EP0971734A1 - Carbohydrate antigens which immunoreact with polyclonal antisera against h. pylori - Google Patents

Abstract

Carbohydrate antigens which immunoreact with H. pylori carbohydrate specific polyconal antisera. Such antigens are produced by DNA contained in ATCC Deposit No. 98373. Such antigens are capable of inducing an immune response against H. pylori.

Description

CARBOHYDRATE ANTIGENS WHICH IMMUNOREACT WITH POLYCLONAL ANTISERA AGAINST H. pylori

This Application claims the benefit of the filing dates of U.S. Provisional Application 60/042,403 and 60/043,976 filed on March 26, 1997 and April 23, 1997, respectively.

This invention relates to antigens and the use and production thereof. More particularly, the present invention is directed to carbohydrate antigens which immunoreact with polyclonal antisera against H. pylori .

Still more particularly, this invention relates to carbohydrate antigens which are derived from H. pylori .

Helicobacter pylori (H. pylori) has been implicated as being a cause of peptic ulcer as a result of an infectious disease caused by H. pylori .

As a result, there has been an increasing interest in H. pylori antigens for potential use in the treatment and/or prevention of H. pylori infection.

According to one aspect of the present invention, there is provided antigens, as well as polynucleotides encoding polypeptideε, which produce such antigens, which antigens immunoreact with H. pylori antisera.

In accordance with another aspect of the present invention, there is provided carbohydrate antigens, as well as fragments and analogs thereof , each of which is immunoreactive with H. pylori carbohydrate specific polyclonal antisera. A procedure for producing such an antisera is described in the Example.

The antigens of the present invention are produced in bacteria by polypeptides encoded by DNA present in clones which are part of ATCC Deposit No. 98373.

As known in the art, bacteria produce carbohydrates, and such carbohydrates are produced in the bacteria by so- called gene clusters (a plurality of genes) which express polypeptides which interact in the bacteria to produce carbohydrates .

The present invention, in part, is directed to polynucleotides which form a gene cluster or operon, which, in bacteria, in particular E. coli . express polypeptides which in the bacteria produce carbohydrate antigens which immunoreact with H. pylori specific antisera. The gene cluster or operon is one which is recovered from H. pylori .

The material deposited as ATCC Deposit No. 98373 on March 26, 1997, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, is a mixture of clones Ml, M2 and M3, each of which is E. coli transformed with Supercos 1 (Stratagene) vector, which vector has inserted therein at the Bam HI restriction site, DNA recovered from H. pylori , which DNA includes a gene cluster which in E. coli produces an antigen which immunoreacts with H. pylori carbohydrate specific polyclonal antisera.

Thus, in accordance with an aspect of the present invention there is provided antigens which immunoreact with H. pylori carbohydrate polyclonal antisera, which antigens are produced by clones Ml, M2 and M3 contained in the deposited material.

All three clones exhibited a mucoid colony phenotype which was lost following deletion of cloned H. pylori DNA from the cosmids, suggesting that they express a recombinant surface exposed exopolysaccharide (rEPS) . The mucoid phenotype is dependent on the environmental conditions during growth; colonies express more rEPS when grown in reduced oxygen or at 30°C or in the presence of glycerol .

Results of transmission electron microscopic studies of M₂ and M₃ clones revealed that numerous small blebs extruding from the cell surface. E. coli had a smooth cell surface with no detectable surface blebs .

Alcian blue fixing and silver staining of PAGE gels revealed high molecular weight carbohydrate which is visible in the stacking gel produced by the clones . Finally, chemical composition analysis of exopolysaccharides derived from the clones, E. coli and H. pylori indicated that the mole percent of fucose and mannose and the fucose/mannose ratio from M,, M₂ and M₃ clones resembles that of H. pylori , rather than E. coli .

The deposit (s) have been made under the terms of the Budapest Treaty on the International Recognition of the deposit of micro-organisms for purposes of patent procedure. The strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent . These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit be required under 35 U.S.C. §112. The sequences of the polynucleotides contained in the deposit materials, as well as the amino acid sequences of the polypeptides encoded thereby, are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

Thus, in one aspect, the present invention is directed to certain antigens produced by a clone deposited as ATCC Deposit No. 98373, which antigens immunoreact with H. pylori carbohydrate specific polyclonal antisera, as well as fragments and/or analogs of such antigens, as well as the polynucleotides forming a gene cluster which produce such antigens, analogs and fragments, which polynucleotides may be DNA and/or RNA.

The DNA may be double-stranded or single-stranded, and if single stranded, may be the coding strand or non-coding (anti-sense) strand. The coding sequence of the gene cluster may be identical to that of a deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides which generate the carbohydrate antigens which immunoreact with H. pylori antiserum as the DNA of the deposited DNA, or an immunoreactive analog or fragment of such antigen.

The polynucleotide which is the gene cluster which generates an antigen which reacts with H. pylori antiserum may include : only the coding sequence , such coding sequence and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence (and optionally additional coding sequence) and non-coding sequence, such as sequence 5' and/or 3' of the coding sequence or a fragment or portion of such polynucleotide which forms the gene cluster.

Thus, the term "polynucleotide encoding a polypeptide" or "polynucleotide forming a gene cluster" encompasses a polynucleotide which includes only coding sequence as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which form a gene cluster which produces fragments, analogs and derivatives of the carbohydrate antigens which react with H. pylori antisera. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide . The present invention also relates to polynucleotide probes constructed from a portion of the polynucleotide of the invention.

Fragments of the polynucleotides of the present invention may be used as a hybridization probe for a library to isolate the full length DNA and to isolate other DNAs which have a high sequence similarity to the genes or portion of the genes forming the gene cluster. Probes of this type preferably have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases. The probe may also be used to identify a DNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promoter regions. An example of a screen comprises isolating the coding region of a gene by using a DNA sequence of a deposited clone, which is part of the operation to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of a polynucleotide of the present invention are used to screen a library of genomic DNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove- described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences in a complementary sense . The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity in a complementary sense between the sequences.

Alternatively, the polynucleotide may have at least 15 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to any part of a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% identity and more preferably at least a 95% identity to one or more polynucleotides which form the gene cluster for generating a carbohydrate antigen which immunoreacts with H. pylori antisera as well as fragments thereof, which fragments have at least 15 bases, preferably at least 30 bases and most preferably at least 50 bases, which fragments are at least 90% identical, preferably at least 95% identical and most preferably at least 97% identical to any portion of a polynucleotide of the present invention.

As used herein, "identity" or "identical," when comparing a second sequence to a first sequence, means that the two sequences are properly aligned and then the second sequence is compared with the first sequence over the length of the first sequence. If any base of the first sequence does not match an aligned base in the second sequence or if the second sequence or the first sequence over the compared aligned length has a base which is not aligned with a base of the other sequence, then each of such occurrences is counted as a difference . The percent identity is then calculated by use of a fraction having as a numerator the difference between the number of bases in the first sequence and the number of base differences and as a denominator the number of bases in the first sequence.

The present invention further relates to antigens which immunoreact with H. pylori antisera and which is produced by one of the deposited clones, as well as fragments, analogs and derivatives of such antigen.

The antigens and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally-occurring polynucleotide or antigen present in a living animal is not isolated, but the same polynucleotide or antigen, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or antigens could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment .

The antigens of the present invention include the immunoreactive antigens produced by DNA of the deposited clones as well as fragments and derivatives thereof which immunoreac ive with H. pylori antigen.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with polynucleotides or vectors of the invention and the production of antigens of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transforτnants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing antigens of the invention by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; etc. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures . In general , the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: the E. coli. lac or trp, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic cells. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampi- cillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomvces , Bacillus subtilis . The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence . Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; pQE70, pQE60, pQE-9 (Qiagen) , pDIO, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

In a further embodiment, the present invention relates to host cells containing the above-described constructs, in particular a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .

The antigens of the present invention, which immunoreact with H. pylori carbohydrate polyclonal antisera, and which are produced by a deposited clone, as well as their fragments, derivatives or analogs, may be used as an immunogen to produce antibodies against H. pylori . These antibodies can be, for example, polyclonal or monoclonal antibodies . The present invention also includes chimeric, humanized, and single-chain antibodies, as well as fragments, thereof. Such antibodies may be produced by procedures generally known in the art .

The antigens or antibodies generated against the antigens may be employed in immunoassays of the type known in the art for detecting the presence of H. pylori and/or antibodies against H. pylori .

The antigens may be employed to induce in a host an immune reaction against H. pylori , preferably in the form of an immunogen wherein the antigen is combined with an appropriate protein.

In accordance with a preferred embodiment in order to improve the immunogenic response to the antigen, the antigen is conjugated to a protein which is known to induce a CD4 T-cell response. Such proteins are known in the art and have been previously employed in the art to induce a T cell response and thereby T-cell dependent memory. Preferred proteins are diphtheria or tetanus toxoids but other proteins having such properties are within the scope of the invention and should be apparent to those skilled in the art from the teachings herein. As known in the art, the carbohydrate antigen may be directly conjugated to the protein or through an appropriate linker or spacer.

Thus, in accordance with another aspect of the present invention, the antigens of the present invention, or polynucleotides forming a gene cluster which produce in bacteria the antigens of the invention may be employed to induce an immune response, in a host, against H. pylori . The host is a mammal, which may be human or non-human. Such antigens and polynucleotides are preferably employed to induce a protective immune response against H. pylori infection and, therefore, may be employed for treatment and/or prevention of H. pylori infection.

An antigen of the present invention, when used for such a purpose, is generally combined with a pharmaceutically effective carrier and is administered in an amount effective to reduce and/or prevent H. pylori infection. In general, such polypeptide would be administered in an amount from 10 μg to 500 mg, and such administration, for example, may be parenteral, oral or intranasal administration.

In accordance with another aspect of the present invention, a polynucleotide, forming a gene cluster of the present invention is employed to produce a recombinant, non-toxigenic organism; in particular, a bacterial organism, which produces an antigen of the present invention. As representative examples of such organisms, there may be mentioned V. cholerae and S . typhi . Thus, for example, a wild-type strain of V. cholerae or S. typhi may be attenuated by procedures known in the art to produce a live bacterial strain which has been attenuated to render the bacteria non-toxigenic. Such non-toxigenic recombinant strains may be employed as a live vaccine for producing, in vivo, an antigen of the present invention to induce a protective immune response.

Although the present invention will be further described with respect to the use of V. cholerae, it is to be understood that such teachings are equally applicable to other organisms .

The non-toxigenic V. cholerae which is employed for producing a recombinant non-toxigenic V. cholerae in accordance with the present invention is V. cholerae of the type which has been developed for potential use as a vaccine against V. cholerae. As known in the art, such V. cholerae is a live, attenuated V. cholerae which has been attenuated to render such V. cholerae non-toxigenic. The V. cholerae which is employed for producing a recombinant vaccine in accordance with the present invention may be an attenuated 01 strain or a non-01 strain.

As known in the art, wild-type V. cholerae strains are attenuated for example, to render same non-toxigenic by a genetic deletion which removes three toxins (cholerae toxin, zonula occludens toxin, and accessory cholerae enterotoxin) , and which may be further attenuated by removal of a colonization factor (core encoded pilin) , and RSI (a site-specific toxin acquisition cassette) . Such attenuated V. cholerae strains have been designated as Peru-2 and Bengal-2.

In another attenuated strain, the strains hereinabove described were engineered to reintroduce into the recA locus the gene encodings ctxB, which yields the strains referred to as Peru-3 and Bengal-3.

Another non-toxigenic strain of V. cholerae which is a spontaneous non-motile filamentous isolate of Peru-3 has also been isolated as designated as Peru-14. Non-motile isolates of Peru-3 and Bengal-3 have also been identified which have been designated as Peru-15 and Bengal-15.

Non-toxigenic strains of V. cholerae are generally known in the art, and such strains may be employed in the present invention. Such strains include but are not limited to: Bengal-2, Bengal -3 , Bengal -15, Peru-2, Peru- 3, Peru-14 , Peru-15, Bah-2, Bah-15, Bang-2, Bang-3 , and Bang- 15.

In accordance with an aspect of the present invention, a non-toxigenic strain of V. cholerae is transformed with a gene cluster of the invention which produces in the organism a carbohydrate antigen which induces an immune response against H. pylori. The polynucleotide is incorporated into a suitable vector for transformation of the V. cholerae. Such vector may be one which integrates the polynucleotide into the chromosome of V. cholerae or may be one which provides for extrachromosomal transformation. Thus, the present invention contemplates a recombinant non-toxigenic V. cholerae in which a polynucleotide gene cluster produces a carbohydrate antigen which induces an immune response against H. pylori is present either extrachromosomally or integrated into the chromosome of the V. cholorae.

Thus, in accordance with the present invention, a polynucleotide gene cluster of the invention is placed in an appropriate vector, and such vector is employed for transforming a non-toxigenic strain of V. cholerae to provide a vaccine for use in the treatment and/or prevention of H. pylori infection.

The vector is provided with an appropriate expression promoter. The vector which is employed is one which functions in V. cholerae to provide for expression of the polypeptides which in the organism produce the carbohydrate antigen which induces an immune response against H. pylori . The promoter, in a preferred embodiment, is derived from V. cholerae. A preferred promoter is a V. cholerae heat shock promoter known as htpGp . The vector or plasmid generally also includes a suitable marker to permit selection of recombinant strains .

Non-toxigenic V. cholerae which has been transformed to produce a carbohydrate antigen which provides an immune response against H. pylori and in particular a protective immune response may be employed as an oral vaccine for treatment and/or prevention of H. pylori infection. The vaccine is employed in an amount effective to treat or prevent H. pylori infection. In general, the recombinant V. cholerae is administered in a dosage of from about 10⁷ to 10⁸ colony forming units. The vaccine may be administered in a single dose or in multiple doses; e.g., biweekly.

The recombinant V. cholerae in accordance with the present invention may be incorporated into an appropriate pharmaceutically acceptable carrier and in particular for oral delivery. The carrier may be a liquid; e.g., and mixture of bicarbonate and ascorbate or a solid such as an enteric coated capsule.

As hereinabove indicated, the non-toxigenic V. cholerae is transformed by use of an appropriate vector, which in one embodiment integrates the polynucleotide expressing the appropriate antigen or immunogen into the chromosome of the V. cholerae . Such vector or plasmid may be employed to facilitate site specific integration onto the chromosome; for example, at either the lacZ, recA or irgA loci (Published PCT Application WO 94/01780) .

In accordance with a preferred embodiment, when integration is desired, the vector is capable of inserting the polynucleotide into the V. cholerae chromosome at the lacZ locus .

Integrating vectors may also be employed for providing transformation in which the appropriate polypeptides are expressed from extrachromosomal DNA. Thus, after the transformation, an appropriate selection procedure is employed to select V. cholerae in which the polynucleotide has been integrated into the chromosome or V. cholerae in which the DNA is maintained as an extrachromosomal plasmid.

Thus, for example, in one embodiment, an attenuated V. cholerae in which the recA gene is deleted is used for preparation of a recombinant V. cholerae in which the polypeptides are expressed from an extrachromosomal plasmid.

In another embodiment, an attenuated V. cholerae which includes the recA gene is used for preparation of a recombinant V. cholerae in which the polypeptides are expressed from DNA integrated into the V. cholerae chromosome .

In both cases, the same vector may be employed, e.g., a plasmid which includes a lacZ sequence. In the case where the recA gene is deleted, the polynucleotide is not integrated into the chromosome; however, when such vector is used for transformation of attenuated V. cholerae which includes recA gene, integration can occur at the lacZ locus and a recombinant V. cholerae in which the DNA is integrated at the lacZ locus can be selected.

Thus in accordance with an aspect of the present invention, there is provided a product and a process for treatment and/or prevention of H. pylori infection wherein a non-toxigenic V. cholerae strain is transformed with a polynucleotide which encodes a polypeptide of the invention which provides an immune response against H. pylori . Such recombinant non-toxigenic V. cholerae strain may be employed as a live oral vaccine for the treatment and/or prevention of H. pylori infection.

In accordance with the present invention, more than one copy of a polynucleotide encoding a polypeptide which induces an immune response may be integrated into the chromosome of V. cholerae . Thus, for example, such polynucleotide may be integrated into more than one locus in the chromosome; e.g., lacZ, recA, and irgA.

Such recombinant products may also be employed in vi tro to produce the carbohydrate antigen.

The invention will be further described with respect to the following example; however, the scope of the invention is not to be limited thereby:

EXAMPLE Characterization of H. pylori Claudio.

H. pylori Claudio strain was streaked on Trypticase Soy agar supplemented with 5% defibrinated sheep blood and incubated at 37°C for 48 to 72h in an anaerobic chamber. Cells were collected from plates with a sterile swab and suspended in l ml Brucella broth. The broth culture was deemed pure by: i) colonial morphology, ii) wet mount microscopy, iii) Gram stain, iv) urease⁺, and, v) agglutination of cells with anti-whole-cell helicobacter polyclonal rabbit sera. The characterized Claudio strain was cell-banked in 10% glycerol and stored in Cryovials in liquid nitrogen.

Harvesting DNA and carbohydrate from Claudio Two hundred ml Brucella broth supplemented with 5% fetal calf serum, TVP antibiotics (Trimethoprim, Vancomycin and Polymixin-B) and anti-fungal Amphotericin, was inoculated with the characterized Claudio cell suspension to OD₆₀₀= 0.05-0.1. The air in the flask was replaced with a gas mixture containing 6% 0₂, 10% C0₂, and 84% N₂. The flask was incubated for 18-24 h at 37°C with shaking (150 rpm) . Cells from this preparation were used to harvest chromosomal DNA (2) and polysaccharide by the method of Westphal and Jann (3) . Chromosomal DNA was digested with Sau3A and BamHI and electrophoresed on 0.7% agarose gel . Southern blot analysis of chromosomal DNA probed with a H. pylori ureAB gene fragment exhibited a strong hybridization signal indicating that the chromosomal DNA was H. pylori in origin. Carbohydrate was prepared by hot phenol- water extraction and purified by ultra-centrifugation. SDS- PAGE/silver staining analysis revealed 0-antigen banding patterns very similar to those observed by Mills et al (1) . Polysaccharide was quantitated by dry weight in order to determine polysaccharide amounts in subsequent immunization and ELISA experiments . Western blot analysis demonstrated that Claudio polysaccharide cross reacted with antibody derived from polyclonal antiserum raised against H. pylori whole-cells in mice. Coomassie stain of a replica gel detected no contaminating protein.

Cosmid cloning of Claudio chromosomal DNA.

A DNA cosmid library of H. pylori Cl chromosomal DNA was made using the SuperCosl cosmid vector (Stratagene) . Chromosomal DNA was partially digested with Sau3A for 3 min. and phosphatased. Vector SuperCosl cosmid DNA was prepared by digestion with Xbal, phosphatase treatment, and digestion with BamHI . Insert and vector DNA were ligated at 4°C overnight and packaged into Gigapacklll Gold packaging extract per manufacturers instructions. Titration of the unamplified cosmid library in E. coli XL1B- MR was 3.4xl0⁴cfu/ml. Amplification of the cosmid library generated clones at 5xl0⁷ cfu/ml. The efficiency of packaging was calculated and in accord with the value indicated in the manufacturer's manual.

Rabbit immunization with H. pγlori Claudio carbohydrate.

A rabbit was immunized intramuscularly with the characterized, purified Claudio polysaccharide. The priming dose was 50ug polysaccharide followed by 3 boosts of lOOug polysaccharide over a period of 2 weeks. The sera was adsorbed twice with E. coli XLIB-MR strain (host strain used to generate the Claudio DNA cosmid library) .

Screening the H. pylori cosmid library using H. pylori- specific polyclonal anti-sera.

Approximately 500,000 cfu were lysed (3% BSA/400 ug/mL lysozyme/1 U/mL DNase in 50 mM Tris-HCl/150 mM NaCl/5 mM MgCl₂) and digested with Proteinase-K before the immunological screening. Anti-Cl polysaccharide polyclonal rabbit sera at 1:15 dilution was used to screen the library by colony immunoblot and an anti-rabbit IgG/HRPO was used as the secondary antibody/conjugate. In the initial screen, a total of 27 immunoreactive colonies were enriched to yield single colonies . Six single colonies maintained cross-reactivity with the polyclonal rabbit sera after passaging on Luria Bertani (LB) agar plate. Those six colonies were streak plated for individual colonies . Nine samples from each of the six immunopositive clones were patched on LB agar plates and re-screened. All the nine progeny patches of the recombinants M₂ and M₃ were immunoreactive whereas only 1/9 of M, was positive. Three cross reactive clones (M₄-M₆) failed to rescreen immunopositive. M,, M₂, and M₃, putative carbohydrate clones, were stored in LB broth supplemented with 10% glycerol at -80°C.

Characterization of the H. pγlori immunoreactive cosmid recombinants M_t , M, and M_?

1. Colonial morphology and cell motili ty: When subcultured on LB (50ug/ml ampicillin) agar plates, all 3 clones, M,, M₂, and M₃ were mucoidy in appearance. In addition, M,_₃ exhibited significantly slower motility when grown in broth culture versus the control E. coli host strain.

2. Western blot analysis of whole-cell lysates : Whole- cell lysates, digested with Proteinase-K, were prepared from cosmid bearing clones grown in broth culture and also from cells grown on agar plates. Western blot analysis using the polyclonal rabbit sera was performed. No reproducible differences were observed between the E. coli host strain and the recombinant clones .

3. Silver staining resul ts : Polysaccharide was prepared from cosmid recombinants by the hot water-phenol extraction procedure (3) . Briefly, cells grown either in broth culture or on agar plates were treated with hot water-phenol, dialyzed, and lyophilized . Results of silver staining of polysaccharide harvested from washed cells grown in LB broth or unwashed cells grown on agar plates indicated no reproducible difference between E. coli and the cosmid recombinants .

4. Hemagglutination assays : The mucoid nature of the cosmid-bearing clones grown on agar plates suggested that these clones might produce an extra-cellular polysaccharide (EPS) . Polysaccharides of several Gram-negative bacteria have been shown to adhere to mammalian cells, including erythrocytes (RBC) . This property is considered a virulence feature of pathogens . Bacteria in which adherence to cells has been shown to be mediated by external polysaccharides include Shigella dysenteriae type 1 (4) , Campylobacter jejuni (5) , Vibrio cholerae (6) , Escherichia coli (7) .

We employed a hemagglutination (HA) assay to further characterize the recombinants. HA of 0.5% sheep RBC (sRBC) occurs if whole cells or supernatant fractions of H. pylori Cl are added to RBC. Washed whole cells of the recombinant clones were HA negative. However, supernatants or purified EPS of the recombinant clones derived from cells grown on LB agar plates and then suspended in PBS are HA positive. The HA titer was determined as the reciprocal of the highest dilution of the antigen that caused agglutination of sRBC. Supernatants of Claudio, M₂ and M₃ exhibited HA titers of 32, 16 and 16 respectively. Boiled or Proteinase-K treated supernatants of the clones remained HA positive, indicating their non-proteinaceous nature. Exopolysaccharide from recombinant clones and E. coli XLIB- MR were harvested from cells grown on LB agar plates . Cells were suspended in PBS and shaken at 200 rpm at room temperature for 2 h. Supernatants were collected and digested with RNaseA (lOOug/ml) , and DNase I (50ug/ml) for 1 hr at 37°C and Proteinase-K (O.lmg/ml) for 4 h at 37°C and subsequently treated with hot water-phenol (3) , dialysed for 48 h and lyophilized. Purified EPS of recombinant clones was white, fluffy, and cotton-like whereas EPS made from E. coli XLIB-MR was granular in texture. The carbohydrate concentration (14) of purified EPS derived from recombinant clones was, M, (5.6mg/ml), M₂ (7.6mg/ml), and M₃ (5.6mg/ml), >2-fold more carbohydrate than E. coli XL1B (2.8mg/ml). EPS was dissolved in distilled water and tested for HA titer. EPS of M_lf M₂, M₃ and E. coli XL1B exhibited HA titers of 4 , 32, 64 and 0 respectively, similar values to the E. coli cosmid supernatants described above.

Inhibition of HA (HAI) is determined using constant amounts of sRBC and antigen (4-fold excess antigen of the calculated HA titer) and a titration of sera. For Claudio the titer was 1:32, therefore a 1:8 dilution of supernatant was added to sRBC. HAI was observed when supernatants of Claudio and M₂and M₃were pre-treated with titrated amounts of anti-polysaccharide rabbit serum. A 1:10 dilution of antisera inhibited HA using M₂ and M₃ supernatants as hemagglutinins and a 1:20 dilution of antisera inhibited HA using Claudio supernatant (200 HAI- and 400 HAI units/ml, respectively) . 5. ELISA analyses :

A) A whole-cell ELISA was performed to further characterize M₂and M₃. The ELISA was done using whole-cells of H. pylori Claudio, M₂and M₃, and E. coli . Cells were grown on LB agar, resuspended in PBS, and bound to microtiter wells (180 ul/well) at concentrations ranging from 10⁴cfu/ml to 10⁹ cfu/ml . Anti-polysaccharide rabbit sera was used as the primary antibody at 1:15 dilution and the conjugate was anti-rabbit IgG-alkaline phosphatase. M₂ and M₃ were highly immunopositive at cell numbers ranging from 1.8xl0⁶-1.8xl0⁸. The E. coli cells bound to the well, negative control , was of negligible OD value at all cell numbers tested. The Claudio whole-cells bound to the plate, OD was 0.83 at a cell number of 1.8xl0⁶. Thus, the anti-polysaccharide sera used to screen the cosmid library and identify the clones is immunoreactive with M₂ and M₃ by ELISA.

B) A polysaccharide ELISA was performed to determine the titer of anti-polysaccharide rabbit sera against Claudio polysaccharide. An Immulon 1 microtiter plate was coated with 2ug/well Claudio polysaccharide . Titrated anti-Claudio polysaccharide rabbit serum was used as the primary antibody and a monoclonal anti-rabbit Ig-alkaline phosphatase as the secondary antibody/conjugate. The rabbit sera exhibited an ELISA titer of >1:5,000. Polysaccharide extracted from M₂ and M₃ clones grown on LB agar plates used in an identical ELISA displayed baseline titers, similar to that of E. coli .

C) A competitive ELISA was employed to determine whether supernatants or whole cell antigens derived from Claudio and cosmid recombinants M₂ and M₃ could inhibit the interaction between anti-Claudio polysaccharide antibodies and polysaccharide antigen (section 5B above) . An Immulon 1 microtiter plate was coated with 2ug/well Claudio polysaccharide. Anti-Claudio polysaccharide sera (1:50) was pre-incubated with two-fold, serial dilutions of antigen containing supernatant at 37°C for lh. The mixture was added to the wells, washed, and monoclonal anti-rabbit Ig-alkaline phosphatase was used as the secondary antibody/conjugate. With no inhibitor, and a 1:50 dilution of anti-polysaccharide sera, the ELISA OD was 0.75. Supernatants derived from M₂ and M₃ inhibited the polysaccharide/anti -polysaccharide interaction significantly greater than the negative control, E. coli ; Claudio supernatants inhibited the antigen/antibody interaction the greatest. Similar results were observed when unwashed, whole-cell extracts were used as inhibitors in place of supernatants .

6. Motility test of the Ml-3 clones and E. coli were also observed on solid agar by mixing cells with 30 ml Luria- Bertani soft agar (0.3% agar) and poured into a petri dish. After 1 h, 15 ml LB agar (1.5%) was poured on top of the solidified soft agar and plates were incubated overnight at 37°C followed by room temperature incubation for 5 days.

In addition, Ml-3 exhibited significantly slower motility in soft agar compared to the E. coli . Similarly, when M-clones were suspended in PBS and observed microscopically, significantly slower motility was observed compared to the cosmid bearing host E. coli .

7. Transmission electron microscopy studies. M2, M3 and E. coli were grown for 18 h at 37°C on LB agar. Cells were fixed with 1.5% glutaraldehyde in carbonate buffer (pH 7.4) , containing CaCl₂ at 4°C. Ruthenium red was added to the fixative to a final concentration of 0.5%. Following fixation, the colonies were scraped off the agar plate with a cotton-tipped wooden stick and collected into glass vials. Cells were washed three times in Sabatini's solution (PBS with 6.8% sucrose) . Samples were then post- fixed with 1% Osmium tetroxide in the same buffer for an additional hour, followed by three washes in Sabatini's solution. Dehydration was done with graded series of alcohol followed by treatment with propylene oxide . Epoxy blocks were polymerized at 37°C overnight and then at 60°C for another 24 h. Ultrathin sections were cut with Sorvall MT2 Ultra Micro tome with a Dupont Diamond knife, stained with uranyl acetate and Reynolds lead citrate, and examined with a transmission electron microscope (JEOL 100CX-II) at accelerating voltage of 60kV.

Transmission electron micrographs revealed major differences in morphology of the cell walls of M2 and M3 clones compared to E. coli . The cell walls of clones M2 and M3 were undulated with numerous small blebs extruding from the cell wall surfaces suggestive of capsule or exopolysaccharide production. E. coli displayed a much smoother cell surface with no visible blebs.

8. Extraction and purification of exopolysaccharide from E. coli recombinant clones. Ml-3 and E. coli were grown on LB agar plates at 37°C for 18 h. Cells from all samples were harvested with a sterile cotton swab and suspended in 30 ml PBS in 50 ml polypropylene centrifuge tubes and shaken for 2 h at 200 rpm at room temperature. Supernatants were collected following centrifugation

(15,000 g for 30 min at 4°C) , digested with RNaseA (100 ug/ml) and DNasel (50 ug/ml, 1 mM MgCl₂) for 1 h at 37°C and with proteinase-K (0.1 mg/ml) for 4 h at 37°C. Suspensions were treated with 90% hot phenol at 68°C for 10 min. , dialyzed against distilled water for 48 h, and lyophilized. Protein and carbohydrate concentrations of the EPS samples were measured by Bradford method and phenol sulfuric acid method, respectively.

9. Chemical composition analysis of purified EPS. Exopolysaccharide (EPS) samples of clones Ml-3, H. pylori Cl and E. coli were tested for both neutral and acidic sugars . For neutral sugar analysis EPS samples were hydrolyzed in 2M TFA at 121°C for 2 h. The hydrolyzed samples were reduced by sodium borodeuteride and acetylated using acetic anhydride and pyridine. Myo-ionositol was added as an internal standard. The samples were run on a GC-MS using a Sp2330 Supelco column. For acidic sugar analysis, the samples were hydrolyzed with freshly prepared 1M methanolic-HCL for 16 h at 80°C. The released sugars were derivatized with Tri-Sil and the samples were run on GC using the Supelco column.

Neutral and acidic sugars composition of the E. coli rEPS samples are presented in Table I below.

Table I Chemical Composition of EPS

Carbohydrate Residue Mole % present

Ml M2 M3 E.c. Cl

Neutral sugar Fucose 50.7 41.7 43.9 13.8 24.7

Mannose 4.0 4.0 3.2 21.9 7.0

Glucose 18.8 18.7 27.3 21.4 39.2

Galactose 26.5 35.6 25.6 42.9 29.1 Acidic sugars

Fucose 33.7 26.2 30.2 3.5 14.1

Mannose 1.9 1.8 1.8 8.7 3.7

Glucose 14.9 4.9 16.1 37.8 40.9

Galactose 37.3 40.8 9.0 46.3 40.5

Glucuronic 12.2 16.3 12.9 3.7 0.8 acid

Fucose/

Mannose

Neutral sugar 12.6 10.4 13.7 0.63 3.5

Ac idi c sugars 17.7 14.6 16.8 0.37 3.8

Exopolysaccharides derived from all three clones, H. pylori Cl and E. coli contained fucose, mannose, glucose, galactose and glucuronic acid. The rEPS clones had significantly higher amounts of fucose in comparison to E. coli, while mannose concentrations of the clones were 4-5 fold less in comaprison to E. coli. Interestingly, the mole percent amounts of fucose and mannose from the rEPS clones resembles that of H. pylori , differing considerably from E. coli and the fucose/mannose ratio of the rEPS clones resembles that of strain Cl rather than E. coli . The other sugars were present in similar amounts (Table-I) .

Sub cloning.

The specific region of the H. pylori DNA contained in the deposited clones which form the gene cluster which produces the carbohydrate antigen is determined by sub-cloning of the cosmid DNA derived from recombinant clones M,, M₂, and M₃. Cosmid DNA is purified by Qiagen midi-prep columns, and subsequently digested with restriction endonucleases (e.g., EcoRl, Bgll, Hindlll, etc.). DNA fragments resulting from such digestions are ligated into a cloning vector (e.g., pBluescript II KS, pKK223-3, pUC19, etc.) and digested with the corresponding restriction endonuclease . The products of the ligations are transformed into E. coli XLIB-MR. Recombinant clones are tested for production of the carbohydrate antigen by colony immunoblot as described previously. The immunoreactive clones are isolated, purified, and screened for phenotypic expressions such as mucoidy on solid agar and agglutination of sRBC by their supernatant or whole cells. This sub-cloning process determines the minimum size of the H. pylori DNA included in a deposited clone for producing in bacteria the carbohydrate antigen which reacts with H. pylori anti sera. The subcloned DNA is then sequenced.

Identification of M_lt M₂, and M₃ recombinant carbohydrate clones .

The deposit made to American Type Culture Collection is a mixture of 3 recombinant clones of Escherichia coli XLIB-MR (:M,, M₂, and M₃) which harbor cosmids expressing Helicobacter pylori carbohydrate antigen. Cells were grown in Luria Bertani (LB) broth supplemented with ampicillin (50 ug/ml) with glycerol added to final concentration of 15%. In order to isolate each clone, a sample of the frozen vial should be streak plated for individual colonies on LB agar supplemented with ampicillin (50 ug/ml) and incubated at 37°C for 18 hr. On agar plates, colonies are mucoid in appearance. Ten to twenty colonies should be picked aseptically, amplified in LB broth supplemented with ampicillin, and harvested for cosmid DNA by alkaline lysis method. The identity of the three different strains can be discerned by analytical restriction digestion of the cosmid DNA with restriction endonuclease Kpnl . The digestion pattern of DNA following digestion with Kpnl is significantly different from each other. Ml produces two DNA fragments of size ranging from 4kb to >23kb, M2 produces 5 fragments of size ranging from 1 to 20kb, and M3 produces 8 fragments of size ranging from 0.5 to >23 kb.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, the invention may be practiced otherwise than as particularly described.

References

1. Mills, S.D., Kurjanczyk, L.A., Penner, J.L. Antigenicity of Helicobacter pylori lipopolysaccharides . J. Clin. Microbiol. 1992; 30: 3175-3180.

2. Sambrook, J., Fritsch, E.F., Manniatiε, T.J. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989.

3. Westphal, 0., and Jann, K. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 1956; 5: 83-91.

4. Qadri, F., Haq, S., Hossain, S.S., Cizner, I., Tzipori, S. The association of hemagglutination and adhesion with lipopolysaccharide of Shigella dysenteriae serotype 1. J. Med. Microbiol. 1991; 2:293-296.

5. McSweegan, E., Waker, R.I. Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucus substrates. Infect. Immun. 1986; 53: 141-148.

6. Chitnis, D.S., Sharma, K.D. , Kamal, R.S. Role of somatic antigen of ViJbrio cholerae in adhesion to intestinal mucosa. J. Med. Microbiol. 1982; 5: 53

7. Cohen, P.S., Arruda, J.C., Williams, T.J., Laux, D.C. Adhesion of a human fecal Escherichia coli strain to mouse colonic mucus. Infect. Immun. 1985; 48: 139-145.

8. Dubois, M. , Gilles, K.A. , Hamilton, J.K., Rebers, P.A. Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956; 28: 350- 356.

Claims

WHAT IS CLAIMED IS:

$ . An antigen, comprising: a carbohydrate antigen which immunoreacts with H. pylori carbohydrate specific polyclonal antisera and which is produced by a clone contained in ATCC Deposit No. 98373 or an analog or fragment of such antigen which immunoreacts with said antisera.

2. The antigen of claim 1 wherein said antigen is produced by a clone contained in ATCC Deposit No. 98373.

3. The antigen of claim 2 wherein the clone is Ml .

4. The antigen of claim 2 wherein the clone is M2.

5. The antigen of claim 2 wherein the clone is M3.

6. An isolated polynucleotide comprising: a polynucleotide operon which is at least 90% identical to the coding sequence of a polynucleotide operon, which in bacteria produces the antigen produced by a deposited clone of ATCC Deposit No. 98373 or a fragment of said antigen, said antigen or fragment being immunoreactive with H. pylori carbohydrate specific polyclonal antisera.

7. The polynucleotide of claim 6 wherein the isolated polynucleotide comprises an operon producing the same antigen as produced by a clone of ATCC Deposit No. 98373.

8. The polynucleotide of claim 6 wherein the clone is Ml.

9. The polynucleotide of claim 6 wherein the clone is M2.

10. The polynucleotide of claim 6 wherein the clone is M3.

11. A process for inducing an immune response in a mammal against H. pylori , comprising: administering to a mammal at least one antigen of claim 1 in an amount effective to induce an immune response against H. pylori .

12. A recombinant bacterial cell containing the polynucleotide of Claim 7.

13. A recombinant vector containing the polynucleotide of Claim 7.

14. A process for producing a carbohydrate antigen, comprising:

producing a carbohydrate antigen in the cell of Claim 12.

15. An immunogen composition comprising: the antigen of Claim 2 conjugated to a protein.