WO2001083532A1

WO2001083532A1 - Helicobacter pylori antigens: fusion of fay 31 and hpa 44

Info

Publication number: WO2001083532A1
Application number: PCT/SE2001/000871
Authority: WO
Inventors: Peter Doig; Jacques Pappo
Original assignee: Astrazeneca Ab
Priority date: 2000-04-29
Filing date: 2001-04-24
Publication date: 2001-11-08
Also published as: AU5280901A; GB0010370D0

Abstract

The present invention concerns a pharmaceutical product, e.g. a vaccine composition or vaccine kit, which comprises Fay31 and HOP38 polypeptides. The invention also concerns a fusion polypeptide comprising both Fay31 and HOP38 amino acid sequences, as well as a nucleic acid encoding such a fusion polypeptide. In addition, the invention concerns a pharmaceutical product comprising separate nucleotide sequences respectively encoding Fay31 and HOP38 polypeptides, Fay31 together with HOP38 provides a synergistic combination that can be used to treat and/or reduce the risk of Helicobacter pylori infection in a mammal.

Description

HELICOBACTER PYLORI ANTIGENS: Fusions of fay 31 and Hpa 44

FIELD OF THE INVENTION

The present invention concerns a pharmaceutical product, eg a vaccine composition or vaccine kit, which comprises Fay31 and HOP38 polypeptides. The invention also concerns a fusion polypeptide comprising both Fay31 and HOP38 amino acid sequences, as well as a nucleic acid encoding such a fusion polypeptide. In addition, the invention concerns a pharmaceutical product comprising separate nucleotide sequences respectively encoding Fay31 and HOP38 polypeptides. Fay31 together with HOP38 provides a synergistic combination that can be used to treat and/or reduce the risk of Helicobacter pylori infection in a mammal.

BACKGROUND OF THE INVENTION

Helicobacter pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet J,: 1273-1275; and Marshall et al, (1984) Microbios Lett. 25: 83- 88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al., (1986) Gut 27: 635-641). Moreover, evidence is accumulating for an etiologic role of H. pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. (Blaser M. J., (1993) Trends Microbiol. I: 255-260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age. (Taylor, D.N. and M. J. Blaser, (1991) Epidemiol. Rev 13: 42-50). H. pylori colonises the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265- 277).

The bacterial factors necessary for colonisation of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of the putative virulence factors include the following: urease, an enzyme that may play a role in neutralising gastric acid pH (Eaton et al, (1991) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee (1991) Microb. Ecol. Hlth. Dis. 4: 121-134; Labigne et al, (1991) J. Bacteriol. 173: 1920- 1931); the bacterial flagellar proteins responsible for motility across the mucous layer. (Hazell et al., (1986) J. Inf. Dis. 153: 658-663; Leying et al., (1992) Mol. Microbiol. 6: 2863-2874; and Haas et al., (1993) Mol. Microbiol. 8: 753-760); Vac A, a bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells (Schmitt, W. and R. - Haas, (1994) Molecular Microbiol. 12(2): 307-319); and several gastric tissue-specific adhesins. (Boren et al., (1993) Science 262: 1892-1895; Evans et al., (1993) J Bacteriol. 175: 674-683; and Falk et al, (1993) Proc. Natl. Acad. Sci. USA 90: 2035-203).

Numerous therapeutic agents are currently available that eradicate H. pylori infections in vitro. (Huesca et. al., (1993) Zbl. Bakt. 280: 244-252; Hopkins, R. j. and J. G. Morris, supra). However, many of these treatments are suboptimally effective in vivo because of bacterial resistance, altered drug distribution, patient non-compliance or poor drug availability. (Hopkins, R. J. and J. G. Morris, supra). Treatment with antibiotics combined with bismuth are part of the standard regime used to treat H. pylori infection. (Malfertheiner, P. and J. E. Dominguez-Munoz (1993) Clinical Therapeutics 15 Supp. B: 37-48). Combinations of a proton pump inhibitors and a single antibiotic have been shown to ameliorate duodenal ulcer disease. (Malfertheiner, P. and J. E. Dominguez-Munoz supra). However, methods employing antibiotic agents can have the problem of the emergence of bacterial strains which are resistant to these agents. (Hopkins, R. J. and J. G. Morris, supra). These limitations demonstrate that new more effective methods are needed to combat H pylori infections in vivo. In particular, the design of new vaccines that may prevent infection by this bacterium is highly desirable.

Reference is made to WO 97/37044, which discloses several Helicobacter pylori sequences, amongst which is Fay31 sequence information. This document also mentions the possibility of using this sequence information for producing a vaccine against Helicobacter pylori infection in a mammal. The SEQ ID NO's relating to Fay31 are as follows: 998, 1052, 326, 817, 188, 679, 187 and 678. These disclosures are specifically incorporated herein by reference, in order to provide further examples (eg, fragments of full-length sequences) that may be used in the present invention.

WO 96/40893 discloses HOP38 sequence information. This document also mentions the possibility of using this sequence information for producing a vaccine against Helicobacter pylori infection in a mammal. The SEQ ID NO's relating to HOP38 are as follows: 764, 1086 and 1537. These disclosures are specifically incorporated herein by reference, in order to provide further examples (eg, fragments of full-length sequences) that may be used in the present invention.

SUMMARY OF THE INVENTION

We have now surprisingly found that Fay31 together with HOP38 provides a synergistic combination that can be used to induce a prophylactic and/or therapeutic immune response in a mammal directed against Helicobacter pylori infection.

Accordingly, the present invention provides a pharmaceutical product comprising first and second isolated polypeptides for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal, wherein a) the first polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) the second polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ

ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

SEQ ID NO's: 1 to 10 represent Fay31 sequence information, while SEQ ID NO's: 11 to 18 represent HOP38 sequence information. It is to be understood that in place of a complete (ie, full-length) Fay31 polypeptide, one can use an immunogenic or antigenic fragment of the complete polypeptide. In place of a complete (ie, full-length) HOP38 polypeptide, one can use an immunogenic or antigenic fragment of the complete polypeptide. Thus, we use the terms "Fay31" and "HOP38" herein to include such fragments. The product can be provided as either a vaccine composition comprising the first polypeptide mixed together with the second polypeptide, or a vaccine kit in which the first polypeptide is provided separately from the second polypeptide.

The present invention also provides :-

a) Use of the first and second polypeptides in the manufacture of a pharmaceutical product, for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

b) Use of the first polypeptide in the manufacture of a pharmaceutical product, for administration simultaneously or sequentially with the second polypeptide to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

c) Use of the second polypeptide in the manufacture of a pharmaceutical product, for administration to a mammal simultaneously or sequentially with the first polypeptide for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

A further aspect of the invention relates to a pharmaceutical product comprising first and second isolated nucleotide sequences for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre- existing Helicobacter pylori infection in the mammal, wherein a) the first nucleotide sequence is identical to, or substantially similar to, a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 ofSEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and b) the second nucleotide sequence is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

The product can be provided as either a vaccine composition comprising the first nucleotide sequence in combination with the second nucleotide sequence, or a vaccine kit comprising an amount of the first nucleotide sequence provided separately from an amount of the second nucleotide sequence.

The present invention also provides :-

a) Use of the first and second nucleotide sequences in the manufacture of a pharmaceutical product, for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre- existing Helicobacter pylori infection in the mammal.

b) Use of the first nucleotide sequence in the manufacture of a pharmaceutical product for administration to a mammal simultaneously or sequentially with the second nucleotide sequence or the polypeptide encoded thereby, for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing

Helicobacter pylori infection in the mammal.

c) Use of the second nucleotide sequence in the manufacture of a pharmaceutical product for administration to a mammal simultaneously or sequentially with the first nucleotide sequence or the polypeptide encoded thereby, for preventing

Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal. Another aspect of the invention relates to a fusion polypeptide for admimsfration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a preexisting Helicobacter pylori infection in the mammal, wherein the polypeptide comprises a) a first amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to

273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) a second amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

The invention also provides use of the fusion polypeptide in the manufacture of a vaccine, for administration to a mammal to prevent Helicobacter pylori infection in the mammal, or to treat a pre-existing Helicobacter pylori infection in the mammal.

An additional aspect of the invention relates to a nucleic acid encoding the fusion polypeptide, wherein the nucleic acid has a) a first nucleotide sequence that is identical to, or substantially similar to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11 ; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and b) a second nucleotide sequence that is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

The invention also provides use of this nucleic acid in the manufacture of a vaccine, for administration to a mammal to prevent Helicobacter pylori infection in the mammal, or to treat a pre-existing Helicobacter pylori infection in the mammal. DETAILED DESCRIPTION OF THE INVENTION

As explained above, in one embodiment, the present invention provides a pharmaceutical product comprising first and second isolated polypeptides for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal, wherein a) the first polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) the second polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

The teπn "isolated" when referring to the "first and second isolated polypeptides" means that apart from each other, the polypeptides have been substantially (most preferably, completely) separated from other Helicobacter pylori polypeptides with which they naturally occur. It is possible, therefore, for example that the pharmaceutical product contains one or two other Helicobacter pylori polypeptides (or fragments thereof) in addition to the first and second polypeptides.

Each of the isolated Fay31 and HOP38 polypeptide antigens used in the invention may be prepared from Helicobacter cells and/or produced by recombinant techniques.

By "substantially similar" when referring to said amino acid sequence of the first polypeptide we mean one or more of the following: (i) the amino acid sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (ii) the amino acid sequence is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues of an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iii) the amino acid sequence differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iv) the amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17. Included in the definition is an immunogenic and/or antigenic amino acid sequence. This may be a full-length HOP38 sequence or a fragment thereof.

A "complement" of a nucleotide sequence refers to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.

In one embodiment, the first polypeptide comprises an amino acid sequence identical to, or substantially identical to, an amino acid sequence selected from SEQ ID NO: 12, 14, 16, and 18.

By "substantially similar" when referring to the amino acid sequence of the second polypeptide we mean one or more of the following: (i) the amino acid sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (ii) the amino acid sequence is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues of an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iii) the amino acid sequence differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iv) the amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9. Included in the definition is an immunogenic and/or antigenic amino acid sequence. This may be a full-length Fay31 sequence or a fragment thereof. In one embodiment, the second polypeptide comprises an amino acid sequence identical to, or substantially identical to, an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8 and 10.

The term "immunogenic" indicates a capability of eliciting a humoral and/or cellular immune response in vitro or in vivo (eg, in a patient) alone or in combination with an adjuvant and/or delivery system.

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

Alternatively, a common assay for T cell proliferation entails measuring tritiated thymidine incorporation. The proliferation of T cells can be measured in vitr'o by determining the amount of ^H-labelled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.

The term "antigenic" refers to the presence ofan antigenic determinant that is recognised by an antibody, B-cell or T-cell receptor (eg, one from a mammal) that also recognises an antigenic determinant or epitope of a respective naturally occurring Helicobacter pylori polypeptide (naturally occurring HOP38 or Fay31). For example, the antigenic determinant may bind to such an antibody or receptor. Standard ELISA or other . immunoassays can be used to detect this. Specific mention is made to the section in WO 96/38475 describing how to identify and analyse epitopes of the Fay31 protein. This can also be applied to HOP38.

The terms "homologous" and "homology" refer to the sequence similarity or sequence identity between two polypeptides or between the nucleotide sequences of two nucleic acid molecules. Generally, a comparison is made when two sequences are aligned to give maximum homology. A suitable algorithm for determining homology is BLAST (NBLAST (for nucleotide sequences) and XBLAST (for amino acid sequences)), utilising default parameters (see http://www.ncbi.nlm.nih.gov).

Preferably, when there is the conservative amino acid replacement of an amino acid with another as shown in the following table, the amino acids are regarded as being homologous to each other.

TABLE 1

CONSERVATIVE AMINO ACID REPLACEMENTS

For a definition of high and low stringency, see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1-6.3.6 and 6.4.1-6.4.10. This definition is incorporated herein by reference as an example of suitable hybridisation conditions for use in the present invention. Stringency of hybridisation is determined by: (a) the temperature at which hybridisation and/or washing is performed; and (b) the ionic strength and polarity of the hybridisation and washing solutions. Hybridisation requires that the two sequences are at least partly complementary; depending on the stringency of hybridisation, however, mismatches may be tolerated. Typically, hybridisation of two sequences at high stringency (such as, for example, in a solution of 0.5X SSC, at 65° C for both the hybridising and washing steps) requires that the sequences be essentially completely homologous. Conditions of intermediate stringency (such as, for example, 2X SSC at 65 ° C for both the hybridising and washing steps) and low stringency (such as, for example 2X SSC at 55° C for both the hybridising and washing steps), require correspondingly less overall complementarity between the hybridising sequences. (IX SSC is 0.15 M NaCI, 0.015 M Na citrate). In a preferred embodiment, one or both of the polypeptides is a fusion protein having a first portion representing Fay31 or HOP38 or a fragment thereof, and a second portion representing a polypeptide (or fragment thereof) other than HOP38 and Fay31. For example, the second portion comprises an amino acid sequence that is not derived from Helicobacter pylori.

The Fay31 and HOP38 antigens can be administered to the patient simultaneously. To this end, the pharmaceutical product of the invention can be provided as a single vaccine composition in which the antigens are mixed together, optionally together with at least one adjuvant and/or antigen delivery system. In another embodiment, the administration of the vaccine composition and an adjuvant can be temporally spaced, such that the end result is the provision of the synergistic combination of antigens in vivo in the presence of the adjuvant. To this end, the pharmaceutical product could be a vaccine kit comprising an amount (eg, a vial) of the antigens mixed together and a separate amount of adjuvant.

The Fay31 and HOP38 antigens can also be administered to the patient sequentially (ie, temporally spaced administration of the antigens in which one antigen is administered before the other), the end result being that both antigens are provided together in vivo in order to provide the synergistic combination that induces a prophylactic and/or therapeutic immune response against Helicobacter pylori infection. For example, to this end a pharmaceutical product can be provided in the form of a kit comprising an amount (eg, a vial) of Fay 31 and a separate amount of HOP38. One or both antigens may be incorporated into a delivery system. One or both antigens can be administered at the same time as a suitable adjuvant. Such an adjuvant can alternatively be administered separately from the antigens. For example, to this end, the pharmaceutical product could be a vaccine kit comprising an amount (eg, a vial) of Fay31, a separate amount of HOP38 and a separate amount of adjuvant.

Preferably, the pharmaceutical product of the invention includes a mucosal adjuvant. Suitable adjuvants are Cholera toxin (CT) and pharmaceutically acceptable derivatives thereof, E. coli heat labile toxin, cytokines and chemokines. Also suitable are nano- and microparticles, ISCOMs, bile, polycations, detergents, aluminium salts, water-in-oil emulsions, liposomes, proteosomes, peptides, carbohydrate polymers and live attenuated bacteria or viruses.

A "mucosal adjuvant" is any agent which results in a specific increase in antigen immunogenicity when the antigen is delivered to a mucosal surface, in association with the agent (see McGhee et al, "The mucosal immune system: from fundamental concepts to vaccine development", Vaccine 10:75 (1992), pp82-85; McGhee et al, Infect. Agents Dis. USA 2:55 (1993), p61, Table 1; and Cox and Coulter, "Advances in adjuvant technology and application", Animal Parasite Control Utilizing Biotechnology 49, 52 (CRC Press, 1992), pp52-53). "Adjuvant" encompasses many types of materials that possess activity in one or more of three broad categories: antigen presentation, antigen targeting and immune modulation (see page 53 of the Cox and Coulter reference).

In an alternative embodiment of the invention, the pharmaceutical product comprises first and second isolated nucleotide sequences for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a preexisting Helicobacter pylori infection in the mammal, wherein a) the first nucleotide sequence is identical to, or substantially similar to, a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 1 1 ; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and b) the second nucleotide sequence is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO:

5; SEQ ID NO: 7; and SEQ ID NO: 9.

Each nucleotide sequence can be part of a longer nucleotide sequence provided by a nucleic acid molecule. The first nucleotide sequence encodes a HOP38 amino acid sequence, while the second nucleotide sequence encodes a Fay31 amino acid sequence.

The term "isolated" when referring to "first and second isolated nucleotide sequences" means: each sequence is not immediately contiguous with coding sequences with which it is immediately contiguous (ie, one at the 5' end and one at the 3' end) when in the naturally-occurring genome of Helicobacter pylori from which the nucleotide sequence is derived; and/or that apart from each other, the nucleotide sequences have been substantially (most preferably, completely) separated from Helicobacter pylori nucleotide sequences encoding amino acid sequences other than HOP38 and Fay31 sequences. Such separation can be achieved using recombinant techniques or by separation from whole Helicobacter pylori cells. It is possible, therefore, for example that the pharmaceutical product contains one or two other Helicobacter pylori polypeptide-encoding nucleotide sequences (or fragments thereof) in addition to the first and second nucleotide sequences.

By "substantially similar" when referring to the first nucleotide sequence we mean one or more of the following: (i) the sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; (ii) the sequence encodes an amino acid sequence that is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iii) the sequence encodes an amino acid sequence that differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iv) the sequence hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17. A nucleotide sequence encoding an immunogenic and/or antigenic HOP38 amino acid sequence is therefore included in the definition. This amino acid sequence may be a full-length HOP38 sequence or a fragment thereof. The definition also includes a first nucleotide sequence that is degenerate as a result of the genetic code to the first nucleotide sequence as defined in the preceding part of this paragraph; or a first nucleotide sequence that is degenerate to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17.

In one embodiment, the first nucleotide sequence is provided by a sequence that is identical to, or substantially identical to, SEQ ID NO: 11, 13, 15 or 17.

By "substantially similar" when referring to the second nucleotide sequence we mean one or more of the following: (i) the sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9; (ii) the sequence encodes an amino acid sequence that is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iii) the sequence encodes an amino acid sequence that differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iv) the sequence hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9. A nucleotide sequence encoding an immunogenic and/or antigenic Fay31 amino acid sequence is therefore included in the definition. This amino acid sequence may be a full-length Fay31 sequence or a fragment thereof. The definition also includes a second nucleotide sequence that is degenerate as a result of the genetic code to the second nucleotide sequence as defined in the preceding part of this paragraph; or a second nucleotide sequence that is degenerate to a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

In one embodiment, the second nucleotide sequence is provided by a sequence that is identical to, or substantially identical to, SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; or SEQ ID NO: 9. The nucleotide sequences can be provided on the same or different recombinant expression vectors (eg, plasmids), together with appropriate transcription regulatory elements (eg, eukaryotic promoters). Suitable vectors and transcription regulatory elements will be apparent to the skilled addressee. Thus, for simultaneous adminisfration, the pharmaceutical product can be a single vaccine composition comprising both the first and second nucleotide 'sequences provided on the same plasmid. Alternatively; or additionally thereto, the composition can comprise the first and second nucleotide sequences provided on respective, different plasmids. For sequential administration, the pharmaceutical product could be a vaccine kit comprising an amount of the first nucleotide sequence provided on a first plasmid, and a separate amount of the second nucleotide sequence provided on a second, different plasmid.

Preferably, the plasmid is contained in a host which acts as a delivery system. This may be a live host cell, such as a live, attenuated bacterial cell, or a virus.

An alternative approach for creating an immune response against the Fay31 and HOP38 antigens is to use the approach known as "nucleic acid vaccination" or "naked DNA" vaccination. It is known in the art that injection into muscle of plasmid DNA encoding an antigen of interest can result in sustained expression of the antigen and generation of an immune response (see e.g. Rabinovich et al. (1994) Science 265, 1401-1404). Several routes of administration are possible, such as parental, mucosal or via a "gene-gun" that delivers tiny amounts of DNA-coated gold beads (Fynan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11478-11482). Mucosal adminisfration is preferred for the present invention using one or more of the plasmids discussed above as the "naked DNA".

Note that irrespective of the actual protocol (simultaneous v. sequential), the result of administration of a pharmaceutical product according to the invention is the provision in vivo of the synergistic combination of Fay31 and HOP38 that induces a prophylactic and/or therapeutic immune response against Helicobacter pylori infection. The preferred adminisfration route is mucosal administration. Examples of suitable mammalian mucosa include the buccal, nasal, tonsillar, gastric, intestinal (small and/or large intestine), rectal and vaginal mucosa. Appropriate corresponding adminisfration routes include oral, nasal, rectal and vaginal adminisfration, with the oral, nasal and rectal routes being the most preferred.

The pharmaceutical products (kits and vaccines) can be used to treat and/or prevent Helicobacter pylori infection in a mammalian-patient by administering an immunologically effective amount of Fay 31 and HOP38 (and/or the nucleotide sequences encoding these) to the patient. The term "immunologically effective amount" means an amount which elicits a response by the patient to the Fay31 and HOP38 antigens which eradicates, suppresses, prevents and/or reduces the risk of the infection in the patient. The present invention can be used for the treatment and/or prophylaxis of Helicobacter (especially Helicobacter pylori) infection in a mammalian host. Thus the vaccine or other pharmaceutical product of the invention can be administered to a patient, eg orally, in order to effect the treatment and/or prophylaxis. By "freatment", we mean the eradication or suppression of an existing Helicobacter infection in the host (in this respect, general reference is made to WO 96/40893). By "prophylaxis", we mean preventing or reducing the risk of the mammal becoming infected by Helicobacter after the vaccine has been administered.

Typically, an appropriate dose of each polypeptide antigen per administration would be approximately lOμg to lOmg, preferably approximately 50μg to 5mg, for oral administration. Suitable dosage forms include a frozen dispersion, freeze-dried particles or a liquid dispersion.

In the method of the present invention, a lipidated form of Fay31 and/or HOP38 can be used (ie, one with one, two or three lipid chains). Most preferably, a fully lipidated form of Fay31 and/or HOP38 is used (ie, one with at least 3 lipid chains, eg three C16 chains). Each lipid chain can be a Cl 2 to C20 lipid chain. C16 and Cl 8 lipid chains are preferred, and most preferably, the antigen has at least one C16 chain and at least one C18 chain. As an alternative to the administration of separate Fay31 and HOP38 polypeptide antigens to the patient, it is possible to administer a fusion polypeptide to the patient for preventing Helicobacter pylori infection, or for treating a pre-existing Helicobacter pylori infection in the patient. The fusion polypeptide comprises a) a first amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) a second amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

In this embodiment, by "substantially similar" when referring to the first amino acid sequence we mean one or more of the following: (i) the amino acid sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (ii) the amino acid sequence is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iii) the amino acid sequence differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iv) the amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17.

In this embodiment, by "substantially similar" when referring to the second amino acid sequence we mean one or more of the following: (i) the amino acid sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (ii) the amino acid sequence is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iii) the amino acid sequence differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iv) the amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

In one example, the first amino acid sequence is provided by a sequence that is identical to, or substantially identical to, SEQ ID NO: 12, 14, 16 or 18.

In one example, the second amino acid sequence id provided by a sequence that is identical to, or substantially identical to, SEQ ID NO: 2, 4, 6, 8 or 10.

The fusion polypeptide may be lipidated, and is preferably administered simultaneously or sequentially with an adjuvant, most preferably a mucosal adjuvant. A delivery system can be used with or without an adjuvant. Details of administration routes, suitable adjuvants and delivery systems are given above.

The invention also relates to a nucleic acid encoding the fusion polypeptide, wherein the nucleic acid has a) a first nucleotide sequence that is identical to, or substantially similar to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions

70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and b) a second nucleotide sequence that is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9. In this embodiment, by "substantially similar" when referring to the first nucleotide sequence we mean one or more of the following: (i) the sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; (ii) the sequence encodes an amino acid sequence that is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 1 4; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iii) the sequence encodes an amino acid sequence that differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; (iv) the sequence hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 ofSEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17. A nucleotide sequence encoding an immunogenic and/or antigenic HOP38 amino acid sequence is therefore included in the definition. This amino acid sequence may be a full-length HOP38 sequence or a fragment thereof. The definition also includes a first nucleotide sequence that is degenerate as a result of the genetic code to the first nucleotide sequence as defined in the preceding part of this paragraph; or a first nucleotide sequence that is degenerate to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17.

By "substantially similar" when referring to the second nucleotide sequence we mean one or more of the following: (i) the sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9; (ii) the sequence encodes an amino acid sequence that is at least 5, 10, 20, 50, 100, 150 or 200 contiguous amino acid residues ofan amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iii) the sequence encodes an amino acid sequence that differs by 1, 2, 3, 5, or 10 residues from an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10; (iv) the sequence hybridises under stringent (high, intermediate or low) conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9. A nucleotide sequence encoding an immunogenic and/or antigenic Fay31 amino acid sequence is therefore included in the definition. This amino acid sequence may be a full-length Fay31 sequence or a fragment thereof. The definition also includes a second nucleotide sequence that is degenerate as a result of the genetic code to the second nucleotide sequence as defined in the preceding part of this paragraph; or a s'econd nucleotide sequence that is degenerate to a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

In one example, the first nucleotide sequence is provided by SEQ ID NO: 11, 13, 15 or 17.

In one example, the second nucleotide sequence is provided by SEQ ID NO: 1, 3, 5, 7 or 9.

The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); the series, Methods in Enzymoloqy (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.) and PCR-A Practical Approach (McPherson, Quirke, and Taylor, eds., 1991). EXPERIMENTAL

Mouse model of Helicobacter pylori infection

A murine model of H. pylori infection was used to assess the efficacy of immunisation with H. pylori antigens, by employing the H. pylori SSI strain for infection of mice. This mouse-adapted H. pylori SSI strain is cagAtvacAL, shows colonisation levels in C57BL/6 mice equivalent to those observed in humans, forms adhesion pedestals, colonises for at least 8 months, and elicits a chronic-active gastritis and mucosal atrophy (Lee et al., Gasfroenterology, 112:1386-1397, 1997). Dose-response studies have shown 100% infection rates with a single inoculation of 10⁶ organisms.

Assessment of gastric H. pylori infection

The presence of H. pylori organisms in gastric tissue was determined by a quantitative urease assay. In this method, a longitudinal segment of antrum, representing approximately VA, of the total antral region was placed in 1 ml of urea broth. After 4 hr, the extent of colour change resulting from urea hydrolysis and increased pΗ was quantitated by spectrophotometric measurement of optical density at 550 nm (Fox et al., Immunol. 88:400-406, 1996; Kleanthous et al, Infect. Immun. 66:2879-2886, 1998).

Defined combinations of Fav31 & ΗOP38 antigens afford synergistic therapy against infection

Groups of mice were immunised intranasally with either purified recombinant Fay31 (182μg), purified recombinant HOP38 (182μg), with both Fay31 and HOP38 (91 μg of each), with 100 μg H pylori whole cell lysate, or treated with buffer alone. All immunisations were performed in the presence of 10 μg CT adjuvant. Full length Fay31 was used, which corresponds to SEQ ID NO: 2. For HOP38, a polypeptide comprising the sequence from position 24 to position 262 of SEQ ID NO: 4 was used. Therapeutic freatment of pre-existing H. pylori infection was assessed by quantitative urease 2 weeks after experimental challenge with 10⁶ CFU of H pylori SSI by gavage in a volume of 200μl. The mice were immunised at weekly intervals for four weeks. For each group, half of the total volume of the formulation was administered into each nostril. The formulations were diluted where appropriate so that each group received the same total volume. Two weeks post last immunisation, the mice were sacrificed and stomach tissue harvested for quantitative urease assay. As table 3 shows, significant therapeutic efficacy was observed by vaccination with a combination of 91 μg of each of ΗOP38 and Fay31.

Table 3

Therapeutic protection by vaccination with a combination of purified recombinant Fay31

& HOP38

The extent of H. pylori infection was assessed by a quantitative urease assay, and is expressed as mean optical density ₅₅₀ ± SEM of duplicate antral samples from n=10 mice/group, by Wilcoxon Rank Sum Test

Claims

CLAIMS:

1. A pharmaceutical product comprising first and second isolated polypeptides for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal, wherein a) the first polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) the second polypeptide comprises an amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

2. The product of claim 1, wherein said amino acid sequence of at least one of the polypeptides has 60% or more homology with the respective selected amino acid sequence.

3. The product of claim 1, wherein the first polypeptide is encoded by a nucleotide sequence that hybridises under stringent conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and the second polypeptide is encoded by a nucleotide sequence that hybridises under stringent conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and

SEQ ID NO: 9.

4. The product of any preceding claim, wherein at least one of the polypeptides is lipidated.

5. The product of any preceding claim, wherein the product is either a vaccine composition comprising the first polypeptide mixed together with the second polypeptide, or a vaccine kit in which the first polypeptide is provided separately from the second polypeptide.

6. The product of any preceding claim , wherein the product includes a mucosal adjuvant and/or delivery system.

7. A fusion polypeptide for administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal, wherein the polypeptide comprises a) a first amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from positions 21 to 270 of SEQ ID NO: 12; positions 24 to 273 of SEQ ID NO: 14; positions 24 to 273 of SEQ ID NO: 16; and positions 1 to 250 of SEQ ID NO: 18; and b) a second amino acid sequence that is identical to, or substantially similar to, an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; and SEQ ID NO: 10.

8. The polypeptide of claim 7, wherein at least one of the amino acid sequences has 60% or more homology with the respective selected amino acid sequence.

9. The polypeptide of claim 7, wherein the first amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11 ; positions 70 to 786 of SEQ ID NO:. 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and the second amino acid sequence is encoded by a nucleotide sequence that hybridises under stringent conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9. lO.The polypeptide of any one of claims 7 to 9, wherein the polypeptide is lipidated.

11. A vaccine comprising the polypeptide of any one of claims 7 to 10 together with a mucosal adjuvant and/or delivery system.

12. A pharmaceutical product comprising first and second isolated nucleotide sequences for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal, wherein a) the first nucleotide sequence is identical to, or substantially similar to, a nucleotide _. sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and b) the second nucleotide sequence is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1 ; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

13. The product of claim 12, wherein at least one of the nucleotide sequences has 60% or more homology with the respective selected nucleotide sequence.

14.The product of claim 12, wherein the first nucleotide sequence hybridises under stringent conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions 70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 of SEQ ID NO: 17; and the second nucleotide sequence hybridises under stringent conditions to the complement of a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

15.The product of any one of claims 12 to 14, wherein each nucleotide sequence is provided in a recombinant expression vector.

16. The product of any one of claims 12 to 15, wherein each nucleic acid is provided in a host cell.

17. The product of any one of claims 12 to 16, wherein the product is either a vaccine composition comprising the first nucleotide sequence in combination with the second nucleotide sequence, or a vaccine kit comprising an amount of the first nucleotide sequence provided separately from an amount of the second nucleotide sequence.

18.A nucleic acid encoding the fusion polypeptide of any one of claims 7 to 10, wherein the nucleic acid comprises a) a first nucleotide sequence that is identical to, or substantially similar to a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 11; positions

70 to 786 of SEQ ID NO: 13; positions 70 to 786 of SEQ ID NO: 15; and positions 82 to 798 ofSEQ ID NO: 17; and b) a second nucleotide sequence that is identical to, or substantially similar to, a nucleotide sequence selected from SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9.

19.The nucleic acid of claim 18, wherein at least one of the nucleotide sequences has 60% or more homology with the respective selected nucleotide sequence.

20. The nucleic acid of claim 18, wherein the first nucleotide sequence hybridises under stringent conditions to the complement of a nucleotide sequence selected from positions 61 to 777 of SEQ ID NO: 1; positions 70 to 786 of SEQ ID NO: 3; positions 70 to 786 of SEQ ID NO: 5; and positions 82 to 798 of SEQ ID NO: 7; and the second nucleotide sequence hybridises under stringent conditions to the complement of a nucleotide sequence selected from positions 873 to 1572 of SEQ ID NO: 9; positions 873 to 1572 of SEQ ID NO: 11; positions 82 to 783 of SEQ ID NO: 13; positions 106 to 804 of SEQ ID NO: 15; and positions 157 to 294 of SEQ ID NO: 17.

21. Use of the first and second polypeptides as recited in of any one of claims 1 to 4 in the manufacture of a pharmaceutical product, for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

22. Use of the fusion polypeptide of any one of claims 7 to 10 in the manufacture of a vaccine, for adminisfration to a mammal to prevent Helicobacter pylori infection in the mammal, or to treat a pre-existing Helicobacter pylori infection in the mammal.

23.Use of the first and second nucleotide sequences as recited in of any one of claims 12 to 16 in the manufacture of a pharmaceutical product, for simultaneous or sequential administration to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

24.Use of the nucleic acid of any one of claims 18 to 20 in the manufacture of a vaccine, for adminisfration to a mammal to prevent Helicobacter pylori infection in the mammal, or to treat a pre-existing Helicobacter pylori infection in the mammal.

25.Use of a first polypeptide in the manufacture of a pharmaceutical product, for adminisfration simultaneously or sequentially with a second^' polypeptide to a mammal for preventing Helicobacter pylori infection in the mammal, or for treating a pre- existing Helicobacter pylori infection in the mammal, wherein the polypeptides are as recited in of any one of claims 1 to 4.

26.Use of the second polypeptide as recited in of any one of claims 1 to 4 in the manufacture of a pharmaceutical product, for adminisfration to a mammal simultaneously or sequentially with the first polypeptide as recited in of any one of claims 1 to 4 for preventing Helicobacter pylori infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

27.Use of a first nucleotide sequence in the manufacture of a pharmaceutical product for administration to a mammal simultaneously or sequentially with a second nucleotide sequence or the polypeptide encoded thereby, for preventing Helicobacter pylori infection in the mammal, of for treating a pre-existing Helicobacter pylori infection in the mammal, wherein the nucleotide sequences are as recited in of any one of claims 12 to 15.

28. Use of the second nucleotide sequence as recited in any one of claims 12 to 15 in the manufacture of a pharmaceutical product for administration to a mammal simultaneously or sequentially with the first nucleotide sequence as recited in any one of claims 12 to 15 or the polypeptide encoded thereby, for preventing Helicobacter pylori- infection in the mammal, or for treating a pre-existing Helicobacter pylori infection in the mammal.

29.The use according to any one of claims 21 to 28, wherein said administration is mucosal administration.

30.The use according to claim 29, wherein said administration is oral adminisfration.

31.The use according to claim 29, wherein said administration is infranasal administration.