies of this invention can be employed in a method for the treatment or prevention of Helicobacter infection in mammalian hosts, which comprises administration of an immunologically effective amount of antibody, specific for antigenic polypeptide as described above. It is proposed that the monoclonal antibodies of the present invention are useful in standard immunochemical procedures, such as ELISA and western blot methods, as well as other procedures which may utilize antibodies specific to H. pylori proteins. While ELISAs are preferred, it will be readily appreciated that such assays include RIAs and other non-enzyme linked antibody binding assays or procedures. Additionally, monoclonal antibodies specific to the particular H. pylori protein or polypeptides are utilized in other useful applications. For example, their use in immunoadsorbent protocols serves to purify native or recombinant H. pylori proteins or variants thereof.

The isolated H. pylori polypeptides of the invention are also used as antigens for raising antibodies and in immunoassays for the detection of anti-19.3 kDa antigen- reactive antibodies. In a variation on this embodiment, samples suspected of containing H. pylori may be screened, in immunoassay format, for reactivity against antibodies specific for 19.3 kDa based polypeptides of this invention. Results from such analyses are then be used to determine the presence of H. pylori and potential infection.

Diagnostic immunoassays include direct culturing of bodily fluids and tissue samples, either in liquid culture or on a solid support such as nutrient agar. A typical assay involves collecting a sample of bodily fluid or tissue sample from a patient and placing the sample in conditions optimum for growth of the pathogen. The determination is then be made as to whether the microbe exists in the sample. Further analysis is carried out to determine the hemolyzing properties of the microbe.

Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot), which U.S. Patents are incorporated herein by reference. Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo. Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the anti-19 kDa antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing an H pylori polypeptide are immobilized onto the well surface and then contacted with the anti-19 kDa antibodies. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.

Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the primary antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if desired.

Competition ELISAs are also used in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. (Antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody). The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely absorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human IgG. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS- Tween).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2.2'-azino-di-(e- ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. Alternatively, the label may be a chemilluminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.

Further embodiments relate to isolated nucleic acid sequences encoding the 19 kDa based polypeptides of this invention, in particular the nucleic acid sequence of SEQ ID. NO. 1. Additional embodiments of the invention relate to the use of this nucleic acid sequence or a nucleic acid sequence being substantially similar to all or a portion thereof. The term substantially similar means having a least 50-70%), more preferably 70-80%), and most preferably 80 or 90% identity to one of said sequences. Such substantially similar nucleic acid sequences hybridize under high stringency southern hybridization conditions with the nucleic acid sequence of SEQ I.D. NO. 1.

The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining high stringency southern hybridization conditions , reference can conveniently be made to Sambrook et al. (1989) at pp. 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency.

It will be appreciated that the sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a Helicobacter antigen as broadly described above.

The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source. Generally, recombinant expression of the nucleic acid sequence of this invention will use a stop codon sequence, such as TAA, at the end of the nucleic acid sequence.

This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter sequences and initiator sequences and a nucleotide sequence which codes for a H. pylori peptide of this invention, the nucleotide sequence being located 3' to the promoter and initiator sequences. In yet another aspect, the invention provides a recombinant DNA cloning vehicle capable of expressing a H. pylori peptide comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a H. pylori peptide, the nucleotide sequence being located 3' to the promoter and initiator sequences. In a further aspect, there is provided a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.

Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989).

In yet further aspects, there are provided fused polypeptides comprising a H. pylori peptide of this invention and an additional polypeptide, for example a polypeptide coded for by the DNA of a cloning vehicle, fused thereto. Such a fused polypeptide can be produced by a host cell transformed or infected with a recombinant DNA cloning vehicle as described above and it can be subsequently isolated from the host cell to provide the fused polypeptide substantially free of other host cell proteins.

Based on the above-identified specific sequences, one may obtain numerous additional isolated nucleic acid sequences encoding the polypeptides of this invention due to the degeneracy of the genetic code. Amino acids and their codons are well- known. Accordingly, using site-directed mutegenesis of one polypeptide of H. pylori, one can generate additional nucleic acid sequences, as desired. These methods of generating nucleic acid sequences and fragments thereof provide a convenient manner in which to generate portions of the polypeptides for fusion molecules.

Using the nucleic acid sequence described herein, one can generate synthetic polypeptides displaying the antigenicity of a H. pylori isolated polypeptide of this invention. As used herein, the term "synthetic" means that the polypeptides have been produced by chemical or biological means, such as by means of chemical synthesis or by recombinant DNA techniques leading to biological synthesis. Such polypeptides can, of course, be obtained by cleavage of a fused polypeptide as described above and separation of the desired polypeptide from the additional polypeptide coded for by the DNA of the cloning vehicle by methods well known in the art. Alternatively, once the amino acid sequence of the desired polypeptide has been established, for example, by determination of the nucleotide sequence coding for the desired polypeptide, the polypeptide may be produced synthetically, for example by the well known Merrifield solid-phase synthesis procedure.

Once recombinant DNA cloning vehicles and/or host cells expressing a desired H. pylori peptide of this invention have been constructed by transforming or transfecting such cloning vehicles or host cells with plamsids containing the corresponding H. pylori nucleic acid sequence, cloning vehicles or host cells are cultured under conditions such that the polypeptides are expressed. The polypeptide is then isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.

This invention also provides for a method of diagnosing an H. pylori infection comprising the step of determining the presence, in a sample, of an amino acid sequence of SEQ ID No.: 3, or preferably any of the isolated H. pylori polypeptides of this invention. Any conventional diagnostic method may be used. These diagnostic methods can easily be based on the presence of an amino acid sequence or polypeptide. Preferably, such a diagnostic method matches for a polypeptide having at least 10, and preferably at least 20, amino acids which are common to the polypeptides of this invention.

The nucleic acid sequences disclosed herein can also be used for a variety of diagnostic applications. These nucleic acids sequences can be used to prepare relatively short DNA and RNA sequences that have the ability to specifically hybridize to the nucleic acid sequences encoding the polypeptides of this invention. Nucleic acid probes are selected for the desired length in view of the selected parameters of specificity of the diagnostic assay. The probes can be used in diagnostic assays for detecting the presence of pathogenic organisms in a given sample. With current advanced technologies for recombinant expression, nucleic acid sequences can be inserted into an expression construct for the purpose of screening the corresponding oligopeptides and polypeptides for reactivity with existing antibodies or for the ability to generate diagnostic or therapeutic reagents.

In preferred embodiments, the nucleic acid sequences employed for hybridization studies or assays include sequences that are complementary to a nucleotide stretch of at least about 10 to about 20 nucleotides, although at least about 10 to 30, or about 30 to 60 nucleotides can be used. Nucleotide stretches of at least 10 nucleotides are beneficial for providing stability and selectivity when testing a clinical sample for Helicobacter infection. A variety of known hybridization techniques and systems can be employed for practice of the hybridization aspects of this invention, including diagnostic assays such as those described in Falkow et al., US Patent 4,358,535. Depending on the application, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward a target sequence. For applications requiring a high degree of selectivity, one will select relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperature of about 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand. For some applications, if one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex. The conditions may be altered by using 0.15M-0.9M salt, at temperatures ranging from about 20°C to about 55°C. In general, it is appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and the method of choice will generally depend on the desired results.

In certain embodiments, one may desire to employ nucleic acid probes to isolate variants from clone banks containing mutated clones. In particular embodiments, mutant clone colonies growing on solid media which contain variants of an H. pylori polypeptide sequence could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to obtain hybridization only between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies. In this manner, small hybridization probes containing short variant sequences of the H. pylori genes of the invention may be utilized to identify those clones growing on solid media which contain sequence variants of the entire genes encoding polypeptides of this invention. These clones can then be grown to obtain desired quantities of the variant nucleic acid sequences or the corresponding antigen.

In clinical diagnostic embodiments, nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids (e.g., amniotic fluid, middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, et.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

The nucleic acid sequences which encode for the H. pylori polypeptides of the invention, or their variants, may be useful in conjunction with PCR™ technology as set out, e.g., in U.S. Patent 4,603,102, one may utilize various portions of any of H. pylori sequences of this invention as oligonucleotide probes for the PCR™ amplification of a defined portion of 1 19kDa H. pylori sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of H. pylori nucleic acid may be detected in a sample utilizing the nucleic acid sequences of this invention.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in the light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1 - Strain sources and culturing

Bacterial strains. H pylori strains PBCC 1105 and PBCC 1107 were isolated from human gastric biopsies obtained from the University of Rochester School of Medicine and Dentistry (Rochester, NY). H pylori strain LET 13 was isolated from human gastric biopsies obtained from the Syracuse Veterans Administration Medical Center (Syracuse, NY). H. pylori strain SSI was originally isolated from a human gastric biopsy, and subsequently adapted to infect mice (obtained from A. Lee, University of New South Wales, Sydney, Australia). The H. pylori strain obtained from American Type Culture Collection (Rockville, MD) is ATCC 43579.

Culturing of Helicobacter strains. Cultures of H. pylori were grown at 37°C on Columbia broth agar plates with 10%> defibrinated horse blood and 10 μg/ml vancomycin in a microaerophilic chamber. Liquid cultures of H pylori strains were grown at 37°C in BHI medium with 4% fetal calf serum and 10 μg /ml vancomycin in flasks infused with a gas mixture of 10% CO₂/ 6% O₂/ 84% N₂ (vol/vol/vol).

Example 2

Purification of the 19 kDa protein. Bacterial cells (ca. 20 g wet wt of H pylori ATCC 43579) were resuspended in 160 ml of 0.05 M HEPES / 10 mM EDTA / 0.4 mM Pefabloc (pH 7.0) by homogenization using a Tekmar Ultra-Turrex tissue homogenizer. The cells were disrupted using a Microfluidizer Model 1 10Y (Microfluidics Corp., Newton, MA). The disrupted cells, including the membrane fraction, were pelleted by centrifugation at 42,000 φm using a Beckman 70Ti rotor for 60 min at 4°C. Following centrifugation, the pellet was resuspended in 160 ml of 0.01 M HEPES / 1.0 mM MgCl₂ / 1.0% TX-100 (pH 7.4) and stirred for 1 hr at room temp. The suspension was centrifuged at 42,000 m using a Beckman 70Ti rotor for 30 min at 4°C. Following centrifugation, the pellet was resuspended in 80 ml of 0.05 M Tris-HCl / 10.0 mM EDTA / 1.0% Zwittergent 3-14 (Z 3- 14) (pH 7.4) and stirred for 2 hr at room temp. The suspension was then centrifuged at 42,000 φm using a Beckman 70Ti rotor for 30 min at 4°C. Following centrifugation, the supernatant containing the 19 kDa protein was collected and stored at -20°C for further purification. The Z 3-14 crude extract was buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column (Pharmacia Biotech Inc., Piscataway, NJ) equilibrated in 0.02 M Tris-HCl / 10.0 mM EDTA / 1.0% Z 3- 14 (pH 8.0). The entire Z 3- 14 crude extract was processed via 2 independent G-25 runs. The buffer exchanged Z 3-14 crude extract preparation was loaded onto a MonoS HR 10/10 column (Pharmacia Biotech Inc.) equilibrated 0.02 M Tris-HCl / 10.0 mM EDTA / 1.0% Z 3-14 (pH 8.0). The 19.3 kDa protein, flowed through the column unretarded. The entire buffer exchanged Z 3-14 crude extract was processed via 3 independent MonoS runs. The column flow-through, containing the 19 kDa protein, from all three runs was pooled and buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column (Pharmacia Biotech Inc.) equilibrated in 0.02 M Tris-HCl / 1.0% Z 3-14 (pH 7.0). The entire column flow-through was processed via 2 independent G-25 runs. Following buffer exchange, the preparation was loaded onto a 50 ml TMAE Fractogel column (EM Separations Technology,

Gibbstown, NJ) equilibrated 0.02 M Tris-HCl / 1.0% Z 3-14 (pH 7.0). Unbound protein was washed through the column with an additional 2 column volumes of equilibration buffer. The 19.3 kDa protein was eluted using a linear NaCl gradient (0-0.3 M NaCl) in 0.02 M Tris-HCl / 1.0% Z 3-14 (pH 7.0) over 20 column volumes. Fractions were screened for 19.3 kDa protein by SDS-PAGE and Western analysis and pooled. The 19.3 kDa protein was precipitated by the addition of 9 volumes of ethanol overnight at -20°C. The resulting suspension was then centrifuged at 9,000 φm using a Beckman SS31 rotor for 30 min at 4°C and the pellet was resuspended in 15 ml of Phosphate Buffered Saline (PBS) / 1.0% Z 3-14. The preparation was then buffer exchanged by passage over a 85 ml Sephadex G-25 (coarse) column (Pharmacia Biotech Inc.) equilibrated in PBS / 1.0% Z 3- 14 (pH 7.4) and stored at -20°C. Example 3

SDS-PAGE and Electroblotting. For the 19 kDa protein from ATCC 43579, SDS- PAGE was carried out as described by Laemmli using 10-20% (w/v) acrylamide gels (Zaxis, Hudson, OH). Proteins were visualized by staining the gels with Pro-Blue (Owl Separation Systems, Woburn, MA). Gels were scanned using a Personal Densitometer SI (Molecular Dynamics Inc., Sunnyvale, CA) and molecular weights estimated using the Fragment Analysis software (version 1.1) and prestained molecular weight markers obtained from Owl Separation Systems. The purity of the protein is >95% as assessed by densitometry and shown in Figure 3 (Note that in Figure 1 , molecular makers are designated as MW, and lanes 1, 2, 3, 4, and 5 contain 0.5ug, 1.0 ug, 2.0 ug, 4.0 ug and 8.0 ug of purified 19 kDa protein). Transfer of proteins to polyvinylidene difluoride (PVDF) membranes was accomplished using a semi-dry electroblotter and electroblot buffers (Owl Separation Systems). Protein transferred to PVDF was also used for N-terminal sequence analysis.

Protein estimation. Protein concentration was estimated by the BCA assay from Pierce (Rockford, IL) using BSA as a standard.

Estimation of molecular weight By MALDI-TOF mass spectrometry. Determination of molecular weight by Matrix Assisted Laser Desoφtion / Ionization - Time of Flight (MALDI-TOF) mass spectrometry (Hillenkamp and Karas) was carried out using a Lasermat 2000 Mass Analyzer (Finnigan Mat, Hemel Hempstead, UK).

MALDI-TOF analysis using 3,5-dimethoxy-4-hydroxy-cinnamic acid matrix in presence of 70%) (v/v) aqueous acetonitrile / 0.1% TFA resulted in identifying a polypeptide with an average molecular mass of 19,299.8 kDa (or 19.3 kDa, which is referenced in these examples as the 19 kDa protein).

Example 4 Amino acid sequence analysis. N-terminal sequence analysis was carried out using an Applied Biosystems Model 477A Protein Peptide Sequencer equipped with an on-line Model 120A PTH Analyzer (Applied Biosystems, Foster City, CA). The phenylthiohydantoin (PTH) derivatives were identified by reversed-phase HPLC using a Brownlee PTH C-18 column (particle size 5 mm, 2.1 mm i.d. x 22 cm 1.; Applied Biosystems). The 19 kDa protein was electroblotted onto PVDF as described above, stained with

Coomassie Brilliant Blue 250 ((Owl Separation Systems), and the first 15 amino acids determined. The following sequence was determined, MLSKDIIKLLNEQVN (SEQ ID NO.3).

Example 5

Immunizations. C57B1/6 mice were vaccinated intragastrically on days 0, 3, 14, and 16 with 100 μg recombinant H. pylori (Ηp) urease (rUre) or native Ηp 19 kDa protein formulated with 10 μg cholera toxin (CT). Control mice received KLΗ admixed with CT, without any vaccine.

Reduction of Colonization in the Mouse Model. Infection was established by the intragastric deposition of one 0.1 ml inoculum of H. pylori strain SS 1 on day 37. The challenge inocula contained a suspension of H. pylori at a concentration of 2 x 10⁸ cfu per ml. The amount of viable H. pylori in the stomachs of mice was determined 4 weeks following challenge (day 64). Four mice were sacrificed by cervical dislocation, their stomachs harvested and split into 2 longitudinal sections. One section from each mouse was homogenized and the homogenate diluted and plated on Columbia agar containing defibrinated horse serum and the appropriate antibiotics. Plates were incubated at 37^°C in a microaerophilic incubator for 5 days. After incubation the number of viable H. pylori isolated were enumerated. Total cfu per gram of stomach tissue were obtained by multiplying colony numbers per plate by the dilution factor and dividing by the weight of the sectioned stomach; numbers were transformed and are expressed as log₁₀cfu per gram homogenized stomach tissue + 1 standard error of the mean. Mice which were vaccinated intragastrically with the 19 kDa protein admixed with CT showed a statistically significant reduction in colonization following challenge with Ηp strain SSI, as compared to controls (See Figure 2), using means comparisons by Turkey-Kramer ΗSD. We note that preliminary experiments using CT with Zwittergent3-14 were comprised apparently by the presence of the Zwittergent3-14 and its likely interaction with the CT.

Example 6

Genetic methods. Isolation of plasmid and chromosomal DNA, agarose gel electrophoresisand restriction enzyme digestion were performed following standard protocols (Sambrook et al.). DNA ligation was performed using a TOPO ligation kit (available from Invitrogen) following the recommended protocol. Selection of PCR primers. PCR primers were synthesized which hybridized to an approximately 500 bp region of the gene encoding the 19 kDa protein. The PCR primers synthesized to amplify the 19 kDa gene from the chromosome of H. pylori strain SSI had the following sequences: FORWARDPRIMER: 5' CCC CGG AAT TCAAAG GAG ATA CTA TGT TAT CAAAAGAC 3' (SEQIDNO.4)

REVERSEPRIMER: 5' CCC CTA GTC TAG ATTAAGATTTCC TGC TTTTAG CG3' (SEQ IDNO.5)

Oligonucleotidesynthesis. Single stranded PCR primers were synthesized on an Applied Biosystems model 380B DNA synthesizer using b-cyanoethyl phosphoramidite chemistry.

DNA sequencing. DNA sequencing was obtained by asymmetric PCR amplification using the fluorescent dye-labeled dideoxynucleotideterminator method (Gyllenstenet al., 1988). The dye-labeled single stranded DNA fragments were separated and identified with an Applied Biosystem model 373A automatic sequencing apparatus. Primary sequence information was analyzed using Mac Vector DNA analysis program (IBI, New Haven CT).

PCR amplification PCR amplificationswere performed in 500 ml MgCl₂; 2.5 units Taq polymerase (Boehringer Mannheim), 200 mM dNTPs; 20 mM oligonucleotide primersand 1-2 mg chromosomal DNA. Templateswere amplifiedfor30 cycles with a 1 sec extension on the annealing time of each cycle in a Perkin-ElmerCetus thermocycler.

Conservation of PCR amplified product. The results of the PCR amplifications indicate that the 500 bp region of the gene encoding the 19 kDa protein is conserved in strains PBCC 1105, PBCC 1107, LET 13, ATCC 43579, and SSI (Figure 3), as shown in lanes 2, 3, 4, 5 and 7, respectively. Lanes 1 and 6 are 1 Kb ladder DNA markers. REFERENCES

References referred to herein above are noted below and are incoφorated herein:

Czinn et al. 1993. Vaccine. 11, 637-642.

Gyllenstein, U.B. and Erlich, M.A. Generation of single stranded DNA the polymerase chain reaction and its application to direct sequencing of the MLA locus. Proc. Nat. Acad. Sci. U.S.A., 1988, 85, 7652-7656.

Hillenkamp, F. and Karas, M. 1990. Mass spectrometry of peptides and proteins by Matrix-Assisted Ultraviolet Laser Desoφtion/Ionization. Methods Enzymol. 193, 280-295

Holmgren, J., Czerkinsky C, Lycke, N. and Svennerholm, A. 1992. Mucosal Immunity: Implications for Vaccine Development. Immunobiol. 184, 157-179.

Kyte and Doolittle. 1992. J. Mol.Biol. 157, 105-137.

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

McGhee, J., Mestecky, J., Dertzbaugh M., Eldridge, J., Hirasawa, M., and Kyono,

H. 1992. The Mucosal Immune System: From Fundamental Concepts to Vaccine Development. Vaccine. 10 (2), 75-88.

Sambrook, Fritsch and Maniatis. Molecular Cloning - A Laboratory Manual. Cold Spring Press (Cold Spring Harbor, New York). 1989.

Sedegah et al. 1994. Immunology. 91, 9866-9870

Wolff et al. 1990. Science 247, 1465-1468

Claims

We claim:

1. A vaccine composition comprising (a) immunologically effective amount of an isolated and purified polypeptide of H. pylori having a molecular weight of about 19 kDa, or a fragment thereof, and (b) one or more of a pharmaceutically acceptable buffer, or diluent, adjuvant or carrier.

2. The vaccine composition of claim 1 wherein the polypeptide has the amino-terminal amino acid sequence of MLSKDIIKLLNEQVN (SEQ I.D. NO. 3).

3. The vaccine composition of claim 1 wherein the polypeptide has a molecular weight of about 19 kilodaltons as measured on a 10% SDS polyacrylamide gel.

4. The vaccine composition of claim 1 wherein the composition comprises a carrier.

5. The vaccine composition of claim 1 wherein said component (b) is an adjuvant.

6. The vaccine composition of claim 1 wherein said adjuvant comprises a liquid.

7. A method of inducing an immune response in a mammal which comprises administering to said mammal the vaccine composition of claim 1.

8. The method of claim 7 wherein said composition is administered parenterally.

9. The method of claim 7 wherein said composition is administered mucosally.

10. An isolated and purified nucleic acid sequence encoding an H. pylori protein having a molecular weight of 19 kilodaltons and having an amino terminal sequence of SEQ ID No. 3

11. An isolated and purified nucleic acid sequence of claim 10, wherein said nucleotide sequence hybridizes under high stringency southern hybridization conditions with a nucleic acid having the nucleotide sequence of SEQ I.D. NO. 1..

12. An isolated and purified nucleic acid sequence comprising a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ I.D. No. 2, or biologically equivalent amino acid sequence thereof.

13. A plasmid containing an isolated and purified nucleic acid sequence encoding an H. pylori protein having a molecular weight of 19 kilodaltons and having an amino terminal sequence of SEQ I.D. NO. 3

14. A plasmid containing an isolated and purified nucleic acid sequence of claim 8, wherein said nucleotide sequence hybridizes under high stringency southern hybridization conditions with a nucleic acid having the nucleotide sequence SEQ I.D. NO. 1.

15. A plasmid containing an isolated and purified nucleic acid sequence comprising a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ. I.D. NO. 2, or biologically equivalent amino acid sequence thereof.

16. A host cell transformed with at least one plasmid, of claim 13.

17. A host cell transformed with at least one plasmid, of claim 14.

18. A host cell transformed with at least one plasmid, of claim 15.

19. A method of preparing a vaccine composition which comprises adding an isolated and purified polypeptide of H. pylori having a molecular weight of about 19 kDa, or fragment thereof, to at least one pharmaceutically acceptable buffer, or diluent, adjuvant or carrier in an amount sufficient elicit an immunological response when provided to an animal or human.

20. A method of producing an H. pylori polypeptide which comprises transforming or transfecting a host cell with the plasmid of claim 13 and culturing the host cell under conditions which permit the expression of said polypeptide by the host cell.

21. A method of producing an H. pylori polypeptide which comprises transforming or transfecting a host cell with the plasmid of claim 14 and culturing the host cell under conditions which permit the expression of said polypeptide by the host cell.

22. A method of producing an H. pylori polypeptide which comprises transforming or transfecting a host cell with the plasmid of claim 15 and culturing the host cell under conditions which permit the expression of said polypeptide by the host cell.

23. A method of diagnosing an H. pylori infection comprising the step of determining the presence, in a sample, of an amino acid sequence of SEQ ID No.: 3.