WO1997002283A1 - Helicobacter antigens - Google Patents

Abstract

The present invention provides an antigenic polypeptide protective against Helicobacter infection together with vaccines containing the polypeptide. The polypeptide has an amino acid sequence of an approximately 18 kDa outer membrane vesicle (OMV) antigen of a Helicobacter spp. such as H. pylori. Fragments of the polypeptide containing the protective epitope of the polypeptide are contemplated as are functionally equivalent variants of the polypeptide.

Description

HELICOBACTER ANTIGENS

This invention relates to antigens protective against Helicobacter infection. Such antigens are suitable for use in vaccines and in methods of therapy and/or prophylaxis of Helicobacter infections, particularly infections by H. pylori.

BACKGROUND

Helicobacter pylori is an etiological agent of chronic gastritis, now known to be one of the most common chronic infections found in man (Blaser, 1992). This bacteria exhibits marked trophism for human gastric epithelium and, once established there, persists indefinitely in the majority of people. While most infected individuals are asymptomatic, a degree of gastric inflammation is always associated with H. pylori infection. A sustained host systemic and mucosal immune response to H. pylori contributes to this inflammation but fails to clear the infection.

Children in developing countries are known to be infected with H. pylori at an early age (Graham et al., 1991; Holcombe et al., 1993; Mitchell et al., 1992; Thomas et al, 1993). It is also known that long term, H. ^y/oπ-induced inflammation is a significant risk factor in the pathogenesis of gastric carcinoma (Forman et al., 1991; Parsonnet et al, 1991; Recavarren-Acre et al., 1991). Immunising these children against H. pylori would represent the most cost-effective intervention available (Bloom, 1989), as well as preventing this risk.

Lee and his colleagues have recently described a murine model of human infection with H. pylori (Lee, et al., 1990). Helicobacter felis, first isolated from the stomach of a cat (Lee, et al., 1988), readily colonises the gastric mucosa of mice. Infection persists for the life-time of the animal, with gastric histologic changes mirroring long-term human infection (Lee, et al, 1993). This model has allowed the study of different immunisation regimes against Helicobacter spp. , measured by their ability to prevent host colonization after challenge. Studies to date have found that a high percentage of these animals can be protected from infection with H. felis by use of an oral vaccine (Chen et al., 1992; Chen et al., 1993; Czinn et al, 1993) consisting of a whole bacterial sonicate plus cholera toxin (Czinn and Nedrud, 1991).

However, given the aim of development a commercial vaccine, it is essential for specific protective antigen(s) to be identified. Currently, the search for a successful H. pylori vaccine candidate is centered around the urease enzyme and its associated heat-shock protein chaperonins, hsp54 and hsp 13. A preliminary study revealed that orogastric administration of urease enzyme from H. pylori generated protection against H. felis challenge (Davin et al., 1993; Michetti et al, 1994). The same enzyme, expressed as an inactive recombinant protein in Escherichia coli, also contains protective determinants (Pappo et al, 1995; Lee et al, 1995). The urease B subunit (Michetti et al, 1994; Ferrero et al., 1994) and heat-shock proteins hspA and hspB (Ferrero et al, 1995) expressed as fusion proteins in Escherichia coli, also generate high levels of protection.

The applicants have however identified a further Helicobacter antigen which generates high levels of protection. This antigen is unrelated to the urease enzyme and heat shock proteins discussed above.

It is the object of this invention to provide a protective composition using this new Helicobacter antigen which will at least offer the public a useful choice.

SUMMARY OF TΗE INVENTION

Accordingly, in one aspect the present invention provides an antigenic polypeptide including an epitope capable of generating a protective immunological response against Helicobacter infection in a susceptible host; said polypeptide having an amino acid sequence of an approximately 18 kDa outer membrane vesicle (OMV) antigen of a Helicobacter organism; or a peptide fragment or variant of said polypeptide including said epitope and having protective immunological activity substantially equivalent to said polypeptide.

Most preferably, said polypeptide has the amino acid sequence of the 18 kDa OMV antigen of H. pylori.

Preferably, said polypeptide has an N4erminal amino acid sequence:

A_rA₂-A₃-N-K-F-X-M-K-A₄-L-Y-A₅-Q-A

wherein A, is K or R; A₂ is D or N; A₃ is F or D; A₄ is A or I; and A₅ is E or K. This sequence is SEQ ID NO 1. Most preferably, the polypeptide also includes one or both ofthe following internal amino acid sequences:

(i) A-E-T-I-Q-A-T-A-D-A; (SEQ ID NO 2) and

wherein A,- is N or V; A₇ is L or R; A₈ is M or V; and Ao is T or K. (SEQ ID NO 3).

In an alternative aspect, the invention provides an anti-idiotypic antibody which mimics the protective epitope of said 18 kDa antigen.

In still a further aspect, the invention provides antibodies specific for the protective epitope of said 18 kDa antigen.

In still a further aspect, the invention provides a vaccine against Helicobacter infection comprising a polypeptide, peptide fragment, variant or anti-idiotypic antibody as defined above, together with an immunologically appropriate adjuvant or carrier.

In yet a further aspect the invention provides a method of protecting a susceptible host against Helicobacter infection comprising the step of administering to said host an amount of a polypeptide, peptide fragment, variant, anti-idiotypic antibody or vaccine as defined above which is protective against such infection.

Conveniently, the host is a human and said polypeptide, peptide fragment, variant, anti- idiotypic antibody or vaccine is administered to protect against H. pylori infection.

DESCRIPTION OF TΗE DRAWINGS

While the invention is broadly as defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto but that it also includes embodiments of which the following description provides examples. In particular, a better understanding ofthe present invention will be gained by reference to the accompanying drawings which show the following:

Fig.l. Immunologic cross-reactivity between 18 kDa outer membrane component expressed by H. felis and different strains of H. pylori. H. felis (Lane 1) or H. pylori (Lanes 2-6) outer membrane vesicles were electrophoresed in 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and immunoblotted with sera from mice immunised with H. felis outer membrane vesicles.

Fig.2. Electron micrograph of negatively stained H. felis outer membrane vesicles, shed from the surface of bacteria during growth in broth culture.

Fig.3. Antibody responses in mice immunised with H. felis antigens, combined either with cholera toxin (in C57B1, A/J and BALB/c strains) or a non-ionic block copolymer adjuvant (in C57B1 and A/J strains). Antimicrobial eradication of established infection in C57B1 and A/J mice resulted in naturally immunised animals. Serum and intestinal secretions were taken after the final immunising dose but before challenge with H. felis.

Fig.4. Immunoblots of individual mouse sera against H. felis outer membrane vesicle components. A/J mice (a) post-H. felis infection; immunised with (b) H. felis sonicate + L121, (c) H. felis sonicate + CT. BALB/c mice (d) immunised with H. felis sonicate + CT, (e) immunised with H. felis outer membrane vesicles, (f) Naive BALB/c controls. 18 kDa antigen is indicated (left).

Fig.5. Three strains of H. pylori (60190, Tx-30a and 84-183) immunoblotted with rabbit antibody to the 66 kDa urease B enzyme subunit. Lanes 1-3: whole cells; lanes 4-6: outer membrane vesicles; lanes 7-9: concentrated broth supernatant (after removal of vesicles). Lane 10: H. pylori water-soluble extract (positive control). 66 kDa antigen is indicated (left).

Fig.6. Immunogold TEM labelling of H. felis in vitro by murine antibodies to the 18 kDa membrane component.

DESCRIPTION OF TΗE INVENTION

As defined above, in its primary aspect, the present invention is directed to the provision of antigens which are host-protective against Helicobacter infections such as H. pylori. Hosts which are susceptible to Helicobacter infection are mammals including humans.

From their investigations, the applicants have identified that oral immunisation with membrane vesicles, shed from the surface oi Helicobacter spp. during growth in broth culture, confer protection against Helicobacter infection. These vesicles lack both the urease B subunit and hsp54 antigen, estabhshing that H. pylori membrane vesicles contain a novel vaccine candidate. More specifically, the applicants have identified an outer membrane polypeptide, which has a molecular weight of approximately 18 kDa, as being involved in protection against Helicobacter infection.

The N-terminal amino acid sequence of the polypeptide has been determined to be as follows:

A_rA₂-A₃-N-K-F-X-M-K-A₄-L-Y-A₅-Q-A

wherein A, is K or R; A₂ is D or N; A₃ is F or D; A₄ is A or I; and A₅ is E or K.

It has further been determined that the polypeptide includes the following internal amino acid sequences:

(i) A-E-T-I-Q-A-T-A-D-A; and

(ii) Y-V-K-A₆-A₇-V-I-A₈-K-Ao

wherein Ag is N or V; A₇ is L or R; A_g is M or V; and Ap is T or K.

In the above sequences, individual amino acids are represented by the single letter code as follows:

Three-letter One-letter

Amino Acid abbreviation symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Asparagine or aspartic acid Asx B Cysteine Cys C

Glutamine Gin Q

Glutamic Acid Glu E

Glutamine or glutamic acid Glx Z

Glycine Gly G Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W Tyrosine Tyr Y

Valine Val V

Unidentified X

The apphcants believe that there exists a family of such 18 kDa polypeptides each being a homologue (or equivalent) ofthe others. In particular, the applicants believe that the 18 kDa OMV antigens of H. felis and H. pylori are homologues or equivalents of each other, and are protective against H. felis and H. pylori infections. Those persons skilled in the art will understand how to identify, isolate and characterise 18 kDa homologues of Helicobacter in general based upon the information provided herein.

The present invention also includes within its scope antigens derived from the native Helicobacter polypeptides identified above where such derivatives have host-protective activity. These derivatives will normally be peptide fragments ofthe native polypeptide which include the protective epitope, but can also be functionally equivalent variants of the native polypeptide modified by well known techniques such as site-specific mutagenesis (see Adelman et al, 1983). For example, it is possible by such techniques to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids known normally to be equivalent are:

(a) A S T P G;

(b) N D E Q;

(c) H R K;

(d) M L I V; and

(e) F Y W.

It is also contemplated that an anti-idiotypic antibody which mimics the protective epitope of the 18 kDa OMV antigen can be employed as a host-protective agent. If required, such antibodies can be prepared using known methodology.

The protective antigens of the invention can be produced by isolation from the shed Helicobacter outer membrane vesicles, using conventional purification techniques. However, it is recognised that for production of the antigen in commercial quantities, production by synthetic routes is desirable. Such routes include the stepwise solid phase approach described by Merryfield (1963) and production using recombinant DNA techniques. The latter route is preferred.

Stated generally, the production ofthe protective antigen ofthe invention by recombinant DNA techniques involves the transformation of a suitable host organism or cell with an expression vector including a DNA sequence coding for the antigen, followed by the culturing of the transformed host and subsequent recovering of the expressed antigen. Such techniques are described generally in Sambrook et al. , "Molecular Cloning", Second Edition, Cold Spring Harbour Press (1987).

An imtial step in the method of recombinantly producing the antigen involves the ligation of a DNA sequence encoding the antigen into a suitable expression vector containing a promoter and ribosome binding site operable in the host cell in which the coding sequence will be transformed. The most common examples of such expression vectors are plasmids which are double stranded DNA loops that replicate autonomously in the host cell. However, it will be understood that suitable vectors other than plasmids can be used.

Preferably, the host cell in which the DNA sequence encoding the polypeptide is cloned and expressed is a prokaryote such as E. coli. For example, E. coli DH5 (Raleigh E A et al, (1988), E. coli K12 strain 294 (ATCC 31446), E. coli B, E. coli X1776 (ATCC 31537), E. coli strain ST9 or E. coli JM 101 can be employed. Other prokaryotes can also be used, for example bacilli such as Baccilus subtilis and enterobacteriaceae such as Salmonella typhimurium, Serratia marcesans or the attenuated strain Bacille Camette- Guerin (BCG) oi Mycobacterium bovis.

In general, where the host cell is a prokaryote, expression or cloning vectors containing replication and control sequences which are derived from species compatible with the host cell are used. The vector may also carry marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli has commonly been transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.

For use in expression, the plasmid including the DNA to be expressed contains a promoter. Those promoters most commonly used in recombinant DNA construction for use with prokaryotic hosts include the β-lactamase (penicillinase) and lactose promoter systems (Chang et α/.,1978; Itakura et al.,1977; Goeddel et α/.,1979) and a tryptophan (tφ) promoter system (Goeddel et α/.,1980; EPO Publ No. 0036776). While these are the most commonly used, other microbial promoters such as the tac promoter (Amann et al, 1983) have been constructed and utilised, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally in operable relationship to genes in vectors (Siebenlist et al, 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al, 1979; Kingsman et α/.,1979; Tschemper et α/.,1980) is commonly used. This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic ofthe yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al, 1968; Holland et al, 1978). Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilisation. Any plasmid vector containing yeast-compatible promoter, origin or replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms such as mammals, plants and insects may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture. Academic Press, Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells and Chinese hamster ovary (CHO) cells. Expression vectors for such cells ordinarily include (if necessary) an origin of repUcation, a promoter located upstream from the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of S V40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al, 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extended from the Hindlll site toward the Bgll site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by construction ofthe vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Upon transformation of the selected host with an appropriate vector, the antigenic polypeptide or peptide encoded can be produced by culturing the host cells. The fusion protein is then recovered. Following recovery ofthe antigenic polypeptide or peptide it is purified as desired. The purification procedure adopted will of course depend upon the degree of purity required for the use to which the polypeptide or peptide is to be put. For most vaccination purposes, separation ofthe fusion protein from most ofthe remaining components ofthe cell culture is sufficient as the antigen can be incorporated into a vaccine in a relatively crude form. However, in cases where a greater degree of purity is desired, the carrier component ofthe fusion protein can be cleaved from the antigenic component. This can be easily achieved through the provision of an appropriate enzyme cleavage site between the carrier component and the antigen.

In addition to the protective antigen(s) of the invention, the present invention provides vaccines against Helicobacter infections. Such vaccines include as the essential component a host protective amount ofthe appropriate Helicobacter polypeptide, peptide fragment, variant or antibody referred to above, together with a suitable adjuvant or carrier.

Examples of suitable adjuvants known to those skilled in the art are saponins (or derivative or related material), muramyldipeptide, trehalose demycollate, Freund's complete adjuvant, Freund's incomplete adjuvant, other water in oil emulsions, double emulsions, dextran, diethylaminoethyl-dextran, potassium alum, aluminium phosphate, aluminium hydroxide, bentonite, zymosan, polyelectrolytes, retinol, calcium phosphate, protamine, sarcosine, glycerol, sorbitol, propylene glycol, fixed oils, non-ionic block copolymers, and synthetic esters of higher fatty acids. Saponins in particular have been found to be effective adjuvants.

In still further embodiments, the vaccine may also be formulated to further include other host-therapeutic agents. Such therapeutic agents include other vaccines, or immunostimulants such as interferons, interleukins or other cytokines.

The vaccine can be admimstered to the host by any of those methods known in the art. For example, one mode of administration of the vaccine is parenteral. The term "parenteral" is used herein to mean intravenous, intramuscular, intradermal and subcutaneous injection. Conveniently, the administration can be by subcutaneous injection.

Preferably, the vaccine incorporating the protective epitope, carrier and optionally an immunostimulant is administered to the host orally as a liquid or by another oral delivery vehicle. Alternatively, the vaccine components can be administered as an aerosol via the oral cavity or respiratory tract.

The amount ofthe vaccine administered to the host to be treated will depend on the type, size and body-weight of the host as well as on the immunogenicity of the vaccine. Conveniently, the vaccine is formulated such that relatively small dosages of vaccine (1-5 ml) are sufficient to be protective.

The vaccine may also be in the form of a live recombinant viral vaccine including nucleic acid encoding the polypeptide, peptide fragment, variant or anti-idiotypic antibody. The vaccine is administered to the host in this form and once within the host expresses the encoded polypeptide, peptide fragment, variant or antibody to induce a host-protective response.

A number of such live recombinant viral vaccine systems are known. An example of such a system is the Vaccinia virus system (US Patent 4603112; Brochier et α/.,1991).

In still a further aspect, the invention provides a method of protecting a host susceptible to infection by a Helicobacter organism. The method of invention includes as its essential step the administration to the host of either the antigenic polypeptide, peptide fragment, variant or anti-idiotypic antibody per se, or of a vaccine as described above.

The present invention also provides IgG antibodies specific for the 18 kDa antigens described above (or to the protective epitope of such antigens). Such antibodies may be polyclonal and be raised by any conventional immunisation protocol, but are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler et al, 1975 and Campbell in "Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas" in Burdon et al. Eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al, 1989.

Such antibodies have potential utility in combatting Helicobacter infection as described above, in antigen purification/extraction procedures and also in monitoring the immune status of individual hosts following vaccination.

The invention will now be illustrated in relation to the H. felis and H. pylori 18 kDa outer membrane proteins. The applicants have found that serum IgG antibodies to the H. felis 18 kDa outer membrane antigen recognise an antigen of similar molecular weight on H. pylori outer membrane preparations (Fig.1.). The applicants therefore believe that the results reported for are analogous to those which would be obtainable using other Helicobacter 18 kDa OMV antigens.

EXPERIMENTAL

A. MATERIALS AND METHODS A.l. Mice. Three genetically disparate strains of mice (C57Bl.H-2b, A/J,H-2a and BALB/c,H2-d) were used for these studies. A.2. Bacterial strains. Helicobacter felis ATCC 49179 (CS 1) and Helicobacter pylori (60190) were used for the animal studies. A further two strains of H. pylori (84- 183 & Tx-30a) were used to characterised the outer membrane. A.3. Bacterial antigens. Bacteria were grown in 2.8% (w/v) Bmcella broth base (Difco), supplemented with 5% fetal calf serum. Cultures were incubated at 37°C in a microaerobic environment (10% hydrogen, 10% carbon dioxide and 80% nitrogen) and were shaken at 120 rpm. The bacteria were harvested from 48-72 hour broth cultures by two centrifugations (10,000 x g, 15 min), washed (three times) and resuspended in PBS (at 4°C). Sonication of these bacteria (on ice) resulted in whole-cell sonicate which was stored at -70°C until used to immunise the mice.

A.4. Outer membrane preparations. Membrane vesicles, shed by the bacteria during growth in broth culture, were collected by ultracentrifugation (100,000 x g, 1 h, 4°C), ofthe culture supernatants. The resulting pellett was washed three times with

PBS (100,000 x g, 1 h, 4°C), as described previously (Blaser et al, 1983). The absence of whole bacteria and flagella in the preparation was confirmed by electron microscopy (Fig.2.).

The protein concentrations of all preparations were measured by a modification of the Lowry method for the estimation of membrane proteins (Markwell et al, 1978). Aliquots of these preparations were stored frozen (~20°C) until used. A.5. Experimental protocols. Study 1

Protocol 1 : C57B 1 and A/J mice were orally infected with 1 x IO⁷ viable H.felis organisms, followed twenty eight days later by a five day regime of colloidal bismuth subcitrate (0.74 mg/day), tetracycline (3mg/day) and metronidazole (2.7 mg/day). The efficacy of this regime was established prior to this study (RAllardyce, unpublished observation). Protocol 2: C57B1 and A/J mice were orally immunised with 1 mg H. felis sonicate (100 μl) on days 1 and 28. L121, a non-ionic block copolymer formulation (lOμl) consisting of 2.5% L121 (BASF Performance Chemicals, Wyandotte, Michigan), 5% squalene (Sigma), 0.2% Tween 80 (Sigma) and 2% MDP (Sigma), was included as adjuvant. This immunising regime has been shown to enhance mucosal immunity with cellular antigens unrelated to H. felis (Allardyce & Rademaker, 1989).

Protocol 3: C57B1, A/J and BALB/c mice each received 1 mg H. felis sonicate plus 10 mg cholera toxin (Sigma) on days 1, 3, 6, 30 and 53, following the protocol successfully used by Chen et al. (1992). Naive Controls: One cage each of C57B 1, A/J and BALB/c mice were kept as controls. These mice were neither infected nor immunised with H. felis.

Collection of samples

Serum and intestinal secretions (Elson et al, 1984) were collected from all animals (including controls) immediately prior to oral challenge with 1 x IO⁸ viable H. felis.

Assessment of protection

Twenty-one days post challenge all mice were sacrificed and 4 gastric biopsies per animal (2 each from the antrum & corpus) were examined for urease activity (Ηazell t α/., 1987).

Study 2

Protocol: BALB/c mice were given four oral immunisations over 1 month, each consisting of 50 μg of H. felis membrane vesicle protein plus 10 μg of cholera toxin (CT). A similar schedule was successfully used by Czinn et al. (1993). Age-matched contiol mice were not immunised. Collection of samples

Four weeks after their last immunisation, mice were bled, then challenged with lxlO⁸ viable H. felis. Sera and intestinal secretions (Νedrud et al, 1987) were collected at necropsy.

Assessment of protection

Twenty one days after challenge all mice were killed. Gastric antral biopsies were assessed by urease activity and by histology. H. felis infection was confirmed if either of these tests was positive (Michetti et al, 1994).

A.6. ELISA. Antigen-coated ELISA plates were prepared by incubating polystyrene assay plates (Νunc-MaxiSorp) with 100 μl/well of the Helicobacter spp outer membrane components(2 μg protein/well) in 0.1 M potassium carbonate buffer (pΗ 9.6) overnight at 4°C (Czinn & Νedrud, 1991). The wells were washed three times with diluent (PBS-0.05% (v/v) Tween 20) and nonspecific binding sites were blocked (0.5% gelatin in diluent) for 2 h at room temperature. Individual sera and intestinal secretions (100 μl/well) were tested at dilutions ranging from neat to 1:500,000. Following incubation at room temperature for 90 min, plates were washed and bound mouse immunoglobulin (serum IgG or intestinal IgA) was detected by the addition of horseradish peroxidase-conjugated goat anti-mouse IgG or IgA (Sigma), diluted 1: 1000. The antibody titre was defined as the reciprocal ofthe highest dilution yielding an A₄₉₂ of 0.05 above wells which contained antigen and which were incubated with the antibody conjugate but without primary antibody (Czinn & Νedrud, 1991).

A.7. SDS-PAGE. The protein profile of the sarkosyl-insoluble outer membrane preparation was compared to tiiat of outer membrane vesicles by SDS-PAGE using a gradient resolving gel of 5%-20% acrylamide. Samples containing 2 μg of protein were mixed with an equal volume of reducing buffer and boiled at 100°C for 3 minutes before being loaded onto the gel. After electrophoresis the gel was fixed and protein present was silver stained by the method of Ηeukeshoven & Dernick (1985).

A.8 Immunoblotting. Outer membrane vesicles (5 μg protein/lane) were fractionated by SDS-PAGE, then electrophoretically transferred to 0.45 μ nitrocellulose membrane. Transfer was optimised using a buffer containing 20% methanol

(Towbin et al, 1979). The blot was washed twice in Tris/saline blotting buffer

(TSBB) for 30 min at RT, then incubated in primary antibody in TSBB for 1 h at RT. After three 5 min washes in TSBB, the blot was incubated in secondary antibody (alkaline phosphatase-conjugated) for 1 h at RT. Reactive bands were visualised as described by Blake et al. (1984), using 5-bromo-4-chloro-3-indolyl phosphate as the alkaline phosphate reagent and Nitro Blue Tetrazolium as the colour development reagent.

A.9 Statistical Analysis. Comparison of antibody titres among experimental groups was evaluated by analysis of variance and the Student-Newman-Keuls test. For protection, recognition of an 18 kDa antigen among groups was evaluated by Fisher's Exact Test.

A.10 Determination of urease activity. The urease activity of the H. pylori cell and membrane vesicle fractions was assessed by the enzymatic hydrolysis of urea in a quantitative spectrophotometric assay, according to the method of Dunn et al. (1990). Briefly, a dilution of sample was added to cuvettes (1-cm light path length) containing 3 ml of a reaction mixture of 31 mM Tris-ΗCl (pΗ 8), 810 μM oxoglutarate, 240 μM NADΗ and 10 mM urea. The reaction was started by adding 96 U of glutamate dehydrogenase and the reduction of NADΗ was followed spectrophotometrically for 10 min at 37°C, with a standard wavelength of 340 nm in a dual beam spectrophotometer. One unit of urease activity was defined as that amount capable of hydrolyzing 1 μM urea per min.

A.l 1 Preparation of 18 kDa immunogen. H. felis membrane vesicles (approx. 1 mg protein) were electrophoresed under reducing conditions on a 12.5% SDS- polyacrylamide gel and the separate components electroblotted to 0.45 μ nitrocellulose membrane. Narrow strips were cut from either edge of the membrane and immunoblotted with murine antibodies to H. felis membrane vesicles. The remaining membrane was stained with India ink, according to the method of Hancock & Tsang (1983). Briefly, the blot was washed in three changes of 0.4% Tween 20/PBS (5 min each), followed by incubation in 0.001% India ink

(diluted in 0.3% Tween 20/PBS) until the lower molecular weight proteins were visualised (approx. 2 h). The 18 kDa antigen was identified by placing the immunoblotted NC membrane strips either side of the India ink-stained blot (Fig.7a). The band, estimated to contain 50 μg protein, was excised and the nitrocellulose cut into very fine pieces prior to sonication (on ice) in PBS. The antigenicity of each preparation was determined by immunoblotting an aliquot of supernatant with murine antisera against H. felis membrane vesicles. The remaining supernatant was homogenised with Freund's complete adjuvant (BBL) to a final concentration of 50% (vol/vol) and the resulting water-in-oil emulsion used to inject 5 mice intra-peritoneally (primary immunisation). A booster, incoφorating Freund's incomplete adjuvant, was prepared the same way and administered to all animals 21 days later. After a further three weeks, two mice in each group were given an additional antigenic stimulus by the implantation ofthe nitrocellulose fragments (which had been stored at -20°C after sonication to remove the 18 kDa antigen) under the skin on their backs.

A12. Preparation of protein for sequencing. SDS-PAGE separated H. pylori (60190) membrane vesicle components (approx. 200 μg protein) were electroblotted to

ProBlott from a 12.5% acrylamide gel. The 18 kDa protein was located on the membrane by staining with 0.1% Coomassie brilliant blue (CBB) in 40% methanol and 1% acetic acid for 1 min, followed by destaining in 50% methanol. The membrane was rinsed in several changes of deionised water, air-dried and submitted for sequence analysis.

B. Results

B.1 Animal Studies. The effectiveness of the three immunising regimes in the lst study to induce protection from challenge with H. felis is shown in Table 1. Protection (resistance to challenge) was clearly associated with the immunising regime and not the strain of animal used. Protocol 1 (eradicated infection) and Protocol 3 (immunised with cholera toxin as adjuvant) were (with one exception) protected. Protocol 2 mice (immunised with the adjuvant L121) were (with one exception) as susceptible to challenge with H. felis as the naive control mice.

Oral immunisation with membrane vesicles shed from the surface of H. felis resulted in 70% protection (Table 1).

Table 1: Protection from H. felis challenge by natural and induced immunisation

Protocol Strain Η-2 n Protected mice post-challenge

Eradicated A J H-2a 8 7/8

Η. felis infection C57B1 H-2b 8 8/8

Η. felis sonicate A/J H-2a 4 4/4*

+ CT C57B1 H-2b 8 8/8

BALB/c H-2d 6 6/6*

Η. felis sonicate A/J H-2a 3 0/3*

+ L121 C57B1 H-2b 8 1/8

Controls A/J H-2a 8 0/8

C57B1 H-2b 3 0/3

BALB/c H-2d 6 0/6

Η. felis outer membrane vesicles BALB/c H-2d 10 7/10

+ CT

Controls BALB/c H-2d 5 0/5

* Animal losses resulted from anaesthesia and collection of intestinal secretory fluids.

B.2 ELISA. Individual pre-challenge sera and intestinal secretions were assayed (ELISA) for antibody response to oral immunisation. The serum IgG responses of the mice protected from subsequent challenge varied. A/J mice, which had been infected, demonstrated significantly greater serum IgG responses than unimmunised control animals (p<0.05). Post-infection C57B1 mice, however, failed to show a significantly elevated serum IgG response. Oral immunisation with H. felis sonicate in the presence of cholera toxin produced a significant serum IgG difference from controls in BALB/c mice (p=0.0103). However, no significant difference in antibody titre was observed in the other two strains of mice (C57B1 and A/J) immunised by this route when compared to controls. Oral immunisation of BALB/c mice with H. felis outer membrane vesicles also resulted in a significant semm IgG response (p=0.0365). No significant differences in intestinal IgA antibody levels were observed in any of the groups of protected mice when compared to controls (Fig.3).

B.3 Immunoblotting. Immunoblotting individual sera from each experimental group of A/J and BALB/c mice against H. felis outer membrane vesicles components (Fig.4) revealed that a serum IgG recognition of an 18 kDa antigen was a significant feature of the antibody response in animals t at were protected from subsequent challenge (p=0.023). Naive animals failed to recognise this antigen. Low antibody titres in C57 mice resulted in no obvious qualitative difference in antibody response between protected and vulnerable animals.

B.4 Characterisation of H. pylori outer membrane vesicles. In contrast to earlier studies of water-soluble surface proteins (Dunn et al, 1990) and sarkosyl-insoluble outer membrane proteins (Ηawtin et al, 1990), no measurable urease activity was detected in any ofthe H. pylori membrane vesicle fractions (Table 2).

Table 2: Urease activity/mg protein

Strain Whole cells Membrane Vesicles

60190 11.1 0.0001

Tx-30a 5.8 0.0004

84-183 5.9 0.0001 urease - negative 0.2 0.0002 mutant

This finding was supported by immunoblotting, which failed to find evidence of either the 66 kDa subunit ofthe urease enzyme (Fig.5) or its associated heat-shock protein chaperonine, hspB (results not shown) in the membrane vesicle fractions. This difference is most likely attributable to lack of cytoplasmic membrane (Murphy, 1989) and peptidoglycan contamination (Mayrand & Grenier, 1989) in membrane vesicle preparations. B.5 Characterisation of 18 kDa antigen. Parenteral immunisation with the 18 kDa antigen isolated from the membrane vesicles of H. felis failed to induce a measurable serum IgG response in any ofthe five mice. To test the hypothesis that this was attributable to insufficient immunogen elution from the nitrocellulose during sonication, the residual nitrocellulose fragments were implanted under the skin of two randomly chosen animals. A similar procedure was carried out on two ofthe animals who also failed to respond to the H. pylori 18 kDa antigen. All four of these animals subsequently mounted a marked antibody response to the respective immunogen. Electron microscopy revealed that these murine antibodies bound to the native antigen on the surfance of either H. pylori or H. felis (Fig.6).

B.6 Protein sequencing. Samples for amino acid sequencing by Edman degradation were submitted to the Protein Microchemistry Facility, Department of Biochemistry, University of Otago. Preliminary amino acid analysis revealed the Ν-terminal ofthe H. pylori 18 kDa peptide was blocked. Partial acid hydrolysis

(70% formic acid for 60 min at 70°C) yielded a tentative Ν-terminal sequence. More rigorous acid hydrolysis yielded 2 intemal sequences of the 18 kDa protein.

Ν terminal Sequence

1 5 10 15

KDFΝKFXMKALYEQA RΝD I K

Intemal sequences

1 5 10

AETIQATADA

1 5 10

YVKΝLVIMKT VR V K

C. DISCUSSION

Serum IgG recognition of an outer membrane vesicle antigen with an apparent molecular weight of 18 kDa (by SDS-PAGE) reveals this antigen to be associated with protection, regardless of the mouse strain. This is important as a genetic basis for host variation in antigen recognition is well documented in mouse models of parasitic infections (Kennedy, 1989). Strains of mice with different major histocompatibility complex (MHC)-haplotype have been shown to vary considerably in antibody repertoire to the same antigenic stimulus, with only those of identical MHC showing similar recognition profiles. The development of a successful vaccine requires that responses against important protective antigens be generated in all individuals, including those whose MHC type is the least reactive.

INDUSTRIAL APPLICATION

The 18 kDa antigens ofthe invention have direct application in the immunoprotection of susceptible hosts against Helicobacter infections such as H. pylori. The epitope-carrying peptide fragments and variants ofthe native antigens, and anti-idiotypic antibodies which mimic the epitope, have equivalent application.

Additional applications of the invention include use of the antibodies specific for the epitope of each 18 kDa antigen in identifying suitable strains oi Helicobacter for vaccine preparation, and in monitoring the immune status of individuals after vaccination.

It will be appreciated by those persons skilled in the art that the above description is provided by way of example only and that all variations and modifications which do not depart from the spirit or scope thereof are contemplated. The invention is limited only by the lawful scope ofthe appended claims.

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Claims

CLAIMS:

1. An antigenic polypeptide including an epitope capable of generating a protective immunological response against Helicobacter infection in a susceptible host, said polypeptide having an amino acid sequence of an approximately 18 kDa outer membrane vesicle (OMV) antigen of a Helicobacter organism; or a peptide fragment or a variant of said polypeptide including said epitope and having protective immunological activity at least substantially equivalent to said polypeptide.

2. A polypeptide or peptide fragment or variant as claimed in claim 1 which has an N-terminal amino acid sequence as listed in SEQ ID No. 1.

3. A polypeptide or peptide fragment or variant as claimed in claim 1 which includes the amino acid sequence as listed in SEQ ID No. 2.

4. A polypeptide or peptide fragment or variant as claimed in claim 1 which includes the amino acid sequence as listed in SEQ ID No. 3.

5. A polypeptide or peptide fragment or variant as claimed in claim 1 which includes the amino acid sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

6. A polypeptide or peptide fragment as claimed in claim 1 isolated from the OMV of a Helicobacter organism.

7. A polypeptide or peptide fragment as claimed in claim 6 which is isolated from the OMV of H. felis or H. pylori.

8. A polypeptide, peptide fragment or variant as claimed in claim 1 which is synthesised or is produced using recombinant DNA techniques.

9. An anti-idiotypic antibody which mimics the protective epitope of the polypeptide of claim 1.

10. An antibody which binds to, and is specific for, the protective epitope of the polypeptide of claim 1.

11. A vaccine against Helicobacter infection which comprises a polypeptide, peptide fragment or variant as claimed in claim 1 or an anti-idiotypic antibody as claimed in claim 9, together with an immunologically appropriate adjuvant or carrier.

12. A method of protecting a susceptible host against Helicobacter infection comprising the step of administering to said host an amount of a polypeptide, peptide fragment or variant as claimed in claim 1, or an amount of an anti-idiotypic antibody as claimed in claim 9, which is protective against such infection.

13. A method of protecting a susceptible host against Helicobacter infection comprising the step of administering to said host an amount of a vaccine as claimed in claim 11 which is protective against such infection.

14. A method as claimed in claim 12 or claim 13 wherein said host is a human and wherein said polypeptide, peptide fragment, variant, anti-idiotypic antibody or vaccine is administered to protect against H. pylori infection.