AU724584B2 - Immunogenic compositions against helicobacter infection, polypeptides for use in the compositions and nucleic acid sequences encoding said polypeptides - Google Patents

Description

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ORIGINAL

COMPLETE SPECIFICATION STAN DARDIDIVISIONAL

PATENT

Invention Title: "IMMUNOGENIC COMPOSITIONS AGAINST H-ELICOBACTER INFECTION, POLYPEPTIDES FOR USE IN THE C OMPOSITIONS AND NUCLEIC ACID SEQUENCES ENCODING SAID POLYPEPTIDES" The following statement is a full description of this invention, including the best method of performing it known to us:- IMMUNOGENIC COMPOSITIONS AGAINST HELICOBACTRR INFECTIQN POLYPEPTIDES FOR USE IN THE COMPOSITIONS AND NUCLEIC ACID SEQUENCES ENCODING SAID POLYPEPTIDES The present invention relates to immunogenic compositions for inducing protective antibodies against Helicobacter spp infection. It also relates to proteinaceous material derived from Helicobacter, and to nucleic acid sequences encoding them.

Antibodies to these proteinaceous materials are also 10 included in the invention.

H. pylori is a microorganism which infects human gastric mucosa and is associated with active chronic gastritis. It has been shown to be an aetiological agent in gastroduodenal ulceration 15 (Petersen, 1991, N. Engl J Med 324 1043-1047) and two recent studies have reported that persons infected with H. pylori had a higher risk of developing gastric cancer (Nomura et al., 1991, N Eng J Med 325 1132- 1136; Parsonnet et al., 1991, N Eng J Med 325 1127- 1131).

In vivo studies of the bacterium and, consequently, work on the development of appropriate preventive or therapeutic agents has been severely hindered by the fact that Helicobacter pylori only associates with gastric-type epithelium from very few animal hosts, none of which are suitable for use as laboratory models.

A mouse model of gastric colonisation has been developed using a helical bacterium isolated from cat gastric mucus (Lee et al., 1988, Infect Immun 56 2 2843-2850; Lee et al., 1990, Gastroenterol 99 1315- 1323) and identified as a member of the genus Helicobacter. It has been named H. fells (Paster et al., 1990, Int J Syst Bacteriol 41 31-38).

To date, only limited information concerning H. fells and the extent of its similarities and differences with H. pylori, is available. The reliability of the mouse model for the development of treatments for H. pylori infection is therefore 9 10 uncertain. Recently, it was shown that H. pylori urease is a protective antigen in the H. felis/mouse model (Davin et al., 1993, Abstract A-304, Gastroenterology (Abstract supplement); Corthesy- Theulaz et al., 1993, Acta Gastro-Enterol. Belgica 15 Suppl., 56 64 (Vith Workshop on Gastroduodenal pathology and H. pylori).

It is therefore an aim of the present invention to provide therapeutic and preventive compositions for use in Helicobacter infection, which furthermore can be tested in laboratory animals.

It is known that H. pylori expresses urease activity and that urease plays an important role in bacterial colonisation and mediation of certain pathogenic processes (Ferrero and Lee, 1991, Microb Ecol Hlth Dis 4 121-134; Hazel et al., 1991).

The genes coding for the urease structural polypeptides of H. pylori (URE A, URE B) have been cloned and sequenced (Labigne et al., 1991, J Bacteriol 173 1920-1931; and French Patent Application FR 8813135), as have the genes coding the "accessory" polypeptides necessary for urease activity in H.

pylori (International Patent Application W093/07273).

Attempts have been made to use nucleic acid sequences from the H. pylori urease gene cluster as probes to identify urease sequences in H. felis.

However, none of these attempts have been successful.

Furthermore, the establishment and maintenance of H.

felis cultures in vitro is extremely difficult, and the large quantities of nucleases present in the bacteria complicates the extraction of DNA.

The inventors have now identified, in the context of the invention, new Heat Shock Proteins or chaperonins, in Helicobacter, which have an enhancing effect on urease activity. Use of the chaperonins in .9 15 an immunogenic composition may induce therefore an enhancement of protection.

Indeed, the genes encoding each of the HspA and HspB polypeptides of Helicobacter pylori have been cloned, expressed independently as fused proteins to the Maltose-Binding Protein (MBP), and purified on a large scale. These proteins have been used as recombinant antigens to immunize rabbits, and in Western immunoblotting assays as well as ELISA to determine their immunogenicity in patients infected with HP The MBP-HspA and MBP-HspB fusion proteins have been shown to retain their antigenic properties. Comparison of the humoral immune response against HspA and/or HspB in patient sera demonstrated that not only HspB but also HspA was recognized by patient sera (29/38 and 15/38), respectively). None of the 14 uninfected patients had antibodies reacting with the Hsps.

According to the invention, an immunogenic composition is provided capable of inducing antibodies against Helicobacter infection, which comprises Heat Shock Protein A (HSP A) also known as a "chaperonin".

HSP A has been elucidated by the inventors in the context of the present invention. Preferably, the chaperonin is from Helicobacter pylori. The HSP A polypeptide is encoded by the hspA gene of plasmid pILL689 (deposited at CNCM on 25 August 1993, under number CNCM 1-1356).

Preferably, the immunogenic composition is capable of inducing protective antibodies.

It is also possible to use, according to the invention, a (HSP A) polypeptide variant in which amino acids of the Figure 6 sequence have been replaced, inserted or deleted, the said variant normally Osee ""exhibiting at least 75%, and preferably at least S. 20 homology with the native HSP A. The variants preferably exhibit at least 75%, for example at least identity with the native HSP A.

The variants may further exhibit functional homology with the native polypeptide. In the case of 25 HSP A "functional homology" means the capacity to enhance urease activity in microorganism capable of expressing active urease, and/or the capacity to block infection by Helicobacter, particularly H. felis and H.

pylori. The property of enhancing urease activity may 30 be tested using the quantitative urease activity assay described below in the examples. Fragments of the HSP A polypeptide having at least 6 amino acids, may be used in the composition. The fragments or variants of HSP A used in the immunogenic composition of the invention are preferably capable of generating antibodies which block the urease enhancing effect normally exhibited by HSP A. This property is also tested using the quantitative assay described in the examples. The presence of the chaperonins in the composition enhances the protection against Helicobacter pylori and felis.

The HSP A immunogenic composition, can be used in the form of a translational fusion protein, for example with the Maltose-Binding-Protein (MBP). The "QIAexpress" system of QIAGEN, USA, may also be used.

Again, the use of the proteins in the form of fusion proteins is entirely optional.

The term "immunogenic composition" signifies, in the context of the invention, a composition comprising a major active ingredient as 9defined above, together with any necessary ingredients o* 20 to ensure or to optimise an immunogenic response, for example adjuvants, such as mucosal adjuvant, etc.

The composition of the invention is advantageously used as a pharmaceutical composition or 25 a vaccine in protecting against Helicobacter pylori and Helicobacter felis, together with physiologically acceptable excipients and carriers and, optionally, with adjuvants, haptens, carriers, stabilizers, etc.

Suitable adjuvants include muanmyl dipeptide (MDP), 30 complete and incomplete Freund's adjuvants (CFA and IFA) and alum. The vaccine compositions are normally formulated for oral administration.

The vaccines are preferably for use in man, but may also be administered innon-human animals, for example for veterinary purposes, or for use in laboratory animals such as mice, cats and dogs.

The immunogenic compositions injected into animals raises the synthesis in vivo of specific antibodies, which can be used for therapeutic purposes, for example in passive immunity.

The invention also relates to the proteinaceous materials used in the immunogenic composition. "Proteinaceous material" means any molecule comprises of chains of amino acids, e.g.

peptides, polypeptides or proteins, fusion or mixed proteins an association of two or more proteinaceous materials, all or some of which may have immunogenic or immunomodulation properties), either S. purified or in a mixture with other proteinaceous or non-proteinaceous material. "Polypeptide" signifies a chain of amino acids whatever its length and englobes 20 the term "peptide". The term "fragment" mens any amino *q acid sequence shorter by at least one amino acid than the parent sequence and comprising a length of amino acids e.g. at least six residues, consecutive in the parent sequence.

25 The peptide sequences of the invention, may for example, be obtained by chemical synthesis, using a technique such as the Merrified technique and synthesiser of the type commercialised by Applied Biosystems.

30 The invention also relates to the proteinaceous material comprising at least the Heat Shock Protein (HSP A) of Helicobacter pylori or a fragment thereof. Preferably the proteinaceous material comprises or consists of HSP A as illustrated in Figure 6 or a polypeptide having at least 75%, and preferably at least 80 or 90%, homology or identity with the said polypeptide or fragment having at least 6 amino acids. A particular fragment of the Helicobacter pylori HSP A polypeptide is the C-terminal sequence: GSCCH T G NHDHKHAKE

HEACCHDHKKH

or a sub-fragment of this sequence having at least six consecutive amino acids. This C-terminal sequence is thought to act as a metal binding domain allowing binding of, for example, nickel.

The proteinaceous material of the invention may also comprise or consist of a fusion or mixed protein including at least the HSP A protein from e Helicobacter or fragment thereof, as defined above W Q Particularly preferred fusion proteins are the Mal-# .fusion proteins and QIAexpress system fusion proteins o o* 20 (QIAGEN, USA) as detailed above.

The invention also relates to monoclonal or polyclonal antibodies to the proteinaceous materials described above.

The invention also concerns monoclonal or Woo 25 polyclonal antibodies to HSP A or fragments as described above. These antibodies may be specific for the Helicobacter pylori chaperonins or, alternatively, they may cross-react with GroEL-like proteins or GroESlike proteins from bacteria other than Helicobacter, 30 depending up the epitopes recognised. Figure 7 shows the homologous regions of HSP A and HPS B with GroESlike proteins and GroEL-like proteins respectively from various bacteria. However it will be appreciated that HSP B is not part of the present invention.

Particularly preferred antibodies are those specifically recognising the HSP A C-terminal sequence having the metal binding function. Again, use of specific fragments for the induction of the antibodies ensues production of Helicobacter-specific antibodies.

The antibodies of the invention may be prepared using classical techniques. For example monoclonal antibodies may be produced by the hybridoma technique or by know techniques for the preparation of human antibodies, or by the technique described by Marks et al., 1991, Journal of Molecular Biology 222 581-597.

The invention also includes fragments of any of the above antibodies produced by enzyme digestion.

Of particular interest are the Fab and F(ab') 2 fragments. Also of interest are the Facb fragments.

The invention also relates to purified 20 antibodies or serum obtained by immunisation of an animal, e.g. a mammal, with the immunogenic composition, the proteinaceous material or fragment, or the fusion or mixed protein of the invention, followed by purification of the antibodies or serum.

25 Also concerned is a reagent for the in vitro detection of H. pylori infection, containing at least these antibodies or serum, optionally with reagents for labelling the antibodies, e.g. anti-antibodies etc.

The invention also relates to a Kit 30 comprising at least the purified antibodies or serum described above, and optionally, appropriate media or *f excipients for administration of the antibodies or serum, or labelling or detection means for the antibodies.

The invention further relates to nucleic acid sequences coding for any of the above proteinaceous materials including peptides. In particular, the invention relates to a nucleic acid sequence characterised in that it comprises: a sequence coding for the proteinaceous material defined above; or (ii) a sequence complementary to sequence or (iii) a sequence capable of hybridizing to sequence or under stringent conditions; or (iv) a fragment of any of sequences (ii) or (iii) comprising at least 10 nucleotides.

0 r" Preferred sequences are those comprising all 20 or part of te sequence of plasmid pILL689 (CNCM I- 1356), for example the sequence of Figure 6, in particular that coding for HSP A or a sequence complementary to this sequence under stringent conditions, or a fragment thereof.

25 High stringency hybridization conditions in the context of the invention are the following: S- 5 x SSC; formamide at 37°C; or: 30 6 x SSC; Denhard medium at 68 0

C

The sequences of te invention also include those hybridizing to any of sequences (ii) and (iii) defined above under non-stringent conditions, that is: 5 x SSC; 0.1% SDS; 30 or 40% formamide at 42°C, preferably The term "complementary sequences" in the context of the invention signifies "complementary" and "reverse" or "inverse" sequences.

The nucleic acid sequences may be DNA or

RNA.

The sequences of the invention may be used as nucleotide probes in association with appropriate labelling means. Such means include radio-active isotopes, enzymes, chemical or chemico-luminescent markers, fluoro-chromes, haptens, or antibodies. The markers may optionally be fixed to a solid support, for example a membrane, or particles.

As a preferred marker, radio-active 20 phosphorous

P

is incorporated at the 5'-end of the probe sequence. The probes of the invention comprise any fragment of the described nucleic acid sequences and may have a length for example of at least nucleotides, for example 60, 80 or 100 nucleotides or 25 more. Preferred probes are those derived from the HSPA gene.

A suitable oligonucleotide primer comprises from 10 to 100 consecutive nucleotides of the above described nucleic acid sequences.

30 The probes of the invention may be used in i the in vitro detection of Helicobacter infection in a biological sample, optionally after a gene amplification reaction. Most advantageously, the probes are used to detect Helicobacter felis or Helicobacter pylori, or both, depending on whether the sequence chosen as the probe is specific to one or the other, or whether it can hybridise to both.

Generally, the hybridisation conditions are stringent in carrying out such a detection.

The invention also relates to a kit for the in vitro detection of Helicobacter infection, characterised in that it comprises:a nucelotide probe according to the invention, as defined above; an appropriate medium for carrying out a hybridisation reaction between the nucleic acid of Helicobacter and the probe; -reagents for the detection of any hybrids formed.

SThe nucleotide sequences of the invention may also serve as primers in a nucleic acid amplification reaction. The primers normally comprise "at least 10 consecutive nucleotides of the sequences described above and preferably at least 18. Typical lengths are from 25 to 30 and may be as high as 100 or more consecutive nucleotides. Such primers are used W 25 in pairs and are chosen to hybridise with the 5' and 3'-ends of the fragment to be amplified. Such an amplification reaction may be performed using for example the PCR technique (European Patent Applications EP200363, 201184 and 229701). The Q-B- 30 replicase technique (Biotechnology, Vol. 6, Oct. 1988) 4* may also be sed in the amplification reaction.

The invention also relates to expression vectors characterised in that they contain nay of the nucleic acid sequences of the invention. A particularly preferred expression vector is plasmid pILL689 (CNCM 1-1356). The expression vectors will normally contain suitable promoters, terminators and marker genes, and any other regulatory signals necessary for efficient expression.

The invention further relates to prokaryotic or eukaryotic host cells stably transformed by the nucleic acid sequences of the invention. As examples of hosts, mention may be made of higher eukaryotes such as CHO cells and cell-lines; yeast, prokaryotes including bacteria such as E. coli, e.g. E. coli HB 101; Mycobacterium tuberculosum; viruses including baculovirus and vaccinia. Usually the host cells will be transformed by vectors. However, it is also possible within the context of the invention, to insert the nucleic acid sequences by homologous •recombination, using conventional techniques.

W 20 By culturing the stably transformed hosts of g* the invention, the Helicobacter urease polypeptide material and, where applicable, the HSP material can be produced by recombinant means. The recombinant Glw proteinaceous materials are then collected and 25 purified. Pharmaceutical compositions are prepared by combining the recombinant materials with suitable excipients, adjuvants and optionally, any other additives such as stabilizers.

The invention also relates to plasmids 30 pILL920 (deposited at CNCM on 20 July 1993, under Accession Number 1-1337) and pILL927 (CNCM 1-1340, deposited on 20 July 1993) constructed as described in deposited on 20 July 1993) constructed as described in the examples below.

Different aspects of the invention are illustrated in the figures It is understood that HSP B and other non-HSP A nucleic acids and/or polypeptides are provided herein for information and are not part of the present invention as claimed.

Figure 1: Transposon mutagenesis and sequencing of pILL205. Linear restriction maps of recombinant cosmid pILL199 and recombinant plasmid pILL205 (and the respective scale markers) are presented. Numbers in parentheses indicate the sized of H. fells DNA fragments inserted into one of the cloning vectors ef^ 14 (pILL575 or pILL570, respectively). The "plus" and "minus" signs within circles correspond to the insertion sites of the MiniTn3-Km transposon in pILL205; "plus" signs indicate that the transposon did not inactivate urease expression, whereas negative signs indicate that urease expression was abolished.

The letters refer to mutant clones which were further characterised for quantitative urease activity and for the synthesis of urease gene products. The location 10 of the structural urease genes (ure-A and ure on pILL205 are represented by boxes, the lengths of which are proportional to the sizes of the respective openreading frames. The arrows refer to the orientation of transcription. The scale at the bottom of the 15 figure indicates the sizes (in kilobases) of the HindIII and PstI restriction fragments. Restriction .sites are represented as follows: B, BamHI; Pv, woo.* PvuII; Bg, BglII; E, EcoRI; H, HindIII; C, ClaI; Ps, PstI. Letters within parentheses indicate that the sites originated from the cloning vector.

Figure 2: Western blot analysis of whole-cell extracts of E. coli HB101 cells harbouring recombinant plasmids were reacted with rabbit polyclonal antiserum (diluted 1:1, 1000) raised against H. felis bacteria. A) extracts were of E. coli cells harbouring: plasmid vector pILL570 (lane recombinant plasmid pILL205 (lane and pILL205 derivative plasmids disrupted in loci and (lanes B) Extracts were of E. coi cells harbouring: recombinant plasmid pILL753 containing the H. pylori ure A and ure B genes (Labigne et al., 1991, supra) (lane and pILL205 derivative plasmids disrupted in loci and (lanes The small arrow heads indicate polypeptides of approximately 30 and 66 kilodaltons which represent putative Ure A and Ure B gene products of H. felis. The large arrow heads in panel B indicate the corresponding gene products of H.

pylori which cross-reacted with the anti-H. felis 10 serum. The numbers indicate the molecular weights (in thousands) of the protein standards.

Figure 3: Nucleotide sequence of the H. fells structural urease genes. Numbers above the sequence 15 indicate the nucleotide positions as well as the amino acid position in each of the two Ure A and Ure B polypeptides. Predicted amino acid sequences for Ure w A (bp 43 to 753) and Ure B (766 to 2616) are shown below the sequence. The putative ribosome-binding site (Shine-Dalgarno sequence, SD) is underlined.

Figure 4: Comparison of sequences for the structural urease genes of H. fellis to: a) the sequence of the two subunits of H. pylori urease (Labigne et al., 1991, supra); b) the sequence of the three subunits of Protaus mirabilis urease (Jones and Mobley, 1989, J Bacteriol 171 6414-6422); c) the sequence of the single subunit of jack bean urease. Gaps (shown by dashes) have been introduced to ensure the best alignment. amino acids identical to those of the H.

felis sequence amino acids shared by the various ureases amino acids unique to the Helicobacter ureases. The percentages relate to the number of amino acids that are identical to those of the H.

felis urease subunits. Helicobacter fells; H.P., Helicobacter pylori; Proteus mirabilis; J.b,, Jack bean.

Figure Restriction map of the recombinant plasmids pILL689, pILL685, and pILL691. The construction of these plasmids is described in details in Table 1. Km within triangles depicts the site of insertion of the kanamycin cassette which led to the construction of plasmids pILL687, pILL688 and pILL696 (Table 2).

15 Boxes underneath the maps indicate the position of the three genetic elements deduced from the nucleotide sequence, namely IS5, Hsp A and Hsp-B.

Figure 6: Nucleotide sequence of the Helicobacter pylori Heat Shock Protein gene cluster. The first number above the sequence indicates the nucleotide positions, whereas the second one numbers the amino acid residue position for each of the HSp A and Hsp B protein. The putative ribosome-binding sequences (Shine-Dalgarno [SD] sites) are underlined.

Figure 7: Comparison of the deduced amino-acid sequence of Helicobacter pylori Hsp A or Hsp B (B) with that of other GroEL-like or GroES-like (B) proteins. Asterisks mark amino acids identical with those in the Helicobacter pylori Hsp A or Hsp B 17 sequences.

Figure 8: Expression of the Helicobacter pylori Hsp A Heat Shock Proteins in E. coli minicells. The protein bands with apparent molecular masses of 58 and 13 kDA, corresponding to the Helicobacter pylori Hs A and Hsp B Heat Shock Proteins are clearly visible in the lanes corresponding to plasmids pILL689 and pILL692 and absent in the vector controls (pILL570 and pACYC177, respectively).

Figure 9: Nucleotide sequence of the Helicobacter felis ure I gene and deduced amino acid sequence.

Figure 15 Comparison of the amino acid sequence of the 99 ure I proteins deduced from the nucleotide sequence of the ure- I gene of Helicobacter fells and that of "Helicobacter pylori.

Figure 11: Genetic code. Chain-terminating, or "nonsense", codons. Also used to specify the initiator formyl-Met-tRNAet The Val triplet GUG is therefore "ambiguous" in that it codes both valine and methionine.

Figure 12: Signification of the one-letter and threeletter amino acid abbreviations.

Figure 13: Purification of H. pylori UreA-MBP recombinant protein using the pMAL expression vector system. Extracts from the various stages of protein purification were migrated on a 10% resolving SDSpolyacrylamide gel. Following electrophoresis, the gel was stained with Coomassie blue. The extracts were 2) non-induced cells; 3) IPTG-induced cells; 4) French press lysate of induced cell extract; 5) eluate from amylose resin column; 6) eluate from anion exchange column (first passage) eluate from anion exchange column (second passage); 1) SDS-PAGE standard marker proteins.

10 Figure 14: Recognition of UreA recombinant fusion proteins by polyclonal rabbit anti-Helicobacter sera.

Protein extracts of maltose-binding protein (MBP, lane 1) felis UreA-MBP (lane and H. pylori UreA-MBP go 15 (lane 3) were Western Blotted using rabbit polyclonal antisera (diluted 1 5000) raised against whole-cell extracts of H. pylori and H. felis. The purified fusion proteins are indicated by an arrow. Putative degradation products of the proteins are shown by an asterisk.

Figure Recognition of UreB recombinant fusion proteins by rabbit antisera raised against purified homologous and heterologous UreB proteins.

Nitrocellulose membranes were blotted with the following extracts 1) standard protein markers; 2) H.

felis UreA-MBP; 3) MBP; 4) H. pylori UreA-MBP. The membranes were reacted with polyclonal rabbit antisera (diluted 1 5000) raised against MBP-fused H. pylori and H. felis Ure B sub-units, respectively. The molecular weights of standard proteins are presented 19 on the left-hand side of the blots.

Figure 16: Western blot analysis of H. pylori and H.

felis whole-cell extracts with- antisera raised against purified UreB MBP-fused recombinant proteins.

SDS-PAGE whole extracts of H. felis (lane 1) and H.

pylori (lane 2) cells we re reacted with polyclonal rabbit antisera raised against purified H. pylori UreB and H. felis UreB MBP-fused proteins (sera diluted 1 10 5000). The difference in gel mobility of the respective non-recombinant UreB sub-units of H. felis and H. pylori can be seen. The numbers on the left refer to the molecular weights of standard marker proteins.

15 Figure 17: Serum IgG responses to MBP (bottom), MBP- SHspA (top) and MBP-HspB (middle) of 28 H. pylori infected patients (squares, left) and 12 uninfected Spatients (circles, right) The, optical density of each serum in the ELISA assay described in Experimental procedures was read at 492 nm, after a nm incubation. The sizes of the symbols are proportional to the number of sera giving the same optical density value.

Figure 18: SDS-PAGE analysis of material eluted from the amylose column (lanes 2 and 3) or from the Ni-NTA column following elution with buffer E (pH lanes 4 and 5; or buffer C (pH lanes 6 and 7. Material eluted from a lysate of MC1061 (PILL933) (lanes 2, 3, and 7) and material eluted from a lysate of MC1061 (PMAL-c2) (lanes 4 and Lane 3 contains the same material as in lane 2 except that it was resuspended in buffer E, thus demonstrating that buffer E is responsible for dimer formation of the MBP-HspA subunit, as seen in lanes 3 and

EXAMPLES

It is understood that reference to HSP B and other non-HSP A nucleic acids and/or polypeptides are provided with the examples herein for information and are not part of the present invention as claimed.

I CLONING, EXPRESSION AND SEQUENCING OF H. FELIS UREASE GENE EXPERIMENTAL PROCEDURES FOR PART I Bacterial strains and culture conditions H. felis (ATCC 49179) was grown on blood agar base no, 2 (Oxoid) supplemented with 5% (v/v) lysed horse blood (BioMerieux) and an antibiotic supplement consisting of 10 ng ml vancomycin (Lederle Laboratories), 2.5 pg ml polymyxin B (Pfizer), 5 pg ml-1 trimethoprim (Sigma Chemical Co.) and 2.5 pg ml 1 amphotericin B Squibb and Sons, I Inc.). Bacteria were cultured on freshly prepared agar 25 plates and incubated, lid uppermost, under microaerobic conditions at 37 0 C for 2-3 days. E. coli t strains HB101 (Boyer and Roulland-Dussoiz, 1969) and MC1061 (Maniatis et al., 1983, Molecular Cloning: A Laboratory Manual. Cold spring Harbor Laboratory, Cold S• 30 Spring Harbor used in the cloning experiments, were grown routinely in Luria broth without glucose added or on Luria agar medium at 37 0 C. Bacteria a grown under nitrogen- limniting conditions were passaged on a nitrogen~1imiting solid medium consisting of ammonium-free M9 minimal medium (pH1 7.4) supplemented with 0.4 D-glucose and 10 mM L-arginine (Cussac et al., 1992, J Bacteriol 174 2466-2473).

DNA manipulations All standard DNA manipulations and analyzes, unless mentioned otherwise, were performed according to the procedures described by Maniatis et al.,1983, supra.

Isolation of H. felis DNA Total genomic DNA was extracted by an 10 sarkosyl-proteinase K lysis procedure (Labigne-Roussel et al., 1988, J Bacterio 170 1704-1708). Twelve blood agar plates inoculated with H. felis were incubated in an anaerobic jar (BBL) with an anaerobic gaspak (BBL 70304) without catalyst, for 1-2 days at 37oC. The 15 plates were harvested in 50 ml of a 15% glycerol 9% sucrose solution and centrifuged at 5,000 rpm (in a Sorvall centrifuge), for 30 min at 40C. The pellet was resuspended in 0.2 ml 50 mM D-glucose in mM Tris-10 mM EDTA (pH 8.0) containing 5 mg ml- 1 20 lysozyme and transferred to a VTi65 polyallomer quick seal tube. A 0.2 ml aliquot of 20 mg ml- 1 proteinase K and 0.02 ml of 5 M sodium per chlorate were added to the suspension. Cells were lysed by adding 0.65 ml of EDTA Sarkosyl, and incubated at until the suspension cleared (approximately 5 min).

The volume of the tube was completed with a CsCl solution consisting (per 100 ml) of 126 g CsCi, 1 ml aprotinine, 99 ml TES buffer (30 mM Tris, 5 mM EDTA, mM NaCI (pH Lysates were centrifuged at 45,000 rpm, for 15-18 h at 180C. Total DNA was collected and dialyzed against TE buffer (10 mM Tris, 22 1 mM EDTA), at 4°C.

Cosmid cloning Chromosomal DNA from H. felis was cloned into cosmid vector pILL575, as previously described (Labigne et al., 1991, supra). Briefly, DNA fragments arising from a partial digestion with Sau3A were sized on a (10 to 40%) sucrose density gradient and then ligated into a BamHI-digested and dephosphorylated pILL575 DNA preparation. Cosmids were packaged into 10 phage lambda particles (Amersham, In Vitro packaging kit) and used to infect E. coli HB101. To screen for urease expression, kanamycin-resistant transductants were replica-plated onto solid nitrogen-limiting medium (see above) containing (20 g ml- 1 kanamycin 15 that had been dispensed into individual wells of microtitre plates (Becton Dickinson). The mictrotitre plates were incubated aerobically, at 370C for 2 days before adding 0.1 ml urease reagent (Hazell et al., 1987, Am J Gastroenterol 82 292-296) to each of the 20 wells. Ureolysis was detected within 5-6 h at 370C by a colour change in the reagent. Several ureasepositive cosmid clones were restriction mapped and one was selected for subcloning.

Subcloninc of H. felis DNA A large-scale CsCl plasmid preparation of cosmid DNA was partially digested Sau3A. DNA fragments (7-11 kb) were electroeluted from an agarose gel and purified using phenol-chloroform extractions.

Following precipitation in cold ethanol, the fragments were ligated into Bg/III-digested plasmid pILL570 (Labigne et al., 1991, supra) and the recombinant plasmids used to transform competent E. coli MC1061 calls. Spectinomycin-resistant transformants were selected and screened for urease expression under nitrogen-rich (Luria agar) and nitrogen-limiting conditions.

Quantitative urease activity Cultures grown aerobically for 2.5 days at 370C were harvested and washed twice in 0.85 (w/v) NaCi. Pellets were resuspended in PEB buffer (0.1 M i 10 sodium phosphate buffer (pH 7.4) containing 0.01 M EDTA) and then sonicated by four 30-sec bursts using a Branson Sonifier model 450 set at 30 W, 50% cycle.

Cell debris was removed from the sonicates by centrifugation. Urease activities of the sonicates 15 were measured in a 0.05 M urea solution prepared in PEB by a modification of the Berthelot reaction (Cussac et al., 1992, supra). Urease activity was expressed as imol urea min-lmg bacterial protein.

Protein determination C 20 Protein concentrations were estimated with a commercial version of the Bradford assay (Sigma Chemicals).

Transposon mutagenesis Random insertional mutations were generated within cloned H. felis via a MiniTn3-Km delivery system (Labigne et al., 1992, Res Microb 143 15-26).

In brief, E. coli HB101 cells containing the transposase-encoding plasmid PTCA were transformed with plasmid pILL570 containing cloned H. felis DNA.

Transposition of the MiniTn3-Km element into the pILL570 derivative plasmids was effected via

J

24 conjugation. The resulting cointegrates were then selected for resolved structures in the presence of high concentrations of kanamycin (500 mgl-1) and spectinomycin (300 mgl-l).

SDS-PAGE and Western blotting Solubilized cell extracts were analyzed on slab gels, comprising a 4.5% acrylamide stacking gal and 12.5% resolving gel, according to the procedure of Laemmli (Laemmli, 1970, Nature 227 680-685).

10 Electrophoresis was performed at 200 V on a mini-slab 9 gel apparatus (Bio-Rad).

Proteins were transferred to nitrocellulose 9* paper (Towbin et al., 1979, Proc Natl Acad Sci 76 4350-4354) in a Mini Trans-Blot transfer cell (Bio- 15 Rad) set at 100 V for 1 h (with cooling).

Nitrocellulose membranes were blocked with 5% (w/v) purified casein (BDH) in phosphate-buffered saline (PBS, pH 7.4) at room temperature, for 2 h (Ferrero et al., 1992, J Bacteriol 147 4212-4217). Membranes were 20 reacted at 4 0 C overnight with antisera diluted in 1% casein prepared in PBS. Immunoreactants were then detected using a biotinylated secondary antibody (Kirkegaard and Perry Lab.) in combination with avidin-peroxidase (KPL). A substrate solution composed of 0.3% 4-chloro-l-naphthol (Bio-rad) was used to visualise reaction products.

DNA Sequencing DNA fragments to be sequenced were cloned into M13mpl8 and M13mpl9 (Meissing and Vieira, 1982, Gene 19 269-276) bacteriophage vectors (Pharmacia).

Competent E. coli JM101 cells were transfected with recombinant phage DNA and plated on media containing X-gal (5-bromo-4-chloro-3-indolyl-

-D-

galactopyranoside) and isopropyi-p-Dthiogalactopyranoside. Plaques arising from bacteria infected with recombinant phage DNA were selected for the preparation of single-stranded DNA templates by polyethylene glycol treatment (Sanger et al., 1977, Proc Natl Acad Sci USA 74 5463-5467). Single-stranded DNA sequenced according to the dideoxynucleotide chain 10 termination method using a Sequenase kit (United States Biochemical Corp.).

Nucleotide sequence accession number "The nucleotide accession number is X69080 .99* (EMBL Data Library).

15 RESULTS OF PART I EXPERIMENTS Expression of urease activity bvy H. felis cosmid clones Cloning of partially digested fragments to 45 kb in size) of H. felis chromosomal DNA into the 20 cosmid vector pILL575 resulted in the isolation of approximately 700 cosmid clones. The clones were subcultured on nitrogen-limiting medium in order to induce urease expression (Cussac et al., 1992, supra).

Six of these were identified as being urease-positive after 5-6 h incubation (as described in the Experimental procedures section). No other ureasepositive cosmid clones were identified, even after a further overnight incubation. Restriction enzyme analysis of 3 clones harbouring the urease-encoding cosmids revealed a common 28 kd DNA fragment. A cosmid (designated pILL199) containing DNA regions at both extremities of the common fragment was selected for subcloning.

Identification of H. felis genes required for urease expression when cloned in B. coli cells To define the minimum DNA region necessary for urease expression in E. coli cells, the ureaseencoding cosmid pILL199 was partially digested with Sau3A and the fragments were subcloned into plasmid pILL570. The transformants were subcultured on 10 nitrogen- rich and nitrogen-limiting media and screened for an urease-positive phenotype. Five transformants expressed urease activity when grown Sunder nitrogen-limiting conditions, whereas no activity was detected following growth on nitrogen- 15 rich medium. Restriction mapping analyses indicated that the urease-encoding plasmids contained inserts of between 7 and 11 kb. The plasmid designated pILL205 V was chosen for further studies.

Random mutagenesis of cloned H. fells DNA 20 was performed to investigate putative regions essential for urease expression in E. coli and to localize the region of cloned DNA that contained the structural urease genes. Random insertion mutants of the prototype plasmid pILL205 were thus generated using the MiniTn3-Km element (Labigne et al., 1992, supra). The site of insertion was restriction mapped for each of the mutated copies of pILL205 and cells harbouring these plasmids were assessed qualitatively for urease activity (Figure A selection of E.

coli HB101 cells harbouring the mutated derivatives of pILL205 (designated to were then used both for quantitative urease activity determinations, as well as for the detection of the putative urease subunits by Western blotting.

The urease activity of E. coli HB101 cells harbouring pILL205 was 1.2 0.5 imol urea min-mg' bacterial protein (Table which is approximately a fifth that of the parent H. felis strain used for the cloning. Insertion of the transposon at sites and resulted in a negative 10 phenotype, whilst mutations at sites h" and *i had no significant effect on the urease activities of clones harbouring these mutated copies of pILL205 S' (Table Thus mutagenesis of pILL205 with the MiniTn3-Km element identified three domains as being 15 required for H. felis urease gene expression in E.

coli cells.

Localization of the H. felis urease structural genes Western blot analysis of extracts of E. coli cells harbouring pILL205 indicated the presence of two 20 polypeptides of approximately 30 and 66 kDa which cross-reacted with polyclonal H. fells rabbit antiserum (Figure 2A). These proteins were not produced by bacteria carrying the vector (pILL570).

Native H. fells urease has been reported to be composed of repeating monomeric subunits with calculated molecular weights of 30 and 69 kDa (Turbett et al., 1992, Infect Immun 60 5259-5266). Thus the and 66 kDa proteins were thought to correspond to the ure A and ure B gene products, respectively.

Interestingly an extract of B. coi cells harbouring 28 the recombinant plasmid pILL763 (Cussac et al., 1992, supra) containing the Helicobacter pyloi cr1 A and ure B genes, expressed two polypeptides with approximate molecular sizes of 30 and 62 kDa which cross-reacted with the anti-H. felis antisera (Figure 2B).

oe.

TABLE 1 Mutagenesis of E. CQ1.i clones and effect on urease activity pILL205 1.2 0.467 a.a a a.

a -a a.

a a.

a a 9 a.

a a.

a a 9* a. a.

9.a* 9 a 9 pILL205: :a negd pILL205-::b 0.74 0.32 :c neg pILL205: :d neg pILL2O5::e 0.54 0.15 pILL2O5: :f neg pILL2O5: :g neg pILL205:4h 1.05 0.25 pILL205::i- 0.93 0.35 a R. Coli cells harboured pILL205 and its derivatives constructed by -transposon mutagenesis. The letters correspond to the insertion sites of the MiniTn3-transposon on pILL2 b Activities of bacteria grown aerobically for 3 days at 37 0 C on solid M9 minimal medium supplemented with 10 mM L-arginine. The values represent the means standard deviations calculated from three determinations.

Urease activity was approximately a fifth as large as that of H. flils wildtype strain (ATCC 49179) i.e. 5.7 0.1 ysmol urea min-Img- 1 protein (Ferrero and Lee, 1991, Supra).

d No activity detected (limit of detection was 1 nmol urea min-'mg- 1 of bacterial protein) Clones harbouring the mutated derivatives of pILL205, in all but one case, expressed the ure A and ure B gene products (Figures 2A, B) Given that several of the mutants mutants and synthesized the urease subunits yet did not produce an active enzyme, it is possible to speculate that accessory functions essential for urease activity may have been disrupted by transposon insertion. In contrast, the mutant designated pILL205::a did not 1 0 produce the ure B product and was urease-negative.

Thus the site of transposon insertion was presumed to be located in the ure B gene. Sequence analyses of the DNA region corresponding to insertion site "a" were undertaken to elucidate potential open reading 15 frames encoding the structural polypeptides of H.

felis urease.

Sequence analyses of H. felis structural urease genes Sequencing of a 2.4 kb region of H. fells DNA adjacent to transposon insertion site resulted S. 20 in the identification of two open reading frames (ORFs) designated ure A and ure B which are transcribed in the same direction (Figure The transposon was confirmed to be located at 240 bp upstream from the end of ure B. Both ORFs commenced with an ATG start codon and were preceded by a site similar to the E. coli consensus ribozome-binding sequence (Shine and Dalgarno, 1974, Proc Natl Acad Sci USA 71 1342-1346). The intergenic space for the H.

felis structural genes consisted of three codons which were in phase with the adjacent open-reading frames.

This suggests that, as has already been observed to be 31 the case for Helicobacter pylori (Labigne et al., 1991, supra), a single mutation in the stop codon of the ure A gene would theoretically result in a fused single polypeptide.

The H. felis ure A and ure B genes encode polypeptides with calculated molecular weights of 26 074 kA and 61 663 Da, respectively, which are highly homologous at the amino acid sequence level to the ure A and ure B gene products of H. pylori. The levels of 10 identity between the corresponding ure A and ure B gene products of the two Helicobacter spp. was calculated to be 73.5% and 88.2% respectively. From the amino acid sequence information, the predicted molecular weights of the ure A and ure B polypeptides from H. felis and H. pylori (Labigne et al., 1991, supra) are very similar. Nevertheless the ure B product of H. felis had a lower mobility than the corresponding gene product from Helicobacter pylori when subjected to SDS-polyacrylamide gel

S

electrophoresis (Figure 2B).

II EXPRESSION OF RECOMBINANT UREASE SUBUNIT PROTEINS FROM H. PYLORI AND H. FELIS ASSESSMENT OF THESE PROTEINS AS POTENTIAL MUCOSAL iMMUNOGENS IN A MOUSE MODEL The aims of the study were to develop recombinant antigens derived from the urease subunits of H. pylori and H. felis, and to assess the immunoprotective efficacies of these antigens in the H. fellis/mouse model. Each of the structural genes encoding the respective urease subunits from H. pylori tf 32 and H. felis was independently cloned and overexpressed in Escherichia coli. The resulting recombinant urease antigens (which were fused to a 42 kDa maltose-binding protein of E. coli) were purified in large quantities from E. coli cultures and were immunogenic, yet enzymatically inactive. The findings demonstrated the feasibility of developing a recombinant vaccine against H. pylori infection.

EXPERIMENTAL PROCEDURES FOR PART 11 10 Bacterial strains, plasmids and growth conditions H. felis (ATCC 49179) was grown on a blood "o agar medium containing blood agar base no. 2 (Oxoid) 0 supplemented with 10% lysed horse blood (BioMerieux) and an antibiotic supplement consisting of vancomycin 15 (10 Lg/mL), polymyxin B (25 Ag/mL), trimethoprim Ag/mL) and amphotericin B (2.5 jg/mL) Bacteria were cultured under microaerobic conditions at 370C for 2 days, as described previously. E. coli strains MC1061 and JM101, used in cloning and expression experiments, 20 were grown routinely at 37 0 C in Luria medium, with or without agar added. The antibiotics carbenicillin (100 Ag/mL) and spectinomycin (100 xg/mL) were added as required.

DNA manipulations and analysis All DNA manipulations and analyses, unless mentioned otherwise, were performed according to standard procedures. Restriction and modification enzymes were purchased from Amersham (France) DNA fragments to be cloned were electroeluted from agarose gels and then purified by passage on Elutip minicolumns (Schleicher and Schull, Germany). Single- 33 stranded DNA sequencing was performed using M13mpl8 and M13mpl9 bacteriophage vectors (Pharmacia, France).

Single-stranded DNA templates were prepared from recombinant phage DNA by polyethylene glycol treatment. Sequencing of the templates was achieved according to the dideoxynucleotide chain termination method using a Sequenase kit (United States Biochemical Corp., Preparation of inserts for cloning using the polvmerase chain reaction (PCR) To clone the urea genes of H. pylori and H.

felis, degenerated 36-mer primers were conceived from 99• the published urease sequences (Labigne et al.,1991, supra; Ferrero and Labigne, 1993, Molec. Microbiol. 9 15 323-333) (primer set refer to Table Purified DNA from E. coli clones harbouring plasmids pILL763 and pILL207 (Table that encoded the structural genes of H. pylori and H. felis ureases, were used as template material in PCR reactions. Reaction samples 20 contained: 10-50 ng of denatured DNA; PCR buffer mmol/L KC1 in 10 mmol/L Tris-HCl [pH dATP, dGTP, dCTP and dTTP (each at a final concentration of 1.25 mmol/L); 2.5 mmol/L MgC1 2 25 pmol of each primer and 0.5 IL Taq polymerase. The samples were subjected to 30 cycles of the following programme: 2 min at 94 0 C, 1 min at 40 0

C.

The amplification products were cloned into the cohesive ends of the pAMP vector (Figure 1) according to the protocol described by the manufacturer ("CloneAmp System", Gibco BRL; Cergy Pontoise, France). Briefly, 60 ng of amplification product was directly mixed in a buffer (consisting of mmol/L KC1, 1.5 mmol/L MgC1 2 0.1% (wt/vol) gelatine in 10 mmol/L Tris-HC1, pH 8.3) with 50 ng of the pAMP 1 vector DNA and 1 unit of uracil DNA glycolsylase.

Ligation was performed for 30 min at 37 0 C. Competent cells (200 AL) of E. coli MC1061 were transformed with pL of the ligation mixture. Inserts were subsequently excised from the polylinker of the pAMP vector by double digestion with BamHl and PstI, and 10 then subcloned into the expression vector pMAL (New England Biolabs Inc., Beverly, USA) chosen for the production of recombinant antigens (pILL919 and PILL920, respectively, Figure 13), as well as in M13mp bacteriophage for sequencing.

15 Amplification of a product containing the SureB gene of H. pylori was obtained by PCR using a couple of 35-mer primers (set Table The PCR reaction mixtures were first denatured for 3 min at 94oC, then subjected to 30 cycles of the following 20 programme min at 94°C, 1 min at 55 0 C and 2 min at 720C. The purified amplification product (1850 bp was digested with EcoRI and PstI and then cloned into PMAL (pILL927, figure Competent cells of E. coli MC1061 were transformed with the ligation reaction.

H. felis ureB was cloned in a two-step procedure, that allowed the production of both complete and truncated versions of the UreB subunit.

Plasmid pILL213 (Table 3) was digested with the enzymes Dral, corresponding to amino acid residue number 219 of the UreB subunit and HindIII. The resulting 1350 bp fragment was purified and cloned into pMAL that had been digested with Xmni and HindIII (pILL219, Figure 2) In order to produce a clone capable of synthesizing a complete UreB protein, PCR primers were developed (set Table 2) that amplified a 685 bp fragment from the N-terminal portion of the ureB gene (excluding the ATG codon), that also overlapped the beginning of the insert in plasmid pILL219. The PCR amplified material was 10 purified and digested with BamHI and HindIII, and then cloned into pMAL (pILL221, Figure 14) A 1350 bp sPstI-PstI fragment encoding the remaining portion of the UreB gene product was subsequently excised from pILL219 and cloned into a linearized preparation of 15 pILL221 (pILL222, Figure 14).

Expression of recombinant urease polypeptides in the vector pMAL The expression vector pMAL is under the control of an inducible promoter (Plac) and contains an 20 open reading frame (ORF) that encodes the production of MalE (Maltose-binding protein, MBP). Sequences cloned in-phase with the latter ORF resulted in the synthesis of MBP-fused proteins which were easily purified on amylose resin. Of the two versions of pMAL that are commercially available, the version not encoding a signal sequence pMAL-c2) synthesized greater amounts of recombinant proteins and was thus used throughout.

E. coli clones harbouring recombinant plasmids were screened for the production of fusion proteins, prior to performing large-scale purification experiments.

Purification of recombinant urease polypeptides Fresh 500 mL volumes of Luria broth, containing carbenicillin (100 Ag/mL and 2% (wt/vol) glucose, were inoculated with overnight cultures mL) of E. coli clones. The cultures were incubated at 370C and shaken at 250 rpm, until the A 600 0.5. Prior to adding 1 mmol/L (final concentration) isopropyl-P- D-thiogalactopyranoside (IPTG) to cultures, a 1.0 mL 10 sample was taken (non-induced cells). Cultures were 0 incubated for a further 4 h at which time another mL sample (induced cells) was taken. The non-induced and induced cell samples were later analyzed by SDS-

PAGE.

15 IPTG-induced cultures were centrifuged at 7000 rpm for 20 min, at 4 0 C and the supernatant discarded. Pellets were resuspended in 50 mL column buffer (200 mmol/L NaC1, 1 mmol/L EDTA in 10 mmol/L TrisHC1, pH containing the following protease 20 inhibitors (supplied by Boehringer, Mannheim, Germany): 2 Amol/L leupeptin, 2 Lmol/L pepstatin and 1 mmol/L phenylmethylsulphonyl fluoride (PMSF). Intact calls were lysed by passage through a French Pressure cell (16 000 lb/in 2 Cell debris was removed by centrifugation and lysates were diluted in column buffer to give a final concentration of 2.5 mg protein/mL, prior to chromatography on a 2.6 cm x cm column of amylose resin (New England Biolabs). The resin was washed with column buffer at 0.5 mL/min until the A 280 returned levels. The MBP-fused recombinant proteins were eluted from the column by washing with column buffer containing 10 mmol/L 1maltose.

Fractions containing the recombinant proteins were pooled and then dialyzed several times at 4 0 C against a low salt buffer (containing 25 mmol/L NaC1 in 20 mmol/L TrisHCl, pH The pooled fractions were then loaded at a flow rate of mL/min onto a 1.6 x 10 cm anion exchange column (HP- Sepharose, Pharmacia, Sweden) connected to a Hi-Load chromatography system (Pharmacia). Proteins were eluted from the column using a salt gradient mmol/L to 500 mmol/L Nal) Fractions giving high absorbance readings at A 280 were exhaustively dialyzed g. against distilled water at 4 0 C and analyzed by SDS--

PAGE.

Rabbit antisera o Polyclonal rabbit antisera was prepared against total cell extracts of H. pylori strain (Labigne et al., 1991, supra) and H. fells (ATCC 49179). Polyclonal rabbit antisera against recombinant protein preparations of H. pylori and H.

felis urease subunits was produced by immunizing rabbits with 100 Mg of purified recombinant protein in Freund's complete adjuvant (Sigma). Four weeks later, rabbits were booster-immunized with 100 Mg protein in Freund's incomplete adjuvant. On week 6, the animals were terminally bled and the sera kept at -200C.

Protein analyzes by SDS-PAGE and Western blotting Solubilized cell extracts were analyzed on slab gels, comprising a 4.5% acrylamide stacking gel and a 10% resolving gel, according to the procedure of Laemmli. Electrophoresis was performed at 200 V on a mini-slab gel apparatus (Bio-Rad, USA).

Proteins were transferred to nitrocellulose paper in a Mini Trans-Blot transfer cell (Bio-Rad) set at 100 V for 1 h, with cooling. Nitrocellulose membranes were blocked with 5% (wt/vol) casein (BDH, England) in phosphate-buffered saline (PBS, pH 7.4) with gentle shaking at room temperature, for 2 h.

Membranes were reacted at 4 0 C overnight with antisera diluted in 1% casein prepared in PBS. Immunoreactants were detected using specific biotinylated secondary antibodies and streptavidin-peroxidase conjugate

S*

(Kirkegaard and Parry Lab., Gaithersburg, USA).

Reaction products were visualized on autoradiographic film (Hyperfilm, Amersham, France) using a chemiluminescence technique (ECL system, Amersham).

r" Protein concentrations were determined by the Bradford assay (Sigma Chemicals Corp., St. Louis,

.USA)

Animal experimentation Six week old female Swiss specific Pathogen- Free (SPF) mice were obtained (Centre d'Elevage R.

Janvier, Le-Genest-St-Isle, France) and maintained on a commercial pellet diet with water ad libitum. The intestines of the animals were screened for the absence of Helicobacter muridarum. For all orogastric administrations, 100 AL aliquots were delivered to mice using 1.0 mL disposable syringes, to which polyethylene catheters (Biotrol, Paris, France) were attached.

Preparation of sonicated extracts and inocula from H.

39 felia cultures H. felis bacteria were harvested in PBS and centrifuged at 5000 rpm, for 10 min in a Sorvall centrifuge (Sorvall, USA) at 4°C. The pellets were washed twice and resuspended in PBS. Bacterial suspensions were sonicated as previously described and were subjected to at least one freeze-thaw cycle.

Protein determinations were carried out on the sonicates.

To ensure a virulent culture of H. felis for protection studies, H. felis bacteria were maintained in vivo until required. Briefly, mice were inoculated three times (with 1010 bacteria/mL), over a period of days. The bacteria were reisolated from stomach biopsies on blood agar medium (4-7 days' incubation in a microaerobic atmosphere at 370C). Bacteria grown for two days on blood agar plates were harvested directly in peptone water (Difco, USA). Bacterial viability and motility was assessed by phase microscopy prior to administration to animals.

Mouse protection studies Fifty Ag of recombinant antigen and 10 Ag cholera holotoxin (Sigma Chemical Corp.), both resuspended in HCo3, were administrated orogastrically to mice on weeks 0, 1, 2 and 3. Mice immunized with sonicated H. felis extracts (containing 400 800 Ig of total protein) were also given 10 Ag of cholera toxin. On week 5, half of the mice from each group were challenged with an inoculum of virulent H. felis.

The remainder of the mice received an additional "boost" immunization on week 15. On week 17 the latter were challenged with a culture of H. fells.

Assessment of H. fells colonization-of the mouse Two weeks after receiving the challenge dose weeks 7 and 19, respectively) mice were sacrificed by spinal dislocation. The stomachs were washed twice in sterile 0.8% NaCi and a portion of the gastric antrum from each stomach was placed an the surf aces of 12 cm x 12 cm agar plates containing a urea indicator medium urea, 120 mg Na 2 HPO,, 80 mg

KH

2

PO

4 1.2 mg phenol red, 1.5 g agar prepared in 100 ML). The remainder of each stomach was placed in formal-saline and stored until processed for histology. Longitudinal sections (4 Atm) of the stomachs were cut and routinely stained by -the Giemsa technique, When necessary, sections were additionally stained by the Haematoxylin-Eosin and Warthin-Starry silver stain techniques.

The presence of HI. felis bacteria in mouse gastric mucosa was assessed by the detection of urease activity (for up to 24 h) on the indicator medium, as well as by the screening of Giemsa-stained gastric sections that had been coded so as to eliminate observer bias. The numbers of bacteria in gastric sections were semi -quantitatively scored according to the following scheme: 0, no bacteria seen throughout sections; 1, few bacteria 20) seen throughout; 2, occasional high power field with low numbers of bacteria; 3, occasional H.P. field with low to moderate numbers 50) of bacteria; and 4, numerous 5) H.P. fields with high numbers of bacteria Mononuclear cell infiltrates were scored as i 41 follows: 0, no significant infiltration; 1, infiltration of low numbers of mononuclear cells limited to the submucosa and muscularis mucosa; 2, infiltration of moderate numbers of mononuclear cells to the submucosa and muscularis mucosa, sometimes forming loose aggregates; and 3, infiltration of large numbers of mononuclear cells and featuring nodular agglomerations of cells.

RESULTS OF PART II EXPERIMENTS Expression of Helicobacter urease polypeptides in E.

coli Fragments containing the sequences encoding the respective UreA gene products of H. felis and H.

pylori were amplified by PCR and cloned in-phase with an ORF encoding the 42 kDa MBP, present on the expression vector pMAL. Sequencing of the PCR Sproducts revealed minor nucleotidic changes that did not, however, alter the deduced amino acid sequences of the respective gene products. E. coli MC1061 cells transformed with these recombinant plasmids (pILL919 and pILL920, respectively) expressed fusion proteins with predicted molecular weights of approximately 68 kDa. Following chromatography on affinity (amylose resin) and anion exchange gel media (Q-Sepharose), these proteins were purified to high degrees of purity (Figure The yield from 2-L cultures of recombinant E. coli cells was approximately 40 mg of purified antigen.

Similarly, the large UreB subunits of H.

pylori and H. fells ureases were expressed in E. coli (plasmids pILL927 and plLL222, respectively) and

I

42 produced fusion proteins with predicted molecular weights of 103 kDa. The yield in these cases was appreciably lower than for the UreA preparations (approximately 20 mg was recovered from 2-L of bacterial culture). Moreover, problems associated with the cleavage of the UreB polypeptides from the MBP portion of the fusion proteins were encountered.

These difficulties were attributed to the large sizes of the recombinant UreB polypeptides.

Analysis of the recombinant urease polypeptides Western blot analyses of the antigen preparations with rabbit polyclonal antisera raised to whole extracts of H. pylori and H. fellis bacteria demonstrated that the antigens retained immunogenicity to the homologous as well as heterologous antisera (Figures 14 and 15). The antisera did not recognize the MBP component alone. Cross-reactivity between the urease polypeptides of H. pylori and H. fells was consistent with the high degrees of identity between the amino acid sequences of these proteins.

Rabbit polyclonal antisera raised against purified recombinant UreA and UreB proteins prepared from H. pylori and H. fells strongly reacted with the urease polypeptides present in whole-cell extracts of the bacteria (Figure 16). As we had already observed, the UreB subunit of H. felis urease migrated slightly higher on SDS-PAGE gels than did that of H. pylori (Figure 16).

Preparation of H. fells inocula used in Immunoprotection studies To ensure the virulence of H. fells ,1 43 bacterial inocula, bacteria were reisolated from H.

felis infected mouse stomachs (see Materials and methods). The bacteria were passaged a minimum number of times in vitro. Stock cultures prepared from these bacteria, and stored at -800C, were used to prepare fresh inocula for other mouse protection studies.

This procedure ensured that the inocula used in successive experiments were reproducible.

Immunization of mice against gastric H. felis infection h Mice that had been immunized for three weeks with the given antigen preparations were divided into two lots and one half of these were challenged two weeks later with an H. felis inoculum containing 10 7 bacteria/mL. One group of animals that had been immunized with recombinant H. felis UreA were also challenged but, unlike the other animals, were not sacrificed until week 19.

Protection at week 20 Eighty-five% of stomach biopsy samples from the control group of mice immunized with H. felis sonicate preparations were urease-negative and therefore appeared to have been protected from H.

felis infection (Table This compared to 20% of those from the other control group of animals given MBP alone. The proportion of urease-negative stomachs for those groups of mice given the recombinant urease subunits varied from 70% (for H. pylori UreB) to (for H. pylori UreA).

The levels of bacterial colonisation by H.

44 felis was also assessed from coded histological slides prepared from gastric tissue. Due to the striking helical morphology of H. felis bacteria, the organisms could be readily seen on the mucosal surf aces of both gastric pit and glandular regions of the stomach.

Histological evidence indicated that the levels of protection in nice was lower than that observed by the biopsy urease test: 25% and 20% of gastric tissue from mice immunized with H. felis sonicate preparations of H. pylori UreB, respectively, were free of H. felis bacteria.

Amongst certain groups of these mice the preponderance of urease-negative biopsies, as well as lower histological scores for bacterial colonisation (unpublished data), suggested that an immunoprotective response had been elicited in the animals. This response, however, may have been insufficient to protect against the inoculum administered during the challenge procedure.

Protection at week 17 The remaining mice, from each group of animals, were boosted on week 15. These mice were challenged at week 17 with an H. felis inoculum containing approximately 100-fold less bacteria than that used previously. Two weeks later all stomach biopsies from the MBP-immunized mice were ureasepositive (Table In contrast, urease activity for gastric biopsies from mice immunized with the recombinant urease subunits varied from 50% for H.

pylori UreA to 100% for H. felis UreB. The latter was comparable to the level of protection observed for the group of animals immunized with H. felis sonicated extracts. Histological evidence demonstrated that the UreB subunits of H. fells and H. pylori protected and 25% of immunized animals, respectively. This compared with a level of 85% protection for mice immunized with H. felis sonicated extracts.

Immunization of mice with recombinant H. pylori UreA did not protect the animals. Similarly, the stomachs of all H. felis UreA-immunized mice, that had been challenged at week 5, were heavily colonised with H.

felis bacteria at week 19 (Table 4).

The urease gastric biopsy test, when compared to histological analysis of gastric tissue sections, gave sensitivity and specificity values of 63% and 95%, respectively. Thus histology proved to be the more accurate predictor of H. fellis infection in the mouse.

Cellular immune response in immunized stomacha In addition to the histological assessment 20 of H. fells colonisation, mouse gastric tissue was also scored (from 0 to 3) for the presence of a mononuclear cell response. In mice immunized with MBP alone, a mild chronic gastritis was seen with small numbers of mononuclear cells restricted to the muscularis mucosa and to the submucosa of the gastric epithelium. In contrast, there were considerable numbers of mononuclear cells present in the gastric mucosae from animals immunized with either the recombinant urease polypeptides, or with H. felis sonicate preparations. These inflammatory cells 46 coalesced to form either loose aggregates, in the submucosal regions of the tissue, or nodular structures that extended into the mucosal regions of the gastric epithelia. The mononuclear cell response did not appear to be related to the presence of bacteria as the .gastric mucosae from the H. felis UreA-immunized mice, that were heavily colonized with H. felis bacteria, contained little or no mononuclear cells.

C i.e C. 0 C C 0* Ce e e *q e C. p p

S

9*im

CC

*0 C C 9** *0 C eOO O&W p p W c *0 CC 0 0 ep *C 44, TABLE 2 The: oligomeric primers used in PCR-based amplification of ureasie-encloding nucleotide sequences., #1 f orw rev CAU CICT* AAA GAA IT(C) TA* GAT(1C) AAA T ATG T TC C TT A*CG A*CG A*G (C)A A (TAT C(IT)ITT C(T)ITT CAT CLJA..

#2 f orw CC GGA GAA TTC ATT AGC AGA AAA GAA TAT GTT 'TCT ATG EcoRI" revl AC GTT CTG CAG CTT ACG AAT AAC TTT TGTI TGC: TTG AGC Ps tI' #3 f orw GGA TC1C AAA AAG ATT TCA CG SBamHI' rev GGAAGC TT C TGC AGTGT GCT TCC CCAGT'C HindIII' PStI' *Degenerated nucleotides in which all possible permutations of 'the genetic code were included G, ()Bracketed nucleoltides indicate that the given nucleotides were: degenerated with the specific base(s) shown.

T Restriction sites introduced in the amplified fragments.

je.

9 9 9* 9 4, *9 4 4 4. 4 4. 4 4 4 4** 9 4 *4 4 4 4e* 4 .44 0% 0' 4. 4 'TABLE 3 Plasmids used Plasmid Vector Relevant phenotype or character Reference p ILL7'63 pILL57O 9.5 kb fragment (Sau3a partial digest of H. pylori Cussac! et al., Ichromosome:) (SpR) 19,911 pILL1919 pILL575 35 kb fragment i(Sau3A partial digest of H. fells Ferrero chromosome) Labigne, 19,93' PILL2O7 pILL57O 11 kb fragment, (Sau3A partial digest of pILL199) This, study pILL919 pMAL -C2 0.8 kb BaznHI-Pstla insert containing a nuclelotilde This study fragment encoding H. fells ureA gene (APR) pILL92O, pMAL,-C'2 0.8 kb BamHI-pStja insert containing PCR product This study _______encoding H. pylori ureA gene pILL92,7 pMAL-c2 1.8, kb EcoRI-pStja PCR fragment encoding H. pyliori ureB This study gene pI'LL 2l3 pUC19 2, kb fragment resulting from Sau3A partial digest of This study (ApR) p ILL2 19 pMAL-,C2 1.4 k-b DraI-HindIIIb' insert containing H. fells ureB This study (bases 657-17,017) pILL221 pMAL-C2 0.7 kb BamHI-PstI PCR fragment encoding H. felis ureB, This study (bases 4-6,67), pILL222 pMAL-C2 1.35 'kb PstI-PstIc fragment encoding H. fells ureB This study (bases 667-1707) from pILL:219 cloned intollinerized !L pILL1221 4 TABLE 4 Protection of mice by immunization with recombinant urease proteins.

MBP

0% (0/10) 0%6 (0/10) 99 9 .9 9 99 9 .9 9 9 9 9=9* 9* *9 9 UreA H. pylori 50 0 (0/10) UreA H. felie'b 12.5 0 (0/10) UreB H. pylori 65 2S (2/8) UreB H. fells 100 60 (5/7) H. fells sonicate 100 85 (7/8) Challenge inoculum dose was 105 bacteria/mouse Mice were challenged on week 5 (with 101 bacteria) and were sacrificed on week 19 III HELICOBACTER PYLORI hspA-B HEAT SHOCK GENE CLUSTER: NUCLEOTIDE SEQUENCE. EXPRESSION AND

FUNCTION

A homolog of the heat shock proteins (HSPs) of the GroEL class, reported to be closely associated with the urease of Helicobacter pylori (a nickel metalloenzyme), has recently been purified from H.

pylori cells by Dunn et al., 1992, Infect. Immun. 1946-1951 and Evans et al., 1992, Infect. Immun. 2125-2127, respectively). Based on the reported Nterminal amino acid sequence of this immunodominant protein, degenerated oligonucleotides were synthesized in order to target the gene (hspB) encoding the GroELlike protein in the chromosome of H. pylori strain 85P. Following gene amplification, a 108-base pair S (bp)-fragment encoding the 36 first amino acids of the HspB protein was purified, and used a probe to identify in the H. pylori genomic bank a recombinant cosmid harboring the entire HspB encoding gene. The hspB gene was mapped to a 3.15 kilobases (kb) BglII restriction fragment of the pILL684 cosmid. The nucleotide sequence of that fragment subcloned into the pILL570 plasmid vector (pILL689) revealed the presence of two open reading frames (ORFs) designated hspA and hspB, the organization of which was very similar to be groESL bicistronic operons of other bacterial species. hspA and hspB encode polypeptides of 118 and 545 amino acids respectively, corresponding to calculated molecular masses of 13.0 and 58.2 kilodaltons (kDa), respectively. Amino acid sequence comparison studies revealed that the H. pylori 51 HspA and HspB protein were highly similar to their bacterial homologs; (ii) that the HspA H. pylori protein features a striking motif at the carboxyl terminus that other bacterial GroEs-homologs lack; this unique motif consists of a series of eight histidine residues resembling metal binding domain, such a nickel binding. Surprisingly, immediately upstream of the gene cluster an IS5 insertion element was found that was absent in the H. pylori genome, and was positively selectioned during the cosmid cloning process. The IS5 was found to be involved in the 9 expression of the hspA and hspB genes in pILL689. The expression of the HspA and HspB proteins from the pILL689 plasmid was analyzed in minicell-producing strain. Both polypeptides were shown to be constitutively expressed in the E. coli cells. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster into an E. coli host strain, an increase of urease activity 20 was observed suggesting a close interaction between the heat shock proteins and the urease enzyme.

Supporting the concept of a specific function for the HspA chaperone, was the fact that whereas a single hspB copy was found in the H. pylori genome, two copies of the hspA were found in the genome, one linked to the hspB gene and one unlinked to the hspB gene. Attempts to construct isogenic mutants of H.

pylori in the hspA and the hspB gene were unsuccessful suggesting that these genes are essential for the survival of the bacteria.

52 EXPERIMENTAL PROCEDURES FOR PART III Bacterial strains, plasmids, and culture conditions The cloning experiments were performed with genomic DNA prepared from H. pylori strain 85P. H.

pylori strain N6 was used as the recipient strain for the electroporation experiments because of its favorable transformability. E. coli strain HB101 or strain MC1061 were used as a host for cosmid cloning and subcloning experiments, respectively. E. coli P678-54 was used for preparation of minicells. vectors and recombinant plasmids used in this study are listed in Table 1. H. pylori strains were grown on horse blood agar plates, supplemented with vancomycin mg/l), polymyxin B (2,500 trimethoprim (5 mg/l), and amphotericin B (4 mg/l). Plates were incubated at 9 S"370C under microaerobic conditions in an anaerobic jar with a carbon dioxide generator envelope (BBL 70304).

E. coli strains were grown in L-broth without glucose (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter; pH 7.0) or on L-agar plates (1.5 agar) at 37 0 C. For measurement of urease activity, the nitrogen-limiting medium used consisted of ammonium-free M9 minimal agar medium (pH 7.4) containing 0.4% D-glucose as the carbon source, and freshly prepared filter-sterilized L-arginine added to the final concentration of 10 mM. Antibiotic concentrations for the selection of recombinant clones were as follows (in milligrams per liter): kanamycin, spectinomycin, 100; carbenicillin, 100.

Preparation of DNA Genomic DNA from H. pylori, was prepared as 53 previously described. Cosmid and plasmid DNAs were prepared by an alkaline lysis procedure followed by purification in cesium chloride-ethidium bromide gradients as previously described.

Cosmid cloning The construction of the cosmid gene bank of H. pylori 85P in E. coli HB101, which was used for the cloning of the H. pylori hspA-B gene cluster, has been described previously.

DNA analysis and cloning methodology Restriction endonucleases, T4 DNA ligase, e DNA polymerase I large (Klenow) fragment, and Taq polymerase were purchased from Amersham, T4 DNA polymerase from Biolabs, and calf intestinal phosphatase from Pharmacia. All enzymes were used 0* according to the instructions of the manufacturers.

o DNA fragments were separated on agarose gels run in Tris-acetate buffer. The 1-kb ladder from Bethesda Research Laboratories was used as a fragment size standard. When necessary, DNA fragments were isolated by. electroelution from agarose gels as previously described and recovered from the migration buffer by means of an Elutip-d minicolumn (Schleicher and Schuell, Dassel, Germany). Basic DNA manipulations were performed according to the protocols described by Sambrook et al.

Hybridization Colony blots for screening of the H. pylori cosmid bank and for identification of subclones were prepared on nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) according to the protocol of Sambrook et al. Radioactive labelling of PCR products was performed by random-priming, using as primers the random hexamers from Pharmacia. Colony hybridizations were performed under high stringency conditions (5 x SSC, 0.1% SDS, 50% formamide, 42 0 C) (1 x SSC; 150 mM NaC1, 15 mM sodium citrate, pH For Southern blot hybridizations, DNA fragments were transferred from agarose gels to nitrocellulose sheets (0.45 ltm pore size; Schleicher Schuell, Inc.), and a.

10 hybridized under low stringency conditions (5 x SSC, *9 "Do 0.1% SDS, 30 or 40% formamide, at 42 0 C with 3 "P-labeled deoxyribonucleotide probes. Hybridization was revealed by autoradiography using Amersham Hyperfilm-MP.

DNA sequencing 15 Appropriate fragments of plasmid DNA were subcloned into M13mpl8/19 vectors. Single stranded DNA was prepared by phage infection of E. col strain JM101. Sequencing was performed by the dideoxynucleotide chain termination method using the United States Biochemicals Sequenase kit. Both the M13 universal primer and additional specific primers (Figure 1) were used to sequence both the coding and non-coding DNA strands. Sequencing of double-stranded DNA was performed as previously described. Direct sequencing of PCR product was carried out following purification of the amplified, electroeluted PCR product through an Elutip-d minicolumn (Schleicher Schuell). The classical protocol for sequencing using the Sequenase kit was then used with the following modifications: PCR product was denatured by boiling annealing mixture containing 200 picomoles of the oligonucleotide used as primer and DMSO to the final concentration of 1% for 3 minutes; the mixture was then immediately cool on ice; the labeling step was performed in presence of manganese ions (mM).

Electroporation of H. pylori In the attempt to construct H. pylori mutants, appropriate plasmid constructions carrying the targeted gene disrupted by a cassette containing a kanamycin resistance gene (aph3'-III), were transformed into H. pylori strain N6 by means of S* electroporation as previously described. Plasmid pSUSlO harboring the kanamycin disrupted flaA gene was used as positive control of electroporation. After electroporation, bacteria were grown on non-selective S" 15 plates for a period of 48 h in order to allow for the expression of the antibiotic resistance and then transferred onto kanamycin-containing plates. The selective plates were incubated for up to 6 days.

S: Polvmerase chain reaction (PCR) PCRs were carried out using a Perkin-Elmer Cetus thermal cycler using the GeneAmp kit (Perkin- Elmer Cetus). Classical amplification reaction involved 50 picomoles (pmoles) of each primer and at least 5 pmoles of the target DNA. The target DNA was heat denatured prior addition to the amplification reaction. Reaction consisted of 25 cycles of the following three steps denaturation (94oC for 1 minute), annealing (at temperatures ranging between 42 and 55°C, depending on the calculated melting temperatures of the primers, for 2 min), and extension (72 0 C for 2 min). When degenerated oligonucleotides 56 were used in non-stringent conditions, up to 1000 pmoles of each oligonucleotide were added, 50 cycles were carried out, and annealing was performed at 42 0

C.

Analysis of proteins expressed in minicells Minicells harboring the appropriate hybrid plasmid were isolated and labeled with methionine p Ci/ml). Approximately 100,000 cpm of acetoneprecipitable material was subjected to sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis in a 10 12.5% gel. Standard proteins with molecular weights ranging from 94,000 to 14,000 (low< molecular-weights kit from Bio-Rad Laboratories were run in parallel.

The gel was stained and examined by fluorography, using En Hance (New England Nuclear).

Urease activity Urease activity was quantitated by the Berthelot reaction by using a modification of the procedure which has already been described. Urease activity was expressed as micromoles of urea hydrolyzed per minute per milligram of bacterial protein.

RESULTS OF PART III EXPERIMENTS Identification of a recombinant cosmid harboring the Helicobacter pylori GroEL-like heat shock protein encoding gene Based on the published N-terminal amino sequence of the purified heat shock protein of H.

pylori, two degenerated oligonucleotides were synthesized to target the gene of interest in the chromosome of H. pylori strain 85P. The first one G CNAA RGARATHAARTTY TCNG 3' where N stands for the four nucleotides, R A and G, Y T and C, N T, C, and A, is derived from for the first 8 amino acids of the protein (AKEIKFSD); the second one 5' CRTTN C K NCCNCKNGGNCC C A T where K G and T, corresponds to the complementary codons specifying the amino acid from position 29 to position 36 (MGPRGRNV, ref). The expected size for the PCR product was 108 base pairs The amplification reaction was performed under low stringency conditions as described in the "Materials and Methods" section, and led to the synthesis of six fragments with size ranging from 400 bp to 100 bp. The three smallest fragments were electroeluted from an acrylamide gal, and purified.

15 Direct sequencing of the PCR products permitted the identification of a DNA fragment encoding an amino S. acid sequence corresponding to the published sequence.

This fragment was therefore labeled and used as probe in colony hybridization to identify recombinant cosmids exhibiting homology to a 51 segment of the H.

pylori GroEL-like encoding gene this gene was further designated hspB. The gene bank consists of 400 independent kanamycin-resistant E. coli transductants harboring recombinant cosmids. Of those one single clone hybridized with the probe, and harbored a recombinant plasmid designated pILL684, 46 kb in size.

The low frequency observed when detecting the hspB gene (1 of 400) was unusual when compared with that of several cloned genes which were consistently detected in five to seven recombinant cosmids. In order to identify the hspB gene, fragments with sizes of 3 to 4 58 kb were generated by partial restriction of the pILL684 cosmid DNA with endonuclease Sau3A, purified, and ligated into the BglII site of plasmid vector Of 100 subclones, x were positive clones, and one was further studied (pILL689); it contains a 3.15 kb insert, flanked by two BglII restriction sites, that was mapped in detail (Figure Using the PCR "P-labeled probe, the 51 end of the hspB gene was found to map to the 632 bp HindIII-SphI central restriction fragment of pILL689, indicating that one could expect the presence of the entire hspB gene in the pILL689 recombinant plasmid.

DNA sequence and deduced amino acid sequence of the H.

pylori hspA-D cone cluster The 3200 bp of pILL689 depicted in Figure were sequenced by cloning into M13mpl8 and M13mpl9, ~the asymmetric restriction fragments BglII-SphI, Sphl- HindIII, HindilI-BglII; each cloned fragment was independently sequenced on both strands 16 oligonucleotide primers (Figure 1) were synthesized to confirm the reading and/or to generate sequences overlapping the independently sequenced fragments these were used as primers in double-stranded-DNA sequencing analyses. The analysis of the sequence revealed two distinct genetic elements. First the presence of two open reading frames (ORFs), depicted in Figure 5, transcribed in the same direction, that were designated hspA and hspB. The nucleotide sequence and the deduced amino acid sequence of the two ORFs are presented in Figure 6. The first codon of hspA begins 323 bp upstream of the leftward Hindlll site of pILL689 (Figure 5) and is preceded by a Shine-Dalgarno ribosome-binding site (RBS) (GGAGAA). The hspA ORF codes for a polypeptide of 118 amino acids. The initiation codon for the hspB ORF begins nucleotides downstream the hspA stop codon; it is preceded by a RBS site (AAGGA). The hspB ORF encodes a polypeptide of 545 amino acids and is terminated by a TAA codon followed by a palindromic sequence resembling a rho-independent transcription terminator 10 (free energy, AG -19.8 kcal/mol) (Figure The Nterminal amino acid sequence of the deduced protein HspB was identical to the N-terminal sequence of the purified H. pylori heat shock protein previously published with the exception of the N-terminal methionine, which is absent from the purified protein and might be post-translationally removed, resulting in a mature protein of 544 amino acids.

The deduced amino acid sequences of H, pylori HspA and HspB were compared to several amino acid sequences of HSPs of the GroES and GroEL class (Figure HspB exhibited high homology at the amino acid level with the Legionella pneumophila HtpB protein (82.9% of similarities), with the Escherichia coli GroEL protein (81.0% of similarities), with the Chlamydia psittaci or C. trachomatis HypB protein (79.4% of similarities), with Clostridium perfringens protein (80.7% of similarities), and to a lesser extent to the GroEL-like proteins of Mycobacterium.

However, like almost all the GroEL homologs, H. pylori HspB demonstrated the conserved carboxyl-terminus glycine-methionine motif (MGGMGGMGGMGGMM) which was recently shown to be dispensable in the E. coli GroEL chaperonin. The degree of homology at the amino acid level between the H. pylori HspA protein and the other GroES-like proteins is shown in Figure 7. The alignment shown features a striking motif at the carboxyl terminus of the H. pylori HspA protein that other bacterial GroES-homologs lack. This unique highly charged motif consists of 27 additional amino acids capable of forming a loop between two double cysteine residues; to the 27 amino acids, 8 are histidine residues highly reminiscent of a metal binding domain.

The second genetic element revealed by the sequence analysis, was the presence of an insertion sequence (IS5) 84 bp upstream of the hspA gene. The nucleotide sequence of this element matched perfectly that previously described for IS5 in E. coli, with the presence of a 16 sequence (CTTGTTCGCACCTTCC) that corresponds to one of the two inverted repeats which flank the IS5 element. Because of the perfect match at the DNA level, we suspected that the IS5 was not initially present in the H. pylori chromosome, but had rather inserted upstream of the hspA-HspB gene cluster during the cloning process, a hypothesis that needed to be confirmed by further analyses.

Identification of the upstream sequence of- the hspA-D gene cluster in H. pylori chromosome The presence of- the IS5 was examined by gene amplification using two oligonucleotides, one being internal to the ISS element and the other one downstream of the IS5 element (oligo #1 and Figure 61 to target a putative sequence in the chromosome of H. pylori strain 85P, (ii) in the initial cosmid pILL684, and (iii) in the 100 subclones resulting of the Sau3A partial restriction of the pILL684 recombinant cosmid. IS5 was absent from the chromosome of H. pylori, and was present in the very first subcultures of the E. coli strain harboring cosmid PILL684. Among the 100 pILL684 subclone derivatives which appeared to contain all or part of the IS5 sequence, we then looked for a subclone harboring the left end side of the IS5 plus the riginal upstream sequence of the hspA-hspB gene cluster. This screening was made by restriction analysis of the different Sau3A partial generated subclones. The restriction map of one (pILL694) of the plasmids fulfilling these criteria is shown in Figure 5. The left end side of the IS5 nucleotide sequence was determined; the presence of a 4-bp duplication CTAA on both side of the 16-bp inverted repeats of the IS5 element (Figure 6) allowed us to confirm the recent acquisition of element by transposition. A 245-nucleotide was then determined that mapped immediately of the IS5 element (shown Figure This consists of a non coding region in which the of a putative consensus heat shock promoter was detected it shows a perfectly conserved -35 region (TAACTCGCTTGAA) and a less consentaneous -10 region (CTCAATTA). Two oligonucleotides and shown on Figure 2) were synthesized which mapped to sequences located on both side of the IS5 element present in the recombinant cosmid; these two oligonucleotides should 62 lead to the amplification of a XXXXbp fragment when the IS5 sequence is present and a fragment in the absence of the IS5. The results of the PCR reaction using as target DNA the pILL684 cosmid, the pILL694 plasmid, and the H. pylori 85P chromosome fit the predictions (results not shown). Moreover, direct sequencing of the PCR product obtained from the H.

pylori chromosome was performed and confirmed the upstream hspA-hspB reconstructed sequence shown in 10 Figure 6 To further confirm the genetic organization of the whole sequenced region, two probes were prepared by gene amplification of the pILL689 plasmid using oligonucleotides #5 and and #7 and #8 (Figure They were used as probes in Southern hybridization experiments under low stringency conditions against an HindIII digest of the H. pylori 85P chromosome. The results demonstrate that no other detectable rearrangement had occurred during the 9.

cloning process (data not shown) These experiments allowed us to demonstrate that whereas a single copy of the hspB gene was present in the chromosome of H.

pylori strain 85, two copies of the hspA gene were detected by Southern hybridization.

Analysis of polypeptides expressed in minicells The pILL689 and the pILL692 recombinant plasmids and the respective cloning vectors pILL570, and pACYC177, were introduced by transformation into E. coli P678-54, a minicell-producing strain. The pILL689 and pILL692 plasmids (Figure 5) contain the same 3.15-kb insert cloned into the two vectors.

pILL570 contains upstream of the poly-cloning site a stop of transcription and of translation the orientation of the insert in pILL689, was made in such way that the transcriptional stop was located upstream of the IS5 fragment and therefore upstream of the hspA and hspB genes. Two polypeptides that migrated with polypeptides having apparent molecular weights of kDa and 14 kDa were clearly detected in minicell experiments from pILL689 and pILL692 (results not shown), whereas they were absent from the 10 corresponding vectors; these results indicated that the hspA and hspB genes were constitutively expressed from a promoter located within the IS5 were constitutively expressed from a promoter located within the IS5 element. Moreover, whereas the amount 15 of polypeptides visualized on the SDS gel was in good agreement with the copy number of the respective vectors, the intensity of the two polypeptidic bands suggested a polycistronic transcription of the two genes.

Attempts to understand the role of the hspA and hspB proteins Two disruptions of genes were achieved in E.

coli by inserting the Km cassette previously described within the hspA or the hspB gene of plasmids pILL686 and pILL691. This was done in order to return the disrupted genes in H. pylori by electroporation, and to select for allelic replacement. The pILL696 resulting plasmid encoded a truncated form of the HspA protein, corresponding to the deletion of the Cterminal end amino acid sequence; in that plasmid the Km cassette was inserted in such way that the promoter 64 of the Km gene could serve as promoter for the hspB downstream gene. The pILL687 and PILL688 plasmids resulted from the insertion of the Km cassette in either orientation within the hspB gene. None of these constructs led to the isolation of kanamycin transformants of H. pylori strain N6, when purified pILL687, pILL688, pILL696 plasmids (Table 2, Figure were used in electroporation experiments, whereas the pSUS10 plasmid used as positive control always did.

10 These results suggest the H. pylori HspA and HspB protein are essential proteins f or the survival of H.

pylori.

Because of the constant description in rz*" the literature of a close association of the HspB protein with the urease subunits; (ii) the unique structure of the HspA protein with the C-terminal sequence reminiscent of a nickel binging domain, and (iii) of the absence of viable hspA and/or hspB mutants of H. pylori, we attempted to demonstrate a role of the H. pylori Hsps proteins in relations with the H. pylori urease by functional complementation experiments in E. coli. Plasmids pILL763 or pILL753 (both pILL570 derivatives, Table 5) encoding the urease gene cluster were introduced with the compatible pILL692 plasmid (pACYC177 derivative) that constitutively expresses the HspA the HspB polypeptides as visualized in minicells. In both complementations, the expression of the HspA and HspB proteins in the same E. coli cell allows to observe a three fold increase in the urease activity following induction of the urease genes on minimum medium supplemented with 10 mM L- Arginine as limiting nitrogen source.

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IV EXPRESSION, PURIFICATION AND IMMUNOGENIC PROPERTIES OF H. PYLORI HSPA AND HSPB EXPERIMENTAL PROCEDURE FOR PART IV Expression and purification of recombinant fusion proteins The MalE-HspA, and MalE-HspB fusion proteins were expressed following the cloning of the two genes within the pMAL-c2 vector as described in the "Results" section using the following primers:- 10 oligo #1 ccggagaattcAAGTTTCAACCATTAGGAGAAAGGGTC oligo #2 acgttctcagTTTAGTTTAGTTTTTTTTGTGATCATGACAGC oligo #3 ccggagaattcGCAAAAGAAATCAAATTTTCAGATAGC oligo #4 acgttctgcaqATGATACCAAAAAGCAAGGGGGCTTAC.

Two liters of Luria medium containing 15 glucose and ampicillin (100 pg/ml) were inoculated with 20 ml of an overnight culture of strain MC1061 containing the fusion plasmid and incubated with shaking at 37 0 C. When the OD600 of the culture reached 0.5, IPTG (at a final concentration of 20 10 mM) was added, and the cells were incubated for a further 4 hours. Cells were harvested by centrifugation (5000 rpm for 30 min at 4°C), resuspended in 100 ml of column buffer consisting of mM Tris-HC1, 200 mM NaCi, 1 mM EDTA supplemented with protease inhibitors [(Leupeptin (2 pM) Pepstatin (2 pm) PMSF (1 mM) Aprotinin (1:1000 dilution)], and passed through a French press. After centrifugation (10,000 rpm for 20 min at 4 0 the supernatant were recovered and diluted (2-fold) with column buffer. The lysate was filtered through a 0.2 pm nitrocellulose filter prior to loading onto a 68 preequilibrated amylose resin (22 x 2.5 cm). The fusion proteins were eluted with a 10 mM maltose solution prepared in column buffer, and the fractions containing the fusion proteins were pooled, dialyzed against distilled water, and lyophilized. Fusion proteins were resuspended in distilled water at a final concentration of 2 mg of lyophilized material/ml, and stored at -20 0 C. Concentration and purity of the preparations were controlled by the 10 Bradford protein assay (Sigma Chemicals) and SDS-PAGE analyses.

S. Nickel binding properties of recombinant proteins E. coli MC1061 cells, containing either the pMAL-c2 vector or derivative recombinant plasmids, 15 were grown in 100 ml-Luria broth in the presence of Scarbenicillin (100 xg/ml). The expression of the genes was induced with IPTG for four hours. The cells were centrifuged and the pellet was resuspended in 2 ml of Buffer A (6 M guanidine hydrochloride, 0.1 M 20 NaH 2 PO4, 0.01 Tris, pH After gentle stirring for one hour at room temperature, the suspensions were centrifuged at 10,000 g for 15 min at 4 0 C. A 1.6 ml aliquot of Nickel-Nitrilo-Tri-Acetic resin (Nickel- NTA, QIA express), previously equilibrated in Buffer A, was added to the supernatant and this mixture was stirred at room temperature for one hour prior to loading onto-a column. The column was washed with ml buffer A, then 30 ml buffer B (8 M urea, 0.1 M Naphosphate, 0.01 M Tris-HCl, pH The proteins were eluted successively with the same buffer as buffer B adjusted to pH 6.3 (Buffer C) pH 5.9 (Buffer D) and 69 pH 4.5 (Buffer E) and Buffer F (6 M guanidine hydrochloride, 0.2 M acetic acid). Fifty jl of each fraction were mixed with 50 Al of SDS buffer and loaded on SDS gels.

Human sera Serum samples were obtained from individuals, 28 were H. pylori-infected patients as confirmed by a positive culture for H. pylori and histological examination of the biopsy, and 12 were 10 uninfected patients. The sera were kindly provided by R. J. Adamek (University of Bochum, Germany).

Immunoblotting Upon completion of SDS-PAGE runs in a Mini- PROTEAN II electrophoresis cell, proteins were 15 transferred to nitrocellulose paper in a Mini Trans-- Blot transfer cell (Bio-Rad) set at 100 V for 1 h (with cooling). Immunostaining was performed as •previously described (Ferrero et al., 1992, supra), except that the ECL Western blotting detection system (Amersham) was used to visualize reaction products.

Human sera and the rabbit antiserum, raised against a whole-cell extract of H. pylori strain 85P, were diluted 1:1000 and 1:5000, respectively, in 1% (w/v) casein prepared in phosphate-buffered saline (PBS, pH 7.4).

Serological methods [enzyme-linked immunosorbent assay, (ELISA)] The following quantities of antigens were absorbed onto 96-well plates (Falcon 3072): 2.5 ig of protein MalE, 5 jig of MalE-HspA, or 2.5 Ag of MalE-- HspB. The plates were left overnight at 4 0 C, then washed 3 times with ELISA wash solution (EWS) PBS containing 0.05% Tween 20]. Saturation was achieved by incubating the plates for 90 min at 37 0

C

in EWS supplemented with 1% milk powder. Wells were again washed 3 times with EWS and then gently agitated for 90 min at 37 0 C in. the presence of human sera (diluted 1:500 in EWS with 0.5% milk powder), under agitation. Bound imunoglobulins were detected by incubation for 90 min at 37 0 C with biotinylated 10 secondary antibody (goat anti-human IgG, IgA or Igm diluted (1:1000] in EWS supplemented with 0.5% milk powder) in combination with streptavidin-peroxidase (1:500) (Kirkegaard and Perry Lab.). Bound peroxidase was detected by reaction with the citrate substrate 15 and hydrogen peroxide. Plates were incubated in the dark, at room temperature, and the optical density at 492 nm was read at intervals of 5, 15 and 30 min in an ELISA plate reader. After 30 min, the reaction was stopped by the addition of hydrochloric acid to a S 20 final concentration of 0.5 M.

RESULTS OF PART IV EXPERIMENTS Construction of recombinant plasmids producing inducible MalE-HspA. and HapB fusion proteins The oligonucleotides #1 and #2 (hspA) and #3 and #4 (hsB) were used to amplify by PCR the entire hspA and the hsaB genes, respectively. The PCR products were electroeluted, purified and restricted with EcoRI and PstI. The restricted fragments (360 bp and 1600 bp in size, respectively) were then ligated into the EcoRI-PstI restricted pMAL-c2 vector to generate plasmids designated pILL933 and pILL934, 71 respectively. Following induction with IPTG, and purification of the soluble protein on amylose columns, fusion proteins of the expected size (55 kDa for pILL933 [Figure 17], and 100 kDa for pILL9334) were visualized on SDS-PAGE gels. Each of these corresponded to the fusion of the MalE protein (42.7 kDa) with the second amino acid of each of the Hsp polypeptides. The yield of the expression of the fusion proteins was 100 mg for MalE-HspA and 20 mg for 10 MalB-HspB when prepared from 2 liters of broth culture.

Study of the antienicity of the HspA and HspB fusion proteins, and of the immunoenicity of HspA and HapB in patients infected with H. pylori 15 In order to determine whether the fusion proteins were still antigenic, each was analyzed by Western blot with rabbit antiserum raised against the MalE protein and a whole-cell extract of H. pylori strain 85P. Both fusion proteins were immunoreactive with antibody to MalE (not shown) and with the anti-H.

pylori antiserum. The anti-H. pylori antiserum did not recognize the purified MalE protein (Figure 18).

These results demonstrated that the fusion proteins retained their antigenic properties; in addition, whereas the HspB protein was known to be immunogenic, this is the first demonstration that HspA per se is immunogenic in rabbits.

In the same way, in order to determine whether the HspA and HspB polypeptides were immunogenic in humans, the humoral immune response against HspA and/or HspB in patients infected with H.

72 pylori was analyzed and compared to that of uninfected persons using Western immunoblotting assays and enzyme-linked immunosorbent assays (ELISA). None, of the 12 sera of the H. pylori-negative persons gave a positive immunoblot signal with MalE, MalE-HspA, or MalE-HspB proteins (Figure 18). In contrast, of 28 sera from H. pylori-positive patients, 12 (42.8%) reacted with the HspA protein whilst 20 (71.4%) recognized the HspB protein. All of the sera that recognized HspA also reacted with the HspB protein.

No association was observed between the immune response and the clinical presentation of the H.

pylori infection although such a conclusion might be premature because of the small number of strains 15 analyzed.

Nickel binding properties of the fused MalE-HspA protein MBP-HspA recombinant protein expressed following induction with IPTG, was purified from a whole cell extract by one step purification on nickel affinity column whereas the MBP alone, nor MBP-HspB exhibited this property. Figure 18 illustrates the one step purification of the MBP-HspA protein that was eluted as a monomer at pH 6.3, and as a monomer at pH 4.5. The unique band seen in panel 7 and the two bands seen in panel 5 were both specifically recognized with anti-HspA rabbit sera. This suggested that the nickel binding property of the fused MBP-HspA protein might be attributed to the C-terminal sequence of HspA which is rich in histidine and cysteine residues.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: INSTITUT PASTEUR STREET: 25-28 rue du Dr Roux CITY: PARIS CEDEX COUNTRY: FRANCE POSTAL CODE (ZIP): 75724 TELEPHONE: 45.68.80.94 TELEFAX: 40.61.30.17 NAME: INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE STREET: 101 rue de Tolbiac CITY: PARIS CEDEX 13 COUNTRY: FRANCE POSTAL CODE (ZIP): 75654 TELEPHONE: 44.23.60.00 TELEFAX: 45.85.07.66 (ii) TITLE OF INVENTION: IMMUNOGENIC COMPOSITIONS AGAINST HELICOBACTER INFECTION, POLYPEPTIDES FOR USE IN THE COMPOSITIONS AND NUCLEIC ACID SEQUENCES ENCODING SAID

POLYPEPTIDES.

(iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: EP 93401309.5 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2619 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: misc feature LOCATION: 31..36 OTHER INFORMATION: /standard_name- "Shine-Dalgarno sequence" 74 (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 756. .759 OTHER INFORMATION: /standard name- "Shine-Dalgarno sequence" (ix) FEATURE: NAME/KEY: CDS LOCATION:- 43. .753 OTHER INFORMATION: /standard name- "lIRE A" (ix) FEATURE: NAME/KEY: CDS LOCATION: 766. .2475 OTHER INFORMATION: /standard -name- "TIRE B" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

I

I.

I

9

I.

9q TCATAGCTTG GCTACCAATA GAAATTCAAT AAGGAGTTTA GC ATG AAA CTA ACC Met Lys Leu Thr 1 CCT AAA Pro Lys 5 GMA CTA GAC Glu Leu Asp AAG TTA ATG CTC Lys Leu Met Leu 10 CGT GGT CTG AAA Arg Gly Val Lys CAT TAT GCG GGC AGA TTG His Tyr Ala Gly Arg Leu

GCA

Ala 102 150 GAA CAA CGC TTG Glu Glu Arg Leu

CTC

Leu MAT TAG ACC GAA Asn Tyr Thr Glu GCG GTC Ala Val GC CTC ATT Ala Leu Ile AGO CTG GCG Ser Val Ala

AGC

Ser COG CGT GTG ATO Cly Arg Val Met

GAA

Glu 45 MAG GCG CGT GAT Lys Ala Arg Asp GGT MAT AMA Gly Aan Lys AA AGAA Lys Lys Glu 198 246 GAT TTG ATG CAA Asp Leu Met Gin

GAA

Glu 60 GGC AGG ACT TGG Gly Arg Thr Trp

CTT

Leu MAT GTG Asn Val ATG GAC GGC GTA Met Asp Gly Val

GCA

Ala 75 AGC ATG ATT CAT Ser Met Ile His

GAA

Glu GTG GGG ATT GMA Val Gly le Glu

GCT

Ala MAC TTC CCC GAT Asn Phe Pro Asp

GGA

Gly 90 ACC MAG CTT CIA Thr Lys Leu. Val

ACT

Thr 95 ATC CAC ACT CCG Ile His Thr Pro

GTA

Val 100 294 342 390 438 GAG CAT MAT GC Glu Asp Asn Gly GAG ATT ACT ATT Asp Ile Thr Ile 120

A

Lys 105 TTA GCC CCC GGC Leu Ala Pro Gly

GAG

Clu 110 GTC T1TC TTA A Val Phe Leu Lys MAT GAG Asn Glu 115 GTG A Val Lys AAC CCC GGC A Asn Ala Gly Lys GAA CC Glu Ala 125 ATT AGC TIC le Ser Leu

A

Lys 130 AAT AAA GGC Asn Lys Gly 135 GAT CGT Asp Arg CCT GTG CAG Pro Val Gin 140 GTG GGA TCA CAT Val Cly Ser His

TTC

Phe 145 CAC TTC TTC His Phe Phe 486 GAA GTG Clu Val 150 AAT AAG CTC TTG Asn Lys Leu Leu

CAC

Asp 155 TTC CAT CGC GCA Phe Asp Arg Ala AAA AGC ITT TGC Lys Ser Phe Cys 160 ITT GAA CCC GGG Phe Glu Pro Gly

AAA

Lys

GAG

Glu

CGC

Arg 165

GAA

Glu CTA GAC ATT GCA Leu Asp ile Ala AAA ACT CTG GAA Lys SerVal Clu 185

TCT

Ser 170 GGA ACA GCG GTG Gly Thr Ala Val

CGC

Arg 175 CTC AlT GAG ATC Leu Ile Asp Ile GGG AAT AAG CGC Cly Asn Lys Arg 180 ATC TAT Ile Tyr 195 582 630 .9 *9 9 9 9 9* p r p.

GGC ITT AAT Gly Phe Asn CTC GGC TIA Leu Cly Leu 215 GGT TGT GAA Gly Cys Glu 230

ICT

Ser 200 TTG GTG CAT CGC Leu Val Asp Arg

CAA

Gln 205 GCC GAT CCC CAT Ala Asp Ala Asp GGT AAA AAA Gly Lys Lys 210 GTA AAC TGC Val Asn Cys 678 AAA CGC GCT AAA Lys Arg Ala Lys CCG ACT AAA GAT Ala Thr Lys Asp 235

GAA

Glu 220 AAA GGT =h cGy Lys Gly Phe ly

ICT

Ser 225 726 774 AMA CAA TAACGAAAAA CC ATG AAA AAC Lys Gin Met Lys Lys 1 ATT TCA Ile Ser CGA AAA GAA TAT Arg Lys Glu Tyr

GTT

Val 10 TCT ATG TAT GCGT Ser Met Tyr Gly CCC ACT Pro-Thr ACC CGG GAT Thr Gly Asp

CGT

Arg GTT AGA CTC CGC Val Arg Leu Cly

GAG

Asp 25 ACT CAT TTG ATC Thr Asp Leu Ile

TTA

Leu 30 CAA GIG GAG CAT Clu Val Glu His

CAT

Asp TGG ACC ACT TAT Cys Thr Thr Tyr

GGT

Cly GMA GAG ATC AAA Clu Glu Ile Lys GGG GC. GGT AAA Cly Gly Gly Lys ACT ATC Thr Ile ITA CAT Leu Asp 870 918 966 CGT CAT GGG Arg Asp Gly TTG GIG CTC Leu Val Leu

ATG

Met AGT CAA ACC AAT Ser Gin Thr Asn

AC

Ser 60 CCT AGC TCT TAT Pro Ser Ser Tyr

GAA

Glu ACT AAC CC CTC ATT GIG GAG TAT ACC Thr Asn Ala Leu lie Val Asp Tyr Thr

GGC

Gly ATT TAG AAA Ile Tyr Lys 1014 1062 CCC GAC Ala Asp ATT GGG ATI AAA lie Gly lie Lys

CAC

Asp GGC AAG ATT GCA Gly Lys Ile Ala ATT CCC AAG GCA Ile Gly Lys Ala

GGC

Gly 100 AAT AAG GAC ATG CAA GAT GGC GTA CAT AAT AAT CT TGC GTA Asn Lys Asp Met Gin Asp Gly Val Asp Asn Asn Leu Cys Val

GGT

Cly 115 1110 CCT GOT ACA Pro Ala Thr GGC ATC GAT Gly lie Asp GCT TTT CC Ala Phe Ala 150 GAG GCT Glu Ala 120 TTG CCA OCT Leu Ala Ala GAG GGC Glu Gly 125 TTC ATT GTA ACC Leu Ile Val Thr GCT GGT Ala Gly 130 1158

ACG

Thr 135 CAT ATT CAC TTT His lie His Phe TCT CCC CAA CAA Ser Pro Gin Gln ATC CCT ACT Ile Pro Thr 145 ACA GGA CCT Thr Gly Pro 1206 1254 AGC GC CTT ACA Ser Gly Val Thr

ACC

Thr 155 ATG ATT GGA CGA Met Ile Gly Gly

GGC

Gly 160 GCG GAT Ala Asp 165 GGC ACG AAT GCG Gly Thr Asn Ala

ACC

Thr 170 ACC ATC ACT CCC Thr Ile Thr Pro

CGA

Cly 175 CGC GCT AAT CTA Arg Ala Asn Leu i 9

II,.

@1

I

i' i 9iI

I.

+i q **r

AAA

Lys 180 ACT ATC T-TG CGT Ser Met Leu Arg

GCA

Ala 185 CCC GAA GAA TAC Ala Clu Glu Tyr

GCC

Ala 190 ATG AAT CTA GGC Met Asn Leu Cly

'TT

Phe 195 TTC CCT AAG GO Leu Ala Lys Cly

AAT

Asn 200 GTG TCT TAC GAA Val Ser Tyr Clu TCT TTA CCC CAT Ser Leu Arg Asp CAG ATT Gin Ile 210 1302 1350 1398 1446 1494 GAA GCA GC Glu Ala Cly CCT GCA CCT Pro Ala Ala 230

GCG

Ala 215 ATT CGT TTT AAA Ile Gly Phe Lys

ATC

Ile 220 CAC GAA GAC TGC His Ciu Asp Trp GGA AGC ACA Cly Ser Thr 225 TAC GAT GTC Tyr Asp Val ATT CAC CAC TGC Ile His His Cys AAT GTC CCC CAT Asn Val Ala Asp

CAA

liu 240 CAA GTG Gin Val 245 GCT ATC CAC ACC Ala lie His Thr

CAT

Asp 250 ACC CTT AAC GAG Thr Leu Asn Glu

GCG

Ala 255 GGC TGT- TA GAA Gly Cys Val Clu

CAC

Asp 260 ACC CTA GAG GCG Thr Leu Glu Ala

ATT

Ile 265 CCC GG CCC ACC Ala Cly Arg Thr

ATC

Ile 270 CAT.ACC TTO CAC His Thr Phe His

ACT

Thr 275 1542 1590 1638 GAA GGG CT CGG Glu Cly Ala Gly

GGT

Cly 280 CGA CAC CCT CCA Cly His Ala Pro

GAT

Asp 285 CT ATC AAA ATC Val ie Lys Met GCA CC Ala Cly 290 GAA TTT AAC Clu Phe Asn AAA AAC ACT Lys Asn Thr 310

ATT

ile 295 CTA CCC CCC TCT Leu Pro Ala Ser

ACT

Thr 300 AAC CCG ACC Asn Pro Thr GM GCC GAG CAC Clu Ala Glu His

ATO

Met 315 GAC ATG TTA ATG Asp Met Leu Met ATT COT TTC ACC lbe Pro Phe Thr 305 GTC TCC CAC CAC Val Cys His His 320 GAT TCC AGG ATT Asp Ser Arg Ile 1686 1734 TTG CAT Leu Asp 325 AAA ACT ATC AAG Lys Ser Ile Lys GAT GTG CAG TTT Asp Val Gin Phe 1782 CGC CCC CAA ACT ATC GCG GCT GMA GAC CAA CTC CAT GAC ATC GGG Arg 340 Pro Gin Thr Ile Ala 345 Ala Glu Asp Gln Leu 350 His Asp Met Cly

ATC

Ile 355 1830 1878 TTT TCT ATC ACC Phe Ser Ile Thr

AGC

Ser 360 TCC GAG TCT CAG Ser Asp Ser Gin

OCT

Ala 365

GAC

Asp ATG GGA CGC OTA Met Gly Arg Val GGC GAG Gly Giu 370 GTG ATC ACA Val Ile Thr GGG CGC TTG Cly Arg Leu 390

CGC

Arg 375 ACT TGG GAG ACA Thr Trp Gin Thr

GCA

Ala 380 AAA MC AAA Lys Asn Lys AAA GAG TTT Lys Glu Phe 385 CGC ATC AAA Arg e Lys AAA GAG GAA AAA Lys Giu Glu Lys

GGC

Gly 395 GAT AAC GAC AAC Asp Asn Asp Asn

TTC

Phe 400

C

Cr !C C CC. C

C

C

C C

C

CC.'

CC

CC

CGC TAC Arg Tyr 405 ATC TCT AAA TAG Ile Ser Lys Tyr

ACC

Thr 410 ATC AAC CCC GG lie Asn Pro Gly

ATC

ile 415 GCG CAT CCC ATT Ala His Giy Ile 1926 1974 2022 2070 2118

TCT

Ser 420 GAG TAT GTC CCC Asp Tyr Val Gly

TCT

Ser 425 CTG GAA GTG GGC Val Giu Val Gly

AAA

Lys 430 TAC GCC GAC CTC Tyr Ala Asp Leu

GTC

Val 435 OTT TOG ACT CCC Leu Trp Ser Pro

GCT

Ala 440 TTC TTT CC ATT Phe Phe Cly Ile

AAG

Lys 445 CCC AAT ATO AT? Pro Asn Met Ile ATT AAG lie Lys 450 GGC GGA TTT Cly Cly Phe CCC ACC OCT Pro Thr Pro 470

AT?

Ile 455 GCG CTG TCT CAA Ala Leu Ser Gin

ATG

Met 460 GGC GAT CCC AAT Gly Asp Ala Asn GCG TOT ATT Ala Ser lie 465 CAC CAT GCC His His Cly 2166 2214

CC.'

c C C C. C GAG CCC OTC TAT Gin Pro Val Tyr

TAO

Tyr 475 COT GAA ATO TTT Arg Glu Met Phe

OGA

Gly 480 AAA AAC Lys Asn 485 AAA TTO GAG ACC Lys Phe Asp Thr

AAT

Asn 490 ATO ACT TTC GTG lie Thr Phe Val CAA GCG OCT TAC Gin Ala Ala Tyr

AAG

Lys 500 GCA GGC ATC AAA Ala Cly le Lys

GAA

Glu 505 GAA CTA GGC CTA Glu Leu Gly Leu

CAT

Asp 510 CCC 0CG GCA COG Arg Ala Aia Pro

CCA

Pro 515 2262 2310 2358 CTC A AAC TGT Val Lys Asn Gys

CGC

Arg 520 MAT ATO ACT AAA Asn lie Thr Lys

AAG

Lys 525 GAC CTC AMA TTG Asp Leu Lys Phe AAC GAT Asn Asp 530 CTG ACC OCA Val Thr Ala

CAT

His 535 ATT CAT GTC AAC Ile Asp Val Asn

CCT

Pro 540 GAA ACC TAT AAG Clu Thr Tyr Lys GTG AAA GTG Val Lys Val 545 2406 GAT GGC AAA GAG GTA ACC TCT AAA Asp Gly Lys Clu Val Thr Ser Lys 550 555 OCA GCA CAT GAA Ala Ala Asp Glu TTG AGC CTA GCG Leu Ser Leu Ala 560 2454 CAA CTT TAT AAT TTG TTC TAGGAGOCTA AGGAGGGGGA TAGAGGGGGT Gin Leu Tyr Asn Leu Phe 565 570 TTATTTAGAG GGGAGTCATT GATTTACCTT TGCTAGTTTA TAATGGATTT MAGAGAGGT TTTTTTCGTG TTTTATACCG CGTTGMAACC CTCAAATCTT TACCAAAAGG ATGGTAA INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 237 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM, Helicobacter felis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 2502 2562 2619 90***9 .9 9 9' 999* p 9 p p.

9 p 99 p p **ea p 9 0* 9 *9e* 9 p p p. 9 Met Lys Leu Thr Pro Lys Glu Leu Asp Gly Thr Asp Leu Val His Leu Leu Phe 145 Arg Glu Gly Lys Gly Thr Lys Lys 130 His Leu Ala Asn Lys Ile Pro Asn 115 Val Phe Ala 20 Val Lys Glu Giu Val 100 Giu Lys Phe 5 Glu Ala Ser Asri Ala Glu- Asp Asn Glu Glu Leu Val Val 70 Asn Asp Ile Lys Val 150 Arg Ile Ala 55 Met Phe Asn Thr Gly 135 Asn Leu Ser 40 Asp Asp Pro Gly Ile 120 Asp Lys Ala 25 Gly Leu Gly Asp Lys 105 Asn Arg Leu Lys 10 Arg Arg Met Val Gly 90 Leu Ala Pro Leu Gly Val Gln Ala 75 Thr Ala Gly Val Asp 155 Val Met Glu Ser Lys Pro Lys Gin 140 Phe Leu Met Leu His Tyr Ala Lys Glu Gly Met Leu dly Glu 125 Val Asp Leu Lys Arg I le Val Glu 110 Ala Gly Arg Asn Ala Thr His Thr Val Ile Ser Ala Tyr Arg Trp Giu Ile Phe Ser His Lys 160 Ser Phe Cys Lys Arg Leu Asp Ile Ala Ser Gly Thr Ala Val 165 170 Arg Phe 175 Glu Lys Asp Ser 225 (2) Pro Gly Glu Glu Lys Ser Val Glu Leu ile Asp Ile Gly Gly Asn 180 185 190 Arg Ile Tyr Gly Phe Asn Ser Leu Val Asp Arg Gin Ala Asp Ala 195 200 205 Gly Lys Lys Leu Gly Leu Lys Arg Ala Lys Glu Lys Gly Phe Gly 210 215 220 Val Asn Cys Gly Cys Glu Ala Thr Lys Asp Lys Gin 230 235 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 569 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE ORGANISM: Helicobacter felis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 6e9*

S

*5S@ 5*

S

0 *5 *5

S

r 50

S

OS S Met 1 Thr Glu Lys Glu Ile Gly Cys Thr Lys Lys Ile Ser Arg Lys Glu Tyr Val Set Met Tyr Gly Pro Thr Gly His Thr Leu Tyr Lys Val Ala 130 Asp Asp Ile Asp Lys Ala Gly 115 Gly Arg 20 Cys Arg Leu Ala Gly 100 Pro Gly 5 Val Thr Asp Val Asp Asn Ala Ile Arg Leu Thr Tyr Gly Met 55 Leu Thr 70 Ile Gly Lys Asp Thr Glu Asp Thr 135 Gly Gly 40 Ser Asn Ile Met Ala 120 His 10 Asp Thr 25 Glu Glu Gin Thr Ala Leu Lys Asp 90 Gin Asp 105 Leu Ala Ile His Asp Ile Asn Ile 75 Gly Gly Ala Phe Leu Lys Ser Val Lys Val Glu Ile 140 Ile Phe Pro Asp Ile Asp Gly 125 Set Leu Gly Ser Tyr Ala Asn 110 Leu Pro Glu Gly Ser Thr Gly Ash Ile Gin Val Gly Tyr Gly Ile Leu Val Gin Gly 160 lie Pro Thr Ala Phe Ala Ser Gly Val Thr Thr Met Ile Gly Gly 145 150 155 Thr Gly Pro Ala Asp 165 Gly Thr Asn Ala Thr Thr lie Thr Pro 170 Gly Arg 175 99**99 9 a **9e a.

a. a. a 9r a a 9 a Ala Leu Asp Gly 225 Tyr Cys Phe Met Pro 305 Cys Ser Met 4 Val Lys 385 Arg His C Asp I Asi Gl Gir 210 Ser Asp Val His Ala 290 Phe His Arg Gly ly 370 ;lu Ile ly .eu i Leu Phe 195 lie Thr Val Glu Thr 275 Gly Thr His Ile Ile 355 Glu' Phe Lys I ile Val I 435 Ly 18( Let Glt Pro Gir Asp 260 Glu Giu Lys Leu Arg 340 Phe Val ly krg er .eu Ser 1 Ala Ala Ala Val 245 Thr Gly Phe Asn Asp 325 Pro Ser Ile Arg Tyr 405 Asp Trp Mei Lyc G13

ALE

23C Ala Leu Ala Asn Thr 310 Lys Gln Ile rhr Leu 390 Ile Cyr er t Leu Gly Ala 215 Ile Ile Glu Gly Ile 295 Glu Ser Thr Thr Arg 375 Lys Ser Val Pro .Arg Asn 200 lie His His Ala Gly 280 Leu Ala Ile Ile Ser 360 Thr Glu Lys Cly Ala 1 440 Ala 185 Val Gly His Thr Ile 265 Gly Pro Glu Lys Ala- 345 Ser rrP ;lu ryr ;er r25 'he Ala Ser Phe Cys Asp 250 Ala His Ala His.

Glu 330 Ala 4 Asp Gin Lys Thr 1 410 Val C Phe C G1 Tyi Lys Let 235 Thr Gly Ala Ser Met 315 Asp Glu Ser rhr 31y 395 le lu :Ty

I

Gli S114 22( Ast Let ArE Pro Thr 300 Asp Val Asp Gln Ala 380 Asp Asn Val Ile .t Pro 205 His Val Asn SThr Asp 285 Asn Met Gin Gln Ala.

365 Asp Asn Pro 4 Gly Lys I 445 Ser Glu Ala Glu lie 270 Val Pro Leu Phe Leu 350 Met Lys Asp Gly Lys Leu Asp Asp Ala 255 His Ile Thr Met Ala 335 His dly Asn Asn I Ile j 415 Tyr 4 Asn to Arg Trp Giu 240 Gly Thr Lys Ile Val 320 Asp Asp k.rg Lys ?he ~00 !a la let Glu Tyr Ala Met Asn 190 Ile Ile 450 Lys dly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn 455 Arm Ala 465 Ser Ile Pro Thr Pro 470 Gin Pro Val Tyr Arg Giu Met Phe Gly 480 Ser Gin 495 His His Gly Lys Asn 485 Lys Phe Asp Thr Asn 490 Ile Thr Phe Val Ala Ala Tyr Ala Pro Pro 515 Lys 500 Ala Giy Ile Lys Giu Leu Gly Leu Asp Arg Ala 510 Asp Leu Lys Val Lys Asn Cys Arg 520 Asn Ile Thr Lys Lys 525 Phe ASn 530 Asp Val Thr Ala His 535 Ile Asp Val Asn Pro Glu Thr Tyr 540.

Ala Ala Asp Giu Lys Leu 560

S

9 5* S S 9 5*

S

*5*S

S.

S i

S

-S.

*SS*

9 5 5955

S

S. S 9 *S59 Val 545 Lys Val Asp Gly Lys 550 Glu Val Thr Ser Ser Leu Ala Gin Leu 565 Tyr Asn Leu Phe INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 2284 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY:- CDS LOCATION: 124. .477 OTHER INFORMATION: /standard-name--."H.

(ix) FEATURE: NAME/KEY:- CDS LOCATION: 506.,.2143 OTHER INFORMATION: /standard name- 11H.

pylori- Hsp A" pylori- Hsp B" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4i ACAA.ACATCA TCTCATATCA GGGACTTGTT CGCACCTTCC CTMMAATGC

GCTATAGTTG

TGTCGCTTAA GAATACTAAG CGCTAAATTT CTATTTTATT TATCAMAACT

TAGGAGAACT

GMA ATO MAG TTT CAA CCA TTA GGA GMA AGG GTC TTA GTA GMA AGA CTT Met Lys Phe Gin Pro Leu Gly Glu Arg Val Leu Val Ciu Arg Leu 1 5 10 1r 120 168 CMA GAA GAG AAC AAA ACC ACT TCA GGC ATC Giu Giu Giu Asn Lys Thr Ser Ser Gly Ile 25 ATC ATC CC? GAT MAC C Ile le Pro Asp Asn Ala AAA GAA AAG Lys Gu Lys AGT GAG GGT Ser Glu Gly

CCT

Pro TTA ATG GGC GTA Leu Met Gly Val AAA GCG GTT AGC Lys Ala Val Ser CAT AAA ATC His Lys lie OCT TTT GGC Ala Phe Gly 264 312 TGC AAA TGC GTT Cys Lys Cys Val

AAA

Lys 55 GAA GGC OAT GTO Glu Gly Asp Val

ATC

lie AAA TAC Lys Tyr AA 0CC OCA GAA Lys Gly Ala Glu

ATC

lie OTT TIA OAT GGC Val Lou Asp Gly GAA TAC ATG GTO Glu Tyr Met Val

CTA

Leu GAA CTA GAA GAO Glu Leu Glu Asp

ATT

lie 85 CTA GOT ATT GTG Leu Gly lie Val TCA GGC TCT TOO Ser Gly Ser Cys

TGT

Cys 360 408 456 508 *r 9e 0 9 S 900 0 0

C

CAT ACA GOT AAT His Thr Gly Asn

CAT

His 100 OAT CAT AAA CAT Asp His Lys His

GCT

Ala 105 AAA GAG CAT GAA Lys Glu His Glu OCT TGC Ala Cys 110 TGT CAT OAT CAC AAA AA CAC Cys His Asp His Lys Lys His 115 TAAAAAACAT TATTATTAAG GATACAAA ATO Met 1 0CA AAA GMA ATC-AAA TTT TCA OAT Ala Lys Glu Ile Lys Ph. Ser Asp 5 OCA AGA AAC CTT Ala Arg Asn Leu TTA TTT GAA Leu Phe Glu GGG CCA AGA Oly Pro Arg CGC GTA AGA Gly Val Arg CAA CTC OAT GAC Gin 1Lu His Asp GTC AAA GTA ACC Val Lys Val Thr

ATO

Met 604 GGC AGO Gly Arg AAC GTG TTO ATC Asn Val Leu lie

CAA

Gin 40 AAA AGO TAT GC Lys Ser Tyr Oly

OCT

Ala CCA AGO AbC ACC Pro Ser lie Thr

A&A

Lys GAC GOC GIG AGC Asp Oly Val Ser

GTG

Val 55 GOT AA GAG ATT Ala Lys Giu lie

GAA

Glu 60 TTA AGT TGC CCC Leu Ser Cys Pro

GTG

Val GCT AAC ATG 0Gc Ala Asn Met Oly

GCT

Ala CAG CTC GTT AAA Gin Lou Vai Lys

GAA

Glu 75 OAT GCG AGC AA Asp Ala Ser Lys ACC OCT Thr Ala 652 700 748 796 844 GAT GCC 0CC Asp Ala Ala ATT TTT AAA Ile Phe Lys 100 OAT 000 ACO ACC ACA 0CG ACC GTG CTG Asp Gly Thr Thr Thr Ala Thr Val Leu OCT TAT AGO Ala Tyr Ser GAG GC TT AGO Glu Gly Leu Arg

AAT

Asn 105 ATO ACG GCT GG GCT AAC CCT ATT Ile Thr Aia Oly Ala Asn Pro Ile 110 GAA GIG Glu Val 115 AAA OGA GGC AIG Lys Arg Oly Met

GAT

Asp 120 AA GCG CCT Lys Ala Pro GM CCG ATO ATT AAT GAG Glu Ala lie lie Asn Olu 125 892

OTT

Leu 130 AAA AAA GCG AGO Lys Lys Ala Ser

AAA

Lys 135 MAA GTG Lys Vai GC GGT AAA GAA GAA ATO Gly Gly Lys 140 Glu Giu le ACC CAA Thr Gin 145 CTC ATC Leu Ile 160 940 988 GTA GCG ACC ATT Val Ala Thr Ile GCA AAO TCC GAT Ala Asn Ser Asp

CAC

His 155 AAT ATC GGG AA Asn Ile Cly Lys GOT GAC GCT Ala Asp Ala GAA OCT AAG Glu Ala Lys 180

ATG

Met 165 GAA AAA CTG GGT Glu Lys Val Gly

AAA

Lys 170 GAO CCC GTG Asp Cly Val ATC ACO CTT GAA Ile Thr Val lu 175 GAA GC ATG CAA Giu Gly Met Gin 190 1036 1084 GO ATT GAA GAT Cly lie Ciu Asp

GAA

Glu 185 TTA GAT GTC GTA Leu Asp Val Val a.

99 9 9* 9 9 a a.

a a..

a *S9 TTT CAT Phe Asp 195 AGA GGC TAO CTC Arg Cly Tyr'Leu

TCC

Ser 200 OCT TAC TTT GTA Pro Tyr Phe Val ACC AAC OCT GAG Thr Asn Ala Giu 205 TTA ACG GAT AAA Leu Thr Asp Lys

AAA

Lys

AAA

Lys 225

ATG

Met 210 ACC OT CAA TTC Thr Ala Gin Leu

GAT

Asp 215 AAC COT TAO ATO Asn Ala Tyr Ile

OTT

Leu 220 1132 1180 1228 ATO TOT AGC ATG lie Ser Ser Met

AAA

Lys 230 GAC ATT CTC CCG Asp le Leu Pro

CTA

Leu 235 CTA GAA AAA ACO- Leu Glu Lys Thr ATG MA Met Lys 240 GAG GGC AAA Glu Cly Lys TWA AOG ACT Leu Thr Thr 260

CCG

Pro 245 OTT TTA ATO ATC Leu Leu lie lie

GCT

Ala 250 GAA GAO ATT GAG Glu Asp Ile Glu GGO GAA GOCT Gly Glu Ala 255 AAT AT- GCA Asn ile Ala 1276 1324 CTA CTG GTG AAT Leu Val Val Asn

AAA

Lys 265 TTA AGA Leu Arg GC GTG TTG Gly Val Leu 270 CG GTT Ala Val 275 AAA GOT CCA GGC Lys Ala Pro Gly Phe 280 CCC GAC AGO AGA Gly Asp Arg Arg AAA GAA ATG CTC Lys Glu Met Leu 285 AGO GA GAA TTG Ser Glu Glu Leu

AAA

Lys

GC

Gly 305

GAO

Asp 290 ATC GOT GTT TTA Ile Ala Val Leu

ACC

Thr 295 GGC GWT CAA GTC Gly Gly Gin Val

ATT

Ile 300 1372 1420 1468 TTG ACT OTA GAA Leu Ser Leu Glu

AAC

Asn 310 GCT GAA GTG GAG Ala Glu Val GCiu

TTT

Phe 315 TTA OGC AAA GCOG Leu Cly Lys Ala AAG ATT Lys lie 320 GTG ATT GAO Val le Asp OAT GAO GTO His Asp Val 340

AAA

Lys 325 GAC AAC ACO ACG Asp Asn Thr Thr

ATO

Ile 330 GTA CAT GGC AAA Val Asp Cly Lys GGC CAT AGO Gly His Ser 335 ATT GCA AGO lie Ala Ser 1516 1564 AAA GAO AGA CTC Lys Asp Arg Val

GCG

Ala 345 CAA ATC AAA ACC Gin lie Lys Thr

CAA

Gln 350 ACG ACA Thr Thr 355 AGC GAT TAC GAC Ser Asp Tyr Asp

AAA

Lys 360 GMA AAA TTG CAA Glu Lys Leu Gln

GMA

Glu 365 AGA TTG GCC A Arg Leu Ala Lys GCG AGT GMA GTG

CTC

Leu 370 TOT GGC GGT GTG Ser Gly Gly Val

OCT

Ala 375 GTG AT? MAA OTG Val Ile Lys Val GGC C 1612 1660 1708 Gl y Ala Ala Ser Glu 380 Val 385 GMA ATO AMA GAG Glu Met Lys Glu

AAA

Lys 390 AAA GAC COG GTG Lys Asp Arg Val

GAT

Asp 395 GAC GCG TTG AGC Asp Ala Leu Ser GCG ACT Ala Thr 400

A

Lys OCO GCG GT Ala Ala Val 405 GMA GMA GOC AT? Glu Glu Gly Ile

GTG

Val 410 ATT GGG GGC GOT Ile Oly Cly Gly GCG 0CC CTC AlaAla Leu 415 OAT GM A Asp Glu Lys

C

S.

C S

C.

C

S

C

S.

.S&9 C CC 'C

C*

C.C

I C C.

I.

*9#e

C

ATT CGC 000 Ile Arg Ala 420 0CC CAA MAA GTO Ala Gin Lys Val

CAT

His 425 TTG MAT TTA CAC Leu Asn Leu His

GAT

Asp 430 OTO 000 Val Gly 435 TAT GMA ATC ATC Tyr Glu Ile Ile

ATG

Met 440 COO 0CC AT? AAA Arg Ala Ile Lys

CC

Ala 445 CCA TTA OCT CMA Pro Leu Ala Gin 1756 1804 1852 1900 1948

ATC

le 450 GCT ATO MAT 0CC Ala Ile Asn Ala

GT

Gly 455 TAT GAT GOC GGT Tyr Asp Gly Gly

GTG

Val 460 GTC GTO MAT GMA Val Val Asn Oiu

GTA

Val 465 GAA AMA CAC GAA Glu Lys His Giu 000 Gly 470 CAT TTT G0? TTT His Phe Gly Phe

MAC

Asn 475 OCT AGC MAT C Ala Ser Asn Gly MAG TAT Lys Tyr 480 OTO GAC ATG Val Asp Met ATC GCT TTA Ile Ala Leu 500

TTT

Phe 485 MAA GMA GGC AT? Lys Giu Gly Ile

ATT

Ile 490 GAO CCC TTA AMA Asp Pro Leu Lys OTA GMA AGO Val Giu Arg 495 TTA ACC ACA Leu Thr Thr 1996 2044 CMA MT 000 OTT Gin Asn Ala Vai

TCG

Ser 505 OTT TCA AGO CTG Val Ser Ser Leu

CT?

Leu 510 GMA 0CC Glu Ala 515 COT OAT Pro Asp 530 ACC GTG CAT GMA Thr Val His Giu

ATO

Ile 520 AMA GMA GM A Lys Olu Giu Lys

GCG

Ala 525 0CC CCA GCA ATG Ala Pro Ala Met 2092 2140 ATO G 000 ATO GC GGA Met Gly Gly Met Oiy Gly 535 ATO OGA 000 ATG Met Gly Cly Met 540 000 000 ATG Gly Giy Met

ATG

Met TMOGCCCCCT TOCTTTTTCO TATOATOToC TTTTMAAATC CATCTTCTAO

MATCCCCCCT

TCTAAMATCC CTTTTTTOOG GGOTGCTTTT GGTTTGATAA AACCGCTCGC TrTTTAAA6C GCGCOMOMA AAACTCTGTT

MOOC

2200 2260 2284 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 545 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM H. pylori (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ala Lys Glu lie Lys Phe Ser Asp Ser Ala Arg Asn Leu Leu Phe 1 5 10 Glu Gly Val Arg Gin Leu His Asp Ala Val Lys Val Thr Met Gly Pro 25 Arg Gly Arg Asn Va l Leu Ile Gin Lys Ser Tyr Gly Ala Pro Ser Ile 35 40 Thr Lys Asp Gly Val Ser Val Ala Lys Glu Ile Glu Leu Ser Cys Pro 5. 0 55 Val Ala Asn Met Gly Ala Gin Leu Val Lys Glu Asp Ala Ser Lys Thr 70 75 Ala Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Tyr 85 90 Set Ile Phe Lys Glu Gly Leu Arg Asn lie Thr Ala Gly Ala Asn Pro 100 105 110 Ile Glu Val Lys Arg Gly Met Asp Lys Ala Pro Glu Ala Ile Lie Asn 115 120 125 Glu Leu Lys Lys Ala Ser Lys Lys Val Gly Gly Lys Glu Glu Ile Thr 130 135 140 Gin Val Ala Thr Ile Ser Ala Asn Ser Asp His Asn Ile Gly Lys Leu 145 150 155 160 Ile Ala Asp Ala Met Glu Lys Val Gly Lys Asp Gly Val Ile Thr Val 165 170 175 Glu Glu Ala Lys Gly Ile Glu Asp Glu Leu Asp Val Val Glu Gly Met 180 185 190 Gin Phe Asp Arg Gly Tyr Leu Set Pro Tyr Phe Val Thr Asn Ala Glu 195 200 205 Lys Met Thr Ala Gin Leu Asp Asn Ala Tyr lie Leu Leu Thr Asp Lys 210 215 220 Lys 225 Ile Ser Ser Mel a a.

9 5 *5* Ur *9 9+ Ua a a.

0*

I

ar y Lys Ala Ala Lys Gly 305 Ile Ser Ser Lys Val 385 Thr Leu Lys Gin Val C 465 Tyr N Arg I Gi Let Ali Asp 29 Leu Val His Thr Leu 370 Glu Lys Ile Jal lie ;lu ral :le 1 cly Lys Pr 24 i Thr Thr Le 260 Val Lys Al 275 Ile Ala Va Ser Leu GlI Ile Asp Ly 32 Asp Val Ly~ 340 Thr Ser Asi 355 Ser Gly G13 Met Lys Glt Ala Ala Val 405 Arg Ala Ala 420 Gly Tyr Glu 435 Ala Ile Asn Lys His Glu Asp Met Phe 485 Ala Leu Gin 500

L

a 1 5 5

I.

Lys Asp 230 iLeu Leu Val Val Pro Gly Leu Thr 295 Asn Ala 310 Asp Asn Asp Arg Tyr Asp Val Ala' 375 Lys Lys 390 Glu Glu Gin Lys I Ile ile b Ala Cly 455 Gly His P 470 Lys Giu G Ii As Ph 28( G13 Glt Thr Val Lys 360 Val sp ;ly lal let 40 'yr 'he ly al e Ile 3 Lys 265 Gly Gly Val Thr Ala 345 Glui Ile tie Arg le His 425 Arg Asp Gly I Ile I 4 Ser I 505

I

I

F

4 t 3 Ala Giu Asp 250 Leu Arg Gly Asp Arg Arg Gin Val Ile 300 Glu Phe Leu 315 Ile Val Asp 330 Gln le Lys Lys Lou Gin ys Val Gly 380 al Asp Asp 395 al Ile Gly ,10 .eu Asn Leu Ia Ile Lys ly Gly Val 7 460 he Asn Ala 1 475 le Asp Pro I 90 al Ser Ser i 11 Va

LY~

28r Se~ G13 G1 3 Thr Glu 365 Ala Ala Gly His '.ka 45 7al ier .eu .eu e Glu 1 Leu 270 s Glu r Glu Lys Lys Gin 350 Arg Ala Leu Gly Asp 4 430 Pro Val I Asn C Lys V 4 Leu L 510 Gl 25 As Mei GlI Ali Gl 3 33- Ile Leu Ser Ser kla ksp eu Lsn vly Val ,eu y Glu n ile t Leu 1 Leu Lys 320 His Ala Ala Glu Ala 400 Ala Glu Ala Glu Lys 480 Glu Thr Ile Lou Pro Lou Lou Giu Lys Thr Met 235 240 Asn Ala V Thr Glu Ala 515 Thr Val His Glu Lys Glu Clu Lys Ala Ala Pro Ala 525 Met Pro Asp Met Gly Gly Met Gly Gly Met Gly Gly Met Gly Gly Met 530 535 540 Met 545 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 118 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM H. pylori (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Si 9 5 9~ #9~ 9- *5~ #9 .59# Met 1 Lys Phe Gin Pro Leu Giv Giu Arg 5 Val 10 Leu Val Glu Arg Leu Glu Glu Glu Asn Glu Lys Pro Lvs Thr Ser Ser Gly Ile 25 ile le Pro Asp Asn Ala Lys Leu Met Gly Val Val 40 Lys Ala Val Ser Ala Phe Gly Lys Glu Gly Cys Lys Cys Val Giu Gly Asp Val Ile Tyr Lys Gl~y Ala Glu Ile 70 Vai Leu Asp Giy Glu Tyr Met Val Leu Giu Leu Giu Asp Ile Leu Gly Ile Val Gly 90 Ser Gly Ser Cys Cys His Thr Gly Asn His Asp His 115 His 100 Asp His Lys His Ala 105 Lys Glu His Giu Ala Cys Cys 110 Lys Lys His INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS:- LENGTH:- 591 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM felis (ix) FEATURE: NAME/KEY:

ODS

LOCATION: 1. .591 OTHER INFORMATION: /standard name- "tIRE In (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TTA GOT CTT GTG TTA TTG TAT OTT C GTC GTG CTO ATC AGC MAC Leu Gly Leu Val Leu Leu Tyr Val Ala Val Val Leu Ile Ser Asn

ATG

Met 1 9 *909 .9

S

9 9* .9 .9 4 9.

4 9 9*9.

S

*9 .9 9 GGA OTT ACT Gly Val Ser MGC TAC TTT Asn Tyr Phe 35

GCC

Gly 20 CTT GCA MAT GTO Leu Ala Asn Val

GAT

Asp 25 CCC MAA AOC Ala Lys Ser OTG GGG GGG GAC Val Gly Gly Asp

TCT

Ser 40 CCA TTC TGT OTA Pro Leu Cys Val MAA CCC ATC ATO Lys-Ala Ile Met ATG TGG TCG CTA Met Trp Ser Leu ACT OT CCA GMA Thr Gly Pro Glu TCA TCT Ser Ser 50 TAT TCC ACT TTC Tyr.Ser Thr Phe

CAC

His 55 CCC ACC CCC CCT Pro Thr Pro Pro

GCA

Ala 96 144 192 240 288 336

GAT

Asp 65 CTC CC CAG GTG Val Ala Gin Val

TCT

Ser 70 CMA CAC CTC AT? Gin His Leu Ile

AAC

Asn 75 TTG TAT CT CCA Phe Tyr-Giy Pro

GCG

Ala ACT OT CTA TTG Thr Gly Leu Leu

TTT

Phe 85 GOT TTT ACC TAC Gly Phe Thr Tyr TAT GCT CCC ATC Tyr Ala Ala Ile MAC MAC Asn Asn ACT TTC MAT Thr Phe Asn

CTC

Leu 100 GAT TOG MAA CCC Asp Trp Lys Pro

TAT

Tyr 105 CCC TOO TAT TG TrC TTT GTA Gly Trp Tyr Cys Leu Phe Vai 110 ACC ATC AAG ACT ATG CCA OCC Thr Ile Asn Thr Ile Pro Ala

GCC

Ala 120 AT? CT? TCT CAC Ile Leu Ser His

TAT

Tyr 125 TCC GAT CCC Ser Asp Ala CT? GAT Leu Asp 130 TTG ATT Phe Ile 145 GAT GAC CG CTG Asp His Arg Leu TOO CTT OCT TOO Trp Leu Ala Trp 150

TTA

Leu 135 GOA ATC ACT GAG Oly le Thr Clii

GC

Gly 140 GAT TOO TOG GCT Asp Trp, Trp Ala 384 432 480 528 TG GA CTT COT MCG

ACT

Ser GOT OTT TTO Oly Val Leu CTA GOT A Leu Gly Lys

CTC

Leu 155 ACT GOT TOO AT? Thr Cly Trp Ile

GAA

lu 160 GyS Ala Leu Gly Lys 165 TTT OTT CCA TOG CTT Phe Val Pro Trp Leu 170 CCC ATC Ala le 175

GTC

Val

CAA

591 Gln

GAG

O iu

CAC

His 89 GOC OTG ATC ACC GCT TOG ATT CCT OCT TOG CTA CTC TTT ATC Oly Val Ile Thr Ala Trp Ile Pro Ala Trp Leu Leu Phe Ile 180 185 190 TGG TCT TGA Trp Ser 195 9 999.94 9 99 .9 9 99 9999 a 99 9 9.

9 99 9* p 99 *99 99 9 *999 9 flee 9*e* .999 9- 9 9 99 9 INFORMATION FOR SEQ ID NO- 8: SEQUENCE CHARACTERISTICS: LENGTH: 199 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM H. felis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Lys Gly Trp Met Leu Gly Leu Val Leu Leu Tyr Val Ala Val Val Leu Ile Ala Trp Gly Oly Ile Leu Ser Trp 145 Ser Ile Ser 50 Pro Pro Asn Phe Asp 130 Trp Asn Met 35 Leu Giu Ala Asfl Val 115 Ala Ala Gly Asn Ser Asp Thr Thr 100 Thr Leu Phe Val Tyr Ser Val Gly Phe Ile Asp Ile Ser Phe Tyr Ala 70 Leu Asn Asn Asp Trp 150 Gly Val Ser 55 Gin Leu Leu Thr His 135 Leu Leu Gly 40 Thr Val Phe Asp Ile 120 Arg Ala Ala 25 Gly Phe Ser Gly Trp 105 Pro Leu Trp Asn Val Asp Ser His Pro Gin His 7.5 Phe Thr 90 Lys Pro Ala Ala Leu Gly Oly Val 155 Asp Pro Thr Ieu Tyr Tyr Ile Ile 140 Leu Ala Lau Pro Ile Leu Gly Leu 125 Thr Trp Lys Cys Pro Asn Tyr Trp 110 Ser Oiu Leu Ser Val Ala Phe Ala Tyr His Gly Thr Lys Met Thr Tyr Ala Cys Tyr Asp Oly 160 Trp Ile Giu Cys Ala Leu Gly Lys Ser Leu Gly Lys Phe Val Pro Trp I AC .LU~1 175 Leu Ala lie Val Giu Gly Val lie Thr Ala Trp Ile Pro Ala Trp Leu 180 185 190 Leu Phe lie Gin His Trp Ser 195 9~*e-~q9

C

C C

C

C.

U CU.

C

iC C

U..

i CC C

C.

a

CC..

CC C

Claims (12)

1. Immunogenic composition capable of inducing antibodies against Helicobacter infection, which comprises the Heat Shock Protein HSP A, encoded by the hspA gene of plasmid plLL689 (CNCM 1-1356), or a polypeptide exhibiting at least 75% homology with the said HSP A, or a fragment of this protein having at least 6 amino acids.

2. Immunogenic composition according to Claim 1 capable of inducing protective antibodies.

3. Pharmaceutical composition for use as a vaccine in protecting against Helicobacter pylori and Helicobacter felis, characterised in that it comprises the immunogenic composition of any of Claims 1 and 2, in combination with physiologically acceptable excipient and possibly adjuvants.

4. Proteinaceous material characterised in that °it comprises at least the Heat Shock Protein (HSP A), of Helicobacter pylori, or a fragment thereof, 20 5. Proteinaceous material according to Claim 4, e "characterised in that it comprises or consists of HSP A having the amino acid sequence illustrated in FIG, 6, or a polypeptide having at least 75%, and preferably at least 80% homology with said polypeptide, or a fragment 25 thereof, comprising at least 6 amino acids. 6, Proteinaceous material according to Claim characterised in that it comprises or consists of HSP A C-terminal sequence: G S CCH T G NH D H K HA K E H E A C C H D H K K H or a fragment comprising at least 6 30 consecutive amino acids of this sequence.

7. Nucleic acid sequence characterised in that it comprises:- a sequence coding for the proteinaceous material of any one of Claims 4 to 6; or (ii) a sequence complementary to sequence or (iii) a sequence capable of hybridizing to sequence or (ii) under stringent conditions; or (iv) a fragment of any of sequences (ii) or (iii) comprising at least nucleotides.

8. Nucleic acid sequence according to Claim 7 characterised in that it comprises all or part of the sequence of plasmid pILL689 (CNCM 1-1356), for example the sequence of FIG. 6, in particular that sequence coding for HSP A, or a sequence complementary to this sequence, or a sequence capable of hybridizing to this oo,* sequence under stringent conditions, or a fragment thereof. 20 9. Expression vector characterised in that it contains a nucleic acid sequence according to Claim 7 or 8. Plasmid pILL689 (CNCM 1-1356).

11. Oligonucleotide suitable for use as a primer 25 characterised in that it comprises from 10 to 100 consecutive nucleotides of the sequence of Claim 7 or 8.

12. Nucleotide probe, characterised in that it *i comprises a sequence according to any one of Claim 7 or 8 with an appropriate labelling means. 0 13. Microorganisms stably transformed by an expression vector according to Claim 9 or

14. Monoclonal or polyclonal antibodies or fragments thereof, to the proteinaceous material of Claims 4 or 5, characterised in that they are either specific for the Helicobacter pylori material or, alternatively, cross-react with GroEL-like proteins or GroES-like proteins from bacteria other than Helicobacter. Monoclonal or polyclonal antibodies according to Claim 14 characterised in that they recognise specifically the HSP A C-terminal sequence.

16. Proteinaceous material comprising a fusion or mixed protein including at least the HSP A protein from Helicobacter or fragment thereof, as defined in any one of Claims 4 to 7.

17. Purified antibodies or serum obtained by immunisation of an animal with the immunogenic composition according to any one of Claims 1 to 3, or with the proteinaceous material or fragment of Claims 4 to 6 or with the fusion or mixed protein of Claim 16.

18. Kit comprising at least the purified antibodies or serum according to Claim 17, and optionally, appropriate media or excipients for administration of the antibodies, or labelling or detection means for the antibodies. .o *oo B, i 'I w*r 0 9. 9 EDITORIAL NOTE No, 75081/98 This specification does not contain page number 94.