| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | July 23, 1996 |
| PATENT TITLE |
Oral treatment of helicobacter infection |
| PATENT ABSTRACT | Method of eliciting in a mammalian host a protective immune response to Helicobacter infection, by orally administering to the host an immunogenically effective amount of Helicobacter antigen. Vaccine compositions are also provided |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | August 22, 1994 |
| PATENT REFERENCES CITED |
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Eaton et al., Campylobacter pylori Virulence Factors in Gnotobiotic Piglets, Infection and Immunity 57:1119-1125, 1989. Eldridge et al., Biodegradable Microspheres: Vaccine Delivery System for Oral Immunization, Current Topics in Microbiology and Immunology 146:59-66, 1989. Elson and Ealding, Generalized Systemic and Mucosal Immunity in Mice After Mucosal Stimulation with Cholera Toxin, J. Immunology 132:2736-2741, 1984. Elson et al., A Levage Technique Allowing Repeated Measurement of IgA Antibody in Mouse Intestinal Secretions, J. Immunological Methods 67:101-108, 1984. Engstrand et al., Inoculation of Barrier-Born Pigs with Helicobacter pylori: a Useful Animal Model for Gastritis Type B, Infection and Immunity 58:1763-1768, 1990. Evans et al., Characterization of the Helicobacter pylori Urease and Purification of its Subunits, Microbial. Pathogenesis 10:15-26, 1991. 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Goodwin et al., Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen. nov. as . . . Helicobacter mustelae comb. nov., Respectively, International J. Systematic Bacteriology 39:397-405, 1989. Gootz et al., Immunological and Molecular Characterization of Helicobacter felis Urease, Infection and Immunity, 62, 793-798, 1994. Graham, Campylobacter pylori and Peptic Ulcer Disease, Gastroenterology 96:615-25, 1989. Gupta et al., Adjuvants--a Balance Between Toxicity and Adjuvanticity, Vaccine 11:293-306, 1993. Guruge et al., Detection of Antibodies to Helicobacter pylori Cell Surface Antigens, Scand. J. Infect. Dis. 22:457-465, 1990. Handt et al., Helicobacter pylori Isolated from the Domestic Cat: Public Health Implications, Infection and Immunity 62:2367-2374, 1994. Heap and Lee, Immunisation and Gastric Colonisation with Helicobacter felis, Microb. Ecol. Health Dis. 4:s119; VIth International Workshop on Campylobacter, Helicobacter and Related Organisms, Oct. 7-10, 1991. Holmgren et al., New Cholera Vaccines, Vaccine 7:94-96, 1989. Hu and Mobley, Purification and N-Terminal Analysis of Urease from Helicobacter pylori, Infection and Immunity 58:992-998, 1990. Hu et al., Purification of Recombinant Helicobacter pylori Urease Apoenzyme Encoded by ureA and ureB, Infection and Immunity 60:2657-2666, 1992. Kazi et al., Cellular and Humoral Immune Responses in Campylobacter pylori-associated Chronic Gastritis, J. Pathology 159:231-237, 1989. Keusch and Bart, Immunization Principles and Vaccine Use, Harrison's Principles of Internal Medicine, 13th Edition vol. 1, pp. 498-511. Labigne et al., Shuttle Cloning and Nucleotide Sequences of Helicobacter pylori Genes Responsible for Urease Activity, J. Bacteriology 173:1920-1931, 1991. Lee et al., Pathogenicity of Helicobacter pylori: A Perspective, Infection and Immunity 61:1601-1610, 1993. Lee et al., A Small Animal Model of Human Helicobacter pylori Active Chronic Gastritis, Gastroenterology 99:1315-1323, 1990. Lee et al., Protection from Helicobacter Infections by Intragastric Immunization with Recombinant Urease, Helicobacter pylori: Basic Mechanisms to Clinical Cure, Poster Abstracts, Nov. 3-6, 1993 (Poster #13). Lycke and Holmgren, Strong Adjuvant Properties of Cholera Toxin on Gut Mucosal Immune Responses to Orally Presented Antigens, Gastroenterology 59:301-308, 1986. Mazanec et al., Immunoglobulin A Monoclonal Antibodies Protect Against Sendai Virus, J. Virology 61:2624-2626, 1987. McGhee et al., The Mucosal Immune System: From Fundamental Concepts to Vaccine Development, Vaccine 10:75-88, 1992. McSweegan et al., Intestinal Mucus Gel and Secretory Antibody are Barriers to Campylobacter jejuni Adherence to INT 407 Cells, Infection and Immunity 55:1431-1435, 1987. Merrifield, Solid Phase Peptide Synthesis. I. The Synthesis of Tetrapeptide, 85:2149-2154, 1963. Mestecky, The Common Mucosal Immune System and Current Strategies for Induction of Immune Responses in External Secretions, J. Clin. Immunology 7:265-276, 1987. Mobley et al., Helicobacter pylori Urease: Properties and Role In Pathogenesis, Proceeding of an International Workshop at Deerhurst, Huntsville, Ontario, Canada. 21-24, Feb. 1991. pp. 39-46. Morris and Nicholson, Ingestion of Campylobacter pyloridis Causes Gastritis and Raised Fasting Gastric pH, Am. J. Gastroenterology 82:192-199, 1987. Nagata et al., Monoclonal Antibodies Against the Native Urease of Helicobacter pylori: Synergistic Inhibition of Urease Activity by Monoclonal Antibody Combinations, Infection and Immunity 60:4826-4831, 1992. Nedrud et al., Combined Oral/Nasal Immunization Protects Mice from Sendai Virus Infection, J. Immunology 139:3484-3492, 1987. Oderda et al., Eighteen Month Follow Up of Helicobacter pylori Positive Children Treated with Amoxycillin and Tinidazole, Gut 33:1328-1330, 1992. Offit and Clark, Protection Against Rotavirus-Induced Gastroenteritis in a Murine Model by Passively Acquired Gastrointestinal But Not Circulating Antibodies, J. Virology 54:58-64, 1985. O'Hagan, Intestinal Translocation of Particulates--Implications for Drug and Antigen Delivery, Advanced Drug Delivery Reviews 5:265-285, 1990. Olivieri et al., Growth of Helicobacter pylori in Media Containing Cyclodextrins, J. Clinical Microbiology 31:160-162, 1993. Orenstein et al., Immunization, Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, 4th Ed. (Churchill Livingstone, New York) Part IV, pp. 2770-2790. Pallen et al., Vaccine against Helicobacter pylori Urease, The Lancet 336:186-187, 1990. Parsonnet et al., Helicobacter pylori Infection in Intestinal- and Diffuse-Type Gastric Adenocarcinomas, J. National Cancer Institute 83:640-642, 1991. Pavlovskis et al., Adjuvant Effect of Escherichia coli Heat-labile Enterotoxin on Host Immune Response . . . Campylobacter Antigens, Microb. Ecol. Health Dis. VIth Int. Workshop on Campylobacter . . . Oct. 7-10, 1991. Peterson, Helicobacter pylori and Peptic Ulcer Disease, New England Journal of Medicine 324:1043-1048, 1991. Rappuoli et al., Development of a Vaccine Against Helicobacter pylori: A Short Overview, Eur. J. Gastroenterol. Hepatol. 5/Suppl. 2, 1993. Rauws et al., Campylobacter pyloridis-Associated Chronic Active Antral Gastritis, Gastroenterology 94:33-40, 1988. Schaedler and Orcutt, Gastrointestinal Microflora, Chapter 14, The Mouse in Biomedical Research, vol. III (Academic Press, Inc. 1983) pp. 341-345. Stacey et al., Local and Systemic Antibody Responses During Helicobacter pylori Infections, Helicobacter pylori and Gastroduodenal Pathology, Pajares et al. (Eds.) (Springer-Verlag, New York, 1993) pp. 165-169. Stuart et al., Further Studies on Urease Production by Proteus and Related Organisms, J. Bacteriol. 49:437-444, 1945. Thomas et al., Effect of Oral Immunization with Helicobacter pylori Antigens on Colonization by H. felis in Mice, Acta Gastro-Enterologica Belgica, VII Int. Workshop on Campylobacter . . . Brussels, Sep. 21-25, 1993. Von Wulffen, Low Degree of Relatedness Between Campylobacter pyloridis and Enteropathogenic Cmpylobacter . . . by DNA-DNA Blot Hybridization and Immunoblot Studies, FEMS Microbiology Letters 42:129-133, 1987. Wegmann et al., Gastrospirillum-hominis-assoziierte Gastritis--Eine Zoonose?, Schwetz med. Wschr. 121:245-254, 1991. Winner III et al., New Model for Analysis of Mucosal Immunity: Intestinal Secretion of Specific Monoclonal . . . Hybridoma Tumors Protects Against Vibrio cholerae Infection, Infection and Immunity 59:977-982, 1991. Wotherspoon et al., Regression of Primary Low-grade B-cell Gastric Lymphoma of Mucosa-associated Lymphoid Tissue Type After Eradication of Helicobacter pylori, The Lancet 342:575-577, 1993. Wyatt et al., Local Immune Response to Gastric Campylobacter in Non-ulcer Dyspepsia, J. Clin. Pathol. 39:863-870, 1986. Abstracts Submitted for Helicobacter Pylori: Beginning the Second Decade and the VIIth Workshop of the European Helicobacter pylori Study Group, Houston, Texas, Sep. 30-Oct. 1, 1994. Hawtin et al., Investigation of the Structure and Localization of the Urease of Helicobacter pylori Using Monoclonal Antibodies, J. General Microbiology 136:1995-2000, 1990. Czinn et al., Serum and Mucosal Immune Response Following Oral Immunization with Helicobacter pylori, Gastroenterology 100:A571, 1991. Czinn et al., Oral Immunization Protects Germ-Free Mice Against Infection from Helicobacter felis, Gastroenterology 102:A611, 1992. Czinn et al., Oral Immunization |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method of eliciting in a mammalian host a protective immune response to Helicobacter infection, comprising orally adminstering to the host an immunogenically effective amount of Helicobacter antigen with a mucosal adjuvant to elicit said protective immune response, wherein the Helicobacter antigen is a whole cell lystate preparation of at least one Helicobacter felis and Helicobacter pylori. 2. A method according to claim 1, wherein said mucosal adjuvant is cholera toxin. 3. A method according to claim 1, wherein said mammalian host is human. 4. A vaccine composition comprising an immunogenically effective amount of a whole cell lysate of Helicobacter antigen and a mucosal adjuvant in association with a pharmaceutically acceptable carrier or diluent, wherein the Helicobacter antigen is a whole cell lysate preparation of at least one of Helicobacter felis and Helicobacter pylori. 5. A Vaccine composition according to claim 4, wherein said mucosal adjuvant is cholera toxin. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
The present invention relates to the treatment of gastric infection in mammals, including humans. More particularly, the present invention relates to a method for the treatment of Helicobacter infection in mammals, including humans, and to vaccine compositions and antibodies suitable for use in such treatment. BACKGROUND OF THE INVENTION Helicobacter pylori (H. pylori) infection of human gastric epithelium is a major factor in the development of gastritis and ulcers and may be a risk factor for the development of gastric cancer.sup.1-3. This slender S-shaped gram negative microorganism is routinely recovered from gastric tissue of adults and children with histologic evidence of gastritis or peptic ulceration. Evidence for a causal relationship between H. pylori and gastroduodenal disease comes from studies in human volunteers, gnotobiotic pigs, and germ-free rodents whereby postulates by Koch were satisfied by creating histologically confirmed gastritis following consumption of viable microorganisms.sup.4-11. Although difficult to treat, when eradication is achieved the underlying gastritis resolves and, in patients with duodenal ulcer disease, the recurrence rate of the ulcer decreases dramatically.sup.12. In spite of in vitro susceptibility to many antimicrobial agents, in vivo long-term eradication of established H. pylori infections with antimicrobial agents is difficult to achieve.sup.18. The microorganism is found within the mucous coat overlying the gastric epithelium. This is a location which does not appear to allow for adequate antimicrobial levels to be achieved when given orally. At the present time, most authorities recommend a "triple therapy", namely a bismuth salt in combination with tetracycline and metronidazole for 2-4 weeks. However, the effectiveness of this or other chemotherapeutic regimens remains suboptimal. At the present time little is known regarding the role of the mucosal immune system in the stomach. The distribution of Ig producing cells in the normal gastric antrum indicates that IgA plasma cells make up 80% of the total plasma cell population. In addition, the number of plasma IgA cells present in the gastric antrum is comparable to other mucous membranes.sup.25,26. Although a number of studies have looked at immunoglobulin levels in various endocrine fluids, no data is available regarding the concentration of immunoglobulins in gastric secretions. Moreover there is only limited data to suggest that patients infected with H. pylori develop specific IgG and/or IgA antibodies in gastric aspiarates.sup.32. Thus once infection is established, neither antibody nor antibiotics are very effective at eradication. Czinn et al have shown that repetitive oral immunizations with H. pylori antigens and cholera toxin result in the inducement of a vigorous gastrointestinal IgA anti-H. pylori response in mice and ferrets.sup.18 However, since mice and ferrets are resistant to H. pylori infection and since no small animal model existed at that time to evaluate protection, it was unknown whether the antibodies so formed were protective. Lee et al have reported the ability to infect germ-free rodents with H. felis and reproducibly document histologic gastritis.sup.9, 10 However no evaluation of protection has been reported. There remains a need therefore for an effective treatment of H. pylori gastric infection, especially in humans. The present invention seeks to fill that need. SUMMARY OF THE INVENTION The present inventors have discovered, surprisingly, that oral immunization of a host with Helicobacter antigen results in the formation of antibodies which are protective against acute infection by Helicobacter microorganisms. The formation of such protective anibodies was not predictable on the basis of prior work since, prior to the present invention, no suitable model existed to evaluate protection. According to one aspect of the present invention, there is provided a method of eliciting in a mammalian host a protective immune response to Helicobacter infection, comprising orally administering to the host an immunogenically effective amount of Helicobacter antigen to elicit the desired protective immune response. According to another aspect of the present invention, there is provided a vaccine composition comprising an amount of Helicobacter antigen effective to elicit a protective human response in a patient, in association with a pharmaceutically acceptable diluent. According to a further aspect of the present invention, there is provided a method of imparting to a mammalian host passive protection to Helicobacter infection, comprising orally administering to the host a immunologically effective amount of a Helicobacter specific IgA antibody to impart the desired passive protection. According to yet another aspect of the present invention, there is provided a murine H. felis specific IgA or IgG monoclonal antibody. According to a yet further aspect of the invention, there is provided a cell line #71-G.sub.5 -A.sub.8. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be further described with reference to the accompanying figures, in which: FIG. 1 is is a bar chart of antibody titers in various sera and secretions of germ free mice after oral immunization with H. felis lysate in association with cholera toxin; and FIGS. 2A and 2B are bar charts of percent of mice infected with H. felis after active immunization (FIG. 2A) and passive immunization (2B) compared with controls. DETAILED DESCRIPTION OF THE INVENTION The present inventors have demonstated that oral immunization in mice using H. felis antigen produces a protective immune response wherein antigen specific protective antibodies are present in gastric secretions. The effect of the protective immune response is that immunized animals when challenged with pathogen do not become infected in comparison to non-immunized animals which do become infected. While not being bound by any theory, the present inventors believe that oral immunization with the H. felis antigen stimulates the common mucosal immune system and perhaps local sites in the gastric mucosa resulting in the appearance of H. felis specific IgA antibodies in the gastric secretions, which prevent H. felis infection. Since H. felis and H. pylori are similar species from the same genus (Helicobacter), it is reasonable to conclude that immunization of for example a germ-free pig with H. pylori antigen plus a mucosal adjuvant such as cholera toxin will be effective in preventing H. pylori infection of the stomach. Since it is a routine matter to conduct pre-clinical trials of candidate vaccines for human use in animal models, it is believed that the methodology of the present invention is effective in humans, especially in the treatment of H. pylori infection in humans. It has been discovered by the present inventors that an H. felis germ-free mouse model can be employed to evaluate antibody protection levels following immunization with H. felis antigen. FIG. 1 relates to the results obtained in experiments with the H. felis germ-free mouse model. Oral immunization of the model with bacterial antigens in association with cholera toxin resulted in elevated serum, gastric and intestinal anti-H. felis antibody titers and protection from acute infection of the stomach by H. felis pathogen. In the experiments, groups of Swiss-Webster germ-free mice (Taconic) were orally immunized 4 or 5 times over a one month period with 2-4 mg of sonicated H. felis lysate plus 10 .mu.g of cholera toxin. The mice were then challenged orally with approximately 10.sup.6 viable H. felis bacteria. The mice were sacrificed and intestinal and gastric secretions collected as described in the following working Examples. Anti-H. felis antibody titers were determined by ELISA. The black solid bars in FIG. 1 represent mean titers (.+-.S. D.) from immunized mice and the open bars represent mean titers (.+-.S. D.) from the control non-immunized mice. The results presented graphically in FIG. 1 are summarized in Table 1 below. TABLE 1 ______________________________________ ANTIBODY TITER (LOG.sub.2) ______________________________________ Serum Gastric Intestine IgA IgG IgA IgG IgA IgG ______________________________________ CONTROL 3.1 3 0 0 1.6 1.1 IMMUNIZED 11.8 16.8 2.1 4.25 4.5 4.4 ______________________________________ H. FELIS INFECTION H. felis (+) H. felis (-) PROTECTION ______________________________________ Control n = 18 14 4 23% Immunized n = 17 4 13 78% ______________________________________ It can be seen from the above results that significantly higher antibody titers are observed for the immunized mice than for the control animals. FIGS. 2A and 2B depict the results of studies to establish the protection against infection by H. felis by conducting active and passive immunization experiments. Referring to the active immunization experiments, gastric biopsies were collected at sacrifice from the H. felis challenged mice in the experiments described above in connection with FIG. 1. The biopsies were scored for the presence of H. felis by rapid urease test and/or culture positivity, described in the following working Examples. FIG. 2A shows the results of pooled data from 3 experiments (n=17 immunized animals and 18 control animals). The black (solid) bars represent challenged immunized mice and the striped bars the control non-immunized mice. It will be seen that from a total of 17 immunized animals, only 4 became infected, as compared to 14 of the 18 control animals. In other words 78% percent of the immunized animals were protected from H. felis infection as compared to 23% of the non-immunized animals The fact that protection was the direct result of IgA antibodies was established by passive immunization of germ-free mice with H. felis specific IgA monoclonal antibodies and comparison of the resulting protection with that exhibited by mice given no antibody or irrelevant antibody (for example Sendai virus specific IgA monoclonal antibody). The results are set forth in FIG. 2B. An IgA monoclonal antibody reactive with H. felis was isolated and subcloned after an immunization protocol similar to that described in FIG. 1. Ascites containing H. felis specific IgA monoclonal antibody produced from the cell line #71-G.sub.5 -A.sub.8, prepared as described in the working Examples, or Sendai virus specific IgA monoclonal antibody or saline were orally administered to germ-free mice at the time of infection with H. felis, and 4, 8, and 24 hours later. Seven days after infection, the mice were sacrificed and gastric biopsies scored for H. felis (n=7 mice received H. felis specific monoclonal antibody and 13 mice received no antibody or Sendai virus specific monoclonal antibody). The black sold bars represent the mice which received the H. felis specific monoclonal antibody and the striped bars represent the mice which received either Sendai virus specific monoclonal antibody or saline (no antibody). These results establish that IgA alone protects against H. felis infection of the gastric mucosa. It is also observed that oral administration of H. felis antigen results in significantly increased levels of anti-H. felis IgG antibodies as well as IgA antibodies. There are a number of possible explanations for this phenomenon. First, it has been observed that cholera toxin can, in some cases, enhance both antigen-specific IgA and IgG responses.sup.22. Secondly, cell traffic studies have shown that mesenteric node IgG lymphocytes are a component of the mucosal immune system and can give rise to mucosal IgG plasma cells which have been observed in gastric mucosa. Thirdly, at least a portion of the observed gastric IgG could be the result of transudation of serum antibody into the gastric lumen secondary to mild to moderate inflammation observed in both control and immunized animals. The above discussion has focussed on the use of H. felis antigen in the treatment of H. felis infection. It will be appreciated however that the present invention is not limited to the treatment of H. felis infection. Thus, the present invention also includes within its scope the treatment or prophylaxis of mammals, including humans, for H. pylori infection, wherein the patient is orally immunized with an immunologically effective amount of H. pylori antigen in order to elicit the formation of protective antibodies to H. pylori pathogen. Preferably, the H. pylori is administered in association with a mucosal adjuvant, for example cholera toxin. Moreover, the present invention includes within its scope the passive immunization of mammals, including humans, against H. pylori infection. This is achieved by orally administering an effective amount of an H. pylori specific antibody to the patient. Preferably an H. pylori specific IgA monoclonal antibody is orally administered to the patient. The vaccine of the invention is administered orally in amounts readily determined by persons of ordinary skill in this art. Thus, for adults, a suitable dosage would be in the range of 10 .mu.g to 10 mg, for example 50 .mu.g to 5 mg. Similar dosage ranges would be applicable for children. As noted above, 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 of antigen plus cholera toxin or its B subunit, microcapsules, or immune stimulating complexes (ISCOM's) or liposomes and attenuated live vectors such as viruses or Salmonella bacteria. 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 .mu.g to 50 .mu.g, for example 10 .mu.g to 35 .mu.g. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. This is something within the skill of a person of ordinary skill in this art. Suitable carriers and diluents are enteric coated capsules and/or 0.2N NaHCO.sub.3 and/or saline. |
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