Title:  Treatment and prevention of helicobacter infection

United States Patent:  6,630,582

Issued:  October 7, 2003

Inventors:  Doidge; Christopher V. (Vincent, AU); Lee; Adrian (Lane Cove, AU); Radcliff; Flona J. (Sydney, AU); Hazell; Stuart L. (Glenfield, AU)

Assignee:  University of New South Wales (Kensington, AU); CSL Limited (Victoria, AU)

Appl. No.:  004014

Filed:  January 7, 1998

Abstract

An antigenic preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, comprises the catalase enzyme of Helicobacter bacteria, particularly the catalase enzyme of H. pylori or H. felis, or an immunogenic fragment thereof.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection, which comprises an at least partially purified preparation of the catalase of Helicobacter bacteria.

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

Accordingly, in a particularly preferred embodiment, the present invention provides an antigenic preparation for use in treatment or prevention of Helicobacter infection, which comprises substantially pure catalase of Helicobacter bacteria. Such a preparation may be prepared as a recombinant catalase by techniques described hereinafter.

In another aspect, the present invention provides an isolated Helicobacter antigen for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises the catalase of Helicobacter bacteria, or an immunogenic fragment thereof.

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

It is to be understood that the present invention extends not only to an antigenic preparation or isolated antigen comprising the catalase of Helicobacter bacteria, but also to antigenic preparations comprising immunogenic fragments of this catalase, that is catalase fragments which are capable of eliciting a specific protective immune response in a mammalian host. Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly monoclonal antibodies which have a protective or therapeutic effect in relation to Helicobacter infection or polyclonal antibodies contained in immune sera from mammalian hosts which have been vaccinated against Helicobacter infection.

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

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

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

In yet another aspect, the invention provides the use of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, in the manufacture of a vaccine composition for the treatment or prevention of Helicobacter infection in a mammalian host.

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

By use of the term "immunologically effective amount" herein in the context of treatment of Helicobacter infection, it is meant that the administration of that amount to an individual infected host, either in a single dose or as part of a series, that is effective for treatment of Helicobacter infection. By the use of the term "immunologically effective amount" herein in the context of prevention of Helicobacter infection, it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, that is effective to delay, inhibit or prevent Helicobacter infection. The effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Preferably, the catalase antigen above comprises an amino acid sequence substantially corresponding to the deduced sequence of the catalase gene from isolate RU1 or isolate 921023 hereinafter (SEQ ID NO.2 or 4), or allelic or other variants thereof. Suitable variants may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to one of the amino acid sequences referred to above, or to a region or part thereof, provided the variant is capable of eliciting a specific protective immune response in a mammalian host.

As described above, the present invention extends not only to the particular catalase antigen of Helicobacter bacteria as described above, but also to immunogenic fragments of the particular antigen, that is fragments of the antigen which are capable of eliciting a specific protective immune response in a mammalian host. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular antigen.

Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.

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

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

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

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

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

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

The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to moeity (1982) at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45^oC. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at .gtoreq.45^oC. for 2-3 hours or high stringent conditions as disclosed by moeity (1982).

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

The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source.

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

Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in moeity (1982).

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

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

Once recombinant DNA cloning vehicles and/or host cells expressing a Helicobacter catalase antigen of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.

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

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

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

The present invention also extends to delivery to the host using a vector expressing the catalase of Helicobacter bacteria, or an immunogenic fragment thereof. Accordingly, in a further aspect this invention provides a preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.

In this aspect, the invention extends to a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.

Further, the invention extends to the use of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof, for the treatment or prevention of Helicobacter infection in a mammalian host.

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

DETAILED DESCRIPTION OF THE INVENTION

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

In a particularly preferred aspect of this invention, an oral vaccine composition comprising substantially pure H. pylori catalase, more preferably recombinant H. pylori catalase, in association with a mucosal adjuvant is used for the treatment or prevention of H. pylori infection in a human host.

The mucosal adjuvant which is optionally, and preferably, administered with the Helicobacter catalase preparation or antigen to the infected host is preferably cholera toxin. Mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the Helicobacter catalase preparation or antigen. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin. Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP muramyl di peptide; acridine and cimetidine.

The Helicobacter catalase preparation or antigen may be delivered in accordance with this invention in ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells. Alternatively, micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors. The Helicobacter catalase preparation or antigen may also be incorporated into oily emulsions and delivered orally. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter⁷.

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

The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

As an alternative to the delivery of the Helicobacter catalase preparation or antigen in the form of a therapeutic or prophylactic oral vaccine composition, the catalase or an immunogenic fragment thereof may be delivered to the host using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the catalase or immunogenic fragment as a foreign antigen. Particularly, bacteria that colonise the gastrointestinal tract, such as Salmonella, Yersinia, Vibrio, Escherichia and Bacille Calmotte Guerin have been developed as vaccine vectors, and these and other examples are discussed by Holmgren et al. ⁸ and McGhee et al. ⁹.

The Helicobacter catalase preparation or antigen of the present invention may be administered as the sole active immunogen in a vaccine composition or expressed by a live vector. Alternatively, however, the vaccine composition may include or the live vector may express other active immunogens, including other Helicobacter antigens such as urease or the lipopolysaccharide (LPS) of Helicobacter bacteria (see International Patent Application No. PCT/AU95/00077), as well as immunologically active antigens against other pathogenic species.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.

Data obtained from Western blots mentioned above, show that H. pylori catalase is recognised by the serum of mice vaccinated with an H. felis antigen preparation (plus cholera toxin adjuvant). These mice can be shown to be protected against H. felis infection. This data indicates the use of H. pylori catalase as a protective antigen in human H. pylori infection, and purified or recombinant catalase may be used as an antigenic component of a therapeutic or prophylactic vaccine, either on its own, or in combination with other antigens, carriers, adjuvants, delivery vehicles or excipients.

Claim 1 of 12 Claims

What is claimed is:

1. An isolated nucleic acid molecule consisting essentially of a sequence of nucleotides which encodes a full length Helicobacter catalase as set forth in SEQ ID NO:1 or SEQ ID NO:3.

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