METHOD OF INDUCING TOLERANCE TO AN ALLERGEN

Abstract

The present invention relates generally to the field of allergies. More particularly, the present invention provides a method for treating an allergy in a subject by inducing tolerance to an allergen associated with the allergy. Medicinal kits useful in protocols to induce tolerance or reduce intolerance in a subject also form part of the present invention.

Claims

1. A method for treating allergy or intolerance to a food allergen in a subject allergic or intolerant to the food allergen, said method comprising orally administering sequentially or simultaneously to said subject a probiotic and the food allergen or an antigenic component thereof, wherein the probiotic is Lactobacillus rhamnosus.

2. The method of claim 1, wherein the food allergen is found in milk, eggs, peanuts, tree nuts, fish, shellfish, soy, wheat, sesame, celery, celeriac, corn, pumpkin or fruit.

3. The method of claim 1, wherein the subject is a human.

4. The method of claim 1, wherein the subject is a non-human animal.

5. The method of claim 4, wherein the non-human animal is selected from the group consisting of a companion animal, a livestock animal and a captured wild animal.

6. The method of claim 1, further comprising administration of a prebiotic.

7. The method of claim 6, wherein the prebiotic is an oligosaccharide or a soluble or insoluble fibre material.

8. The method of claim 1, wherein the probiotic and the food allergen are provided in different formulations.

9. A method for inducing tolerance to a food allergen in a subject with an allergy or intolerance to the food allergen, said method comprising orally administering sequentially or simultaneously to said subject a probiotic and the allergen or an antigenic component or fragment or analog thereof, in an amount effective to induce tolerance to the allergy, wherein the probiotic is Lactobacillus rhamnosus.

10. The method of claim 9, wherein the food allergen is found in milk, eggs, peanuts, tree nuts, fish, shellfish, soy, wheat, sesame, celery, celeriac, corn, pumpkin or fruit.

11. The method of claim 9, wherein the subject is a human.

12. The method of claim 9, wherein the subject is a non-human animal.

13. The method of claim 12, wherein the non-human animal is selected from the group consisting of a companion animal, a livestock animal and a captured wild animal.

14. The method of claim 9, further comprising administration of a prebiotic.

15. The method of claim 14, wherein the prebiotic is an oligosaccharide or a soluble or insoluble fibre material.

16. The method of claim 9, wherein the probiotic and the food allergen are provided in different formulations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

[0035] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, is understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0036] As used in the subject specification, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to “a biotic agent” includes a single biotic agent, as well as two or more biotic agents (which includes two or more probiotics or prebiotics or a probiotic and a prebiotic); reference to “an allergen” includes a single allergen, as well as two or more allergens; reference to “the invention” includes single and multiple aspects of an invention; and so forth.

[0037] The present invention provides a medicinal protocol for treating a subject with an allergy by generating tolerance in the subject to an allergen. The protocol comprises providing the subject with a biotic agent and the allergen or a modified form thereof to which tolerance is desired.

[0038] “Inducing tolerance” includes reducing sensitivity to an allergen or an allergen associated with an allergy. Hence, it encompasses reducing sensitivity to an allergy as well as reducing intolerance to an allergen-induced allergy.

[0039] Hence, the present invention provides a method for treating allergen intolerance in a subject, the method comprising administering sequentially or simultaneously to the subject, a biotic and the allergen or an antigenic component or fragment or analog thereof in an amount effective to induce tolerance to the allergen.

[0040] The allergen is provided to initiate and/or boost and/or maintain an immune response. Reference to an “allergen” includes any substance which is capable of stimulating a typical hypersensitivity reaction in atopic subjects. Allergens contemplated herein include any substance in food, drugs, perfume, plants, the environment or biological systems (e.g. prokaryotic or eukaryotic cells or viruses), as well as chemical allergens. Types of allergens include animal products (e.g. cats, fur and dander, cockroach calyx, dust mite excretion); drugs (e.g. penicillin, sulfonamides, salicylates (also found naturally in numerous fruits), local anaesthetics); foods (e.g. celery, celeriac, corn or maize, eggs (typically albumen, the white), fruit, pumpkin, legumes (e.g. beans, peas, peanuts, soybeans), milk, seafood, sesame, soy, tree nuts (e.g. pecans almonds), wheat, insect stings (e.g. bee sting venom, wasp sting venom, mosquito stings); mold spores, latex, metal; and plant pollens (e.g. grass—ryegrass, timothy-grass, weeds—ragweed, plantago, nettle, Artemisia, vulgaris, chenopodium album, sorrel, trees—birch alder, hazel, hornbeam, aesculus, willow, poplar, platanus, tilia, olea).

[0041] The present invention is particularly directed to food allergens such as found in milk, eggs, peanuts, tree nuts, fish, shellfish, soy and wheat.

[0042] In one embodiment, the present invention is directed to inducing tolerance to legume allergens and in particular peanut allergens.

[0043] Reference to the “allergen” includes the allergen in a purified or substantially purified or isolated form or when incorporated as part of a substance such as food, a biological system, or chemical composition. Furthermore, the allergen to be administered may also be a modified form including an antigenic derivative or component or homologue or chemical analog. An “allergen” encompasses a mixture of allergens as well as genetically modified or chemically modified allergens.

[0044] The present invention is directed to inducing tolerance or reducing sensitivity to an allergen or an allergy associated with the allergen as well as reducing intolerance to an allergen-induced allergy.

[0045] The term “biotic” encompasses both a probiotic and a prebiotic. A probiotic is generally a live eukaryotic or a prokaryotic organism which has a beneficial property when given to a subject. In one aspect, the probiotic complements the existing microflora in the subject. Hence, the probiotic agent is a live microorganism which can confer a health benefit to a host subject. The probiotic agent may be a culture of microorganisms or provided in a dietary supplement or may be freeze dried and reconstituted prior to use. A prebiotic is an agent which facilitates or confers growth, maintenance and/or beneficial properties of or on the subject's microflora. A prebiotic includes an oligosaccharide and soluble or insoluble fibre material. A probiotic and a prebiotic may also be sequentially or simultaneously administered.

[0046] Examples of probiotic agents include species of Lactobacillus, Escherichia, Bacillus, Bifidobacterium, Saccharomyces and Streptococcus.

[0047] Particularly useful probiotic agents are from the genus Lactobacillus such as Lactobacillus acidophilus NCFM, Lactobacillus casei, Lactobacillus casei Shirota, Lactobacillus casei immunitass, Lactobacillus johnsonii, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius and Lactobacillus helvetirus.

[0048] Lactobacillus rhamnosus GG (LGG) is considered herein to be a particularly useful probiotic agent.

[0049] The microorganisms may be naturally occurring, attenuated or genetically modified to introduce new or to alter existing traits. In one embodiment, the probiotic has been genetically modified to introduce an allergen gene or part or fragment or portion thereof which is expressed to produce recombinant microorganisms which release or expose the subject's immune system to the allergen or an antigenic fragment thereof. Hence, the probiotic and allergen may be given to the subject as a single entity. As indicated above, a probiotic and a prebiotic may also be administered, together with the allergen or the allergen may be produced by the probiotic.

[0050] Hence, the present invention provides a method for inducing tolerance in a subject to an allergen, the method comprising administering to the subject an amount of allergen or antigenic fragment, compound or analog thereof and a biotic agent effective to induce tolerance in the subject to the allergen.

[0051] In another embodiment, the present invention contemplates a method for inducing tolerance in a subject to an allergen, the method comprising administering to the subject an amount of probiotic modified to produce an allergen or fragment or homolog thereof in an amount effective to induce tolerance in the subject to the allergen.

[0052] In one particular embodiment, the allergen is a legume such as a peanut.

[0053] Hence, another aspect of the present invention contemplates a method for inducing tolerance in a subject to a legume allergen, the method comprising administering to the subject an amount of the legume allergen or an antigenic fragment, component or analog thereof and a biotic agent effective to induce tolerance in the subject to the legume allergen.

[0054] One or more allergens may be administered generally at an amount which does not cause distress to the subject such as in the form of anaphylaxis. As indicated above, the legume allergen may also be produced by a probiotic form of the biotic.

[0055] In a particular embodiment, probiotic is a species of Lactobacillus.

[0056] Another aspect of the present invention contemplates, therefore, a method for inducing tolerance in a subject to a legume allergen, the method comprising administering to the subject an amount of the legume allergen or an antigenic fragment, component or analog thereof and a biotic agent is a probiotic agent selected from the list consisting of L. acidophilus NCFM, L. casei, L. casei Shirota, L. casei immunitass, L. johnsonii, L. lactis, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius and L. helvetirus effective to induce tolerance in the subject to the allergen.

[0057] Yet another aspect of the present invention contemplates, therefore, a method for inducing tolerance in a subject to a legume allergen, the method comprising administering to the subject an amount of the legume allergen or an antigenic fragment, component or analog thereof and a biotic agent is a prebiotic agent selected from the list consisting of an oligosaccharide and a fibre effective to induce tolerance in the subject to the allergen.

[0058] In a particular embodiment, the probiotic is L. rhamnosus or a modified form thereof. In another particular embodiment, the prebiotic is an oligosaccharide or a soluble or insoluble fibre.

[0059] Another aspect of the present invention contemplates a method for inducing tolerance in a subject to a peanut allergen, the method comprising administering to the subject an amount of the peanut allergen or an antigenic fragment, component or analog thereof and a biotic agent effective to induce tolerance in the subject to the peanut allergen.

[0060] The present invention further contemplates, a method for inducing tolerance in a subject to a peanut allergen, the method comprising administering to the subject an amount of the peanut allergen or an antigenic fragment, component or analog thereof and a biotic agent selected from the list consisting of L. acidophilus NCFM, L. casei, L. casei Shirota, L. casei immunitass, L. johnsonii, L. lactis, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius and L. helvetirus effective to induce tolerance in the subject to the allergen.

[0061] Yet another aspect of the present invention provides a method for inducing tolerance in a subject to a peanut allergen, the method comprising administering to the subject an amount of the peanut allergen or an antigenic fragment, component or analog thereof and a prebiotic agent selected from the list consisting of an oligosaccharide and a fibre effective to induce tolerance in the subject to the allergen.

[0062] As indicated above, the fibre may be soluble or insoluble.

[0063] A method is also provided for reducing a subject's sensitivity to an allergy, the method comprising administering to the subject a biotic agent in conjunction with an allergen associated with the allergy or an antigenic fragment or component or analog of the allergen for a time and under conditions sufficient for a level of tolerance to be induced in the subject.

[0064] The “administering” includes sequential and simultaneous administration of a probiotic and/or prebiotic and allergen.

[0065] Whilst not intending to limit the present invention to any one theory or mode of action, the combined effect of the biotic agent with allergen immunotherapy is proposed to increase Th1 responses compared to Th2 responses.

[0066] The present invention also provides a medicinal kit comprising in compartmental form a first compartment or series of compartments comprising biotic agents and a second compartment or series of compartments comprising an allergen or source of allergens or antigenic fragments, components or analogs thereof with instructions for use.

[0067] The instructions for use include a medicinal protocol to use the biotic agents in conjunction with an allergen or source of allergen to induce tolerance or reduced sensitivity to an allergen.

[0068] An “effective amount” or “therapeutically effective amount” means an amount necessary at least partially attain the desired immunological effect of tolerance or to delay the onset or inhibit progression or halt altogether, the onset of progression of an allergic response to an allergen in the subject in need of treatment. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The “effective amount” relates to the allergen and biotic, individually or combined. Conveniently, administration of at least the allergen includes a “rush” amount followed by a “maintenance” amount. Examples of effective amounts range from 0.05 mg to 2000 mg per day, week or month. For peanut allergens, 0.1 mg to 300 mg per day is effective.

[0069] The present invention in a particular aspect is directed to treatment of a subject in need thereof rather than prophylaxis. That said, in another aspect, a prophylactic component is contemplated.

[0070] As used herein, the terms “treating” or “treatment” encompass the administration of an agent which induces tolerance to an allergen.

[0071] The present invention is further directed to a method for inducing a level of tolerance to an allergy in a subject, the method comprising providing to the subject effective amounts of a biotic and an allergen associated with the allergy.

[0072] As indicated above, a single allergen may be provided or multiple allergens are provided.

[0073] The “level” of tolerance includes complete tolerance or an increased threshold in the amount of allergen to which a subject may be exposed prior to inducing an adverse allergic reaction.

[0074] The term “subject” as used herein refers to an animal, particularly a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the methods of the present invention. Generally, the subject is in need of treatment as the present invention is particularly directed to treatment of an allergen-induced allergy. Genetic testing of subjects or embryos in utero may also identify subjects at risk of developing an allergy. A subject regardless of whether a human or non-human animal or embryo, may be referred to as an individual, subject, animal, patient, host or recipient. The present invention, therefore, has both human and veterinary applications. For convenience, an “animal” specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. The non-human animal may also include a companion animal such as a dog or cat or captured wild animal.

[0075] The present invention extends to any subject having an allergy or predisposed to an allergic reaction. Hence, the subject may have a family history, genetic trait or predisposition to the development of an allergy and accordingly may be administered doses of the probiotic and allergen to induce some level of tolerance to the allergen.

[0076] The biotic and allergen are given in conjunction with each other. Insofar as the biotic is a probiotic, the allergen or a genetically modified form or fragment thereof, may be produced by the microorganism.

[0077] By “in conjunction” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. The term “in conjunction” also includes the use of two or more allergens in the same therapeutic protocol. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. The biotic and allergen may be administered in any order. The probiotic form of the biotic may also produce the allergen. The biotic (i.e. probiotic and/or prebiotic) may be sequentially or simultaneously administered with the allergen.

[0078] As used herein “administering” or “administration” or “providing” an agent to a subject includes delivery via any route such as oral, subcutaneous, sub lingual, nasal, intravenous, anal or intra-peritoneal routes. The biotic may be given over a period of time prior to the allergen vice versa. Alternatively, both agents may be given at approximately the same time.

[0079] Standard formulations may be employed for each or either of the biotic and allergen. As indicated above, the biotic may be in freeze dried form which is then reconstituted prior to use or the biotic may be given as a dietary supplement. The freeze dried formulation may also comprise the allergen in a similar form. The biotic may also be given with a source of allergen such as milk, eggs, bread, soy and the like.

[0080] The present invention further provides diagnostic assays to monitor immune mechanisms underlying tolerance. Examples of immune mechanisms include monitoring IgE, IgG4 and IgA levels as well as regulatory T-cell levels (Tregs).

[0081] The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Effects of Probiotic and Allergen

Subject Recruitment, Treatment and Sample Collection

[0082] Eleven healthy non-atopic adults were treated with 1.8×10.sup.1° CFU LGG (Dicoflor, Dicofarm SpA, Rome) daily for 7 days. ten ml venous blood was harvested prior to treatment and at day 7. Blood samples were collected in polypropylene tubes containing heparinized RPMI tissue-culture medium (Invitrogen, Carlsbad, Calif.).

Preparation of Blood Samples and Cell Culture

[0083] Mononuclear cells were separated by density centrifugation and cryopreserved for future batched analyses (Dunstan et al, J Allergy Clin Immunol 112:1178-1184, 2003). Two million cells/ml were incubated with or without antigen in AIM-V serum free medium (Invitrogen) for 48 h, or in RPMI with 10% v/v autologous plasma for 6 days in proliferation assays. Heat killed LGG (HKL) was prepared by incubating LGG at 75° C. for 45 minutes and was used at 5:1 HKL to mononuclear cell ratio. OVA (Sigma, St Louis, Mo.) was used at 100 μg/ml. IFNγ-1b (Boehringer Ingelheim, Germany) and LPS (Sigma) were both used at 10 ng/ml. All cell culture reagents were tested for endotoxin contamination (Cape Cod Associates, E. Falmouth, Ma.). OVA required endotoxin removal over polymyxin B columns prior to use (Pierce, Rockford, Ill.).

Flow Cytometry

[0084] Cell pellets were stained with fluorochrome-conjugated monoclonal antibodies in 50 μl staining volumes. Lineage cocktail-FITC (anti-CD3, 14, 16, 19, 20, 56), HLA-DR-Peridinin chlorogphyll protein (PerCP), CD123-PE and CD11c- Allophycocyanin (APC) were used to identify DC phenotypes as CD11c.sup.hiCD123w.sup.lo myeloid DC (mDC), CD123.sup.hiCD11c.sup.lo plasmacytoid DC (pDC) and CD11c.sup.hiCD123w.sup.lo immature DC (iDC), CD3-APC, CD4-PerCP, CD25-PE-Cy7 (BD Bioscience, San Jose, Calif.), CD25-FITC and FoxP3-PE (E-Bioscience, San Diego, Calif.) were used to identify CD25.sup.hiFoxP3.sup.hiT-cell populations. CFSE was used as a cell tracking dye, and aminostiblbamidine methanesulfonate as a viability dye (Molecular Probes, Eugene, Oreg.). CBMC or PBMC were incubated with fluorochrome-labeled antibodies or isotype controls for 30 minutes, and for intracellular staining cells were subsequently permeabilized, fixed and stained with FoxP3-PE antibody or isotype control (E-Bioscience). Data were acquired on a 4-color LSR 111 (BD Bioscience) and analyzed with FACSDiva v4.1 software using well defined gating strategies.

ELISA

[0085] Concentrations of IL-10, IFN-γ, IL-13, IL-12p40 and TNF-α were determined n CBMC culture supernatants harvested at 48 h by multiplex cytokine bead assay using a Luminex 100 analyzer (Luminex Corporation, Austin, Tex.). Anti-cytokine beads and matched anti-cytokine biotinylated reporters were used according to the manufacturer's instructions (Millipore, Billerica, Ma.). Data were analyzed with Luminex IS 2.3 Software using a five-parameter regression formula to calculate sample concentrations from standard curves. concentrations of TGF-β1 were evaluated using a commercial human TGF-β1 ELISA kit according to the manufacturer's protocol (BD Biosciences). Supernatants were analyzed undiluted in duplicate with recombinant cytokine as a positive and culture medium as a negative control. TGF-β1 concentrations were determined based on a standard curve generated using the KCjunior v1.40.3 program (Bio-Tek Instruments, Winooski, Vt.) with a four-parameter equation. ELISA data were analyzed both as dichotomous data-detected versus not detected; and as continuous data—mean level in each group.

Real Time PCR

[0086] RNA was extracted using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed to cDNA using the Superscript First Strand Synthesis System and oligo(dT) primers (Invitrogen). All reactions included a ‘RT minus’ control with no reverse transcriptase to control for the possibility of contaminating DNA. FoxP3 and IL-4 mRNA were quantified by real time PCR using FAM-labeled Taqman Gene Expression Assays on an ABI Prism 7300HT system (Applied Biosystems, Foster City, Calif.). Eukaryotic translation elongation factor 1 alpha (EEF1A1) which is stably expressed in human mononuclear cell cultures was used as a reference gene (Hamalaninen et al, Anal Biochem 299:63-70, 2001). The mean level of gene expression in cDNA samples was expressed as a ratio to mean EEF1A1 expression.

Statistics

[0087] The clinical trial was designed with a sample size of 250 in order to have 90% power to detect a 40% difference in eczema risk between probiotic and placebo groups. Secondary outcomes included immune outcomes reported herein. All available CBMC samples were evaluated and primary analyses were by intention to treat. Data were assessed using histograms and skewed data were logo transformed. Parametric paired data were analyzed using the paired t-test, and non-parametric paired data using Wilcoxon signed rank test and Sign test.

[0088] Parametric unpaired data were analyzed using the independent t-test, and non-parametric unpaired data using Mann Whitney U test. Continuous data are presented as arithmetic means±1 SEM, or medians with inter-quartile ranges. Categorical data were analyzed using X2 test or Fisher's exact test. P value<0.05 was considered statistically significant, with due caution in interpreting the results of multiple comparisons. Where the significance of findings was unclear a sensitivity analysis was undertaken by excluding participants in whom: (1) treatment compliance data (returned capsule counts) were not available (n=9); (ii) capsule counts suggested compliance levels <50% (n=2); or (iii) no treatment capsules were taken due to premature delivery between randomization and 36 weeks gestation (n=1). analyses were performed using SPSS v 16.0 for Windows (SPSS Inc., Chicago, Ill.).

Effects of LGG Treatment on BPMC Proliferation

[0089] The effects of orally administered LGG were evaluated in healthy adults. PBMC were harvested from 11 adults prior to and upon completion of 7 days LGG treatment. Treatment was associated with a 30% reduction (95% CI 11 to 50%; P=0.03) in mean CD4.sup.+ T-cell proliferative response to heat killed LGG (HKL) compared with proliferative responses from the same subjects before LGG treatment. In contrast, there was no changed in CD4.sup.+ T-cell proliferation to OVA (P=0.2) or medium alone (P=0.06) after LGG treatment.

Effects of LGG Treatment on DC Phenotype

[0090] DC phenotype was investigated in cultured PBMC harvested from adults before and after LGG treatment. Plasmacytoid DC (pDC) increased from 3.20% to 5.29% of total DDC (P=0.02) after LGG treatment, in PBMC cultured for 48 h with HKL. A trend towards increased ratio of pDC to myeloid (mDC) was also seen after LGG treatment in PBMC cultured with HKL (mean ratio 0.36 pre-treatment, 0.58 post-treatment; P=0.07). LGG treatment was not associated with any significant change in DC phenotype in PBMC cultured with OVA or medium alone.

[0091] The data show that oral administration of LGG to healthy adults leads to systemically detectable changes in T-cell proliferative responses and DC phenotype. Both the decrease in CD4.sup.+ T-cell proliferation and the increase in pDC numbers in PBMC cultured with HKL are consistent with antigen-specific tolerance induction (Colonna et al, i Nat Immunol 5:1219-1226, 2004).

[0092] 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. 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 of any two or more of said steps or features.

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