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WO1992019970A1 - Methode de detection et de traitement des maladies causees par des allergenes bacteriens - Google Patents

Methode de detection et de traitement des maladies causees par des allergenes bacteriens Download PDF

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Publication number
WO1992019970A1
WO1992019970A1 PCT/US1992/003284 US9203284W WO9219970A1 WO 1992019970 A1 WO1992019970 A1 WO 1992019970A1 US 9203284 W US9203284 W US 9203284W WO 9219970 A1 WO9219970 A1 WO 9219970A1
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WIPO (PCT)
Prior art keywords
pylori
composition
allergen
protein
ige
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PCT/US1992/003284
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English (en)
Inventor
Emanuel Calenoff
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Emanuel Calenoff
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Publication date
Application filed by Emanuel Calenoff filed Critical Emanuel Calenoff
Priority to AU20036/92A priority Critical patent/AU667040C/en
Priority to JP4511878A priority patent/JPH06507494A/ja
Publication of WO1992019970A1 publication Critical patent/WO1992019970A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)

Definitions

  • This invention is related to the field of allergens, more specifically to the identification of bacterial allergens, and to their use in disease diagnosis and treatment.
  • a number of idiopathic recurrent diseases are of unknown etiology. Some of these diseases are thought to be linked to infection by a microorganism. However, the-causal relationship between the microorganism and the disease is often not established. The twin disorders of chronic gastritis and peptic ulcer disease are within this category.
  • Chronic gastritis and peptic ulcer disease are diseases of major significance. Five to ten percent of all individuals develop chronic gastritis and or gastroduodenal ulcers in their lifetime. Ulcer disease is a common cause of morbidity. The annual prevalence of symptomatic peptic ulcer disease in the United States of America is approximately 18 per 1,000 adults (or 4,500,000 people). Approximately 350,000 new cases of peptic ulcer disease are diagnosed each year.
  • Diagnosis of these diseases is usually performed by gastroduodenal endoscopy, an invasive and costly procedure. Treatment encompasses oral medication, dietary controls, and surgery. Rarely is it definitive, and these chronic conditions often have cycles of improvement and relapse.
  • the identification of protein allergens of H. pylori associated with gastritis/ulcer disease allows for a relatively non-invasive detection of the disease by a modified RAST test. It also allows for treatment of the disease by immunotherapy, using purified protein allergens.
  • one aspect of the invention is a method of measuring IgE which binds immunologically to a bacterial allergen.
  • Serum suspected of containing the IgE is reacted with an extract of the bacteria coupled to a solid support, followed by washing and reacting with labelled anti-IgE, and detecting labeled anti-IgE bound to the solid support, the improvement comprising reacting the serum with a composition capable of removing IgA and IgG from the serum prior to the reacting with the bacterial extract coupled to the solid support, wherein the amount of composition used is sufficient to remove IgA and IgG which interferes with the IgE binding to the bacterial allergen.
  • Another aspect of the invention is a method of preparing purified protein allergen from bacteria comprising: (a) treating bacteria containing a protein allergen with acetone to remove lipid components; (b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benazamidine; (c) separating a protein containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition comprised of proteins which are of molecular weight at least about 5 1,000; and (e) separating the proteins of the composition of (d) by ion-exchange chromatography.
  • Still another aspect of the invention an immunotherapeutic method of treating an individual for a disease resulting from an allergic reaction to a
  • Another aspect of the invention is a method of determining whether an individual has an allergic response to Helicobacter pylori, the method comprising: (a) providing serum from an individual suspected of containing IgE to H. pylori allergens; (b) providing a
  • composition consisting essentially of H. pylori protein allergens; (c) reacting the serum of (a) with the composition of (b) under conditions which allow immunological binding between IgE and an allergen to which it is directed; and (d)detecting IgE-allergen
  • Still another aspect of the invention is a composition consisting essentially of protein allergens of H. pylori.
  • Another aspect of the invention is a protein allergen of H. pylori coupled to a solid substrate.
  • Yet another aspect of the invention is a method of treating an individual for H. pylori induced gastritis comprising introducing into the individual a composition comprised of a polypeptide which contains one or more epitopes that are immunologically identifiable with immunogenic epitopes of H. pylori, wherein the polypeptide is in an amount sufficient to relieve an allergic reaction to H. pylori in the individual, and wherein the composition is further comprised of a suitable excipient.
  • Still another aspect of the invention is a diagnostic kit comprised of a polypeptide containing at least one epitope which is immunologically identifiable with an H. pylori epitope, packaged in a suitable container, and a means for detecting immunological complexes formed between the polypeptide and IgE in the biological sample, if any.
  • compositions comprised of a structural analog of an epitope of an H. pylori allergen, wherein the structural analog binds to an IgE paratope.
  • compositions comprised of a purified polyclonal antibody directed to a polypeptide allergen of H. pylori.
  • composition comprised of a monoclonal antibody directed to a polypeptide allergen of H. pylori.
  • Figure 1 is a graph showing the effect of scrubbing serum with Protein A on the detection of anti- 3 pylori IgE in a modified RAST test.
  • Figure 2A is a graph showing the serum IgE levels of IgE directed to subfractions of 2£. pylori protein allergens in healthy individuals (controls) .
  • Figure 2B is a graph showing the serum IgE levels of IgE directed to subfractions of H. pylori protein allergens in gastritis patients.
  • Figure 3 is a plot of the net total IgE immunological reactivity of serum from control and gastritis patients using all available H. pylori protein fractions isolated from an HPLC DEAE column.
  • Figure 4 is a plot of the net total IgE immunological reactivity of serum from control and gastritis patients with the proteins in fractions 59, 64, 66, 68, 72 and 74 of the HPLC DEAE column.
  • the present invention stems from the discovery that individuals with chronic gastritis or gastroduodenal ulcers have serum IgE specific for protein allergens of H. pylori, implicating hypersensitivity to this microorganism in the etiology of the diseases.
  • H. pylori is most likely an innocuous colonizer of the gastric mucosa. It dwells just beneath the protective mucous layer and probably feeds from it without much harm to the host or to the host's protective defenses against the gastric acid.
  • the inflammatory process recognized in chronic gastrtitis results in those individuals who possess the genetic proclivity toward allergy and then have the necessary MHC II antigen framework for presenting the H. pylori allergenic proteins as allergens.
  • a qualitative and/or quantitative reduction in the secretion of protective mucus by the goblet cells probably occurs thus making the underlying mucosa vulnerable.
  • immunoassays to detect an H. pylori induced allergic reaction in individuals.
  • these immunoassays utilize purified protein allergens, and are preferable to endoscopy since they may be performed in vitro and are relatively non-invasive.
  • the discovery allows for a novel treatment of these diseases; i.e., immunotherapy with compositions comprised of at least one purified protein allergen of H. pylori. and/or with an allergoid of a protein allergen of H. pylori. 15
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of protein purification, microbiology, molecular biology, and immunology, which are within the skill of the art. Such techniques are explained fully in
  • allergen refers to an antigen that gives rise to allergic sensitization by IgE antibodies.
  • allergoid refers to a chemically
  • the term "individual”, as used herein, refers to a vertebrate, particularly members of the mammalian species, and includes but is not limited to domestic
  • allergy denotes an altered state of immune reactivity, usually denoting hypersensitivity.
  • Immunologically identifiable 35_ with/as refers to the presence of epitope(s) and polypeptides(s) which are also present in the designated polypeptide(s) . Immunological identity may be determined by antibody binding and/or competition in binding; these techniques are known to those of average skill in the art, and are also illustrated infra.
  • epitope refers to an antigenic determinant of a polypeptide.
  • An epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 5 such amino acids, and more usually, consists of at least 8-10 such amino acids.
  • Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • a polypeptide is "immunoreactive" when it is “immunologically reactive” with an antibody, i.e., when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
  • An “immunoreactive" polypeptide may also be “immunogenic".
  • the term “immunogenic polypeptide” is a polypeptide that elicits a cellular and/or humoral immune response, whether alone or linked to a carrier in the presence or absence of an adjuvant.
  • an antibody refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site.
  • An antibody combining site may be formed from a heavy and/or a light chain domain (VH and VL, respectively) , which form hypervariable loops which contribute to antigen binding.
  • a “paratope” is an antibody combining site for an epitope, the simplest form of an antigenic determinant.
  • antibody includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalent antibodies, the Fab proteins, and single domain antibodies.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • polypeptide does not connote the method by which the molecule was made, and thus includes naturally occurring molecules, as well as molecules made by chemical or recombinant synthesis.
  • a "biological sample” refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents.
  • Coupled refers to attachment by covalent bonds or by strong non-covalent interactions (e.g., hydrophobic interactions, hydrogen
  • Covalent bonds may be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like.
  • support refers to any solid or semi- solid surface to which a desired polypeptide.
  • 15 supports include glass, plastic, metal, polymer gels, an the like, and may take the form of beads, wells, dipsticks, membranes, and the like.
  • label refers to any atom or moiety which can be used to provide a detectable ,_ (preferably quantifiable) signal, and which can be attached to a polynucleotide or polypeptide.
  • treatment refers to prophylaxis and/or therapy.
  • immunogenic refers to an agent used
  • an individual's allergic sensitivity to H. pylori is
  • IgE 30 determined by detecting IgE specific to g. pylori allergens. Any method of detecting IgE specific for an allergen known in the art may be used.
  • one or more polypeptides comprised of epitopes immunologically
  • 3-5.. identifiable with epitopes of allergens are coupled to a solid substrate.
  • a biological sample suspected of containing IgE specific for allergens from the material is reacted with the allergen-substrate complex, and IgE that reacted immunologically with the allergen of the complex is detected.
  • An example of this kind of assay is the Radioallergosorbent (RAST) test.
  • an allergen extract is coupled to cellulose particles or paper discs.
  • Patient's serum containing IgE antibody or a standard serum is reacted with the allergen-coupled immunosorbent.
  • labeled anti-IgE is reacted with the immunosorbent.
  • the label on the ' separated sorbent is determined and is a measure of the amount of specific serum IgE antibodies to that allergen.
  • the RAST test is modified to increase its sensitivity by removing IgG and/or IgA antibodies which may interfere with IgE binding to the allergen. This is particularly critical when measuring serum IgE specific to H. pylori allergens.
  • Reactants capable of remvoving IgG and/or IgA are known in the art, and include, for example. Protein G, anti-human IgG and anti-human IgA, as well as Protein A. For convenience, these reactants may be affixed to a solid substrate, including, for example, Sepharose. The amount of the reactants used is sufficient to removing interfering IgG and IgA, but not the IgE which is to be detected. The determination of the desired amount is by methods known to those of skill in the art.
  • a method of removing interfering IgG and/or IgA antibodies by incubation of the serum with Protein A is discussed in the Examples, infra. Generally, the amount of Protein A which is used is sufficient to prevent the blocking antibodies from competing with the IgE having the same specificity.
  • the modified RAST test may also include the use _ of purified protein allergens.
  • Methods of purifying proteins are known in the art, and include, for example, differential extraction, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for Q example, Methods in Enzymology for a variety of methods for purifying proteins.
  • An example of a purification procedure which separates protein allergens of H. pylori from carbohydrates, lipids, and nucleic acids is presented in the Examples.
  • polypeptides comprised of one or more epitopes which are immunologically identifiable with epitopes of H. pylori allergens may be packaged in diagnostic kits.
  • Diagnostic kits include the polypeptides in suitable containers and a means for detecting immunological complexes formed between the polypeptide and IgE in the biological sample, if any.
  • the polypeptides may be affixed to a solid 0 substrate.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular diagnostic protocol, for example, standards, buffers, as well as instructions for conducting the test using the kit ingredients.
  • individuals suspected of having a propensity for, or suffering from H are suspected of having a propensity for, or suffering from H.
  • pylori induced gastric disease are treated with substances which reduce the allergic response to the microorganism.
  • Treatment may be with, for example, a composition containing purified protein allergens, or with recombinant polypeptides or anti- idiotype antibodies which are immunologically identifiable with the protein allergen by virtue of one or more immunogenic epitopes which are immunologically cross-reactive with those on H. pylori protein allergen.
  • One or more allergens contained within DEAE fractions 59, 64, 66, 68, 72 and 74, the preparation of which is described in Example 1, may be particularly suitable.
  • Treatment may also be with, for example, allergoids of H.. pylori protein allergens.
  • Methods of preparing allergoids from antigens are known in the art.
  • mild formalin or glutaraldehyde treatment of the antigen reduces the allergenicity (IgE formation) without affecting the antigenicity (IgG "blocking" antibody formation) .
  • Treatment may also be with, for example, compositions containing at least one structural analog of an epitope of a protein allergen, which binds to the corresponding IgE paratope.
  • Structural analogs are organic molecules which are capable of assuming the appropriate charge distribution and hydrophobic/hydrophilic characteristics to allow binding to the paratope in a fashion which mimics the immunologic binding of the epitope.
  • the treated individual receives injections of a composition comprised of one or more relevant allergens continuously.
  • Treatment is begun at a dosage low enough to avoid any local or systemic reactions, and frequent injections, usually once or twice a week are administered at increasing dosages until the highest dose the patient can tolerate without excessive local or systemic reactions is reached.
  • This is a maintenance dose, which is then continued at less frequent intervals, usually every 2-6 weeks depending upon the individual's response.
  • the actual dosage and treatment regimen will depend upon the individual treated, and will be determined by the person administering the treatment.
  • the immunoreactive polypeptides are prepared into vaccines.
  • Vaccines may be prepared from one or more immunogenic polypeptides. If recombinant, these polypeptides may be expressed in a variety of host cells (e.g., bacteria, yeast, insect, or mammalian cells), or alternatively may be isolated from the bacterial preparations.
  • vaccines which contain an immunogenic polypeptide(s) or structural analogs of epitopes as active ingredients, is known to one skilled in the art.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or.the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-
  • thr-MDP L-threonyl-D-isoglutamine
  • CGP 11637 N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine
  • nor-MDP N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosph oryloxy)-ethylamine
  • CGP 19835A referred to as MTP-PE
  • RIBI which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an H. p
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, .sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspen ⁇ sions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of about 5 micrograms to about 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • a polypeptide containing one or more epitopes immunologically identifiable with epitopes of an H. pylori allergen are used to prepare antibodies to H. pylori epitopes, using the polypeptide as an immunizing agent, and methods known to those of skill in the art.
  • the antibodies prepared may be purified polyclonal antibodies, single-chain antibodies, monoclonal antibodies, antibody fragments, and the like. These antibodies may be used, for example, for purification by affinity chromatography polypeptides of interest. More specifically, they can be used to purify polypeptides containing epitopes immunologically identifiable with epitopes of fl. pylori allergens, including the allergens themselves.
  • antibodies to H. pylori epitopes may be used for the preparation of anti-idiotype antibodies.
  • These anti-idiotype antibodies are comprised of a region which mimics the epitope of the allergen.
  • Anti-idiotype may be synthesized using methods known in the art, and will usually use antibodies directed to fl. oylori epitopes as an immunizing agent.
  • Anti-idiotype antibodies may be useful in immunotherapy of individuals sensitive to fl. pylori allergens, as well as for the purification of and/or detection of antibodies directed to fl. pylori antigens containing epitopes which immunologically cross-react with the anti-idiotype antibodies.
  • the immunogenic polypeptides prepared as described above are used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing an H. pylori epitope(s). Serum from the im ⁇ munized animal is collected and treated according to known procedures.
  • polyclonal antibodies to an H. pylori epitope contains antibodies to other antigens
  • the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987) IMMUNOCHEMICAL METHODS IN CELL /ND MOLECULAR BIOLOGY (Academic Press, London) .
  • polyclonal antibodies may be isolated from an individual previously infected with fl. pylori. and purified by the methods discussed above.
  • Monoclonal antibodies directed against fl. pylori epitopes can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Im ⁇ mortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. (1980) HYBRIDOMA TECHNIQUES PRINCIPLES AND PRACTICE, SECOND EDITION (Springer-Verlag, N.Y.).; Hammerling et al.
  • Antibodies both monoclonal and polyclonal, which are directed against H. pylori epitopes are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy.
  • Monoclonal antibodies may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
  • anti-idiotype antibodies may also be useful for treatment, vaccination and/or diagnosis of H. pylori induced gastritis and/or gastroduodenal ulcers, as well as for an elucidation of the immunogenic regions of H. pylori antigens
  • H. pylori ATCC strain 43504; ATCC, Bethesda, MD, USA
  • H. pylori obtained from the American Type Culture Collection, ATCC No. 43504, was removed aspectically from its vial, suspended in 1 ml sterile Difco Brucella broth, and transferred by an in inoculating loop to 3 separate Brucella Agar plates (Anaerobe systems, San Jose, CA) .
  • the plates were incubated at 35 deg C for 5 days in a microaerophilic atmosphere of 85% N,, 10% CO., and 5% 0,. After incubation the plates were removed and examined. Tiny grayish-white colonies were observed. Microscopic examination of a Gram-stained smear showed large oxbow- shaped and loops of Gram-negative rods (5 microns) , which are typical of H. pylori. fl. pylori in colonies from the 5 day plates were transferred to a fresh set of Brucella plates, and the plates were incubated microaerophilically at 35 deg C for 3 to 5 days. After 3 days a more luxuriant growth of H. pylori colonies occurred. These colonies were used as the inoculum for a broth seed culture.
  • a broth seed culture was prepared by transferring to several 10 ml screw-capped tubes 5 ml sterile Brucella broth with 5% horse serum (GIBCO BRL) , and colonies collected by swab from the plates. All tubes were incubated at 35 deg C under a microaerophilic atmosphere for 3 to 5 days. If a heavy degree of turbidity was observed in the tubes after this period, the culture was examined for purity by microscopic examination of a Gram stained slide. The broth seed culture was used as an inoculum for one liter of sterile Difco Brucella broth containing 5% horse serum.
  • the inoculated culture was grown in a 3 liter flask by incubation at 35 deg C in a microaerophilic atmosphere for 3 to 5 days. When a moderate degree of turbidity was observed, the culture was checked for purity as described above. One liter of culture generally yielded an unwashed cell amount of about 2.0 grams.
  • the living organisms from the liter culture were pelleted by centrifugation at 3,000 RPM, 4 deg C for 15 minutes. They were attenuated and grossly defatted by suspension and vortexing in ice cold acetone for 15 minutes. The attenuated bacteria was then repelleted by similar centrifugation. The pellet was resuspended in 20 ml of cold buffer containing 50 mM sodium phosphate, pH 7.3, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 micrograms/ml PMSF and 100 micrograms/ml of benza idine. Ten mL or 150-210 micron, acid washed glass beads (Sigma, St.
  • the supernatant was centrifuged for l hr at 100,000 g. 4 deg C, in a Beck an SW 40Ti rotor (Beckman, Palo Alto, CA, USA) . To the resulting supernatant was added 0.456 gm/ml of RbCl (Aldrich Chemical Co., Milwaukee, Wis. , USA). The solution was then centrifuged at 4 deg C for 48 hrs. in a Beckman 70 Ti rotor (the first 24 hrs at 65,000 RPM and the second 24 hrs at 48,000 RPM). The supernatant contents of each gradient tube were collected in ten equal fractions beginning at the bottom of each tube. The pellet in each tube representing most of the residual complex carbohydrates and nucleic acids containing in the pregradient supernatant was discarded.
  • each gradient fraction was dialyzed against 20 mM sodium phosphate buffer, pH 7.0, at 4 deg C using dialysis tubing with a 1,000 MW cutoff. An approximation the protein content per fraction was made by ⁇ g spectrophotometry at a wavelength of 280 nm. Ninety percent of the detected protein was found in fractions 2 through 6, inclusive; these fractions were pooled. The pooled fractions were then loaded onto a Bio-Sil DEAE analytical anion exchange HPLC column (BioRad, Richmond,
  • the equilibrating buffer (Buffer A) was 20 mM Sodium phosphate, pH 7.0.
  • the salt containing buffer (Buffer B) was 20 mM sodium phosphate, pH 7.0, with 1.0 M NaCl.
  • Example 2 A Modified RAST Procedure for Detecting IgE Specific to H. pylori Allergens
  • IgE specific for H. pylori allergens was assayed for using a modified RAST procedure. Part of the procedure was essentially as described by Nalebuff et al. (Nalebuff et al., Otolarygol Head Neck Surg 89:271 (1981).). More specifically, an aliquot of 100 microliters of serum was incubated overnight with an appropriate allergen disc and washed three times with 50 mM phosphate buffered saline (PBS), pH 7.3, containing 0.1% Tween 20. This was followed by a second overnight incubation with I 125-labelled anti-IgE specific zor the
  • De-2 determinant After being washed and prior to being counted, the allergen discs were placed into fresh tubes in a gamma counter for the amount of time previously selected by a time control.
  • the time control consists of 25 units of WHO-standardization IgE that is run against a PRIST anti-IgE disc for the time needed for the IgE to bind 25,000 counts. This time is used in the counting of all subsequent tests.
  • Scrubbing was by incubation with recombinant Protein A/Sepharose (Zymed, S. San Francisco, CA, USA) . More specifically, two ml of serum per one ml of Protein A/Sepharose were incubated with agitation for 1 hr. The suspension was then centrifuged at 1500 RPM for 15 min. and the serum supernatants collected.
  • Figure 3 shows a plot of the net total IgE immunological reactivity of serum from control and gastritis patients using all available fl. pylori protein fractions isolated from an HPLC DEAE column.
  • Figure 4 is a plot of the net total IgE immunological reactivity of serum from control and gastritis patients with the proteins in fractions 59, 62, 65, 70, 64, 68, 71, 73, and 74.
  • Polypeptides containing one or more epitopes immunologically identifiable with epitopes of fl. pylori proteins are useful in the diagnosis of fl. pylori induced gastric diseases, and may also be useful for treatment of these diseases.
  • These polypeptides are also useful for the production of antibodies, both purified polyclonal and monoclonal, directed towards epitopes of fl. pylori.
  • the antibodies in their turn, are useful in the purification of polypeptides containing one or more epitopes immunologically identifiable with epitopes of fl. pylori proteins.
  • Monoclonal antibodies are useful in the production of anti-idiotype antibodies, which in turn, are useful for the detection of antibodies containing specific epitopes of fl. pylori, and may also be useful in the production of vaccines for H. pylori induced diseases.
  • the methods described herein use one or more polypeptides containing one or more epitopes of fl. pylori. and detect IgE directed to fl. pylori allergens. These methods, particularly the modified RAST method, are useful for the diagnosis of fl. pylori induced gastric diseases.

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Abstract

L'invention fournit des matériaux et des méthodes utiles pour le diagnostic et le traitement du trouble gastrique causé par H.pylori. Sont décrits des polypeptides contenant un ou plusieurs déterminants antigéniques identifiables imunologiquement avec des déterminants antigéniques de protéines H.pylori. Ces polypeptides sont utiles pour les analyses visant à mesurer l'IgE dans les prélèvements biologiques provenant d'individus présumés infectés. Ils servent également à traiter par immunothérapie les individus infectés.
PCT/US1992/003284 1991-04-26 1992-04-21 Methode de detection et de traitement des maladies causees par des allergenes bacteriens WO1992019970A1 (fr)

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Cited By (13)

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EP0590138A1 (fr) 1992-04-13 1994-04-06 Inc. Oravax Traitement par voie orale de l'infection provoquee par l'helicobacter
WO1994029696A1 (fr) * 1993-06-09 1994-12-22 Quidel Corporation Titrages en une etape specifiques d'un antigene
WO1995017677A1 (fr) * 1993-12-20 1995-06-29 Calenoff Emanuel J Methodes et preparations pour le diagnostic et le traitement d'affections dues aux antigenes de micro-organismes
WO1996012965A1 (fr) * 1994-10-20 1996-05-02 Genelabs Diagnostics Pte Ltd. Methodes de diagnostic de l'helicobacter pylori et necessaires correspondants
WO1996026740A1 (fr) * 1995-02-27 1996-09-06 Enteron Limited Partnership Procedes et compositions de vaccins individualises contre les affections liees aux antigenes de micro-organismes
EP0794434A1 (fr) * 1995-09-22 1997-09-10 Asahi Denka Kogyo Kabushiki Kaisha Procede d'analyse pour allergene
US5871749A (en) * 1993-07-27 1999-02-16 Csl Limited Therapeutic treatment of H. pylori associated gastroduodenal disease
US5972336A (en) * 1992-11-03 1999-10-26 Oravax Merieux Co. Urease-based vaccine against helicobacter infection
US6406703B1 (en) 1993-07-27 2002-06-18 Csl Limited Treatment of H. pylori associated gastroduodenal disease
EP1872792A1 (fr) * 2006-06-29 2008-01-02 Biotech Tools S.A. Méthode pour la production d'un allergène hydrolysé
RU2568242C2 (ru) * 2012-04-24 2015-11-10 Федеральное государственное бюджетное учреждение науки Институт молекулярной биологии им В.А. Энгельгарда Российской академии наук (ИМБ РАН) Способ обработки образца крови при проведении иммунологического анализа аллерген-специфических и общих иммуноглобулинов е
RU2587327C2 (ru) * 2014-11-19 2016-06-20 Государственное бюджетное образовательное учреждение высшего профессионального образования "Российский национальный исследовательский медицинский университет им. Н.И. Пирогова" Министерства здравоохранения Российской Федерации (ГБОУ ВПО РНИМУ им. Н.И. Пирогова Минздрава России) Способ диагностики in vitro специфической сенсибилизации организма к бактериальным аллергенам
US9884111B2 (en) 2010-02-12 2018-02-06 Laboratorios Leti, S.L. Process for producing an allergen extract

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US4870053A (en) * 1980-09-19 1989-09-26 Roussel Uclaf Novel compositions and processes
US4849337A (en) * 1983-01-31 1989-07-18 Minnesota Mining And Manufacturing Company Assaying allergen specific IgE levels with fluorogenic enzyme labeled antibody
EP0282018A2 (fr) * 1987-03-13 1988-09-14 Hoechst Aktiengesellschaft Procédé pour isoler des protéines de membrane de base de tissus humains et animaux
EP0451800A1 (fr) * 1990-04-13 1991-10-16 Abbott Laboratories Attachement d'allergènes à une phase solide

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Gastroenterology, Volume 101, No. 1, issued July 1991, ACETI et al., "Basophil-Bound and Serum Immunoglobulin E Directed Against Helicobacteri pylori in Patients with Chronic Gastritis", pages 131-137, especially Abstract on page 131, page 133, left column, second full paragraph and page 136, left column first full paragraph. *
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See also references of EP0582672A4 *
The New England Journal of Medicine, Volume 324, No. 15, issued 11 April 1991, PETERSON, "Helicobacter Pylori and Peptic Ulcer Disease", pages 1043-1048. *
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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567594A (en) * 1991-04-26 1996-10-22 Enteron, L.P. Methods and compositions for the detection and treatment of diseases associated with antigens of microorganisms
US5538729A (en) * 1992-04-13 1996-07-23 Oravax, Inc. Oral treatment of helicobacter infection
EP0590138A1 (fr) 1992-04-13 1994-04-06 Inc. Oravax Traitement par voie orale de l'infection provoquee par l'helicobacter
US5972336A (en) * 1992-11-03 1999-10-26 Oravax Merieux Co. Urease-based vaccine against helicobacter infection
WO1994029696A1 (fr) * 1993-06-09 1994-12-22 Quidel Corporation Titrages en une etape specifiques d'un antigene
US6129923A (en) * 1993-07-27 2000-10-10 Csl Limited Treatment of H. pylori associated gastroduodenal disease
US6406703B1 (en) 1993-07-27 2002-06-18 Csl Limited Treatment of H. pylori associated gastroduodenal disease
US5871749A (en) * 1993-07-27 1999-02-16 Csl Limited Therapeutic treatment of H. pylori associated gastroduodenal disease
WO1995017677A1 (fr) * 1993-12-20 1995-06-29 Calenoff Emanuel J Methodes et preparations pour le diagnostic et le traitement d'affections dues aux antigenes de micro-organismes
WO1996012965A1 (fr) * 1994-10-20 1996-05-02 Genelabs Diagnostics Pte Ltd. Methodes de diagnostic de l'helicobacter pylori et necessaires correspondants
WO1996026740A1 (fr) * 1995-02-27 1996-09-06 Enteron Limited Partnership Procedes et compositions de vaccins individualises contre les affections liees aux antigenes de micro-organismes
EP0794434A1 (fr) * 1995-09-22 1997-09-10 Asahi Denka Kogyo Kabushiki Kaisha Procede d'analyse pour allergene
EP0794434A4 (fr) * 1995-09-22 2003-06-11 Asahi Denka Kogyo Kk Procede d'analyse pour allergene
EP1872792A1 (fr) * 2006-06-29 2008-01-02 Biotech Tools S.A. Méthode pour la production d'un allergène hydrolysé
WO2008000783A1 (fr) * 2006-06-29 2008-01-03 Biotech Tools S.A. Procédé de production d'un allergène hydrolysé
EP3308799A1 (fr) * 2006-06-29 2018-04-18 ASIT BioTech S.A. Procédé de production d'allergène hydrolysé
US9884111B2 (en) 2010-02-12 2018-02-06 Laboratorios Leti, S.L. Process for producing an allergen extract
US11246923B2 (en) 2010-02-12 2022-02-15 Laboratorios Leti, S.L. Unipersonal Process for producing an allergen extract
RU2568242C2 (ru) * 2012-04-24 2015-11-10 Федеральное государственное бюджетное учреждение науки Институт молекулярной биологии им В.А. Энгельгарда Российской академии наук (ИМБ РАН) Способ обработки образца крови при проведении иммунологического анализа аллерген-специфических и общих иммуноглобулинов е
RU2587327C2 (ru) * 2014-11-19 2016-06-20 Государственное бюджетное образовательное учреждение высшего профессионального образования "Российский национальный исследовательский медицинский университет им. Н.И. Пирогова" Министерства здравоохранения Российской Федерации (ГБОУ ВПО РНИМУ им. Н.И. Пирогова Минздрава России) Способ диагностики in vitro специфической сенсибилизации организма к бактериальным аллергенам

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EP0582672A4 (en) 1994-06-29
EP0582672A1 (fr) 1994-02-16

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