Methods and protocols to develop food-induced allergic and anaphylactic responses in a porcine animal model

Abstract

Provided herein is an in vivo swine model system, and methods for making and using the same, for an allergic condition, comprising: a pig sensitized to trigger an immune response for at least one of: allergy symptoms or anaphylaxis, wherein the allergy symptoms at triggered by repeated tape stripping (TS) of the skin and application of an allergen and an antigen, or wherein an immune response by intraperitoneal injections (IP), or both, with an allergen and an antigen once a week for up to six weeks.

Description

METHODS AND PROTOCOLS TO DEVELOP FOOD-INDUCED ALLERGIC AND ANAPHYLACTIC RESPONSES IN A PORCINE ANIMAL MODELCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 595,130, filed November 1, 2023, the entire contents of which are incorporated herein by referenceTECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of animal models, and more particularly, to novel methods and protocols for developing food-induced allergic and anaphylactic responses in a porcine animal model and uses thereof.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under R01AH35197 awarded by the National Institutes of Health NIAID. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] None.BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with animal models for allergic reactions to, e.g., peanuts.

[0006] Peanut allergy is one of the most common types of food allergy. It affects millions of people worldwide and is the leading cause of anaphylaxis1,2. It is estimated that approximately 1% of the US population, especially children under five years of age, is living with peanut allergy3. Strict avoidance of peanuts, patient education, and medications such as epinephrine that provide temporary relief are the mainstay of a peanut allergy management plan. In 2020, an oral immunotherapy, Palforzia, was approved by the FDA to treat peanut allergy in patients aged 4 to 17 years. However, due to frequent adverse effects4,5, the therapy has received poor adoption by the patients, and Nestle, the company that currently owns the technology is considering selling it off6. As such, there is an urgent need for animal models to develop new and improved peanut allergy treatments, to better understand disease etiology, and to establish superior biomarkers.

[0007] The mouse is a commonly used model organism at the preclinical level; however, the mouse does not fully mimic human allergic disease, thus, a higher / larger animal model is needed that more closely mimics a human immune response.

[0008] Two such animal models are taught by Helm, et al., A neonatal swine model for peanut allergy, J Allergy Clin Immunol. 2002 Jan; 109(1): 136-42. doi: 10.1067 / mai.2002.120551, and by Rupa, et al., Porcine IgE in the context of experimental food allergy: purification and isotype- specific antibodies, Vet Immunol Immunopathol. 2008 Oct 15; 125(3-4):303-14. doi: 10.1016 / j.vetimm.2008.05.028.Epub 2008 Jun 8. However, both studies report mostly mild allergic reactions in pigs, which were inconsistent and difficult to reproduce.

[0009] Another model system is taught by Ratcliff, et al., A swine model of soy protein-induced food allergenicity: implications in human and swine nutrition, Animal Frontiers, Volume 9, Issue 3, July 2019, Pages 52-59, doi.org / 10.1093 / af / vfz025, in which a subset of pigs was found that naturally develops allergies to soy, making them an ideal model for soy allergies. Significant challenges arise from this method, which included that the pigs must be mated for many generations to select for potentially allergic pigs. For example, researchers mated allergic pigs for up to 8 generations to get pigs sensitized to soy. The allergic reaction was limited to soy allergens.

[0010] Thus, there is a need for an animal model to better understand the pathogenesis and to develop diagnostic and therapeutic tools for allergies, such as food or environmental allergens. As such, a large animal model that closely mimics a human allergic response and / or anaphylaxis and causes a consistent allergic response, and does not require multi -generational breeding is needed.SUMMARY OF THE INVENTION

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to an in vivo swine model system for an allergic condition, comprising: a pig sensitized to trigger an immune response for at least one of: allergy symptoms or an immune response, wherein the allergy symptoms are triggered by repeated tape stripping (TS) of the skin and application of an allergen and an immunomodulator, or wherein the immune response is triggered by intraperitoneal injections (IP) with the allergen and the immunomodulator once a week for at least 4 weeks, or both. In one aspect, the allergen is selected from the group consisting of food allergens, house dust mite, animal dander, feathers, plant antigens, molds, and household or industrial chemicals, plant, pollen, animal, arthropod, insect, fungus, or venom allergen, an aeroallergen, pollen allergen, grass allergen, tree allergen, animal allergen, dust mite allergen, cockroach allergen, ormold or fungi allergen, Hymenoptera allergen, or combinations thereof, or an extract derived from the allergic source material, or purified from the extract, synthesized chemically or biologically, or a food allergy allergen selected from a groundnut, peanut, milk, egg, tree nut, seed, fish, shellfish, crustacean, cereal, legume allergy, hazelnut, cashew, walnut, pecan, brazil nut, macadamia, chestnut, pistachio, coconut, almond, sesame, soy, kidney bean, black bean, common bean, chickpea, pea, cow pea, lentil or lupine allergy or a mustard seed allergy, or a combination thereof, wherein the allergen is whole or a fragment thereof of the allergen. In another aspect, the food allergen is a peanut allergen that comprises Ara hl, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hl 1, Ara hl 2, Ara hl 3, a peptide fragment thereof, or a combination thereof. In another aspect, the immunomodulator stimulates T helper type 2 (Th2) cells that promote B cell class switching to IgE. In another aspect, the immunomodulator is selected from at least one of: steroids, bacterial components, metal hydroxides, natural polymers, cytokines, nucleic acid components, and other small molecules that trigger B cell class switching to IgE. In another aspect, the immune response is anaphylaxis. In another aspect, the allergen is a respiratory allergen in humans. In another aspect, the pig is a miniature porcine strain that is a Hanford, Yucatan, Micro-Yucatan, or Gottingen. In another aspect, the TS and IP are applied at the same time, simultaneously, or alternating weekly.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of preparing an in vivo swine model system for an allergic condition, comprising: sensitizing a pig with a protocol that leads to at least one of: allergy symptoms or anaphylaxis, wherein the allergy symptoms at triggered by repeated tape stripping (TS) of the skin and application of a reservoir comprising the allergen and an immunomodulator, or wherein an immune response by intraperitoneal injections (IP) with an allergen and an antigen once a week. In one aspect, the administration is at least 4, 5, 6, 7, 8, or more weeks. In another aspect, the method further comprises, following sensitization, exposing the pig to an allergen; and measuring an immune reaction in the pig to the allergen. In another aspect, the sensitization triggers systemic allergen-specific IgG and IgE responses. In another aspect, the allergen is selected from the group consisting of food allergens, house dust mite, animal dander, feathers, plant antigens, molds, and household or industrial chemicals. In another aspect, the allergen is a plant, pollen, animal, arthropod, insect, fungus, or venom allergen, an aeroallergen, pollen allergen, grass allergen, tree allergen, animal allergen, dust mite allergen, cockroach allergen, or mold or fungi allergen, Hymenoptera allergen, or combinations thereof, or an extract derived from the allergic source material, or purified from the extract, or synthesized chemically or biologically, and is whole or a fragment thereof of the allergen. In another aspect, the allergen is a food allergy allergen toselected from a groundnut, peanut, milk, egg, tree nut, seed, fish, shellfish, crustacean, cereal, legume allergy, hazelnut, cashew, walnut, pecan, brazil nut, macadamia, chestnut, pistachio, coconut, almond, sesame, soy, kidney bean, black bean, common bean, chickpea, pea, cow pea, lentil or lupine allergy or a mustard seed allergy, or a combination thereof. In another aspect, the food allergen is a peanut allergen that comprises Ara hl, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hl 1, Ara hl 2, Ara hl 3, a peptide fragment thereof, or a combination thereof. In another aspect, the allergen is associated with asthma in humans. In another aspect, the in vivo testing is selected from at least one of: efficacy of one or more candidate drug(s) for a treatment of asthma; in vivo screening or testing of anti-inflammatory drugs, therapies, and / or procedures; or in vitro screening assays for the development of new antiinflammatory or anti-eosinophil degranulation drugs. In another aspect, the immunomodulator stimulates T helper type 2 (Th2) cells that promote B cell class switching to IgE. In another aspect, the immunomodulator is selected from at least one of: steroids, bacterial components, metal hydroxides, natural polymers, cytokines, nucleic acid components, and other small molecules that trigger B cell class switching to IgE. In another aspect, the method further comprises the step of repeated administration of antigen into the mammary gland of a mammal, thereby to induce a specific allergic response characterized by the recruitment of inflammatory cells into the mammary gland of the mammal. In another aspect, the method further comprises the step of repeated administration of antigen into the lung of a mammal, thereby to induce a specific allergic response characterized by the recruitment of inflammatory cells into the lung of the mammal. In another aspect, the method further comprises the step of collection of the inflammatory cells. In another aspect, the method further comprises at least one step selected from: measuring asthma; examining the effects of chronic allergen exposure. In another aspect, the method further comprises providing the pig with one or more candidate drugs for anti-allergic drug testing. In another aspect, the method further comprises at least one of: identifying processes or molecules differentially active or expressed in “activated” and “non-activated” eosinophils and / or other inflammatory cells; identifying processes and molecules involved in the recruitment of eosinophils and / or other inflammatory cells; identifying processes and molecules involved in degranulation of eosinophils and / or other inflammatory cells; in vivo screening and testing of anti-inflammatory drugs, therapies, and / or procedures; or in vitro screening assays for the development of new antiinflammatory or anti-eosinophil degranulation drugs. In another aspect, the pig is a miniature porcine strain that is a Hanford, Yucatan, Micro- Yucatan, or Gottingen. In another aspect, the pigs are not allergic to soy or soy antigens. In another aspect, the pigs are not the result of multi- generational breeding.

[0013] As embodied and broadly described herein, an aspect of the present disclosure relates to an in vivo swine model system for an allergic condition comprising: a pig that is sensitized to an allergen that leads to at least one of: allergy symptoms or anaphylaxis; wherein the pig is sensitized to trigger the allergy symptoms by repeated tape stripping (TS), wherein the pig is sensitized to trigger the anaphylaxis by intraperitoneal injections (IP) with an allergen and an antigen once a week for up to six weeks, or both.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0015] FIGS. 1A to 1C show the experimental timeline, allergen dose, and post-sensitization plasma analysis- (FIG. 1 A) Three to four weeks old mini pigs were sensitized through repeated tape stripping method (TS) or intraperitoneal injections (IP) of PE mixed with CT on a weekly basis up to six weeks. One week later, all pigs were challenged orally, and IP. The skin test was performed one week later to assess their hypersensitivity to peanuts. Pigs were rested for around a month before assessing their sensitivity a second time. Post second challenge, pigs were allowed to rest again for a month before proceeding to the third and final challenge. Each challenge point included one oral followed by one IP challenge with PE, and a skin hypersensitivity test, each separated one week apart. At the end of the study, pigs were euthanized, and tissue samples were collected for analysis. (FIG. IB, FIG. 1C) post sensitization plasma analysis. Anti -PE IgG at different plasma dilutions (FIG. IB) and IgE antibody response at 1 :20 pig plasma dilution (FIG. 1C). ELISA was used to measure anti -PE antibody responses in the form of optical density at 492 nm. Individual pig plasma was used in the analysis. The t-test was used to compare between sensitized and control groups. Bars denote Mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

[0016] FIGS. 2A to 21 are graphs that show the results from the first peanut challenge- Sensitized pigs were challenged through oral route with PE (2 g PE / pig). (FIG. 2A) clinical score, (FIG. 2B) drop in body temperature, (FIG. 2C) PE-specific IgG, and (FIG. 2D) PE-specific IgE analysis was conducted in pig plasma collected post oral challenge. Two weeks later, pigs were challenged through IP route with PE (200 mg / pig). (FIG. 2E) clinical score, (FIG. 2F) drop in body temperature, (FIG. 2G) PE-specific IgG, (FIG. 2H) PE-specific IgE analysis in blood collected post IP challenge. (FIG. 21) percent survival post IP challenge. In addition, the following were also determined: clinical condition in IP, and control pigs (not shown). ELISA was used tomeasure anti-PE antibody response in the form of optical density at 492 nm. Individual pig plasma was used in the analysis. The t-test was used to compare sensitized and control pigs. Bars denote Mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

[0017] FIG. 3. First assessment of skin hypersensitivity- Skin hypersensitivity reaction in pigs sensitized through the TS route, IP route, control unsensitized pigs was assessed. Ekhistamine, P:Peanut protein extract, R:rice protein and S:saline solution were injected intradermally in each quadrant drawn on the belly skin of mini pigs. (FIG. 3) Wheal and flare reaction diameter (WFD) was measured post 15 min of injection with help of a digital caliper. One-way ANOVA was used for statistical comparisons between the groups. Bars denote Mean ± SD. **** p < 0.0001.

[0018] FIGS. 4A to 4H are graphs that show the results from the second peanut challenge. Post first peanut challenge, all pigs were rested for a month before proceeding for the second challenge. Pigs were challenged first orally with (2 g PE / pig). (FIG. 4A) Change in body temperature, (FIG. 4B) clinical score, (FIG. 4C) anti-PE IgG, and (FIG. 4D) anti-PE IgE analysis in blood collected post oral challenge. Pigs were rested one week before proceeding to the second IP challenge. (FIG. 4E) Clinical score, (FIG. 4F) change in body temperature, (FIG. 4G) anti-PE IgG, and (FIG. 4H) anti-PE IgE analysis in blood collected post second IP challenge. Clinical conditions observed during second IP challenge in TS, and IP-sensitized pigs were measured (data not shown). ELISA was used to measure anti-PE antibody responses in the form of optical density at 492 nm. Individual pig plasma was used in analysis. The t-test was used to compare between TS and IP- sensitized pigs. Bars denote Mean ± SD. ns: not significant.

[0019] FIG. 5 shows the second assessment of skin hypersensitivity. Skin hypersensitivity reaction in pigs sensitized through the TS route, and IP routes. Ekhistamine, P:Peanut protein extract, R:rice protein and S: saline solution in each quadrant drawn on belly skin of mini pigs. (FIG. 5) WFD was measured 15 min post injection with the help of a digital caliper. The t-test was used to compare between TS and IP-sensitized pigs. Bars denote Mean ± SD. ns: not significant.

[0020] FIGS. 6A to 6G are graphs that show the third peanut challenge. The sensitized pigs were rested around one month before proceeding to the third and final challenge. First, pigs were challenged orally with (2 g PE / pig). (FIG. 6A) Change in body temperature, (FIG. 6B) anti-PE IgG, (FIG. 6C) anti-PE IgE analysis in blood collected post third oral challenge. One week later, pigs were challenged through IP route. (FIG. 6D) Clinical score, (FIG. 6E) change in body temperature, (FIG. 6F) anti-PE IgG, (FIG. 6G) anti-PE IgE analysis in blood collected post third IP challenge. ELISA was used to measure anti-PE antibody responses in the form of opticaldensity at 492 nm. Individual pig plasma was used in analysis. The t-test was used to compare between TS and IP-sensitized pigs. Bars denote Mean ± SD. ns: not significant.

[0021] FIG. 7 shows the results from the third assessment of skin hypersensitivity. Skin hypersensitivity reaction in TS, and IP-sensitized pigs. H:histamine, P:Peanut protein extract, R:rice protein and S: saline solution in each quadrant drawn on belly skin of mini pigs (data not shown). (FIG. 7) WFD was measured 15 min post injection with the help of a digital caliper. The t-test was used to compare between TS and IP-sensitized pigs. Bars denote Mean ± SD. ns: not significant.DETAILED DESCRIPTION OF THE INVENTION

[0022] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0023] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0024] The present invention is a novel pig model to study and understand allergic reactions to food in humans. The model of the present invention is helpful in the discovery of new therapeutic targets and the development of new treatments against food allergy and anaphylaxis. Some key advantages of using large animal models of the present invention include: their outbred nature, allowing studies that are more comparable to humans, the ability to conduct serial experiments within the same cohort of animals, and their relative longevity, allowing more relevant investigations into chronic disease as well as the long-term evaluation of specific therapies.

[0025] The present invention includes two different sensitization protocols, one is based on a tape stripping (TS) method, and another is based on intraperitoneal (IP) injection of an allergen. The unique feature of the approaches of the present invention is that both methods achieved 100% success in inducing allergic responses in the pigs, which is unlike previous reports where the reactions in pigs were inconsistent. An important modification was made in the time interval forthe application of the allergen, and the inventors have studied TS to induce allergies in a porcine model for the first time. Furthermore, in the IP injection group, the pigs showed different reactions than the pigs in the TS group. The neonatal piglets that were injected through the IProute exhibited lethargy, skin rashes, vomiting, and a drop in body temperature, while in the TS group, the pigs showed an anaphylactic reaction within minutes of challenge.

[0026] Pigs more closely display food allergy symptoms like a human. In this regard, pigs are noteworthy because their gastrointestinal system, skin, general physiology, and anatomy are closer to humans than that of mice. Using the novel pig model for food allergies, the present invention can be used to test other allergens, including food and respiratory allergens. Overall, inventors significantly improved the ability to use the pig as an animal model to study food allergies.

[0027] Very few studies are available with the pig as an animal model to study peanut allergies, and in fact, the inventors are aware of just two such studies - Helm et al. (2OO2)10and Rupa et al. (2008)11. Helm, et al., were perhaps the first to report a peanut allergy sensitization protocol in neonatal farm pigs via a hyperimmunization approach that involves IP injections of a crude peanut extract mixed with cholera toxin (CT) given at specific time intervals. The dose timeline included three injections given to neonatal pigs on day 7, 8, 9 after birth and then booster doses on days 17 and 25. Upon oral challenge with peanuts, 8 of 14 sensitized pigs (57%) were reported to present with grade 2 clinical symptoms including vomiting, malaise, lethargy, skin rashes, tremors, and convulsions; and 3 of 14 pigs were reported to require an epinephrine injection to manage the allergic reaction. Sensitized pigs were also reported to have developed a positive reaction towards a skin sensitivity test10. Rupa, et al., sensitized the pigs just three times on days 14, 21 and 35 after birth. They reported low to mild clinical symptoms after oral challenge, with only 1 out of 5 animals requiring an epinephrine injection11.

[0028] The term “allergen” refers to an immunogenic molecule (or a combination thereof) involved in an allergic reaction contained in food or air or venom or other sources. The allergen exposure might occur via ingestion of food, respiratory route, skin contact, eye contact, or contact at other parts of the body. The allergen in one embodiment is a lipid, carbohydrate, protein, peptide, polypeptide, or a combination thereof. In one embodiment, the allergen is a native food preparation, a food extract, or a purified protein, polypeptide, and / or peptide composition. The allergen may be in a natural state, or produced artificially (e.g., by recombinant and / or enzymatic techniques, and or de novo synthesis for instance). The allergen in one embodiment is structurally altered or modified to improve its stability or immunogenicity. The allergen in another embodiment is in admixture with one or more other constituents, such as an adjuvant or a stabilizerto stabilize the formulation or allergen or both. The allergen may be a mixture of several molecules (e.g., an extract such as a peanut protein extract). The allergen may be present in combination with other allergens, or in combination with other molecules from the food that are not immunogenic.

[0029] The invention may be used with any food or food allergens such as, without limitation, groundnut, peanut, milk, egg, tree nuts and seeds (such as but not limited to: hazelnut, cashew, walnut, pecan, brazil nut, macadamia, chestnut, pistachio, coconut, almond, sesame, mustard), fish, shellfish, crustaceans, cereals (e.g., wheat, corn, oat, barley, rye, rice, sorghum, spelt), legumes (e.g., soy, kidney bean, black bean, common bean, chickpea, pea, cow pea, lentils, lupine), or mixtures thereof.

[0030] In one embodiment, the allergen is a peanut allergen or a combination of peanut allergens. The peanut allergen in one embodiment is in the form of a peanut protein extract. Thirteen peanut allergens (Ara hl through Ara hl3) have been recognized by the Allergen Nomenclature Subcommittee of the International Union of Immunological Societies. In one embodiment, the peanut allergen comprises one or more of Ara hl, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hl 1, Ara hl2 or Ara hl 3, or a peptide fragment of one of the foregoing, or a combination thereof. In a further embodiment, the peanut allergen comprises Ara hl, Ara h2, Ara h3, a peptide fragment thereof, or a combination thereof. In yet another embodiment, the peanut allergen comprises Ara hl, a peptide fragment thereof, or multiple peptide fragments thereof.

[0031] Peanut Flour (PF) for use as an allergen composition can be obtained commercially, for example, from the Golden Peanut Company (Alpharetta, Ga.). The PF can be defatted, and can comprise in one embodiment, Florunner, Virginia, or Spanish PF, or a combination thereof. In one embodiment, the PF comprises equal parts Florunner, Virginia and Spanish PF. In another embodiment, roasted peanuts are used as a source of allergen for the allergen composition. Peanut extract for use as an allergen composition in another embodiment, can be obtained commercially, for example, from Greer Labs (Lenoir, N.C.).

[0032] As used herein, the terms “immunomodulator” or “immunomodulatory” refer to those agents, molecules, or compounds that stimulate T helper type 2 (Th2) cells that promote B cell class switching to IgE. Non-limiting examples of immunomodulators include: steroids (i.e., Glucocorticoids); bacterial components (i.e., Cholera toxin, Lipopolysaccharides, Staphylococcal enterotoxin B, Complete and incomplete Freund’s adjuvant); metals hydroxides (i.e., Alum); natural polymers (i.e., Carrageenan); cytokines e.g.: interleukin-4 (IL-4), IL-5, IL-9, IL-13 and IL-25 (also known as IL-17E), Thymic stromal lymphopoietin (TSLP), etc., nucleic acidcomponents (i.e., cyclic-di-GMP), and / or other chemicals (i.e., Acrolein) or activators of the GATA3, STAT6 pathways, such as those taught by Paul and Zhu, “How are Tn2-type immune responses initiated and amplified?”, Nat Rev Immunol. 2010 Apr; 10(4): 225-235, doi: 10.1038 / nri2735, relevant portions incorporated herein by reference.

[0033] Examples of toxins for use as an antigen with the present invention include one or more toxins selected from, e.g., tetanus toxin or toxoid, tetanus toxin Fragment C, diphtheria toxin or toxoid, cholera toxin or toxoid, Staphylococcus aureus toxin, exotoxins or toxoids, Escherichia coli heat-labile enterotoxin, Pseudomonas aeruginosa exotoxin A; bacterial outer membrane proteins, serotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB), porins; keyhole limpet hemocyanin (KLH), hepatitis B virus core protein, thyroglobulin, albumins, and ovalbumin; pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding proteins, peptidyl agonists of TLR-5.

[0034] Materials and Methods. Animals and housing. Twelve, four-day-old mini pigs (Yucatan Miniature Swine) were acquired from Sinclair Research BioResources (MO, USA). Pigs were acclimated for twelve days in the animal care facility at Texas Tech University before starting the study. Animals were housed in climate-controlled environment conditions in open stainless-steel pens with plastic-coated expanded metal flooring. Pigs were provided ad libitum water through a lixit. Mini pigs were fed on Birthright™ standard baby pig milk replacer (Product No. -8229, Ralco Agriculture Marshall, MN) until weaned from milk at approximately 21 days of age. Creep feeding started from 7 days old until 7 days post-weaning with a mini-pig starter diet (Lab Diets 5080, Lab Diet, St. Louis, MO) and later switched to a mini-pig standard grower diet (Lab Diet 5L80, Lab Diet, St. Louis, MO). All diets were peanut-free and meal-fed to meet nutritional requirements. The pigs and housing environment were enriched with rotating toys, peanut-free treats, and radio music. All animal experiments were performed under approved protocols and ethical guidelines of the Institutional Animal Care and Use Committee at Texas Tech University, USA.

[0035] Sensitization protocols. The sensitization method is summarized in FIG. 1A. Pigs were sensitized to peanuts through TS or IP injections. For IP injections, peanut extract (PE) Img (Greer Laboratories, Inc.) mixed with the immunomodulator, in this example, cholera toxin (CT) 100 pg (Sigma Aldrich, MO) was injected every week for up to six weeks total. For TS, the pigs were anesthetized using an isoflurane air mixture and then a small portion of hair from the back region of pigs were removed with the help of a shaving razor. Skin was then vigorously cleaned with 70% ethanol to remove dirt and allow proper adhesion of tape. Subsequently, a tape (Comply™Steam indicator Tape, 3M) was stuck to the hairless skin and removed. This was repeated up to 12-15 times on the same skin area (measuring about 1 x 1 cm2) until a characteristic shine was observed on the skin. Tape-stripping (TS) is done to sequentially remove layers of the skin. After TS, a mixture of PE (1 mg) and CT (100 pg) was applied to the cleaned area. This was done by first sticking a square-like hollow foam-tape reservoir (Acrysure Next Gen PE Foam, Mactac) disc to the TS skin, and then a mixture containing PE and CT was dispensed into this reservoir disc. The disc was covered with another tape to confine the liquid in the reservoir. Another larger tape (Waterproof Transparent Dressing, CVS or Tagaderm, 3M) was applied on top to better secure the reservoir. After this procedure, pigs were removed from anesthesia and monitored until awake. The adhesive system was allowed to stick on skin around 24h, and then it was removed. This procedure was performed every week for up to six weeks in total.

[0036] Allergen challenge and assessment of clinical reactions. Sensitized pigs were challenged orally with 20 g PE mixed in frozen banana paste. One week after oral challenge, pigs were challenged through the IP route by injecting 0.2 g PE dissolved in 3 ml sterile saline solution. After each challenge, body temperature and clinical scores were recorded. Body temperature was recorded before and after the challenge every 15 min for 2 h. To record temperature, a temperature sensor (LifeChip with Bio-Thermo Technology) was implanted on the left side of neck muscles, and read with help of Universal Worldscan Reader (HomeAgain, Merck & Co., Inc.). Pigs were monitored for any signs of allergic reaction. Clinical signs of allergic reaction were scored as discussed10,12. In brief, 0 = no signs; 1 = immobility, lethargy, malaise; 2 = scratching, rash, coughing, gagging, stomach contractions; 3 = diarrhea, emesis; 4 = increase in respiratory rate, neck extension; 5 = forced expiration; 6 = confluent cutaneous reddening, cyanosis, anaphylaxis. Epinephrine (Img / lOOlb) was given intramuscularly to control severity of allergic reactions. Blood samples were collected before and after the challenge of analyzing the molecular markers.

[0037] Skin hypersensitivity test (SHT). SHT was performed one week after each challenge. To facilitate the injection of allergens and in making readouts, animals were anesthetized, and then the flank region was shaved with the help of an electric hair trimmer and further cleaned with a razor blade. Four squares were drawn with a pen on the shaved flank. A tuberculin syringe was used for intradermal injections of PE (100 pg / lOOpl), histamine as a positive control (100 pg / lOOpl), rice protein (100 pg / lOOpl) as an irrelevant allergen considered as a negative control, and base saline solution (100 pl) also as a negative control in each square. Fifteen minutes later whealflare inflammatory diameter (WFD) was measured with calipers and digital pictures were taken to observe the change in skin morphology.

[0038] Blood collection and plasma analysis. At each time point, blood was collected via jugular vein puncture (FIG. 1 A). Whole blood was collected in EDTA containing blood collection tubes and kept on ice until it was centrifuged at 10,000 x g for 15 minutes for plasma separation. Plasma was separated and stored at -80°C for analysis. The ELISA technique was used to measure PE- specific antibodies in plasma. Briefly, ELISA plates were coated overnight with PE at 50 pg / ml in 0.1 M carbonate buffer (50 pl / well). Plates were then washed with ELISA buffer (IxPBS with Tween 0.05%, v / v), incubated with 100 pl / well blocking solution (2%, milk solution) for 2h, and washed again with ELISA buffer. Plasma diluted in phosphate-buffered saline with Tween 20 (PBST) was added to each well (50 pl / well). After 1.5 h, plates were washed with ELISA buffer, and secondary antibody, a goat anti-pig IgG HRP conjugated (BIO-RAD-AHP865P) (50 pl / well) was added into the plate for 1.5 h to detect IgG. For IgE detection, the secondary antibody used was mouse anti-pig IgE (Cloud Clone Corp.-MAA545Po21), which was added to the plate for 1.5 h and the plates were then washed. Further another secondary HRP-conjugated goat anti-mouse IgG (50 pl / well) was added into the plate and incubated for 1.5 h. Plates were washed again with PBST and 50 pl / well peroxidase substrate solution (OPD powder in phosphate citrate buffer pH 5.O+H2O2) was added to each well. The reaction was stopped after 12 min by adding 50 pl / well 3 M H3PO4 and the plates were read at 492 nm.

[0039] Statistical analyses. Statistical analysis was conducted using Graphpad Prism 6 software. A one-way ANOVA test was used for statistical calculations between the treatment groups. Significance was considered for p < 0.05 for a 95% confidence interval.

[0040] Anti PE-specific antibody response. Both TS or IP routes of sensitization were able to induce systemic PE-specific IgG and IgE antibodies in the mini pigs. The PE-specific IgG response was higher in both TS and IP groups in comparison to the control group (FIG. IB), however, the IP group had a higher IgG response than the TS group. The anti-PE IgE response was also higher in both TS and IP groups than the control group (FIG. 1C), however, there was no statistical difference between IP and TS groups. Overall, both modes of sensitization were able to induce systemic PE-specific antibody responses.

[0041] Allergic response to first oral and IP challenge.

[0042] Oral challenge: None of the sensitized pigs responded significantly to an oral PE challenge and did not exhibit any major clinical reaction or significant drop in body temperature (FIGS. 2 A, 2B). Nonetheless, sensitized pigs had significantly higher systemic PE-specific IgG and IgE antibody responses than the control group (FIGS. 2C, 2D).

[0043] IP challenge: Interestingly, upon IP challenge with PE, both TS and IP sensitized pigs responded and displayed clinical symptoms including anaphylaxis (FIG. 2E) and a significant drop in body temperature (FIG. 2F). Moreover, both groups retained an elevated level of PE-specific IgG and IgE antibodies in comparison to the control pigs (FIGS. 2G, 2H).

[0044] The clinical reactions observed during the first IP challenge are discussed in detail in the following paragraphs.

[0045] TS group with IP challenge: The pigs developed severe allergic symptoms including anaphylaxis during the first IP challenge. Five minutes post-injection, all pigs started to vomit and presented symptoms of uneasiness and lethargy. The pigs vomited several times during the whole observation time (120 min post IP challenge). Around 30 min post-injection, out of four, two pigs suffered from cutaneous anaphylaxis as their skin started to turn purple. Three out of four pigs showed forced respiration with a significant drop in body temperature (FIG. 2F). Due to reaching a humane endpoint, the pigs with forced respiration were treated with injections of epinephrine and dexamethasone. Despite the intervention, one of the pigs died due to a significant drop in body temperature (FIG. 21). About 120 min after IP challenge, the skin color of the pigs that showed cutaneous anaphylaxis turned back to normal, however, they continued to suffer from drop in body temperature. After about 6 h of the IP challenge, the sick pigs gained their baseline body temperature (~103°F) though they were still lethargic with mild skin rashes. The next day, pigs were active and looked healthy without any allergic symptoms.

[0046] IP group with IP challenge: Similarly, IP-sensitized pigs showed strong food allergy symptoms during the first IP challenge. All pigs started to vomit in the first 5 min after IP challenge, and continuously vomited several times in the next 15-30 min. They were lethargic and exhibited malaise during this time. Within 15 min of IP challenge their body temperature dropped in comparison to the control group (FIG. 2F). At 60 min post-challenge, intense skin rashes started appearing in all pigs. In one of the pigs, the rashes faded down at 120 min post-challenge and completely diminished in the next hour. However, another pig still had prominent skin rashes around the neck and head region. At 6 h post IP challenge, all pigs mostly returned to normalcy, however, they continued to be lethargic. In one of the pigs, the body temperature went down considerably and did not rise back up. That pig died at 8 h post-challenge despite medical intervention (FIG. 21). The following day, the remaining pigs were normal and healthy without any allergic symptoms.

[0047] Control (ctl) group- All control pigs were normal and healthy during both the oral and the IP challenges and presented none of the allergic or anaphylactic symptoms (FIG. 2F).

[0048] Response to SHT after first oral and IP challenges. To assess skin hypersensitivity, all pigs were tested against intradermally injected PE. Histamine was used as a positive control, and rice protein and saline as the negative controls. In the TS group, the wheal and flare reaction diameter (WFD) from PE intradermal injection was 16±0.6 mm while from the histamine positive control was 21±4 mm. The negative control injections of rice protein produced a WFD of 2±3 mm and it was negligible for saline injections (FIG. 3). The color of WFD was dark red, and the reaction spot persisted until 72 h post-injection. This positive and strong reaction indicates the presence of peanut hypersensitivity in TS-sensitized pigs. Likewise, IP-sensitized pigs showed a positive reaction with a WFD of 21±5 mm from a PE intradermal injection while from histamine it was 17±1 mm. The rice protein had a WFD of 3±5 mm, and a negligible WFD from saline injection (FIG. 3). The reaction spots from PE injection were dark red and persisted until 72 h post-injection. As expected, the control pigs did not respond to PE except for a mild reaction that developed in one pig, which resolved quickly (FIG. 3). The histamine’s WFD was 18±4 mm in control pigs (FIG. 3), however, other injections (rice antigen, saline) did not induce any response in the control pigs (FIG. 3).

[0049] Sensitized pigs retained peanut hypersensitivity during the second challenge. To check the longevity of sensitization, pigs were rested for a month and further challenged a second time through oral and IP routes.

[0050] Oral challenge: Like the first oral challenge, pigs did not respond to the second oral challenge (FIG. 4A). No major change was observed in body temperature (FIG. 4B). However, both TS and IP groups of sensitized pigs still retained a high level of PE-specific IgG and IgE antibodies in serum (FIGS. 4C, 4D).

[0051] TS group with IP challenge: Out of three, one pig showed cutaneous anaphylaxis 15 min post allergen injection and its skin color turned slightly purple (FIG. 4E). The skin redness remained up to 60 min and completely diminished at 120 min post-injection. In another pig, the skin rashes appeared at 30 min and resolved fully at the 120 min mark. However, in the third pig, the skin rashes appeared later at around 60 min post challenge and resolved fully at the 120 min mark (FIG. 4E). All pigs vomited several times and were lethargic. There was a significant drop in body temperature recorded in one of the pigs, which returned to baseline at the 120 min mark (FIG. 4F). For the other two pigs, the body temperature remained close to the baseline. None of the pigs reached the humane endpoint. All pigs were active and normal without any allergic symptoms the day after the challenge. Pigs were rested one week before proceeding to the secondIP challenge. (FIG. 4E) Clinical score, (FIG. 4F) change in body temperature, (FIG. 4G) anti-PE IgG, and (FIG. 4H) anti-PE IgE analysis in blood collected post second IP challenge.

[0052] IP group with IP challenge: Pigs again exhibited peanut hypersensitivity during the second IP challenge, however, their clinical symptoms were less severe. Two out of the three pigs started vomiting at 15 min post challenge and vomited several times over the next 30 min (FIG. 4E). All pigs were lethargic and less active at the 120 min mark. The skin rashes started to appear in two of three pigs at 30-45 min post-challenge, however, the rashes faded at the 60 min mark. All pigs returned to normalcy within 120 min of the challenge. A few degrees drop in body temperature was observed; however, the change was not statistically different from the TS group (FIG. 4F).

[0053] SHT: Both groups of sensitized pigs retained peanut hypersensitivity during the second SHT. In TS pigs, the WFD was 16.5±2 mm for PE, 21.7±0.4 mm for histamine, 2.5 ± 4.4 mm for rice protein, and negligible reaction for saline injection (FIG. 5). Likewise, IP pigs had a WFD of 17.3 ±3 mm for PE, 23.7 ±3 mm for histamine, 2.8±4.9 mm for rice protein, and there was no reaction to saline injection (FIG. 5).

[0054] Sensitized pigs retained peanut hypersensitivity during the third challenge. To further assess the longevity of peanut hypersensitivity, pigs were rested for one more month before proceeding to the third and final challenge.

[0055] Oral challenge: As in previous oral challenges, pigs responded poorly to the third oral challenge without any major change in body temperature (FIG. 6 A). While they still retained PE- specific IgG and IgE responses in the blood (FIG. 6B, 6C), the IgE levels were slightly lower as compared to the 2 months prior samples, indicating that the IgE levels might be reducing in the resting phase.

[0056] TS group with IP challenge: One pig out of three showed cutaneous anaphylaxis at 15 min post allergen injection (FIG. 6D). However, another pig showed moderate skin rashes in the abdominal area in the first 15 min. The rashes remained up to 30 min and then started fading off at the 45 min mark. In the third pig, the skin rashes appeared slightly later around 60 min, and persisted till 120 min. There was no breathing trouble or systemic anaphylaxis observed during the challenge in any of the pigs.

[0057] IP group with IP challenge: All pigs were active without any symptoms till 30 min. However, two out of three pigs started vomiting at 60 min, and the pigs became lethargic and less active. The skin rashes started appearing on the skin of all pigs around 60 min post challenge and resolved at the 120 min mark. None of the pigs experienced any symptoms of severe anaphylaxis.Moreover, no significant change in body temperature, anti-PE IgG and IgE responses observed between TS and IP groups during third IP challenge (FIGS. 6E, F, G).

[0058] SHT: Both groups of sensitized pigs retained peanut hyperreactivity as their skin showed a positive inflammatory reaction to PE injected intradermally. In TS pigs, the WFD was 14.6 ±2.2 mm for PE, 16.6±1.7 mm for histamine, and negligible for rice protein and saline injections (FIG. 7). Likewise in IP pigs, the WFD was 14.8±0.5 mm for PE, 17.4 ±1.8 for histamine, and negligible for rice protein and saline injections (FIG. 7).

[0059] The present invention includes developing an animal disease model able to mimic the human clinical symptoms and disease etiology to facilitate investigations into the mechanism of the disease process, identify and develop new drug targets, develop an effective treatment, and develop diagnostic biomarkers.

[0060] The pig intestinal physiology is anatomically and histologically similar to humans14. Their microflora is also more diverse than rodent models, and they are outbred like humans. These characteristics make pigs an important model because they better recapitulate the human genetic variability as opposed to inbred mice. The neonatal pig resembles humans in terms of acute symptoms including diarrhea, bleeding, weight loss, cutaneous erythema, and respiratory distress including other immunological functions14,15. Thus, the pig has been considered as an animal model for peanut allergy10,11and egg allergy16.

[0061] Importantly, when the inventors followed the protocol of Helm, et al., which is the first such published protocol10, the inventors observed that after oral challenge with peanut proteins the severity of allergic symptoms in the pigs was mild and the number of responders was low. Because allergen challenge via the IP route delivers the peanut proteins directly into the systemic compartment, it can generate a high severe allergic reaction21, therefore, the inventors also attempted an IP challenge on the sensitized pigs. The IP challenge did increase the severity of clinical symptoms a little, however, none of the pigs exhibited severe reactions. The inventors tried this protocol a total of six independent times over a course of about two years, but the results were unsatisfactory. A summary of these results is in Table 1. It is noteworthy that in some cohorts the inventors saw almost no allergic reaction even after IP challenges. A similar observation has been made by Rupa, et al., who noticed in their egg allergy model in a pig that out of three litters they tried, one simply failed to produce sensitized pigs16.

[0062] Therefore, the inventors set out to improve the protocol for generating a pig model of peanut allergy with two goals in mind: (i) induce sensitization with a high response rate, and (ii) induce more consistent and high severity of allergic response upon allergen challenge. Theinventors chose the miniature porcine strain Yucatan over the domestic farm strains such as Landrace due to its ease of handling and slow growth rate. Moreover, the Yucatan mini pigs can maintain a smaller size at maturity22, therefore, live procedures such as blood collections and injections can be more easily conducted. This is especially important for allergy experiments because the studies can extend several months even up to a year, first to sensitize the pigs, and then to investigate the effect of different treatments.

[0063] Table 1. Comparison of methods and outcomes comparing prior methods with the present invention.

[0064] Sensitization routes *IP dose [0.5 mg peanut protein extract (PE) + lOOpg CT)], **IP (0.5 mg PE+10 mg Alum),$Oral (1 g Peanut Flour + 100 pg CT),$$IP+Oral [IP (0.5 mg PE + 100 pg CT) + Oral (1g Peanut Flour + 100 pg CT)],

[0065] ^Challenge Dose- 20 g peanut flour, ^Challenge Dose- 25 g peanut flour, §IP-challenge dose 0.2 gm PE was given to the sensitized pigs.

[0066] In allergy research, the development of an animal model depends on several factors including the type of allergen, exposure route, adjuvant type, dose and frequency of allergen, and genetic background of the animal23. Through numerous studies in Landrace pigs (Table-1), the inventors observed that the oral route of sensitization is not superior to the IP route for creating sensitization. Therefore, in the current study, since the IP route offers more control over the allergen dose and is also easier to perform as compared to the oral gavage, the inventors decided to use it for allergen administration. The IP route is also well-established for inducing food allergen sensitization in mice24. Two IP injections at weekly intervals are sufficient to induce allergic sensitization24, however, in pigs, Helm, et al. used five injections (three on consecutive days followed by two more weekly injections)10, while Rupa, et al. gave three injections separated by 7 and 14 days11. Activation of immune response takes longer than a day, therefore, the inventors decided to space the IP injections by a week and decided to increase the number of IP injections with the goal of increasing the peanut-specific IgE titers and for enhancing the severity of allergic hypersensitivity. In one aspect, the present invention does not use five injections (three on consecutive days followed by two more weekly injections). In another aspect, the present invention does not use three injections separated by 7 and 14 days.

[0067] Another route the inventors selected for creating peanut sensitization is the skin. The skin is a unique immunological organ of the body that acts as a protective barrier to prevent the entry of microbes, prevents water loss, and excludes the entry of different allergens from the environment25,26. A mechanical disruption in the skin barrier may allow the entry of these microbes or allergens. Recently, mechanically damaged skin has been utilized as an alternate route to achieve food sensitization and anaphylaxis reaction in animal models25,26. Indeed, even human studies, which have attempted to use tape-striped (TS) skin for allergen immunotherapy observed that too much stripping can cause disease exacerbation rather than producing a therapeutic effect27. Therefore, the inventors used the tape-stripping process to mechanically disrupt the skin barrier to induce peanut sensitization in mini pigs.

[0068] To develop peanut hypersensitivity, the inventors selected cholera toxin (CT) as an adjuvant for sensitization. CT is known to stimulate type 2 inflammatory response and generates allergy-inducing immune response when mixed with the allergen27. The mixture of CT and an allergen induces robust production of systemic allergen-specific IgE antibodies via the secretion of cyclic adenosine monophosphate (cAMP) from antigen-presenting dendritic cells29,30. Indeed, the inventors observed an elevated level of systemic peanut-specific IgE antibody response.

[0069] For the allergen challenge, both groups of sensitized pigs displayed severe allergic and anaphylactic symptoms during the IP challenge. Although the IP route is not ideal to mimic the human allergen challenge, it provides better control of allergen dosage and produces consistency in developing food allergy symptoms. In the present study, it was observed severe peanut hypersensitivity in both IP- and TS-sensitized groups with a hundred percent rate of incidence. The common clinical reactions observed in sensitized pigs were vomiting, malaise, lethargy, skin rashes, aggressive scratching, and a significant drop in body temperature. Previous porcine studies have reported mild and inconsistent allergic reactions in sensitized pigs10,11.

[0070] The TS pigs showed severe food-allergic clinical symptoms including anaphylaxis during the first challenge and moderate clinical reactions in the following challenges. These results in TS pigs are new and have not been reported before. The TS-sensitized pigs of the present invention displayed severe symptoms of lethargy, malaise, cutaneous anaphylaxis, forced respiration, and systemic anaphylaxis. One pig died due to anaphylaxis even after intervening with two epinephrine injections.

[0071] Peanut hypersensitivity in sensitized pigs was further verified through skin hypersensitivity testing (SHT), which mimics the skin prick test in humans. SHT has been a simple tool in preclinical studies to diagnose IgE-mediated food allergies31. In SHT, the inflammatory reaction due to intradermal injection of an allergen in SHT indicates the presence of type 1 hypersensitive reaction. The injected allergen in the skin cross-links the allergen-specific IgE antibodies which bind to the IgE receptors located on the mast cells. The cross-linking of IgE antibodies in presence of allergens triggers the degranulation of the mast cells to release the histamine which is responsible for the wheal and flare reaction32. The results of skin tests are positive and in line with previous studies in atopic dogs33,34. There was a direct correlation between the positive SHT and the level of allergen-specific IgE in serum until the last IP challenge. Allergic reactions were observed up to 2 months after stopping the allergen sensitization protocol, indicating the persistence of allergen sensitivity.

[0072] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0073] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0074] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0075] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0076] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0077] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. Theskilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0078] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0079] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0080] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

[0081] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0082] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCES

[0083] 1 Warren, C., Lei, D., Sicherer, S., Schleimer, R. & Gupta, R. Prevalence and characteristics of peanut allergy in US adults. J Allergy Clin Immunol 147, 2263-2270 e2265 (2021). doi.org: 10.1016 / j .jaci.2020.11.046

[0084] 2 Lyons, S. A. et al. Prevalence of Food Sensitization and Food Allergy in Children Across Europe. J Allergy Clin Immunol Pract 8, 2736-2746 e2739 (2020). doi.org: 10.1016 / j .jaip.2020.04.020

[0085] 3 Sicherer, S. H., Munoz-Furlong, A., Burks, A. W. & Sampson, H. A. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J Allergy Clin Immunol 103, 559-562 (1999). doi.org: 10.1016 / s0091-6749(99)70224-l

[0086] 4 Dunlop, J. H. Oral immunotherapy for treatment of peanut allergy. J Investig Med 68, 1152-1155 (2020). doi.org: 10.1136 / jim-2020-001422

[0087] 5 Erlich, D. Peanut Allergen Powder (Palforzia) for Peanut Allergy. Am Fam Physician 105, 20-21 (2022).

[0088] 6 Meier, C. (ed Christoph Meier) 1-2(www.nestle.com / media / mediaeventscalendar / allevents / 2022-nestle-investor-seminar, 2022).

[0089] 7 Kanagaratham, C., Sallis, B. F. & Fiebiger, E. Experimental Models for Studying Food Allergy. Cell Mol Gastroenterol Hepatol 6, 356-369 e351 (2018). doi.org: 10.1016 / j .jcmgh.2018.05.010

[0090] 8 Liu, T., Navarro, S. & Lopata, A. L. Current advances of murine models for food allergy. Mol Immunol 70, 104-117 (2016). doi.org: 10.1016 / j. molimm.2015.11.011

[0091] 9 Shakya, A. K. et al. Microneedles coated with peanut allergen enable desensitization of peanut sensitized mice. J Control Release 314, 38-47 (2019). doi.org: 10.1016 / j.jconrel.2019.09.022

[0092] 10 Helm, R. M. et al. A neonatal swine model for peanut allergy. J Allergy Clin Immunol 109, 136-142 (2002). doi.org: 10.1067 / mai.2002.120551

[0093] 11 Rupa, P., Hamilton, K., Cirinna, M. & Wilkie, B. N. Porcine IgE in the context of experimental food allergy: purification and isotype-specific antibodies. Vet Immunol Immunopathol 125, 303-314 (2008). doi.org: 10.1016 / j.vetimm.2008.05.028

[0094] 12 Radcliffe, J. S. et al. A swine model of soy protein-induced food allergenicity: implications in human and swine nutrition. Anim Front 9, 52-59 (2019). doi.org: 10.1093 / af / vfz025

[0095] 13 Schulke, S. & Albrecht, M. Mouse Models for Food Allergies: Where Do We Stand? Cells 8 (2019). doi.org: 10.3390 / cells8060546

[0096] 14 Dearman, R. J. & Kimber, I. Animal models of protein allergenicity: potential benefits, pitfalls and challenges. Clin Exp Allergy 39, 458-468 (2009). doi.org: 10.1111 / j .1365- 2222.2008.03194.x

[0097] 15 Rupa, P., Schmied, J. & Wilkie, B. N. Porcine allergy and IgE. Veterinary immunology and immunopathology 132, 41-45 (2009). doi.org: 10.1016 / j.vetimm.2009.09.013

[0098] 16 Rupa, P., Hamilton, K., Cirinna, M. & Wilkie, B. N. A neonatal swine model of allergy induced by the major food allergen chicken ovomucoid (Gal d 1). Int Arch Allergy Immunol 146, 11-18 (2008). doi.org: 10.1159 / 000112498

[0099] 17 Landers, J. J. et al. Targeted allergen-specific immunotherapy within the skin improves allergen delivery to induce desensitization to peanut. Immunotherapy 14, 539-552 (2022). doi . org: 10.2217 / imt-2021 -0206

[0100] 18 Monteiro-Riviere, N. A., Bristol, D. G., Manning, T. O., Rogers, R. A. & Riviere, J. E. Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species. The Journal of investigative dermatology 95, 582-586 (1990).

[0101] 19 Barbero, A. M. & Frasch, H. F. Pig and guinea pig skin as surrogates for human in vitro penetration studies: A quantitative review. Toxicology in Vitro 23, 1-13 (2009). doi.org:doi.org / 10.1016 / j.tiv.2008.10.008

[0102] 20 Summerfield, A., Meurens, F. & Ricklin, M. E. The immunology of the porcine skin and its value as a model for human skin. Molecular Immunology 66, 14-21 (2015). doi.org:doi.org / 10.1016 / j.molimm.2014.10.023

[0103] 21 Gertie, J. A. et al. Oral anaphylaxis to peanut in a mouse model is associated with gut permeability but not with Tlr4 or Dock8 mutations. J Allergy Clin Immunol 149, 262-274(2022). doi.org:10.1016 / j.jaci.2021.05.015

[0104] 22 Stricker-Krongrad, A., Shoemake, C. R. & Bouchard, G. F. The Miniature Swine as a Model in Experimental and Translational Medicine. Toxicol Pathol 44, 612-623 (2016). doi.org: 10.1177 / 0192623316641784

[0105] 23 Bonnegarde-Bernard, A. et al. Routes of allergic sensitization and myeloid cell IKKbeta differentially regulate antibody responses and allergic airway inflammation in male and female mice. PLoS One 9, e92307 (2014). doi.org: 10.1371 / joumal. pone.0092307

[0106] 24 Dearman, R. J. & Kimber, I. A mouse model for food allergy using intraperitoneal sensitization. Methods 41, 91-98 (2007). doi.org: 10.1016 / j.ymeth.2006.07.008

[0107] 25 Ballegaard, A. R., Madsen, C. B. & Bogh, K. L. An Animal Model for Wheat Allergy Skin Sensitisation: A Comparative Study in Naive versus Tolerant Brown Norway Rats. Int Arch Allergy Immunol 178, 106-118 (2019). doi.org: 10.1159 / 000493802

[0108] 26 Bartnikas, L. M. et al. Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food-induced anaphylaxis. J Allergy Clin Immunol 131, 451- 460 e451-456 (2013). doi.org: 10.1016 / j .jaci.2012.11.032

[0109] 27 Nesovic, L. D., Shakya, A. K. & Gill, H. S. Treating allergies via skin - Recent advances in cutaneous allergen immunotherapy. Adv Drug Deliv Rev 190, 114458 (2022). doi.org: 10.1016 / j. addr.2022.114458

[0110] 28 Marinaro, M. et al. Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. J Immunol 155, 4621-4629 (1995).

[0111] 29 Mattsson, J. et al. Cholera toxin adjuvant promotes a balanced Thl / Th2 / Thl7 response independently of IL-12 and IL-17 by acting on Gsalpha in CDl lb(+) DCs. Mucosal Immunol 8, 815-827 (2015). doi.org: 10.1038 / mi.2014.111

[0112] 30 Snider, D. P., Marshall, J. S., Perdue, M. H. & Liang, H. Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin. J Immunol 153, 647-657 (1994).

[0113] 31 Justo, X., Diaz, I., Gil, J. J. & Gastaminza, G. Prick test: evolution towards automated reading. Allergy 71, 1095-1102 (2016). doi.org: 10.1111 / all.12921

[0114] 32 Olivry, T., Dunston, S. M., Murphy, K. M. & Moore, P. F. Characterization of the inflammatory infiltrate during IgE-mediated late phase reactions in the skin of normal and atopic dogs. Vet Dermatol 12, 49-58 (2001). doi.org: 10.1046 / j.1365-3164.2001.00230.x

[0115] 33 Buchanan, B. B. et al. Thioredoxin-linked mitigation of allergic responses to wheat.Proc Natl Acad Sci U S A 94, 5372-5377 (1997). doi.org: 10.1073 / pnas.94.10.5372

[0116] 34 Carmona-Gil, A. M., Sanchez, J. & Maldonado-Estrada, J. Evaluation of Skin Prick-Test Reactions for Allergic Sensitization in Dogs With Clinical Symptoms Compatible With Atopic Dermatitis. A Pilot Study. Front Vet Sci 6, 448 (2019). doi.org: 10.3389 / fvets.2019.00448

Claims

What is claimed is:

1. An in vivo swine model system for an allergic condition, comprising: a pig sensitized to trigger an immune response for at least one of: allergy symptoms or an immune response, wherein the sensitization is induced by repeated tape stripping (TS) of the skin and application of an allergen with or without an immunomodulator, or wherein the sensitization is induced by intraperitoneal injections (IP) with the allergen with or without an immunomodulator once a week for at least 3 weeks, or both TS and IP.

2. The model of claim 1, wherein the allergen is selected from the group consisting of food allergens, house dust mite, animal dander, feathers, plant antigens, molds, and household or industrial chemicals, plant, pollen, animal, arthropod, insect, fungus, or venom allergen, an aeroallergen, pollen allergen, grass allergen, tree allergen, animal allergen, dust mite allergen, cockroach allergen, or mold or fungi allergen, Hymenoptera allergen, or combinations thereof, or an extract derived from the allergic source material, or purified from the extract, synthesized chemically or biologically, or a food allergy allergen selected from a groundnut, peanut, milk, egg, tree nut, seed, fish, shellfish, crustacean, cereal, legume allergy, hazelnut, cashew, walnut, pecan, brazil nut, macadamia, chestnut, pistachio, coconut, almond, sesame, soy, kidney bean, black bean, common bean, chickpea, pea, cow pea, lentil or lupine allergy or a mustard seed allergy, or a combination thereof, wherein the allergen is whole or a fragment thereof of the allergen.

3. The model of claim 2, wherein the food allergen is a peanut allergen that comprises Ara hl, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hl 1, Ara hl2, Ara hl 3, a peptide fragment thereof, or a combination thereof.

4. The model of claim 1, wherein when the immunomodulator is present it stimulates T helper type 2 (Th2) cells that promote B cell class switching to IgE.

5. The model of claim 4, wherein when the immunomodulator is present it is selected from at least one of: steroids, bacterial components, metal hydroxides, natural polymers, cytokines, nucleic acid components, and other small molecules that trigger B cell class switching to IgE.

6. The model of claim 1, wherein the immune response is anaphylaxis.

7. The model of claim 1, wherein the allergen is a respiratory allergen in humans.

8. The model of claim 1, wherein the pig is a miniature porcine strain that is a Hanford,Yucatan, Micro- Yucatan, or Gottingen.

9. The model of claim 1, wherein the TS and IP are applied at the same time, simultaneously, alternating weekly.

10. A method of preparing an in vivo swine model system for an allergic condition, comprising: sensitizing a pig with a protocol that leads to at least one of: allergy symptoms or anaphylaxis, wherein the allergy sensitization is induced by repeated tape stripping (TS) of the skin and application of a reservoir comprising the allergen with or without an immunomodulator, or wherein sensitization is induced by intraperitoneal injections (IP) with an allergen with or without an immunomodulator once a week, or both.

11. The method of claim 10, wherein the administration is at least 3, 4, 5, 6, 7, 8, or more weeks.

12. The method of claim 10, further comprising, following sensitization, exposing the pig to an allergen; and measuring an immune reaction in the pig to the allergen.

13. The method of claim 10, wherein the sensitization triggers systemic allergen-specific IgG and IgE responses.

14. The method of claim 10, wherein the allergen is selected from the group consisting of food allergens, house dust mite, animal dander, feathers, plant antigens, molds, and household or industrial chemicals.

15. The method of claim 10, wherein the allergen is a plant, pollen, animal, arthropod, insect, fungus, or venom allergen, an aeroallergen, pollen allergen, grass allergen, tree allergen, animal allergen, dust mite allergen, cockroach allergen, or mold or fungi allergen, Hymenoptera allergen, or combinations thereof, or an extract derived from the allergic source material, or purified from the extract, or synthesized chemically or biologically, and is whole or a fragment thereof of the allergen.

16. The method of claim 10, wherein the allergen is a food allergy allergen to selected from a groundnut, peanut, milk, egg, tree nut, seed, fish, shellfish, crustacean, cereal, legume allergy, hazelnut, cashew, walnut, pecan, brazil nut, macadamia, chestnut, pistachio, coconut, almond, sesame, soy, kidney bean, black bean, common bean, chickpea, pea, cow pea, lentil or lupine allergy or a mustard seed allergy, or a combination thereof.

17. The method of claim 10, wherein the food allergen is a peanut allergen that comprisesAra hl, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hl 1, Ara hl 2, Ara hl 3, a peptide fragment thereof, or a combination thereof.

18. The method of claim 10, wherein the allergen is associated with asthma in humans.

19. The method of claim 10, wherein the in vivo testing is selected from at least one of: efficacy of one or more candidate drug(s) for a treatment of asthma; in vivo screening or testingof anti-inflammatory drugs, therapies, and / or procedures; or in vitro screening assays for the development of new anti-inflammatory or anti-eosinophil degranulation drugs.

20. The method of claim 10, wherein when the immunomodulator is present it stimulates T helper type 2 (Th2) cells that promote B cell class switching to IgE.

21. The method of claim 10, wherein when the immunomodulator is present it is selected from at least one of: steroids, bacterial components, metal hydroxides, natural polymers, cytokines, nucleic acid components, and other small molecules that trigger B cell class switching to IgE.

22. The method of claim 10, further comprising the step of repeated administration of antigen into the mammary gland of a mammal, thereby to induce a specific allergic response characterised by the recruitment of inflammatory cells into the mammary gland of the mammal.

23. The method of claim 10, further comprising the step of repeated administration of antigen into the lung of a mammal, thereby to induce a specific allergic response characterized by the recruitment of inflammatory cells into the lung of the mammal.

24. The method of claim 10, further comprising the step of collection of the inflammatory cells.

25. The method of claim 10, further comprising at least one step selected from: measuring asthma; the examining the effects of chronic allergen exposure.

26. The method of claim 10, further comprising providing the pig with one or more candidate drugs for anti-allergic drug testing.

27. The method of claim 10, further comprising at least one of: identifying processes or molecules differentially active or expressed in “activated” and “non-activated” eosinophils and / or other inflammatory cells; identifying processes and molecules involved in the recruitment of eosinophils and / or other inflammatory cells; identifying processes and molecules involved in degranulation of eosinophils and / or other inflammatory cells; in vivo screening and testing of anti-inflammatory drugs, therapies, and / or procedures; or in vitro screening assays for the development of new anti-inflammatory or antieosinophil degranulation drugs.

28. The method of claim 10, wherein the pig is a miniature porcine strain that is a Hanford, Yucatan, Micro- Yucatan, or Gottingen.

29. The method of claim 10, wherein the pigs are not allergic to soy or soy antigens.

30. The method of claim 10, wherein the pigs are not the result of multi -generational breeding.

31. An in vivo swine model system for an allergic condition comprising: a pig that is sensitized to an allergen that leads to at least one of: allergy symptoms or anaphylaxis; wherein the pig is sensitized by repeated tape stripping (TS) for at least 3, 4, 5, or 6 weeks, wherein the pig is sensitized by intraperitoneal injections (IP) for at least 3, 4, 5, or 6 weeks, or both.

Images