WO1997003359A1 - Helicobacter clpb - Google Patents

Abstract

The invention features Helicobacter ClpB polypeptides, and methods and compositions for preventing and/or treating Helicobacter infection using these polypeptides. The figure shows a schematic representation of the nucleotide sequence of the insert of plasmid pCP6 which encodes Helicobacter ClpB.

Description

HELICOBACTER CLPB Background of the Invention This invention relates to methods and compositions for preventing and/or treating Helicobacter infection.

Helicobacter is a genus of spiral, gram-negative bacteria which colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably, H. pylori , H. heilmanii , H. felis, and H. mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been found to infect humans, but at lower frequencies than H. pylori .

Helicobacter infects over 50% of adult populations in developed countries, and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections of humans worldwide. Infection with H . pylori results in chronic stomach inflammation in all infected subjects, although the clinical gastroduodenal diseases associated with Helicobacter generally appear from several years to several decades after the initial infection. H. pylori is the causative agent of most peptic ulcers and chronic superficial (type B) gastritis in humans. H. pylori infection is also associated with atrophy of the gastric mucosa, gastric adenocarcinoma, and non-Hodgkin's lymphoma of the stomach (see, e.gr., Blaser, J. Infect. Dis. 161:626-633, 1990; Scolnick et al . , Infect. Agents Dis. 1:294-309, 1993; Goodwin et al . , "Helicobacter pylori , " Biology and Clinical Practice , CRC Press, Boca Raton, FL, 465 pp, 1993; Northfield et al . , "Helicobacter pylori , " Infection , Kluwer Acad. Pub., Dordrecht, 178 pp, 1994) .

If untreated, H. pylori infection and the associated gastritis persist lifelong, despite systemic and local immune responses to the bacterium in the infected host (Crabtree et al . , "Host responses," in Helicobacter pylori Infection , Northfield et al . (Eds.), Kluwer Acad. Pub., Dordrecht, pp. 40-52, 1991; Kist "Immunology of Helicobacter pylori , " in Helicobacter pylori in peptic ulceration and gastritis , Marshall et al . (Eds.), Blackwell Sci. Pub., Oxford, pp. 92-110, 1991; Fox et al . , Infect. Immun. 61:2309-2315, 1993). Conventional treatment of peptic ulcer disease associated with H. pylori infection involves the use of one or more antibiotics combined with a proton pump inhibitor or an H₂-receptor antagonist. Such treatment regimens are unsuccessful in 10% to 70% of patients. Moreover, successful eradication of H. pylori infection with antibiotics does not prevent subsequent reinfection. The most effective conventional treatment is a triple therapy with bismuth, metronidazole, and either amoxiciUin or tetracycline. The triple therapy treatment is complicated by a complex and prolonged dosing regimen, a high incidence of side-effects, poor compliance, and emergence of resistant bacterial strains (Hentschel et al . , N. Engl. J. Med. 328:308-312, 1993).

Summary of the Invention We have identified a novel Helicobacter polypeptide, designated Helicobacter ClpB, which may be used, e . g. , in methods and compositions for preventing and/or treating Helicobacter infection.

Accordingly, the invention features a method for preventing or treating Helicobacter infection in a mammal involving administering to the mammal Helicobacter ClpB, or an immunogenic fragment or derivative thereof. In this method, administration may be to a mucosal (e . g. , intranasal, oral, ocular, gastric, rectal, vaginal, intestinal, or urinary tract) surface of the mammal, or parenteral (e . g. , intravenous, subcutaneous, intraperitoneal, or intramuscular) . In addition to Helicobacter ClpB (or an immunogenic fragment or derivative thereof), an adjuvant, e . g. , a cholera toxin, Escherichia coli heat-labile enterotoxin (LT) , or a fragment or derivative thereof having adjuvant activity, may be administered to the mammal. Mammals that may be treated using the method of the invention include, but are not limited to, mammals such as humans, cows, horses, pigs, dogs, cats, sheep, and goats.

The invention also features a method of preventing or treating Helicobacter infection in a mammal involving administering to a mucosal (e.g., oral) surface of the mammal an antibody (e.g., a monoclonal antibody) which recognizes Helicobacter ClpB.

Substantially pure Helicobacter ClpB polypeptide, for example, a polypeptide containing amino acid sequences substantially identical to the amino acid sequences shown in Fig. 2 (SEQ ID NO:2), is also included in the invention. (The nucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO:2) sequences in Fig. 2 contain errors, for example, a stop codon is present at amino acid 752. Thus, the invention features a polypeptide having an amino acid sequence which is substantially identical to the ClpB amino acid sequence encoded by the insert in the plasmid contained in the bacteria deposited with the NCIMB as XLOL HP CP6, see below) . Purified DNA which encodes Helicobacter ClpB polypeptide is also included in the invention. The purified DNA of the invention may contain a nucleotide sequence which is substantially identical to that of the insert in the plasmid contained in the bacteria deposited with the NCIMB as XLOLR HP CP6, see below. The invention also features a nucleotide sequence which is substantially identical to the nucleotide sequence shown in Fig. 2 (SEQ ID NO:l). The DNA may be contained in a vector, for example, a plasmid vector, and/or a cell.

The invention also includes a vaccine composition containing, in addition to Helicobacter ClpB (or immunogenic fragment or derivative thereof) , a pharmaceutically acceptable diluent or carrier (e .g. , water, a saline solution (e.g., phosphate-buffered saline), or a bicarbonate solution (e .g. , 0.24 M NaHC0₃)). An adjuvant, e . g. , a cholera toxin, Escherichia coli heat-labile enterotoxin (LT) , or a fragment or derivative thereof having adjuvant activity, may also be included in the vaccine composition of the invention.

A method of producing a recombinant Helicobacter ClpB polypeptide is also included in the invention. In this method, a cell transformed with DNA encoding Helicobacter ClpB polypeptide, and positioned for expression in said cell, is cultured under conditions for expressing the DNA. Recombinant Helicobacter ClpB polypeptide is then isolated from the cell. Helicobacter ClpB polypeptide made by this method is also included in the invention.

A substantially pure antibody (e.g., a monoclonal antibody, e . g. , an IgA or IgG antibody) that specifically binds Helicobacter ClpB polypeptide, or a fragment or derivative thereof, is also featured in the invention. This antibody may be used in a method for detecting Helicobacter in a sample involving contacting the sample with the antibody and detecting immune complexes formed between the antibody and the sample as an indication of the presence of Helicobacter in the sample.

By a "substantially identical" polypeptide sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e . g. , valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function (e .g. , the specific antigenicity) of the polypeptide.

Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to the sequences of Fig. 2 (SEQ ID NO:2), or the ClpB sequence encoded by the insert of XLOLR HP CP6 (see below) . For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.

Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "protein" or "polypeptide" is meant any chain of amino acids, regardless of length or post- translational modification (e . g. , glycosylation or phosphorylation) .

By "substantially pure" is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e . g. , the ClpB polypeptide (or fragment or derivative thereof) or ClpB-speσific antibody. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e .g. , column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

By "purified DNA" is meant DNA that is not immediately contiguous with both of the sequences (e.g., the coding sequences) with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. By a "substantially identical" nucleic acid is meant a nucleic acid sequence which encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e . g. , valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide. Preferably, the encoded sequence is at least 45%, more preferably 60%, and most preferably 85% identical at the amino acid level to the amino acid sequences of Fig. 2 (SEQ ID NO:2) (or the amino acid sequence encoded by the insert in XLOLR HP CP6, see below) . If nucleic acid sequences are compared a "substantially identical" nucleic acid sequence is one which is at least 85%, more preferably 90%, and most preferably 95% identical to the sequence of Fig. 2 (SEQ ID NO:l) (or the ClpB amino acid sequence encoded by the insert in XLOLR HP CP6, see below) . The length of nucleic acid sequence comparison will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Again, homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .

By "purified antibody" is meant antibody which is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.

By "specifically binds" is meant an antibody which recognizes and binds a ClpB polypeptide but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) which naturally includes ClpB polypeptide. An antibody which "specifically binds" ClpB is sufficient to detect a ClpB protein product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation) .

By "positioned for expression" is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of Helicobacter ClpB protein) .

Other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments thereof, and from the claims.

Detailed Description The drawings are first described. Drawings

Fig. l is a schematic representation of the nucleotide sequence of the insert of plasmid pCP6 (SEQ ID N0:1), which insert encodes Helicobacter clpB (SEQ ID NO:2) . The putative promoter is indicated by the arrow at position 5. The ribosome binding site (RBS; position 116) , start codon (ATG; position 126) , and stop codon (TGA; position 2695) , are indicated. The numbering starts at the beginning of the pCP6 insert. The bold arrow indicates the 5' to 3' orientation of the gene. Fig. 2 is a schematic representation of the nucleotide sequence of (SEQ IDD NO:l), and the amino acid (SEQ ID NO:3) encoded by, the insert of plasmid pCP6, which insert encodes Helicobacter ClpB. The start (ATG) and stop (TAG) codons are indicated by the boxes.

Methods and Compositions for Preventing and/or Treating Helicobacter Infection

We have isolated a Helicobacter gene that encodes a polypeptide (designated Helicobacter ClpB) of approximately 87 kD which, based on amino acid sequence comparisons, is a member of the Clp protein family. This family consists of three sub-groups: ClpA, ClpB, and ClpC, which, although differing in size and sequence organization, share extensive regions of homology. Several of the Clp proteins identified thus far are heat shock proteins (hsps) , which are thought to function as regulatory subunits of ATP-dependent proteases, and as molecular chaperones. Helicobacter ClpB can be used in vaccination methods for preventing and/or treating Helicobacter (e . g. , H. pylori , H. f lis , or H. heilmanii) infection in a mammal (e.g., a human). In these methods, Helicobacter ClpB, or an immunogenic fragment or derivative thereof, is administered to a mucosal (e.g., intranasal, oral, ocular, gastric, rectal, vaginal, intestinal, and urinary tract) surface of a mammal, or is administered parenterally (e.g., by intravenous, subcutaneous, intraperitoneal, or intramuscular routes) . Any of a number of adjuvants that are known to one skilled in the art may be co-administered with the Helicobacter ClpB polypeptide. For example, a cholera toxin (CT) or the heat-labile enterotoxin of Escherichia coli (LT) , or a fragment or derivative thereof having adjuvant activity, may be used in mucosal administration. An adjuvant such as RIBI (ImmunoChem, Hamilton, MT) or aluminum hydroxide may be used in parenteral administration.

Helicojacter ClpB polypeptides which may be used in the vaccination methods of the invention may be prepared using any of several standard methods. For example, standard recombinant DNA methods may be employed (see, e.g., Ausubel et al . , Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994). In these methods, a suitable host cell is transformed with an appropriate expression vector containing all or part of a Helicobacter ClpB-encoding nucleic acid (e.g., DNA or RNA) fragment (see Fig. 2 for the nucleotide (SEQ ID NO:2) and amino acid sequences of Helicobacter ClpB; also bacteria strain XLOLR HP CP6, which was deposited with the NCIMB (see below) contains a DNA sequence encoding ffelicojacter ClpB) . Nucleic acid fragments encoding Helicobacter ClpB may be obtained from plasmid pCP6, which was deposited in a bacterial strain (E. coli XLOLR; Stratagene, Ltd., Cambridge, UK) with the National Collections of Industrial & Marine Bacteria (NCIMB) in Aberdeen, Scotland, on July 4, 1995, and designated with NCIMB accession number 40748. XLOLR HP CP6 contains a vector, designated pCP6, which is a pBK-CMV plasmid vector (Stratagene, Ltd., Cambridge, UK) containing the H. pylori ClpB gene inserted into the BamHI site of the polylinker. pCP6 was obtained by excision from a bacteriophage vector Lambda Zap II Express (Stratagene, Inc. , Cambridge, UK) containing the H. pylori ClpB gene. Standard methods of molecular biology may also be used to obtain the Helicobacter ClpB gene (see below, and Ausubel et al . , supra) .

Any of a variety of expression systems may be used to produce recombinant Helicobacter ClpB polypeptides. For example, ClpB polypeptides may be produced in a prokaryotic host (e . g. , E. coli) or in a eukaryotic host (e.g., yeast cells (e.g., saccharomyces cerevisiae) , mammalian cells (e.g., C0S1, NIH3T3, or JEG3 cells), or arthropod cells (e.g., Spodoptera frugiperda (SF9) cells) ) . Such cells are available from a number of different sources known to those skilled in the art, e.g., the NCIMB or the American Type Culture Collection (ATCC), Rockville, MD (also see, e . g. , Ausubel et al . , supra) . The transfection/transformation method used, and the choice of expression vector, will depend on the host system selected, as is described by, e .g. , Ausubel et al . , supra . Expression vectors (e.g., plasmid or viral vectors) can be chosen from, e.g., those described in Cloning Vectors : A Laboratory Manual (Pouwels et al . , 1985, Supp. 1987; also see, e.g., Ausubel et al . , supra) . Helicobacter ClpB polypeptides, particularly short fragments, may also be produced by chemical synthesis, e.g., by the method described in Solid Phase Peptide Synthesis , 1984, 2nd edn., Stewart et al., Eds., Pierce Chemical Co., Rockford, IL, and by standard in vitro translation methods. In addition, Helicobacter ClpB may be purified from Helicobacter cultures, using standard methods.

In addition to native, full length, Helicobacter ClpB, polypeptide fragments of ClpB, or ClpB polypeptides (or polypeptide fragments of ClpB) containing mutations, may be used in the invention, provided that the antigenicity of the polypeptide is retained. Fragments of Helicobacter ClpB polypeptides are made by standard methods, including, e .g. , recombinant, chemical synthetic, or proteolytic methods (see, e . g. , Ausubel et al . , supra) . Generally, ClpB polypeptide fragments for use in the methods of the invention should be at least 12 amino acids in length, in order to maintain antigenicity. Genes encoding fragments of Helicobacter ClpB, and/or ClpB polypeptides containing mutations, are made using standard methods (see, e.g., Ausubel et al . , supra) .

Fragments and derivatives of Helicobacter ClpB which are included in the invention may be screened for antigenicity and/or therapeutic efficacy using standard methods in the art, e .g. , by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H. felis-mouse model system (see, e.g., Czinn et al . , Vaccine 11(6) :637- 642, 1993; Lee et al . , European Journal of Gastroenterology and Hepatology 7:303-309, 1995).

Fusion proteins containing Helicobacter ClpB (or a fragment or derivative thereof) fused to, e .g. , an adjuvant (e.g., cholera toxin (CT) or the Escherichia coli heat-labile enterotoxin (LT) , or a fragment or derivative thereof having adjuvant activity) , are also included in the invention, and can be prepared using standard methods (see, e.g., Ausubel et al . , supra) . In addition, the vaccines of the invention may be covalently coupled or chemically cross-linked to adjuvants (see, e.g., Cryz et al . , Vaccine 13:67-71, 1994; Liang et al . , J. Immunology 141:1495-1501, 1988; and Czerkinsky et al . , Infection and Immunity 57:1072-1077, 1989). The amount of vaccine administered depends on, e.g., the particular vaccine antigen, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection and/or treatment) , as can be determined by one skilled in the art. In general, the vaccine antigens of the invention are administered in amounts ranging between, e.g., 1 μg and 100 mg. If adjuvants are administered with the vaccines, amounts ranging between, e.g., 1 ng and 1 mg may be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by 3 booster doses at weekly intervals.

Vaccine compositions of the invention contain ClpB polypeptide, or immunogenic fragments or derivatives thereof, in a pharmaceutically acceptable carrier or diluent (e.g., water, a saline solution (e.g., phosphate- buffered saline), or a bicarbonate solution (e.g., 0.24 M NaHC0₃) ) . The carriers and diluents used in the invention are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington 's Pharmaceutical Sciences , a standard reference text in this field, and in the USP/NF. Adjuvants may also be included in these compositions.

Antibodies against Helicobacter ClpB may be used in passive immunization methods for protecting and/or treating mammals (e.g., humans) from Helicobacter (e.g., H. pylori , H. felis , or H. heilmanii ) infection. Monoclonal antibodies against Helicobacter ClpB are produced using standard immunological methods (see, e.g., Coligan et al . , Eds., Current Protocols in Immunology, John Wiley & Sons, Inc., New York, New York, 1994).

Antigens for use in these methods may be obtained, e.g., by expression of the Helicobacter ClpB gene in, e . g. , E. coli , using standard methods (see, e.g., Ausubel et al . , supra) . Antibodies of any isotype, e .g. , IgA and IgG, may be used in the invention. In addition to monoclonal antibodies, purified polyclonal antibodies, single chain antibodies, chimeric antibodies (e.g., human/murine chimeric antibodies), humanized antibodies (e.g., humanized murine monoclonal antibodies) , and Fab fragments which recognize Helicobacter ClpB may be used in the invention. Antibodies which recognize Helicobacter ClpB may be identified using standard immunological assays, e.g., Western blot analysis and ELISA (see, e .g. , Coligan et al . , supra) . Antibodies may be screened for therapeutic efficacy using, e.g., the H. fells-mouse model (see, e.g., Czinn et al . , supra ; Lee et al. , supra ) .

In the passive immunization methods of the invention, antibodies (e.g., monoclonal antibodies) which recognize Helicobacter ClpB are administered to a mucosal (e.g., oral or intragastric) surface of a mammal. The amount of antibody to be used in this method is readily determined by one skilled in the art.

The ClpB polypeptides, nucleic acids, and antibodies of the invention may also be used for detecting the presence of anti-tfelicoJbaσter antibodies, Helicobacter nucleic acids, or Helicobacter polypeptides, respectively, in biological samples, using standard methods (e.g., Western blot analysis, ELISA, and nucleic acid hybridization methods; see, e.g., Ausubel et al . , supra ; Coligan et al . , supra) . Thus, nucleic acid fragments, e.g., RNA or DNA fragments of at least 10, preferably at least 12, more preferably at least 15, and most preferably at least 18 nucleotides, which hybridize to nucleic acid which encodes Helicobacter ClpB, may be used in diagnostic methods, and are included in the invention.

Isolation of the Helicobacter ClpB Gene

The following methods were used to isolate the gene encoding Helicobacter ClpB.

Methods:

Preparation of Chromosomal DNA and Construction of a Genomic Library

Genomic DNA was prepared from H. pylori NCTC 11637 (ATCC accession number 43504) using the method described by Leying et al . (Mol. Microbiol. 6:2863-74, 1992). An expression library containing this H. pylori genomic DNA was constructed in the bacteriophage vector Lambda Zap II Express (Stratagene, Inc. , Cambridge, UK) by ligating size-selected (2-10 kb) Sau3A partially-digested fragments of the genomic DNA into Bamffl-digested lambda arms. The ligated DNAs were then packaged into phage heads in vitro. Analysis of a random selection of clones showed that the average insert size was 4.5 kb.

Preparation of Antiserum

Rabbit antiserum was prepared against H. pylori by standard methods using live, whole cells of H. pylori Roberts strain as antigens (Luke et al . , FEMS Microbiol. Lett. 71:225-230, 1990; Roberts strain was obtained from Dr. C. Penn, School of Biological Sciences, University of Birmingham, Birmingham, UK) . Screening of the Gene Library

The bacteriophage library was plated on E. coli

XLl-Blue MRF (Stratagene, Ltd. , Cambridge, UK) to give approximately 1.4 x IO³ plaques per 90 mm plate (4.0 x IO³ plaques in total) . The plaques were lifted onto nitrocellulose and screened with a 1:200 dilution of anti-H. pylori whole cell antiserum, using standard methods (see, e . g. , Maniatis et al . Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) . Immunodetection was performed using a mouse anti-rabbit IgG alkaline phosphatase-conjugated antibody (Sigma Chemical Co. ,

St. Louis, MO) .

In Vivo Excision of Recombinant Plasmid pBK-CMV from Lambda Zap II Express

In vivo excision of recombinant pBK-CMV from Lambda Zap II Express was carried out according to the methods provided by the manufacturer (Stratagene, Ltd. , Cambridge, UK). E. coli XLOLR (Stratagene, Ltd., Cambridge, UK) was used for plating excised phagemids. SDS-PAGE and immunoblotting

E. coli XLOLR clones were harvested from LB broth cultures, washed in PBS, and resuspended in cracking buffer (49 mM Tris, pH 6.7, containing 2.5% SDS, 1.3% 2- mercaptoethanol, 5% glycerol, and 0.002% bromophenol blue) . Samples were boiled for 5 minutes, and 1-2 μg of protein was applied to an SDS polyacrylamide gel. SDS- PAGE and immunoblotting were carried out using standard methods (see, e.g., Maniatis et al . , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) . Creation of Nested Deletions

Nested deletions were created using the Erase-A- Base kit (Promega Corporation, Madison, WI) , according to the instructions provided by the manufacturer. Hybridization

A l.l kb internal Hindlll fragment was excised from low melting point agarose and radiolabelled with ³ P- dCTP (Amersham, UK) using the Megaprime kit (Amersham, UK) , according to the instructions provided by the manufacturer. This fragment was used to probe Haelll- digested genomic DNA from both cagA+ and cagA- strains of H. pylori (H . pylori strains 84, 114, 20, 74, and 113 from St. Bartholomew's Hospital, London; and strains 945 and 781 from St. James's Hospital, Dublin) . Results: Screening of the Library and Identification of CP6

A total of 4 x IO³ bacteriophages were screened, and 54 clones which reacted with the antiserum were identified. One clone, designated CP6, which expressed an immunoreactive polypeptide of approximately 87 kD, was selected for further study. Plasmid DNA (designated pCP6) was rescued from this clone, as is described above, and was found to contain an insert of 3.2 kb. Localization of the Gene and DNA Sequence Analysis

A series of nested deletions were created in CP6 with insert sizes ranging from 3-0.3 kb. Immunoblot analysis revealed that clones with insert sizes of less than 3 kb no longer expressed the 87 kD protein, indicating that most of the insert DNA contained coding sequence. Analysis of the Nucleotide Sequence

A total of 2,755 basepairs of the insert were sequenced. An open reading of 2,571 basepairs was identified, coding for a protein of 857 amino acids. The predicted molecular weight of this protein is 94 kD, which is close to the estimate of 87 kD, which was based on electrophoretic mobility. A schematic representation of the nucleotide sequence of the gene is shown in Fig. 1 (SEQ ID NO:l). Numbering starts at the beginning of the insert. The structural gene has an ATG initiation codon at position 126 and a stop codon at position 2695. A potential ribosome binding site is present six bases upstream from the start codon. The regions with the closest homology to the E. coli σ70 promoter sequences were identified (TTGAGA at position 5 and TATTTT at position 26) . The G+C content of the gene is 41%, which is slightly higher than the G+C content (34 to 37%) reported for the entire H. pylori genome (Maniatis et al . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) . Analysis of the Predicted Amino Acid Sequence

Comparison of the predicted amino acid sequence with known protein sequences on the Swiss protein database (using the BLAST program; see, e.g., Ausubel et al . , supra) showed a high degree of homology with the Clp family of proteins which are thought to function as molecular chaperones and as ATPase subunits of ATP- dependent proteases. Several of these proteins are listed in Table 1. The Clp proteins share two large, highly conserved sequence blocks, each centered around a nucleotide-binding domain. Alignment of the H . pylori predicted amino acid sequence with these proteins showed that the H. pylori sequence has a high level of identity with the Clp proteins in the two conserved sequence blocks (Table 1) and also contains two potential nucleotide binding regions. Hybridization analysis

DNA fragments from 7 different strains of H. pylori (3 cagA+ and 4 cagA-) hybridized with a CP6 probe, and marked restriction fragment length polymorphism was observed. Table 1. Clp proteins related to the H . pylori ClpB amino acid sequence

Organism Protein name Length % identity with (No. of amino acids) H. pylori predicted amino acid sequence (in conserved blocks)

Escherichia coli ClpB 857 65

Bacteroides nodosus ClpB 860 65

Trypanosoma brucei Clp 868 61

Pisum sativum (pea) ClpA 922 63

Ly coper si con CD4B 923 63 00 esculentum (tomato) I

Ly coper sicon CD4A 926 63 esculentum (tomato)

Saccharomyces HSP7 811 62 cerevisiae

Saccharomyces MSP104 908 50 cerevisiae

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: OraVax, Inc. (ii) TITLE OF INVENTION: HELICOBACTER CLPB (iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson P.C.

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/499,222

(B) FILING DATE: 07-JUL-1995

(C) CLASSIFICATION:

(viii) ATTORNE /AGENT INFORMATION:

(A) NAME: CLARK, PAUL T.

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 06132/017WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617/542-5070

(B) TELEFAX: 617/542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2755 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AATATTGAGA GTTGTCTAAA AAGCGTATTT TGGAGAGTYT GTCATTAATG TGAGCGTTTT 60

AAAAACCTTT GAGGGTTAAA ATAGTGTAAA ATAGTAAAGA TTTTAAAACT CAAAAAGGAT 120

TGATAATGAA TTTATTTGAA AAAATGACTG ACCAATTGCA TGAGGCTTTA GACAGCGCGC 180

TCGCTTTAGC TTTACACCAT AAAAACGCTG AAGTAACGCC CTTGCACATG CTTTTTGCCA 240

TGCTCAATAA CTCCCAAGGC ATCCTCATTC AAGCCTTACA AAAAATGCCT GTGGATATTG 300 AAGCTTTAAA ACTTAGCGTT CAAAGCGAGC TGAATAAGTT TGCTAAAGTT TCACAAATCA 360

ATAAGCAAAA TATCCAATTA AACCAAGCTC TAATCCAAAG TTTAGAAAAC GCTCAAGGCT 420

TGATGGCTAA AACGGGCGAT TCTTTCATCG CTACCGATGT GTATCTTTTG GCGAACATGA 480

GCCTTTTTGA AAGCGTTTTA AAGCCTTATT TAGACACTAA GGAATTGCAA AAAACTTTAG 540

AATCTTTAAG AAAAGGCGCG ACTATTCAAG GTAAAAGCGA TGATTCTAAT TTGGAAAGTT 600

TAGAAAAATT TGGCATTGAT TTGACGCAAA AAGCCTTAGA AAATAAGCTG GATCCCGTGA 660

TCGGAAGAGA TGAAGAAATC ATTCGCATGA TGCAAATTTT GATCAGAAAA ACAAAAAATA 720

ACCCTATTTT ACTGGGCGAG CCTGGAGTGG TGAAAACGGC GGTTGTGGAA GGGTTAGCCC 780

AACGCATTGT GAATAAGGAA GTGCCTAAAA CGCTTTTAAA CAAACGAGTC ATCGCTTTAG 840

ATTTAAGCTT GTTGGTGGCT GGAGCGAAAT ACAGAGGCGA GTTTGAAGAG CGCCTGAAAA 900

AGGTGATTGA AGAAGTTAAA AAAAGCGCGA ATGTGATTTT ATTCATTGAT GAAATCCACA 960

CGATCGTAGG GGCTGGGGCT AGTGAGGGGG GCATGGATGC GGCTAATATT TTAAAACCCG 1020

CGCTCGCTAG GGGGGAATTG CACACGATTG GAGCGACCAC CTTGAAAGAA TACCCCAAGT 1080

ATTTTGAAAA AGACATGGCG CTACAAAGGC GTTTCCAACC CATTTTACTC AATGAGCCTA 1140

GCATCAATGA AGCTTTACAG ATTTTAAGGG GGTTAAAAGA AACTTTAGAA ACGCACCATA 1200

ATATCACCAT CAATGACTCC GCGCTCATAG CGAGCGCTAA ACTCTCTAGC CGTTATATCA 1260

CCGATAGGTT TTTACCCGAT AAAGCGATTG ATTTGATTGA TGAGGGGGCG GCTCAATTAA 1320

AAATCCAAAT GGAATCAGAG CCGGCCAAAC TCTCTAGCGT GAAGCGCTCC ATTCCAAGAC 1380

TGGAAATGGA AAAACAAGCC CTTGAAATGG AAAAAAAGGA AAGCAACCAT AAACGCATGC 1440

AAGAAATCCT TAAAGAATTG AGCGATTTGA AAGAAGAAAA AATCCAATTA GAAGCGCAAT 1500

TTGAAAACGA AAAAGAAGCG TTCAAAGAAA TTTCACGCTT GAAAATGGAA ATGGAAAGCT 1560

TGAAAAAAGA GGCTGAGAGG TTTAAGCGCA ATGGGGATTA CCAGCAAGCG GGTGAAATTG 1620

AATACTCTAA AATCCCTGAA AATHAAAAGA AAGAAGAAGA ATTGCAACGT AAATGGGAAG 1680

CGATGCAACA AAACGGGGCG TTGTTGCAAA ACGCTTTAAC CGAAAACAAC ATCGCTGAGA 1740

TCGTGAGCCA ATGGACGCAT ATCCCGGTCC AAAAAATGCT CCAAAGCGAA AAAAATAGGG 1800

TTTTAAACAT TGAAAGCGAA TTGCAAAAAA GAGTGGTGGG GCAAGAAAAA GCGATCAAAG 1860

CGATCGCTAA AGCGATTAAA AGGAATAAGG CCGGACTTAG CGATAGCAAT AAACCCATAG 1920

GGAGTTTCCT CTTTTTAGGG CCAACAGGCG TGGGTAAAAC CGAGAGCGCT AAAGCCTTGG 1980

CGCAATTCTT GTTTGATAGC GATAAAAATC TTATAAGAAT TGACATGAGC GAATATTTGG 2040

AAAAGCATGC CATAAGCCGT CTTATTGGGC CCGCTCCTGG GTATGTGGGC TATGAAGAAG 2100

GCGGGCAGTT GACCGAAGCG GTGCGCAGAA AGCCTTATAG CGTGGTGCTG TTAGATGAAG 2160

TGGAAAAAGC CCATCCAGAT GTGTTTAACC TCTTGTTGCA GGTTTTAGAT GAAGGGCATT 2220 TAACCGATAG TAAGGGCGTG AGGGTGGATT TCAAAAACAC GATTTTGATT TTAACTAGCA 2280

ATGTAGCTAG CGGCGCGCTT TTGGAAGAAA ATTTGAGCGA AGCCGCCAAA CAAAAAGCGA 2340

TTAAAG GAG CTTGAGCAAT TCTTCAAGCC GGAATTTTTA AACCGCTTAG ATGAAATCAT 2400

CTCCTTTAAC GCCCTAGATA GTCATGCTGT CATTAATATC GTGGGGATAC TCTTTGAAAA 2460

CATTCAAAAA AAAGCGCTTG AAAGGGGCAT TAATATAACT TTAGACGAAG AGGCAAAAGA 2520

ATTGATCGCT GAAGCGGGAT TTGACAGATT TTATGGCGCT AGACCCCTAA AGCGTGCACT 2580

CTATGAAATG GTAGAAGACA AGCTCGSTGA ACTCATTTTA GAGGATAAAA TTAAAGAGAA 2640

TGGSAGCGKG GGATYWGYGG CAGAAMATCA CGAGATTGTG CCTAAGATTA AGTGAAGTCT 2700

GGCTATCCTC AAAANTAAGA AATGGTCATT TTGRGGAAAA GGATTGCAAT GATGT 2755

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 751 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asn Leu Phe Glu Lys Met Thr Asp Gin Leu His Glu Ala Leu Asp 1 5 10 15

Ser Ala Leu Ala Leu Ala Leu His His Lys Asn Ala Glu Val Thr Pro 20 25 30

Leu His Met Leu Phe Ala Met Leu Asn Asn Ser Gin Gly Ile Leu Ile 35 40 45

Gin Ala Leu Gin Lys Met Pro Val Asp Ile Glu Ala Leu Lys Leu Ser 50 55 60

Val Gin Ser Glu Leu Asn Lys Phe Ala Lys Val Ser Gin Ile Asn Lys 65 70 75 80

Gin Asn Ile Gin Leu Asn Gin Ala Leu Ile Gin Ser Leu Glu Asn Ala 85 90 95

Gin Gly Leu Met Ala Lys Thr Gly Asp Ser Phe Ile Ala Thr Asp Val 100 105 110

Tyr Leu Leu Ala Asn Met Ser Leu Phe Glu Ser Val Leu Lys Pro Tyr 115 120 125

Leu Asp Thr Lys Glu Leu Gin Lys Thr Leu Glu Ser Leu Arg Lys Gly 130 135 140

Ala Thr Ile Gin Gly Lys Ser Asp Asp Ser Asn Leu Glu Ser Leu Glu 145 150 155 160

Lys Phe Gly Ile Asp Leu Thr Gin Lys Ala Leu Glu Asn Lys Leu Asp 165 170 175 Pro Val Ile Gly Arg Asp Glu Glu Ile Ile Arg Met Met Gin Ile Leu 180 185 190

Ile Arg Lys Thr Lys Asn Asn Pro Ile Leu Leu Gly Glu Pro Gly Val 195 200 205

Val Lys Thr Ala Val Val Glu Gly Leu Ala Gin Arg Ile Val Asn Lys 210 215 220

Glu Val Pro Lys Thr Leu Leu Asn Lys Arg Val Ile Ala Leu Asp Leu 225 230 235 240

Ser Leu Leu Val Ala Gly Ala Lys Tyr Arg Gly Glu Phe Glu Glu Arg 245 250 255

Leu Lys Lys Val Ile Glu Glu Val Lys Lys Ser Ala Asn Val Ile Leu 260 265 270

Phe Ile Asp Glu Ile His Thr Ile Val Gly Ala Gly Ala Ser Glu Gly 275 280 285

Gly Met Asp Ala Ala Asn Ile Leu Lys Pro Ala Leu Ala Arg Gly Glu 290 295 300

Leu His Thr Ile Gly Ala Thr Thr Leu Lys Glu Tyr Pro Lys Tyr Phe 305 310 315 320

Glu Lys Asp Met Ala Leu Gin Arg Arg Phe Gin Pro Ile Leu Leu Asn 325 330 335

Glu Pro Ser Ile Asn Glu Ala Leu Gin Ile Leu Arg Gly Leu Lys Glu 340 345 350

Thr Leu Glu Thr His His Asn Ile Thr Ile Asn Asp Ser Ala Leu Ile 355 360 365

Ala Ser Ala Lys Leu Ser Ser Arg Tyr Ile Thr Asp Arg Phe Leu Pro 370 375 380

Asp Lys Ala Ile Asp Leu Ile Asp Glu Gly Ala Ala Gin Leu Lys Ile 385 390 395 400

Gin Met Glu Ser Glu Pro Ala Lys Leu Ser Ser Val Lys Arg Ser Ile 405 410 415

Pro Arg Leu Glu Met Glu Lys Gin Ala Leu Glu Met Glu Lys Lys Glu 420 425 430

Ser Asn His Lys Arg Met Gin Glu Ile Leu Lys Glu Leu Ser Asp Leu 435 440 445

Lys Glu Glu Lys Ile Gin Leu Glu Ala Gin Phe Glu Asn Glu Lys Glu 450 455 460

Ala Phe Lys Glu Ile Ser Arg Leu Lys Met Glu Met Glu Ser Leu Lys 465 470 475 480

Lys Glu Ala Glu Arg Phe Lys Arg Asn Gly Asp Tyr Gin Gin Ala Gly 485 490 495

Glu Ile Glu Tyr Ser Lys Ile Pro Glu Asn Xaa Lys Lys Glu Glu Glu 500 505 510 Leu Gin Arg Lys Trp Glu Ala Met Gin Gin Asn Gly Ala Leu Leu Gin 515 520 525

Asn Ala Leu Thr Glu Asn Asn Ile Ala Glu Ile Val Ser Gin Trp Thr 530 535 540

His Ile Pro Val Gin Lys Met Leu Gin Ser Glu Lys Asn Arg Val Leu 545 550 555 560

Asn Ile Glu Ser Glu Leu Gin Lys Arg Val Val Gly Gin Glu Lys Ala 565 570 575

Ile Lys Ala Ile Ala Lys Ala Ile Lys Arg Asn Lys Ala Gly Leu Ser 580 585 590

Asp Ser Asn Lys Pro Ile Gly Ser Phe Leu Phe Leu Gly Pro Thr Gly 595 600 605

Val Gly Lys Thr Glu Ser Ala Lys Ala Leu Ala Gin Phe Leu Phe Asp 610 615 620

Ser Asp Lys Asn Leu Ile Arg Ile Asp Met Ser Glu Tyr Leu Glu Lys 625 630 635 640

His Ala Ile Ser Arg Leu Ile Gly Pro Ala Pro Gly Tyr Val Gly Tyr 645 650 655

Glu Glu Gly Gly Gin Leu Thr Glu Ala Val Arg Arg Lys Pro Tyr Ser 660 665 670

Val Val Leu Leu Asp Glu Val Glu Lys Ala His Pro Asp Val Phe Asn 675 680 685

Leu Leu Leu Gin Val Leu Asp Glu Gly His Leu Thr Asp Ser Lys Gly 690 695 700

Val Arg Val Asp Phe Lys Asn Thr Ile Leu Ile Leu Thr Ser Asn Val 705 710 715 720

Ala Ser Gly Ala Leu Leu Glu Glu Asn Leu Ser Glu Ala Ala Lys Gin 725 730 735

Lys Ala Ile Lys Glu Ser Leu Ser Asn Ser Ser Ser Arg Asn Phe 740 745 750

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 116 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Arg Glu Leu Glu Gin Phe Phe Lys Pro Glu Phe Leu Asn Arg Leu Asp 1 5 10 15

Glu Ile Ile Ser Phe Asn Ala Leu Asp Ser His Ala Val Ile Asn Ile 20 25 30 Val Gly Ile Leu Phe Glu Asn Ile Gin Lys Lys Ala Leu Glu Arg Gly 35 40 45

Ile Asn Ile Thr Leu Asp Glu Glu Ala Lys Glu Leu Ile Ala Glu Ala 50 55 60

Gly Phe Asp Arg Phe Tyr Gly Ala Arg Pro Leu Lys Arg Ala Leu Tyr 65 70 75 80

Glu Met Val Glu Asp Lys Leu Xaa Glu Leu Ile Leu Glu Asp Lys Ile 85 90 95

Lys Glu Asn Xaa Ser Xaa Gly Xaa Xaa Ala Glu Xaa His Glu Ile Val 100 105 110

Pro Lys Ile Lys 115

What is claimed is:

Claims

1. A vaccine for preventing Helicobacter infection in a mammal, said vaccine comprising Helicobacter ClpB, or an immunogenic fragment or derivative thereof, formulated for administration.

2. The vaccine of claim 1, wherein said mucosal surface is intranasal.

3. The vaccine of claim 1, wherein said mucosal surface is oral.

4. The vaccine of claim 1, wherein said Helicobacter ClpB, or said immunogenic fragment or derivative thereof, comprises an adjuvant.

5. The vaccine of claim 4, wherein said adjuvant is selected from the group consisting of a cholera toxin, Escherichia coli heat-labile enterotoxin (LT) , and fragments and derivatives thereof having adjuvant activity.

6. A vaccine for treating Helicobacter infection in a mammal, said vaccine comprising Helicobacter ClpB, or an immunogenic fragment or derivative thereof, formulated for therapeutic administration to a mucosal surface of said mammal.

7. The vaccine of claim 6, wherein said mucosal surface is intranasal.

8. The vaccine of claim 6, wherein said mucosal surface is oral.

9. The vaccine of claim 5, wherein said Helicobacter ClpB, or said immunogenic fragment or derivative thereof, comprises an adjuvant.

10. The vaccine of claim 9, wherein said adjuvant is selected from the group consisting of a cholera toxin,

Escherichia coli heat-labile enterotoxin (LT) , and fragments and derivatives thereof having adjuvant activity.

11. A composition for preventing Helicobacter infection in a mammal, said composition comprising an antibody which recognizes Helicobacter ClpB, formulated for administration to a mucosal surface of said mammal.

12. The composition of claim 11, wherein said mucosal surface is oral.

13. A composition for treating Helicobacter infection in a mammal, said composition comprising an antibody which recognizes Helicobacter ClpB, formulated for administration to a mucosal surface of said mammal.

14. The composition of claim 13, wherein said mucosal surface is oral.

15. Substantially pure Helicobacter ClpB polypeptide.

16. The polypeptide of claim 15, wherein said polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 2.

17. Purified DNA encoding the polypeptide of claim 15.

18. The purified DNA of claim 17, wherein said purified DNA comprises a nucleotide sequence substantially identical to the nucleotide sequence shown in Fig. 2.

19. A vector comprising the purified DNA of claim 17.

20. A cell comprising the purified DNA of claim 17.

21. A vaccine comprising the polypeptide of claim 15 in a pharmaceutically acceptable carrier or diluent.

22. A method of producing a recombinant Helicobacter ClpB polypeptide, said method comprising the steps of: a. providing a cell transformed with DNA encoding said Helicobacter ClpB polypeptide, said DNA being positioned for expression in said cell; b. culturing said transformed cell under conditions for expressing said DNA; and c. isolating said recombinant Helicobacter ClpB polypeptide.

23. HelicoJacter ClpB polypeptide produced by the method of claim 22.

24. A substantially pure antibody that specifically binds Helicobacter ClpB polypeptide.

25. The substantially pure antibody of claim 24, wherein said antibody is a monoclonal antibody.

26. A method of detecting Helicobacter in a sample, said method comprising the steps of: a. contacting said sample with the antibody of claim 24; and b. detecting immune complexes formed between said antibody and said sample as an indication of the presence of Helicobacter in said sample.