AU762563B2 - Multimeric, recombinant urease vaccine - Google Patents

Description

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Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): ORAVAX, INC Invention Title: MULTIMERIC, RECOMBINANT UREASE VACCINE The following statement is a full description of this invention, including the best method of performing it known to me/us: la MULTIMERIC. RECOMBINANT UREASE VACCINE Background of the Invention This invention relates to methods and compositions for preventing and/or treating Helicobacter infection.

Helicobacter is a genus of spiral, gram-nagative bacteria which colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably, H. pylori, H. heilmanii, H. felis, and H.

mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been found to infect humans, but at lower frequencies than H. pylori.

Helicobacter infects over 50% of adult populatiois 15 in developed countries, and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections of humans worldwide.

Infection with H. pylori results in chronic stomach inflammation in all infected subjects, although the clinical gastroduodenal diseases associated with Helicobacter infection generally appear from several years to several decades after the initial infection. H.

pylori is the causative agent of most peptic ulcers and chronic superficial (type B) gastritis in humans. H.

pylori infection is also associated with atrophy of the gastric mucosa, gastric adenocarcinoma, and non-Hodgkin's lymphoma of the stomach (see, Blaser, J. Infect.

Dis. 161:626-633, 1990; Scolnick et al., Infect. Agents Dis. 1:294-309, 1993; Goodwin et al., Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, 465 pp, 1993; Northfield et al., Helicobacter pylori, Infection, Kluwer Acad. Pub., Dordrecht, 178 pp, 1994).

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the lb references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

H:\Pcabral\Keep\speci\42567.00.doc 13/09/02 2 Summary of the Invention We have shown that vaccine compositions containing multimeric, recombinant Helicobacter urease are effective in preventing and treating Helicobacter infection. In addition, we have shown that a composition containing H. pylori urease and a mucosal adjuvant, in combination with an antibiotic and a bismuth salt, is more effective at treating Helicobacter infection than any of these components alone, or any combinations of three or less of these components.

Accordingly, in a first aspect, the invention features a vaccine for inducing an immune response to Helicobacter H. pylori) in a patient a human patient) that is at risk of developing, but 15 does not have, a Helicobacter infection (a "protective" immune response), or is already infected by Helicobacter (a "therapeutic" immune response). This vaccine contains multimeric complexes of recombinant, enzymatically inactive Helicobacter H. pylori) urease, and a pharmaceutically acceptable carrier or diluent sterile water or a 2% weight/volume sucrose solution). The vaccine may contain a multimeric complex containing eight Urease A subunits and eight Urease B subunits, a multimeric complex containing six Urease A subunits and six Urease B subunits, a multimeric complex containing four Urease A subunits and four Urease B subunits, or a mixture thereof. In addition, the vaccine may contain a mucosal adjuvant, such as the heat-labile enterotoxin of enterotoxigenic Escherichia coli a cholera toxin a Clostridium difficile toxin, or a subunit or derivative thereof lacking toxicity but still having adjuvant activity a fragment, mutant, or toxoid), or a mixture thereof. For example, a fragment containing the 794 carboxyl-terminal amino acids of C. difficile Toxin A 3 (see, Dove et al., supra, for the sequence of C.

difficile Toxin A) may be used. Further, the multimeric complexes of recombinant, enzymatically inactive Helicobacter urease may be freeze-dried prior to use in the vaccines of the invention.

In a second aspect, the invention features a method of inducing a protective and/or therapeutic mucosal immune response to Helicobacter H. pylori) in a patient a human patient). In this method, a composition containing an immunogenically effective amount of multimeric complexes of recombinant, enzymatically inactive Helicobacter H. pylori) urease is administered to a mucosal surface an oral or intranasal surface; or to a rectal surface by, 15 anal suppository) of the patient. The composition may contain a multimeric complex containing eight Urease A subunits and eight Urease B subunits, a multimeric complex containing six Urease A subunits and six Urease B subunits, a multimeric complex containing four Urease A subunits and four Urease B subunits, or a mixture thereof. The composition may be administered without gastric neutralization by, sodium bicarbonate.

In addition to the multimeric complexes of recombinant, enzymatically inactive Helicobacter urease, the composition used in this method may further contain a mucosal adjuvant. For example, the composition may contain the heat-labile enterotoxin of enterotoxigenic Escherichia coli a cholera toxin a Clostridium difficile toxin, or a subunit or derivative thereof lacking toxicity but still having adjuvant activity fragments, mutants, or toxoids having adjuvant activity), or mixtures thereof. For example, a fragment containing the 794 carboxyl-terminal amino acids of C. difficile Toxin A (see, Dove et al., supra, for the sequence of C. difficile Toxin A) may be used.

4 Further, the multimeric complexes of recombinant, enzymatically inactive Helicobacter urease in the composition may be freeze-dried prior to use in this method.

In a third aspect, the invention features a composition for treating Helicobacter H. pylori) infection in a patient a human). This composition contains a Helicobacter H. pylori) antigen urease), and an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof. An example of a Helicobacter urease antigen which may be used in this composition is one containing multimeric complexes of recombinant, enzymatically inactive Helicobacter urease, as is described above. Other 15 Helicobacter antigens which may be used include, e.g., HspA, HspB, AlpA, AlpB, ClpB, and the antigen recognized by monoclonal antibody IgG 50 (see below). This composition may further contain a mucosal adjuvant (or a combination of mucosal adjuvants), such as those which are listed above.

Antibiotics which may be included in this composition include, but are not limited to, amoxicillin, clarithromycin, tetracycline, metronidizole, and erythromycin; and bismuth salts which may be included are, bismuth subcitrate and bismuth subsalicylate.

Antisecretory agents which may be included in the composition are, proton pump inhibitors omeprazole, lansoprazole, and pantoprazole), H 2 -receptor antagonists ranitidine, cimetidine, famatidine, nizatidine, and roxatidine), and prostaglandin Analogs misoprostil or enprostil).

In a final aspect, the invention features a method for treating Helicobacter H. pylori) infection in a patient a human patient). In this method, a composition containing a Helicobacter H.

5 pylori) antigen the antigens listed above), and an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof, is administered to the patient. Any standard mode of administration, or a combination of standard modes, may be employed. For example, mucosal administration administration to an oral or intranasal surface, or administration to a rectal surface by, anal suppository) may be used.

A composition administered to a mucosal surface in this method may further contain a mucosal adjuvant (or a combination of mucosal adjuvants), the mucosal adjuvants which are described above. Antibiotics, bismuth salts, and antisecretory agents which may be included in the composition used in this method are 15 described above.

By "vaccine" is meant a composition containing at least one antigen an antigen from a pathogenic microorganism, such as H. pylori) which, when administered to a patient, elicits or enhances an immune response to the antigen that is effective in the Sprevention of disease disease caused by infection by the microorganism), or in the treatment of a preexisting disease.

By "immunogenically effective amount" is meant an amount of an antigen multimeric complexes of "e recombinant, enzymatically inactive Helicobacter urease, or any of the other antigens listed above) which is effective to elicit an immune response a humoral or a mucosal immune response) when administered To a patient, such as a human patient.

By "Helicobacter" is meant any bacterium of the genus Helicobacter, particularly a Helicobacter which infects humans H. pylori). By "urease" is meant an enzyme H. pylori urease) which catalyzes the 6 conversion of urea into ammonium hydroxide and carbon dioxide.

By "multimeric complex" is meant a macromolecular complex of polypeptides urease polypeptides). The polypeptides may be associated in the complex by a variety of intermolecular interactions, such as covalent bonds disulfide bonds), hydrogen bonds, and ionic bonds.

By "mucosal adjuvant" is meant a compound an immunomodulator) which non-specifically stimulates or enhances a mucosal immune response production of IgA antibodies). Administration of a mucosal adjuvant in combination with an immunogenic composition facilitates the induction of a mucosal immune response to the antigen 15 in the composition.

By "antibiotic" is meant a compound derived from a mold or bacterium that inhibits the growth of other .i microorganisms.

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

Detailed Description The drawings are first described.

Drawings 25 Fig. 1 is a diagram showing the restriction enzyme map of the 2.5 kb PCR product cloned from pSCP1. The numbers above the map indicate nucleotide positions using the first base pair (bp) of BL1 as nucleotide #t.

Locations of ureA and ureB genes and the direction of transcription of these genes are shown. The locations of restriction enzyme cleavage sites are shown below the genes. The numbers next to the name of each restriction enzyme indicate the locations of the first base pair in the recognition sequence for the enzyme indicated.

7 Fig. 2 is a diagram of the genetic map of the expression plasmid pORV214. The ureA and ureB genes are shown as open bars with arrows. The solid arrow and solid bar represent the T7 promoter and terminator sequences, respectively. Thin lines represent pBR322 DNA. The phage and plasmid origins of replication on the plasmid are shown by large shaded arrows, denoted "fl origin" and "ori," respectively, and indicate the direction of DNA synthesis. The genes encoding kanamycin resistance (kan) and the lactose repressor (lacl) are shown as shaded bars. The BamHI and EcoRI sites used for cloning the PCR product are indicated. Arrows within bars representing lacI, ureA, ureB, and kan genes indicate the direction of transcription.

15 Fig. 3 is a schematic representation of the nucleotide sequence of the H. pylori locus encoding the ureA and ureB genes, as well as the transcriptional regulatory sequences from pORV214 (SEQ ID NO:1). The predicted amino acid sequences of ureA (SEQ ID NO:2) and ureB (SEQ ID NO:3) are shown above the DNA sequence.

Numbers to the left of the sequence correspond to nucleotide positions and those on the right indicate amino acid positions. The sequences corresponding to the PCR primers are underlined and the BamHI and EcoRI sites 25 used to clone the PCR product are indicated. The predicted transcriptional initiation site and direction of transcription are shown with an arrow. The ribosomal binding site (RBS) is indicated. The sequences which regulate transcriptional initiation (T7 promoter and lac operator) and termination are underlined and labeled.

Fig. 4 is a graph showing analytical size exclusion HPLC for recombinant urease.

Fig. 5 is a series of graphs showing the bacterial score versus the levels of serum IgA, serum IgG, and 8 fecal IgA antibodies from mice immunized with recombinant H. pylori urease and cholera toxin (CT).

Fig. 6 is a series of graphs showing the bacterial score versus the levels of serum IgA, serum IgG, and fecal IgA antibodies from mice immunized with recombinant H. pylori urease and enterotoxigenic E. coli heat-labile toxin.

Fig. 7 is a table showing the experimental protocol used for comparing intranasal and oral immunization routes for recombinant urease.

Fig. 8 is a series of graphs showing the serum IgG (Serum serum IgA (Serum fecal IgA (Fecal and salivary IgA (Sal A) levels in groups of mice treated according to the protocol illustrated in Fig. 7.

Fig. 9 is a table showing the experimental protocol used for comparing intranasal and intragastric immunization routes for recombinant urease.

Fig. 10 is a series of graphs showing the results of urease tests carried out on the mice treated according to the protocol illustrated in Fig. 9.

Fig. 11 is a graph showing that rUrease (10 Mg) given by the intranasal route with CT (5 gg) is at least as effective as rUrease (25 pg) given by the oral route with CT (10 Mg) in preventing infection with H. felis.

Fig. 12 is a graph showing that immunization of mice with rUrease 10 Mg LT reduces or eradicates established H. felis infection. When these animals were reinfected with H. felis, they were protected against challenge.

Fig. 13 is a graph showing the mean antibody response of mice 4 and 10 weeks after therapeutic immunization with either recombinant H. pylori urease and CT, or CT alone.

Fig. 14 is a graph showing numbers of IgA antibody secreting cells in gastric mucosa of immunized and 9 unimmunized mice before, and at intervals after, infection with H. fells.

Fig. 15 is a graph showing the clearance of H. felis infection in mice previously immunized with recombinant H. pylori urease and CT.

Cloning of the ureA and ureB genes The structural genes encoding urease, ureA, and ureB, have been cloned (Clayton et al., Nucl. Acids. Res.

18:362, 1990; Labigne et al., J. Bacteriol. 173:1920- 1931, 1991), and the recombinant urease encoded by these genes has been purified (Hu et al., Infect. Immun.

60:2657-2666, 1992). For use in the present invention, urease was cloned from a clinical isolate of H. pylori (CPM630) obtained from a clinical specimen provided by Dr. Soad Tabaqchali, St. Bartholomew's Medical College, ;.University of London. A genomic DNA library of strain CPM630 was prepared in the lambda phage vector EMBL3.

Plaques were screened for reactivity with rabbit anti-Helicobacter urease polyclonal antibody, and a single reactive plaque was isolated. This clone contained a 17 kb SalI fragment that encoded the ureA and ureB genes. The 17 kb fragment was subcloned onto pUC18 and designated pSCP1. A 2.7 kb TaqI fragment was subcloned (pTCP3) and completely sequenced. The 2.7 kb 25 TaqI fragment encoded both ureA and ureB.

The primers BL1 (CGG GAT CCA CCT TGA TTG CGT TAT GTC T; SEQ ID NO:4) and BL2 (CGG AAT TCA GGA TTT AAG GAA GCG TTG; SEQ ID NO:5) were used to amplify and clone a kb fragment from pSCP1. BL1 and BL2 correspond to nucleotides 2605-2624 of GenBank accession number M60398 (the BL1 primer) and nucleotides 2516-24998 of EMBL accession number X17079 (the BL2 primer). A restriction enzyme map of the 2.5 kb fragment PCR product is shown in Fig. 1. The 2.5 kb fragment contains the entire coding 10 region of ureA and ureB, as well as translational start signals from H. pylori upstream of ureA.

Expression of recombinant urease The purified 2.5 kb PCR fragment containing the genes encoding ureA and ureB was digested with EcoRI and BamHI and inserted into the expression vector pET24+ (Novagen, Madison, Wisconsin) to produce the plasmid pORV214 (Fig. pET24+ contains the colEl origin of replication, the filamentous phage (fl) origin of replication for single strand rescue, and the kanamycin resistance gene of Tn903. The fragment was inserted downstream of the T7 promoter, which provides transcription initiation for the urease genes. Lactose operator (lacO) sequences are present between the T7 promoter and the cloning sites to provide inducible expression of the urease genes. A T7 transcription terminator sequence is located downstream of the cloning sites. The vector also contains the lactose repressor gene (lacl) to ensure complete repression of expression.

20 Other sequences present in the vector are derived from pBR322, which served as the backbone for vector construction.

The initial ligation mixture, containing the kb PCR EcoRI-BamHI fragment and the pET24+ vector 25 digested with EcoRI and BamHI, was used to transform XL1-Blue (Stratagene, La Jolla, CA) prepared by the CaCl 2 method. XL1-Blue is an E. coli strain that does not express T7 RNA polymerase. Kanamycin resistant-colonies were directly screened by PCR using urease specific primers. Plasmid DNA from several positive colonies was extracted using Qiagen minispin columns (Qiagen, Chatsworth, CA) and checked for the correct restriction digestion pattern.

11 Purified pORV214 DNA was used to transform the E. coli strain BL21-DE3 (Novagen, Madison, Wisconsin) prepared by the CaC1 2 method. BL21-DE3 is an E. coli B strain that is lysogenized with lambda phage DE3, a recombinant phage that encodes T7 RNA polymerase under the control of the lavUV5 promoter. BL21 is deficient in ion and ompT proteases, as well as the hsdSB restriction/modification system and dcm DNA methylation.

DNA was prepared from kanamycin resistant colonies with Qiagen mini spin columns and screened by restriction enzyme analysis using BamHI and EcoRI to confirm the presence of the plasmid. Urease expression was assessed by examination of BL21-DE3 (pORV214) cell lysates by SDS-PAGE and Western blot. Several positive clones had the correct restriction endonuclease digestion pattern and expressed urease.

A single clone containing pORV214 was selected, grown on LB plates containing kanamycin (50 gg/ml), harvested, and stored at -80 0 C in LB containing glycerol. A research cell bank was prepared by growing a sample from the glycerol stock on LB plates containing kanamycin, selecting an isolated colony, and inoculating a LB broth culture containing kanamycin. This culture was grown to OD 600 of 1.0, pelleted, and resuspended in an equal volume of LB containing 50% glycerol. These research cell bank (RCB) aliquots (100 Al) were then stored at -80°C. A master cell bank (MCB) was similarly prepared using an isolated colony from the research cell bank, and a manufacturer's working cell bank (MWCB) was prepared using an isolated colony from the MCB.

The MCB and MWCB cells were viable, kanamycin resistant, and displayed a normal E. coli colony morphology. T7 RNA polymerase expression and lambda phage lysogeny was confirmed using appropriate tests, which are well known in the art. Urease expression was 12 IPTG-inducible in the MCB and MWCB cells, as determined by examination of cultures grown in the presence of IPTG, and analysis of lysates from these cell cultures on SDS-PAGE. Production of 60 kD and 29 kD proteins (UreB and UreA, respectively) by the MCB and MWCB cells increased with the incubation time.

Plasmid DNA was isolated from the MCB and MWCB cells and was tested by restriction enzyme analysis, restriction fragment length polymorphism (RFLP), and DNA sequence analysis to confirm plasmid structure. The MCB contained a plasmid with the appropriate restriction endonuclease digestion pattern with no deletions or rearrangements of the plasmid. Likewise, there were no differences between the RFLP fingerprint of pSCP1 and the RFLP fingerprints of plasmid DNA from MCB and MWCB, o indicating that the urease genes had not undergone detectable 50 bp) deletions or rearrangements in the ':cloning process or in the manufacture of the cell banks.

The coding regions of ureA and ureB, and the sequences of the promoter and termination regions of the plasmid isolated from MCB cells, were sequenced.

Sequencing reactions were performed using the Di-Deoxy Cycle Sequencing Kit, according to the manufacturer's instructions (Applied Biosystems, Inc., Foster City, CA), o 25 using fluorescent-labeled dideoxynucleotides. The sequences of the ureA and ureB genes, with predicted protein sequences, as well as the DNA sequences of flanking regions are shown in Fig. 3.

Large-scale production of recombinant urease The fermentor(s) to be used was cleaned and sterilized according to approved procedures. The culture medium contained 24 g/L yeast extract, 12 g/L tryptone, 6-15 g/L glycerol, in RO/DI water. Since pORV214 was sufficiently stable in the absence of antibiotics, no 13 antibiotics were present in the large-scale fermentation cultures. Antifoam was added and the unit was sterilized in place.

For production in the 40 liter fermentor, a vial of the MWCB was thawed and used to inoculate a four liter shaker flask containing one liter of LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl) without antibiotics. The culture was shaken at 37 0 C for 16-24 hours. The inoculum was then transferred to the liter fermentor (30 liter working volume) containing the production media described above. Fermentation was carried out with aeration and agitation until the cell density determined by OD 6 0 0 was approximately 8-10.

Isopropyl A-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.1 mM, and induction was allowed to proceed for 16-24 hours. The cells were harvested by centrifugation and the wet paste was aliquoted into polypropylene storage containers and stored at -80°C. For production at the 400 liter scale .e (300 liter working volume), the inoculum and culture in the 40 liter vessel were prepared as described above.

When the 40 liter vessel reached an OD 600 of approximately 5.0, it was used to inoculate the 400 liter fermentor.

The culture was further incubated to an OD 600 of 8-10.

25 The culture was then induced, harvested, and stored, as is described above. This procedure required 2-3 generations more than the 40 liter process.

Purification of recombinant H. pylori urease Containers of cell paste produced by thefermentation processes described above were removed from storage, thawed at room temperature, and resuspended in mM sodium phosphate/l mM EDTA buffer, pH 6.8. The resuspended cells were disrupted by extrusion through a narrow orifice under pressure .(Microfluidizer Cell 14 Disruptor). The disrupted cells were centrifuged at 4 0

C

to sediment cell debris and the supernatant containing soluble urease was collected.

A solution of 3 M sodium chloride was added to the cell supernatant to a final concentration of 0.1 M. The supernatant was then applied to a DEAE-Sepharose column equilibrated in 20 mM sodium phosphate/1 mM EDTA, and the pass-through was collected for further processing. The pass-through was diafiltered into 20 mM sodium phosphate/I mM EDTA, pH 6.8, with a 100 kD cutoff membrane to remove low molecular weight contaminants and to reduce ionic strength.

This material was applied to a second DEAE-Sepharose column equilibrated in 20 mM sodium phosphate/1 mM EDTA. The pass-through was discarded and the column rinsed with 20 mM sodium phosphate/1 mM EDTA, pH 6.8. The bound material was eluted with approximately three column volumes of 0.1 M NaCl/l mM P-mercaptoethanol. Effective elution of the bound urease was controlled by the volume and flow rate of the elution buffer.

The partially purified urease was diafiltered against 25 mM Tris-HCl, pH 8.6, and applied to a Q-Sepharose column. The column was washed with approximately two column volumes of the same buffer and the pass-through containing highly purified urease was .collected. The pass-through from the Q-Sepharose step was then concentrated and diafiltered into 2% sucrose in water for injection (WFI), pH Characterization of the antigenicity and subunit composition of purified recombinant H. pylori urease To compare the antigenicity and subunit composition of recombinant H. pylori urease to native H. pylori urease, native H. pylori urease was purified 15 and used as an antigen to produce polyclonal anti-urease antibodies, as well as mono-specific polyclonal anti-UreA, anti-UreB, and anti-urease holoenzyme antibodies.

Native H. pylori urease was purified using a modification of the procedure reported by Hu and Mobley (Infect. Immun. 58:992-998, 1990). H. pylori strain ATCC 43504 (American Type Culture Collection, Rockville, MD) was grown on Mueller-Hinton agar (Difco Laboratories, Detroit, MI) containing 5% sheep red blood cells (Crane Labs, Syracuse, NY) and antibiotics (5 g/ml trimethoprim, 10 ig/ml vancomycin and 10 U/ml polymyxin B sulfate) (TVP, Sigma Chemical Co., St. Louis, MO).

Plates were incubated 3-4 days at 37 0 C in 7% CO 2 and humidity, and bacteria were harvested by centrifugation.

The bacteria were suspended in water or 20 mM phosphate, 1 mM EDTA, 1 mM A-mercaptoethanol (pH 6.8) containing protease inhibitors, lysed by sonication and clarified by centrifugation. The clarified supernatant was mixed with S 20 3 M sodium chloride to a final sodium chloride concentration of 0.15 M and passed through DEAE-Sepharose (Fast Flow). Active urease that passed through the column was collected, concentrated in a Filtron Macrosep 100 centrifugal filtration unit, and then passed 25 through a Superose-12 or Superdex 200 size exclusion column. Size-exclusion chromatography was performed using Pharmacia FPLC pre-packed columns. The fractions containing urease activity were pooled, concentrated, and further purified by FPLC anion-exchange chromatography on a Mono-Q sepharose column prepacked by Pharmacia. The bound urease was eluted using a sodium chloride gradient.

The fractions with urease activity were pooled and concentrated using Macrosep 100 centrifugal filters from Filtron Inc. For some lots, a final purification was 16 achieved by analytical size-exclusion FPLC on Superose-12 columns.

Polyclonal antiserum to H. pylori urease was produced by immunizing three female New Zealand white rabbits with purified native H. pylori urease. The animals were pre-bled to confirm non-immune status and then immunized subcutaneously with 150 ig urease in complete Freund's adjuvant. Two booster doses of 150 jg each were administered subcutaneously 27 and 45 days later with Freund's incomplete adjuvant. After confirmation of the immune response by ELISA and Western blotting against purified urease, the animals were exsanguinated. The blood was clotted at 4 0 C overnight and serum was harvested by centrifugation. Serum IgG was purified by ammonium sulfate precipitation overnight at 4 0 C. The precipitate was resuspended in PBS and dialyzed to remove ammonium sulfate. The anti-urease titer of the IgG from each animal was found to be 1:107 and the antibodies from the three animals were pooled.

20 The protein concentration was determined to be 17.3 mg/ml. Aliquots of 0.2 ml each were prepared and ges stored at -80 0

C.

Mouse polyclonal ascites against H. pylori urease holoenzyme ("MPA3") was prepared by injecting five mice 25 subcutaneously with 10 Ag native urease holoenzyme in complete Freund's adjuvant on Day 0. The mice were boosted on Days 10 and 17, bled on day 24 to confirm anti-urease immune response, and boosted again on Day 26.

On Day 28 the mice were injected intraperitoneally with Sarcoma 180 cells. A final intraperitoneal booster dose of 10 ig urease was given on Day 31, and ascitic fluids collected seven days later.

The ascitic fluids were incubated for two hours at room temperature and then at 4 0 C for 16 hours; clots were disrupted by vortexing and removed by centrifugation at 17 10,000 rpm for 10 minutes. After overnight incubation at 4 0 C in plastic tubes, the fluids formed solid clots.

These were homogenized, diluted five-fold with PBS, and re-clarified by centrifugation at 10,000 rpm for 10 minutes. Thirty-six ml of diluted ascitic fluids were collected and frozen at -20 0 C in 300 pl aliquots.

Western blot analyses confirmed that the antibody reacts with UreA and UreB subunits. An endpoint titer of 1:300,000 was achieved in ELISA against urease.

Mouse polyclonal ascites against H. pylori UreA ("MPA4") was prepared by injecting mice subcutaneously with native UreA subunit H. pylori urease isolated by electroelution from SDS-PAGE gels. Subsequent steps in the preparation of anti-ureA ascites were performed as described above for generation of antibodies against the holoenzyme.

Mouse polyclonal ascites against native H. pylori UreB subunit ("MPA6") was prepared by injecting mice subcutaneously with native UreB isolated by electroelution from SDS-PAGE gels. Subsequent steps in the preparation of anti-UreB ascites were performed as described above for generation of antibodies against the holoenzyme.

MAB71 is an IgA monoclonal antibody against H. felis urease which recognizes a protective epitope on the B subunit. Preparation of this antibody is described in Czinn et al., J. Vaccine 11(6):637-642, 1993, and the hybridoma cell line which produces it was deposited with the ATCC and designated deposit number HB 11514-- The antibodies described above were used in Western blot experiments to characterize the purified recombinant S. H. pylori urease.

18 SDS-PAGE and Western blot analysis of recombinant urease Recombinant urease was first analyzed by SDS-PAGE run under reducing conditions. Two major protein bands (29 kD and 60 kD) and several lighter bands (approximately 38 kD) were evident. To identify the proteins in these bands, Western blots were performed using the anti-urease and anti-urease subunit antibodies described above. The 60 kD protein reacted with MPA3 (anti-urease holoenzyme) and MPA6 (anti-UreB), but not with MPA4 (anti-UreA). The 29 kD protein reacted with MPA3 (anti-urease holoenzyme) and MPA4 (anti-UreA), but not with MPA6 (anti-UreB). The lighter 38 kD band reacted with MPA3 anti-urease holoenzyme) and MPA6 (anti- UreB), indicating that this protein is a portion of UreB.

Two faint high molecular weight (>150 kD) bands were evident in SDS-PAGE gels. Both bands reacted faintly with antibodies to both UreA and UreB, indicating that a minor portion of recombinant urease subunits form covalent units resistant to sulfhydryl reduction under the conditions used. No other protein bands were apparent in Coomassie-stained gels. Thus, all proteins detected in the purified product are either UreA, UreB, or a derivative of UreA or UreB.

The wet Coomassie-stained SDS-PAGE gel was scanned using an Ultroscan XL laser densitometer and a Gel Scan XL software program (Pharmacia-LKB Biotechnology, Piscataway, NJ). The densitometry data were consistent with a 1:1 molar ratio of the UreA:UreB subunits-, as expected from the structure of native H. pylori urease.

30 UreA and UreB accounted for more than 95% of the total protein present in the purified urease preparation. The estimated average value for the combined UreA UreB peaks was 95.2% 1.2%.

19 Analytical size-exclusion HPLC of purified recombinant urease The purity and molecular composition of purified recombinant urease was determined by analytical size-exclusion high performance liquid chromatography.

Chromatography was performed using a Beckman System Gold HPLC consisting of Pump 126, diode array dual wavelength detector 168, System Gold Software Version V7.11, Progel-TSK G4000SWXL (5 mm x 30 cm column (Beckman Instruments, Brea, California), and SWXL guard column from Supel Co. Chromatography was performed under isocratic conditions using 100 mM phosphate, 100 mM sodium chloride buffer, pH 7.0. The column was calibrated using molecular weight markers from Pharmacia-LKB.

A typical HPLC separation profile of representative purified bulk sample is shown in Fig. 4.

The purified urease product shows a prominent protein peak with a retention time between that of thyroglobulin (MW 669 kD) and ferritin (MW 440 kD). An apparent molecular weight of 550-600 kD was estimated based on a series of runs. This peak was tentatively designated as hexameric urease. The area of this hexameric urease was "at least 70% of the total protein in different lots of 25 product.

A second prominent protein peak with a lower retention time (higher molecular weight) was also detected. The area of this peak ranged from 5-20%. This peak, with a molecular weight >600 kD, was designated as octomeric urease. The total area of the octomeric plus hexameric urease peaks was over These two characteristic peaks were isolated for further characterization. The two peaks were purified by HPLC from a preparation of reconstituted, freeze-dried K 35 urease and stored at 4 0 C for over one week. During 20 storage for this period of time, octomeric urease increases while the hexameric form decreases. The separated peak fractions were analyzed by reducing SDS-PAGE and ELISA reactivity with monoclonal and polyclonal antibodies to urease. On SDS-PAGE, both peaks showed urease A and B bands in comparable ratios. In addition, both showed nearly identical immunoreactivity in ELISA with polyclonal anti-urease holoenzyme antibodies (MPA3) and a monoclonal anti-UreB antibody (MAB71).

Enzymatic (urea hvdrolytic) activity of recombinant urease Three methods were used to investigate the enzymatic activity of recombinant urease: urease-specific silver staining following electrophoresis, the pH-sensitive phenol red urea broth assay, and direct detection of ammonia. Urease-specific silver staining, described by deLlano et al. (Anal.

Biochem. 177:37-40, 1989), is based on the reaction of urease with urea to produce ammonia. The reaction leads to a localized increase in pH which facilitates a photographic redox reaction leading to disposition of metallic silver. Enzymatic activity of native H. pylori urease was detected with only a 1.0 pg sample. In 25 contrast, 20 Ag purified recombinant urease exhibited no urease activity.

The pH-sensitive phenol red broth assay, which is well known in the art, is based on a change in pH due to ammonia generation as a result of urea hydrolysis.

30 Urease activity was demonstrated with as little as 0.2 ig/ml purified H. pylori urease. In contrast, no urease activity was associated with purified recombinant urease, even at concentrations up to 750 Ag/ml.

O

21 Direct estimation of ammonia produced by hydrolysis of urea by urease was quantitated using Nesslers' reagent (Koch et al., J. Am. Chem. Society 46:2066-2069, 1924). Urease activity of native H. pylori urease was detectable at a concentration of 1 Ag/ml. No activity was detected in assays containing up to 500 -g/ml purified recombinant urease.

Based on the results of the urease-specific silver staining assay, the pH-sensitive phenol red broth assay, and direct estimation of ammonia, there is no detectable urease activity in the recombinant urease product.

Protective and therapeutic efficacy of purified recombinant urease The H. felis-mouse model was used to test the efficacy of recombinant H. pylori urease in the prevention and treatment of Helicobacter infection. In this model, colonization of the stomach is readily established and is accompanied by gastric inflammation.

This animal model is a well established system for the study of Helicobacter, and has been used extensively in laboratory investigations of the pathogenesis and treatment of Helicobacter-induced disease (see, e.g., Fox et al., Infect. Immun. 61:2309-2315, 1993; Goodwin and Worsley, Helicobacter pylori, Biology and Clinical 25 Practice, CRC Press, Boca Raton, FL, 465 pp, 1993).

Antigenic cross-reactivity between H. pylori and H. felis ureases allows use of the human vaccine candidate of the invention, recombinant H. pylori urease (rUre),-to immunize animals infected, or subsequently challenged, with H. felis.

Both germ-free and conventional mice are susceptible to infection by H. felis, and develop life-long infection of the gastric epithelium, characterized by infiltration of inflammatory cells 22 (Fox et al., supra). Dose response studies indicated that 100% of Swiss-Webster specific pathogen-free

(SPF)

mice become infected after a single oral challenge with 104 H. felis. Unless otherwise specified, a single dose of 107 was used to infect mice prior to therapeutic immunization or challenge mice after prophylactic immunization. A challenge of -103 times the infectious dose (I.D.

90 with this Helicobacter represents a severe test of immunity.

Assays for castric infection Several methods were used to detect Helicobacter in gastric tissue, including measurement of gastric urease activity, histologic examination, and culture of gastric tissue. Gastric urease activity was measured both qualitatively (presence or absence) and quantitatively. In the qualitative assay, stomachs were divided longitudinally into two halves from the gastroesophageal sphincter to the pylorus. One longitudinal piece, representing approximately 1/4 of the stomach, was placed in 1 mL of urea broth (0.1 g yeast extract, 0.091 g monopotassium phosphate, 0.095 g disodium phosphate, 20 g urea, and 0.1 g/L phenol red, pH A distinctive color change (due to hydrolysis of urea by the enzyme, production of ammonia, and 25 increased pH) after four hours incubation at room temperature indicated a positive result. For quantitative determinations, urease activity was determined by measuring absorbance at 550 nm of .clarified urea broth incubated with whole stomach sections-for 4 30 hours. This assay can detect as few as 1-2 x 104 H. felis/0.1 g stomach tissue. This assay provides the same sensitivity as commercially available urease test kits used for human samples. Commercial kits have proven to be 100% specific and 90-92% sensitive compared to 23 biopsy/histology (Szeto et al., Postgrad. Med. J. 64:935- 936, 1988: Borromeo et al. J. Clin. Pathol. 40:462-468, 1987).

Quantitative gastric urease assays by spectrophotometric measurement of A 550 were slightly more sensitive than visual determinations, and allowed estimation of the severity of infection. The cut-off value for a negative urease assay was defined as 2 standard deviations above the mean A 550 for unchallenged/uninfected mice. The cut-off for a positive gastric urease assay was defined by 2 standard deviations below the mean A 550 of unimmunized/challenged mice.

Individual animals with low-grade infections had values intermediate between the negative and positive cut-offs.

Visual grading of the urease response identified 11/12 of the positive samples.

Histologic examination was performed by fixing stomach tissue in 10% formalin. The tissue was then embedded in paraffin, sectioned and stained with a modified Warthin-Starry silver stain (Steiner's stain; Garvey, et al. Histotechnology 8:15-17, 1985) to visualize H. felis, and by hematoxylin and eosin stain to assess inflammatory responses in the tissue. Stained sections were examined by an experienced pathologist 25 blinded to the specimen code.

A semi-quantitative grading system was used to determine the number of bacteria and intensity of inflammation. The system used is a modification of the widely-accepted Sydney System for histological characterization of gastritis in humans (Price, Gastroenterol. Hepatol. 6:209-222, 1991). Full-thickness mucosal sections were examined for the intensity of inflammation (increase in lymphocytes, plasma cells, neutrophils and presence of lymphoid follicles) and depth 35 of infiltration of these cells and graded on a scale of 24 Grading the density of H. felis was accomplished by counting the number of bacterial cells with typical spiral morphology in an entire longitudinal section of gastric antrum (or corpus, if specified). Grades were assigned according to a range of bacteria observed (0 none; 1+ 1-20 bacteria; 2+ 21-50 bacteria; 3+ 51-100 bacteria; and 4+ >100 bacteria).

Route of immunization The effect of the route of administration upon the efficacy of the vaccine of the invention was examined in the mouse infection model. Six to eight week-old female specific pathogen free (SPF) Swiss-Webster mice were immunized with 200 gg recombinant H. pylori urease, either with or without 0.24 M NaHCO 3 The recombinant urease was co-administered with 10 pg of cholera toxin (CT) as a mucosal adjuvant in all animals. Intragastric (IG) immunization was performed by delivering the antigen in 0.5 ml through a 20-gauge feeding needle to anesthetized animals. Oral immunization was performed by delivering the antigen in a 50 pl volume via a pipette tip to the buccal cavity of unanesthetized animals. For parenteral immunization, mice received 10 Mg recombinant urease subcutaneously. Freund's complete adjuvant was used in the first subcutaneous immunization and Freund's 25 incomplete adjuvant was used in subsequent boosters. For all routes of administration, a total of four doses of vaccine were administered at seven day intervals. Mice were challenged with 107 H. felis two weeks after the final vaccine dose, and necropsied two weeks after 30 challenge. Gastric H. felis infection was detected by urease activity and histology. Protection in an individual mouse was defined by a negative urease assay and by a 0 or 1+ bacterial score by histology.

.i 25 Oral and intragastric administration of recombinant urease provided significant protection to challenge with H. felis (86-100%) (Table Oral administration was effective both with and without coadministration of NaHCO 3 with the recombinant urease.

Intragastric administration was more effective when the recombinant urease was co-administered with NaHCO 3 Parenteral injection of the vaccine antigen was least effective. Immunized mice had significantly lower numbers of bacteria in gastric tissue after challenge than unimmunized controls. IgA antibody responses in serum and secretions were highest in mice immunized by the oral route. IgA antibodies were not elicited by parenteral immunization.

eoo** e. o *C C 0 Table 1 Recombinant H. Rylori urease protects mice from challenge with H. fells after mucosal but not Parental immunization

VACCINE

ROUTE 0F ADJUVANT BICARBONATEa PROTECTED IMMUJNI ZATION PRMEC /TOTAL)

UREASE

ASSAY HSOOy PBS oral CT No 02A1 (08 07 200 yg rUre oral CT No 100 100 200 pg rUre oral CT -Yes 100 86 200 pg rUre Aintragastric CT No 75 38 (3/8) 200 pg rUre intragastric CT Yes 100 100 10 pg rUre 1subcutaneous Freund's I NA 38 25 (2/8) a 0.24 M sodium bicarbonate was administered with vaccine and adjuvant.

b Percent protected (number mice with 0-1+ bacterial score/number tested).

*p<0.01, Fisher' s Iexact test, compared to mice given CT alone.

27 The effect of immunization route upon the anti-urease antibody response was examined in mice.

Swiss-Webster mice were immunized four times at ten day intervals with either: 1) 200 pg recombinant purified H.

pylori urease with 10 pg CT, either with or without NaHCO 3 by oral administration; 2) 200 Ag recombinant purified H. pylori urease and 10 pg CT with NaHC03, by intragastric administration; or 3) 10 pg recombinant purified H. pylori urease with Freund's adjuvant by subcutaneous administration. One week after the fourth vaccine dose, mucosal and serum antibody responses were examined by ELISA using microtiter plates coated with pg of native H. pylori urease. Serum samples were diluted 1:100 and assayed for urease-specific IgA and IgG. Fresh fecal pellets, extracted with a protease inhibitor buffer (PBS containing 5% non-fat dry milk, 0.2 pM AEBSF, 1 pg aprotinin per ml, and 10 AM leupeptin), were examined for fecal anti-urease IgA antibody. In some experiments, fecal antibody values 20 were normalized for total IgA content determined by ELISA, with urease-specific fecal IgA expressed in

A

405 units/mg total IgA in each sample. Saliva samples were collected after stimulation with pilocarpine under ketamine anesthesia, and tested for urease-specific IgA at a dilution of No significant differences between antibody responses of mice immunized orally with or without NaHCO 3 were detected, and the data were pooled for analysis.

Mice given subcutaneous antigen developed urease-specific S 30 serum IgG, but serum and fecal IgA responses were elicited only when antigen was delivered by mucosal routes (oral or intragastric).

Mice immunized either orally or intragastrically with urease/CT were challenged with 107 H. felis. The pre-challenge antibody levels of orally or 28 intragastrically immunized mice were correlated with histologically-determined bacterial scores after H. felis challenge (Fig. Although IgA responses varied considerably between individual mice, on the whole, mice with higher serum and fecal IgA levels had reduced bacterial scores. These data indicate a role for IgA in suppression of, and protection from, infection. In contrast, serum IgG antibodies did not correlate with protection. While some protected mice had no detectable IgA antibodies, high levels of anti-urease IgA were not observed in animals that developed 4+ infections after challenge. These results not only support a role for mucosal immune responses in protection, but also suggest that immune mediators other than fecal and serum IgA play a role in eradication of H. felis infection.

These data show that: i) mucosal immunization is required for protective immunity; ii) the oral route of immunization is as effective, or more effective, than the i intragastric route; iii) neutralization of gastric acid 20 with NaHCO 3 is required for effective intragastric (but not oral) immunization; and iv) parenteral immunization does not stimulate mucosal immunity or provide effective protection against challenge.

Schedule of immunization 25 Alternative immunization schedules were compared in order to determine the optimal immunization time-table to elicit a protective mucosal immune response.

Swiss-Webster mice were immunized with 100 Ag recombinant urease in two, three, or four oral administrations, on a 30 schedule shown in Table 2. The mucosal adjuvant CT pg) was co-administered with the recombinant urease.

As assessed by qualitative gastric urease assay, mice which received four weekly doses of antigen exhibited the highest levels of protection. Significant protection was 29 also observed in mice given three doses of antigen on days 0, 7, and 21. Secretory IgA antibody responses were highest for mice given a total of four doses of recombinant urease at one week intervals. On the basis of protection ratio and antibody responses, the latter schedule was selected for further evaluation of the therapeutic and prophylactic efficacy of the vaccine.

e 30 Table 2 Effect of different schedules of oral immunization on the prophlactic efficacy of urease vaccine VACCINE DOSE/ SCHEDULE (DAY IMMUNIZATION OF PROTECTED oROUP IMMUNIZTION) (#/TTAL) b 1 25 0, 7, 14, 21 70 (7/10)* 2 50 0, 14 20 (2/10) 3 50 0, 14 30 (3/9) 4 33.3 0, 7, 21 40 (4/10)* 50, 25, and 0, 7, 21 60 (6/10)* 6 None 0, 7, 14, 21 0 (0/10) a Each group of mice were immunized orally with a total dose of 100 gg recombinant H. pylori urease. CT (10 Mg) was used at each immunization, suspended in sterile distilled water, as the mucosal adjuvant. Group 6 received CT alone on the same schedule as group 1 and acted as the control. Groups 4 and 5 compared the effect of booster doses administered after two previous weekly immunizations.

b Percent protected (No. protected/No. tested) from a 10 7 challenge dose of H. felis two weeks post-immunization.

Mice were sacrificed two weeks after challenge and protection was determined by the qualitative gastric urease assay.

p<0.05, Fisher's exact test, compared to mice-given CT alone.

The effect of different immunization schedules upon anti-urease antibody production was examined in mice. Antibody responses of mice immunized by one of the five different immunization schedules described in 31 Table 2 were examined. Significant protection was observed in mice that received vaccine in four weekly doses or two weekly doses with a boost on day 21.

Mice immunized by either of these two immunization schedules also had the highest average immune responses.

The serum IgG and salivary IgA levels were highest in mice vaccinated on a schedule of four weekly doses.

Dose-protection relationship Graded doses of recombinant urease were orally administered to mice to determine the minimal and optimal doses required for immunization and protection.

Recombinant urease doses of 5, 10, 25, 50, and 100 Mg were administered to groups of eight mice by the oral route with 10 Ag CT in PBS. The antigen was given on a schedule of four weekly doses. As assessed by gastric urease and histologic bacterial score, significant protection of mice against challenge with H. fells was observed at all doses, with no significant differences between dose groups (Table A dose response effect 20 was clearly demonstrated in serum and mucosal antibody responses to recombinant urease, with the highest immune response at the 100 Mg dose level.

Table 3 Recombinant H. pylori grease at doses of 5 auq or more. protects mice against challenge with H. fells

PROTECTEDA'

ROUTE OF (#PROTECTED/TOTAL) VACCINE ADJUVANT IMMUNIZATION UREASE ASSAY HISTOLOGY none 10 gg CT oral 0 0 (0/7) gg rUre 10 jtg CT oral 86 71 gg rUre 10 Ag CT oral 88 63 Ag rUre 10 jgg CT Oral 100 100 jig rUre 10 jig CT oral 100 88 L100 tig rUre 10 jig CT oral 100 71 A Percent protected (number mice with 0-20 bacteria per section/number tested) as determined by examination of silver-stained stomach sections.

*Comparison to sham-immunized controls, Fisher's exact test, p:50.02.

33 The effect of recombinant urease dosage upon the anti-urease antibody response was examined in mice.

Swiss-Webster mice were immunized orally with graded doses 5, 10, 25, 50, or 100 Mg) of recombinant urease plus 10 ig CT as a mucosal adjuvant. IgA antibody responses in serum, feces, and saliva increased with the amount of recombinant urease administered. A 100 gg dose of recombinant urease produced the highest antibody levels.

Swiss-Webster mice were orally immunized with graded doses 5, 25, or 100 Mg) of recombinant urease, with 25 Mg enterotoxigenic E. coli heat-labile toxin (LT) as a mucosal adjuvant. One group of mice received 25 pg recombinant urease and 10 Mg CT as a mucosal adjuvant for comparison. The mice were immunized orally 4 times every 7 days. Serum, feces, and saliva were collected 10 to 13 days after the last immunization and urease-specific antibody levels were determined by ELISA. Mice immunized with recombinant urease and LT developed serum and 20 secretory antibodies against urease, with a clear doseresponse effect. Strong salivary IgA antibody responses were observed in these animals.

Mice immunized with urease and LT as described above were challenged with 107 H. felis (see Fig. 4).

25 The pre-challenge antibody responses of the urease/LT immunized mice were correlated with histologicallydetermined bacterial scores (Fig. Fully protected animals (0 bacterial score) and those with low-grade infections bacterial score) had higher levels of antibodies in all compartments than animals with more severe infections. A few protected mice had no detectable immune response, suggesting that immune mediators other than IgA antibodies play a role in eradication of H. felis infection.

34 The ability of high doses of recombinant urease to elicit a mucosal immune response was examined by administering intragastrically 1 gg, 200 pg, or 5 mg without adjuvant. One group of mice were intragastrically immunized with 200 gg urease plus 10 Mg CT as a control. Blood, feces, and saliva were collected to 8 days after the last immunization. Animals were then challenged with 107 H. felis 10 days after the last immunization and sacrificed 14 days after challenge.

H.

felis infection was detected by urease activity in stomach tissue.

High doses of recombinant urease elicited urease-specific serum IgG, but elicited comparatively low levels of mucosal antibodies as detected by ELISA.

Animals which exhibited a urease-specific serum IgG response were fully susceptible to H. fells challenge, indicating that serum IgG does not play a role in protection.

In summary, data from the above experiments 20 investigating different administration routes, administration schedules, and mucosal adjuvants S. demonstrate that, when administered with an effective mucosal adjuvant, oral or intragastric administration of recombinant urease at relatively low doses elicits 25 secretory IgA antibody and serum IgA and IgG responses.

Secretory IgA antibody provides protection, while serum IgG responses do not. When protection is measured by histological bacterial counts, animals with higher IgA antibody titers were fully protected, or had 30 significantly reduced infections, as compared to animals with lower IgA antibody titers. Animals which did not exhibit detectable IgA antibody levels developed severe infections after challenge. Antibody responses were dose-dependent and differed by the schedule of administration of antigen. The highest levels were 35 achieved at antigen doses of 100-200 Ag. Administration of four doses of antigen at one week intervals provided the optimal schedule for immunization. An additional administration route, intranasal, was investigated, as follows.

Intranasal vaccination with recombinant urease Swiss Webster mice were immunized either orally or intranasally (IN) with recombinant urease or formalin-fixed urease. The amounts of antigen and adjuvant, routes of administration (IN or oral), and immunization schedules are shown in Fig. 7. The formalin-fixed urease (Form-ure) was prepared according to the following protocol: a vial containing 1 mg recombinant urease is reconstituted with 150 pL of RO/DI water; 50 tL of formalin (37% formaldehyde), diluted 1:1000 in RO/DI water, is added to the vial (final concentration of urease is 5 mg/ml); and *o the vial is incubated at 35 0 C for 48 hours.

20 Serum IgG, IgA, fecal IgA and salivary IgA responses in IN CT group were higher than the oral CT group, despite higher doses of rUrease and adjuvants that were used for oral immunization (Fig. 7 and Table 4).

Protection was measured by the urease test, as well as by determining bacterial scores on stomach tissues of sacrificed animals. One hundred percent protection was found in the IN group, as compared to 80% in the oral group, when protection was assayed by urease test. Seven of eight of the orally immunized mice were positive for S 30 bacteria on their stomach tissues, whereas only 1/10 of the mice in the IN group were positive for bacteria (see groups 3 and 6 in Table 4 and Fig. 8).

In a second experiment, mice were immunized either intragastrically (IG) or intranasally with rUrease co- 36 administered with LT as mucosal adjuvant (see Fig. 9 for details of the experiment and the immunization schedule).

In this experiment, serum IgG, serum IgA, and salivary IgA responses in the IN LT group were higher than in the IG LT group, despite higher doses of antigen and adjuvant that were used for IG immunizations (Fig. 10 and Table 5, groups 4 and Mice in the IN LT group were fully protected against H. felis challenge (10/10 compared with 3/9 untreated group), as determined by urease test (see groups 4 and 5 of Table e oe e ee Table 4 A- I ~Jtt. ~t.I .t S t~.t Intranasa± immunization with recomb~inant urease and CnOlera toxin Urease hours Mouse treatment route/adjuvant serum IgG serum IgA fecal IgA salivary IgA urtas Bact. Path.

M 10 pg/HCHO IN/none 3.398 3.318 3.377 3.339 1A2 3.218 0.786 3.249 1A3 2.535 0.893 3.153 lA4 2.390 1.065 3.311 lAS 3.009 2.647 0.617 3.244 Al 2.994 2.068 0.497 3.329- 1B2 2.611 -0.010 3.235 183 3.306 1.823 0.323 3.295 1B4 2.931 0.522 3.372 lBS 3.175 0.832 0.803 2.439 2A1 10 pg IN/none 3.021 2.562 2.090 3.204 2A2 2.806 1.475 3.304 2A3 1.943 0.584 3.288 2A4 2.804 2.291 3.363 2.926 3.264 0.118 3.418+ 281 3.220 3.505 1.634 3.415 282 2.708 0.410 3.336 2B3 1.814 0.411 3.253 2134 3.090 2.672 3.266 3.055 2.076 1.193 3.263 3A1 10 pg IN/CT 2.969 0.405 0.192 1.670 -0 2 3A2 2.871 0.097 0.154 0.526 -0 2 3A3 2.984 0.677 0.081 0.473 3A4 3.092 0.563 1.824 3.398- 0.256 1.052 0.587 -1 2 3B1 0.148 0.142 0.586 -0 2 3B2 3.068 0.388 0.867 2.410 -0 2 3B3 0.255 0.468 0.778 -0 2 3B4 0.985 1.086 1.172 -0 2 0.186 1.904 1.317 -0 2 4A1 100 pg/HCHO oral/none -0.006 0.024 0.021 0.009 4A2 0.097 0.079 0.041 4A3 0.097 0.056 -0.019 4A4 3.195 0.598 0.190 2.097 0 3 1.271 0.027 0.034 0.010 48Ol 0.096 0.045 0.165 0.309 4B2 0.021 0.033 0.043 0.059 483 1.109 0.055 0.080 0.019 484 0.006 0.022 0.083 0.017 0.013 0.027 0.039 0.010 SMl 10 oral/none 0.032 0.042 0.151 0.030 5A2 0.756 0.077 2.957 0.156 5A3 2.362 0.075 0.104 0.262 0.012 0.034 0.146 0.065 0.035 0.163 0.013 5B1 0.042 0.089 0.017 5B2 0.017 0.076 0.049 5B3 0.058 .0.058 0.182 5B4 0.030 0.093 0.093 1.672 0.059 0.060 0.093 6A1 25pg- oral/CT 0.698 0.267 0.076 0.387 1 2 6A2 0.065 0.089 0.206 1 2 6A3 0.013 0.098 0.011 3 2 6A4 2.957 0.354 0.185 2.999 1 2 0.019 0.042 0.026 0 2 0.000 0.022 0.041 0.005 1 2 6B2 0.009 0.002 0.067 0.069 2 2 6B3 0.022 0.041 0.048 4 2 6B4 0.010 0.009 -0.'021 0.023 4 2 2.633 0.090 0.047 0.647 1 2 7A1 none 0.018 0.074 0.019 4 1 U U U *jU

U

U U

U

U

*UU

U. U *U *U U U 7A2 -0.025 0.042 -0.004 4 2 7A3 0.019 0.082 0.018 4 2 7A4 0.028 0.024 -0.002 4- 2 0.011 0.005 0.041 0.029 X 7B1 0.004 0.089 -0.005 4 1 7B2 -0.003 -0.009 0.008 4 2 7B3 0.024 0.066 0.167 4 2 7B4 0.018 0.063 0.018 4 1 0.023 0.111 0.011 plate I controls 3.280 1.651 3.315 0.040 0.065 0.056 plate 2 controls_______ 3.289 1.700 3.298 0.034 -0.004 0.019 S S S. S. S S S S S S SSS *S S Table S Intranasal immunization with recombinant urease and LT Urease 2 week sacrif ice House tTreatment serum IgG serum IgA salivary IgA Absorbance...

1AN 25 pg Sx IN 3.530 0.350 0.340 0.802 l1AL 200 pg 2x IG 3.263 0.154 1.544 0.768 l1AR 0.630 0.204 0.805 -1ALL 0.318 0.251 0.806 -lBN 0.401 0.112 0.726 -lBL 0.724 2.015 0.168 lER 0.225 0.426 0.748 lELL 0.591 0.719 0.807 -lBRR __3.262 0.141 0.202 0.675 -2AN 25 pg 5x IN 3.164 0.054 0.014 0.814 -2AL 200 pg lx IG 3.232 0.231 0.747 0.823 2AR 3.236 0.981 0.412 0.695 2ALL 3.303 0.122 0.832 0.813 2 BN 0.690 0.993 0.747 2 EL 1.002 0.840 0.704 -2ER 1.377 .0934 0.161 2BLL .0458 0.191 0.748 2 BRR 0.129 0.134 0.720 3AM 25 pg 2x IN 0.165 0.031 0.001 0.791 3AL 200 pg 2x IG 3.364 0.332 0.356 0.824 3AR 0.031 -0.003 0.810 -3ALL 0.450 0.541 0.798 3ARR 3.358 0.158 0.001 0.675 3 8N 0.276 0.212 0.820 3BL 3.174 2.463 2.838 0.839 3BR 3.274 0.607 1.517 0.481 -3BLL 3.284 1.279 2.593 0.829 3 BRR 3.254 0.427 1.684 0.852 4AN 25 pg 4x IN 3.375 0.118 0.212 0.111 4AL with 2 pg LT 3.482 0.346 0.546 0.107 4AR 3.237 3.302 0.109 4ALL 3.067 3.229 0.103 4ARR 1.625 2.156 0.106 4BN I3271.263 2.943 0.224 4BL 0.496 2.029 0.139 4BR 0.809 2.307 0.114 4BLL 1.019 3.346 0.106 4BRR 3.474 0.674 1.683 .0.119 S S S S 555 55.

S S S 5* S 55 S S S S S S S

S

SAN none 0.075 0.023 0.001 0.126 0.005 0.016 -0.002 0.881 0.008 -0.001 0.839 SARR 0.005 0.007 0.000 0.834 SBN 0.016 0.001 0.134 0.009 0.004 0.814 0.014 0.007 0.833 SBLL 0.019 0.008 0.155 0.012 0.003 0.818 6AN 25 pg 5x IN 3.353 0.680 0.498 6AL 200 pg lx IG 3.470 0.248 0.100 6AR 1.236 0.741 _6ALL 0.409 0.322 6ARR 1.036 1.056 6BN 2.749 2.014 6BL 0.360 6BR 0.749 1.882 6BLL 't3.347 0.275 0.030 6ERR 0.311 0.989 7AN 25 pg 4x IN 3.415 0.756 7AL with 2 pg LT 13.428 13.080 3.287 0 0 0* I* 0 0 0 0 0** 0 A 0* 0 0 0 *0 *t* 00 *0 *0 0 *00 0 00* 0 7AR 2.054 3.188 7ALL 1.010 3.240 7ARR 0.713 3.243 7BN 3.310 3.081 3.274 7BL 3.439 2.298 3.222 7BR 3.359 1.026 1.757 7BLL 3.370 1.441 3.025 7BRR 3.330 0.689 BAN 200 pg 4x IG 0.281 0.031 8AL with 10 pg LT 3.234 0.925 1.872 BAR 0.827 2.751 BALL 0.547 0.395 BARR 0.382 1.144 8BN 1.189 1.880 BBR 1.608 2.536 8BBLL 0.438 0.599 9AN none I0.011 0.003 0.000 9AL 0.005 9AR 0.002 0.001 9ALL 0.013 9ARR 0.019 -0.002 S S S 55 S S SC S S S 555 *55 a

S~*

S*

S

S

S 55 a 5 55 C. 0 a. 0 5* 9BN 0.010 -0.001 981. 0.020 0.008 9BR 0.031 0.012 -0.002 9ELL 0.005 0.016 0.005 9BRR 0.019 0.011 0.o000 46 Further support for the efficacy of intranasal immunization is shown in Fig. 11. Briefly, this experiment showed that rUrease (10 pg), administered by the intranasal route with CT (5 Ag), is at least as effective as rUrease (25 pg) given by the oral route with CT (10 gg) in preventing infection with H. felis.

Selection of a mucosal adiuvant E. coli heat-labile toxin (LT) is a multisubunit toxin which is closely related, both biochemically and immunologically, to CT. Because the toxicity of LT is lower than CT, LT is a practical mucosal adjuvant for use in humans (Walker and Clements, Vaccine Res. 2:1-10, 1993).

Mice were orally immunized with 5 Mg, 25 pg, or 100 pg recombinant urease and 25 pg recombinant LT (Swiss Serum Vaccine Institute, Berne, Switzerland) for a total of four weekly doses. Control mice received LT only or 25 pg urease vaccine with CT. Mice were challenged two weeks after the fourth dose and necropsied two weeks 20 after challenge.

Gastric urease assays indicated significant protection or suppression of the infection in immunized animals at all vaccine doses (Table Histological assessment confirmed significant reductions in bacterial 25 scores in mice which received 5 5 pg urease and LT.

Protection was directly correlated with dose as determined by both gastric urease assay and histological examination, with the highest protection conferred by 100 pg of recombinant urease co-administered with LT.

30 Antibody determinations confirmed a dose-response relationship, with highest mucosal immune responses at the 100 pg dose. When recombinant urease was administered at equivalent doses (25 pg), LT was superior to CT as a mucosal adjuvant. These data indicate that 47 while CT enhances the mucosal immune response to orally administered recombinant urease, LT is a better mucosal adjuvant and is thus the preferred over CT for coadministration with recombinant urease.

Table 6 E. coli heat-labile enterotoxin (LT) as a mucosal adiuvant for immunization with recombinant H. pylori urease

PROTECTED

VACCINE ADJUVANT PROTECTED/TOTAL) a UREASE ASSAY HISTOLOGY None 25 pg LT 0 0 (0/9) pg rUre 10 Ag CT 70 60 (6/10)* pg rUre 25 pg LT 787 56 Ag rUre 25 pg LT 90 80 (8/10)* 100 pg rUre 25 pg LT 100 100 15 a Percent protected (number of mice with negative urease assay or bacterial score 0-1+/number tested) determined by bacterial scores of silver-stained stomach sections.

Comparison to sham-immunized controls, Fisher's exact test, p<0.03.

To determine the adjuvant activity of lower doses of LT, mice were orally immunized with 25 pg of recombinant urease administered with 1, 5, 10, or 25 pg of LT. Similar protection ratios and antibody responses were observed at all LT doses.

Several studies were performed to assess-adjuvants other than CT and LT, and to determine whether the requirement for a mucosal adjuvant could be eliminated by administration of antigen alone at a high dose. Cholera toxin B subunit (CTB) was compared with CT as a mucosal adjuvant for urease immunization. No protective effect 48 was observed when 200 jg urease was co-administered intragastrically with 100 ig CTB (Calbiochem, La Jolla, CA) in 0.24 M sodium bicarbonate, whereas the same amount of urease with 10 pg CT gave 100% protection, as determined by gastric urease activity.

An orally-active semi-synthetic analogue of muramyl dipeptide, GMDP, (N-acetylglucosaminyl-(bl-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine), was co-administered with 25 Mg urease at doses of 2, 20, and 200 gg. GMDP co-administered with recombinant urease failed to protect mice against H. felis challenge.

Large doses (200 Mg, 1 mg, or 5 mg) of recombinant urease with or without CT were intragastrically administered to mice once a week for four weeks. The intragastric route was required because volumes for high antigen doses exceeded those that could be given orally in a reproducible fashion. NaHCO 3 was co-administered to neutralize gastric acid. A total of four doses of 20 antigen were administered at ten day intervals. Blood, feces, and saliva were collected five to eight days after the last immunization. Animals were challenged with 1 x 10' H. felis, and infection was determined by urease activity in stomach tissue.

25 In the absence of CT, high antigen doses did not confer protection against H. felis challenge, whereas controls given 200 Mg of recombinant urease with CT were significantly protected (Table Urease-specific serum IgG was induced at the high recombinant urease doses 30 without adjuvant, but serum, fecal, and salivary IgA responses were absent or minimal. Histological examination of coded specimens from animals given 5 mg doses of urease revealed no differences in bacterial scores or leukocytic infiltrates, compared with sham-immunized animals.

5 S S S

S

S..

S S 55 S

S

S S Llitate~ nrotection by recombinant urease Table 7 A mucosal adjuvant fac~ Llitates nrotection by recombinant urease Mean antibody levels (±BD) before challengea %protected Vaccine Adjuvant (#protected/ Serum Serum Focal Balivary IgG IgG IqA IgA none PBS 0 0.01 0.03 0.01 0.02 (±0.03) 2) 200 pg rUre 10 pg CT 88 >2.97 >1.02 0.16 0.59 (±0.73) 200 Mg rUre none 0 >2.06 0.07 0.02 0.06 (±0.13) 1 mg rUre none 0 >3.28 0.12 0.02 0.18 (±0.40) mg rUre none 0 >2.68 >0.22 0.132 0.08 (±0.11) 2) a Mean ureAs6-specific serum IgG or I gA expressed as A 4 0 5 units of serpm (diluted 1:100), fecal extract (diluted 1:20), or saliva (diluted Means are shown as when individual values of 4.0 (the maximum A 4 0 5 reading) were included in calculating the mean -values.

Comparison to sham-immunized controls, Fisher's exact test, p=0.005.

50 Therapeutic immunization of mice with H. felis gastritis The efficacy of the recombinant urease vaccine in treating Helicobacter infection in mice was evaluated by intragastrically infecting Balb/c mice with 107 H. felis, and, four weeks after infection, orally administering to the infected mice four weekly doses of 100 pg recombinant urease with 10 pg LT. Control mice received LT only (Fig. 12).

Ten of the mice in each group were necropsied four weeks after the final immunization for examination of the degree of Helicobacter infection by quantitative gastric urease. Nine out of ten Balb/c mice were free from infection, as measured by quantitative urease assay, whereas all controls were infected.

At four weeks, twelve animals receiving LT and animals receiving urease LT were reinfected with H. fells. Ten weeks after the challenge, the animals were sacrificed to determine the extent of infection by .quantitative urease assay. Of nine animals which were 20 given urease LT, but not reinfected, 5 were still clear of infection as determined by gastric urease activity. All twelve LT-treated animals which were rechallenged were infected. Thirty-seven of the 40 mice which were given urease LT, and then re- 25 challenged with H. felis, were protected as determined by e reduced gastric urease activity. This experiment shows that urease vaccination not only eradicates an existing Helicobacter infection, but also protects the host against reinfection.

30 Five of fourteen immunized Swiss-Webster mice were cured or had reduced infections, whereas all control animals were infected, although the differences in infection ratios between groups was not significant (p=0.26, Fisher's exact test, two-tailed). Infection in Swiss-Webster mice was more severe than in Balb/c mice, 51 as measured by higher mean gastric urease activity in unimmunized animals (p 0.0001, one-way ANOVA), possibly explaining the lower cure rates in Swiss-Webster versus Balb/c mice. Differences in susceptibility of mouse strains to H. fells has been noted (Sakagami et al., Am.

J. Gastroenterol 89:1345, 1994).

By histologic assessment, all unimmunized Balb/c mice had 4+ infections 100 bacteria/section), whereas reduced bacterial scores were seen in 43% of immunized mice at four weeks. At ten weeks, four of six had reduced urease activity, although only one of six had a reduced bacterial score.

The role of antibodies in Helicobacter therapy The role of anti-urease antibodies in Helicobacter therapy, the clearance of H. felis from infected mice, was examined by first infecting Balb/c mice with 107 H. felis. Four weeks after infection, the mice were orally immunized with 200 pg recombinant urease plus 10 pg CT. Control mice were given 10 pg CT only.

20 Antigen was administered 4 times at one week intervals.

Animals were sacrificed 4 and 10 weeks after the last immunization, and serum and fecal samples were collected for ELISA.

S* .Mice infected with H. fells produced serum anti- 25 urease IgG antibodies, but no secretory anti-urease IgA response was detected. However, infected animals immunized with urease/CT exhibited high secretory antiurease IgA antibody responses (Fig. 13). There-was no significant difference in urease specific mucosal IgA 30 levels between immunized mice that remained infected and those with reduced bacterial scores.

These data indicate that H. felis infection does not elicit a secretory anti-urease response. Thus, suppression of the IgA antibody response may play a role 52 in the ability of H. felis to evade clearance by the immune system. In contrast, immunization of H. felisinfected mice with urease and a mucosal adjuvant resulted in strong mucosal anti-urease responses, which correlated with clearance of the H. felis infection.

Correlation of rotection aainst Helicobacter infection with qastric immune responses Several of the experiments using the mouse infection model showed that some animals rendered resistant to infection by recombinant urease vaccine lacked detectable antibody responses, or had low antibody levels, in serum, saliva, or feces. Thus, the immune response was measured in the gastric mucosa itself, in order to determine whether measurement of an immune response could be more precisely correlated with protection.

Immune responses in gastric mucosa were assessed by detecting IgA antibodies and IgA-positive antibody secreting cells in intestinal and gastric murine tissue 20 by immunohistochemistry. Portions of the stomach containing pylorus-proximal duodenum, antrum, corpus, and i. cardia were mounted in OCT compound, flash-frozen, and cryosectioned. Sections (7 pm thick) were fixed in cold acetone, and IgA-positive cells were identified by 25 staining with biotinylated monoclonal anti-IgA, followed by avidin conjugated to biotinylated glucose oxidase (ABC-GO, Vector Laboratories, Burlingame, CA), and counterstained with methyl green. Urease-specific antibody secreting cells (ASC) were identified by 30 sequentially incubating sections with recombinant urease, rabbit anti-urease, biotinylated donkey anti-rabbit Ig (Amersham, Arlington Heights, IL), ABC-GO, TNBT, and methyl green. Control sections were incubated without urease or urease plus rabbit anti-urease to determine 53 reactivity with the donkey secondary reagent and background endogenous glucose oxidase activity.

Cryosections of cell pellets from a hybridoma which produces a monoclonal IgA antibody against H. felis ureB (MAB71) and an irrelevant IgA monoclonal (HNK20) against F glycoprotein of respiratory syncytial virus served as positive and negative controls, respectively.

Swiss-Webster mice were immunized orally with four weekly doses of 100 pg recombinant urease plus adjuvant Control mice received adjuvant only. Groups of three mice each were necropsied at 3, 7, 14, or 21 days after the last immunization. Peyer's patches were removed from the intestines and lamina propria lymphocytes (LPL) were isolated by separation on a 40-70% Percoll gradient. IgA-positive B cells were detected by ELISPOT assays in 96-well filter plates coated with 1 pg/well recombinant urease and blocked with bovine serum albumin. Ten-fold serial dilutions of LPL were added to the wells, starting at 1 x 106 cells. IgApositive ASC were detected with a biotinylated anti-mouse IgA reagent followed by streptavidin-alkaline phosphatase, and positive cells were counted by microscopy.

Anti-urease IgA-positive ASC were found by ELISPOT *25 in intestinal lamina propria as early as three days after the last immunization, peaked at seven days, and diminished thereafter (Table Urease-specific ASC represented -10% of the total IgA-positive cells observed on immunohistochemistry. Two-color immunofluorescence 30 microscopy confirmed that urease-specific ASC were also IgA-positive. These observations confirm that an intense anti-urease IgA response occurs at the level of the intestinal mucosa after oral antigenic stimulation. The chronology and kinetics of this response are similar to those described for other oral vaccines 54 (Czerkinsky et al., Infect. Immun. 59:996-1001, 1991; McGhee and Kyono, Infect. Agents Hum. Dis. 2:55-73, 1993).

Table 8 Rinetics of induction of anti-urease IqA secreting cells after oral immunization with recombinant urease NUMBER OF ASC/10 6 LAMINA PROPRIA LYMPHOCYTES

IMMUNIZATION

DAY 3 a DAY 7 DAY 14 DAY 21 recombinant 17 c 7400 10 2 urease CTb PBS CT 1 200 0 0

S..

S.

S S

S

S

*5 a Day after the last oral immunization.

b Cholera toxin.

c Average value from duplicate wells containing intestinal lymphocytes from three separate mice.

To determine whether IgA-positive cells are recruited into the gastric mucosa, stomachs of orally immunized mice were examined by immunohistochemistry, as is described above. IgA-positive cells were virtually absent in gastric mucosa of immunized only and control 20 mice, indicating that the stomach is immunologically "silent" until stimulated by Helicobacter challenge.

The role of the stomach as an immunological effector organ in challenged, immunized mice was examined. Mice were given four weekly oral doses of 200 Ag recombinant urease with 10 gg CT. Control mice received CT only. One week after immunization, the mice were challenged. Mice were necropsied prior to challenge and at 7, 14, 28, 70, and 133 days after challenge.

Prior to challenge, no IgA-positive ASC were found. At all time intervals after challenge, IgApositive ASC were present in large numbers in the gastric 55 mucosae of immunized mice, with a peak at seven days (Fig. 14). The number of IgA-positive ASC greatly exceeded that in unimmunized (CT only) mice, especially 7-28 days after challenge. The anatomical localization of IgA-positive ASC also differed, with immunized mice having cells throughout the mucosa, in the lamina propria, and around the crypts, but rarely under the surface epithelium. Urease-specific and IgA-positive ASC revealed that the majority of urease-specific cells in gastric mucosa were IgA-positive.

These observations indicate that the gastric mucosa of animals primed by prior immunization becomes immunologically activated only after antigenic stimulation by H. felis. The resulting tissue response is characterized by rapid, intense, and long-lasting recruitment of IgA-positive B cells, many of which are urease specific. This response is quantitatively greater than in immunologically naive mice after challenge.

Moreover, the localization of IgA-positive cells in 20 immunized mice differs from that in immunologically naive mice that are challenged and become persistently infected with H. felis. The enhanced IgA-positive ASC response in gastric mucosa suggests a basis for the protection conferred by immunization against H. felis challenge.

These data are concordant with studies of cholera vaccine, in which immunological memory responses triggered within hours after bacterial challenge were sufficient to provide protection (Lycke and Holmgren, Scand. J. Immunol. 25:407-412, 1987).

.t S S S. S S. S

S

9r

S

It 00e.

t Correlation of the gastric immune response and bacterial load The relationship between the gastric tissue immune response and bacterial infection was defined at the structural level. Swiss-Webster mice were immunized with 56 4 weekly doses of 200 pg recombinant urease with 10 gg CT. One week after the last immunization, the mice were challenged with 107 H. felis. Animals were sacrificed 0, 1, 7, 14, 28, 70, and 133 days after challenge, and H. felis colonization was assessed by light and electron microscopy.

Within 24 hours after challenge, both immunized and unimmunized mice had substantial numbers of H. felis within the lumen of gastric pits (Fig. 15). Within seven days after challenge, the bacteria were cleared from the immunized mice, but were still present in high numbers in the gastric pits and the lumens of unimmunized mice.

Bacteria were also associated with the apical membrane of mucus-secreting cells of the unimmunized mice. The clearance of bacteria from the gastric mucosa of immunized mice corresponded to the appearance of IgApositive ASC and anti-urease ASC in the gastric tissue.

These results suggest the following sequence of events: 1) challenge of immunized mice results in transient colonization of gastric epithelium with H. felis; 2) unimmunized animals remain infected, while animals immunized with recombinant urease clear the bacteria from the stomach during the first week after challenge; and 3) clearance of infection is associated *25 with the recruitment of IgA-positive urease-specific ASC to gastric mucosa. A similar mechanism may be responsible for the clearance of bacteria from the gastric mucosa of chronically infected animals that are subjected to therapeutic immunization.

30 Antiqenic conservation of urease among strains of H. pylori The ability of various antisera to bind multiple clinical isolates of H. pylori was tested. The antisera included MPA3, a hyperimmune rabbit serum prepared 57 against purified H. pylori urease, and sera and secretions (gastric wick samples and saliva) from mice immunized with recombinant urease. Antibody preparations were tested by immunoblotting for recognition of the homologous H. pylori strain (Hp630), ATCC 43504 type strain, and five clinical isolates from ulcer patients at St. Bartholomew's Hospital, London, collected within the last five years.

All antisera recognized the UreA and UreB subunits of all H. pylori strains, as well as purified native and recombinant H. pylori ureases, native and recombinant H. felis urease, and native H. mustelae urease. In addition, urease-specific IgA antibodies in gastric secretions and saliva of immunized mice reacted with both UreA and UreB of all H. pylori strains, as well as heterologous ureases. Immunologic recognition was greater for UreB than for UreA. Sham-immunized mice showed no reactivity with any urease subunits. These results demonstrate that H. pylori strains express 20 ureases that are highly conserved at the antigenic level.

Thus, antigenic variation among H. pylori strains is not a significant factor for development of a recombinant urease vaccine.

Combination methods and compositions for treating Helicobacter infection Gastroduodenal infections, such as Helicobacter infection, may be treated by oral administration of a vaccine antigen H. pylori urease) and a mucosal S adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof. Examples of such compounds which may be administered with the vaccine antigen and the adjuvant are: antibiotics, including, macrolides, tetracyclines, P-lactams, 58 aminoglycosides, quinolones, penicillins, and derivatives thereof (specific examples of antibiotics that may be used in the invention include, amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin); antisecretory agents, including, H 2 -receptor antagonists cimetidine, ranitidine, famotidine, nizatidine, and roxatidine), proton pump inhibitors omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs misoprostil and enprostil), and anticholinergic agents pirenzepine, telenzepine, carbenoxolone, and proglumide); and bismuth salts, including colloidal bismuth subcitrate, tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and pepto-bismol (see, Goodwin et al., Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395, 1993; Physicians' Desk Reference, 4 9 t h edn., Medical Economics Data Production Company, Montvale, New Jersey, 1995). In 20 addition, compounds containing more than one of the above-listed components coupled together, e.g., ranitidine coupled to bismuth subcitrate, may be used.

Amounts of the above-listed compounds used in the methods and compositions of the invention may readily be determined by one skilled in the art. In addition, one skilled the art may readily design treatment/immunization schedules. For example, the non-vaccine components may be administered on days 1-14, and the vaccine antigen adjuvant may be administered on days 7, 14, 21, and 28.

In addition to urease, other Helicobacter antigens may be used in the methods and compositions of the invention involving combination therapy. For example, Helicobacter heat shock proteins A or B (HspA or HspB; WO 94/26901), Helicobacter adhesion lipoprotein A (AlpA; The nucleotide sequence encoding AlpA is set forth in SEQ ID 59 NO:6 and the corresponding amino acid sequence of AlpA is in SEQ ID NO:7), Helicobacter H. pylori) clpB (clpB is encoded by the insert in the plasmid in bacterial strain XLOLR HP CP6, which was deposited with the National Collections of Industrial Marine Bacteria in Aberdeen, Scotland, and designated with NCIMB accession number 40748), or the 16-19 kD Helicobacter antigen recognized by monoclonal antibody IgG 50 (IgG may be obtained from hybridoma cell line #50-G 6

-B

7 which was deposited with the ATCC and designated with ATCC accession number HB-11952) may be used. In addition, Helicobacter surface-exposed or secreted antigens identified using the transposon shuttle mutagenesis method of Haas et al. may be used as vaccine antigens in the invention (Haas et al., Gene 130:23-31, 1993; Haas et al., Abstract from the VI th Workshop on Gastroduodenal Pathology and Helicobacter pylori, Brussels,. September 21-25, 1993; Odenbreit et al., Gut, An International Journal of Gastroenterology and Hepatology, Abstract from 20 the VIII th International Workshop on Gastro-duodenal Pathology and Helicobacter pylori, Edinburgh, Scotland, July 7-9, 1995). In this method, cloned H. pylori genes are mutagenized by transposon insertion mutagenesis in Escherichia coli. The inactivated genes are reintroduced into H. pylori by DNA transformation, and allelic replacement results in the generation of corresponding H.

pylori mutants. The transposon used in one variation of this method carries blaM, which is a 5'-truncated version e of the f-lactamase gene. Use of this transposoTn 30 facilitates tagging of genes encoding leader peptidecontaining exported proteins, and thus direct selection of clones containing mutants of exported proteins cell surface proteins such as adhesins and cytotoxins, as well as other potential cell surface virulence factors), which are strong candidates for vaccine antigens. In 60 addition to Helicobacter antigens, antigens from non- Helicobacter gastroduodenal pathogens may be used in methods and compositions for preventing and/or treating infection caused by their respective pathogens.

Polypeptide fragments containing protective and/or therapeutic epitopes of the above-listed antigens may also be used in the invention.

Adjuvants which may be used in the methods and compositions of the invention include mucosal adjuvants, the heat-labile enterotoxin of E. Coli a cholera toxin, a C. difficile toxin, or derivatives fragments, mutants, or toxoids having adjuvant activity), or combinations thereof. For example, a fragment containing the 794 carboxyl-terminal amino acids of C. difficile Toxin A (see, Dove et al., supra, for the sequence of C. difficile Toxin A) may be used.

The amounts of.vaccine antigen and adjuvant which may be used in these compositions and methods of the invention are described above.

20 The efficacy of a composition containing a vaccine pylori urease LT), an antibiotic (amoxicillin), and .i a bismuth salt (pepto-bismol) in the treatment of Helicobacter infection is illustrated below.

Combination therapy for Helicobacter infection of mice 25 One month after infection with -107 H. felis, mice were treated with amoxicillin (1.5 mg/30 g mouse) and/or pepto-bismol (0.2 mg/30 g mouse) once per day for 14 days (days 1-14), and/or a vaccine composition containing 100 pg recombinant H. pylori urease 5 Ag LT on days 7, 30 14, 21, and 28 (see Table 9 for an illustration of the combinations used). Helicobacter infection in these mice was measured by quantitative gastric urease assay (see above) 1 week and 4 weeks after termination of the treatment (day 28). As is illustrated in Table 9, the 61 most effective treatment regimen, achieving 100% clearance, involved administration of a vaccine (urease LT), an antibiotic (amoxicillin), and a bismuth salt (pepto-bismol).

Table 9 Mice Cleared of H. fells Infection treatment 1 week 4 weeks amoxicillin 60% pepto-bismol 0% 0% amoxicillin pepto-bismol 80% vaccine 70% vaccine amoxicillin 89% 56% vaccine pepto-bismol vaccine amoxicillin pepto-bismol 100% 100% (vaccine urease adjuvant) Identification of human patients for administration of recombinant H. pylori urease vaccine The recombinant H. pylori urease vaccine of the invention may be administered to uninfected individuals as a prophylactic measure or to individuals infected with Helicobacter as an antibacterial therapy. Individuals S 25 selected for prophylactic administration of recombinant urease include any individual at risk of Helicobacter infection as based upon age, geographical location, or the presence of a condition which renders the individual susceptible to Helicobacter infection. Individuals at particularly high risk of infection, or who would be most severely affected by infection, include individuals in 62 developing countries, infants and children in developing and in developed countries, individuals with naturally or artificially low gastric acid pH, submarine crews, and military personnel.

Individuals who may receive the recombinant H. pylori urease vaccine as a therapeutic include those individuals with symptoms of gastritis or other gastrointestinal disorders which may be associated with H. pylori infection. The clinical symptoms associated with gastritis, an inflammation of the stomach mucosa, include a broad range of poorly-defined, and generally inadequately treated, symptoms such as indigestion, "heart burn," dyspepsia, and excessive eructation. A general discussion of gastritis appears in Sleisenger and Fordtran, In Gastrointestinal Disease, 4th Edn., Saunders Publishing Co., Philadelphia, PA, pp. 772-902, 1989.

Individuals who have a gastrointestinal disorder may also be treated by administration of the vaccine of the invention. Gastrointestinal disorders includes any 20 disease or other disorder of the gastrointestinal tract of a human or other mammal. Gastrointestinal disorders include, for example, disorders not manifested by the presence of ulcerations in the gastric mucosa (nonulcerative gastrointestinal disorder), including chronic 25 or atrophic gastritis, gastroenteritis, non-ulcer dyspepsia, esophageal reflux disease, gastric motility disorders, and peptic ulcerdisease gastric and duodenal ulcers). Peptic ulcers involve ulceration and formation of lesions of the mucous membrane of the 30 esophagus, stomach, or duodenum, and is generally S" characterized by loss of tissue due to the action of digestive acids, pepsin, or other factors.

Alternatively, it may be desirable to administer the vaccine to asymptomatic individuals, particularly where the individual may have been exposed to H. pylori or has 63 a condition rendering the individual susceptible to infection.

Infection with Helicobacter can be readily diagnosed by a variety of methods well known in the art, including, by serology, 13 C breath test, and/or gastroscopic examination.

Preparation of purified recombinant urease for administration to patients The process for formulation of recombinant urease involves combining urease with a stabilizer, a carbohydrate mannitol) and freeze drying lyophilizing) the product. This process prevents degradation by aggregation and fragmentation. In addition, the product is stable for months following lyophilization. The process which leads to instability involves formation of disulfide bonds between protein subunits, and is effectively inhibited by lyophilization.

Recombinant urease is freeze-dried following the final purification step. The purified protein product 20 (approximately 4 mg/ml) is dialyzed against 2% sucrose, and this solution is transferred to lyophilization vials.

The vialed solution is either: frozen in liquid nitrogen, and then placed into the lyophilizer, or (2) cooled to 4 0 C, and then placed in the lyophilizer, where it is frozen to -40 0 C, or lower. Lyophilization is carried out using standard methods. The freeze-dried product may be reconstituted in water.

S" Mode of administration to human patients Recombinant H. pylori urease is administered to a mucosal surface of the individual in order to stimulate a mucosal immune response effective to provide protection to subsequent exposure to Helicobacter and/or facilitate 64 clearance of a pre-existing Helicobacter infection.

Preferably, recombinant urease is administered so as to elicit a mucosal immune response associated with production of anti-urease IgA antibodies and/or infiltration of lymphocytes into the gastric mucosa. The recombinant urease may be administered to any mucosal surface of the patient. Preferable mucosal surfaces are intranasal, oral, and rectal by use of an anal suppository). In addition to being administered to a single mucosal surface, the vaccine of the invention may be administered to combinations of mucosal surfaces oral+rectal, oral+intranasal, or rectal+intranasal). In the case of oral administration, it is preferable that the administration involves ingestion of the vaccine, but the vaccine may also be administered as a mouth wash, so that an immune response is stimulated in the mucosal surface of the oral cavity, without actual ingestion of the vaccine. Alternatively, a systemic mucosal immune response may be achieved by 20 administration of the vaccine to a mucosal surface of the eye in the form of, an eye drop or an intraocular implant.

Dosages of recombinant H. pylori urease administered to the individual as either a prophylactic 25 therapy or an antibacterial therapy can be determined by Sone skilled in the art. Generally, dosages will contain between about 10 Ag to 1,000 mg, for example, between about 10 mg and 500 mg, 30 mg and 120 mg, 40 mg and mg, or 60 mg recombinant H. pylori urease.

At least one dose of the recombinant H. pylori urease will be administered to the patient, for example, at least two doses, at least four doses, or with up to six or more total doses administered. It may be desirable to administer booster doses of the recombinant urease at one or two week intervals after the last 65 immunization, generally one booster dose containing less than, or the same amount of, recombinant H. pylori urease as the initial dose administered. For example, the vaccine regimen may be administered in four doses at one week intervals. The priming and booster doses may be administered to the same or different mucosal surfaces.

In the case of different mucosal surfaces, for example, an oral priming dose may be followed by intranasal or rectal boosters, an intranasal priming dose may be followed by oral or rectal boosters, or a rectal priming dose may be followed by oral or intranasal boosters.

Recombinant H. pylori urease may be coadministered with a mucosal adjuvant. The mucosal adjuvant may be any mucosal adjuvant known in the art which is appropriate for human use. For example, the mucosal adjuvant may be cholera toxin (CT), enterotoxigenic E. coli heat-labile toxin or a derivative, subunit, or fragment of CT or LT which retains adjuvanticity. The mucosal adjuvant is co- 20 administered with recombinant H. pylori urease in an amount effective to elicit or enhance a mucosal immune response, particularly a humoral and/or a mucosal immune response. The ratio of adjuvant to recombinant urease may be determined by standard methods by one skilled in the art. For example, the adjuvant may be present at a ratio of 1 part adjuvant to 10 parts recombinant urease.

A buffer may be administered prior to administration of recombinant H. pylori urease in order *.io to neutralize or increase the pH of the gastric acid.

30 Any buffer that is effective in raising the pH of gastric acid and is appropriate for human use may be used. For example, buffers such as sodium bicarbonate, potassium bicarbonate, and sodium phosphate may be used. In the case of oral administration, the vaccine may be buffer- 66 free, no amount of a pH-raising buffer compound effective to significantly affect gastric acid pH is administered to the patient either prior to, or concomitant with, administration of the vaccine recombinant urease.

The vaccine formulation containing recombinant urease may contain a variety of other components, including stabilizers, flavor enhancers sugar), or, where the vaccine is administered as an antibacterial therapeutic, other compounds effective in facilitating clearance and/or eradication of the infecting bacteria.

For prophylactic therapy, the vaccine containing recombinant H. pylori urease may be administered at any time prior to contact with, or establishment of, Helicobacter infection. Because the vaccine can also act as an antibacterial therapy, there is no contraindication for administration of the vaccine if there is marginal evidence or suspicion of a pre-existing Helicobacter v* infection an asymptomatic infection).

20 For use of the vaccine as an antibacterial therapy, recombinant H. pylori urease may be administered .at any time before, during, or after the onset of ia symptoms associated with Helicobacter infection or with gastritis, peptic ulcers or other gastrointestinal 25 disorder. Although it is not a prerequisite to the initiation of therapy, one may confirm diagnosis of Helicobacter infection by, a 13 C breath test, serology, gastroscopy, biopsy, or another Helicobacter detection method known in the art. The progress-of immunized patients may be followed by general medical evaluation, screening for H. pylori infection by serology, 13 C breath test, and/or gastroscopic examination.

67 Example of administration of recombinant H. pylori urease to a human A vaccine composed of 60 mg of recombinant H.

pylori urease in a total volume of 15 ml of water containing 2% weight/volume sucrose, pH 7.5 is orally administered to the patient. Administration of the vaccine is repeated at weekly intervals for a total of 4 doses. Symptoms are recorded daily by the patient. To determine adverse effects, physician interviews are performed weekly during the period of vaccine administration, as well as 1 week and 1 month after the last immunization. Anti-urease'antibodies are measured in serum and saliva, and antibody-secreting cells are monitored in peripheral blood collected 7 days after the last immunization.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The entire disclosure in the complete specification of our Australian Patent Application No. 55764/96 is by this cross-reference incorporated into the present specification.

*oo 68 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: OraVax, Inc.

(ii) TITLE OF INVENTION: MULTIMERIC, RECOMBINANT UREASE (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Fish Richardson P.C.

STREET: 225 Franklin Street CITY: Boston STATE: MA COUNTRY: USA ZIP: 02110-2804 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US96/----- FILING DATE: 23-APR-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/431,041 FILING DATE: 28-APR-1995

CLASSIFICATION

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/568,122 FILING DATE: 06-DEC-1995

CLASSIFICATION

(viii) ATTORNEY/AGENT INFORMATION: NAME: Clark, Paul T.

REGISTRATION NUMBER: 30,162 REFERENCE/DOCKET NUMBER: 06132/020001 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 542-5070 TELEFAX: (617) 542-8906 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2735 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

VACCINE

.:0 0: TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA TTCATCCACC TTGATTGCGT TATGTCTTCA AGGAAAAACA CTTTAAGAAT AGGAGAATGA GATGAAACTC ACCCCAAAAG 120 69

AGTTAGATAA

GCATTAAGCT

CGAGAGCTGG

CAGATGATGT

TTCCTGATGG

TTCCTGGTGA

TTAGCGTGAA

TCTTTGAAGT

ACATTGCGAG

TTGACATTGG

ACAACGAAAG

AAAGCGATGA

AAGAATATGT

?ACTTGATCGC

GCGGTAPIAAC

ATTTAATTAT

GTATTAAAGA

GCGTTAAAAA

TCG;TAACGGC

CAGCTTTTGC

CTAATGCGAC

AAGAATATTC

TAGCCGATCA

CTCCTTCTGC

TCCACACAGA

GACGCACTAT

TTAAAGTAGC

CCGTGAATAC

GCATTAAAGA

PAAGACACTTT

GCCGTGTGGG

GTTGATGCTC

TAACTATGTA

TAAAAAGACT

GATGGATGGC

GACTAAACTC

GTTGTTCTTA

AGTTAAAAAT

GAATAGATGC

CGGGACAGCG

CGGTAACAGA

CAAAAAAATT

CAACTATGTA

TTCTATGTAT

TGAAGTAGAA

CCTAAGAGAA

CACTAACGCT

TGGCAAAATC

CAATCTTAGC

TGGTGGTATT

AAGCGGtGTA

TACTATCACT

TATGAATTTA

AATTGAAGCC

AATCAATCAT

CACTTTGAAT

GCACACTTTC

CGGTGAACAC

AGAAGCAGAG

AGATGTTCAG

GCATGACATG

TGAAGTTATC

CACTACGCTG

GAAGCAGTAG

GCGGCTGAAT

GTGGCAAGCA

GTAACCGTGC

AAAAATGAAG

GTTGGCGACA

CTAGACTTTG

GTAAGATTTG

AGAATCTTTG

GCTTTACACA

AAAACAATTA

GGTCCTACTA

CATGACTACA

GGCATGAGCC

TTAATCGTGG

GCTGGCATTG

GTAGGTCCTG

GACACACACA

ACAACCATGA

CCAGGCAGAA

GGTTTCTTGG

GGTGCGATTG

GCGTTAGATG

GAAGCCGGTT

CACACTGAAG

AACATTCTTC

CACATGGACA

TTCGCTGATT

GGGATTTTCT

ACTAGAACTT

GAGAATTGOC

CTTTGATTAG

TGATGCAAGA

TGATCCATGA

ATACCCCTAT

ACATCACTAT

GACCGGTTCA

ACAGAGAAAA

AGCCTGGCGA

GATTTAACGC

GAGCTAAAGA

AGGAGTAAGA

CAGGCGATAA

CCATTTATGG

AATCTAACAA

ATTACACCGG

GTAAAGGCGG

CTACTGAAGC

TCCACTTCAT

TTGGTGGTGG

GAAATTTAAA

CTAA.AGGTAA

GCTTTAAAAT

TTGCGGACAA

GTGTAGAAGA

GCGCTGGCGG

CCGCTTCCAC

TGCTTATGGT

CAAGGATCCG

CAATCACCAG

GGCAAACAGC

TAAAAAACGC

TGCCCATATT

AGGGCGCACT

AGTGGGTATT

TGAGGCCAAT

CAACGAAGGC

AATCGGCTCA

AACTTTCGGT

AGAAAAATCC

ATTGGTTGAT

GCGTGGTTTT

AATGAAAAAG

AGTGAGATTG

CGAAGAGCTT

CCCTAGCAAA

TATTTATAAA

TAACAAAGAC

CTTAGCCGGT

TTCACCCCAA

AACCGGTCCT

ATGGATGCTC

CGCTTCTAAC

TCACGAAGAC

ATACGATGTG

CACTATGGCT

CGGACACGCT

TAACCCCACC

GTGCCACCAC

CCCTCAAACC

TTCTGACTCT

TGACAAAAAC

AAAGAAAAAG

ATGGAAGAAG

CTTTTAAAAC

GAAGCGATGT

GGTAAATTAG

AAAAAAGCCG

CACTTCCATT

AAACGCTTAG

GTAGAATTGA

AGACAAGCAG

CATGGCGCTA

ATTAGCAGAA

GGCGATACAG

AAATTCGGTG

GAAGAGTTGG

GCGGATATTG

ATGCAAGATG

GAAGGTTTGA

CAAATCCCTA

GCTGATGGCA

AGAGCGGCTG

GATGCGAGCT

TGGGGCACCA

CAAGTCGCTA

GCTATTGCTG

CCTGATaTTA

ATCCCTTTCA

TTGGATAAAA

ATTGCGGCTG

CAAGCGATGG

AAGAAAGAAT

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 1 0 TTGGCCGCTT GAAAGAAGAA AAAGGCGATA ACGACAACTT CAGGATCAAA CGCTACTTCT 70 CTAAATACAC CATTAACCCA GCGATCGCTC ATGGGATTAG CGAGTATGTA GGTTCAGTAG

AAGTGGGCAA

ACATGATCAT

TCCCTACCCC

ACGATGCAAA

TAGGACTTGA

TGCAATTCAA

TGGATGGCAA

GCATTTTCTA

ACAAGCTTGC

AAGCCCGAAA

TTGGGGCCTC

AGTGGCTGAC

CAAAGGCGGA

ACAACCGGTT

CATCACTTTT

AAGACAAGTG

CGACACTACT

AGAAGTAACT

GGATTTTTTA

GGCCGCACTC

GGAAGCTGAG

TAAACGGGTC

TTGGTATTGT

TTCATTGCGT

TATTACAGAG

GTGTCTCAAG

TTGCCGGTAA

GCTCACATTG

TCTAAACCAG

GGAGCAACGC

GAGCACCACC

TTGGCTGCTG

TTGAGGGGTT

GGAGTCCAGC

TAAGCCAAAT

AAATGTTCGC

CGGCTTATGA

AAAATTGCAG

AAGTCAATCC

CCAATAAAGT

TTCCTTAAAT

ACCACCACCA

CCACCGCTGA

TTTTG

ATTCTTTGGC

GGGCGATGCG

TCATCATGGT

CAAAGGCATT

AAATATCACT

TGAAACTTAC

GAGCTTGGCG

CCTGAATTCG

CTGAGATCCG

GCAATAACTA

GTGAAACCCA

AACGCTTCTA

AAAGCTAAAT

AAAGAAGAAT

AAAAAAGACA

CATGTGTTCG

CAACTCTTTA

AGCTCCGTCG

GCTGCTAACA

GCATAACCCC

2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2735 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHAR.ACTERISTICS: LENGTH: 238 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Leu Thr Pro Lys Glu Leu Asp Lys Lys Leu Met Leu His Tyr Ala Gly Glu Leu Val Glu Ala Ala Gly Lys Ala Lys Lys Arg Lys Glu 25 Ala Gly Ile. Lys Val Ala Leu Ile Hie Ile Met Glu Gly Leu Asn Tyr Glu Ala Arg Arg Thr Leu Lys Thr Ala Leu Lys Ala 55 Met Leu Met Gln Glu Pro Asp Asp Val Val 70 Met Asp Gly Val Ala 75 Thr Gly Ile Glu Ala Phe Pro Asp Gly 90 Leu Ser Met Ile His Glu Lys Leu Val Thr Val Pro Glv Glu Leu Phe His Thr Pro Leu Lys Asn 115 I le 100 Glu Glu Ala Asn Gly Lys 105 Asn Val 110 Ala Val Ser Asp Ile Thr I le 120 Glu Gly Lys Lys 125 Val Lye 130 Val Lys Asn Val Gly Asp Arg Pro Val 135 Gln Ile Gly Ser His 140 71 Phe His Phe Phe Glu Val Asix Arg Cys Leu 145 150 Thr Phe Gly Lys Arg Leu Asp Ile Ala Ser 165 170 Glu Pro Gly Glu Glu Lys Ser Val Glu Leu 180 185 Arg Arg Ile Phe Gly Phe Aen Ala Leu Val 195 200 Glu Ser Lys Lys Ile Ala Leu His Arg Ala 210 215 Gly Ala Lys Ser Asp Asp Aen Tyr Val Lys 225 230 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 566 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Asp 155 Gly Ile Asp Lys Thr 235 Phe Thr Asp Arg Glu 220 Ile Arg Val Gly 190 Ala Gly Glu Glu Lye 160 Arg Phe 175 Gly Aen Asp hen Phe His Pro Thr Met Lys Lye Ile Ser Arg Lys Glu Tyr Val Ser Met Tyr Gly a Thr Glu Lys Glu Ile Gly Ser Thr Ile 145 Gly His Thr Leu Tyr Lys Vai Ala 130 Pro Asp Lys Asp -Tyr Leu Arg Asp Leu Lye Ala Gly Giy 100 Gly Pro 115 Gly Gly Thr Ala Val Thr Glu Ile Asp Aen Ala Ile Phe Arg Ile Gly Ile 70 Ile Lye Thr Asp Ala 150 Leu Tyr Met 55 Thr Gly Asp Glu Thr 135 Ser Gly Gly 40 Ser Aen Ile Met Ala 120 His Gly Asp 25 Glu Gin Ala Lye Gin 105 Leu Ile Val Thr Glu Ser Leu Asp 90 Asp Ala His Thr Asp Lau Aen Ile 75 Gly Giy Gly Phe Thr 155 Lau Lys Asn Val Lye Val Glu Ile 140 Met Ile Phe Pro Asp Ile Lye Gly 125 Ser Ile Ala Glu Gly Gly Ser Lye Tyr Thr Ala Gly Asn Asn 110 Leu__Ile Pro Gin Gly Gly Val Gly Glu Gly Ile Lau Val Gin Gly 160 Thr Gly Pro Ala Asp Gly Thr Aen Ala Thr 165 170 Thr Ile Thr Pro Gly Arg 175 72 Arg Asn Leu Gly Asp Gin 210 Gly Thr 225 Tyr Asp Cys Val Phe His Val Ala 290 Pro Phe 305 Cys His Ser Arg Met Gly *Val Gly 370 Glu 385 Arg Ile His Gly Asp Leu Ile Ile 450 *Ala Ser S *465 His His Leu Phe 195 Ile Thr Val Giu Thr 275 Gly Thr His Ile Ile 355 Giu Phe Lys Ile Val 435 Lys Ile Gly Lys 180 Leu Glu Pro Gin Asp 260 Glu Giu Val Leu Arg 340 Phe Val Giy Arg Ser 420 Leu Giy Pro Lys Asp 500 Trp, Met Leu Arg Ala Ala Giu Giu Tyr Ser Met Asn Ala Ala Ser Vai 245 Thr Gly His Asn Asp 325 Pro Ser Ile Arg Tyr 405 Glu Trp Gly Thr Ala 485 Lys Giy Ala 230 Ala Met Ala Asn Thr 310 Lys Gin Ile Thr Leu 390 Leu Tyr Ser Phe Pro 470 Lys Gly Ala 215 Ile Ile Ala Gly Ile 295 Giu Ser Thr Thr Arg 375 Lye Ser Val Pro Ile 455 Gin Tyr Asn 200 Ile Asn His Ala Gly 280 Leu Ala Ile Ile Ser 360 Thr Giu Lye Gly Ala 440 Ala Pro Asp 185 Ala Gly His Thr Ile 265 Gly Pro Giu Lye Ala 345 Ser Trp Glu Tyr Ser 425 Phe Leu Val Ala Giu 505 Ser Phe Ala Asp 250 Ala His Ala His Glu 330 Ala Asp Gin Lye Thr 410 Val Phe Ser Tyr Asn 490 Asn Lye Leu 235' Thr Gly Ala Ser Met 315 Asp Glu Ser Thr Gly 395 Ile Glu Gly Gin Tyr 475 Ile Asp Ile 220 Asp Leu Arg Pro Thr 300 Asp Val Asp Gin Ala 380 Asp Asn Val Val Met 460 Arg Thr Ala 205 His Val Asn Thr Asp 285 Aen Met Gin Thr Ala 365 Asp Asn Pro Giy Lye 445 Gly Glu Phe 190 Ser Giu Ala Glu Met 270 Ile Pro Leu Phe Leu 350 Met Lys Asp Ala Lye 430 Pro Asp Met Val1 Leu Asp ABp Ala 255 His Ile Thr Met Ala 335 His Gly .Asn Asn Ile 415 Val Aen Ala Phe Ser 495 Ala Trp Lye 240 Gly Thr Lye Ile Val 320 Asp Asp Arg Lye Phe 400 Ala Ala Met Asn Ala 480 Gin Ala Ala Tyr Lye Gly Ile Lys Glu Leu Gly Leu Glu Arg Gln 73 Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gin 515 520 525 Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Pro Glu Thr Tyr His 530 535 540 Val Phe Val Asp Gly Lys Glu Val Thr Ser Lys Pro Ala Asn Lys Val 545 550 555 560 Ser Leu Ala Gin Leu Phe 565 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CGGGATCCAC CTTGATTGCG TTATGTCT 28 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CGGAATTCAG GATTTAAGGA AGCGTTG 27 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 1557 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGATAAAAA AGAATAGAAC GCTGTTTCTT AGTCTAGCCC TTTGCGCTAG CATAAGTTAT S GCCGAAGATG ATGGAGGGTT TTTCACCGTC GGTTATCAGC TCGGGCAAGT CATGCAAGAT 120 S* GTCCAAAACC CAGGCGGCGC TAAAAGCGAC GAACTCGCCA GAGAGCTTAA CGCTGATGTA 180 ACGAACAACA TTTTAAACAA CAACACCGGA GGCAACATCG CAGGGGCGTT GAGTAACGCT 240 TTCTCCCAAT ACCTTTATTC GCTTTTAGGG GCTTACCCCA CAAAACTCAA TGGTAGCGAT 300 GTGTCTGCGA ACGCTCTTTT AAGTGGTGCG GTAGGCTCTG GGACTTGTGC GGCTGCAGGG 360 74

ACGGCTGGTG

CTCCCTAGCT

AACACCAATT

TTTAATGCGA

AGTGGTGCGA

GGCCAACAAA

GAAGCTTTCA

TTTACAGGTT

AAAAACACCA

GCTGTGCAAG

GATAAAATCA

GCCGGGAACA

CAATTCTTCG

GGAGCGAGCG

GGGACTGATG

GGCTTTTTTA

CCCAATGTGA

GGTATGAGGA

GTGGAATTTG

AC!CGTGA.AGT

GCACTTCTCT

TGACTGACAG

TCCCCAACAT

TGAATAAGGc

CTGGTTCAGA

ATATCTTAAA

ACTCTGCCGT

TGGTGCA.AGG

TTAGCGGGAG

GGCGCGCTAG

ATGCGCTCAA.

GCCGTTCA.AC

GGAAGAAAAG

TGGGCTTTAG

TGTTGTATAA

GCGGTATCCA

AATTGCATGG

TGAACTTCGG

GCGTAGTGGT

ATTTCCGTCC

TAACACTCAA

GATTTTAAGC

GCAACAACAG

TTTAGAGAAT

TGGTCAAACT

TAACGCAGCG

AGCCGCCAAC

CATTATTGAT

TGCGGTTATT

TCAGCTCCCT

TAATCAAGTG

GAATATTTTA

GAATATCGGT

ATCCACTCAA.

CATCTTTAGC

ATTAGCCGGT

GAAAATCAAT

TAAGTTGGAC

GCCTACGATT

TTATAGCGTT

AGCACTTGCA

ACGATCGGCA

CTCACCTACT

AAGAATGGAA

TACTCCACAC

AACTTGCTCA

ATTGGGAATA

CAATCTCAAG

AGCGCTGGGA

AACGCTCTTT

AGAAGCATGC

AACGGGTTTT

TTGCGCTATT

AATAATGTAG,

CGCTCCTATC

GAGACCTTCC

AACACGCACT

GGGAAATCCA

TATAACACTT

TATTGGTCTT

CCGTTGCGGG

GCCAGACTAA

TGAATGCGGG

CTAGTAGTGC

AAGCTATCCA

AGCAAGATGA

AGGAATTCAA

CGGTTTATAA

TAAACTCCAA

ATAACGCGCA

CTTACTTGCC

ACACCAAAAT

ATGGTTTCTT

GGTTATACAC

AAAACCGCTC

AATCCACGCT

TCCAGTTCC!T

ACCGCCACAA

ATTACAAATC

ATGGGTATTC

CTATTACTGG

CTACGGCACG

GAATGTGTTT

TAGTGGAACT

ATACCTTCAA

ATTGCTCTTA

TTCAGCCGCT

CGAGCTCACT

CCAAGCTAAC

AGTAACTTTG

CCAATTCAGA

AGGCTATAAG

TTCTTATAAc

TTATGGGGTG

TGTGGATATG

CAGAGATGAC

CTTTGACTTC

CCAGCACACG

AGCAGGGACT

ATTCTAA

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1557 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 5.18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Ile Lys Lys Asn Arg Thr Leu Phe Leu Ser Leu Ala Le u Cys Ala 1 5 10 Ser Ile Ser Tyr Ala Glu Asp Asp Gly Gly Phe Phe Thr Val Gly Tyr 20 25 Gin Leu Gly Gin Val Met Gin Asp Val Gin Asn Pro Gly Gly Ala Lys 40 Ser Asp Glu Leu Ala Arg Glu Leu Asn Ala Asp Val Thr Asn Asn Ile 55 Leu Asn Ann Asn Thr Gly Gly An 70 Phe Asn Ser Thr Thr 145 An Gly Gly Gin Ile 225 Giu Asn Gin Val Arg 305 Asp Pro Phe Ile Ser Giy Giy Gin 130 Asp Thr An Thr Thr 210 Leu Ala Ser Ala Ile 290 Ala Lys Gin Tyr Giy 370 Gin Ser Thr 115 Ser Arg An Vai Ser 195 Tyr An Phe Ala Val 275 Ser Ser Ile Phe Thr 355 Leu Tyr Asp 100 Cys Thr Ile Phe Phe 180 Ser Ser Asn An Ala 260 Tyr Ala Gin An Arg 340 Lys Arg Leu Val Ala Cys Leu Pro 165 Phe Ala Thr Ala Ser 245 Phe An Gly Leu Ala 325 Ala Ile Tyr Tyr Ser Al a Thr Ser 150 An An Ser Gin Ala 230 Ala Thr Giu Ile Pro 310 Leu Gly Gly Tyr Ser Ala Ala Val 135 Thr Met Ala Gly Ala 215 An Val Gly Leu An 295 An An An Tyr Gly 375 Leu An Gly 120 Ala Ile Gin Met Thr 200 Ile Leu Ala Leu Thr 280 Ser Ala Asn Ser Lys 360 Phe Ile Ala Leu Gly 90 Ala Leu 105 Thr Ala Gly Tyr Gly Ser Gin Gin 170 Asn Lys 185 Ser Gly Gin Tyr Leu Lys Ala An 250 Val Gin 265 Lys An Ann Gin Leu Tyr Gin Val 330 Arg Ser 345 Gin Phe Phe Ser Gly 75 Ala Leu Gly Tyr Gin 155 Leu Aia Ala Leu Gin 235 Ile Gly Thr Ala An 315 Arg Thr Phe Tyr Ala Tyr Ser Gly Trp 140 Thr Thr Leu Thr Gin 220 Asp Gly Ile Ile An 300 Ala Ser An Gly An 380 Leu Pro Gly Thr 125 Leu An Tyr Giu Gly 205 Gly Glu An Ile Ser 285 Ala Gin Met Ile Lys 365 Gly Ser Thr Ala 110 Ser Pro Tyr Leu An 190 Ser Gin Leu Lys Asp 270 Gly Val Val Pro Leu 350 Lys Ala An Lys Val

LOU

Ser Gly An 175 Lys Asp Gin Leu Giu 255 Gin Ser Gin Thr Tyr 335 An Arg Ser Ala Leu Gly An Leu Thr 160 Aia An Gly An Leu 240 Phe Ser Ala Gly Leu 320 Leu Zly An Val Giy Phe Arg Ser Thr 385 Gin Ann Ann Val Giy 390 Leu Tyr Thr Tyr Gly Val 395 400 Gly Ser Phe Ile Aen 465 Val.

Ser Ser Thr Val.

Gin Aen 450 Phe Glu Ala Tyr Asp Asp Ser 435 hen Gly Phe Gly Gly 515 Leu 405 Gly Leu His Leu Val 485 Thr Ser Tyr Phe Arg Phe Asp 470 Val Val.

Phe Ile Ser Asp 440 Phe Lye Pro Tyr 76 ie Ser 410 .y Ile ,u Phe .r hen ir Ile 490 ie Arg Arg Gin Val.

Asp Arg 475 Tyr Pro Ser Leu Lys Phe 460 His hen Tyr Tyr Ala Leu 445 Gly hen Thr Ser Gin Gly 430 His Met Gin Tyr Vai 510 hen 415 Giu Giy Arg His Tyr 495 Tyr

Claims (64)

1. A vaccine for inducing a mucosal immune response to Helicobacter in a patient, said vaccine comprising: multimeric complexes of recombinant, enzymatically inactive Helicobacter urease; and a pharmaceutically acceptable carrier or diluent.

2. The vaccine of claim 1, comprising a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A subunits and four Urease B subunits, or a mixture thereof.

3. The vaccine of claim 1, comprising a multimeric complex comprising eight Urease A subunits and 20 eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a S. multimeric complex comprising four Urease A subunits and four Urease B subunits.

4. The vaccine of claim 1, further comprising a mucosal adjuvant. The vaccine of claim 1, wherein said mucosal adjuvant is the heat-labile enterotoxin of enterotoxigenic 30 Escherichia coli, a cholera toxin, a Clostridium difficile toxin, a subunits or derivative thereof lacking toxicity but still having adjuvant activity, or a mixture thereof.

6. The vaccine of claim 4, wherein said mucosal adjuvant is the heat-labile enterotoxin of enterotoxigenic Escherichia coli, or a subunit or derivative thereof lacking toxicity but retaining adjuvant activity. \\melb-files\homeS\cintae\Keep\speci\div 55764.96.doc 19/06/00 78

7. The vaccine of claim 4, wherein said mucosal adjuvant is a cholera toxin, or a subunit or derivative thereof lacking toxicity but retaining adjuvant activity.

8. The vaccine of claim 4, wherein said mucosal adjuvant is a Clostridium difficile toxin, or a subunit or derivative thereof lacking toxicity but retaining adjuvant activity.

9. The vaccine of claim 8, wherein said mucosal adjuvant comprises the carbohydrate binding domain of Clostridium difficile Toxin A.

10. The vaccine of claim 1, wherein said multimeric complexes of recombinant, enzymatically inactive Helicobacter urease have been freeze-dried.

11. The vaccine of claim 1, wherein said 20 Helicobacter urease is Helicobacter pylori urease.

12. A composition for treating a gastroduodenal infection in a patient, said composition comprising an antigen from a gastroduodenal pathogen, and an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.

13. The composition of claim 12, wherein said gastroduodenal pathogen is a Helicobacter.

14. The composition of claim 13, wherein said Helicobacter is Helicobacter pylori. The composition of claim 13, wherein said antigen is a urease.

16. The composition of claim 13, wherein said \\melb_files\home$\cintae\Keep\speci\div 55764.96.doc 19/06/00 79 antigen comprises multimeric complexes of recombinant, enzymatically inactive Helicobacter urease.

17. The composition of claim 13, comprising a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A subunits and four Urease B subunits, or a mixture thereof.

18. The composition of claim 13, comprising a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A subunits and four Urease B subunits.

19. The composition of claim 12, wherein said Helicobacter infection is Helicobacter pylori infection.

20. The composition of claim 12, further comprising a mucosal adjuvant.

21. The composition of claim 20, wherein said 25 mucosal adjuvant is the heat-labile enterotoxin of enterotoxigenic Escherichia coli, a cholera toxin, a Clostridium difficile toxin, a subunit or derivative S* thereof lacking toxicity but still having adjuvant activity, or a mixture thereof.

22. The composition of claim 20, wherein said mucosal adjuvant is the heat-labile enterotoxin of enterotoxigenic Escherichia coli, or a subunit or derivative thereof lacking toxicity but still having adjuvant activity.

23. The composition of claim 20, wherein said \\melb-files\homeS\cintae\Keep\speci\div 55764.96.doc 19/06/00 80 mucosal adjuvant is a cholera toxin, or a subunit or derivative thereof lacking toxicity but still having adjuvant activity.

24. The composition of claim 20, wherein said mucosal adjuvant is a Clostridium difficile toxin, or a derivative thereof lacking toxicity but still having adjuvant activity.

25. The composition of claim 24, wherein said mucosal adjuvant comprises the carbohydrate binding domain of Clostridium difficile Toxin A.

26. The composition of claim 12, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetracycline, metronidizole, and erythromycin.

27. The composition of claim 12, wherein said 20 bismuth salt is selected from the group consisting of bismuth subcitrate and bismuth subsalicylate.

28. The composition of claim 12, wherein said antisecretory agent is a proton pump inhibitor.

29. The composition of claim 28, wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole.

30. The composition of claim 12, -wherein said antisecretory agent is an H2-receptor antagonist.

31. The composition of claim 30, wherein said H2-receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine. \\melb_files\homeS\cintae\Keep\speci\div 55764.96.doc 19/06/00 81

32. The composition of claim 12, wherein said antisecretory agent is a prostaglandin analog.

33. The composition of claim 32, wherein said prostaglandin analog is misoprostil or enprostil.

34. A method of inducing a mucosal immune response to Helicobacter in a patient, said method comprising administering to a mucosal surface of said patient a composition comprising an immunogenically effective amount of multimeric complexes of recombinant, enzymatically inactive Helicobacter urease. The method of claim 34, wherein said composition comprises a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A "subunits and four Urease B subunits, or a mixture thereof.

36. The method of claim 34, wherein said S" composition comprises a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B 25 subunits, and a multimeric complex comprising four Urease A 6 subunits and four Urease B subunits.

37. The method of claim 34, wherein said mucosal surface is intranasal, oral, or rectal.

38. The method of claim 34, wherein said composition is administered without gastric neutralisation.

39. The method of claim 34, wherein said composition further comprises a mucosal adjuvant. The method of claim 39, wherein said mucosal \\melbfiles\home$\cintae\Keep\speci\div 55764.96.doc 19/06/00 82 adjuvant is the heat-labile enterotoxin of enterotoxigenic Escherichia coli, a cholera toxin, a Clostridium difficile toxin, a subunit or derivative of one of said toxins lacking toxicity but still having adjuvant activity, or a mixture thereof.

41. The method of claim 40, wherein said mucosal adjuvant comprises the carbohydrate binding domain of Clostridium difficile Toxin A.

42. The method of claim 34, wherein said multimeric complexes of recombinant, enzymatically inactive Helicobacter urease have been freeze-dried.

43. The method of claim 34, wherein said Helicobacter urease is Helicobacter pylori urease.

44. The method of claim 34, wherein said Helicobacter to which said mucosal immune response is 20 raised is Helicobacter pylori. The method of claim 34, wherein said patient is at risk of developing, but does not have, a Helicobacter infection.

46. The method of claim 34, wherein said patient is infected by Helicobacter.

47. A method of treating a gastroduodenal 30 infection in a patient, said method comprisi-ng administering to said patient an antigen from a gastroduodenal pathogen, and an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.

48. The method of claim 47, wherein said gastroduodenal pathogen is a Helicobacter. \\melb.files\homeS\cintae\Keep\speci\div 55764.96.doc 19/06/00 83

49. The method of claim 48, wherein said Helicobacter is Helicobacter pylori.

50. The method of claim 48, wherein said antigen is urease.

51. The method of claim 48, wherein said antigen comprises multimeric complexes of recombinant, enzymatically inactive Helicobacter urease.

52. The method of claim 51, wherein said antigen comprises a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A subunits and four Urease B subunits, or a mixture thereof.

53. The method of claim 51, wherein said antigen 20 comprises a multimeric complex comprising eight Urease A subunits and eight Urease B subunits, a multimeric complex comprising six Urease A subunits and six Urease B subunits, and a multimeric complex comprising four Urease A subunits and four Urease B subunits.

54. The method of claim 47, wherein said gastroduodenal infection is a Helicobacter infection. The method of claim 54, wherein said 30 Helicobacter infection is Helicobacter pylori infection.

56. The method of claim 47, further comprising administering an adjuvant to said patient.

57. The method of claim 56, wherein said adjuvant is a mucosal adjuvant. \\melb-files\home\cintae\Keep\speci\div 55764.96.doc 19/06/00 84

58. The method of claim 57, wherein said mucosal adjuvant is the heat-labile enterotoxin of enterotoxigenic Escherichia coli, a cholera toxin, a Clostridium difficile toxin, a subunit or derivative of one of said toxins lacking toxicity but still having adjuvant activity, or a mixture thereof.

59. The method of claim 58, wherein said mucosal adjuvant comprises the carbohydrate binding domain of Clostridium difficile Toxin A. The method of claim 47, wherein said antibiotic is amoxicillin, clarithromycin, tetracycline, metronidizole, or erythromycin.

61. The method of claim 47, wherein said bismuth salt is bismuth subcitrate or bismuth subsalicylate.

62. The method of claim 47, wherein said 20 antisecretory agent is a proton pump inhibitor.

63. The method of claim 62, wherein said proton pump inhibitor is omeprazole, lansoprazole, or pantoprazole.

64. The method of claim 47, wherein said antisecretory agent in an H2-receptor antagonist. The method of claim 64, wherein said H2- 30 receptor antagonist is ranitidine, cimetidin.e, famotidine, nizatidine, or roxatidine.

66. The method of claim 47, wherein said antisecretory agent is a prostaglandin analog.

67. The method of claim 66, wherein said prostaglandin analog is misoprostil or enprostil. \\melb files\homeS\cintae\Keep\speci\div 55764.96.doc 19/06/00 85

68. The method of claim 47, wherein said antigen is administered to a mucosal surface of said patient.

69. The method of claim 68, wherein said mucosal surface is oral, intranasal, or rectal. A vaccine for inducing mucosal immune response to Helicobacter substantially as herein described.

71. A composition for treating gastroduodenal infection substantially as herein described.

72. A method of inducing mucosal immune response to Helicobacter substantially as herein described.

73. A method of treating gastroduodenal infection substantially as herein described. Dated this 19th day of June 2000 ORAVAX, INC By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and 25 Trade Mark Attorneys of Australia e a *e \\melb_files\home$\cintae\Keep\speci\div 55764.96.doc 19/06/00