WO2005113603A1 - Eradication of helicobacter infection by activation of stomach mast cells - Google Patents

Abstract

The present invention relates to a novel method and novel compositions for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal. According to the invention a disease caused by or associated with Helicobacter in a mammal is prevented and/or treated by administering to said mammal a preventive and/or therapeutically effective amount of a composition capable of activating mast cells in the stomach of said mammal. Furthermore, the invention provides a composition capable of activating mast cells in the stomach of a mammal which leads to an increase of the expression of mast cell proteases 1 and/or 2 or related mast cell activation markers as well as a method for eradicating Helicobacter from the stomach of a mammal.

Description

Eradication of Helicobacter infection by activation of stomach mast cells

Field of the invention

The present invention relates to a novel method and to novel compositions for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal. Furthermore, it relates to a novel method for eradicating Helicobacter from the stomach of a mammal.

Background of the invention

Helicobacter pylori (H. pylori) is a gram-negative, spiral bacterium that colonizes the gastric mucosa of at least 50% of the world's population and plays a causative role in the development of chronic gastritis as well as in gastric and duodenal ulcers (Blaser MJ. and Parsonnet J. "Parasitism by the slow bacterium Helicobacter pylori leads to altered gastric homeostatis and neoplasia" J. Clin Invest. 1994; 94, pp 4-8). This infection also represents a risk factor for the development of gastric adenocarcinomas and mucosa-associated lymphoid tissue (MALT) lymphomas. The clinical course of H. pylori infection, in particular the occurrence of complications such peptic ulcers or malignancies, is influenced by both microbial and host factors (El-Omar EM et al. "Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms" Gastroenterology 2003 ; 124, pp 1193-1201). Since the first culture of H. pylori 20 years ago, the diagnosis and treatment of upper gastroduodenal diseases have changed dramatically. Peptic ulcer disease is now approached as an infectious disease, in which elimination of the causative agent cures the condition. Effective antimicrobial therapy based on associations of antibiotics is available but its effectiveness is impaired by its costs and by growing antibiotic resistance (Suerbaum S and Michetti P. "Helicobacter pylori infection" N Engl J Med 2002; 347, pp 1175-1186).

H. pylori triggers vigorous humoral and cellular immune responses in the systemic as well as mucosal compartments. In spite of this response, the vast majority of infected hosts are unable to clear the infection that persists for decades. Prophylactic and therapeutic vaccination has been partly successful in animal models (Michetti P. et al. "Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease" Gastroenterology 1994; 107, pp 1002-1011) but translation to a human vaccine has remained difficult, in part because the immunology of the stomach is still poorly understood.

Vaccination against H. pylon is an important goal in the prevention of gastroduodenal diseases, including ulcers and gastric malignancies. Despite the proven ability of vaccination to prevent or reduce Helicobacter infection in murine models, the precise mechanisms of protection have remained obscure. Mucosal and systemic immunizations with Helicobacter extracts or purified recombinant proteins generate antigen-specific serum, salivary and intestinal antibody responses as well as cellular immunity (Lee CK. Et al. "Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis" J Infect Dis 1995; 172, pp 161-172). Furthermore, after immunization and challenge, gastric IgA and T cell responses can be measured. The requirement of major histocompatibility complex class II for protection in mice implies a role for CD4⁺ T cells (Ermak TH et al. "Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class H-restricted responses" J Exp Med 1998; 188, pp 2277-2288).

There is evidence that mast cells can modulate and/or initiate adaptive immunity (Henz BM et al. "Mast cells as initiators of immunity and host defense" Exp Dermatol. 2001 Feb; 10(1), pp 1-10). Mast cells are derived from multipotential stem cells in bone marrow, and are abundant in mucosal tissues. Mast cells are in close contact with the external environment in sites such as the respiratory and gastrointestinal tracts, and the skin. Mast cells are known to be the primary responders in allergic reactions, orchestrating strong responses to minute amounts of allergens. On a more beneficial note, mast cells or their products have been shown to be pivotal in mediating leukocytes recruitment in vivo, to play a role in defense against bacterial and parasite infections. Mast cells secrete cytokines in response to both immune and nonimmune stimulations. These cytokines can influence both T-cell and B-cell development and function, and include IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL-16, TNFα and chemokines (Mecheri S, David B. "Unravelling the mast cell dilemma: culprit or victim of its generosity?" Immunol Today 1997; 18, pp 212-215). Mast cells can present antigens to T cells in MHC a class-ll restricted fashion (Mekori YA, "Metcalfe DD Mast cell-T cell interactions" J Allergy Clin Immunol 1999; 104, pp 517- 523). In addition, it has been shown that mast cells are activated at the site of antigen encounter and migrate to draining lymph nodes to initiate T cell recruitment (Wang HW. et al. "Mast cell activation and migration to lymph nodes during induction of an immune response in mice" J Clin Invest. 1998; 102, pp 1617-1626). A recent study shows that the recruitment of T cell in draining lymph nodes is mediated by the secretion of TNFα by mast cells (McLachlan JB. et al. "Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection" Nat Immunol. 2003; 4, pp 1199-1205).

The failure of the natural immune response to clear Helicobacter infection remains, however, unexplained. Indeed activated CD4⁺ T cells and mast cells are found in the gastric mucosa of H. pylori infected patients. A likely explanation is that Helicobacter down regulates the local host immune response, thus preventing the development of efficient CD4⁺ T cell (Gebert B. et al. "Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation" Science 2003; 301, pp 1099-1102) and mast cell responses (Lutton DA et al. "Modulatory action of Helicobacter pylori on histamine release from mast cells and basophils in vitro" J Med Microbiol. 1995; 42, pp 386- 393). In support of this hypothesis, Gebert et al. recently showed that H. pylori VacA blocks proliferation and activation of T cells and abrogates nuclear translocation of the nuclear factor of activated T cells NFAT, a global regulator of immune response genes, possibly inducing a local immune suppression.

Although many antigens have been identified and tested in animal models with success to eradicate Helicobacter infection, none of the clinical studies testing vaccine candidates in human have been successful.

For example, International Patent Applications WO94/09823 (Michetti et al.) and WO95/22987 (Michetti et al.) disclose the use of a Helicobacter pylori urease specific IgA monoclonal antibody in vaccine compositions for passive immunization. The specifications of these two patent applications disclose the use of IgA since it has been shown that there is a correlation between protection and IgA response whereas serum IgG antibodies are believed not to play a role in protection against bacterial infections of the gut. In addition, these patent applications disclose as therapeuthic tool to prevent bacterial colonisation the inhibition of enzymatic and/or adhesive activities of urease by passive administration of monoclonal antibodies.

Nonetheless, International Patent Application WO97/003360 (Nedrud et al.) recently describes methods of preventing and / or treating Helicobacter infection with an IgG monoclonal antibody directed to a Helicobacter antigen of 19kD. This patent application discloses as therapeutic tool to prevent bacterial colonisation the inhibition of the activity of the bacterial associated 19kD antigen. This attempt seems not to have been confirmed in humans.

These failures highlight the necessity to understand in animal model the immune mechanisms that clear Helicobacter infection following vaccination. The identification of such mechanisms will allow the build up of a human vaccine strategy. In this strategy, one need to identify the best antigens derived from Helicobacter, the appropriated adjuvants to formulate the vaccine and lastly the best route of vaccine administration. The fact that the above-described methods failed to prevent and / or to treat Helicobacter infections is explained by the misunderstanding of the complex role of the mucosal immune system in the stomach. More particularly, the role of mast cells in the mucosal immune system in the stomach has probably been underestimated since up to now, it is believed that these cells only play a negative role in the immune response. Thus, activation of mast cells for their use in the treatment of Helicobacter infections has never been envisaged. In addition, the identification of the immune mechanisms leading to the clearance of Helicobacter after vaccination will generate diagnostic tools allowing the follow up of the immune status of the vaccinated people.

The present invention shows that the activation of mast cells is a crucial step in the cure of Helicobacter infection which can overcome the failure of the natural immune response. The object of the present invention is to provide an improved approach for the prevention and/or treatment of diseases caused by or associated with Helicobacter in a mammal which does not have the above-mentioned disadvantages. Therefore, what is needed is a composition and method for the prevention, treatment and eradication of Helicobacter infection in an mammal in particular human that is selective for Helicobacter, that affects non-growing as well as growing Helicobacter, that does not induce drug-resistant strains of Helicobacter, that has minimal side effects, that encourages patient compliance and that can be provided at a reasonable cost.

Summary of the invention

This and further objects have been achieved with a novel method and novel compositions for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal. According to the invention a disease caused by or associated with Helicobacter in a mammal is prevented and/or treated by administering to said mammal a preventive and/or therapeutically effective amount of a composition capable of activating mast cells in the stomach of said mammal. Furthermore, the invention provides a composition and the use thereof for activating mast cells in the stomach of a mammal as well as a method for eradicating Helicobacter from the stomach of a mammal.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

Brief description of the figures

Fig. 1. Kinetics of Helicobacter clearance after vaccination. Balb/c mice were treated intranasally (at day 0, 7, 14, and 28) with either 30μg of urease + 10μg of cholera toxin (CT) as adjuvant, or CT alone. At day 42, the mice were challenged with H. felis (5x10⁷) and sacrificed 4 or 5 days post challenge. At sacrifice, stomachs were recovered for urease testing (A) or quantification of bacterial density by histology (B, cresyl violet staining). In another set of experiments, mice were immunized as described above and challenged at day 42 and 44 with 5x10⁸ H. pylori 49 (Hp49) and sacrificed at day 49. At sacrifice, stomachs were recovered for urease testing (C) and for colony forming unit counts (D). * 5 mice per group, p<0.02: ** 10 mice per group p<0.0001 ; *** 6 mice per group p<0.003; **** 6 mice per group p<0.003 (Mann-Whitney test).

Fig. 2. Mast cells hyperplasia in the stomach of urease + CT vaccinated mice during the Helicobacter clearance. Flow cytometry analysis of lymphoid cell populations recovered from the stomachs of vaccinated (CT+urease) or CT control mice (CT) at day 4 post H. felis challenge. (A): percentages of CD4⁺ cells, (B): percentages of CD3OD117⁺ cells. (C): cresyl violet staining of a stomach section of a urease + CT vaccinated mouse at day 5 post H. felis challenge. Relative expression of the mMCP- 1 (E) and 2 (D) mRNA in total mRNA extracts from stomachs of vaccinated (CT+urease) or CT vaccinated mice (CT) at day 5 post H. felis challenge. Results are shown as ratios between mRNA copy numbers of mMCP-1 or mMCP-2 and mRNA copy numbers of the housekeeping gene GAPDH. mMCP-1 levels were determined by ELISA at day 5 post H. felis challenge in the serum of vaccinated (CT+urease) or CT control mice (CT) (F). * 5 mice per group, p<0.02; ** 5 mice per group, p<0.008; *** 6 mice per group, p<0.005; **** 4 mice per group, p<0.005; ***** 5 mice per group, p<0.008 (Mann-Whitney test).

Fig. 3. Critical role of mast cells in

Wild type WBB6 F1 mice were vaccinated (at day 0, 7, 14, and 28) either with urease + CT or CT alone. At day 42, mice were challenged with H. felis (5x10⁷). At sacrifice (day 56), urease tests were performed on gastric samples (A). W/W^v mast cell deficient mice were vaccinated, challenged and sacrificed. At sacrifice, urease tests were performed on gastric specimens (B). W/W^v mast cell deficient mice were vaccinated as described above and at day 35 they were reconstituted with bone marrow-derived mast cells. At day 42, the mice were challenged with H. felis and at day 56 sacrificed to perform urease tests on gastric specimens (C). Balb/c mice were vaccinated as described above and challenged with H. felis at day 42. The group of CT+urease vaccinated mice were CD4⁺ cell-depleted (CT+urease CD4⁺ depletion) or injected with control antibody (CT+urease) (12). At sacrifice (day 47), urease tests were performed on gastric specimens (D). W/W^v mice were immunized, mast cell repopulated, challenged at day 42 with H. felis and CD4⁺ cells depleted or injected with control antibody as described above. At sacrifice (day 47), urease tests were performed on gastric specimens (E). * 12 mice per group, p<0.001 ; ^** 12 mice per group, p<0.001 ; *** 12 or 10 mice per group, p<0.01 ; **** 5 mice per group; p<0.008; ****^* 5 mice per group p<0.04 (Mann-Whitney test).

Fig. 4. Serum anti-urease antibody titers (Iog10) of WBB6 F1 mice W/W or +/W^{( )} at day 42 post immunization. Mice were administered (at day 0, 7, 14, and 28) with either urease + CT or CT alone and bled at day 42. Serial dilutions of immune serum were tested for the presence of anti-urease specific antibodies by the ELISA procedure. The antibody titre was defined as the Iog10 of the greater dilution of serum giving an OD two fold higher than of the OD value of the pre-immune serum, diluted at 1 :200. * 9 and 13 mice per group p = 0.11 , no significant difference (Mann- Whitney test).

Fig. 5. Neutrophil depletion at the time of Helicobacter challenge does not preclude vaccine efficacy. Balb/c mice were vaccinated with CT+urease or CT administered as described previously. 12 hours before H. felis challenge, mice were either injected with anti-neutrophil depleting antibody (RB6-8C5) or rat control IgG (250μg/mouse intraperitoneal injection). Antibody injections were repeated at day 1 and 4 post challenge. At day 5 post challenge, the mice were sacrificed to perform the urease test on gastric specimens. * 7 mice per group p<0.008; ** 7 mice per group p<0.002 (Mann-Whitney test).

Fig. 6. TNFα produced by mast cells is dispensable for efficacy of anϋ-Helicobacter vaccination. As described previously, W/W mice were immunized with CT+urease and repopulated with mast cells derived from bone marrow of B6 TNFα -/- mice or B6 wild type mice. Mice were challenged with H. felis and, 12 days later, sacrificed and their stomachs evaluated by urease tests. * 7 mice per group p=0.9 no significant difference (Mann-Whitney test).

Fig. 7. IL-9 transgenic mice do not clear Helicobacter infection spontaneously. An IL- 9 transgenic mouse develops a massive mast cell infiltration in the stomach (S3). IL- 9 transgenic and non-transgenic mice (8 week-old) were infected with H. felis and, 14 days later, sacrificed and their stomachs evaluated by urease tests. * 9 mice per group p=0.9 no significant difference (Mann-Whitney test).

Fig. 8. Balb/c mice developing a gastric mastocytosis do not clear Helicobacter infection spontaneously. Balb/c mice (8 week-old) were injected daily with rlL-3 from day 4 to day 0, infected with H. felis and, 14 days later, sacrificed and their stomachs evaluated by urease tests. * 6 mice per group p=0.07 no significant difference (Mann-Whitney test).

Fig. 9. Represents the sequence comparison with human germ line V genes. Sequence comparison of the deduced amino acid sequences of the isolated H. pylori binders (A9 and A8) and the antiurease scFv F11 revealed close homologies (between 90 to 100%) of all scFvs to their germ line V genes. The anti-H. pylori scFvs has identical heavy chains of the VH4 family which were most closely related to germ line gene DP-71 (VH4-59) and closely related kappa light chains with the highest homology to germ line gene DPK9 (O2/O12). The heavy chain of antiurease scFv F11 belonged to the VH5 family and was most closely related to germ line gene DP- 73 (VH5-51). The lambda light chain (VL3 family) showed the highest homology (100%) to germ line gene DPL-16 (V2-13). % ID, identity to the germ line gene; dots denote sequence identity, and dashes denote gaps in the sequence (Reiche et al. "Generation and characterization of Human Monoclonal scFv antibodies against Helicobacter pylori antigens"; Infection and Immunity, Aug. 2002, Vol. 70, N°8, p. 4158-4164).

Fig. 10. Effect of mast cell activation on in vitro bacterial clearance. Activated mast cells kill more efficiently Helicobacter pylori in vitro.

Fig. 11. Effect of mast cell activation on in vivo bacterial clearance. In vivo mast cells activation is critical to eradicate Helicobacter following vaccination.

Fig. 12. Effect of the administration of leukotriene B4 on the course of H. felis infection. Detailed description of the invention

As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

"Helicobacter" is a genus of spiral, gram-negative bacteria which colonize the gastrointestinal tracts of mammals. H. pylori infection is present in about 50% of all humans (Hunt, R.H. Eradication of Helicobacter pylori infection .Am. J. Med. 100 (suppl 5A):42S-51S, 1996). Several species colonize the stomach, most notably, H. pylori, H. heilmanii, H. felis, and H. mustelae. Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been found to infect humans, but at lower frequencies than H. pylori. The term "Helicobacter", according to the present invention includes, but is not limited to, Helicobacter mustelae (H. mustelae), Helicobacter felis (H. felis), Helicobacter heilmanii (H. heilmanii) and Helicobacter pylori (H. pylori).

"Gastroduodenal diseases" or "diseases" caused by or associated with Helicobacter infection in a mammal include acute, chronic, and atrophic gastritis, peptic ulcer disease including both gastric and duodenal ulcers, gastric cancer or gastric lymphoma, chronic dyspepsia with severe erosive gastroduodenitis, refractory non- ulcer dyspepsia, intestinal metaplasia, mucosal atrophy, and low grade MALT lymphoma. Helicobacter infection is also the principle cause of asymptomatic chronic gastritis. More preferably, the disease caused by or associated with Helicobacter infection is duodenal or gastric ulcers, gastritis or gastric carcinoma.

"Treatment" or "treating" and "preventing" or "prevention" refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of gastric cancer, the therapeutically effective amount of the drug may: reduce the number of bacteria which are involved in the development of gastric cancer and consequently the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing Helicobacter bacteria, it may be cytostatic and/or cytotoxic. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, preferably by at least 50 percent, more preferably by at least 70 percent, most preferably by at least 80 percent, in particular by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, a group of cancer cells or a tumor, or other feature of pathology.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Example of cancer includes, but is not limited to gastric carcinoma.

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. "Passive therapy" refers to the administration of compounds to mammal leading to the clearance of the bacteria without generating specific immune responses in the administered mammal.

"Active therapy" refers to the administration of vaccine or active compounds that normally generate specific immune responses in the administered mammal leading to the clearance of the bacteria.

"Antibody" refers to a class of plasmaproteins produced by the B-cells of the immune system after stimulation by an antigen. Mammal (i.e. Human) antibodies are immunoglobulins of the Ig G, M, A, E or D class. The term "antibody" as used for the purposes of this invention includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, and anti-idiotypic antibodies. The antibodies may be naturally derived from any animal, synthesized in bacteria or another non-animal source, chemically synthesized, or genetically synthesized.

"Antigen" is a compound with the ability to elicit an immune response. Antigens are typically proteins but they can also contain sugars or lipid moieties. Antigens have a molecular weight of generally more than 10 000 Dalton.

A "Vaccine" is a preparation containing an immunogen able to activate the B-cell arm and eventually the T-cell arm of the immune system. "Vaccination" or "Vaccinating" refers to the administration of a vaccine.

The "Antigen/antibody interaction" is the interaction of a specific antibody with the surface of the corresponding antigen. This interaction is reversible and the complex can dissociate depending o the strength of the binding.

The term "urease peptides" refers to, but is not limited to, any urease or subunit of urease, either naturally occurring or obtained by recombinant DNA techniques, as well as a digested fragment or peptide thereof, fusion proteins comprising the whole urease, subunits, or fragments thereof, or truncated urease constructs. Also included within the term "urease peptides", are proteins or peptides that display epitopes sufficiently homologous to epitopes displayed by Helicobacter urease such that antibodies that recognize epitopes displayed by Helicobacter urease will recognize epitopes displayed by said peptides or proteins.

An "Adjuvant" is a substance or a mixture of substances which is added to the vaccine in order to enhance its efficiency to elicit cellular responses and/or antibodies or generate a specific class of antibodies such as, for example, IgM immunoglobulins or antibodies able to bind complements. Substances that will be used as adjuvants are, for example, mineral oils, derivatives of aluminium or compounds of mycobacteria. The composition of this invention may be used with or without adjuvant.

Despite the proven ability of immunization to prevent Helicobacter infection in mouse models, the precise mechanisms of protection have remained obscure. In pioneer experiments, Applicant demonstrates that mast cells are required for the stomach eradication of Helicobacter. Surprisingly, it has been found that anti- Helicobacter vaccination of mice that are deficient in mast cells do not lead to the elimination of the bacteria from the stomach. Furthermore, the involvement of mast cells in the elimination of Helicobacter from the stomach after vaccination was definitively proved. Applicant reconstituted mast cell deficient mice with bone marrow derived mast cells and showed that vaccination of these reconstituted mice lead to Helicobacter eradication. Moreover, by conducting fluorescent cell activated analyses, immunocytochemistry and molecular biology techniques, Applicant found that the mast cells accumulate in the stomach during the immune responses elicited by the vaccine and leading to elimination of Helicobacter.

Applicant has surprisingly found increased percentages of mast cells in the gastric mucosa during vaccine-induced clearance of Helicobacter

mice. This was associated with increased expressions of mRNA encoding granule-stored mast cell protease 1 and 2 (mMCP-1 and 2) in the stomach and elevated serum mMCP-1 protein levels. Immunized mast cell deficient mice (W W^v) were not protected from Helicobacter colonization, but vaccinated W/W^v mice re-populated with mast cells cleared the infection. These experiments show that mast cells are remarkably and unexpectedly critical for the cure of diseases caused by or associated with Helicobacter infection.

The present invention provides evidence that mast cells are required to clear Helicobacter infection after immunization. Indeed, the results presented here indicate that an early influx of mast cell takes place in the gastric mucosa in immunized mice following Helicobacter challenge. These mast cells are activated since elevated mMCP-1 protein levels are present in the serum of these mice. The lack of immunization mediated protection in mast cell deficient W/W mice and the restitution of this protection upon reconstitution of the mast cell population in these mice underline the critical role of mast cells for the immune mediated gastric protection against Helicobacter infection.

It is known that Helicobacter nfected individuals develop both humoral and cellular responses against the bacteria but are unable to clear the infection. Unexpectedly, the present invention has evidenced that mast cells are necessary to eliminate Helicobacter from the stomach after vaccination.

The method of the present invention for treating a disease caused by or associated with Helicobacter in a mammal which comprises administering to said mammal a preventive and/or therapeutically effective amount of a composition capable of activating mast cells in the stomach of said mammal which preferably further comprises the step of vaccinating the mammal with an appropriate antigen derived from Helicobacter prior to administering the composition to said mammal. In case the individual is vaccinated prior to administering the composition, the time period between the vaccination and the administration of the composition is usually between 4 to 10 days after the last immunization, preferably 5 days or more.

The vaccine consists for example in urease peptides or other antigen preparations derived from Helicobacter and can be administered as described below for the pharmaceutical composition of the present invention.

For example, the administration of immunological combinations or vaccines of the present invention may be made as a single dose or as a dose repeated once or several times after a certain period. The appropriate dosage varies according to various parameters, for example the individual treated (adult or child), the vaccinal antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and if present, the type of adjuvant and the desired effect (e.g. protection or treatment), as can be determined by persons skilled in the art. In general, the vaccination with an antigen according to the invention may be administered in a quantity ranging from 10μg to 500 mg, preferably from 1 mg to 200 mg. In particular, it is indicated that a parenteral dose should not exceed 1 mg; preferably 100 μg. Higher doses may be prescribed for e.g. oral use. Independently of the formulation, the quantity of protein administered to man by the oral route is for example of the order of 1 to 10 mg per dose, and at least 3 doses are recommended at 4-week intervals.

The composition capable of activating mast cells in the stomach of a mammal can lead to a specific or unspecific activation of the mast cells in the stomach of infected patients which clears Helicobacter infection. In particular, the composition capable of activating mast cells in the stomach of a mammal may be used as an agent for treating a disease caused by or associated with Helicobacter in said mammal.

Usually, the activation of mast cells in the stomach of a mammal leads to an increase of the expression of mast cell proteases 1 and/or 2 or related Mast cell activation markers such as Tryptase in human. The increase of expression level of mast cell proteases 1 and/or 2 caused by the activation depends on the amount of administration and other factors as described below. Usually, the increase is two fold up to 1000 fold, preferably 10 fold as compared to the level of expression without activation. The increase of expression can be detected in the blood of the individual and therefore can serve as an indirect mean for detecting the clearance of Helicobacter in the stomach of a mammal.

This method of detection further comprises the step of administering the pharmaceutical composition as described below prior to the detection of the blood level increase of the expression of mast cell proteases 1 and/or 2 or other related Mast cell activation markers such as Tryptase in human. One possible and also the preferred method leading to specific mast cell activation is the administration of a composition consisting essentially of IgE and/or IgG antibodies binding to or specific to antigens derived from Helicobacter, whereas the IgE and/or IgG antibodies induce the activation of said mast cells.

For the preparation of IgE and/or IgG antibodies binding to or specific to antigens derived from Helicobacter, urease can be used and is the preferred antigen. Indeed, the urease protein is the main antigenic protein produced by the bacteria.

More preferably IgE antibodies binding to or specific to urease derived from Helicobacter are used. One sequence of an IgE antibody binding to or specific to urease is illustrated in Fig. 9. Antigens according to the invention may be used either for the preparation of IgE and/or IgG antibodies binding to or specific to antigens derived from Helicobacter or for the preparation of a vaccine. Therefore, the antigen may be the same or different.

Other immunoglobulin isotypes can also be considered to activate mast cells. Preferably, the antibody should be a monoclonal antibody, however, polyclonal antibodies or a mixture of monoclonal antibodies binding to antigens derived from Helicobacter can be used. For the specificity of the monoclonal antibody, either urease or other Helicobacter derived antigens can be used.

Preferred antigens for use in the invention are Helicobacter (e. g., H. pylori or H. felis) proteins or other components (e. g., lipopolysaccharides or carbohydrates) that either are purified from bacterial cultures or are produced using standard recombinant or chemical synthetic methods. Preferred antigens are proteins or portions of proteins (i. e., peptides or polypeptides). Methods for identifying immunogenic fragments of polypeptide antigens are known in the art, and can be employed in preparing antigens for use in the methods of the invention (Stumiolo et al., Nature Biotechnology, "Generation of Tissue-Specific and Promiscuous HLA Ligand Databases Using DNA Microarrays and Virtual HLA Class II Matrices", June 1999). Additional antigens that can be used in the invention are whole bacteria and non-purified protein preparations, such as Helicobacter lysats.

Further antigens from Helicobacter which can be used are vacuolating cytotoxin VacA, the neutrophil activating protein or uncharacterised preparations of the bacterium.

The antigens used in the invention can be produced as fusion proteins, i. e., polypeptides containing amino acid sequences corresponding to two or more proteins (or fragments thereof), which are normally separate proteins, linked together by a peptide bond (s). Fusion proteins generally are synthesized by expression of a hybrid gene containing nucleotides encoding each of the individual polypeptides that make up the fusion protein. An example of an antigenic fusion protein included in the invention is one that contains a CT or an LT toxin adjuvant (e. g., toxin A or B subunit, or a fragment or derivative thereof having adjuvant activity) fused to an H. pylori antigen, e.g., urease. Another type of fusion protein included in the invention consists of an antigen fused to a polypeptide (e. g., glutathione S-transferase (GST)) that facilitates purification of the fusion protein. Proteins used as antigens in the invention can also be covalently coupled or chemically cross-linked to adjuvants using standard methods.

The most preferred Helicobacter antigens for use in the invention are urease peptides and derivatives thereof. Most preferred are enzymatically inactive, recombinant multimeric urease complexes, produced as described in Lee et al., WO 96/33732, which is hereby incorporated by reference. A number of other immunogenic Helicobacter antigens can also be administered according to the invention, e. g., catalase (WO 95/27506), HspA and HspB (WO 94/26901), lactoferrin receptor (WO 97/13784), p76 (WO 97/12908), p32 (WO 97/12909), BabA and BabB (WO 97/47646), AlpA (WO 96/41880), AlpB (WO 97/11182), as well as the antigens described in WO 96/38475, WO 96/40893, WO 97/19098, WO 97/37044, and WO 98/18323. The immunogenic antigens can be used alone (with the adjuvant) or in "cocktails" of two or more antigens. Other compounds (biological or chemical) which lead to unspecific activation such as Mastprom ("Augmentation of Mast cell Bacterial Activity by the anti-leukemic drug 4- (3'bromo-4'-hydoxyphenyl)-amino-6,7-dimethoxyquinazoline" Ravi et al. Leukemia and lymphoma 2002, vol 43 pp 1329-1332), C48/80, Mastroparan, stem cell factor, C3a, Substance P, Neuropeptide Y and/or related chemical or biological agents can be used as potent composition able to cure the Helicobacter infection.

In addition, Applicant has surprisingly found that both CD4⁺ T cells and mast cells are collaborating to clear Helicobacter infection following vaccination procedure, this means that one can stimulate relevant immune cells against H. pylori with the appropriate vaccine formulation or drugs acting on the physiological mechanisms leading to the bacterial clearance from the gastric mucosa.

Both mast cells and CD4⁺ cells are essential to confer protection against Helicobacter following vaccination. There is a bi-directional dialogue between mast cells and CD4⁺ T cells, this bi-directional dialogue leads to the maturation of both cell types and to the modulation of the physiological functioning of the organ where this mast cell-CD4⁺ T cell dialogues are taking place.

Therefore molecules derived from mast cells and/or CD4+ T cells modulating the functioning and/or maturation of both cell types and/or other cells of the Helicobacter infected stomach can be seen as particularly interesting drugs to cure Helicobacter infection. Other cells presented in Helicobacter infected stomach can be of epithelial, neuronal, muscular, bone marrow derived, or other origins.

Mast cells derived molecules:

Mast cells produce three main classes of mediators: preformed granule-associated mediators; newly generated lipid mediators; and a variety of cytokines and chemokines. Of the granule-associated mediators, proteases are important constituents, including tryptase and chymase. Mast cell granules also contain vasoactive amines, proteoglycans or lipids mediators, such as histamine or leukotrienes. Mast cells produce several antimicrobial peptides also known as defensins. A. Leukotrienes:

Leukotrienes (LT) are metabolites of arachidonic acid formed from the 5- lipoxygenase (5-LOX) pathway and exert potent vasoactive and pro-inflammatory effects. LTs play a pivotal role in the pathophysiology of asthma, LTs also play a physiological role in the host defence against microbial infections. The activation of 5- LOX is calcium-dependent, and 5-LOX acts together with 5-LOX-activating protein (5-LOX-AP) to form LTA₄. LTA₄ is unstable and is rapidly converted to either LTB₄ or cysteinyl-LTs, LTC₄, LTD₄, and LTE₄. 5-LOX is predominantly expressed by cells of myeloid origin, particularly neutrophils, eosinophils, macrophages/monocytes, and mast cells. It is however the first time that Leukotreines derived from mast cells (or agonist receptors of leukotreines) are surprisingly used in the present invention for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

B. Histamine:

It has been reported that H. pylori infection of histamine-deficient (histidine decarboxylase knockout) mice lead to lower local inflammation process and lower systemic antibody responses directed toward Helicobacter in comparison with wild type mice. Since histamine is known to modulate the immune response (by acting on T, B and dendritic cells) and the physiology of the stomach, Applicant came to the finding that histamine can be directly or indirectly involved in Helicobacter clearance of vaccinated mice. It is however the first time that Histamine derived from mast cells (or agonist receptors of histamine) is surprisingly used in the present invention for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

C. mMCP-1: mMCP-1 is a chymase expressed in mast cells located in the intestine and stomach mucosa. mMCP-1 is involved in the increased intestinal epithelial paracellular permeability, via the degradation of the tight junctional complexes, observed in the intestinal clearance of the nematode Trichinella spiralis (T. spiralis) infection. The targeted deletion of mMCP-1 leads to delayed expulsion of T. spiralis. During mast cell activation in the stomach mucosa of vaccinated mice, mMCP-1 release in the extra cellular space may degrade the tight junction complexes. This tight junction complexes degradation may allow to the access of plasma derived molecules (such as antibodies, complements, etc..) and/or immune cells with direct bactericidal activity such as mast cells, neutrophils, macrophages to Helicobacter niche leading to the bacterial eradication. It is however the first time that mMCP-1 derived from mast cells is surprisingly used in the present invention for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

D. Tryptase:

Protease-activated receptors (PARs) are G protein-coupled 7-transmembrane domain receptors that are activated by proteolytic unmasking of the cryptic tethered ligand present in the N-terminal domain. PAR-2 is a receptor activated by trypsin, mast cell tryptase, coagulation factors Vila and Xa, and others. Trypsin and mast cell beta-tryptase activate PAR2. On activation, PAR-2 triggers neurally mediated mucus secretion, enhances mucosal blood flow, suppresses acid secretion, and increases pepsinogen secretion. Modification of mucus, mucosal blood flow, and acid secretion may influence the survival of Helicobacter in the stomach. It is however the first time that Mast cell beta-tryptase is surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

E. Cathelicidins:

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils, epithelia cells and mast cells and can act as natural antibiotics by directly killing a wide range of microorganisms. Caths are expressed in mast cells, because these cells have been previously associated with inherent antimicrobial activity. Cultured murine mast cells contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cathelicidins, and this expression was inducible by LPS. Mast cells derived from cathelicidins deficient animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS. Applicant also tested the ability of mast cells generated from the bone marrow to kill H. pylori in vitro. It is however the first time that cathelicidins derived from the mast cells are surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal. Molecules involved in the mast cell-CD4⁺ T cell crosstalks.

F. The mast cells modulate the CD4⁺ T cell response. Mast cells are also important to initiate the recruitment and/or the proliferation of anti-urease CD4⁺ T cells in the infected stomach of vaccinated mice. Mast cells are known to modulate the T-helper- cell differentiation; mast cells can drive the maturation of the Th2 cells, whereas NK cells drive the development of Th1 cells. Therefore, the absence of Th2 cells in the stomach mucosa may be responsible for the absence of bacterial clearance in vaccinated W/W^v mice. IL-4 secreted by the mast cells is the major factor driving the Th2-cell differentiation. It is however the first time that IL-4 or other molecules driving the Th2 cells development derived from the mast cells are surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

G. The CD4⁺ T cells modulate the mast cells:

Applicant findings show that the accumulation and maturation of mast cells found in the stomach mucosa of vaccinated mice infected with Helicobacter is driven by the vaccine-primed memory CD4⁺ T cells which are induced to proliferate and differentiate by the Helicobacter infection.

Mast cell hyperplasia is dependent on several cytokines, including IL-3, IL-4 and IL- 10, which determine not only the growth, but also mast cells phenotype. It is the first time that IL-3, IL-4, IL-10 and/or other molecules driving the mast cell hyperplasia derived from the CD4⁺ T cells are surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

H. LTβR-LTβR ligands :

Mast cells can undergo degranulation during T cell-mediated inflammatory processes. Moreover, in some morphological studies mast cells were found to reside in close proximity to T cells in inflamed tissues and allergic reactions. This close apposition between mast cells and T cells has led investigators to propose a functional relationship between these two cell populations that might facilitate elicitation of the immune response. Lymphotoxin-β (LTβ) receptor (LTβR) is a member of the TNF receptor super family. Mast cells express LTβR at the mRNA as well as at the protein level. Moreover, LTβR expressed on mast cells can transduce a co-stimulatory signal in T cell-dependent mast cell activation leading to IL-4, IL-6, TNF-α, chemokines macrophage inflammatory protein 2 and RANTES release. Applicant has found that LTβR-LTβR ligands interactions between mast cells and T cells can lead to bacterial clearance and thus it is the first time that LTβR-LTβR ligands are surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

In addition, it has recently been shown that mast cell-T cell interactions leading to T cell activation can occur via the OX40L-OX40 ligand receptor interactions. MCs express OX40L on their surface and can induce T cell proliferation in an OX40L- dependent manner. Applicant has found that the OX40L-OX40 ligands receptor interaction between mast cells and T cells can lead to bacterial clearance thus, it is the first time that OX40L-OX40 ligands receptor are surprisingly used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

Furthermore, It has been already described that some of the secreted mediators during the mast cell activation can both influence surrounding cells but also the mast cell themselves. For exemple TNF-α secreted by the mast cells can activate the chemokines secretion of the activated mast cells (McLachlan JB, Abraham SN. 2001 Studies of the multifaceted mast cell response to bacteria. Current opinion in Microbiology 4:260-266). In addition, it has been described that IL-4 secreted by mast cells can upregulate expression of functional FcεR at the surface of the mast cells (Austen KF, Boyce JA. 2001. Mast cell lineage development and phenotypic regulation. Leukemia Research 25: 511-518). These autocrine functions of the mast cell-derived mediators can be potentially useful to modulate the mast cells activation status in Helicobacter infected stomach and can be involved in the vaccine-induced bacterial eradication.

Therefore, the present invention also encompass a method for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal leading to specific mast cell activation by the administration of a composition comprising molecules derived from mast cells and/or CD4⁺ T cells. Molecules derived from mast cells essentially consist in Leukotrienes, Histamine, mMCP-1 , tryptase, cathelicidins and a mixture thereof. Preferably, Leukotrienes derived from mast cells is Leukotriene B4.

According to the present invention, molecules derived from CD4⁺ T cells essentially consist in molecules involved in the mast cell-CD4⁺ T cell crosstalks. For example, molecules involved in the mast cell-CD4⁺ T cell crosstalks are selected among the lists comprising IL-3, IL-4, IL-10, LTβR- LTβR ligands, OX40L-OX40 ligands.

Another subject matter of the present invention is a method for activating mast cells in the stomach of a mammal comprising, administering to said mammal a preventive and/or therapeutically effective amount of a composition consisting essentially of IgE and/or IgG antibodies binding to or specific to antigens derived from Helicobacter whereas the IgE and/or IgG antibodies induce the activation of said mast cells which preferably further comprises the step of vaccinating the mammal with an appropriate antigen derived from Helicobacter prior to administering the composition to said mammal. The composition may also comprise alone or in combination with antibodies, molecules derived from mast cells and/or CD4+ T cells as defined above.

A further subject matter of the present invention is a pharmaceutical composition comprising as an active substance a pharmaceutically effective amount of the composition as defined above, optionally in combination with pharmaceutically acceptable carriers, diluents and adjuvants. Preferably, the pharmaceutical composition of the present invention comprises pharmaceutically acceptable carriers, diluents and adjuvants. Such acceptable carriers, diluents and adjuvants should be non-toxic and should not interfere with the efficacy of the active ingredient. This pharmaceutical composition is preferably used as an agent for the preparation of a medicament for the prevention or treatment of diseases caused by or associated with Helicobacter in a mammal, more preferably for the prevention or treatment of duodenal or gastric ulcers, gastritis or gastric carcinoma.

The pharmaceutical composition can be in any suitable form, e.g. in the form of a solution, suspension, powder, lyophilisate, ointment or tincture. The composition can be administered by any suitable method, e.g. per injection (systemically or locally). The precise nature of the carrier or other material will depend on the route of administration, which may be intra-nasal, oral, rectal, or by injection, e.g. intramuscular, transdermal, sub-cutaneous or intravenous. Preferably, the route of administration is oral.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogenfree and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

A suitable mucosal adjuvant is cholera toxin. Others, which may be used, are non- toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the urease antigen plus cholera toxin or its B subunit. Other suitable delivery methods include biodegradable microcapsules or immuno-stimulating complexes (ISCOMs) or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) viruslike particles, e.g., bluetongue. The amount of mucosal adjuvant employed depends on the type of mucosal adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 μg to 50 μg, for example 10 μg to 35 μg. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsules to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in this art.

The respective pharmaceutically effective amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific composition used, especially if the composition additionally contains an antisecretory agent and/or an antibiotic effective against Helicobacter infection. The treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks. The pharmaceutically effective amount of a dosage unit of the composition usually is in the range of 0.001 ng to 100 μg per kg of body weight of the patient to be treated.

Pharmaceutical compositions used in accordance with the present invention are prepared for storage by mixing a pharmaceutically effective amount of the composition as defined above having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN(TM), PLURONICS(TM) or polyethylene glycol (PEG).

The active ingredients or agents may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.

The pharmaceutical composition or formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide antibodies which bind to different antigens derived from Helicobacter or to provide antibodies (with the same specificity or with different specificities) which bind different receptors on mast cells in the one formulation. Alternatively, or additionally, it will become apparent that the pharmaceutical composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. Preferably, the pharmaceutical composition may further comprise a cytokine, anti-hormonal agent, immune modulators and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

Preferably, the composition of the invention, capable of activating mast cells in the stomach of a mammal for the use as agent for treating a disease caused by or associated with Helicobacter in said mammal comprises molecules derived from mast cells and/or CD4⁺ T cells as defined above.

Further anti-Helicobacter agents compositions of the invention may be administered in conjunction with an antisecretory agent and/or an antibiotic effective against Helicobacter pylori. These components offer rapid relief from any existing H. pylori infection, thereby complementing of immunotherapy.

These may be administered in the same composition according to the present invention, but will typically be administered separately. They may be administered at the same time, but they will generally follow a separate administration protocol e.g. daily. They may be administered by the same route as disclosed above, but they will generally be administered orally. They may be administered over the same timescale as disclosed above, but they will generally be administered from shortly before (e. g. up to 5 to 14 days before) the first dose of the composition of the invention up to shortly after (e. g. up to 5 to 14 days after) the last dose of the composition according to the present invention.

Preferred antisecretory agents are proton pump inhibitors (PPIs), H2 receptor antagonists, bismuth salts and prostaglandin analogs.

Preferred PPIs are omeprazole (including S-and B-forms, Na and Mg salts etc.), lansoprazole, pantoprazole, esomeprazole, rabeprazole, heterocyclic compounds, imidazo pyridine derivatives, fused dihydropyrans, pyrrolidine derivatives, benzamide derivatives, alkylenediamine derivatives ect. Preferred H2-receptor antagonists are ranitidine, cimetidine, famotidine, nizatidine and roxatidine.

Preferred bismuth salts are the subsalicylate and the subcitrate, and also bismuth salts of antibiotics of the moenomycin group.

Preferred prostaglandin analogs are misoprostil and enprostil.

Preferred antibiotics are tetracycline, metronidazole, clarithromycin and amoxycillin.

It is contemplated that, the pharmaceutical composition comprising as an active substance a pharmaceutically effective amount of the composition as defined above may be used to treat various diseases or disorders. Generally, the disease or disorder to be treated is caused by or associated with Helicobacter in a mammal. In particular, the present invention provides a method of treating or preventing duodenal or gastric ulcers, mucosal atrophy, gastrite, gastric carcinoma or gastric lymphoma. More preferably, the disease caused by or associated with Helicobacter is duodenal or gastric ulcers, gastrite or gastric carcinoma.

For the prevention or treatment of disease and especially cancer, the appropriate dosage of the composition as described above will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition and the discretion of the attending physician. The pharmaceutical composition is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of composition is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. The preferred dosage of the composition will be in the range from about 0.005 mg/kg to about 1.0 mg/kg. Thus, one or more doses of about 0.05 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses of the composition). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosing regimen comprises administering an initial loading dose of about 0.4 mg/kg, followed by a weekly maintenance dose of about 0.2 mg/kg of the composition. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

As an example of embodiment, the administration of IgE anti-urease antibodies may be carried out as follows. Systemic injection or local administration of isotype E antibodies that are directed to urease activates stomach mast cells and leads to the recruitment and activation of T CD4+ cells specific to Helicobacter resulting in its eradication from the stomach mucosa.

Isotype E anti-urease antibodies are prepared by usual technique of generating monoclonal antibodies. Antibodies are selected for their specificity (B epitope), their isotype (IgE) as well as their affinity for urease. Different form of isotype E antibodies are produced and selected on their ability to eliminate Helicobacter \n vivo.

Doses comprised between 1 μg to 500 μg are injected intravenously. Doses that are injected by tube-feeding are comprised between 100 μg and 500 μg. For the gastric administration, antibodies are solubilized in a carbonate buffer in order to neutralize the gastric acidity. Galenic forms protecting antibodies from the gastric acidity may also be used.

Mice are infected with Helicobacter felis or Helicobacter pylori, two weeks after infection; the isotype E anti-urease antibody is administered. A kinetic of the infection's disappearance is carried out; mice are sacrificed 2, 3, 5, 7, 10 and 12 days after the antibody's administration. Blank or control mice are infected by Helicobacter and an IgE isotype antibody directed against an irrelevant antigen is administered.

Alternatively, the administration of agents capable of activating mast cells may be carried out as follows. Systemic injection or local administration of activator agents of the stomach mast cells lead to the recruitment and activation of T CD4+ cells that are specific to Helicobacter resulting to its eradication from the stomach mucosa.

These activator agents for example the Mastprom (4-(3'bromo-4'-hydroxylphenyl)- amino-6,7-dimethoxyquinazoline) are systemically or locally administered (10mg/kg). For gastric administration, the activators are solubilized in a carbonate buffer in order to neutralize the gastric acidity. Galenic forms protecting antibodies from the gastric acidity may also be used.

Mice are infected by Helicobacter felis or H. pylori and the activator is administrated two weeks after infection. A kinetic of the infection's disappearance is carried out; mice are sacrificed 2, 3, 5, 7, 10, and 12 days after the antibody's administration. Blank or control mice are infected by Helicobacter in the absence of an activator.

It is another embodiment of the invention to provide a kit for the prevention or treatment of diseases caused by or associated with Helicobacter in a mammal, said kit comprising the pharmaceutical composition of the present invention, optionally with reagents and/or instructions for use.

The kit of the present invention may further comprise a separate pharmaceutical dosage form comprising an antisecretory agent and/or an antibiotic effective against Helicobacter and combinations thereof.

Generally, the Kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice, such as gastric cancer. In one embodiment, the label or package inserts indicates that the composition of the present invention can be used to prevent or treat diseases caused by or associated with Helicobacter infection. More preferably, the disease caused by or associated with Helicobacter infection is duodenal or gastric ulcers, gastritis or gastric carcinoma.

Alternatively, or additionally, the Kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In a further embodiment, the invention provides a method for eradicating Helicobacter pylori from the stomach of a mammal by administering to said mammal an effective amount of a composition capable of activating mast cells in the stomach of said mammal. The composition which can be used its way of administration and the effective amount is as desribed above. The eradication is usually obtained by the method of the present invention if the urease breath test is negative.

The invention also encompass a method for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal comprising, administering to said mammal a preventive and/or therapeutically effective amount of a composition comprising molecules derived from mast cells and/or CD4⁺ T cells. Molecules derived from mast cells and/or from CD4⁺ T cells are as described above.

Finally, the composition comprising molecules derived from mast cells and/or CD4+ T cells (as described above) can also be used for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal or in the manufacture of a medicament for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

Examples

Example 1

Materials and Methods

Mice.

WB/ReJ-Kit^w/+ and C57BL/6J-Kit^w7+ mice were purchased from the Jackson

Laboratory (Bar Harbor, ME) and mated in our SPF animal facility. We used either male or female 6 to 8 week-old W/W^v and WBB6F1 +/+ mice. Female Balb/c mice (6 to 8 week-old) were purchased from Harlan, Horst, The Netherlands. This study was approved by the State of Vaud Veterinary Office (authorization no. 836.6).

Immunization

CD4⁺ cells in vivo depletion and mastocytosis induction. Mice were immunized intranasally four times at 1-week intervals with 30 μg of recombinant H. pylori urease (kindly provided by Acambis, Cambridge, Mass.) combined with 5 μg of cholera toxin (CT) (Calbiochem, Lucerne, Switzerland). 100μg of depleting monoclonal antibody (GK1.5) was injected intra-peritoneally at days -2, -1 , 0, and +3 of bacterial challenge. As a control, mice were administered with 100μg of purified rat IgG (Sigma, 1-4131). CD4 cell depletion was confirmed at sacrifice (day +5), by flow cytometry analysis of the lymphoid cell population recovered from the lymph nodes draining the stomach. In the control rat IgG- injected mice, 30.7% of the total lymphoid population stained positive with the anti-CD4 monoclonal antibody H 129.19, as compared to 0.8% of the lymphoid population in GK1.5 injected mice. To induce mastocytosis, Balb/c mice were injected daily with rlL-3, as described in S1. Control mice were injected with PBS.

Bacteria and infection

H. pylori P49 was grown on GC agar plates supplemented with IsoVitaleX and horse serum or in brain heart infusion broth supplemented with 0.25% yeast extract and 10% horse serum, under microaerophilic conditions (BHI). H. pylori P49, kindly provided by Harry Kleanthous (Acambis, Cambridge, Mass.), is a human clinical isolate adapted to mice. The H. felis strain ATCC 49179 was grown biphasically under microaerophilic conditions. H. felis and H. pylori P49 infections were performed by orogastric intubation with polyethylene tubing under light anesthesia with halothane (Halocarbon Laboratories, River Edge, NJ). The tubing was introduced at a fixed distance of 4.5 cm from the incisors. Mice were treated once with 5x10⁷ H. felis or twice (with a 2-day interval) with 5x10⁸ H. pylori P49, administered intragastrically in 200μl of BHI.

Assessment of Helicobacter colonization and histology

Rapid urease test (Jatrox-test; Procter & Gamble, Weiterstadt, Germany) was used to assess the infection status. Briefly, stomachs were retrieved and cut along the lesser and greater curve to obtain identical halves. One half was immersed in 500 μL of supplier's suspension and incubated at 37°C for 3 hours. Specimens were centrifuged, and the supernatant was used for spectrophotometric quantification at an optical density of 550 nm. The other half was processed for histology and fixed in neutral buffered 10% formalin, embedded in paraffin, and routinely processed. Sections of 5-μm thickness were stained with cresyl violet and hematoxylin and eosine. The number of H. felis was assessed in 10 glandular crypts in the antral and fundal mucosa on coded sections. CFU (only for Hp49): immediately after collection, one half of the stomach was immersed in 0.2 mL BHI and homogenised with a fitted plastic pestle in a sterile Eppendorf tube. Serial 10-fold dilutions of the homogenate were then plated on Brucella agar plates supplemented with 0.25% yeast extract, 5% sheep red blood cells (all from Difco Laboratories, Detroit, Ml), and 1% Skirrow supplement SR 69 (Oxoid Ltd., Basingstoke, England). Plates were incubated for 3- 4 days in microaerophilic conditions (6% 02, 10% CO2, and 84% N2), and CFU counted. Identification of /-/, pylori was based on the appearance of colonies on plates, Gram stains, and urease activity. Results were expressed as number of colony-forming units per half stomach.

Determination of the anti-urease antibody response

Microtiter plates were coated with 0.5 μg recombinant urease. Serum samples were serially diluted and plated. Specific antibodies were detected with biotinylated rabbit anti- mouse immunoglobulin G IgG (Amersham, Dϋbendorg, Germany) used at a dilution of 1 :500, followed by incubation with streptavidin-bound horseradish peroxidase (AP- Biotech) at a dilution of 1 :5,000 (Dako, Zug, Switzerland). Immune complexes were revealed with o-phenylenediamine (Sigma), in the presence of 0.03% H₂O₂ as a substrate, and plates were read (at 492nm) after 15 min of incubation.

Quantification of mMCP-1 m-MCP1 serum concentrations were assayed using the RF6.1 monoclonal-based enzyme-linked immunosorbent assay (Wastling JM. et al. "Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1" Am J Pathol. 1998; 153, pp 491-

504).

Preparation of lymphoid cell suspension from the stomach

Stomachs were isolated and cut longitudinally in half. After washing (NaCI, 9g/l), one half was cut into small pieces using a scalpel. Stomach tissue fragments were then incubated in PBS 1mM EDTA for 20 min under gentle stirring at room temperature. After centrifugation (1500 rpm, 10 min, 4°C), tissue fragments were incubated at 37°C for 15 min under stirring conditions (150 rpm) with 10 ml of RPMI 1640 (Gibco, Invitrogen corporation, Carlsbad, California) 10% foetal calf serum (heat inactivated, Biological Industries, Beit Haemek) and 0.5mg/ml type IV collagenase (Sigma C- 5138). The preparations were then passed through two mesh tea strainers (70μM and 40μM) to separate cell suspension from the undigested tissue. The cell suspension was centrifuged at 1500 rpm for 10 min (4°C) and recovered cells were washed twice with 20 ml of fresh 10% FCS RPM1 1640. Cells were ressuspended in 20 ml 10% FCS RPMI 1640, and 10 ml Ficoll-PaqueTM plus (Amersham Biosciences Corp, Amersham, Dϋbendorg, Germany) was added in the bottom of the preparation (50 ml Falcon, Becton Dickinson and Co., Mountain View, CA) and centrifuged 10 min at 2200 rpm (4°C). The lymphoid cells were recovered from the interface between Ficoll and 10% FCS RPM1 1640. The cells were washed twice with 20 ml of fresh 10% FCS RPMI 1640.

Flow cytometry

Lymphoid cells were first incubated with purified anti-mouse CD16/CD32 (Clone 2.4G2) to block unspecific binding and incubated with the following rat anti-mouse antibodies: anti-mouse CD117 (c-kit) (Clone 2B8); anti-mouse CD3 (Clone 145-2C11); anti-mouse CD4 (H129.19); anti-mouse CD19 (1D3); anti-CD8α (53-6.7); or purified rat lgG2a or lgG2b. Rat anti-mouse antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA). Samples were analysed using a Coulter EPICS XL-MCL (Miami, FL). Dead cells were excluded by a combination of forward and side scatter.

Culture of bone marrow-derived mast cells

Mouse Bone marrow cells (BMC) were recovered from 6- to 8-wk-old WBB6F1-+/+. Freshly isolated BMC were suspended in 25-cm2 flasks (Corning, Corning, NY) at a concentration of 1 x 10⁵ cells/ml of enriched medium (RPM1 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 μM 2-ME (Sigma, St. Louis, MO), and 10% FCS to sustain cell viability (Biological Industries, Beit Haemek) supplemented with 100 U/ml of IL-3 (R&D Systems Inc. Minneapolis). Cells were fed with fresh medium and cytokines weekly and were dispersed to maintain a cell concentration below 1 x 10⁶ cells/ml. Bone marrow-derived mast cells were recovered after three weeks of culture. Before injection into mice, the recovered cell population was characterized by flow cytometry analysis (all the cell suspensions obtained were composed of 98 to 99 % CD117⁺ cells). 1x10⁶ mast cells were injected intravenously to repopulate W/W^v mice.

Quantitative PCR RNA extraction was performed on one stomach half using the Rneasy Mini kit (Qiagen, Valencia, CA, USA). RNA (1μg) was reverse transcribed into cDNA using Oligo(dT) (ThermoSript RT-PCR System, Invitrogen Corporation/Life technologies, Carlsbad, CA). For every reaction, one RNA sample was run without ThermoScript RT to provide negative control in the subsequent PCR (RT). To minimise variation in the reverse transcription reaction, all the RNA samples from a single experimental set-up were reverse transcribed simultaneously. PCR amplification was performed on the MyiQ icycler (Biorad, Hercules, CA), using 96-well microtiter plates (Biorad). For each sample (RT⁺ or RT^"), the PCR reaction was performed in duplicate with the iQ™ SYBR Green Supermix (Biorad). Samples were heated at 95°C for 3 minutes and then subjected to 35 cycles consisting of denaturing (95°C, 15s) and primer annealing and extension for 60s at 60°C (GAPDH), 63.1 (mMCP-2) or 65 °C (mMCP- 1). Melt curves of the amplified products were performed to identify the amplicon. The used primers were are followed GAPDH (900nM, 4mM MgCI₂): 5'- GCTAAGCAGTTGGTGGTGCA-3' and 5'-TCACCACCATGGAGAAGGC-3', mMCP-1 (350nM, 3mM MgCI₂): 5'-GGAAAACTGGAGAGAAAGAACCTAC-3' and 5'- GACAGCTGGGGACAGAATGGGG-3' (J. M. Wastling et al., Am. J. Pathol. 153, pp 491 ; 1998) , mMCP-2 (350nM, 3mM MgCI₂): 5'-ATTTCATTGCCTAGTTCCTCTGAC- 3' and 5'-CAGGATGAGAACAGGCTGGGAT-3'. Quantification of input cDNA from the unknown samples was performed by including a standard curve. To construct the standard DNA, amplicons generated by RT-PCR using the same primers as described above were purified on silica columns (QiAquick PCR purification, Qiagen), and cloned into pGEM-Teasy (Promega Corp., Madison WI). Ligated fragments were transformed into DH5α-competent cells and plasmid DNA were prepared using silica cartridges (Qiagen). The exact sequence of cloned amplicons was analyzed by cycle sequencing. DNA plasmid concentrations were measured by optical density spectrophotometry and the corresponding copy number was calculated using the equation: 1μg of 1000 bp DNA = 9.1 x 10¹¹ molecules. Serial 10-fold dilutions of plasmids ranging from 10⁷ to 10² DNA copy were used as standard curve in each PCR run. To minimise inter-assay variability, all samples analysed from a single experiment were performed in the same 96-well microtiter plate.

Example 2 Applicant has explored the cellular events associated with Helicobacter clearance from the stomach following vaccination. Applicant has first studied the kinetics of H. felis clearance in wild type mice vaccinated intranasally with urease + cholera toxin (CT). At day 4 post challenge, infection, as defined by positive urease tests on gastric mucosa samples, was detected in 4 out of 5 urease + CT vaccinated mice (Fig. 1A). At day 5 post challenge, only 2 out of 10 urease + CT vaccinated mice showed positive urease tests, whereas 10 out of 10 control mice vaccinated with CT alone were positive. Gastric histological analysis confirmed the presence of H. felis in CT vaccinated mice, but not in urease vaccinated mice (Fig. 1 B). When vaccinated mice were challenged with H. pylori, urease tests were decreased at day 5 post challenge in urease + CT vaccinated mice as compared to CT vaccinated controls (Fig. 1C). Furthermore, quantitative cultures of - , pylon from gastric samples showed decreased colony forming unit (CFU) counts in urease + CT vaccinated animals, as compared with controls (Fig. 1 D). Taken together, these kinetic studies indicate that vaccination-mediated clearance of Helicobacter begins on day 4-5 after bacterial challenge.

Based on these results, pplicant analyzed the cell populations recovered from the gastric mucosa at day 3, 4, 5, 8, 11 and 19 post challenge, by flow cytometry. Stomach samples were minced and then digested by collagenase treatment. The lymphoid cell population was recovered by Ficoll gradient, and stained with monoclonal antibodies directed towards CD3, CD4, CD8, CD19, and CD117. Applicant has found an increased percentage of CD4⁺ cells in the gastric lymphoid population of urease + CT vaccinated mice at days 3, 4, 5, 8, and 11 post challenge, as compared to CT vaccinated controls (Fig. 2A). This result is in agreement with the known role of CD4⁺ T cells in vaccination-mediated protection against Helicobacter spp (Kleanthous H, Myers GA, Georgakopoulos KM, Tibbitts TJ, Ingrassia JW, Gray HL, Ding R, Zhang ZZ, Lei W, Nichols R, Lee CK, Ermak TH, Monath TP. Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection. Infect Immun. 1998; 66:2879-86). Interestingly, Applicant also found significantly increased gastric mast cell populations (CD3^"CD117⁺) in urease + CT vaccinated mice at days 3 and 4, as compared to CT controls (Fig. 2B). Using histological analysis, mast cells were identified both in the epithelium and the muscularis mucosae of challenged mice (either urease + CT or CT vaccinated, Fig. 2C), but percentages of mast cells appeared similar in both groups. Therefore, to determine whether the mast cell population was increased in the stomach mucosa after urease vaccination, applicant has measured mRNA expression of the mast cell proteases 1 and 2 (mMCP-1 and 2) by quantitative PCR. Preformed mMCP-1 and mMCP-2 are stocked in cytoplasmic granules of mast cells located in the gastric and intestinal mucosa in mice. Expression levels of both mMCP-1 and 2 were found to be highly increased in the stomach of urease + CT vaccinated mice at day 5 post challenge as compared to CT vaccinated controls (Fig. 2D and 2E). In addition, serum mMCP-1 protein levels were significantly increased at the same time point in urease + CT vaccinated mice at day 5 post challenge, in comparison to CT administered mice (Fig. 2F), suggesting that degranulation of mast cells occurred only in urease + CT vaccinated mice. This was confirmed by the presence of degranulated mast cells in the stomach of urease + CT vaccinated mice (data not shown). Taken together, these results show that within 4 to 5 days after challenge, both CD4⁺ and mast cells accumulate in the gastric mucosa of urease + CT vaccinated mice.

Example 3

As CD4⁺ T cells are already known to be critical in vaccination-induced protection against Helicobacter, Applicant undertook to determine whether mast cells were required for immune mediated protection after Helicobacter vaccination. To this aim, Applicant immunized and challenged mast cell deficient Kit(W)/Kit(W-v) double mutant mice (W/W ) (Geissler EN et al. "The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene" Cell 1988; 55, pp 185-192). Since these mutant mice are on an F1 background (WBB6F1), Applicant first tested whether non-mast cell deficient F1 mice can clear Helicobacter infection after vaccination. As shown in Fig. 3A, non-mast cell deficient mice (+/+, +/W or +/W ) (or wild type F1) were fully protected from bacterial challenge after urease + CT vaccination. In sharp contrast, W/W mice were not protected from H. felis colonization after vaccination; indeed only 1 out of 12 urease + CT vaccinated W/W^v mice had negative urease tests (Fig. 3B). Applicant measured serum antibody responses in wild type F1 and W/W^v mice and found that both groups of mice were able to mount a good antibody response against urease, suggesting that the immunization protocol was efficient in both types of mice (Fig. 4). This result suggests that urease + CT vaccinated mice lacking mast cells are unable to clear Helicobacter infection. To challenge this interpretation, Applicant injected cultured wild type F1 bone marrow-derived mast cells to urease + CT vaccinated W/W^v mice and challenged them with Helicobacter 1 week later. In comparison to mast cell- reconstituted W/W mice vaccinated with CT, mast cell- reconstituted urease + CT vaccinated W/W^v mice recovered the ability to clear Helicobacter after vaccination. Indeed, 8 out of 10 mast cell-reconstituted urease + CT vaccinated W/W^v mice had negative urease tests, whereas 5 out of 5 mast cell-reconstituted W/W^v mice vaccinated with CT alone had positive urease tests (Fig. 3C). In order to show that the immune response leading to Helicobacter clearance in mast cell-reconstituted urease + CT vaccinated W/W^v mice shares the characteristics previously described in Balb/c mice (Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R, Lee CK, Kleanthous H, Monath TP "Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class ll-restricted responses" J Exp Med 1998; 188, pp 2277-2288), Applicant depleted the CD4⁺ population in Balb/c and in mast cell- reconstituted W/W^v mice and looked at their ability to clear Helicobacter infection. As expected, CD4⁺ cell depletion of vaccinated Balb/c mice led to the loss of bacterial clearance (Fig. 3D). The same observation was made in mast cell-reconstituted W/W^v vaccinated mice; depletion of CD4⁺ cells also led to the loss of bacterial clearance (Fig. 3E). These results indicate that the immune response leading to Helicobacter clearance in Balb/c and in mast cell-reconstituted W/W^v mice involves mast cells as well as CD4⁺ T cells.

Example 4

Mast cells have been shown to be critical in bacterial clearance in a murine model of bacterial peritonitis (Echtenacher B, Mannel DN, Hultner L. "Critical protective role of mast cells in a model of acute septic peritonitis" Nature 1996; 381 , pp 75-77). In this model, mast cells contribute to bacterial clearance by triggering the recruitment of polymorphonuclear leukocytes, in part via a secretion of TNFα during the acute phase of the bacterial infection, as well as by direct phagocytosis. Thus, Applicant tested whether recruitment of polymorphonuclear leukocytes was responsible for the clearance of Helicobacter observed in urease + CT vaccinated mice. Applicant injected anti-neutrophil monoclonal depleting antibody to vaccinated mice and challenged the animals with H. felis. Surprisingly, it has been observed that there were a significant reduction of the Helicobacter colonization in urease + CT vaccinated mice depleted in neutrophils (Fig. 5). In addition, preliminary data show that reconstitution of urease + CT vaccinated W/W^v mice with cultured bone marrow- derived mast cells from TNFα-/- mice still led to the clearance of Helicobacter (Fig. 6). These results suggest that urease + CT vaccination-mediated clearance of Helicobacter at day 5 post challenge does not depend on the recruitment of polymorphonuclear leukocytes or on the TNFα production by mast cells. Mast cells and CD4⁺ T lymphocytes with a Th2 cytokine secretion phenotype have been described as collaborating to expel intestinal parasites (Lawrence CE. Et al. "Is there a common mechanism of gastrointestinal nematode expulsion?" Parasite Immunol. 2003; 25, pp 271-281). In the model according to the present invention, mast cell recruitment is reminiscent of the recruitment that takes place during parasite infection of the intestinal epithelium in mice. A similar recruitment and collaboration between mast cells and CD4⁺ T cells leads to Helicobacter clearance in the stomach during the vaccine-induced immune response.

In this study, Applicant found mast cell hyperplasia and activation already at day 3 to 4 post-challenge in vaccinated mice. It is possible that a similar pattern of collaboration between mast cells and CD4⁺ T cells takes place in vaccinated mice during Helicobacter infection. Activated mast cells and recruited CD4⁺ T cells potentially collaborate either in the draining lymph node or in the gastric mucosa, leading to the clearance of Helicobacter infection. The result of this interaction is probably not limited to a CD4⁺ T cell-mediated proliferation of mast cells in the gastric mucosa, as a non-specific increase in the gastric mast cell population did not lead to the clearance of Helicobacter infection in the absence of vaccination. Indeed, increased gastric mastocytosis, which occurs in IL-9 transgenic mice as well as in IL- 3 treated mice, failed to promote H. felis clearance in non-vaccinated animals (Fig. 7, Fig. 8).

Example 5

Effect of mast cell activation on in vitro bacterial clearance Mouse bone marrow derived mast cells were incubated with Helicobacter pylori in vitro for 6 hours in micro-aerophylic conditions. Following incubation, the cell suspension was plated on agar plates to numerate the number of viable bacteria present in the medium at the end of incubation. Remarkably, when mast cells were activated with Mastprom or urease + anti-urease IgE antibodies, the bacteria were killed more efficiently by the mast cell population (Fig. 10). This result demonstrates that activation of mast cells leads to increase capability of the mast cells to kill Helicobacter.

Table 1

Example 6

Effect of mast cell activation on in vivo bacterial clearance

Applicant compared the ability of two vaccination protocols to eradicate Helicobacter infection. Mice were vaccinated intranasally with urease and cholera toxine, one day before Helicobacter infection; one group of mice was intravenously injected with hyper-immune serum containing high titers of IgE anti-urease antibodies. When both groups of mice were challenged with Helicobacter pylori, one can see that mice which were injected with anti-urease IgE antibodies eradicate Helicobacter infection more efficiently (see Fig. 11).

Table 2

Example 7

Effect of the administration of leukotriene B4 on mice that were pre-infected two weeks before with H. felis (Fig. 12). Mice were infected with H. felis (5 10⁷) at day 0, two weeks after bacterial infection, the mice were left untreated (unmanipulated) or intragastrically administered with 1μg of leukotriene B4 or with 50μl of vehicle. At sacrifice (one week after leukotriene B4 administration), urease tests were performed on gastric samples to probe for the bacterial infection. Mice injected with leukotriene B4 were statistically less infected than the unmanipulated or vehicle administered mice (p<0.05, Mann Whitney test).

Claims

Claims

1. A method for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal comprising, administering to said mammal a preventive and/or therapeutically effective amount of a composition capable of activating mast cells in the stomach of said mammal.

2. The method according to claim 1, further comprising, vaccinating the mammal with an appropriate antigen derived from Helicobacter prior to administering the composition to said mammal.

3. The method according to claim 1 or 2, wherein the activation of mast cells in the stomach of a mammal leads to an increase of the expression of mast cell proteases 1 and/or 2.

4. The method according to any of claims 1 to 3, wherein the composition consists essentially of IgE and/or IgG antibodies specific to antigens derived from Helicobacter, whereas the IgE and/or IgG antibodies induce the activation of said mast cells.

5. The method according to any of claim 1 to 4, wherein the composition consists essentially of IgE antibodies specific to urease derived from Helicobacter, whereas the IgE antibodies induce the activation of said mast cells by binding to IgE-specific immunoglobulin receptors of said mast cells.

6. The method according to claim 1 or 2, wherein the composition comprises molecules derived from mast cells and/or CD4+ T cells.

7. The method of claim 6, characterized in that molecules derived from mast cells essentially consist in Leukotrienes, Histamine, mMCP-1 , tryptase, cathelicidins and a mixture thereof.

8. The method of claim 7, wherein Leukotrienes derived from mast cells is Leukotriene B4.

9. The method of claim 6, characterized in that molecules derived from CD4+ T cells essentially consist in molecules involved in the mast cell-CD4+ T cell crosstalks.

10. The method of claim 9, wherein molecules involved in the mast cell-CD4+ T cell crosstalks are selected among the lists comprising IL-3, IL-4, IL-10, LTβR- LTβR ligands, OX40L-OX40 ligands.

11. The method according to any of claim 1 to 10, wherein the disease caused by or associated with Helicobacter is duodenal or gastric ulcers, mucosal atrophy, gastrite, gastric carcinoma or gastric lymphoma.

12. The method according to claim 11 , wherein the disease caused by or associated with Helicobacter is duodenal or gastric ulcers, gastrite or gastric carcinoma.

13. A method for activating mast cells in the stomach of a mammal comprising, administering to said mammal a preventive and/or therapeutically effective amount of a composition consisting essentially of IgE and/or IgG antibodies specific to antigens derived from Helicobacter whereas the IgE and/or IgG antibodies induce the activation of said mast cells.

14. The method of claim 13, further comprising, vaccinating the mammal with an appropriate antigen derived from Helicobacter prior to administering the composition to said mammal.

15. The method according to claim 13 or 14, wherein the composition consists essentially of IgE antibodies specific to urease derived from Helicobacter whereas the IgE antibodies induce the activation of said mast cells.

16. A composition capable of activating mast cells in the stomach of a mammal for the use as agent for treating a disease caused by or associated with Helicobacter in said mammal.

17. The composition of claim 16, consisting essentially of IgE and/or IgG antibodies specific to antigens derived from Helicobacter whereas the IgE and/or IgG antibodies induce the activation of said mast cells.

18. The composition according to claim 16 or 17, consisting essentially of IgE antibodies specific to urease derived from Helicobacter whereas the IgE antibodies induce the activation of said mast cells by binding to IgE-specific immunoglobulin receptors of said mast cells.

19. The composition of claim 16, selected from the group comprising: Mastprom, C48/80, Mastroparan, stem cell factor, C3a, Substance P, Neuropeptide Y and/or related chemical or biological agents.

20. The composition of claim 16, comprising molecules derived from mast cells and/or CD4+ T cells.

21. The composition of claim 20, characterized in that molecules derived from mast cells essentially consist in Leukotrienes, Histamine, mMCP-1 , tryptase, cathelicidins and/or a mixture thereof.

22. The composition of claim 21 , wherein Leukotrienes derived from mast cells is Leukotriene B4.

23. The composition of claim 6, characterized in that molecules derived from CD4+ T cells essentially consist in molecules involved in the mast cell-CD4+ T cell crosstalks.

24. The composition of claim 9, wherein molecules involved in the mast cell-CD4+ T cell crosstalks are selected among the lists comprising IL-3, IL-4, IL-10, LTβR- LTβR ligands, OX40L-OX40 ligands.

25. A pharmaceutical composition suitable for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal, comprising as an active substance a pharmaceutically effective amount of the composition of claims 16 to 24 optionally in combination with pharmaceutically acceptable carriers, diluents and adjuvants.

26. The pharmaceutical composition of claim 25, further comprising an antisecretory agent and/or an antibiotic effective against Helicobacter.

27. The pharmaceutical composition of claim 26, wherein the antisecretory agent is a proton pump inhibitor, a H2 receptor antagonist, a bismuth salt or a prostaglandin analog.

28. The pharmaceutical composition of claims 25 to 27, wherein its route of administration is intra-nasal, oral, sub-cutaneous, intra-muscular, rectal, transdermal or intravenous.

29. The pharmaceutical composition of claim 28, wherein its route of administration is oral.

30. The use of the pharmaceutical composition of claims 16 to 24, for the preparation of a medicament for the prevention or treatment of diseases caused by or associated with Helicobacter in a mammal.

31. The use of claim 30, wherein diseases caused by or associated with Helicobacter are duodenal or gastric ulcers, gastrite or gastric carcinoma.

32. A kit for the prevention or treatment of diseases caused by or associated with Helicobacter in a mammal, said kit comprising the pharmaceutical composition of claims 16 to 24, optionally with reagents and/or instructions for use.

33. A method for eradicating Helicobacter from the stomach of a mammal comprising, administering to said mammal an effective amount of a composition capable of activating mast cells in the stomach of said mammal.

34. The method according to claim 33, further comprising vaccinating the mammal with an appropriate antigen derived from Helicobacter prior to administering the composition to said mammal.

35. The method according to claim 33 or 34, wherein the activation of mast cells in the stomach of a mammal leads to an increase of the expression of mast cell proteases 1 and/or 2.

36. The method according to any of claim 33 to 35, wherein the composition consists essentially of IgE and/or IgG antibodies specific to antigens derived from Helicobacter whereas the IgE and/or IgG antibodies induce the activation of said mast cells.

37. The method according to any of claim 33 to 36, wherein the composition consists essentially of IgE antibodies specific to urease derived from Helicobacter whereas the IgE antibodies induce the activation of said mast cells.

38. A method for detecting the presence of Helicobacter in the stomach of a mammal, said method comprising, the administration of the pharmaceutical composition of claims 16 to 24 for the activation of the mast cells in the stomach of said mammal and, the detection of the blood level increase of the expression of mast cell proteases 1 and/or 2.

39. A method for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal comprising, administering to said mammal a preventive and/or therapeutically effective amount of a composition comprising molecules derived from mast cells and/or CD4+ T cells.

40. The method of claim 29, characterized in that molecules derived from mast cells essentially consist in Leukotrienes, Histamine, mMCP-1 , tryptase, cathelicidins and a mixture thereof.

41. The method of claim 30, wherein Leukotrienes derived from mast cells is Leukotriene B4.

42. The method of claim 29, characterized in that molecules derived from CD4+ T cells essentially consist in molecules involved in the mast cell-CD4+ T cell crosstalks.

43. The method of claim 29, wherein molecules involved in the mast cell-CD4+ T cell crosstalks are selected among the lists comprising IL-3, IL-4, IL-10, LTβR- LTβR ligands, OX40L-OX40 ligands.

44. Use of a composition comprising molecules derived from mast cells and/or CD4⁺T cells for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.

45. Use of a composition comprising molecules derived from mast cells and/or CD4⁺ T cells in the manufacture of a medicament for preventing and/or treating a disease caused by or associated with Helicobacter in a mammal.