OA10372A - Urease-based vaccine and treatment for helicobacter infection - Google Patents

Description

i Μ>!ηϊα&Γ.Λ-- i il W ΙώΛί! 010372

WVTWT ATTCRXKT OOCIŒT MO. 0δ132/3Χ1ΟΑ1UP.EA5E-3ASED VACCIME AND ΤΡ.ΕΑΤΜΕΝΤ

FOP. HELICQBACTEP- INFECTION

The présent invention relates to the prévention,and treatæent of gastric infection in mammals, includinghumans. More particularly, the présent invention relatesto a vaccine suitable for use in the prévention andtreatment of Hélicobacter infection in mammals, includinghumans, and to a method of treatment of humans sufferingfrom gastric infection, its conséquences such as chronicgastritis or peptic ulcer, and prévention of gastriccancer.

Background of the Invention Hélicobacter infections of human gastricepithelium cause gastritis, are a major factor in thedevelopment of peptic ulcers and gastric lymphoma, andmay be a risk factor for the development of gastriccancer. (Blaser, M.J. "Gastric Campylobacter-likeOrganisms, Gastritis and Peptic Ulcer Disease"Gastroenterology, vol. 93, 371-333 (1987); Graham, D.Y. "Campylobacter pylori and Peptic Ulcer Disease"Gastroenterology, vol. 196, 615-625 (1989); Parsonnet, J.et al. "Helicobacter pylori Infection in Intestinal andDiffuse-Type Gastric Adenocarcinomas" J. Natl. CancerInst., vol. 93, 640-643 (1991); Wotherspoon, A.C., et al., "Régression of Primary Low-Grade B-Cell GastricLymphoma of Mucosa-Associated Lymphoid Tissue Type AfterEradication of Hélicobacter pylori," Lancet, vol. 342,575-577 (1993)). The most frequent infection agent is

Helicobacter pylori. followed by a much lower frequency 010372 sy Hélicobacter heilmanli. Hélicobacter pylori is asler.der S-shaped grara négative micrcorganism, wnich isroutinely recovered frcm gastric bicpsies of adules andchildren with histologie evidence of gastritis or pepticulcération. Evidence for a causal relationship betweenHélicobacter pylori and gastroduodenal disease cornes fromstudies in human volunteers, patients with ulcers andgastric cancer, gnotobictic pigs, and germ-free redents.Regardinq etiology, Koch's postulâtes were satisfied bycreating histologically confirmed gastritis in previouslyuninfected individuals following consumption of viablemicroorganisms . (Marshall, B.J. et al. "Attempt toFulfill Koch's Postulate for pyloric Campylobacter" Med.J. Aust., vol. 142, 436-439 (1985); Morris, A. et al. "Ingestion of Campylobacter pylorifis Causes Gastritisand Raised Fasting Gastric pH" Ara. J. Gastroenterol.,vol. 82, 192-199 (1987); Engstrand, L. et al. "Inoculation of Barrier-Born Pigs With Helicobacterpylori: A Useful Animal Model for Gastritis Type B:

Infect. Immun., vol. 53, 1763-1768 (1990); Fox, J.G. et al. "Gastric Colonization by Campylobacter pylori Subsp.Mustelae in Ferrets" Infect. Immun., vol. 56, 2994-2996(1988); Fox, J.G. et al. "Hélicobacter mustelae-Associated Gastritis in Ferrets: An Animal Model ofHélicobacter pylori Gastritis in Humans"

Gastroenterology, vol. 99, 352-361 (1990); Lee, A. et al."A Small Animal Model of Human Hélicobacter pylori ActiveChronic Gastritis" Gastroenterology, vol. 99, 1315-1323 (1990); Fox, J.G. et al. "Hélicobacter Felis Gastritis in 3 010372

Gnotobiotic Rats: An Animal Model of Hélicobacter pyloriGastritis" Infect. Immun., vol. 59, 785-791 (1991);

Eaton, K.A., et al. "Campylobacter pylori VirulenceFactors in Gnotobiotic Piglets" Infect. Immun., vol. 57, 5 1119-1125 (1989)), and by treatment to eradicate Hélicobacter pylori· with resolution of the gastritisand, in patients with peptic ulcer disease, a decrease inthe récurrence rate. (Peterson, W.L. "Hélicobacterpylori and peptic Ulcer Disease" N. Engl. J. Med., vol. 10 324, 1043-1048 (1991)).

Gastroduodenal diseases thought to be associated with Hélicobacter infection include acute, chronic, andatrophie gastritis, peptic ulcer disease including bothgastric and duodenal ulcers, gastric cancer, chronic 15 dyspepsia with severe erosive gastroduodenitis, refractory non-ulcer dyspepsia, intestinal metaplasia,and low grade MALT lymphoma. Hélicobacter infection isalso the principle cause of asymptomatic chronicgastritis. 20 in spite of in vitro susceptibility to many antimicrobial agents, in vivo éradication of establishedHélicobacter pylori infections with antimicrobial agentsis often difficult to achieve. (Czinn, S.J. and Nedrud,J.G. "Oral Immunization Against Hélicobacter pylori" 25 Infect. Immun., vol. 59, 2359-23S3 (1991)). The microorganism is found within the mucous coat overlyingthe gastric epithelium and in gastric pits. These arelocations which do not appear to allow for adéquateantimicrobial levels to be achieved even when antibiotics 4 010372 are given orally at high doses. At the présent tome,most authorities recomroend a "triple therapy", namely abismuth sait in combination with drugs sucn as tétracycline and metronidacole for 2-4 weeks. Hovever,the effectiveness of this or other chemotherapeuticregimens remains suboptimal. Recently, a Nationalïnstitutes of Health panel of medical experts reccmmendeda triply therapy with bismuth, tétracycline andmetronidacole, administered for two weeks for treatmentof peptic ulcers (Cimons, M., "Drug Combination FcundEffective on Peptic Ulcers," L.A. Times at A14 (Feb. IC,1994)) . However, this treatment is commonly associatecwith diarrhea and it may produce serious adverse drugreactions. (See. Dick-Hegedus, E. and Lee, A., "Use of aMouse Model to Examine Anti-Helicobacter pylori Agents,"Scand. J. Gastroenterol., vol. 26, 909-915 (1991)).

Treatment with antibiotics also may not solve the problemof reinfection and there is evidence for a high incidenceof reinfection in some studies (Coelho, L.G., et al.,"Duodenal Ulcer and Eradication of H. pylori in aDeveloping Country: An 18-month Follow-Up Study," Scand.J. Gastroenterol. vol. 27, 362-66 (1992)). Thereforethere is a great need for a vaccine that can be usée totreat infection and to prevent future infections.

At the présent time little is known regarding therôle of the mucosal immune Systems in the stomach. Thedistribution of immunoglobulin (Ig) producing cells inthe normal gastric antrum indicates that IgA plasma cellsmake up 80% of the total plasma cell population. In 010372 - 5 - addition, the number of plasma IgA cells présent in thegastric antrum is comparable to other mucus membranes.(Brandtzaeg, P. "Rôle of J Chain and Secretory Componentin Receptor-Mediated Glandular and Hepatic Transport ci 5 Immunoglobulins in Man" Scand. J. Immunol., vol. 22, 111-146 (1985); Brandtzaeg, P. et al. "Production and Sécrétion of Immunoglobulins in the GastrointestinalTract" Ann. Allergy, vol. 59, 21-39 (Nov., 1987)). Anumber of studies in humans (Wyatt, J.I. et al. "Local 10 Immune Response to Gastritis Campylobacter in Non-ulcerDyspepsia" J. Clin. Path., vol. 39, 863-870 (1986)), andin animal modèle (Fox, J.G. et al. "Hélicobactermustelae-Associated Gastritis in Ferrets: An AnimalModel of Hélicobacter pylori Gastritis in Humans" 15 Gastroenterology, vol. 99, 352-361 (1990); Fox, J.G. etal. "Hélicobacter felis Gastritis in Gnotobiotic Rats:

An Animal Model of Hélicobacter pylori Gastritis" Infect.Immun., vol. 59, 785-791 (1991); Fox, J.G. et al. "Localand Systemic Immune Responses in Murine Hélicobacter 20 felis Active Chronic Gastritis," Infect. & Immun, vol. 61, 2309-15 (1993)), hâve demonstrated spécifie IgG and

IgA responses in sérum and in gastric sécrétions inresponse to Helicobacter infection. However, theobservation that Hélicobacter pvlori infection persists 25 as a chronic infection for years, despite inducing alocal and systemic immune response, is not encouragingthe development of immunization strategies.

Lee et al. hâve reported the ability to infectgerm-free rodents with Helicobacter felis, a bacterium 0 1 0372 - 6 - closely related to Helicobacter pylori. and reproduciblydocument histologie gastritis. (Lee, A. et al. "A. SmallAnimal Model of Human Hélicobacter pylori Active ChromeGastritis" Gastroencerology, vol. 99, 1315-1323 (1390) ;

Fox, J.G. et al. "Hélicobacter felis Gastritis inGnotobiotic Rats: An Animal Model of Hélicobacter pyloriGastritis" Infect. Immun., vol. 59, 735-791 (1991)).

Since then, this bacterium-'nost pairing nas been acceptéeas a good model to study Helicobacter-mediated gastritesand its initiating factors. (Lee, A. et al. "Pathogenicity of Hélicobacter pylori: A Perspective"Infect. Immun., vol. 61, 1601-1610 (1993)). Infection of mice with H, felis results in a similar pathologieresponse to that found in humans infected with H, pylort;both types of infections resuit in active, chronicgastritis. (Lee et al., Gastroenterology, vol. 99, pp.1315-1323 (1990)). Researchers hâve found that Hélicobacter felis has the same susceptibility to antimicrobial therapy as Hélicobacter pylori, and the K.felis/mouse model has been used to develop new treatmentsagainst H, pylori infection. (Dick-Hegedus, E. and Lee,A., "Use of a Mouse Model to Examine Anti-Helicobacterpylori Agents," Scand. J. Gastroenterol., vol. 26, 909- 915 (1991); Chen et al., "Immunization Against Gastric

Helicobacter Infection in a Mouse/Helicobacter felisModel," Lancet, vol. 339, p.1120 (1992)). Czinn et al.hâve shown that répétitive oral immunization with a crudelysate of Hélicobacter py1ori plus choiera toxin adjuvantinduces a vigorous gastrointestinal IgA anti-Hélicobacter 7 010372 pylori response in mice and ferrets. (Czinn, S.J. andNedrud, J.G. "Oral Immunisation Against Hélicobacterexlazi" Infect. Immun., vol. 59, 2359-2363 (1991)). Ir. addition, Chen et al. and Czinn et al. hâve recentlyreported that oral immunization with a crude lysate of H félis induced protection against H, felis infection inmice. (Chen, et al. "Immunization Against GastricHélicobacter Infection in a Mouse/Helicobacter felisModel, "(letter) Lancet, vol. 339, 1120-1121 (1992);Czinn, S. et al. "Oral Immunization Protects Germ-FreeMice Against Infection from Hélicobacter felis"Proceedings of the DDW, American GastroenterologicalAssociation, 1321, A-331 (May 10-13, 1992); Czinn et al.,Vaccine, vol. il, 637-642 (1993)). The exact nature ofthe antigen(s) responsible for the induction of thisprotection, however, had not been determined, and noinformation suggested that the protective antigen(s) ofH, felis that induced protection against this pathogenwould induce a cross - reactive protection extending toanother Hélicobacter species.

We hâve demonstrated for the first time thatHelicobacter pylori and H, felis shared antigenicdéterminants by obtaining monoclonal antibodies afteroral immunization of mice with either Helicobacter pylorior H. felis sonicates and showing that sortie of theseantibodies, directed against Hélicobacter pylori, wouldcrossreact with H. felis and vice versa, (Michetti, ?. etal. "Specificity of Mucosal IgA Response in Balb/C MiceFollowing H. felis or Hélicobacter pylori Challenges"

01 0372 - 8 -

Proceedings of the DDW, American Gastroenterological al. "Hélicobacter pylore Urease Elicits ProtectionAgainst H. felis Infection in Mice" Proceedings ot theDDW, American Gastroenterolcgical Association, 1212, A-304 (May 16-19, 1993)), but the oasis for these cross- reactivities were unknown.

Basée on the homology existing between the different knewn urease amino acid sequences, it has beenproposed that jack bean urease could be used as a vaccineagainst Hélicobacter pylori. (Pallen, M.J. and Clayton,C.L. "Vaccination Against Hélicobacter pvlori Urease"Lancet, vol. 336, 186-7 (1990)). Nevertheless, despite the homology among the different urease sequences, cross-reactivity is not the rule. Guo and Liu hâve shown yearsago that ureases of Proteus mirabilis. Proteus vulgarisand Providencia rettgeri show cross-reactivity to eachother, while ureases of jack bean and Morganella morganliare immunologically distinct from the three formerureases. (Guo, M. and Liu, P.V. "Serological

Specificities of Ureases of Proteus Species" J. Gen.Microbiol., vol. 136, 1955-2000 (1965)). So, even if an antigenic cross-reactivity of Hélicobacter oylori ureasewith other Helicobacter ureases was a reasonable postulate, no data existed demonstrating that this was really the case until we showed that some H. felis monoclonal antibodies cross-reacted with Hélicobacter pvlori urease. (Davin, C. et al. "Hélicobacter pylori

Urease Elicits Protection Against H. felis Infection in 9 010372

Mice" Proceedings of the DDW, American

Gastroenterological Association 1213, A-304 (May 16-19,1993)) . J. Pappo has further demonstrated that micewhich hâve been infected by H. felis produce antibodieswhich crossreact with Hélicobacter pylori urease but notjack bean urease (J. Pappo, unpublished data, 1993) . Thefact that jack bean urease does not fall in the sameimmunological category than Helicobacter urease suggeststhat jack bean urease may not be useful for immunizationagainst Hélicobacter infections, the way it was done ferenteric bacteria. (Pimentai, J.L. and Cook, M.E. "Improved Growth in the Progeny of Hens Immunized withJackbean Urease" Poultry Sci., vol. 64, 434-439 (1938).

Furthermore, attempts to immunité mice against H, felisinfection by oral or intraperitoneal delivery of jackbean urease resulted in the production of antibodiesagainst jack bean urease, but failed to protect the micefrom infection. (Chen, M. et al. "Failure ofImmunization Against Hélicobacter Using Jack BeanUrease," Acta Gastroenterol. Belg., vol. 56, 94 (1993)).

The use of an antigen that is the reaction productof urease and glutaraldehyde is described in U.S. PatentNo. 4,837,017, "Urease Antigen Product and Process,"issued June 6, 1989, to LeVeen et al. The patentdescribes the use of the antigen to reduce ammoniatoxicity cause! by urea splitting organisms. LeVeer. etal. disclose the injection of glutaraldehyde treatedjack bean urease into the bloodstream. The LeVeen patentdoes not disclose the administration of the urease 10 010372 antigen to the mucosal surface of a mammal in order testimulate antibody production by the local immune System.Furthermore, there is no évidence in the spécificationthat the injection of a jack bean urease antigen couldprevent Hélicobacter infection or be used to treatgastroduodenal infection by Hélicobacter.

Eaton et al. hâve shown that mutant H. pvloricultures with weak urease activity are unable to infectgnotobiotic piglets. (Eaton et al., "Essential Rôle ofUrease in the Pathogenesis of Gastritis Induced byHélicobacter pvlori in Gnotobiotic Piglets,"

Gastroenterology, vol. 98, A654 (1990)). Eaton does net describe the use of a urease antigen as a vaccine toprevent Hélicobacter infection or as a method of treatingHélicobacter infection.

The use of Hélicobacter pylori urease, or ofrelated ureases, as a vaccine against Hélicobacter pyloriinfection has previously been proposed by A. Labigne, andincorporated among the claims of a patent filed onOctober 6th, 1988 by Pasteur Institute, Paris, France.(Labigne, A. "Sequences of Nucléotides Coding for aProtein Having an Urease Activity". EPO patent application #EPO 367 644 Al, 1989. International

Publication # WO 90/04030, 1990) . The specification of this document contains, however, no evidence ofvaccination of any mammal against any Helicobacterinfection with urease. This part of the PasteurInstitute patent, therefore, has not been reduced topractice, and the related claims (claims 27 and 28, page 11 010372 16) should not be considered as valid. Furthermore, triedaims of this document relate to a protein presenting aurease activity, and it will be understood frcm theexperiments described below that enzymatic activity ofthe urease-based vaccine is not required to induceprotection after oral immunization.

Moreover, while sequence homology with otherbacterial ureases might support the use of urease as avaccine candidate against Hélicobacter pvlori infection,the current knowledge of human Hélicobacter pvloriinfection would certainly not. First, despite the factthat infected individuals often mount a strong antibodyresponse to urease, the anti-urease immune response doesnot resuit in clearance or control of the infection.Second, Hélicobacter pvlori is able to transport ureaseout of the cell and to shed it from its surface, (Evans, D.J. et al. "Urease-Associated Heat Shock Protein ofHélicobacter pvlori11 Infect. Immun., vol. 60, 2125-2127(1992), Ferrero, R.L. and Lee, A., "The Importance ofUrease in Acid Protection for the Gastric-GolonizingBacteria Hélicobacter pvlori and Hélicobacter felis sp.nov." Microb. Ecol. Health Dis., vol. 4, 121-134 (1991)), thus urease may not represent an appropriate target forthe development of a protective mucosal immune response.Indeed, mucosal immune protection is thought to be mainlymediated by secretory IgA, the agglutinating activity ofwhich would be impaired when the recognized antigen canbe shed by the target pathogen. Third, urease appears tobe toxic for épithélial cells in culture, and has been 12 0 1 0372 suspected to play a rôle in mucus dégradation and inpeptic ulcération in vivo (Mégraud, F. et al., "FurtherEvidence of the Toxic Effect of Ammonia Produced byHelrcobacter pvlori Urease on Human Epithelial Cells,"Infect. & Immun., vol. 60, 1858-63 (1992); Murakamr, M.et al. "Gastric Ammonia has a Potent Ulcerogenic Actionon the Rat Stomach," Gastroenterology 1993, vol. 1C5,1710-15), thus its use as antigen may be toxic.

Nevertheless, we reasoned that this antigen couldbe a potentially efficient vaccine if: first, we would deliver it orally at asufficiently high dose to elicit a stronger immuneresponse than the naturally occurring one second, the amount of antibodies produced wouldbe high enough to bind ail the urease, shed or not shed third, we would use subunits of urease or amolecular species that was non toxic.

Another aspect of the invention describes the useof antibodies directed against urease to prevent and totreat Helicobacter infection. European Patent

Application No. 91310049.1, filed by Kunio Ando onOctober 31, 1991, claiming priority on Japanese Patent

Application No. 296609/90 filed November 1, 1990, titled "A Method for Producing a new Medicine for Both Treatingand Preventing Peptic Ulcer Diseases and Gastritis andThus Formulated Medicines," describes the oraladministration of polyclonal antibodies derived frorabovine colostrum and bovine sérum to patients with activechronic gastritis type B and to patients with duodenal 13 010372 ulcer. The Ando application describes the use of an antibody préparation directed against many antigerts, including Hélicobacter pylori. and does not disclcse theuse of an antibody directed against urease to treat orprevent Hélicobacter pylori infection. The use ofantibodies to treat gastric disease in gnotobioticpiglets was described in United States Patent Nos. 5,258,178 and 5,260,057, issued to Cordle and Schallerand titled "Method and Product for the Treatment ofGastric Disease." The Cordle and Schaller patentsdescribe the use of an antibody préparation that does tôtsolely contain antibodies directed against Hélicobacterpylori, and does not disclose the use of an antibodydirected against urease to treat or prevent Hélicobacterpylori infection. Nagata et al. describe the préparationof a monoclonal antibody directed against Hélicobacterpylori that inhibits urease activity. (Nagata, K., etal . , "Monoclonal Antibodies Against the Native Urease cfHélicobacter pylori: Synergistic Inhibition of UreaseActivity by Monoclonal Antibody Combinations," Infect,and Immun., Vol. 60, 4826 (1992)). Nagata et al. do netdescribe the use of monoclonal antibodies directedagainst urease to prevent or to treat Hélicobacter pyloriinfection.

Very few examples of therapeutic vaccines areavailable in the literature. Most of them are related toparentéral immunisations aimed to stimulate the host'simmune System against malignant tumors, to moduiate theimmune System in autoimmune diseases such as rheumatoid 14 01 0372 arthritis or as desensitization in allergy States.Therapeutic vaccination procedures against differentinfections were also performed, most of them via aparentéral route cf immunizatiou. They includedimmunisations against leprosy in humans (Zaheer S. A. eral. "Combined Multidrug and Mycobacterium w VaccineTherapy in Patients with Multibacillary Leprosy" J.

Infect Dis., vol. 167, 401-410 (1993), Mukherjee A. et al., "Histopathological Monitoring of an

Immunotherapeutic Trial with Mycobacterium w," Int. JLepr. Other Mycobact. Dis., vol. 60, 28-35 (1992)) , in complémentation of antibiotic therapy, vaccinationagainst Phythiosis insidiori, a mycological infection, inhorses (Mendoza L, et al., "Evaluation of Two Vaccinesfor the Treatment of Phythiosis insidiori in Horses"Mycopathologia, vol. 119, 89-95 (1992)), an uncontrolled study on the use of an autovaccine in chronic osteomyelitis (Sologub V.V. "Expérience in Using auAutovaccine in Treating Patients with ChronicOsteomyelitis" Vrach, Delo, 122-125 (1992)) and systemicimmunization against Campylobacter fétus infection offemale cattle (Schurig, G.G.D., et al., "Bovine Ver.erealVibriosis: Cure of Génital Infection in Females by

Systemic Immunization," Infect. & Immun., Vol. 11, 245- 51 (1975)) . To date, only one oral immunotherapy study aimed at stimulating the mucosal immune System in crderto treat (and to prevent récurrence of) a mucosalinfection has been performed, for urinary tract infection(Schulman C.C., et al. "Oral Immunotherapy of Récurrent . '*-‘αΙΑ·»· 010372 15

Urinary Tract Infections: A Double-Blind Placebo-Controlled Multicenter Study" J Urol., vol. 150, 917-921(1993)). In that study, Schulman et al. used a lysate ofselected E. coli strains, tcgether with an concomitanttreatment of antibiotics, chemotherapeutics or urinarytract disinfectants to treat the acute infection at entryin the study. Therefore, no study has demonstrated sofar the effectiveness of a therapeutic vaccine, used as amcnotherapy, administered to the mucosal immune System,against a bacterial disease.

The novelty of a therapeutic vaccine againstHélicobacter infection also cornes from the observationChat H. pylori persists as a chronic infection in thegastric cavity for years, despite inducing a vigorouslocal and systemic immune response. This observation wasconceptually already an obstacle to the development of aprophylactic vaccine against Hélicobacter infection, butwas even more an obstacle to the development of atherapeutic immunization.

In summary, there remains a need for effectivetreatment and prévention of Hélicobacter pylori-inducedgastric infection in humans. Recent data suggested thepossibility to generate a vaccine against this infection,but hâve not provided a clear identification of definedantigen(s), common to ail strains of Hélicobacter pylori,that could be incorporated into a safe and effectivevaccine.

In this invention, we hâve identified the urease antigen of Hélicobacter pylori as a candidate vaccine and 16 010372 demonstrated its efficacy in an animal model. We hâvealso démonstrated the use of the Hélicobacter pyloriurease antigen for the treatment and éradication ofHélicobacter infection. We hâve further demcnstratedthat the B subunit of urease alone (ure B) is effectiveas a vaccine useful for the prévention of and treatmentof Hélicobacter infections. These results wereunexpected in the light of the natural history ofHélicobacter infections.

Summarv of the Invention

We hâve determined that it is useful to immunitéanimais with Hélicobacter urease peptides for bothprophylactic and therapeutic treatment. Immunization ofanimais with Hélicobacter urease peptides preventsinfection by Hélicobacter and eradicates infection inpreviously infected animais. This method, and thevaccine compositions, are useful for the prévention andtreatment of gastroduodenal disease associated withHélicobacter infection.

We hâve discovered that immunity can be induced inmammals susceptible to gastrointestinal Hélicobacterinfection by exploiting urease epitopes displaved on orabout the surface of Hélicobacter organisms and usingthem as a vaccine target. The immunity can be induced byimmunization with native urease, but can also be inducedwith recombinant urease subunit, produced as anenzymatically inactive, therefore non-toxic fora. Theinvention provides a method of inducing immunity to 17 010372 Hélicobacter infection by administering to a mucosalsurface of a mammal a polyaminoacid préparation, i.e., amixture of peptides and/or proteins, together with anappropriate adjuvant. This polyaminoacid préparationprésents a plurality of epitopes characteristic of andexhibited by a urease enzyme endogenous to the infectingHélicobacter organism. The administration of thepolyaminoacid préparation may be performed by the oralroute.

The active ingrédient of the préparation maycomprise natural or biosynthetic epitopes and may takevarious forms. A non exhaustive list of possiblepréparations includes purified, naturally occurring orrecombinantly produced urease préparations of bacterialor other origin, digests of urease, fusion proteinscomprising urease epitopes, truncated forms of ureaseenzyme, or peptides homologous with the amino acidsequence of urease. Since development of immunitydépends on induction of humoral and/or cellular immuneresponses which bind to the infecting Hélicobacterorganism, preferred préparations are those which mostclosely duplicate the epitopes of the urease endogenousto the infecting organism. For example, préparationsdisplaying the epitopes of urease of Hélicobacter pyloriare preferred for administration in humans susceptible toHélicobacter pylori. and préparations displaying theepitopes of urease of H. felis are preferred for theadministration in humans susceptible to H. felis.

However, in accordance with an important aspect of the ·» J» A h.·' Λ 010372 - 18 - invention, it has been discovered that urease from aheterologous species may be used. For exampie, we hâveshown that H. felis infection in mice can be prevented byadministration of urease frcm Hélicobacter pylori. Thus, H. pylori urease can be used to protect against H. pylorias well as H. felis. H. felis is an occasional cause ofhuman infection and disease. (Wegman, W. et al.,

Schweig. Med. Wochenschr. vol. 121, 245-54 (1391)).

According to a first embodiment of the présentinvention, a method is provided of eliciting rn amammalian host a protective immune response teHélicobacter infection. According to a second embodimentof the présent invention, a method is provided oftreating a mammalian host that is infected withHélicobacter.

Thus, in a first aspect, the présent inventionprovides a method of eliciting in a mammalian host aprotective immune response to Hélicobacter infection.

The method comprises the step of administering to amucosal surface of the mammal, including humans, animmunologically effective amount of a urease antigen,preferably Hélicobacter pylori urease, capable ofeliciting such a protective immune response. The term"comprising" is used herein as it is recognized in the art.

According to one aspect of the invention, there is provided a method of eliciting in a mammalian host a protective immune response to Hélicobacter infection wherein an immunologically effective amount of a urease 19 'ilK·= sses, 010372 antigen capable of eliciting such a protective immuneresponse, preferably Hélicobacter pylori urease orHélicobacter pylori urease 3 subunit, is administered toa mucosal surface of the host.

In a second aspect, the présent invention providesa method of eliciting in a mammalian host a protectiveimmune response to Helicobacter infection. The methodcomprises the step of administering to a mucosal surfaceof the mammal, including humans, an immunologicallyeffective amount of recombinant, enzymatically inactive,urease B subun.it as antigen, preferably recombinantHélicobacter pylori urease B subunit, capable ofeliciting such a protective immune response.

The invention also includes within its scope thetreatment or prophylaxie of mammals, including humans,for Hélicobacter infection, wherein an immunologicallyeffective amount of a urease, or its subunits, capable ofeliciting a protective immune response to Helicobacterinfection, is administered to a mucosal surface of apatient. Preferably, the urease is Hélicobacter pyloriurease or Hélicobacter pylori urease B subunit, and theurease is preferably administered in particulate form inassociation with a hydroxylated calcium phosphate, forexample hydroxyapatite. Moreover, it is preferred toadminister the Hélicobacter pylori urease in associationwith a mucosal adjuvant, the B subunit of choiera tcxin,muramyl dipeptide or other such adjuvants.

According to another aspect of the présentinvention, there is provided a vaccine composition 20 010372 suitable for prévention of Hélicobacter infection,comprising an effective amcunt of a urease antigen,preferably Hélicobacter pylori urease or Hélicobacterpylori urease B subunit, or recombinant Hélicobacterpylori urease subunits, capable of eliciting in a host aprotective immune response to Hélicobacter infection, onassociation with a pharmaceutically acceptable carrier ordiluent. Suitable carriers and diluents will berecognized by those skilled in the art and can be roundin, for example Reminoton's Pharmaceutical Sciences (18thed., 1990) .

The vaccines of the invention are administered inamounts readily determined by persons of ordinary sxillin this art. Thus, for adults a suitable dosage will bein the range of 10 μ g to 100 mg, for example 50 μα to 5Cmg. A suitable dosage for adults will also be in therange of 5 μg to 500 mg. Similar dosage ranges will beapplicable for children. Carrier Systems in humans mavinclude enteric release capsules protecting the antigenfrom the acidic environment of the stomach, and includir.gurease antigen in an insoluble form as fusion proteons.The vaccine can be administered as a primary prophylacticagent in adults or in children, as a secondary prévention, after successful éradication of Hélicobacterpylori in an infected host, or as a therapeutic agent inthe aim to induce an immune response in the host susceptible to contribute to the éradication of Hélicobacter pylori. 21 01 0372

As noted above, a suitable mucosal adjuvant ischoiera toxin. Others whic’n may be used are non-toxicdérivatives of choiera toxin, including its B subunit,and/or conjugates or genetically engineered fusions ofthe urease antigen plus choiera toxin or its B subunit.Other suitable delivery methods include biodégradablemicrocapsules or immuno-stimulating complexes (ISCOMs) orliposomes, genetically engineered attenuated live vectorssuch as viruses or bacteria, and recombinant (chimeric)virus-like particles, e.g., bluetongue. The amount ofmucosal adjuvant employed dépends on the type of mucosaladjuvant used. For example, when the mucosal adjuvant ischoiera toxin, it is suitably used in an amount of 5 /xgto 50 /ig, for example 10 μα to 35 μg. When used in theform of microcapsules, the amount used will dépend on theamount employed in the matrix of the microcapsules toachieve the desired dosage. The détermination of thisamount is within the skill of a person of ordinary skillin this art.

Suitable carriers for the vaccines of theinvention are enteric coated capsules and polylactide-glycolide microspheres. Suitable diluents are 0.2N NaHC03and/or saline.

Particulate hydroxylated calcium phosphate (HCP)is especially useful as a carrier for the Hélicobacterpylori urease to be applied to mucosal surfaces. It isbelieved that the Hélicobacter pylori urease-hydroxylatedcalcium phosphate conjugate is transported acrossepithelium where it raises a poly Ig immune response. 22 010372

Preferably, the hydroxylated calcium phosphate is in theform of microparticles suitable for transport across theepithelium, particularly by cells specialized for thispurpose (M cells). A preferred form of hydroxylatedcalcium phosphate is hydroxyapatite, a commerciallyavailable crystalline hydroxylated calcium phosphateCa10 (PO4) 6 (OH) 2 ·

Commercially available hydroxyapatite generallyconsists of slab-like crystals that are cnemically andphysically analogous to inorganic hydroxyapatite innormal bone tissue. Ingestion of hydroxyapatite shouldtherefore be safe, as evidenced by the existence ofnutritional calcium/phosphorus suppléments derived fromground bone, which are designed to be ingested.Commercially-high resolution hydroxyapatite (fromCalBiochem) consists of crystals varying widely in size.Crystals over Ιμπι in length are unlikely to be taken upby M cells. Therefore, for use in the invention,commercial hydroxyapatite crystals are broken into small,relatively uniform crystalline fragments such as bysonication. Preferably, a substantial proportion of thehydroxyapatite is présent as fragments of about 0.01-Ι.Ομπι. Fragmentation may be measured either by électronmicroscopy or light scattering, using standardtechniques.

Preferred modes of administration of theHélicobacter pylori urease antigen are orally, nasally,rectally or ocularly. Oral administration can provide 23 010372 delivery to other G.I. (gastrointestinal) mucosa including the intestinal mucosa.

The vaccines of the présent invention may beadministered to a mucosal surface in the form of a_naérosol, suspension, capsule and/or suppository. Themethod of administration will be readily apparent to aperson of ordinary skill in this art and can be fcund in,for example, Remington's Pharmaceutical Sciences (I8thed., 1990) .

According to a further aspect of the présentinvention, there is provided a method of imparting to amammalian host passive protection to Hélicobacterinfection, comprising administering to a mucosal surfaceof the host an immunologically effective amount of aurease-specific antibody produced in a host immunizedwith a urease, preferably Hélicobacter pylori urease orHélicobacter pylori urease B subunit, capable ofeliciting a protective immune response to Hélicobacterinfection.

The présent invention further includes the passiveimmunization of mammals, including humans, againstHélicobacter infection. This is achieved byadministering to a mucosal surface of the patient an-effective amount of a urease spécifie antibody,preferably an effective amount of a Hélicobacter pyloriurease spécifie IgA monoclonal antibody.

Since the urease of Hélicobacter pylori is shown to represent the antigen involved in inducing protective immunity, a further aspect of the invention is the use of 24 010372 Hélicobacter pylori urease as a diagnostic reagent tomeasure the immune response of pensons who hâve receiveda vaccine based on urease or to détermine whether anmdividual is immune or susceptible (and thus in need ofvaccination). The présent invention also includes theuse of urease and urease-specific antibodies to ccnstructassays and kits for diagnosis of Hélicobacter immunity,assessment of Hélicobacter susceptibility, and définitionof immune responses to vaccines.

In a third aspect, the invention provides a methcdof treating gastroduodenal disease in a mammal. Thismethod comprises the step of administering a therapeutically effective amount of a composition comprising Hélicobacter urease peptides. The gastroduodenal diseases included within the scope cf theinvention include, but are not limited to, gastrites,peptic ulcer disease, including both gastric and duodenalulcers, gastric cancer, chronic dyspepsia with severeerosive gastroduodenitis, refractory ulcer dyspepsia,intestinal metaplasia, low grade MALT lymphoma,Hélicobacter infection, Hélicobacter pylori infection andHélicobacter félis infection. The term "urease peptides"refers to, but is not limited to, any urease or subcnitof urease, either naturally occurring or obtained byrecombinant DNA techniques, as well as a digestedfragment or peptide thereof, fusion proteins comprisingthe whole urease, subunits, or fragments thereof, ortruncated urease constructs. Also included within theterm "urease peptides," are proteins or peptides that ; , j»£*u.'U&i&'iâS&QBBC. 25 010372 display epitopes sufficiently homologous to epitopes displayed by Helicobacter urease such that antibcdiesthat recognize epitopes displayed by Hélicobacter ureasewill recognize epitopes displayed by said peptides orproteins.

In a more particular related aspect, the inventionprovides a method of treating gastroduodenal diseasecaused by Hélicobacter infection. The Hélicobacterinfection may be, but is not limited to, Hélicobacterpv.lori or H, felis infection.

More particularly, the invention provides a methodof treating gastroduodenal disease caused by Hélicobacterinfection whereby the composition comprising Hélicobacterurease peptides is administered to a mucosal surface.

Without limiting the type of mucosal surface used foradministration, the mucosal surface may be oral, nasal,rectal, or ocular.

The invention can also feature the administrationof a composition comprising Hélicobacter urease peptidesin association with a mucosal adjuvant. The mucosaladjuvant may be selected from, but is not limited to,choiera toxin, procholeragenoid, choiera toxin B subunit,fungal polysaccharides including, but not limited to,schizophyllan, muramyl dipeptide, muramyl dipeptidedérivatives, phorbol esters, microspheres, non-

Helicobacter pylori bacterial lysâtes, labile toxin ofBscherichia coli. block polymers, saponins, and ISCOMs.

Other mucosal adjuvants will be recognized to those inthe art and can be found in, for example, Azuma, I., - 25 - 010372

3 Ncn-S ΜΟί imuncaenr

Interieukins ο and 6 on Rat Tear rg

Itnmunology, vol. 73, 19-23 (1991) ; Adam, A. & ledersr, "Muramvl Deotides mmunomoau îa tors ATLAS O( SCIENCE 205 (1988) ; Cléments, J.D., et al. "Adjuvant

Activity of Escherichia poli Heat-labile Enterotoxin a: 10 Effect on the Induction of Oral Tolérance in Mice toUnrelated Protein Antigens" Vaccine, vol. 6, 269 (1388^

Ben Ahmeida, et al. "ImmunoDotentiation of loc; and Systemic Humoral Immune Responses by ISCOMs,

Liposomes and FCA: Rôle in Protection Against Influença 15 A in Mice" Vaccine, vol. il, 1302 (1993); and Gupta, R. h. et al. "Adjuvante -- A Balance Between Toxicity andAdjuvanticity" Vaccine, vol. 11, 290-308 (1993) .

The mucosal adjuvant may also be genetically orchemically linked to the urease peptides. Examples of 20 this type of fusion peptide are known to those skilled inthe art and can also be found in Czerkinsky et al., "OralAdministration of a Streptococcal Antigen Coupled teChoiera Toxin 3 Subunit Evokes Stron.g Antibody Responsesin Salivary Glande and Extramucosal Tissues" Infect. 25 Immun., vol. 57, 1072-77 (1989); Nashar et al., "Current

Progress in the Development of the B Subunits of ChoieraTcxin and Eschericiiia Col 1 Heat-Labile Enterotoxin as

Carriers for the Oral Oelv/erv of Keteroicgous .cens and Epitopes" Vaccine, vol. 11, 235-40 (1993); and j ,,4,. . v_.i 27 010372

Dertzbaugh and 3lscn, "Comparative Ef fect iveness of tr.e7oxm 3 Srfcrr.it and Alkaiine Phosphatase as

Cral Vaccines," Infect. Immun., vol. 61, 46- _arr. *3 £ G - 55 (1292) . ?or example, the urease 3 subunit could be expressed as a chimeric protein that is geneticallyimkeu to the choiera toxin B subunit through the use ofa DNA expression vector containing the ure B nucléotidesequence linked to the choiera toxin 3 subunit nucléotidesequer.ee.

In another related aspect, the method involves theadministration of a composition comprising Helicobacterurease peptides where such composition is delivered inparticulate form. The composition may be delivered inparticulate form through association with a carrier. Thecarrier may be a hydroxylated calcium phosphate, ferexample, hydroxyapatite. The term "hydroxyapatite"refers to, but is not limited to, its meaning asrecognized by those skilled in the art to mean a tribasiccalcium phosphate, also known as hydroxylated calciumphosphate or calcium hydroxide phosphate. This is onlyan example, and is not meant to be limiting as to thetype of carrier that may be used.

In another related aspect, the administered dosageof the composition comprising Hélicobacter ureasepeptides may range from 100 ;zg to 1 g, for example, 0.14mg per kg of body weight. Those of skill in the art willrecognice that the optimal dose may be more or lessdependmg upon the patient1 s body weight, disease, theroute of administration, and other factors. The dosage

S3S 0 1 0372 28 level is readiiy àeterminable by standard methods. Thenumber cf doses will dépend ucon the disease, theformulâtron, and efficacy data frcm clinical trials. Forexample, the dosage may ne administered cver 3 to S dosesfor a primary immunisation schedule over 1 month. Thiscourse of treatment is an example and is not meant to be1imiting.

In one related aspect, the method involves theadministration of a composition comprising Helicobacterurease peptides wherein the composition is administeredin association with a microsphere carrier. Such amicrosphere carrier may be, for example, but is notlimited to, a polylactide-coglycolide biodégradablemicrosphere carrier.

In another related aspect, the method involves theadministration of a composition comprising theHe1icobacter urease peptides wherein such compositioncomprises a recombinant live vector which expresses aHélicobacter urease peptide. Those skilled in the artwill recognize that such live vector may be, for exemple,a bacterial or a viral vector. For example, without anylimitation, the live vector may be selected from thegroup consisting of Salmonella typhimurium, Salmonellatyphi. Shigella. Bacillus, Lactobacillus, BCG,

Escherichia coli, Vibrio cholerae , Campylobacter, yeast,Herpes virus, Adenovirus, Poliovirus, Vaccinia, andAvipox. In addition, a carrier System which expresses aHelicobacter urease peptide, such as Bluetongue virus-like particles, Rotavirus-like particles, and Ty 29 01 0372 particles, may be used :o deliver the urease peptide. Ina preferred aspect, the iive vectcr or the carrier Systemmay be administered to a mucosal surface. A preferred embodiment of the présent inventioncomprises a method of treating a huraan infected withHélicobacter pylori. comprising orally administering atherapeutically effective amount of a compositioncomprising the ure B subunit of Hélicobacter pyloriurease, in association with a mucosal adjuvant selectedfrom the group consisting of choiera toxin, procholeragenoid, choiera toxin B subunit, fungal polysaccharides, including schizophyllan, muramyl, dipeptide, muramyl dipeptide dérivatives, phorbol esters,liposomes, microspheres, non-Helicobacter pyloribacterial lysâtes, labile toxin of Escherichia coli,block polymers, saponins, and ISCOMs, the compositionbeing administered in a particulate form in associationwith hydroxyapatite. The composition may also beadministered with concurrent oral administration of achewable 1.0 g NaHCO, tablet. For the purposes of thisinvention, the term "in association with" includes anytype of association, including but not limited to, aChemical or genetic association, such as that présent ina fusion protein.

In a fourth aspect, the invention provide a methodof treating gastroduodenal disease in a mammal,comprising administering a therapeutically effectiveamount of a composition comprising an antibody thatrecognizes Hélicobacter urease. The gastroduodenal 0 1 0372 - 30 - disease included within the scope cf the invention arereferenced acove. These methods use the abcve-ref ere.aceccomposition comorisino Hélicobacter urease peptides toeiicit an antibody response in a mammal . The antibodies 5 produced by the immunized mammal are isolated and administered to the subject mammal. The préparation cfantibodies that reccgnize a given antigen, such as theacove-referenced composition, is known te those skilledin the art. For example, polyclonal and monoclonal IC antibodies can be prepared following the disclosure inHarlow, E. & Lane, D., Antibodies; A Laboratory Manuel(1988).

In a more particular related aspect, theadministered antibody is a monoclonal antibody. The 15 préparation of monoclonal antibodies is known to thoseskilled in the art.

More particularly, the administered antibody is anIgA antibody, either a polyclonal or monoclonal IgAantibody. The préparation of monoclonal IgA antibodies 20 is known to those skilled in the art and may be found :n,for example, Winner, L., et al., "New Mode! for Analysasof Mucosal Immunity: Intestinal Sécrétion of SpécifieMonoclonal Immunoglobulin A from Hybridoma TumorsProtects Against Vibrio Cholerae Infection" Infect, and 25 Immun., vol. 59, 977-982 (1991); and Weltzin, R., et al.,"Binding and Transepithelial Transport of Immunoglobulinsby Intestinal M Cells; Démonstration Using MonoclonalIgA Antibodies Against Enteric Viral Proteins" J. CellBiol, vol. 108, 1673-1685 (1989). 31 010372

The terri "antibody" as used for the purposes ofthis invention ir.cludes, but is not limited to,polyclonal antibodies, rr.onocional antibodies, and anti-idiotypic antibodies. The antibodies may be naturallyderived from any animal, synthesized in bacteria oranother non-animal source, chemicaliy synthesized, orgenetically synthesized.

In a preferred aspect of the invention, a methodis provided of treating a human infected withHélicobacter pylori, comprising administering atherapeutically effective amount of a compositioncomprising an IgA monoclonal antibody that recognizes theure B subunit of Hélicobacter pylori urease.

In a fifth aspect, the invention provides acomposition useful in the therapeutic treatment ofgastroduodenal disease, comprising Hélicobacter ureasepeptides. The gastroduodenal diseases included withinthe scope of the invention, as well as the Hélicobacterurease peptides and mucosal adjuvants included within thescope of the invention, are referenced above.

In a related aspect, the composition comprisesHélicobacter urease peptides and a mucosal adjuvant.

In another related aspect, the composition is inparticulate form. The composition may exist inparticulate form through association with a carrier. Thecarrier may be, for example, hydroxyapatite, asreferenced above. This is only an example, and is notmeant to be limiting as to the type of carrier that maybe used. 010372 J Z.

More particuiarly, the composicicn is présent mparticuiats fcm, in a liquid suspension. ïn a related aspect, the composition compriseselic oc a et sr urease peptides in association with a Ξ microsphere carrier. Such microsphere carrier may be,for example, but is not limitée to, a polylactide-coglycolide biodégradable microsphere. The ureasepeptides may be encapsuiated in che biodégradableraicrospheres. Polylactide-coglycolide microspneres 0 slowly hydrolyse in the presence of water and beccn.ewater-soluble, thus delivering the peptides that areincorporated in the microsphere.

In another related aspect, the compositioncomprises Helicobacter urease peptides wherein the 5 composition comprises a recombinant live vector whichexpresses a Hélicobacter urease peptide. Those skiiledin the art will recognize that such live vector may be,for example, a bacterial or a viral vector. For exemple,without any limitation, the live vector may be selected 0 from the group consisting of Salmonella typhimurium,

Salmonella typhi . Shiciella, Bacilias , Lactobaci1lus . BCG,Escherichia cο1i, Vibrio cholerae . Campylobacter, yeast,Herpes virus, Adenovirus, Poliovirus, Vaccinia, andAvipox. In addition, a carrier System which expresses a 5 Helicobacter urease peptide, such as Bluetongue virus-lihe particles, Rotavirus-like particles, and Typarticles, may be used to deliver the urease peptide.

In a preferred embodiment, a composition isprovided that is useful in the theraüeutic treatment of fs ' ’ 010372 '33 - Hélicobacter. nylorl infection ci a huraan, corrçrising theure 3 subun.it cf Hélicobacter tylcri urease, a mucosaladjuvant selectec from a group ccnsisting of choieratoxin, procr.oleragenoid, choiera toxm B subunit, fungal 5 polysaccharides, includmg schizopnyllan, muramyl dipeptide, muratr.yl dipeptide dérivatives, phorbol esters,liposomes, microspneres, non-Helicobacter pylcri bacterial lysâtes, labile toxin of Escherichia coli,block polymers, saponins, and ISCOMs, and further 1' comprising hydroxyapatite, wherem the composition is ma particulate fort in a liquid suspension. The term "inassociation with" is definec as referenced above.

In a sixth aspect, a composition is providedthat is useful in the therapeutic treatment of 15 gastroduodenal disease, comprising an antibody thatrecognizes Helicobacter urease. The gastroduodenaldisease included within the scope of the invention, aswell as the antibodies included within the scope of theinvention, are referenced above. 20 In a preferred embodiment of the invention, a composition is provided that is useful in the therapeutictreatment of gastroduodenal disease, comprising an IgAmonoclonal antibody that recognizes the ure B subunit ofHelicobacter oylori urease. 25 While not being bound by any theory, the présent inventors believe that administration of the urease antigen, or B subunit thereof, to a mucosal surface stimulâtes the common mucosal immune System and perhaps local sites in the gastric mucosa inducing an immune 010372 - 34 - response, ir.ciuding the appearar.se of Hel iccbacter pvl crispécifie IgA. ar.tibodies in the aastric sécrétions, whichprevent Helicobacter infection. Secacse of this relief,the terms "ittiunioaticn" and "vaccine" are used herein ovtheir common meanings as recogncced b’/ those skilled mthe art and also to ir.dicate metheds and compositionsused for the treatment of Hel iccbacter infection, withcutlimitation to these meanings, and without being bour.d ttany theory regarding the mechanis- of treatment.

Since it is a routine matter to conduct pre-clinical trials of candidate vaccines for human use inanimal models, it is believed that the methodolcgy cf theprésent invention is effective in humans, especially inthe prévention and treatment of peptic ulcers, gastrites,gastric maiignancies and other conditions arisir.g as aresuit of the présence of Hélicobacter pylori and/or H.teljs.

Based on the dosage and course of treatment thatsuccessfully eradicated infection in the mouse mcdel, apreferred range of dosage would be 100 gg to 10 g ofHélicobacter urease peptides. Those skilled in the artwill recognize the appropriate dosage level to test fromresearch reported for other oral vaccines such as, forexample, the research performed with Escherichia colilysate (6 mg dose daily up to a total of 540 mg' and withan enterotoxigenic E. ccli purified antigen (4 doses cf 1ma) . (Schulman et al,, "Oral Immunctherapy of RécurrentUrinary Tract Infections: A Double - Elind Placebo-Controlled Multicenter Study," J. Urci., vol. 150, 9L7- 35 010372 921 (1993); Boedeker et al., "Saiety, Immunogenicity andEfficacy in Human Volunteers of Biodégradable,

Biocompatible Microspneres Contamine Colonization FactorArtigen/Il (CFA/II) as an Enterai Vaccine AgainstEnterotoxigenic E. coli (ETEC) ".AmericanC-astroenterological Asscc., vol. 999, A-222 (1993) .

Wrthout intending any limitation as te the course cftreatment, the treatment could te administered over 3 to8 doses for a primary immunisation schedule over 1 month.(Boedeker, American Gastroenterciogicai Assoc., vol. 88 Ξ,A-222 (1993) ) . A recommended method of immunisation isadministration of the Helicobacter urease peptidecomposition in the form of a liqutd alone or suspensioncontaining Na2HCO, or similar material to temporariiyneutralize gastric acid. The neutralizing material mayalso be delivered separately at the time the peptidecomposition is administered, such as in the form of achewable Na2HCO3 tablet. Alternattvely, the compositioncan be administered in the form of enteric-coatedcapsules. These methods will likely avoid the problem cfdégradation of the urease composition during its passagein the upper gastrointestinai tract. These methods arereviewed in, for example, Levine à Norlega, "Vaccines tePrevent Enteric Infections," Ballreres Clin. Gastro.,vol. 7, 501-517 (1993) .

Although a high dose of choiera toxin is notpreferred as a mucosal adjuvant fer human use, othermucosal adjuvants recognized by these skilled in the art, 010372 - 3 ο and as referenced accve, wiil ce useful for treatrsr.: tfhumans .

It ·,·ί”11 de readilv acDaracc to one skilled ir. cfeart cnac varyir.ç substitutions and mcdif icaticns ma y bemade to tne invention dicciosed hersm withcut deçartmofrora the scope and spirit of the invention. 3irx>3 ;- .D*?s?,ct~lPH_l·c ~ .DxT'-iwi nos

The invention wiil now de further descrtbed w:threference to the accompanving drawings, in whichFigures 1 through 8B are graphies! représentations of therésulta set forth in Tables 1 through 3.

Figure 1 is a graphie représentation of theresults from Table 1 of tests for antibodies in sérum(IgG) and intestinal sécrétion (IgA) in mice that werenot proteoted after immunisation with urease.

Figure 2 is a graphie représentation of theresults from Table 1 of tests for antibodies in sérum(IgG) and intestinal sécrétion (IgA) in mice that wereprotected after immunization with urease.

Figure 3 is a graphie représentation of theresults from Table 1 of the tests for antibodies in sérum.(IgG) and intestinal sécrétion (IgA) in mice that werenot protected after immunization with Hélicobacter pylcnsonicate.

Figure 4 is a graphie représentation of theresults from Table 1 of the tests for antibodies in sérum.(IgG) and intestinal sécrétion (Iç.A; in mice that were 010372 - 37 - crotected af;er iramunizatior. with Hélicobacter pylorisonicate.

Figure 5 is a graphie représentation of therésulté set eut in Table 2 ccmparing the level ofprotection ofctained with Hélicobacter pylori urease ascompared to that obtained with Hélicobacter pylorisonicate and with choiera toxin.

Figures 6A and 65 are graphie représentations ofthe results set out in Tables 5 and 6 which measuresurease activity in mice that were challenged after oralimmunization with recombinant urease A and B subunits.

Figure 7 is a graphie représentation of theresults set out in Table 8 which measures gastric tissueurease activity as a reflection of Hélicobacter infectionin mice that were subsequently treated with the Hélicobacter pylori ure B subunit, choiera toxin andhydroxyapatite only (sham-immunized), or untreated.

Figures 8A and 8B are graphie représentations ofthe results set out in Table 9 which measures gastrictissue urease activity as a reflection of Hélicobacterinfection in mice that were subsequently treated with theHélicobacter pylori ure B subunit, or choiera toxin andhydroxyapatite only (sham immunized. For Figure 8A, themice were sacrificed and urease assays performed 2.5weeks after the last immunization. For Figure 8B, themice were sacrificed and urease assays performed 8 weeksafter the last immunization. 010372 - 38 - ver.*: mvenlors n; a;scoverea mat ora_ aCTir.istrat ;sr. ~ o x:e or pclyamircacid préparationsexhibitmg the spitcces of Hélicobacter oylori urease gives rase te a protective immunological respcr.se againstH. f elis m rr.ice, an animal mode! of generaliy-acceptedvalue for the study of the immune resconse to

He 1_1 cQ-ba,cter infection (Lee, A. et al. "A Small AnimalModel of Kuman Hélicobacter nylon Active Chronic

Gastritis" Gastroenterology, vol. 99, 1315-1323 (1950)) , and a recogniced pathogen causing gastritis in hutnars(Wegtnan, W. et al., Schweig, Med. Wochenschr, , vol. 121,245-54 (1991)) . The effect of the protective immune reuponse is that iinmunized animais, when challer.ged withpathogen, hâve a greatlv reduced incidence of infection,in conroarison to non-immunized. animais. Furthermore, tbs inventors hâve discovered that oral immunization in miceusing Hélicobacter pylori urease B subunit, produced asan enzymatically-inactive recombinant protein, gives riseto a protective immunological respense in mice against H.f e1i s . The effect of the protective immune respense isthat immunized animais, when challer.ged with pathogen,hâve also a greatly reduced incidence of infection, inccmparison to non-immunized animais which do becor.sinfected. clearance of the

The présent inventors hâve discovered that oral administration of He !. icobacr or urease peptides to mice infect.ed with H . f e 1 i. s results in the 010372 - 39 - infection. This resuit indicates that the oraladministration of He 1 icotac t e r trease peotides is aneffective therapy for the treatment of Hélicobacterinrecticn ir. mammals. The oral administration of = Hélicobacter pylori urease 3 subunit, prcduced as an enzymatically-inactive recombinant protein significantlydecreases the level of H, f e 1 i s infection in infectedraice, in comparison to control infected mice.

The following examples are offered by way ofIC illustration and are not intended to limit the invention in any manner.

Bacterial Cultures and Urease Purification

The strain of Hélicobacter pylori used in thestudy originates from a patient with a duodenal ulcer, 15 and has been subcultured on BHI agarose plates tohoinogeneity. He 1 i cobac t e r py 1 or i is cultured in asuitable medium, typically BHI (Brain-Heart Infusion)medium, containing 0.25% yeast extract and 10% fêtai calfsérum and supplemented with 0.4% yeast extract and 10% 20 fêtai calf sérum and supplemented with 0.4% Campylobactersélective complément (Skirrow supplément; Oxoid 69). Thebacteria are incubated under microaerophilic conditionsat 37°C in bottles that are sealed and shaken at 37°C for2 to 3 days to produce a liquid culture. A culture may 25 also be prepared in agarose plates consisting of BHI with 0.25% of yeast extract and 5% of sheep blood under microaerophilic conditions at 37°C for 3 days. The quantity of bacteria is determined by optical density of 40 010372 the BHI solution at 660 nm, with one optical density unitcorresponding to 108 bacteria. Cultures on aaarose platesare first resuspended in 154 mM NaCl.

One currently preferred source of polyaminoaciddisplaying urease epitopes is purified urease, e.g.,Hélicobacter pylori urease obtained by following thegeneral method of Dunn et al. J. Biol. Chem. 265, 9464-9469, modified as described below. Following culturing,the Hélicobacter pylori is harvested in water, spun,vortexted and spun again to produce a supernatant. Thesolution containing the urease activity of Hélicobacterpylori (assessed by rapid urease test, see below) is thenchromatographed on a CL-6B sizing column and thefractions which présent a strong urease activity arepooled and dialyzed overnight and again chromatographedon an anion exchanger gel. The fractions are eluted inincreasing NaCl buffer and the collected fractions with astrong urease activity are individually submitted to anSDS gel followed by Coomassie staining. Two distinctbands corresponding to a molecular weight of about 63 andabout 28 kDa are identified as urease. The fractionscontaining urease are pooled to give purifiedHélicobacter pylori urease having a purity in the régionof 95% to 99%. B. Oral Immunization with Urease Purified from Hélicobacter pylori

While it is preferred to employ purifiedHélicobacter pylori urease obtained as described above asthe antigenic material, it will be understood that it is 41 010372 also possible to use, as the antigenic matériel, anyurease or subunit of urease, either naturaily occurringor obtained by recombinant DNA techniques, as well asdigested fragment thereof, fusion proteins comprising thefragments or the whole urease, truncated ureaseconstructs, or other peptide or protein préparationsexhibiting urease epitopes which are capable of elicitinga protective immune response to Hélicobacter infection(See below). Thus, it is possible to employ a ureasehaving a substantiel homology with respect to Hélicobacter pylori urease and which is effective inraising a cross-protective immune response toHélicobacter. An example of suer, a urease is jack beanurease, which possesses about 70% homology withHélicobacter pylori urease. Although it was once thoughtthat jack bean urease would be an effective antigen toprevent infection by Helicobacter. this is no longerbelieved to be the case. (See Chen, M. et al. "Faiiureof Immunization Against Hélicobacter Usina Jack BeanUrease," Acta Gastroenterol. Belg., Vol. 56, 94 (1993)).

The invention is therefore not limited to the use ofintact urease, and covers the use of any polyaminoacidpréparation which displays urease epitopes sufficier.tlyhomologous to Helicobacter pylori urease to generate aprotective immunological response in a host toHelicobacter infection. A suitable urease must hâvesufficient homology to H. pylori urease to elicit aprotective immune response against Hélicobacterinfection. Typically, a urease having a homology of 42 010372 greater than 70%, for example, 80-90% homology, withrespect to Hélicobacter pyiori urease, may be employeà asthe urease antigen in the invention. A non-limiting list of sources cf potentiallyuseful urease préparations includes endcgenous ureaseenzymes of the different Hélicobacter species, ureasefrom other bacteria such as Klebsiella pneumoniae orProteus mirabilis, and, by analogy, any other ureasewhich the condition that these ureases share cross-reactive epitopes with Hélicobacter pyiori urease. Theurease genes of ail the organisms mentioned aboverepresent a potential tool for expressing recombinanturease products as a whole protein or as a part thereof. A non-limiting list of potentially useful ureasepréparations includes peptides generated from purifiedurease (the sources are mentioned above), using physicaland/or Chemical cleavage procedures (i.e., CnBr) and/orproteolytic cleavage (using proteases, e.g., V8-protease,trypsin or others); or peptides syntnesized chemicallyand retaining cross reactive epitopes with urease.

Other sources of potentially useful epitopesinclude epitopes identified by their crossreactivity withurease, as the resuit of screening with anti-ureaseantibodies. These peptides can be naturally occurringpeptides or peptides resulting from Chemical synthesis.Furthermore such peptides can resuit from the expressionof recombinant random oligonucleotides.

Another source of potentially useful epitopesincludes epitopes similar to urease as a resuit of the 43 0 1 0372 génération of anti-idiotypie antibodies to urease. Suchanti-idiotypie antibodies, generated in anyimmunocompétent host, are obtained by immunization ofthis host with anti-urease antibodies, with the goal et Ξ generating antibodies directed against anti-ureaseantibodies, which share structural homologies withurease.

The discussion herein focuses on the use of ureasenaturally produced by He1icobacter pylori (section 3). IG However, it will be appreciated that the urease or subunits or constructs thereof mentioned above, capableof eliciting the desired protective immune response, maybe produced by recombinant DNA techniques well known inthe art. The efficacy of particular préparations may be 15 determined by routine administration using animal models,oral administration of the candidate vaccine, andchallenge with pathogen using a protocol substantiallvsimilar or identical to the procedure described below.

It will be recognized by those skilled in the art 20 that other methods may be used to administer the

Helicobacter urease peptides. For example, the ureasepeptides may be administered as part of a microspherecarrier formulation. Without limiting the type ofmicrosphere carriers used, one example would be the 25 administration of the urease peptides as part of a polylactide-coglycolide biodégradable microsphere carrierformulation.

Another method of administering the Helicobacterurease peptides would be to express the urease peptides

Alti' ίι>-- - 010372 44 in a recombinant form in a live vector, for example, abacterial or a viral vector. To construct such a livevector, nucléotide sequer.ces ccding for urease peptideswould be incorporated into the genetic material of a live 5 vector. Such live vector would be administered to anindividual for the purpose of preventing Helicobacterinfections and aise for the purpose of treating anindividual already infected with Hélicobacter. Thcseskilled in the art will recognize that examples of IC appropriate live vectors include Salmonella tyohimurium,Salmonella typhi, Shioella, Bacillus, Lactobacl1lus., BC3,Escherichia col i , Vibrio cholerae, CampylobaclLer, yeast,Herpes virus, Adenovirus, Poliovirus, Vaccinia, andAvipox. In addition, a recombinant carrier System which 15 expresses a Hélicobacter urease peptide, such as

Bluetongue virus-like particles, Rotavirus virus-likeparticles, or Ty particles may be used to deliver theurease peptide. The aforementioned list is not meant tobe limiting. Preferably, the live vector or the carrier 20 System would be administered mucosaily such that the recombinant urease peptides expressed by the live vectorwould be administered to a mucosal surface.

Tables 1 and 2 below and Figures 1-5 describe therecuits obtained when mice were oraily immunized with 25 purified Hélicobacter pylori urease. In this first experiment, administration of the Hélicobacter pyloreantigen was carried out by oraily administering to themice Hélicobacter pylori urease purified as described in section A, and coupled to hydroxyapatite crystals, used 010372 - 45 - as a carrier to enhance M cells bindina and uptake.

Choiera toxin (Sigma) was given as a mucosal adjuvant.

In this experiment, groups of female SFF BALB/c six-weekold mice were each orally immunized with 30 μ g of 5 purified Hélicobacter pylori urease coupled to 1 mg ofhydroxyapatite plus 10 pg of choiera toxin adjuvant atday 0, 7, 14 and 21. The mice were then challenged twice with 108 H, felis, at day 28 and 30. For comparisonpurposes, similar female SPF BALB/c six-week old mice IC were orally immunized with whole Hélicobacter pylori lysate (sonicate) and 10 /xg choiera toxin at day 0, 7, 14 and 21. The mice were challenged at day 28 and 30 withH. .felis. The Hélicobacter pylori sonicate was preparedby collecting Hélicobacter pylori from cell cultures, 15 pelleting by centrifugation and resuspending the pelletin 0.9% sodium chloride followed by sonication.

As a control, female SPF BALB/c six-week old micewere orally sham-immunized with 10 /xg of choiera toxinand 1 mg of hydroxyapatite at day 0, 7, 14 and 21. The 20 mice were then challenged at day 28 and 30 with H. felis.Ail mice were housed and immunized in parallel. Ail micesubject to the study were sacrificed on day 35. C. Oral Immunjzation with Recombinant Urease Subunits of Hélicobacter pylori, 25 Genes encoding the structural A and B subunits of Hélicobacter pylori urease were obtained by polymerase cham reaction (PCR) cloning according to standard procedures, based on previcusly published sequences.

££^S®B®ï2â&2âa&HL 010372 \ - 46 - (Clayton, C.L. et al. "Nucléotide Sequence of Two Genesfrom Hélicobacter pylori Encoding for Urease Subunits"

Nucleic Acid Res., vol. 18, 362 (1990)). These geneswere inserted in a vector (named pEV40) designed for highexpression and easy purification of foreign genes in E.g_Q.l_i · Briefly, the foreign gene is inserted downstreamof a thermo-repressible promoter, and in frame of asequence encoding for a repeat of six histidines. AnampR gene is présent on this vector for sélection oftransformants. Under the appropriate températureconditions, the recombinant protein obtained issupplemented by six histidines at the N-terminal, whichallow for a one-step affinity purification on a nickelcolumn. Both Hélicobacter pvlori recombinant urease Aand B subunits were expressed separately in E, coli, andpurified on a nickel column to >95% purity.

While it is preferred to employ recombinantHélicobacter pvlori urease obtained as described above asthe antigenic material, it will be understood that it isalso possible to use, as the antigenic material, anyurease or subunit of urease obtained by recombinanttechniques (e.g., fusion proteins) expressing antigenicsites of urease, which is capable of eliciting a protective immune response to Hélicobacter infection.

Thus, it is possible to employ in a construct a urease gene having a substantial homology with respect to Hélicobacter pylori urease and which is effective in raising a cross-protective immune response to Hélicobacter. Examples of such a urease is jack bean t - k <ί 010372 - 47 - urease, which possesses about 70% homology withHélicobacter .pylori urease, or H. felis urease, whichpossesses about 88% homology with Hélicobacter pyloriurease. The invention is therefore not limited to the 5 use of Hélicobacter pylori urease genes and their geneproducts, and covers the use of any recombinant urease,or the subunits thereof, which is sufficiently closeantigenically to generate a protective immunologicalresponse in a host to Hélicobacter infection. The 10 invention includes within its scope the use of any ureaseor subunit of urease, either naturally occurring orobtained by recombinant DNA techniques, as well as adigested fragment or peptide thereof, fusion proteincomprising the whole urease, subunit or fragment thereof, 15 or truncated urease construct which is effective inreducing the level of Helicobacter infection in aninfected mammal. Typically, a recombinant urease havinga homology of 70-95% homology, for example, 80-90%homology, with respect to Hélicobacter pylori urease, may 20 be employed as the recombinant urease antigen in theinvention.

The discussion herein focuses on the use ofrecombinant Hélicobacter pylori urease A and B subunitsproduced by E. coli (section C). However, it will be 25 appreciated that recombinant urease or subunits or constructs thereof mentioned above, capable of elicitingthe desired protective immune response, may be producedusing other recombinant DNA techniques and other Λ 010372 - 48 - eukaryotic or prokaryotic expression vectors well kncwnin the art.

Tables 3, 4 and 5 below and Figure 6 describe theresults obtained w'nen mice were orally immunized withrecombinant Hélicobacter pylori urease subunits producedin E. coli. in this experiment, administration ci the Hélicobacter pylori antigen was carried out by orallyadministering to the mice reccmbinant Hélicobacter pyloriurease A or B subunits produced in E, coli and purifiedas described above, and coupled to hydroxyapatitecrystals, used as a carrier to enhance M cell binding anduptake. Choiera toxin (Sigma) was given as a mucosaladjuvant. In this experiment, groups of female SPFBALB/c six-week old mice were each orally immunized with30 gg of recombinant Hélicobacter pylori urease Asubunit, coupled to 1 mg of hydroxyapatite plus IC gg ofchoiera toxin adjuvant at day 0, 8, 14 and 21. The mice were then challenged with 10H H. fells, at day 32, 34 and 36. For comparison purposes, similar female SPF BALB/csix-week old mice were orally immunized with 30 gg ofrecombinant Hélicobacter pylori urease B subunit coupledto hydroxyapatite plus 10 gg choiera toxin at day 0, 8,

14 and 21. The mice were challenged three times, at day32, 34 and 36, with H, felis. As a control, female SPF BALB/c six-week old mice were each orally sham-immunizedwith 10 gg of choiera toxin and 1 mg of hydroxyapatite atday 0, 8, 14 and 21. The mice were then challenged at day 32, 34 and 36 with H. felis. Ail mice subject to the study were immunized and challenged in parallel. Animais 010372 - 49 - were sacrificed on day 48 (12 days after challenge) or 10weeks after challenge. EL·. Analysis of Gastric Biopsies, Blood, and

Intestinal Sécrétions 5 Biopsies were taken from the stomach and blood was obtained from the heart. The intestines were removed andwashed with 1 mM PMSF (Boeringher) in PBS buffer teobtain intestinal sécrétions for ELISA analysis.

To evaluate protection against H, felisIC colonization, gastric biopsies from each animal were screened for the presence of H. felis by assessing rapidurease activity by the Jatrox HP test (Rohm Pharma),according to the supplier's directions. Briefly, gastricbiopsies are immersed in 0.5 ml supplier's mixture of 15 urea and phénol red, a pH indicator. A non-commercialversion of the urea and phénol red mixture will berecognized by those skilled in the art and may contain,for example, Bacto yeast extract (o.lg), monopotassiumphosphate (0.091g), disodium phosphate (0.095g), urea 20 (20g), and Bacto phénol red (0.01g) at a final pH of 6.5 at 25°C. Urease activity generates ammonia andbicarbonate from urea, and is followed by thecolorimétrie change of the solution towards a higherabsorbance at 550 nm. Urease activity was quantified by 25 spectrophotometric analysis. Other methods of assayingurease activity will be recognized by those skilled inthe art and may be found in, for example, Mobley, H.L.T. &amp; Hausinger, R.P., "Microbial Ureases: Significance, 50 010372 Régulation, and Molecular Characterization, " Micrcb.Reviews, Vcl. 52, 35-103 (1933} .

Gastric biopsies of each animal included in theexperiment described in section 3 were also cuitured enEHI agarose niâtes, supplemented as above, for thedétection of B. f e 1 i s . After incubation for 3 to 10 daysm microaerophilic conditions, the presence of H. f g 1 i swas confirmée by Gram staining and détermination cfurease activity. As a very significant corrélation wasobtained for the détection of H. f e1i s in gastricbiopsies between urease tests and H, felis culturesduring the first set of experiments (See Table 3), onlygastric biopsy urease tests were performed for thedétection of H, felis in the experiment described insection C. Détection of H. f e1i s was confirmed bymicroscopy by two independent investigators, using twodifferent colorations (acridine orange and cresylviolet) .

Blood samples were allowed to clôt for 3 hours atRT (roora température), and sérum harvested and frozen at-20°C, until further analysis. Intestinal sécrétionswere spun for 5 minutes at 4°C to remove débris, and keptfrozen at -20°C. Sérum and intestinal samples of eachanimal were analyzed by EL.ISA for évaluation of anti-Hellcobacter activity, according to standard procedures.Briefly, 96-well plates were coated with a sonicate ofBelLcobacter pylori. followed by saturation with 5% fat-free milk. Samples were serially diluted from 1:1 to1:1000 and incubated overnight at 4°C on 3LISA plates. 51 010372

Siotinylated anti-mouse IgG (sérum) and anti-mouse IgA,followed by streptavidin-Korseradish peroxidase was usedfer the détermination of the antibody levels.

The résulta of H. f g 1 i s challenges following5 immunizations with purified Hélicobacter oylori urease are set out in Tables 1-3 and Figures 1-4 and the resuitsof H. .£ e1i s challenges following immunizations withrecombinant Hélicobacter pylori urease A and E subunits are set out in Tables 4-6 and Figures 5 and 6.

Table 1 mouae nurnber Immuniiation urease test culture Gram Immunoglobuline Sérum Intestinal sécrétion 12h ig Ig G ig IgA 1 Urease+HF + H. fells 27 0 25 253 2 Ureaae+HF 0 0 264 273 221 246 3 Ureaee+HF 0 0 84 44 318 354 4 Ureaae+HF + H. fells 81 42 12 5 5 Urease+HF 0 0 98 137 126 234 6 Urease+HF + 0 968 2093 31 22 7 Urease+HF 0 0 98 0 96 34 8 Urease+HF 0 0 247 1010 214 128 9 Urease+HF 0 0 N.D . N.D. 48 23 10 Urease+HF 0 0 50 0 124 99 11 Urease 0 0 319 205 44 53 12 Urease 0 0 14 0 86 87 13 Urease 0 0 0 0 0 0 14 Urease 0 0 0 0 43 61 15 Urease 0 0 53 0 110 127 16 Urease 0 0 140 63 21 37 17 Urease 0 0 84 240 114 280 18 Urease 0 0 N.D. N.D. 93 148 i 19 Urease 0 0 45 0 135 216 ' 20 Urease 0 0 261 197 161 261 ; 21 CT+HF 0 0 0 0 0 2 2 7 | CT + HF ·( H. fells 63 0 310 303 01 0372 - 52 -

Table 1 Continued 25 CT+HF + fi. felis 197 250 250 440 26 CT+HF + felis J 105 135 214 138 27 CT+HF + H. felis 140 47 109 55 28 CT+HF + 0 0 0 16 15 29 CT+HF N. felis 0 0 0 0 30 CT+HF 4- fi. felis N.D. N.D. N.D. N.D. 31 HP Bonicate+HF + H. felis 0 0 76 103 32 HP 3onicate+HF fi. felis 77 0 11 33 33 HP sonicate+HF + fi. felis 549 748 57 36 34 HP sonicate+HF 0 0 660 153 180 286 35 HP sonicate+HF + fi. felis 730 192 0 5 36 HP sonicate+HF + fi. felis 32 0 5 64 37 HP eonicate+HF 0 0 400 400 312 1149 38 HP Bonicate+HF + fi. felis 1007 1360 149 26 39 HP sonicate+HF 0 0 220 186 133 122 40 HP sonicate 0 0 873 1016 352 514 41 HP aonicate 0 0 727 899 126 191 42 HP sonicate 0 0 109 68 44 83 43 HP sonicate 0 0 147 949 167 97 44 HP sonicate 0 0 845 1094 246 64 45 HP sonicate 0 0 1217 1198 210 157 46 HP sonicate 0 0 81 0 256 218 47 HP sonicate 0 0 329 210 241 276 48 HP sonicate 0 0 1049 737 197 211 010372

In Table 1, which refers to the experimentdescribed in section 3, "h" means hour, "Ig" means immunoglobulin, "ND" means "not determined", "urease -K?" means that the mice were immunized with urease(coupled to hydroxyapatite, with choiera toxin) and ther.challenged with H. félis, "urease" means that the micewere immunized with urease (coupled to hydroxyapatite,with choiera toxin) and not challenged, "CT+HF" meansthat the mice were sham-immunized with choiera toxin andchallenged with H, felis, "HP sonicate + HF" means thatthe mice were immunized with Hélicobacter pylori sonicatewith choiera toxin and challenged by H, felis, and "HPsonicate" means that the mice were immunized withHélicobacter pylori sonicate with choiera toxin and notchallenged. In Table 1, the numbers for the ar.tibodyresults are given as a measure of absorbance at 595 nmmultiplied by 1000. The background measured in absenceof the antibodies, was subtracted.

The results of experiment described in section 3obtained on the basis of the gastric biopsies ureasetests and on Gram staining of H. felis cultures are setout in Table 2 . Infection was defined by mice with oneor more markers of colonization by H. felis, includingurease or Gram staining of cultures. Ά ν5^:' · 0 1 0372 TA3LE 2

Immunization Chai ler.ge % infected % protected Urease H. felis 3/10 (3 0%) 7/10 (70%) * Sonicate H. felis 6/9 ( 66%) 2/9 (33%) ** CT H. felis 9/10 (90%) 1/10 (10%) * p=0.0I98 (two tailed Fisher exact test) comp, to CT contre! ** ρ-0.303 (twc tailed Fisher exact test) comparée, to CT control 10 It will be seen from the results set out in Tables 1 and 2 that statistically significant protection againstH. felis challenge is obtained with oral immunizationusing Hélicobacter oylori urease as compared to thatobtained using either Hélicobacter oylori sonicate or 15 choiera toxin. Referring to Table 2, it will be seen that from a total of 10 immunized animais, only 3 becameinfected, as compared to 6 of the animais immunized withHélicobacter pylorl sonicate and 9 of the animaisimmunized with choiera toxin. Table 2 shows that 70% of 20 the animais were protected from challenge by H. felis ascompared to 33% of the animais immunized withHélicobacter pylorl sonicate and 10% of the animaisimmunized with choiera toxin and then subjected to H.fe1is challenge. In other words, 90% of the control mice 25 exposed to H. f e1i g became infected by that pathogen whereas, in contrast, in mice immunized with Helicobacter oylori urease 23 davs before exposure to H. felis. the infection rate was only 30%. 55 010372

This represents a significant réduction in infection(p=0.019S in the Fisher exact test, as compared to thecontrol mice,. When the mice were orally immunized withHélicobacter pylori sonicate, the infection rate vas 67%(not sianificant versus the control). The protectionobtained usina Hélicobacter pylori urease is unexpectedand could not hâve been predicted on the basis of theresults observed using Hélicobacter pylori sonicate.

Referring to Figures 1-4, Figure 1 representsgraphically the results of tests for antibodies in sérum(IgG) and intestinal sécrétion (IgA) in mice not protected after immunization with urease. These are micenumbers 1, 4 and 6 appearing in Table 1, and constituteGroup A. Figure 2 shows the antibody responses of micethat were protected after immunization with urease (GroupB), i.e. mice 2, 3, 5 and 7-10.

Figures 3 and 4 relate to the results obtainedwith mice numbers 31-39. Figure 3 (Group C) depictsantibody responses of mice not protected afterimmunization with Hélicobacter pylori sonicate (micenumbers 31, 32, 33, 35, 36 and 38) and Figure 4 (Group D.depicts the antibody responses of mice protected afterimmunization with Hélicobacter pylori sonicate (micenumbers 34, 37 and 39) . It is of interest to note with respect to Figures 3 and 4 that the IgA antibodyresponses (but not IgG) are higher in the mice exhibitingprotection than in the mice that are not protected,suggesting a corrélation between protection and IgAresponse. Sérum IgG responses did not exhibit a 0 10372 56 corrélation. Mucosal IgA but notplay a rôle in protection againstthe gut. (McGhee, J.R. and Kyonoin Vaccine Development: MucosalInfect Agents Dis., vol. 2, 55-73 sérum IgG are known tobacterial infections cf, H. "New PerspectivesImmunity to Infections"(1993)). 010372 TABLE 3 + Urease Test

Total 16

Urease Test H. felis 16 culture (+) H. felis 2 culture (-)

Total 18

Two-tailed Fisher's Exact Test: p<0.00001

Table 3 shows that a very significant corrélation exista between the results of urease tests perfortr.ed or.gastric biopsies and the identification of H. felis bycultures. Ail animais shown in Table 3 that werepositive for urease activity, were H, felis positive byhistopathology. As cultures detected less often H. felisinfection than urease tests, urease tests were preferredfor the diagnosis of H. felis infection in mice in thenext experiments, due to its better sensitivity. Thisapproach allowed the duplication of urease tests withlarger fragments of the stomach of each mouse, and afurther increase in the sensitivity of the urease test.Furthermore, the use of the method with the highestsensitivity prevents an overestimation of the protectionobtained by the vaccine préparation to be tested. >Jhenpositive culture is used as the standard for infection,the protection induced after urease immunization duringthe experiment depicted in section 3 is as significant as 30 30 32 48 58 010372 with the cotdoined use of urease cestversus p=O.CLS;.

The results of the experiment ;section C (reccrafainant urease subunit

5 basis of the gasfric biopsies ureasein Table 4, Ξ and 6 and depicted m F and culture p=0.321 s described in s), obtained on the tests, are setigure 6. out 59 010372

In Table 1, which refers ta the experiment described in section B, "h" means hour, "Ig” mear.s immunoglobulin, "ND" means "non determined", "urease -+· HF" means that the mice were immunized with urease 5 (coupled to hydroxyapatite, with choiera toxin) and thenchallenged with H. felis, "urease" means that the micewere immunized with urease (coupled to hydroxyapatite,with choiera toxin) and not challenged, "CT+HF" meansthat the mice were sham-immunized with choiera tcxin anc 10 challenged with H. felis, "HP sonicate + HF" means thatthe mice were immunized with Hélicobacter pylori sonicatawith choiera toxin and challenged by H. felis. and "H?sonicate" means that the mice were immunized withHélicobacter pylori sonicate with choiera toxin and net 15 challenged. In Table 1, the numbers for the antibodyresults are given as a measure of absorbance at 595 nmmultiplied by 1000. The background measured in absenceof the antibodies, was subtracted.

The results of experiment described in section B 20 obtained on the basis of the gastric biopsies urease tests and on Gram staining of H. felis cultures are set out in Table 2. Infection was defined by mice with one or more markers of colonization by H. felis, including urease or Gram staining of cultures. 60 t- 1 MU*.*» > ’{W.ï3*r ii&amp;u 010372 TABLE 2

Immunization challenge % infected % protected

Urease H. felis 3/10 (30%) 7/10 (70%) * Sonicate H. felis 6/9 (66%) 3/9 (33%) ** CT H. felis 9/10 (90%) 1/10 (10%) * p=0. 0198 ( two tailed Fisher exact test) compa to CT control ** p=0.303 (two tailed Fisher exact test) ccnpared to CT control

It will be seen from the results set out in Tables1 and 2 that statistically significant protection againstH., felis challenge is obtained with oral immunizationusing Hélicobacter pylori urease as compared to thatobtained using either Hélicobacter ovlori sonicate orchoiera toxin. Referring to Table 2, it will be seenthat from a total of 10 immunized animais, only 3 becazeinfected, as compared to 6 of the animais immunized withHélicobacter ovlori sonicate and 9 of the animaisimmunized with choiera toxin. Table 2 shows that 70% cfthe animais were protected from challenge by H. felis ascompared to 33% of the animais immunized with Hélicobacter ovlori sonicate and 10% of the animaisimmunized with choiera toxin and then subjected to H.felis challenge. In other words, 90% of the control miceexposed to H. felis became infected by that pathogenwhereas, in contrast, in mice immunized with Hélicobacterpylori urease 28 days before exposure to H. felis, theinfection rate was only 30%.

This represents a significant réduction in infection(p=0.0198 in the Fisher exact test, as compared to thecontrol mice). When the mice were orally immunized withHélicobacter pylori sonicate, the infection rate was 67%(not significant versus the control) . The protectionobtained using Hélicobacter ovlori urease is unexpecred * ,ΛΪ 010372 - 61 - and could not hâve been predicted en the basis of theresults observed using Hélicobacter oylori sonicate.

Referring to Figures 1-4 , Figure 1 représentagraphically the results of tests for antibodies in sérum 5 (IgG) and intestinal sécrétion (IgA) in mice not protected after immunization with urease. These are micanunbers 1, 4 and 5 appearing in Table 1, and constituteGroup A, Figure 2 shows the antibody responses of micethat were protected after immunization with urease (Group 10 B), i.e. mice 2, 3, 5 and 7-10.

Figures 3 and 4 relate to the results obtained with mice numbers 31-39. Figure 3 (Group C) depictsantibody responses of mice not protected afterimmunization with Hélicobacter oylori sonicate (mice 15 numbers 31, 32, 33, 35, 36 and 38) and Figure 4 (Group D)depicts the antibody responses of mice protected afterimmunization with Hélicobacter oylori sonicate (micenumbers 34, 37 and 39) . It is of interest to note with respect to Figures 3 and 4 that the IgA antibody 20 responses (but not IgG) are higher in the mice exhibitingprotection than in the mice that are not protected,suggesting a corrélation between protection and IgAresponse. Sérum IgG responses did not exhibit acorrélation. Mucosal IgA but not sérum IgG are known to 25 play a rôle in protection against bacterial infections ofthe gut. (McGhee, J.R. and Kyono, H. "New Perspectivesin Vaccine Development: Mucosal Immunity to Infections"Infect Agents Dis., vol. 2, 55-73 (1993)).

The results of the corrélation between the 30 détection of H. fe1is in gastric biopsies by urease testsand by cultures are set out in Table 3.

62 010372 TABLE 3

Urease Test +

Urease Test

Total H. felis 16 16 culture (+) H. felisculture (-) 30 32

Total 30 48

Two-tailed Fisher's Exact Test: p<O.00001

Table 3 shows that a very significant corrélation exists between the results of urease tests performed ongastric biopsies and the identification of H. felis bycultures. Ail animais shown in Table 3 that werepositive for urease activity, were H. felis positive byhistopathology. As cultures detected less often H. felisinfection than urease tests, urease tests were preferredfor the diagnosis of H. felis infection in mice in thenext experiments, due to its better sensitivity. Thisapproach allowed the duplication of urease tests withlarger fragments of the stomach of each mouse, and afurther increase in the sensitivity of the urease test.Furthermore, the use of the method with the highestsensitivity prevents an overestimation of the protectionobtained by the vaccine préparation to be tested. Whenpositive culture is used as the standard for infection,the protection induced after urease immunization duringthe experiment depicted in section B is as significant aswith the combined use of urease test and culture (p=0.021versus p=0.01&amp;).

The results of the experiments described in section C (recombinant urease subunits), obtained on the basis of the gastric biopsies urease tests, are set out in Table 4, 5 and 6 and depicted in Figure 6. 63 0 1 0372 TABLE 4 immunisation mice n3 Urease test Inf sc CT 20 0.49 Sacrificed 12 days 21 0.21 post challenge 22 0.62 + 23 0.57 * 24 0.55 4- 50 0.50 ~r 51 0.37 * 52 0.29 - 53 0.79 * 5 4 0.32 - ure Λ + HAP + CT 40 0.67 - Sacrificed 12 days 41 0.43 * post challenge 42 0.42 * 43 0.65 - 44 0.56 * 45 0.52 * 46 0.33 * 47 0.63 - 48 0.22 * 49 0.37 ure B + HAP + CT 25 0.15 - Sacrificed 12 days 26 0.07 post challenge 27 0.03 - 28 0.64 * 29 0.13 30 0.02 - 31 0.66 32 0.00 - 33 0.79 + 34 0.15 - ure A + HAP + CT 68 0.00 - Sacrificed 10 weeka 69 0.07 - post challenge 70 0.42 + 71 0.00 - 72 0.00 - ure B + HAP + CT 73 0.37 + Sacrificed 10 weeks 74 0.00 - post challenge 75 0.37 + 76 0.00 77 0.00 - 78 0.00 - 79 0.39 80 0.00 31 0.37 82 0.00 -

In Table 4, "CT" means choiera toxin; "UreA" meansrecombinant Hélicobacter pylori urease A subunit; "Ure Ξ"means recombinant Hélicobacter ovlori urease B subunit;and "HAP" means hydroxyapatite crystals. Mice 20 to 54were sacrificed 12 days post challenge and mice 6S ro 32, 64 010372 10 weeks (106 days) post challenge. The results of theurease test performed from biopsies of the stomach ofeach animal are expressed as OD values at 550 nm. TheO.D. value for the assay solution alone (0.075 O.D.) was 5 subtracted as background from the O.D. value obtained foreach mouse. The positive and négative signs depicts thefinal status of infection of each animal, according tothe positivity or negativity of the urease test fordétection of H. felis. Positivity: OD550 values >0.2. 10 The 0.2 value was chosen to define a positive infectionbecause at this value, the color change to the Jatroxsolution could be observed with the naked eye.

Umî..». - 65 - TABLE 5 010372

Protection as measured 12 days post challenge

Immunization Challenoe Urease A subunit H. felis 5 Urease B subunit H. felis CT H. felis % infected 10/10 (%)3/10 (30%) % protected 0/10 (0%)7/10 (70%) * 10/10 (100%) 0/10 (0%) * p=0.003l (two tailed Fisher exact test) comparedto CT control TABLE 6 10

ProtectionImmunizationUrease A subunitUrease B subunit as measured 10 weeks post challenge

Challenge % infectedH. felis 1/5 (20%)H. felis 4/10 (40%) % protected 4/5 (80%)6/10 (60%) 15 * p=o. 003 (two tailed Fisher exact test) compared to CT control ** p=0 .01 (two tailed Fisher exact test) compared to CT control

It will be seen from the results set eut in Tables4, 5 and 6 that statistically significant protection 20 against H. felis challenge is obtained with oral immunization using recombinant Hélicobacter pylori ureaseB subunit as compared to that obtained using eitherrecombinant Hélicobacter pylori urease A subunit orchoiera toxin. Referring to Table 4, it will be seen 25 that, 12 days post challenge, from a total of 10 immunized animais, onlv 3 were found infected in theurease B subunit grcup, as compared to ail 10 animaisimmunized with Hélicobacter ovlori A subunit of ureaseand 10 out of 10 of the animais immunized with choiera 30 toxin. Table 4 shows that 70% of the animais were

protected from challenge by H. felis as compared to 0% ofthe animais immunized with Hélicobacter ovlori urease A 66 010372 subunit and 0% of the animais immunized with choieratoxin and then subjected to H. felis challenge. In otherwords, 100% of the control mice challenged with H. felisbecame infected whereas, in contrast, in mice immunizedwith recombinant Hélicobacter ovlori urease B subunit theinfection rate was only 30%. This represents a significant réduction in infection (p=0.0031, Fisherexact test) as compared to the control mice. The factthat the protection observed with Hélicobacter ovloriurease is entirely conferred by immunization with the Bsubunit of urease, and that the A subunit has no sucheffect, was not expected on the basis of our experimentwith purified urease. This définition of the rôles ofthe 2 structural subunits of urease in the development ofa protective immune response is therefore novel. Theprotection obtained using recombinant urease subunits,which are enzymatically inactive, also teaches that nontoxic forms of urease can be used as oral vaccine againstHélicobacter infection. Furthermore, these resultsstrongly suggest that récognition of the active site isnot required for protection, as an inactive urease Bsubunit is very unlikely to induce antibodies that willrecognize and inhibât the catalytic site of nativeurease.

Referring to Table 6, it will be seen that, whenmice 20 are sacrificed 10 weeks post infection, 60% (6mice out of 10) of the animais immunized with urease Bsubunit and 80% (4 mice out 5) of the animais immunizedwith Hélicobacter ovlori urease A subunit were protectedagainst H. felis infection. The fact that protectionobtained through immunization with urease B subunit lastsover time and that immunization with urease A induces aprotection which is delayed compared to the one inducedby urease B subunit could not be expected from ourexperiment with purified urease or with other experiment 67 010372 performed earlier. The fact that urease A subunitinununization confers protection definitively proves thatrécognition of the active site is not required forprotection. 5 Figure 6 summarizes results obtained after oral inununization with recombinant urease A and B subunits(described in Table 5 and 6) . A second set of mice was immunized and assayed forinfection with Hélicobacter felis at 10 weeks post 10 challenge according to the protocol described in Section C. In this exaraple, twelve mice were sham immunized withchoiera toxin alone, twelve mice were immunized withrecombinant ure A subunit, and ten mice were immunizedwith recombinant ure B subunit. The mean urease activity 15 level found in stomach samples mice that were immunizedwith ure B, but not infected (uninfected mice Controls),was 0.045, and this amount was subtracted as backgroundfrom each O.D. value obtained. Mice were considered tobe infected when the O.D. value was greater than twice 20 the standard déviation of values obtained with theuninfected mice Controls; the standard déviation was0.022. The data obtained from this experiment arepresented in Table 7. 68 010372 TABLE 7 immunization mice no* Urease test Infection CT 135 0.40 4 Sacrificed 10 weeks 136 0.28 4 post challenge 137 0.25 4- 133 0.10 4 139 0.10 4- 140 0.34 4- 141 0.41 142 0.36 -h 143 0.46 4- 144 0.40 4- 145 0.40 4- 146 0.51 4 ure A + HAP + CT 161 0.12 4 Sacrificed 10 weeks 162 0.47 4 post challenge 163 0.00 - 164 0.00 - 165 0.02 - 166 0.01 - 167 0.01 - 168 0.37 4 169 0.00 - 170 0.39 4 171 0.47 4 172 0.00 - ure B + HAP + CT 151 0.00 - Sacrificed 10 week3 152 0.00 - post challenge 153 0.00 - 154 0.03 - 155 0.00 - 156 0.00 - 157 0.02 - 158 0.00 - 159 0.01 - 160 0.00 Using this alternative method of analysis, the data presented in Table 4 were reanalyzed along with the data in Table 7. Instead of subtracting the background O.D. value obtained with the urease assay solution alone (0.075 O.D.), the raean urease level obtained from the uninfected mice Controls was used as the background level. 69 010372

The mean urease level obtained from uninfectedmice sacrificed at twelve days was 0.089. Mice numbered20-54 were considered infected when the O.D. value wasgreater than twice the standard déviation of values 5 obtained with the uninfected mice Controls; the standarddéviation was 0.008. The background level subtractedfrom the O.D. values of mice sacrificed at ten weeks was0.045, and mice were considered infected when the O.D.value was greater than 0.044. 10 Using this alternative method of analysis, at twelve days post challenge, no effect was seen after shamimmunization or after immunization with the ure Asubunit. However, only a low-grade infection (O.D. value< 0.22) was observed in 70¾ of the animais immunized with 15 the ure B subunit (p < 0.02, Mann Whitney U-test, compared to sham immunized control). When mice weresacrificed ten weeks post challenge, 59% (10/17) of themice immunized with the ure A subunit were protectedagainst H. felis infection (p = 0.0019, two-tailed 20 Fisher's exact test, when compared to sham-immunizedmice). Furthermore, 80% (16/20) of the mice immunizedwith the ure B subunit were protected against H. felisinfection (p = 0.00002, two-tailed Fisher's exact test,when compared to control, sham-immunized mice). Under 25 this alternative analysis, the ure A subunit also elicitsprotective immunity. Under either method of analysis,the results.obtained demonstrate that immunization with 70 - 010372 recombinant Hélicobacter pylori urease subunits elicitsprotective immunity against Hélicobacter infection.

EXAMPLES

The invention will now be further described by5 reference to the following non-limiting examples. a) The Bacterial Strains H. felis was provided by J. Fox (Division ofComparative Medicine, Mass. Institute of Technology,Boston, USA). Hélicobacter pylori was isolated from 10 patients with ulcer disease (CHUV, Lausanne,

Switzerland) . b) Bacterial cultures

Liquid Culture - Bacteria were cultured on BHI(Brain-Heart Infusion, BioMerieux) liquid medium 15 containing 0.25% of yeast extract (Difco) and 10% offêtai calf sérum (Inotech) supplemented with 0.4% ofCampylobacter sélective complément (Oxoid). The bacteriawere incubated under microaerophilic conditions at 37°Cand shaken at 37°C for 2 to 3 days. 20 Frozen Culture - The bacteria were cultured in liquid media, then assayed for urease activity, and evaluated for morphology by Gram staining and for motility by microscopy. The bacteria were then 71 010372 centrifugea and resuspended at a concentration of 30 O.D.per rai in BHI plus 20% glycerol and frozen at -8O°C.

Just before use, frozen stocks were thawed on ice, washedin 20 ml BHI, centrifugea, and resuspended at a 5 concentration of 1 O.D. to 1.5 O.D. per 200 μΐ in 5 mMHaHCO3.

Culture on Agarose niâtes - The bacteria verecultured on agar plate consisting of BHI with 0.25% ofyeast extract and 5% of sheep blood under raicroaerophilic 10 conditions at 37°C for 3 days.

Quantification - The quantity of bacteria vasdeterrained by the optical density of the BHI solution at660 nm (1 optical density unit (O.D.) corresponding to108 bacteria). The number of viable bacteria is œeasured 15 on the number of colony forraing units. c) Préparation of sonicates Hélicobacter pylori was collected from 31 bloodagar plates in 0.15 M NaCl and spun 5 minutes at 1400g at4°C. The pellet was resuspended in 3 ml of NaCl and 20 sonicated for 4 minutes. The amount of protein was evaluated by a Bradford assay (BioRad Kit, according tosupplier). 010372 - 72 - d) Coupling of immunogen to HydroxyapatiteImmunogen (urease or subunit thereof) was incubated for i hour at 4°c with hydroxyapatite. l.C mgof hydroxyapatite was used fer 30 ug cf immunoger. per 5 mouse. At the end of the incubation, 10 pg cf choieratoxin was added in a final volume of 200 pl PBS. e) Challenge with Hélicobacter felis

Mice were lightly anesthetized prior tointragastric challenge with Hélicobacter felis. H. felis 10 in a total volume of 200 μΐ were delivered to the stomachs of the respective mice using Silicon tubzngattached to a hypodermic syringe. EXAMPLE 1 a) Extraction 15 Hélicobacter uylori from 30 blood agar plates was harvested in 0.15 M MaCl on ice. The solution was spur.for 5 minutes at 1400 g at 4°C. The pellet was resuspended in 20 ml of II20 and vortexed for 45 seconds(maximum speed). The extract was then spun for 20 20 minutes at 6700 g at 4 °C. The supernatant was recovered and the quantity of protein was evaluated (see"Quantification" above) and precipitated with 70i cf ammonium sulfate. , a1’57’/ 010372 - 73 - b) Purification of urease

The solution was chromatographed on a Sepharose CL-6B coluan (Pharmacia) with PBS (phosphate bufferedsaline) as mobile phase. The 22 collected fractions 5 which presented a strong urease activity were pooled anddialyzed overnight at 4°C against 3 liters of PEB (20 mMphosphate buffer, pH 7) and then chromatographed on a QSepharose fast flow (Pharmacia) with PEB as mobile phase.The fractions were eluted by a 0 to 500 mM NaCl gradient. 10 Ten of the collected fractions with strong ureaseactivity were individually subjected to an SDS gelfollowed by Coomassie staining. The 6 fractionspresenting 2 distinct bands corresponding to MW-63 andMW-28 KDa were pooled and were considered as the purified 15 urease. EXAMPLE 2 (see also section B)

Mice employed in the immunization studies were lightly anesthetized with ether prior to intragastricimmunization. And then, a sonicate préparation or 20 purified urease, hydroxyapatite and choiera toxin vas suspended in PBS and 200 μΐ were delivered to the stomachof the respective mice uoing polyethylene tubing attachedto a hypodermic syringe. This procedure will be referredto as oral immunization. 25 Three oral immunization protocols were evaluated.

These are described below. ·>,-· ,«d*siu.A...»à. 010372 - 74 -

Protocol Bl - Vaccination with Purified urease

Fenale 3ALB/C six-week old nice (20) were orally imnunized with 30 ug of purified Hélicobacter pyloriurease and 1 mg of hydroxyapatite and 10 ug of choiera 5 toxin at day 0, 7, 14 and 21. Ten nice were chalienged atday 28 and 30 with 5xl07 and 103 H. fe11s fron liçuidculture.

Protocol B2 - Vaccination with Hélicobacter sonicates

Female BALB/c six-week old rnice (20) were orally10 immunized with 2 mg of Hélicobacter pylori sonicate solution at day 0, 7, 14, and 21. Ten mice were chalienged at day 28 and 30 with 5xl07 and 108 H. félis.

Protocol B3 - Control

Female BALB/c six-week old mice were orally15 immunized with 1 mg hydroxyapatite and 10 gg of choiera toxin at day 0, 7, 14 and 21. The mice were chalienged at day 28 and 30 with 5xl07 and 108 H. felis.

At day 35 ail mice were sacrificed and biopsiesfrom the stomach were taken as well as intestinal 20 sécrétions and blood.

Protection and évaluation

To evaluate protection, biopsies were screer.sd for the urease activity by the Jatrox HP test (Rohm Pharma), according to the instructions of the supplier. The 010372 - 75 - urease is quantified by a spectrophotometric measurementat 550 nn. The biopsies were also cultured for thepresence of H. felis and the presence of H. felis vasdeternined by Gram staining. Gastric antral biopsies 5 were honogenized and diluted (1:10 and 1:1000) in 0.15 MNaCl and plated onto blood agar plates and incubatedunder microaerophilic conditions at 37°C for 4 to 10days.

ELISA 10 Intestinal sécrétions and blood were analyzed by ELISA for the évaluation of antibody titer. The ELISAwas carried out as follows. Polystyrène plates (96wells) were coated with 1 pg/well of purified urease at37°C for 2 hrs. Non-specific binding sites were blocked 15 with 5% powdered milk in PBS 0.1% Tween at 37°C for 30 minutes. The plates were washed once with PBS 0.1%Tween. Blood samples were tested at dilution 1:1000 andintestinal sécrétions at 1:1. 100 μΐ of each sample were added to the antigen coated plates. After 2 hrs of

20 incubation, plates were washed 3 times with PBS containing 0.1% Tween. Anti-mouse biotinylated wholeantibody from goat and anti-mouse IgA, IgG, and IgMbiotinylated (Amersham) were added (100 μΐ) at dilution1:500, except for IgA (1:250), and incubated at 37°C for 25 1 hr. The places were washed 3 times with PBS 0.1% Tween and 100 μΐ of 1:1000 dilution cf streptavidin Horseradish 76 010372 perczidase in PBS containing 0.1¾ Tveen were added andincubated at 37°C for 30 minutes. The plates were washed3 times and 50 μΐ of 1:50 dilution cf o-pheny1-diamine incitrate buffer pH 5.0 with 1 μΐ/ml of 30% H202 were added 5 and incubated at room température for 20 minutes. Theabsorbance at 495 nm was measured in each well. EXAMPLE 3 (see also section C)

Mice employed in the immunisation studies werelightly anesthetized with ether prior to intragastric 10 immunisation. Then, 30 /zg recombinant Hélicobacter py lori urease A or B subunit produced in E. coli, boundwith hydroxyapatite, and supplemented with choiera toxinwas suspended in PBS and 200 μΐ were delivered to thestomach of the respective mice using a polyethylene 15 tubing attached to a hypodermic syringe. This procedurewill be referred to as oral immunization.

Three oral immunisation protocols were evaluated.

These are described below.

Protocol ci - Vaccination with recombinant urease A 2 0 subunit

Female BAL3/c six-week. old mice (10) were crallyimmunized with 30 gg of purified recombinant Hélicobacterpylori urease A subunit and 1 mg of hydroxyapatite and 10 nq of choiera toxin at day 0, 3, 14 and 21. Ten mice were 77 010372 challenged at day 32, 34 and 36 with 10a H. felis fromliquid culture.

Protocol C2 - Vaccination vith recombinant urease B subunit

Female BALB/c six-week old mice (10) were orallyimmunized with 30 yq of recombinant Hélicobacter pvloriurease B subunit and 1 mg of hydraxyapatite and 10 p,g ofchoiera toxin at day 0, 8, 14, and 21. Ten mice werechallenged at day 32, 34 and 36 with 103 H. felis fromliquid culture.

Protocol C3 - Control

Female BALB/c six-week old mice were orallyimmunized with 1 mg of hydroxyapatite and 10 μ-q ofchoiera toxin at day 0, 8, 14 and 21. The mice werechallenged at day 32, 34 and 36 with 10a H. felis.

At day 42, or at day 106, mice were sacrificed andmultiple biopsies from the stomach were taken.

Protection and évaluation

To evaluate protection, biopsies of the corpus andantrum of the stomach were screened for urease activityby the Jatrox HP test (Rohm Pharma) according to theinstructions of the supplier. The urease is quantifiedby a spectrophotometric neasurement at 550 nm. The total 0 1 0372 - 73 - of corpus and antrum OD values were added to obtain afinal OD value for each mouse. EXAMPLE 4

To détermine whether imnunization with urease5 peptides would be an effective treatment of Helicobacrer infection animais, mice were first challenged with H.felis and then immunized with Hélicobacter pylori ure Bsubunit. The utility of immunisation with Hélicobacterpylori ure B subunit to treat Hélicobacter infection is 10 demonstrated in both Example 4 and Example 5. a) Infection of mice with H. felis

Female BALB/c six-to-eight week old mice werechallenged with H. felis at days 1 and 3 with 1 O.D. offrozen culture. The mice were challenged at day 5 with 15 1.53 O.D. of liquid H. felis culture. b) Vaccination with recombinant Hélicobacter pylori urease B subunit

Eight H. felis infected mice were orallv immunizedwith 30 ug of recombinant Hélicobacter pylori urease B 20 subunit, l mg of hydroxyapatite and 10 ug of choiera toxin (holoenzyme obtained from Calbiochem) at days 23, 30, 37, and 44. 010372 - 79 - c)

Controls

Ten H. felis infected nice were orally immunizedwith 1 mg of hydroxyapatite and 10 /xg of choiera toxin atdays 23, 30, 37, and 44. These mice were designated as"sham" immunized. Seven H. felis infected mice were notsubject to immunization. d) Sacrifice and Evaluation

At day 70, ail mice were sacrificed. The stomachswere removed and eut in half longitudinally. To evaluateprotection, one-half of the stomach of each mouse (bothcorpus and antrum sections) was screened for ureaseactivity 3-4 hours post-sacrifice, by the Jatrox HP test(Rohm Pharma) according to the instructions of thesupplier. The urease was quantitated by a spectrophotometric mcasurement at 550 nm. Ten naive(uninfected) BALB/c mice served as Controls. These micewere sacrificed at day 70 and the stomach samplesevaluated for urease activity to détermine the backgroundlevel of urease activity. The mean background value, 0.033 O.D., was subtracted from the O.D. value for eachmouse. The standard déviation in urease activity amongthe naive mice was 0.025 O.D. The résulte of the ureaseassays are set out in Table 3 and depicted in Figure 7.

In Figure 7, the dashed line indicates the O.D. value for twice the standard déviation in urease activity among the naive mice. 010372

Treatment

Untreated

Ure B + HAP + CT - 80 - TABLE 8

Sham (HAP+CT only)

Mouse Mo. Urease test Infsetion T_ 0.42 + 2 0.36 + 3 0.41 4 0.33 4· 5 0.38 + 6 0.52 + 7 0.48 + 8 0.51 + g 0.48 + 10 0.61 4- 11 0.44 + 12 0.51 + 13 0.48 4- 14 0.44 +· 15 0.58 + 16 0.48 4- 17 0.53 + 18 0.36 4· 19 0.01 - 20 0.43 4- 21 0.07 +/- 22 0.39 + 23 0.10 +/- 24 0.38 4· 25 0.06 +/-

In Table 3, "ure B" raeans recombinant Hélicobacterpylori urease B subunit as referenced above; "CT" meanschoiera toxin; "HAP" means hydroxyapatite crystals; and"untreated" means the raice were challenged with H. felis,but received no subséquent immunization. The results ofthe urease test performed from biopsies of the stomach ofeach animal are expresse! as O.D. values at 550 nm. Thebackground value subtracted from the O.D. value for eachmouse was 0.033. The infection is rated as positive(indicated by a "+") where the O.D. value at 550 nm isover twice the standard déviation found with the naive 31 010372 mouse Controls (0.05 O.D.). A "4-/-" indicates that theo.D. value is greater than background, but less than0.03, and that the value is reduced compared ta the sham-immunized Controls.

It will be seen from the results set out in Tahle7 and in Figure 7 that administration of the Hélicobacterpylori ure B subunit to nice infected with H. felis resulted in the clearance of infection in one out ofeight mice. According to another, less conservative,interprétation of these results, defining a positiveresuit as a value greater than 0.2, four out of eightrnice (numbers 2, 4, 6, and 8) treated with ure B would befound to be protected from infection. However, undereither of the two définitions of a positive resuit thesemice exhibited a reduced urease activity as compared tothe untreated and the sham-immunized mice. Using the0.05 value to define infection, the level of infection inure B immunized mice as compared to sham-immunized miceexhibits a statistically significant (p value of lessthan 0.004) réduction in the level of infection. Thus,the results of Example 4 indicate that administration ofure B to mice infected with H. felis results in a reduced level of infection. 010372 - 82 - EXAMPLE 5 a) Infection of mice with H. felis

Female BALB/c five-to-eight week old nice werechallenged with H. felis at day 1 with 1 O.D. of liquid 5 culture. The mice were challenged at day 3 with 0.8 O.D.H. felis cultured on agarose plates. The mice werechallenged at day 5 with 1 O.D. of H. felis cultured onagarose plates. b) Vaccination with recombinant Hélicobacter pylori1° urease B subunit H. felis infected mice were orally immunized with30 pg of recombinant Hélicobacter pylori urease Bsubunit, 1 mg of hydroxyapatite and 10 of choieratoxin (holoenzyme obtained from Calbiochem) at days 23, 15 30, 37, and 44. These mice were designated as "immunized." c) Controls H. felis infected mice were orally immunized with1 mg of hydroxyapatite and 10 pg of choiera toxin at days 20 23, 30, 37, and 44. These mice were designated as "sham" immunized. d) Sacrifice and Evaluation

At day 62, ten immunized mice and six shamimmunized mice were sacrificed. At eight weeks after the 25 last immunization, ten immunized and eight sham immunized 33 010372 mice were sacrificed. Stomach samples were screened forurease activity as described in Example 4. The meanbackground found in 29 naive BALB/c mice, sacrificed atvarions times, was 0.04 O.D. This level of activity vasused as a background measurenent and subtracted from theO.D. value for each mouse. The standard déviation inurease activity among the naive mice was 0.02 O.D. Theresults of the urease assays are set out in Table 9 anddepicted in Figure 8. In Figure 8, the dashed lineindicates the O.D. value for twice the standard déviationin urease activity among naive mice. e) Analysis of Blood and Fecal Samples

Blood Sample Collection

Blood samples were allowed to clôt for 3 hours atroom température, and sérum harvested and frozen at-20°C, until further analysis.

Fecal Pellet Collection

Fresh fecal pellets (3-3) were collected in 1.5 mleppendorf tubes containing 500 μΐ of PBS +5% non-fat drymilk + protease inhibitors (AEBSF 0.2 mM, Aprotinin 1pg/ml, Leupeptin 10 μΜ, Bestatin 3.25 μΜ) . The sampleswere frozen at -20°C until use. They were then thawed onice, mashed, and centrifuged at 10,000g for 10 minutes at4°C to yield a supernatant free of débris, and yellowishbrown in appearance. 010372 - 84 -

ELISA Sérum and fecal pellet samples of each animal wereanalyzed by ELISA for évaluation of anti-urease activity,according to standard procedures. 5 Fecal pellets and blood were analyzed by ELISA fer the évaluation of antibody titer. Polystyrène plates (96wells) were coated with 0.5 bg/well of purified

recombinant urease at 37°C for 2 hrs. Hon-specificbinding sites were blocRed with 5% powdered milk in PBS 10 containing 0.1¾ Tween at 37°C for 30 minutes. The plateswere washed once with PBS containing 0.1¾ Tween. Blocdsamples were tested at a dilution of 1:200 and fecalpellets at a dilution of 1:1. 100 μΐ of each sample were added to the antigen coated plates. After 2 hrs of

15 incubation, plates were washed 3 times with PBS containing 0.1% Tween. Anti-mouse biotinylated wholeantibody from goat (Amersham) and anti-mouse IgA coupledto Horseradish peroxidase (Serotec) were added (100 μΐ)at a dilution of 1:500 and incubated at 37°C for 1 hr. 20 The plates were washed 3 times with PBS containing 0.1%Tween and, for blood samples only, 100 μΐ of a 1:500dilution of streptavidin and Horseradish peroxidase inPBS containing 0.1% Tween were added and incubated at37°c for 30 minutes. The plates were washed 3 times and 25 50 μΐ of a 1:50 dilution of o-phenyl-diamine in citrate buffer, pH 5.0, with 1 μΐ/ml of 30% H202 were added and -ι'·ίΐι-%ιίΐ 010372 - 35 - incubated at roon température for 20 minutes. Theabsorbance at 495 nm was measured in each well.

For the évaluation of total IgAs in fecal pellets,the ELISA was carried out as described above except that 5 the plates were coated with 1 pg/ml of goat anti-mouseIgA (SIGMA) and the fecal supernatant was tested at 1:200 dilution. - 86 - 0 1 0372 TABLE 9

Sacrif ica Treatner.t Mouse Mo. Urease test Infection 2.5 weeks Sham 1 0.53 + (HAP+CT only) 2 0.51 -a. 3 0.50 4. 4 0.54 - 5 0.47 -r 6 0.36 4- Ure B + HAP + CT 7 0.02 - 8 0.06 9 0.01 - 10 0.00 - 11 0.02 - 12 0.02 - 13 0.02 - 14 0.44 15 0.00 - 16 0.03 - 8 weeks Sham 17 0.30 + (HAP+CT only) 18 0.27 + 19 0.29 + 20 0.20 + 21 0.27 + 22 0.27 + 23 0.37 + 24 0.31 + Ure B +HAP+CT 25 0.26 + 26 0.30 + 27 0.00 - 23 0.17 + 29 N. D. 30 0.00 - 31 0.00 - 32 0.00 - 33 0.00 - 34 0.00 — 87 010372 TA3LE 10

Sacrifice 2.5 weeks 8 weeks * p = 0.007 (tvsham control **p = 0.014 (tvsham control

Treatnent ure Bsham ure Bsham -tailed Fisher exact -tailed Fisher exact % cleared 8/10 (80%)* 0/6 6/9 (67%)** 0/9 test) compared to test) compared to

In Tables 9 and 10, "ure B" means recombinant Hélicobacter pylori urease B subunit, as referencedabove, "CT" means choiera toxin; and "HAP" meanshydroxyapatite crystals. "Sacrifice" means the date ofsacrifice, as measured from the last immunization at day44. "N.D." indicates that no data were available. The results of the urease test performed from biopsies of thestomach of each animal are expressed as O.D. values at550 nm. The infection is rated as positive (indicated bya "+") where the O.D. value at 550 nm is over twice thestandard déviation found in the mature mice (0.04 O.D.).The background value subtracted from the O.D. value foreach mouse was 0.04 O.D. In Table 10, "% cleared" indicates clearance of infection as measured by theurease assay.

It will be seen from the results set out in Tables 9 and 10, and in Figure 8, t hat administration of the VV' UMWÜ » Λ. Α&amp;>-ί>-ί 010372 - 88 - Hélicobacter nylori ure B subunit to mice infected withH. felis resulted in the clearance of infection in eightout of ten mice as assayed when the mice were sacrificed2.5 weeks after the last immunization. When the assaywas performed when the nice were sacrificed eight weeksafter the last immunisation, the infection was cleared insix out of nine mice. If the higher value of 0.2 ischosen to define infection as referred to in thediscussion of Table 8, under this method of interprétation, treatment with the ure B subunit resultedin clearance of infection in nine out of ten micesacrificed at 2.5 weeks.

Table 10 indicates that statistically significanttherapeutic treatment of H. felis infection is obtainedwith oral immunization using recombinant Hélicobacterpylori urease B subunit as compared to that obtained bysham immunization with choiera toxin and hydroxyapatitealone. Table 10 shows that, when measured at 2.5 weeksafter the last immunization, 80% of the infected micewere cleared of infection. When measured at eight weeksafter the last immunization, 67% of the infected micewere cleared of infection. îlone of the sham-immunized mice were cleared of infection when measured at either2.5 or eight weeks after the last immunization. Thesignificance of the réduction in the percentage of micecleared of infection when measured at eight weeks ascompared to 2.5 weeks after the last immunization is " 89 " 0103 72 unclear at présent. TABLE 11

Mouse No. ürease Test Total anti-ureB Icfs in Blood TotalicfAs in Feces ureB Ictas in Feces ureB iqAs in Feces/ Total IdAs in Feces Ex. 4 : 8 0.51 0.27 0.77 0.05 0.06 9 0.48 0.23 0.65 0.02 0.03 10 0.61 0.25 0.43 0.08 0.18 ! 11 0.44 0.36 0.69 0.03 0.04 1 12 0.51 0.31 0.58 0.00 0.00 j 13 0.48 0.32 0.67 0.11 0.17 14 0.44 0.27 0.51 0.03 0.05 ! 15 0.58 0.29 0.82 0.07 0.08 ) 16 0.48 0.30 0.77 0.18 0.23 | 17 0.53 0.30 0.53 0.09 0.18 i 18 0.36 0.22 0.51 0.00 î o.oo | 19 0.01 0.47 0.62 0.47 0.75 fj 20 0.43 0.35 0.57 0.15 0.26 fl 21 0.07 0.25 0.64 0.11 0.17 lj 22 0.39 0.27 0.50 0.15 0.30 23 0.10 0.27 1.08 ND ND 24 0.38 0.28 0.66 ND ND 25 0.06 0.28 0.53 ND ND | Ex. 5: 7 0.02 0.11 0.61 0.06 0.11 8 0.06 0.13 0.62 0.02 0.03 9 0.01 0.15 0.62 0.04 0.07 10 0.00 0.15 0.59 0.03 0.05 11 0.02 0.19 0.60 0.15 0.24 12 0.02 ! 0.16 0.60 0.00 -0.01 - 90 - 010372

Mouse No. ürease Test Totalant i-ureB Iqs in Blood Total IqAs in Feces ureB IqAs in Feces ? ureB IqAs 1in Feces/. 2Total 1IqAs in iFeces j 13 0.02 0.19 0.66 0.07 J o.ii ! 14 0.44 0.18 0.62 0.00 0.00 15 0.00 0.10 0.64 0.08 0.12 ! 16 0.03 0.14 0.62 0.29 0.46 25 0.26 0.35 0.98 0.07 0.07 26 0.30 0.38 1.12 0.04 0.03 27 0.00 0.39 1.15 0.01 0.01 28 0.17 0.33 0.97 0.02 0.02 29 N.D. * 0.38 1.00 0.08 0.08 30 0.00 0.34 0.69 0.04 0.06 31 0.00 0.38 0.76 0.08 0.10 32 0.00 0.37 0.58 0.02 0.04 33 0.00 0.38 1.07 0.22 0.21 34 0.00 0.35 0.64 0.06 0.09 91 010372

In Table il, the mice that were treated with ureB, used in Examples 4 and 5, were evaluated for theantibody content of the blood and feces. The mousenumbers used correspond to the numbers used in Tables 8and 9. The urease test results correspond to the resultsreported in Tables 8 and 9. "ND*" indicates that no data was available because one of the stomachs, from micenumbered 18-21, was lost. The inventors are unable todétermine which of the sets of data from mice numbered18-21 is incomplète. Therefore, the "ND" is not meant tobe specifically assigned to mouse number 21.

In the assays of the mice from Example 4, and themice numbered 1-10 from Example 5, the inventors haddifficulty obtaining adéquate samples for analysis.

The clearance of H. felis infection by oraladministration of the Hélicobacter pylori ure B subunitwas not expected and is therefore novel. The resultsdescribed herein also teach that the ure B subunit can be used to treat Hélicobacter infection.

One skilled in the art will readily appreciatethat the présent invention is well adapted to carry outthe objects and obtain the ends and advantages mentioned,as well as those inhérent therein. The urease peptides,mucosal adjuvants, carriers, and antibodies, along withthe methods, procedures, treatments, and assays, areexemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses .. »1: tiàîWi' 010372 - 92 - will occur to those skilled in the art. which are encompassed within the spirit cf the invention as definedby the scope of the clains.

Claims (29)

1. -93 - 01 0372 SUBSTITUTS CLAIMS

   1. Use of a composition comprising a recombinantHélicobacter antigen in the préparation of a médicamentfor treating Hélicobacter infection in a mammal.
2. 2. The use of claim 1, wherein said Hélicobacterinfection is Hélicobacter pylori infection.
3. 3. The use of claim 1, wherein said recombinantHélicobacter antigen comprises a Hélicobacter pyloriantigen.
4. 4. The use of claim 1, wherein said recombinantHélicobacter antigen comprises a Hélicobacter urease, ora fragment thereof.
5. 5. The use of claim 1, wherein said recombinantHélicobacter antigen comprises a subunit of aHélicobacter urease, or a fragment thereof.
6. 6. The use of claim 1, wherein said compositionfurther comprises a mucosal adjuvant.
7. 7. The use of claim 6, wherein said mucosaladjuvant comprises the heat-labile toxin of Eschericbiacoli, or a fragment thereof.
8. 8. The use of claim 6, wherein said mucosaladjuvant comprises the B subunit of the heat-labile toxinof Escherichia coli, or a fragment thereof.
9. 9. The use of claim 1, wherein said mammal is a human. -94 - 010372
10. 10. The use of claim 1, wherein said compositionis administered to said mammal orally, nasally, orrectally.
11. 11. Use of an antibody that recognizes aHélicobacter antigen in the préparation of a médicamentfor the treatment of Hélicobacter infection in a mammal.
12. 12. The use of claim 11, wherein saidHélicobacter infection is Hélicobacter pylori infection.
13. 13. The use of claim il, wherein said mammal is a human.
14. 14. The use of claim 11, wherein saidHélicobacter antigen comprises a Hélicobacter pyloriantigen.
15. 15. The use of claim 11, wherein saidHélicobacter antigen comprises a Hélicobacter urease.
16. 16. The use of claim 11, wherein said antibody isa monoclonal antibody.
17. 17. The use of claim 11, wherein said antibody isan IgA antibody.
18. 18. A composition useful in the treatment ofHélicobacter infection in a mammal, said compositioncomprising a recombinant Hélicobacter antigen.
19. 19. The composition of claim 18, wherein saidrecombinant Hélicobacter antigen comprises a Hélicobacterpylori antigen. 010372 -95 -
20. 20. The composition of daim 18, wherein saidHélicobacter antigen comprises a Hélicobacter urease, ora fragment thereof.
21. 21. The composition of claim 18, wherein saidHélicobacter antigen comprises a subunit of aHélicobacter urease, or a fragment thereof.
22. 22. The composition of claim 18, wherein saidcomposition further comprises a mucosal adjuvant.
23. 23. The composition of claim 18, wherein saidmucosal adjuvant comprises the heat-labile toxin ofEscherichia coli, or a fragment thereof.
24. 24. The composition of claim 18, wherein saidcomposition comprises the B subunit of the heat-labiletoxin of Escherichia coli, or a fragment thereof.
25. 25. A composition useful for the treatment ofHélicobacter infection in a mammal, said compositioncomprising an antibody that recognizes a Hélicobacterantigen.
26. 26. The composition of claim 25, wherein saidHélicobacter antigen comprises a Hélicobacter pyloriantigen.
27. 27. The composition of claim 25, wherein saidHélicobacter antigen comprises a Hélicobacter pyloriurease.
28. 28. The composition of claim 25, wherein saidantibody is a monoclonal antibody. -96- 010372
29. 29. The composition of claia 25, wherein saidantibody is an IgA antibody. 198903.ail