Protective helicobacter antigens Number:6,762,295 from the United States Patent and Trademark Office (PTO) owispatent Protective Helicobacter antigens, especially H. pylori antigens, and the use of these antigens for the treatment of or prevention of, gastroduodenal disease associated with H. pylori infection.<H helicobacter, Protective, Protective, heli, 6762295.html, Patent, pto, 6762295

Title: Protective helicobacter antigens

Abstract: Protective Helicobacter antigens, especially H. pylori antigens, and the use of these antigens for the treatment of or prevention of, gastroduodenal disease associated with H. pylori infection.

Patent Number: 6,762,295 Issued on 07/13/2004 to Doidge, et al.

Inventors: Doidge; Christopher Vincent (Box Hill, AU), Lee; Adrian (Lane Cove, AU), Radcliff; Fiona Jane (Sydney, AU), Hocking; Dianna Margaret (Flemington, AU), Webb; Elizabeth Ann (Eltham, AU)
Assignee: CSL Limited (Parkville, AU)
The University of New South Wales (Kensington, AU)

Appl. No.: 08/945,038

Filed: December 23, 1997

PCT Filed: April 19, 1996

PCT No.: PCT/AU96/00225

PCT Pub. No.: WO96/33220

PCT Pub. Date: October 24, 1996

Foreign Application Priority Data


Apr 21, 1995 [AU]			PN2575
Jul 03, 1995 [AU]			PN3931
Jan 16, 1996 [AU]			PN7565

Current U.S. Class: 536/23.7 ; 435/252.33; 435/320.1; 435/6; 435/69.1; 536/23.1

Current International Class: C07K 14/195 (20060101); C07K 14/205 (20060101); C07K 16/12 (20060101); A61K 39/00 (20060101); A61K 38/00 (20060101)

Field of Search: 424/184.1 435/6,69.1,172.2,172.3,252.1,252.33,320.1 514/44 536/23.1,23.7

References Cited [Referenced By]

U.S. Patent Documents


5262156	November 1993	Alemohammad
5527678	June 1996	Blaser et al.

Foreign Patent Documents


WO 93/07273	Apr., 1993	WO
9405771	Mar., 1994	WO
WO 95/03824	Feb., 1995	WO
WO 95/22563	Aug., 1995	WO
WO 96/01272	Jan., 1996	WO

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E.B. Drouet et al., Characterization of an Immunoreactive Species-Specific 19-Kilodalton Outer Membrane Protein from Helicobacter pylori, J. Clin. Microbiol. vol. 29, No. 8, 8/91, pp. 1620-1624. .
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Primary Examiner: Smith; Lynette R. F.
Assistant Examiner: Portner; Ginny Allen
Attorney, Agent or Firm: Foley & Lardner

Parent Case Text

This application is a national phase application based on PCT/AU96/00225, filed Apr. 19, 1996, which claims priority to PN 2575, filed Apr. 21, 1995, PN 3931, filed Jul. 3, 1995 , and PN 7565, filed Jan. 16, 1996.

Claims

What is claimed is:

1. An isolated nucleic acid molecule which encodes a Helicobacter antigen having a molecular mass of approximately 36 kDa, said nucleic acid molecule consisting essentially of the sequence of nucleotides of SEQ ID NO:3.

2. A recombinant DNA molecule comprising an expression control sequence operatively linked to the nucleic acid molecule according to claim 1.

3. The recombinant DNA molecule according to claim 2, wherein said expression control sequence comprises a promoter sequence and an initiator sequence, and said sequence of nucleotides is located 3' to the promoter and initiator sequences.

4. A recombinant DNA cloning vector comprising the recombinant DNA molecule according to claim 2.

5. The recombinant DNA cloning vector according to claim 4, wherein said vector is a plasmid.

6. The host cell transfected or transformed with the recombinant DNA molecule according to claim 1.

7. A host cell according to claim 6, wherein said host cell is E. coli.

8. A preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vector which is a host cell capable of expressing a Heilcobacter antigen having a molecular mass of approximately 36 kDa, wherein said host cell comprises a heterologous nucleic acid molecule consisting essentially of the sequence of nucleotides which encodes SEQ ID NO:4.

9. A preparation according 8, wherein said vector is a bacterium that colonizes the gastrointestinal tract of the mammalian host.

10. A preparation according to claim 8, wherein said vector is a bacterium selected from the group of Salmonella, Shigella, Yersinia, Vibrio, Escherichia and Bacillus Calmette-Guerin (BCG).

11. An isolated nucleic acid molecule which encodes a Helicobacter antigen having a molecular mass of approximately 36 kDa, said nucleic acid molecule consisting essentially of the sequence of nucleotides which encodes SEQ ID NO:4.

Description

FIELD OF THE INVENTION

This invention relates to protective Helicobacter antigens, especially H. pylori antigens, and in particular to the use of these antigens for the treatment of, or prevention of, gastroduodenal disease associated with H. pylori infection.

BACKGROUND OF THE INVENTION

Helicobacter pylori is a gram negative, spiral bacterium which infects the lining of the human stomach. It is widely distributed, chronically infecting perhaps half the world's population. The bacterium spreads from person to person by oral-oral or faecal-oral transmission, there being no recognised environmental reservoir.

Infection with the bacterium causes an inflammation of the gastric mucosa, or stomach lining. Usually this does not resolve, and infection and inflammation are believed to persist for many decades. Often this is not associated with symptoms, however this chronic infection is associated with an increased risk of a number of sequelae. A significant portion of those infected develop peptic ulceration of the duodenum or stomach, when the infection process disrupts the usual protective mechanisms the stomach has against its own digestive products. Also, long periods of infection increase the risk of the development of adenocarcinomas or lymphomas of the stomach wall.

Therefore, prevention or treatment of H. pylori infection has the potential to prevent considerable mortality and morbidity resulting from the sequelae of chronic infection.

In early experiments, H. pylori did not infect conventional laboratory animals. However, a laboratory mouse model of H. pylori infection, using the closely related organism, Helicobacter felis, has been developed (Lee et al., 1990; Dick-Hegedus and Lee, 1991). This model has proven very useful in screening new antimicrobial therapeutic regimes.

H. felis is a spiral shaped bacterium that shares a very close DNA homology with H. pylori. The bacterium colonises the mouse stomach in a similar manner to the way that H. pylori colonises the human stomach. The main ecological niche is gastric mucus, and colonisation is mainly seen in the antrum of the stomach. In germfree mice, H. felis infection induces a gastritis that is very similar to the human H. pylori infection, with a chronic inflammation of mononuclear cells accompanied by a polymorphonuclear leucocyte infiltration. Infection with either organism results in the induction of a similar raised systemic humoral immune response against H. pylori and H. felis respectively (Lee et al., 1990).

The H. felis model has proved to be very predictive of the efficacy of anti-H. pylori therapy in humans. Thus, monotherapy with agents with high in vitro activity such as erythromycin show no significant in vivo effect against H. felis in mice, just as erythromycin has no ant-H. pylori effect in humans, despite its high antimicrobial effects in vitro. In contrast, the triple therapy regimens of a bismuth compound, metronidazole, and tetracycline or amoxycillin lead to a very high eradication rate in H. felis infected mice (Dick-Hegedus and Lee, 1991). Such therapies are among the most successful human anti-H. pylori regimens.

The H. felis model has also been used to demonstrate that mice can be orally immunised with Helicobacter antigens, either to protect them from becoming infected (Chen et al, 1992), or to treat them when they are already infected so as to eradicate the infection (Doidge et al, 1994). Antigens that have been used in these vaccines include disrupted cellular preparations from either H. felis or H. pylori, and the bacterial enzyme urease from H. felis or H. pylori or subunits thereof, produced from E. coli clones expressing all or part of the H. pylori urease molecule (Michetti et al, 1994; see also International Patent Publications Nos. WO 90/04030, WO 93/07273 and WO 94/09823). H. pylori heat shock protein (Hsp or HSP) has also been shown to be a protective antigen (Ferrero et al., 1995).

International Patent Publication No. WO 93/18150 (Application No. PCT/EP93/00472) discloses vaccines or therapeutic compositions comprising one or more of recombinant H. pylori cytotoxin (CT or VacA), H. pylori cytotoxin-associated immunodominant antigen (CAI or CagA) or H. pylori heat shock protein, optionally together with H. pylori urease. International Patent Publication No. WO 95/27506 (Application No. PCT/FR95/00383) discloses an anti-H. pylori immunising composition containing a substantially purified H. pylori catalase as the active ingredient; and International Patent Publication No. WO 95/14093 (Application No. PCT/EP93/03259) discloses an immunogenic composition capable of inducing protective antibodies against Helicobacter infection which comprises at least one urease structural polypeptide from H. pylori or H. felis and optionally a urease-associated heat shock protein or chaperonin from Helicobacter.

The fact that antigens derived from H. pylori can be used to protect mice from H. felis infection suggests that there are cross-reactive, and cross-protective antigens between the two species. That is, that there are molecules present in H. pylori, which can induce immune responses in mice that recognise targets on H. felis, thus protecting the mice from H. felis infection. If an immune response to these H. pylori molecules will protect mice from H. felis infection, it is likely that similar immune responses will protect humans from H. pylori infection, or if already infected, cure them of it. Urease has been demonstrated to be such a cross-protective molecule in the H. felis mouse model (Michetti et al, 1994).

In work leading to the present invention, in order to identify further cross-reactive and cross protective antigens, a DNA library from an H. pylori strain has been constructed and screened with serum from mice that had been orally immunised with a vaccine prepared from disrupted H. felis cells and a mucosal adjuvant, with the aim of identifying E. coli clones expressing H. pylori proteins recognised by anti-H. felis antibodies and of subsequently identifying the antigenic protective H. pylori proteins.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an at least partially purified preparation of at least one Helicobacter antigen selected from the group consisting of: (i) an antigen having a molecular mass of approximately 19 kDa which is processed into a mature form having a molecular mass of approximately 17 kDa; (ii) an antigen having a molecular mass of approximately 13 kDa; (iii) an antigen having a molecular mass of approximately 36 kDa; (iv) an antigen having a molecular mass of approximately 50 kDa; (v) an antigen having a molecular mass of approximately 29 kDa; and (vi) immunogenic fragments of any of antigens (i) to (v) above which are capable of eliciting a specific protective immune response in a mammalian host.

In another aspect, the present invention provides an isolated Helicobacter antigen for use in the treatment or prevention of Helicobacter infection in a mammalian host, selected from the group consisting of: (i) an antigen having a molecular mass of approximately 19 kDa which is processed into a mature form having a molecular mass of approximately 17 kDa; (ii) an antigen having a molecular mass of approximately 13 kDa; (iii) an antigen having a molecular mass of approximately 36 kDa; (iv) an antigen having a molecular mass of approximately 50 kDa; and (v) an antigen having a molecular mass of approximately 29 kDa; and (vi) immunogenic fragments of any of antigens (i) to (v) above which are capable of eliciting a specific protective immune response in a mammalian host.

Each of the above antigens is further characterised by being reactive with anti-H. felis antibodies.

Preferably, antigen (i) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone B4.6 hereinafter (SEQ ID NO.10), or allelic or other variants thereof; antigen (ii) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone C3.5 hereinafter (SEQ ID NO.2), or allelic or other variants thereof; antigen (iii) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone E2.5 hereinafter (SEQ ID NO.4), or allelic or other variants thereof; antigen (iv) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone G3.8 hereinafter (SEQ ID NO. 6), or allelic or other variants thereof; and antigen (v) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone H5.1 hereinafter (SEQ ID NO. 8), or allelic or other variants thereof.

Suitable variants may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to one of the amino acid sequences referred to above, or to a region or part thereof, provided the variant is capable of eliciting a specific protective immune response in a mammalian host.

The term "at least partially purified" as used herein denotes a preparation in which the content of the particular antigen is greater, preferably at least 30% greater and more preferably at least 50% greater, than the content of the antigen in a whole cell sonicate of Helicobacter bacteria. Preferably, the preparation is one in which the antigen is "substantially pure", that is one in which the content of the particular antigen is at least 80%, more preferably at least 90%, of the total Helicobacter antigens in the preparation.

The term "isolated" as used herein denotes that the antigen has undergone at least one purification or isolation step, and preferably the antigen is in a form suitable for use in a vaccine composition.

It is to be understood that the present invention extends not only to the particular antigens of Helicobacter bacteria as described above, but also to immunogenic fragments of the particular antigen(s), that is fragments of the antigen(s) which are capable of eliciting a specific protective immune response in a mammalian host. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular antigen(s). Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.

In another aspect, the present invention provides a vaccine composition for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant, together with one or more pharmaceutically acceptable carriers and/or diluents.

In yet another aspect, the present invention provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant.

In a related aspect, this invention provides the use of a vaccine composition comprising an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant, for the treatment or prevention of Helicobacter infection in a mammalian host.

By use of the term "immunologically effective amount" herein, 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. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, 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.

Preferably, but not essentially, the antigenic preparation of this invention is orally administered to the host, and is administered in association with a mucosal adjuvant. However, the invention also extends to parenteral administration of this antigenic preparation.

The present invention also extends to an antibody, which may be either a monoclonal or polyclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above. Such antibodies may be produced by methods which are well known to persons skilled in this field.

In this aspect, the invention further provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises passive immunisation of said host by administration of an effective amount of an antibody, particularly a monoclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above.

The Helicobacter antigenic preparation or isolated antigen of this invention may be prepared by purification or isolation from natural sources, such as a whole cell sonicate of Helicobacter bacteria. Alternatively, however the antigenic preparation or isolated antigen may be prepared by synthetic, preferably recombinant, techniques. When prepared by recombinant techniques, the antigen may have an amino acid sequence substantially identical to the naturally occurring sequence or may contain one or more amino acid substitutions, deletions and/or additions thereto provided that following such alterations to the sequence, the molecule is still capable of eliciting a specific protective immune response against the naturally occurring Helicobacter antigen. A similar immunogenic requirement is necessary for any fragments or derivatives of the antigen whether made from the recombinant molecule or the naturally occurring molecule. Accordingly, reference herein to a Helicobacter antigen is considered reference to the naturally occurring molecule, its recombinant form and any mutants, derivatives, fragments, homologues or analogues thereof provided that such molecules elicit a specific protective immune response against the naturally occurring Helicobacter antigen. Also included are fusion molecules between two or more Helicobacter antigens or with other molecules including fusion molecules with other molecules such as glutathione-S-transferase (GST) or .beta.-galactosidase.

The present invention also extends to an isolated nucleic acid molecule encoding a Helicobacter antigen of the present invention, and preferably having a nucleotide sequence as set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or being substantially similar to all or a part thereof. The term "substantially similar" means having at least 40-50%, more preferably at least 60-70%, and most preferably at least 80% identity. A "part" in this context means a contiguous series of at least 15 nucleotides, and more preferably at least 25 nucleotides.

According to this embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides which encodes a Helicobacter antigen as broadly described above, and hybridises under low stringency conditions to all or part of a nucleic acid sequence set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or to a complementary form thereof.

In another aspect, this invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or a part thereof.

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 the level of stringency, reference can conveniently be made to Manratis, et al. (1982) which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45.degree. C. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at .+-.45.degree. C. for 2-3 hours or high stringent conditions as disclosed by Maniatis, et al. (1982).

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.

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 Helicobacter antigen, 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 Helicobacter antigen comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a Helicobacter antigen, 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 is provided fused polypeptides comprising a Helicobacter antigen 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.

The present invention also extends to synthetic polypeptides displaying the antigenicity of a Helicobacter antigen 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 Helicobacter antigen of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.

Isolated polypeptides comprising, or containing in part, amino acid sequences corresponding to a Helicobacter antigen may be used to raise polyclonal antisera by immunising rabbits, mice or other animals using well established procedures. Alternatively, such polypeptides may be used in the preparation of monoclonal antibodies by techniques well known in the art.

In addition, the polypeptides in accordance with this invention including fused polypeptides may be used as an active immunogen in the preparation of single or multivalent vaccines by methods well known in the art of vaccine manufacture for use in the treatment or prevention of Helicobacter infection in a mammalian host.

Alternatively, the polypeptides in accordance with the present invention including fused polypeptides may be used as antigen in a diagnostic immunoassay for detection of antibodies to Helicobacter in a sample, for example, a serum sample from a human or other mammalian patient. Such immunoassays are well known in the art, and include assays such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", is to be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, the antigenic preparation or isolated antigen of this invention comprises H. pylori or H. felis antigen(s). Preferably also, this antigenic preparation or isolated antigen is used in a vaccine composition for oral administration which includes a mucosal adjuvant.

In a particularly preferred aspect of this invention, an oral vaccine composition comprising an antigenic preparation or isolated antigen comprising H. pylori antigen(s) as broadly described above, in association with a mucosal adjuvant, is used for the treatment or prevention of H. pylori infection in a human host.

The mucosal adjuvant which is optionally, and preferably, administered to the infected host with the Helicobacter antigenic preparation of this invention, is preferably cholera toxin. 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. These may be added to, or conjugated with, the Helicobacter antigenic preparation. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin. 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.

The Helicobacter antigenic preparation or isolated antigen of this invention may be delivered in accordance with this invention in ISCOMS.TM. (immune stimulating complexes), ISCOMS.TM. 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 antigenic preparation or isolated antigen may also be incorporated into oily emulsions and delivered orally. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter, (1992).

Other adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in the prophylactic or therapeutic vaccine composition of this invention. The vaccine composition may, for example, be formulated in enteric coated gelatine capsules including sodium bicarbonate buffers together with the Helicobacter antigenic preparation or isolated antigen and cholera toxin mucosal adjuvant.

The formulation of such prophylactic or therapeutic vaccine compositions is well known to persons skilled in this 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. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. 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. Supplementary active ingredients can also be incorporated into the compositions.

The Helicobacter antigenic preparation or isolated antigen 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, VacA, CagA or catalase, as well as immunologically active antigens against other pathogenic species.

As an alternative to the delivery of the Helicobacter antigenic preparation or isolated antigen in the form of a therapeutic or prophylactic vaccine composition, the antigen or an immunogenic fragment thereof may be delivered to the mammalian host 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 antigen of immunogenic fragment as a foreign polypeptide. Particularly, bacteria that colonise 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).

Accordingly, the present invention also extends to delivery to the host using a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof. Accordingly, in a further aspect this invention provides a preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof.

In this aspect, the invention extends to a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of a vaccine vector expressing an isolated Helicobacter antigen as broadly described above or an immunogenic fragment thereof.

Further, the invention extends to the use of a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof, for the treatment or prevention of Helicobacter infection in a mammalian host.

Further features of the present invention are more fully described in the following Examples. It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and B, 2A and B show cloned H. pylori proteins expressed from E.coli XLOLR; (1A) analysed on 4-20% gradient SDS-polyacrylamide gels, and visualised by CBB stain. Lane M, Molecular weight standards (kDa); Lane 1, Family A; Lane 2, Family B; Lane 3, Family C; Lane 4, uncharacterized protein; Lane 5, Family F; Lane 6, Family G; Lane 7, Family H; Lane 8, Negative Control, E.coli XLOLR; Lane 9, Positive Control, Helicobacter pylori total cell proteins; (1B) corresponding Western blot samples, lane order the same as for panel A; analyzed on 4-20% gradient SDS-polyacrylamide gels, and visualized by CBB stain. Lane M, Molecular weight standards (kDa); Lanes 1 and 3, uncharacterized protein, Lane 2, Famile E; Lane between lanes 3 and 4, uncharacterized protein; lane 4, negative control. (2B) corresponding Western Blot samples, lane order the same as for panel A.

EXAMPLE 1

Identification of E.coli clones expressing H. pylori proteins recognised by anti-H. felis antibodies.

A. Materials and Methods

Bacterial strains

Helicobacter pylori strain HP921023 was used as the DNA donor for preparing the gene library. Escherichia coli strain ER1793 (New England Biolabs) was the host used for phage infection and plating of Lambda ZAP Express. E.coli strains XLl-Blue MRF' and XLOLR (Stratagene) were used for excision of phagemid pBK-CMV and protein expression of cloned genes.

Isolation of H. pylori chromosomal DNA

Whole cell DNA from H.pylori was prepared essentially as reported by Majewski and Goodwin (1988).

Anti-sera preparation

Mouse anti-sera was raised against Helicobacter felis by four ora-gastric immunisations at weekly intervals. Each vaccine dose consisted of 1 mg (protein) of sonicated H. felis cells and 10 ug of cholera toxin. Blood was collected and serum pooled. This serum was adsorbed with 50% v/v E.coli extract (Promega) containing 5% w/v skim milk and 0.05% v/v Tween 20 in TBS at a final dilution of 1:100. The preparation was incubated at room temperature for 4 hours prior to immunoscreening to eliminate or reduce nonspecific reactivity of antisera with host proteins. The specificity of the sera was confirmed by dot blot and Western blotting, using dilutions of whole cells of H. pylori for positive control and E.coli XLOLR as the negative control.

Bacterial growth conditions

For infection with Lambda ZAP Express, strain ER1793 cells were initially grown in Luria-Bertani (LB) broth supplemented with 0.2% w/v maltose and 10 mM MgSO4 at 30.degree. C. Following infection, cells were maintained in LB broth at 37.degree. C. for 15 minutes and then plated on NZY agar medium and incubated at 42.degree. C. for 4 hours then at 37.degree. C. overnight. For phagemid excision and plasmid isolation E.coli strains XL1-Blue and XLOLR were grown in LB broth at 37.degree. C., and transformed XLOLR cells selected on LB/Kanamycin plates (50 .mu.g/ml) at 37.degree. C.

Construction of H. pylori gene library

An H.pylori expression library was constructed, using standard procedures (Sambrook et al, 1989), in the Lambda ZAP Express vector (Stratagene) which had been predigested with BamHI and the terminal 5' phosphates removed with calf intestinal phosphatase. Genomic DNA partially digested with Sau3AI, was fractionated by gel electrophoresis and DNA fragments between 6 to 12 kb were isolated. This DNA was ligated with 1.0 .mu.g of BamH1-digested lambda arms. Recombinant phage DNA was packaged in vitro using Gigapack II extract (Stratagene). The library was titrated by infecting E.coli strain ER1793 or XL1-Blue MRF' cells with aliquots of packaged phage and plated onto indicator plates containing IPTG and X-Gal. The ratio of nonrecombinant phage to recombinant phage was 1:5. The titre of the recombinant library was calculated to be 1.times.10.sup.6 pfu per .mu.g of lambda DNA.

Antibody screening of H.pylori genomic library

A portion of the library was screened by plaque immunoblot assay. A total of 10,000 plaques were plated (2,000 bacteriophage plaques per plate), and lifted onto Hybond-C extra nitrocellulose filters (Amersham) to be processed as per Sambrook et al (1989). The filters were screened with a 1:100 dilution of anti-H. felis mouse sera, at room temperature overnight. After being washed in 0.05% v/v Tween 20 in TBS, filters were incubated in 1:2000 conjugated goat anti-mouse immunoglobulin G-conjugated horse radish peroxidase for 1.5 hours. Filters were washed as previously described above and the colour reaction was developed with TMB substrate (KPL Inc.). When a positive phage clone was identified, an agar plug containing the plaque was picked and phage eluted into SM buffer. To obtain plaque purity the process of infecting bacteria, replating and immunoscreening was repeated.

In vivo excision of plasmid pBK-CMV from Lambda ZAP Express vector

In vivo excision of pBK-CMV containing H.pylori DNA from Lambda ZAP Express was achieved by infecting E.coli strain XL1-Blue MRF' simultaneously with Lambda ZAP Express containing insert DNA and ExAssist helper phage M13. Excised phagemids were packaged as filamentous phage particles and secreted from host cells, which were subsequently heat killed. The phagemids were rescued by infecting XLOLR cells and plating onto LB/Kanamycin (50 .mu.g/ml) plates. Bacterial colonies appearing on plates contained pBK-CMV double-stranded phagemid with the cloned DNA insert from H.pylori. These colonies were then analysed for protein expression.

SDS-PAGE and Western blot analysis of proteins

The total proteins produced by cloned H. pylori DNA in E.coli XLOLR were analysed by standard SDS-PAGE and Western Blot techniques (Sambrook et al 1989; Towbin et al 1979). 10 ml cultures of XLOLR containing expression plasmid were grown in supermedium at 37.degree. C. overnight. Cultures were divided in two and one induced with IPTG to a final concentration of 1 mM, with continued incubation for 2-4 h. Aliquots of 1 ml were collected, cells pelleted by centrifugation and resuspended in 10 mM Tris-HCl (pH 8). Cells were mixed with equal volume of SDS sample reducing buffer and boiled for 10 minutes. Proteins were resolved by electrophoresis on 4-20% gradient Tris-glycine gels (Novex) and stained with coomassie brilliant blue (CBB). A gel run in parallel was electrotransferred onto nitrocellulose membrane (BioRad), for detection of immunoreactive proteins of H.pylori using anti-H. felis mouse sera as described above.

For molecular mass estimation, the Coomassie Blue stained wet gel was scanned with a Molecular Dynamics model 300A densitometer and the apparent molecular mass determined relative to standard proteins using Image Quant version 3.3 software.

Protein N-terminal sequence determination

Proteins to be N-terminal sequenced were separated by SDS-PAGE and transferred onto PVDF membrane (Novex) in IxCAPS electroblotting buffer and then stained with 0.1% w/v CBB in 50% v/v methanol, and destained in 50% v/v methanol until protein bands were visible. The bands corresponding to immunopositive proteins identified by western blot, were excised and sequenced. Amino acid sequencing was performed on an Applied Biosystems Inc., model 473A sequencer at the Centre of Animal Biotechnology, School of Veterinary Science, University of Melbourne. Additional sequencing was provided by Auspep Pty. Ltd.

DNA preparation and sibling analysis of clones

Plasmid DNA was isolated by the alkaline lysis method (Sambrook et al, 1989) from cultures of E.coli XLOLR clones carrying different H.pylori DNA inserts. Restriction enzyme digestions were performed as recommended by the enzyme manufacturer (Promega Inc.). Restriction fragments of cloned H. pylori DNA to be used as probes were resolved by gel electrophoresis in 0.8% agarose, stained with ethidium bromide, excised from gel and purified with a Bresaclean kit for nucleic acid purification (Bresatec Ltd). The SalI/NotI fragments of 2.5-7.0 kb in size were labelled with (.sup.32 P)d-ATP using Random Primers DNA labelling kit (Gibco BRL).

For cross-hybridization analysis, to determine related clones, cell suspensions of XLOLR clones were dotted onto nitrocellulose and treated as per the manufacturers protocol (Amersham). Filters were hybridized at 65.degree. C., overnight in a solution containing 2xPE, 1% w/v skim milk and 7% w/v SDS. After hybridisation, filters were subjected to one 15 min wash in 2xSSC, 0.1% w/v SDS, at room temp and two 30 min. washes in 2xSSC, 0.1% w/v SDS at 65.degree. C. The hybridisation results were visualised by autoradiography on Kodak Biomax film.

B. Results and Discussion

In order to clone potential protective antigens of Helicobacter pylori, a genomic library of strain HP921023 was constructed in the lambda expression vector Lambda ZAP Express. The library was screened for immunoreactivity with sera from mice vaccinated with Helicobacter felis in an attempt to detect clones expressing H.pylori antigens that cross-reacted with H. felis antigens. Approximately 10,000 plaques were screened using the anti-H. felis mouse serum. Fifty immunopositive clones with varying signal intensities were recognised by the mouse sera. These were picked, purified and the expression plasmid pBK-CMV excised for further characterisation of the cloned DNA and the encoded proteins. The proteins expressed by these recombinant plasmids were analysed by SDS-PAGE (FIGS. 1A and 2A) and Western blotting (FIGS. 1B and 2B).

The molecular mass of cloned proteins recognised by the mouse sera ranged from approx. 13 kDa to approx. 62 kDa. A pattern emerged where by clones could be grouped into families based on the protein profile and protein size (see Table 1 below). Families were named alphabetically for convenience (eg.family A, B, C etc.). Family A consists of five members, identified by two predominant proteins of approx. 62 kDa and approx. 33 kDa (FIG. 1B, Lane 1). Family B has 14 related clones expressing two proteins of approx. 19 kDa and approx. 17 kDa (FIG. 1B, Lane 2). The smaller of the two proteins tends to be produced in greater amounts than does the approx. 19 kDa protein. Depending upon the culture conditions, the approx. 19 kDa protein may be present in equivalent amounts to the approx. 17 kDa protein or noticeably less. This may explain why the approx. 17 kDa protein is often observed as being a stronger immunopositive band than the approx. 19 kDa when visualised by Western blotting. Family C has 10 members characterised by a small protein of approx. 13 kDa in size (FIG. 1B, Lane 3) which is often more easily distinguished on Western blot than on CBB stained gel. Clonal variation in expression levels of the protein exist and the signal on blots can vary from weak to strong. Family E is represented by one clone that encodes a protein of approx. 36 kDa (FIG. 2, Lane 2). Family F is also represented by one clone which expresses an abundant amount of an approx. 55 kDa protein (FIG. 1B, Lane 5). Of all the cloned proteins, this protein is the most strongly recognised by the anti-H. felis mouse sera when observed on a Western blot. Family G has 2 members that express an approx. 50 kDa protein (FIG. 1B, lane 6) which is not produced in a quantity that can be easily visualised on a CBB stained gel over and above the equivalent sized host E.coli protein (FIG. 1A, lane 6). However, antibodies in the mouse sera clearly demonstrate binding to this protein and not to the E.coli proteins run in lane 8. Given that this cloned H.pylori protein is not expressed in high amounts but is quite immunopositive, it may well be an important antigen in eliciting a strong immune response to Helicobacter pylori infection. Lane 7 (FIGS. 1A and 1B) contains the only representative of family H, an approx. 29 kDa protein which is poorly expressed and gives a weaker signal than other family proteins on a Western blot. Lane 8 FIG. 1 and Lane 4, FIG. 2 comprises the negative control, E.coli XLOLR bearing expression plasmid pBK-CMV without H.pylori insert. Absorption of the mouse sera with E.coli extract largely prevented non-specific binding to host cell proteins. Depending upon the length of development time of the substrate a maximum of six E.coli proteins were recognised throughout all the lanes compared with a plethora of host cell proteins appearing on blots probed with unabsorbed mouse sera (data not shown). A dominant host cell protein is recognised at 37 kDa. Lane 9 comprises the positive control, total cell proteins of Helicobacter pylori with.about.10 immunopositive bands ranging in size from 11 to 95 kDa. Results of the sibling DNA analysis (data not shown) confirmed the Western blot data that seven families of cloned H.pylori proteins exist.

The clones were screened for the presence of urease since the genomic DNA used in the generation of the library was obtained from a Ure B positive strain of H.pylori, and urease is a known protective antigen which already has been cloned (Michetti et al, 1994). Hybridization with oligonucleotide probes to Ure A and Ure B genes revealed five clones to be positive for both urease A and urease B DNA sequences (Table 1). All the urease positive clones belong to family A. No other clones existing outside of family A were urease positive. Identity of the approx. 62 kDa and approx. 33 kDa proteins was confirmed by N-terminal sequencing. Protein homology searches in the database Swiss-Prot/GenPeptide identified 100% homology of the 15 amino acid residues of the approx. 62 kDa protein with the Urease B subunit of Helicobacter pylori. The 18 amino acid sequence of the approx. 33 kDa protein was found to have 94.4% homology with the Urease A subunit, with only one mismatched amino acid residue.

Preliminary N-terminal sequence has also been obtained for family B, family C, family F and family G. The protein sequence of the approx. 19 kDa protein of family B has been found to correspond to the membrane-associated lipoprotein antigen (Lpp20) of Helicobacter pylori (Kostrzynska et al., 1994).

No significant homology was found in the data base to the approx. 13 kDa protein of family C or the approx. 36 kDa protein of family E.

Protein sequence data for the 55 kDa protein from family F was found to have 80% homology with the first 15 N-terminal amino acids of the heat shock protein 60 (Hsp60) sequence of Helicobacter pylori, with only three residues unmatched. This finding supports the Western blotting results and explains the high signal intensity of this immunoreactive band, as Hsp60 is known to elicit a strong antibody response.

TABLE 1 Summary of cloned H. pylori antigen families. N-terminal sequences were compared with those in the Swiss-Prot/GenPeptide database. Protein Protein No. Urease Molecular SEQ Identity of Hybridization Mass Protein N-terminal ID (from Family Clones Oligo A & B (kDa) Sequence NO. database) A 5 Yes .about.62 MKKISRKEYV 11 Urease B sub-unit .about.33 MKLTPKELDKLMLHRAGE 12 Urease A sub-unit B 14 No .about.19 MLNQVLLKLGMSVKAAMV 13 Lpp20 .about.17 Not determined Mature Lpp20 C 10 No .about.13 MISKEEVLEYIGSLS 14 Unknown E 1 No .about.36 Not determined Unknown F 1 No .about.55 AKEIKFVDAARNLFF 15 Hsp 60 G 2 No .about.50 MFGFKQLQLQFSQKV 16 Unknown H 1 No .about.29 Not determined Unknown Subsequently, DNA sequencing has identified some errors in the N-terminal amino acid sequences determined above.

EXAMPLE 2

Selected representative clones from cloned H. pylori antigen families C, E, G, H and B (Table 1) have been sequenced as follows:

(i) Clone C.3.5 (SEQ ID NO. 1 and 2)

The strategy used to sequence the 4423 bp insert in clone C3.5 included a combination of procedures which are summarized below.

1. Plasmid DNA was prepared using a modified alkaline lysis procedure.

2. Nested deletions were generated from both the T7 and T3 ends using ExoIII and S1 nuclease.

3. Deletion clones were size-selected for DNA sequencing by electrophoresis on agarose gels.

4. DNA sequencing was performed using standard dideoxynucleotide termination reactions containing 7-deaza dGTP. 7-deaza dITP was used, if necessary, to resolve severe GC band compressions. [.sup.35 S]dATP was used as the label.

5. Sequencing reactions were analysed on 6% polyacrylamide wedge gels containing 8M urea. All samples were loaded in the order G-A-T-C.

6. Internal sequencing primers were synthesised as necessary.

(ii) Clone E2.5 (SEQ ID NO. 3 and 4)

The strategy used to sequence the 2435 bp insert in clone E2.5 included a combination of procedures which are summarised below.

1. The NotI/SalI fragment was blunt-ended, cloned into the EcoRV site of pBluescript II SK.sup.+ (Stratagene) and used to transform XL1-Blue cells.

2. Plasmid DNA was prepared using a modified alkaline lysis procedure. The deletion clones were generated from both the original clone and the EcoRV subclone.

3. Plasmid DNA was prepared using a modified alkaline lysis procedure.

4. Deletion clones were size-selected for DNA sequencing by electrophoresis on agarose gels.

5. DNA sequencing was performed using standard dideoxynucleotide termination reactions containing 7-deaza dGTP. 7-deaza dITP was used, if necessary, to resolve severe GC band compressions. [.sup.35 S]dATP or [.sup.33 P]dATP were used as the label.

6. Sequencing reactions were analysed on 6% polyacrylamide wedge gels containing 8M urea. All samples were loaded in the order G-A-T-C.

7. Internal sequencing primers were synthesised as necessary.

(iii) Clone G3.8 (SEQ ID No. 5 and 6)

The strategy used to sequence the 6081 bp BamHI insert in clone G3.8 included a combination of procedures which are summarised below.

1. Nested deletions were generated from both the T7 and T3 ends using ExoIII and S1 nuclease.

2. Plasmid DNA was prepared using a modified alkaline lysis procedure.

3. Deletion clones were size-selected for DNA sequencing by electrophoresis on agarose gels.

4. DNA sequencing was performed using standard dideoxynucleotide termination reactions containing 7-deaza dGTP. 7-deaza dITP was used, if necessary, to resolve severe GC band compressions. [.sup.35 S]dATP was used as the label.

5. Sequencing reactions were analysed on 6% polyacrylamide wedge gels containing 8M urea. All samples were loaded in the order G-A-T-C.

6. Internal sequencing primers were synthesized as necessary.

(iv) Clone H5.1 (SEQ ID NO. 7 and 8)

The strategy used to sequence the 1199 bp insert in clone H5.1 included a combination of procedures which are summarised below.

1. The SalI/NotI fragment was blunt-ended and cloned into the EcoRV site of pBluescript II SK.sup.+ (Stratagene) and used to transform XL1-Blue cells.

2. Nested deletions were generated from both the T7 and T3 ends using ExoIII and S1 nuclease.

3. Plasmid DNA was prepared using a modified alkaline lysis procedure.

4. Deletion clones were size-selected for DNA sequencing by electrophoresis on agarose gels.

5. DNA sequencing was performed using standard dideoxynucleotide termination reactions containing 7-deaza dGTP. 7-deaza dITP was used, if necessary, to resolve severe GC band compressions. [.sup.35 S]dATP was used as the label.

6. Sequencing reactions were analysed on 6% polyacrylamide wedge gels containing 8M urea. All samples were loaded in the order G-A-T-C.

7. Internal sequencing primers were synthesised as necessary.

(v) Clone B4.6 (SEQ ID NO. 9 and 10)

The strategy used to sequence the 4518 bp insert in clone B4.6 included a combination of procedures which are summarised below:

1. Plasmid DNA was prepared using a modified alkaline lysis procedure.

2. Nested deletions were generated from both the T7 and T3 ends using Exo III and S1 nuclease.

3. Deleted clones were size-elected for DNA sequencing by electrophoresis on agarose gels.

4. DNA sequencing was performed using standard dideoxynucleotide termination reactions containing 7-deaza dGTP. 7-deaza dIPT was used, if necessary, to resolve severe GC band compressions. [.sup.35 S]dATP was used as the label.

5. Sequencing reactions were analysed on 6% polyacrylamide wedge gels containing 8M urea. All samples were loaded in the order G-A-T-C.

6. Internal sequencing primers were synthesized as necessary.

EXAMPLE 3

Subcloning, Expression, Purification, and Testing of Recombinant H. pylori Antigens in an H. pylori Mouse Model

1. Development of the H. pylori Mouse Model

1.1 Introduction

A human strain of H.pylori has been adapted to survive in the mouse gastric mucosa thus producing a useful model of H.pylori infection. This model was used for these vaccine studies. Detailed below is the method of derivation of this strain, characteristics of the mouse model and the methods used to demonstrate the effectiveness of the recombinant antigens of the present invention.

1.2 Mouse adaptation

A number of biopsies and fresh clinical isolates of H. pylori were obtained from patients. Homogenised biopsies and/or fresh clinical isolates were inoculated per os into specific pathogen free (SPF) BALB/c mice. Gastric samples from the infected mice were examined by direct phase microscopy and urease assay. One group of animals, inoculated with a mixture of four clinical isolates, were found to be colonised with spiral-shaped bacteria which gave a positive urease result. Gastric mucus from the colonised animals was cultured on blood agar base containing 5% horse blood and vancomycin (100 .mu.g/ml), polymyxin B (3.3 .mu.g/ml), bacitracin (200 .mu.g/ml), nalidixic acid (10.7 .mu.g/ml) and amphotericin B (50 .mu.g/ml). Representative colonies were examined by phase contrast microscopy and urease and catalase activity was determined. DNA was extracted from those colonies found to have characteristics of H. pylori i.e. spiral-shaped, urease and catalase positive. Isolates were confirmed as belonging to the Helicobacter genus by a Helicobacter specific PCR. To identify which of the four clinical isolates had colonised the mice, RAPD's were performed. Resulting finger prints from the original human clinical isolates and the mouse isolates were compared. The results of this comparison showed that all mice had been colonised with only one of the four clinical isolates originally inoculated into the mice. The human and mouse isolates were also found to be vacA and cagA positive by PCR. The mouse isolates were subsequently passaged through mice an additional three times.

One of the isolates, designated HpM8, obtained from a SJL mouse colonised with the original culture and a homogenate from an infected mouse was selected as our standard mouse adapted culture. This isolate has been called the "Sydney Strain" of H.pylori (The strain has been redesignated Syd1 and has been deposited in the culture collection of the School of Microbiology & Immunology at The University of New South Wales. (World Directory of Collections of Cultures of Microorganisms. Registration Number 248).

1.3 Mouse strain specificity

Isolate Syd1 was found to colonise a number of strains of mice including BALB/c, DBA, SJL, C3H/He, C3H/HeJ, C57BL/6 and Quackenbush/Swiss. The bacteria were found to colonise all regions of the mouse stomach i.e. the antrum, body and cardia equivalent region, with the bacteria preferentially colonising the border region between the antrum and body mucosa in some strains of mice. The colonisation pattern was found to vary depending upon the strain of mouse inoculated. BALB/c mice were selected for the present study. Electron microscopy revealed a close association of the bacteria with the epithelial surface, occasionally forming adhesion pedestals as seen with human infections. For routine assay of colonisation, urease reactivity was shown to correlate well with bacterial count and so was used as the assay method for H. pylori colonisation.

2. Subcloning Antigen Coding Regions into E.coli Expression Vectors

The specific antigen coding sequences from H.pylori cloned families B,C,E,G and H were isolated by PCR amplification of representative clones using oligonucleotides designed to contain appropriate restriction endonuclease sites to enable cloning into particular expression vectors (Table 2).

Amplified products from families B,C and E were cloned into the XmaI/BgIII sites of pGEX-STOP vector (a modified version of pGEX-4T-1 (Pharmacia) in which a termination codon has been inserted close