Treatment of immune disorders by administration of antigen-specific preparations

Abstract

To provide formulations for mucosal administration to induce immune tolerance to an antigen in a subject.SOLUTION: Provided herein is a mucoadhesive carrier for use in inducing immune tolerance to an antigen in a subject, comprising non-living bacterial particles and an antigen, where the mucoadhesive carrier is administered to the subject by trans-mucosal administration.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of medicine and immunology. The present invention relates to the treatment of immune-mediated disorders, such as autoimmune and allergic diseases, by administration of formulations consisting of combinations of antigens, tolerizing agents, and mucoadhesive carriers optimized for each immune disease indication. [Background technology]

[0002] The immune system has the task of discriminating between self and non-self. The mucosal immune system, present along the respiratory, gastrointestinal, and genitourinary tracts, is additionally burdened by the coexistence of numerous bacterial and harmless antigens, such as food, airborne antigens, or commensal flora. A key feature of the mucosal immune system is its ability to tolerate these antigens while retaining the ability to effectively eliminate pathogens. Systemic introduction of antigens, whether by injection or injury, results in local infiltration of inflammatory cells and production of specific immunoglobulins. In contrast, antigens introduced to mucosal surfaces, such as the oral cavity, gastrointestinal tract, and genitourinary tract, induce active inhibition of immune responses to these antigens systemically. The specific induction of these controlled responses by administration of antigens through mucosal surfaces of the oral cavity and gastrointestinal tract is known as oral tolerance. Oral administration of antigens can result in systemic unresponsiveness, which is an attractive alternative to immunosuppressive medical inventions (such as steroids) that have undesirable side effects. Mucosal-mediated tolerance induction has been proposed as a disease-modifying therapeutic strategy for autoimmune, allergic, and inflammatory diseases.

[0003] The following discussion of the background of the invention is provided merely to aid the reader in understanding the invention and is not admitted to describing or constituting prior art to the invention.

[0004] Autoimmune and allergic diseases or immune-mediated disorders such as anti-drug antibodies (ADA) impose a tremendous burden on patients and society, reducing quality of life and resulting in significant costs. Furthermore, there is no adequate treatment that is socially justified or without acceptable side effects. Current treatments for autoimmune diseases are primarily palliative and are generally immunosuppressive or anti-inflammatory. Treatment with steroids or NSAIDs suppresses the inflammatory symptoms of many autoimmune diseases. Intravenous immunoglobulin therapy is used for some autoimmune diseases. More specific immunomodulatory therapies such as the TNFα antagonist etanercept have been shown to be useful in the treatment of rheumatoid arthritis (RA). Nevertheless, these immunotherapies are associated with an increased risk of adverse effects, such as increased susceptibility to infection. Celiac disease, an autoimmune-like immune-mediated disorder characterized by chronic small intestinal inflammation induced by gluten (glutenin and gliadin), can only be effectively treated by a lifelong socially restricted diet that excludes foods containing wheat, rye, or barley. A strict gluten-free diet will heal the intestine, but the intolerance to gluten is permanent.

[0005] Thus, induction of antigen-specific oral tolerance therapy is an attractive therapeutic approach. Oral tolerance was first described in 1911, but it was only since the late 1970s that researchers began to address the mechanisms involved (Mayer, Shao, Nat Rev Immunol. 2004, 4:407-419). Several mechanisms have been proposed for the development of oral tolerance, ranging from deletion of antigen-specific T cells to induction of immune deviation and anergy, as well as suppression by Tregs (Mucida et al., J Clin Invest. 2005, 115:1923-1933). Targeted and more efficient delivery of molecules for therapeutic and prophylactic applications is a priority for the pharmaceutical industry. Effective strategies would reduce the required dose, increase safety, and improve efficacy by concentrating molecules at the desired site of action. Using mucosal routes for delivery of drugs and vaccines offers several logistical and biological advantages over injections. Oral mucosal delivery, such as via the sublingual or buccal route, is particularly attractive as the ease of administration makes it highly suitable for individuals who have difficulty swallowing tablets, such as children. Furthermore, with regard to immunotherapy, there is a strong scientific rationale for the preventative effect of targeting the developing immune system of children, and immunotherapy via the oral mucosal route would make this possible.

[0006] Concerning allergen sublingual immunotherapy (SLIT), considerable progress has been achieved in the last few years in our understanding of the physiology of the oral immune system. This strengthened scientific background has confirmed the validity of the sublingual pathway to induce antigen-specific tolerance and further highlighted the advantage of targeting oral antigen-presenting cells such as dendritic cells (DCs) and Langerhans cells (oCLs) to better mobilize allergen-specific regulatory immune mechanisms. It has now been established that SLIT is a safe oral mucosal treatment for allergy, which is now widely accepted as a valid alternative to subcutaneous immunotherapy (SCIT), and more than 20 years of clinical studies have confirmed the validity of the sublingual pathway to induce long-term allergen-specific tolerance and disease-modifying effects. The excellent safety profile of SLIT can be explained by the fact that oral tissues are largely free of pro-inflammatory cells such as mast cells and eosinophils as well as other danger signals, and that antigen-presenting cells (APCs), responsible for antigen capture, processing and subsequent presentation of induced peptides to naive T cells and oral APCs, display a tolerogenic phenotype. As a result, the initial response to antigen initiated by oral APCs results in tolerance that allows the antigen to be administered whole without systemic inflammatory responses, unlike the situation when administered via intravenous, intradermal, or intradermal routes, for example. However, challenges remain regarding efficacy and the need for daily administration over 3 years of treatment, which leads to low compliance and adherence rates. The low efficacy, which requires 3 years of treatment, is explained by the short retention time of the therapeutic agent when formulated as drops or tablets in the oral mucosa. Allam et al. (J Allergy Clin Immunol. 2010, 126:638-645) demonstrated dose- and time-dependent binding of allergens by oral mucosal Langerhans cells (oLCs) in human oral mucosa, indicating that dose intensity determines the efficiency of AIT.

[0007] Harmless bacteria used in fermented foods and probiotics have been shown to have antigen-specific tolerogenic properties and can therefore be used as tolerizing agents to improve the efficiency of antigen-specific oral mucosal immunotherapy (Huibregtse et al., Gastroenterolgy 2007, 133: 517-528; Huibregtse et al., J. Immunology 2009, 183: 2390-2396; Takiishi et al., J Clin Invest. 2012, 122: 1717-1725; Hsiao, KC et al., Lancet Child & Adolescent 2017, 1: 97-105). Examples include dietary lactic acid bacteria such as Lactococcus lactis and Lactobacillus rhamnosus, or the beneficial intestinal bacteria Escherichia coli Nissle. However, current approaches use live bacteria, which in some cases have been genetically modified to produce the antigen of interest in situ. Such approaches are associated with several disadvantages, some of which are practical in nature, and some of which are regulatory obstacles. Practical challenges include the need to keep the bacteria alive or viable in the formulation and during administration until they reach the target cells in the mucosal tissue, limitations on the expression of antigens by the recombinant bacteria, degradation of antigens by host bacterial proteases, and the need for new master cell banks for each antigen-specific treatment. Regulatory challenges include the difficulty of defining the exact antigen dose, the presence of recombinant DNA, and containment of the recombinant bacteria. Even if these practical and regulatory challenges were overcome, for example by simply mixing live non-recombinant bacteria with antigen, the short retention time of such preparations in the oral cavity would still limit the efficacy of treatment, necessitating long treatment regimens, and therefore low compliance and adherence rates would remain a challenge.

[0008] Thus, there remains a problem in the art of effectively inducing tolerance to antigens in general, and doing so via the oral mucosal delivery route in particular. Summary of the Invention [Problem to be solved by the invention]

[0009] Surprisingly, it was found that antigens eliciting immune responses in patients suffering from, for example, allergic rhinitis and allergic asthma, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, Sjogren's disease, neuromyelitis optica, food allergies, or celiac disease, when combined with non-viable bacterial particles derived from food-grade lactic acid bacteria or beneficial gut bacteria in a mucoadhesive carrier of hydrogel or electrospun fibers and delivered via the oral mucosal route, induced antigen-specific and long-lasting immune tolerance in short-term treatment. It was observed that oral mucosal administration of such antigens using formulations comprising non-viable bacterial particles incorporated in the mucoadhesive hydrogel or electrospun fibers allows delayed release, better presentation, and extended exposure time of pharmacologically active substances, resulting in significantly better suppression of antigen-specific immune responses compared to oral mucosal administration of the antigen alone or in combination with the bacterial particles. As a result, it was observed that oromucosal administration of such antigens simply as a formulation with non-viable bacterial particles resulted in significantly better suppression of antigen-specific immune responses compared to oromucosal administration of the antigen alone.

[0010] We demonstrate that embodiments of the present invention can induce oral tolerance with higher efficiency than monotherapy with antigen or bacterial particles or mucoadhesive hydrogel / electrospun fiber alone. Administration of the antigen composition according to the present invention strongly promoted the activation of antigen-specific regulatory T cells in vivo and ex vivo. Specifically, oral mucosal administration of gliadin or gliadin-derived peptides, which are immunodominant in DQ2- and / or DQ8-mediated T cell responses, in combination with a mucoadhesive carrier of hydrogel / electrospun fiber and bacterial particles induces local and systemic suppression of DQ2- and DQ8-restricted T cell responses. Treatment resulted in an antigen-specific reduction in the proliferative capacity of splenocytes and lymph node cells, which was strongly dependent on the production of IL-10 and TGF-β and was associated with a significant induction of Foxp3+ regulatory T cells. This approach of antigenic bacterial particles in a mucoadhesive hydrogel / electrospun fiber carrier has the potential to enhance oral tolerance even in the setting of established hypersensitivity, which may be applicable to the treatment of celiac disease and other autoimmune and / or allergic diseases. The efficacy of the present invention has also been demonstrated in models of autoimmune and allergic diseases, as well as for immune inactivation of biological therapies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Throughout this disclosure, various publications, patents, and patent publications are referenced by an identifying citation. The disclosures of these publications, patents, and patent publications are hereby incorporated by reference.

[0012] General method The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are explained in detail in such references as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by M.J. Gait, 1984); "Animal Cell Culture" (edited by R.I. Freshney, 1987); the series "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., 1987, and periodical publications), "Polymerase Chain Reaction" (edited by Mullis et al., 1994); and "Current Protocols in Immunology" (edited by J.E. Coligan et al., 1991).

[0013] definition As used herein, certain terms may have the meanings defined below. As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells and mixtures thereof. Similarly, the use of "compound" for the treatment or preparation of a medicament described herein contemplates the use of one or more compounds of the invention for such treatment or preparation, unless the context clearly dictates otherwise.

[0014] As used herein, the term "comprising" is intended to mean that the composition or method includes the recited element, but does not exclude others. When used to define compositions and methods, "consisting essentially of" means excluding other elements that give essential meaning to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants from the isolation and purification methods, and phosphate buffered saline, preservatives, and other pharma- ceutically acceptable carriers. "Consisting of" means excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these conjunctions are within the scope of the invention.

[0015] invention We demonstrate that oral mucosal administration of immunodominant antigens formulated with bacterial particles in mucoadhesive patches, films, or hydrogels induces potent suppression of local and systemic T cell responses. Treatment resulted in an antigen-specific reduction in the proliferation capacity of splenocytes and lymph node cells, which was highly dependent on IL-10 and TGF-β production and was associated with a significant induction of Foxp3+ regulatory T cells. This approach of antigen-formulated bacterial particles has the potential to enhance oral tolerance even in the setting of established hypersensitivity. It can therefore be applied to the treatment of celiac disease and other autoimmune and / or allergic diseases. The efficacy of the present invention was also demonstrated in animal models of autoimmune and allergic diseases, as well as ex vivo models derived from human immune disorders.

[0016] The object of the present invention is achieved by a preparation for the administration of at least one antigen, preferably a purified antigen, or a biological substance comprising one or more antigens, with or without bacterial particles, hereinafter referred to as "antigen-containing substance" incorporated into a mucoadhesive patch, film or hydrogel. In this way, the overall solubility and dissolution rate of such antigen-containing substance is optimized by techniques known in the art to increase its bioavailability. The effect is most pronounced in variants whereby the antigen-containing substance is directly incorporated into the material, e.g. nanofibers as disclosed in WO 2014 / 131376 or electrohydraulically obtained fibers as disclosed in WO 2015 / 189212, and formulated into a mucoadhesive patch, including but not limited to the mucoadhesive patches disclosed in WO 2012 / 097763 and WO 2016 / 051159. In a preferred embodiment, the mucoadhesive patch is a patch as disclosed in WO 2016 / 051159. In another embodiment of the invention, the antigen-containing material is formulated into a bioadhesive film such as, for example, US 2015 / 125495 and WO 2016 / 079246. In a further embodiment of the invention, the antigen-containing material is formulated into a hydrogel matrix and preferably a biodegradable mucoadhesive hydrogel, as disclosed, for example, in "Advances in Physicochemical Properties of Biopolymers" Part 2 (Mansuelli, Renard, eds., 2017, eISBN: 978-1-68108-544-9).

[0017] The most suitable biocompatible materials for producing the layer of the mucoadhesive patch, film or hydrogel are biocompatible polymers, in particular polyvinyl alcohol (PVA), polylactide (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyvinylpyrrolidone (PVP), polyurethane (PUR), polyacrylic acid (PAA), their copolymers, such as cellulose derivatives, such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), polyethylene oxide (PEO), polyethylene glycol / polyvinylcaprolactam / polyvinylacetate copolymers, silk fibroin, chitosan, alginate and hyaluronic acid derivatives, mixtures thereof, as well as a large number of polymerizable monomers, mixtures of at least two of these. To facilitate the release of the antigen-containing substance from the layer, it is possible to produce the layer from a biodegradable material. The advantage of this variant is that the layer degrades within the recipient's body after or during release of the antigen-containing material, so there is no need for further removal of the layer.

[0018] In one embodiment, the polymer layer may contain plasticizers such as polyols, phthalates, or citrates to increase the plasticity of the layer, and / or absorption enhancers such as acetylcysteine, surfactants to enhance particle penetration, chelating agents, fatty acids, polyols, dextran sulfate, sulfoxides, Azone®, (lyso)phosphatidylcholine, methoxysalicylate, menthol, aprotonin, dextran sulfate, cyclodextrin, 23-lauryl ether.

[0019] In a first aspect, the present invention relates to a mucoadhesive carrier comprising a non-viable bacterial particle and an antigen for use in inducing immune tolerance to an antigen in a subject, wherein the mucoadhesive carrier is administered mucosally to the subject. Mucosal administration of a mucoadhesive carrier is understood to include contacting the mucoadhesive carrier with the mucous membrane or mucosa of the subject. It is further understood herein that induction of immune tolerance is understood to include increasing or strengthening already existing (too weak) tolerance.

[0020] Preferably, the present invention relates to a method for inducing immune tolerance to an antigen comprising the oromucosal administration of said antigen by means of a bacterial particle-mucoadhesive patch, film or hydrogel formulation.

[0021] The terms "immune tolerance", "immunological tolerance", "tolerance", or "desensitization" are defined herein as reducing the immunological reactivity of a subject to an antigen, thereby rendering a sensitized or hypersensitive subject hyposensitive, insensitive, or non-responsive to a given antigen to which the subject is tolerized. Immune tolerance can occur, for example, by exposing a mucosal surface to a tolerogenic antigen-BLP-containing mucoadhesive carrier composition as defined herein. Mucosal administration of high and low doses of antigen results in immune tolerance, which subsequently reduces the immune response upon systemic administration of the antigen. There are thought to be at least two mechanisms of immune tolerance. Tolerance to high doses of antigen appears to occur through inactivation or clonal deletion of Th1 and Th2 cells. In contrast, tolerance to low doses of antigen results in bystander immune suppression mediated by stimulation of Treg cells, which produce inhibitory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and TGF-β. See Anderson, Jabri (Current Opinion in Immunology 2013, 25:410-417) and Rayner, Isaacs (Seminars in Arthritis & Rheumatism (2018), doi: https: / / doi.org / 10.1016 / j.semarthrit.2018.09.008).

[0022] Preferably, the present invention relates to the use of a bacterial particle-mucoadhesive patch, film or hydrogel formulation of an antigen for the preparation of a medicine, medical food or nutritional food for oral mucosal administration to treat an immune response-related disease in a patient, wherein said antigen is released in the oral mucosal tissue of said patient.

[0023] Preferably, the antigen is delivered by a bacterial particle-antigen formulated in a mucoadhesive patch, film, or hydrogel.

[0024] Preferably, the invention relates to the use of antigens formulated with bacterial particles for the preparation of medicaments for oromucosal administration to induce immune tolerance.

[0025] Preferably, said immune tolerance is induced in a patient. Said patient is preferably a human or an animal. Said animal is preferably a mammal, preferably selected from the group consisting of mouse, rat, rabbit, cat, dog, pig, cow, sheep and horse. Preferably, said mammal is a human. Preferably, said immune tolerance is mucosal tolerance.

[0026] mucous membrane As used herein, a mucosa may be any mucosa, such as oral mucosa, rectal mucosa, urethral mucosa, vaginal mucosa, ocular mucosa, pulmonary mucosa, and nasal mucosa.

[0027] Oral mucosal administration as used throughout this application includes targeted delivery to the oral mucosa. Oral mucosal administration includes buccal, sublingual, and gingival delivery routes. Preferably, said oral mucosal delivery is buccal or gingival administration and said tolerance is oral tolerance.

[0028] Oral mucosal tolerance, as used herein throughout this application, is the inhibition of a specific immune response in an animal (including a human) to an antigen after said animal has been exposed to said antigen via the oral mucosal route. Preferably, said mucosal tolerance is systemic tolerance.

[0029] The present invention relates to a method or use as described herein, wherein said induction of immune tolerance is at least 1.5-fold, preferably 2-fold, more preferably 3-fold or more effective compared to before said induction. Alternatively, said antigen is tolerated at least 1.5-fold, 2-fold, 3-fold or more compared to before said induction. The induction of immune tolerance can be measured by methods known in the art. Preferably, said induction of immune tolerance can be measured by modulation of cytokine levels in said animal. Thus, modulation may be an increase in cytokine levels, for example an increase in said cytokine levels, for example IL-10 or TGF-β, of at least 1.5-fold, 2-fold, 3-fold or more compared to before said induction. Alternatively, said modulation is a decrease in certain cytokine levels, for example IL-12, IL-17, and IFN-γ, for example a decrease in said cytokine levels, of at least 1.5-fold, 2-fold, 3-fold or more compared to before said induction. The cytokine to be regulated may be selected from any relevant cytokine, preferably said cytokine is selected from the group consisting of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, IL-23, TNF-α, IFN-γ, IFN-α, MCP-1, TGF-β, RANK-L, and Flt3L.

[0030] Tolerizing Compounds In the present invention, non-viable bacterial particles can stimulate the immune system to enhance an antigen-specific tolerogenic immune response while simultaneously delivering the antigen to the intended site, for example the oral mucosa.

[0031] Tolerogenic compositions comprising non-viable bacterial particles according to the invention can be prepared and tested for immunogenicity, efficacy and safety employing the techniques disclosed in WO 02 / 101026, which is incorporated herein by reference.

[0032] Preferably, the non-viable bacterial particles for use according to the invention are derived from beneficial intestinal bacteria. More preferably, the non-viable bacterial particles are derived from (probiotic) Lactobacillus, Lactococcus, Bifidobacterium or Escherichia coli Nissle. The majority of the non-viable bacterial particles for use according to the invention are derived from at least one of Lactococcus lactis MG1363, Lactobacillus rhamnosus GG, and Escherichia coli Nissle 1917.

[0033] Antigen-containing formulations may be based on particles derived from inactivated Lactococcus lactis, a safe bacterium traditionally used in the food industry, for example in cheese production (particles also elsewhere described as Gram-positive enhancer matrix or bacteria-like particles, referred to herein as "BLP"). BLP activates the innate immune system through Toll-like receptor 2 (TLR2, Ramirez et al., Mucosal Immunology 2010,3:159-171). TLR2 exerts downstream effects on myd88. TLR2 has recently been shown to be important for the downregulation of microbe-induced myd88 transcription (Koch et al., Nature Comm 2018,9:4099). myd88 transcription has been linked to the control of inflammatory signaling. Therefore, commensal bacteria such as lactobacilli in general, and in particular BLP, may constitute suitable tolerogenic compounds. BLPs are obtained by acidic heat treatment of L. lactis bacteria or any other Gram-positive bacteria, resulting in non-viable bacterial particles consisting mainly of peptidoglycan surface and without recombinant DNA. The preparation of BLPs is disclosed in WO 02 / 101026. The antigenic polypeptides of the invention may simply be mixed with the BLPs or may be loaded onto the BLPs. This method employs a non-covalent coupling technique called Protan technology, disclosed in WO 02 / 101026 and WO 2012 / 128628. The resulting antigen-associated BLPs (antigen mixed with or bound to the BLPs) form part of a therapeutic formulation (e.g. mucoadhesive patch, mucoadhesive gel, capsule, tablet, or liquid) that is delivered to humans through the mucosal layer of the oral cavity or other parts of the gastrointestinal tract without the need for injection. Preferably, the therapeutic formulation comprising the BLP and antigen is delivered to humans through the mucosal layer of the oral cavity using a mucoadhesive patch, film, or hydrogel.Even more preferably, the therapeutic formulation comprising BLP and an antigen comprises additional tolerizing compounds known in the art, such as cytotoxic T-lymphocyte antigen 4-immunoglobulin G (Maazi et al., 2013, Clin Exp Immunol 172:113-120), Zebularine (WO 2008 / 147283), retinoic acid (Coombes et al., 2007, J Exp Med 204:1757-1764), rapamycin (Maldonado, von Adrian 2010, Adv Immunol 108:111-165), vitamin D3 (Taher et al., 2008, J Immunol 180:5211-5221), and / or D-mannose (Zhang et al., 2017, Nature Med 23:1036-1047).

[0034] In another embodiment of the invention, tolerogenic compositions comprising non-viable bacterial particles may be prepared from empty bacterial envelopes, i.e. so-called bacterial ghosts (BG). The antigen-containing formulation may be based on BG derived from E. coli Nissl 1917, a safe bacterium used in the pharmaceutical product Mutaflor® for gut beneficial bacteria, or any other safe Gram-negative bacterium. BG is produced by controlled heterologous expression of genes that trigger partial lysis of the cell membrane, as disclosed in WO 0053163 and WO 2015 / 124717, which are incorporated herein by reference. An example of such a lytic gene is gene E of the bacteriophage PhiX174, which encodes a polypeptide that is inserted into the cell membrane complex of Gram-negative bacteria and that, upon oligomerization, leads to the formation of a transmembrane tunnel structure through the inner and outer membranes. The internal diameter of this tunnel structure may be 40-200 nm or 500-1000 nm, depending on the lysis conditions. The cytoplasmic material of the cell is released through this tunnel, leaving behind an empty cell envelope with its original morphology, which can be loaded with antigens and thus act as a delivery vehicle (WO 0053163, WO 0154672, WO 2005 / 011713). The antigenic polypeptides of the present invention can be simply mixed with BG, loaded into the empty cell envelope, or co-expressed with bacteriophage lysin and anchored to the cell envelope by techniques known in the art (WO 9906567, WO 0044878). Additionally, BG can be prepared from gram-positive bacteria using a chimeric EL lysis gene (US Pat. No. 5,075,223).

[0035] BG is highly suitable as a carrier or targeting vehicle for antigens or antigen-containing substances. Since only BG contains the desired antigen, a high degree of loading and therefore a high degree of antigen tolerization efficiency can be achieved. Although BG is also an option as a carrier, the tolerization effect can also be achieved by simply mixing antigen with BG. Overall, bacterial ghosts are non-viable bacterial particles that no longer contain recombinant DNA, and therefore are safe tolerization materials (WO 03 / 006630, WO 2009 / 090093). The resulting antigen-associated BG (antigen mixed with BG, loaded into BG, or bound to BG) constitutes part of a therapeutic formulation (e.g., mucoadhesive patch, mucoadhesive gel, capsule, tablet, or liquid) that is delivered to humans through the mucosal layer of the oral cavity or other parts of the gastrointestinal tract without the need for injection. Preferably, the therapeutic formulation containing BG and antigen is delivered to humans through the mucosal layer of the oral cavity using a mucoadhesive patch, film, or hydrogel. Even more preferably, the therapeutic formulation comprising BG and an antigen comprises additional tolerizing compounds known in the art, such as cytotoxic T-lymphocyte antigen 4-immunoglobulin G (Maazi et al., 2013, Clin Exp Immunol 172:113-120), Zebularine (WO 2008 / 147283), retinoic acid (Coombes et al., 2007, J Exp Med 204:1757-1764), rapamycin (Maldonado, von Adrian 2010, Adv Immunol 108:111-165), vitamin D3 (Taher et al., 2008, J Immunol 180:5211-5221), and / or D-mannose (Zhang et al., 2017, Nature Med 23:1036-1047).

[0036] In a further embodiment of the invention, the tolerogenic composition comprising non-viable bacterial particles may be prepared from a culture of chemically sterilized non-recombinant food grade lactic acid bacteria or beneficial enterobacteria. The antigen-containing formulation may be based on non-viable particles derived from chemically sterilized food grade gram-positive and gram-negative bacteria, such as, but not limited to, Lactococcus, Lactobacillus, or Escherichia coli. In a preferred embodiment of the invention, chemically sterilized Lactococcus lactis, Lactobacillus rhamnosus, or Escherichia coli Nissl 1917 are used. Chemical sterilization of bacteria is known in the art. In a preferred embodiment of the invention, chemical sterilization is achieved by the use of an alcohol solution. Examples of alcohol solutions include ethanol and isopropyl alcohol, and in particular a solution of 60%-90% (v / v) alcohol and 10-40% (v / v) purified water is a rapid antimicrobial agent for bacteria. To achieve complete sterilization, it is preferred that the contact time of the bacteria with the alcohol solution is 12 hours or more.

[0037] In another preferred embodiment, chemical sterilization is achieved by the use of β-propiolactone. In the method used, β-propiolactone is preferably added at a final concentration of 0.01% to 0.1% (v / v), more preferably 0.025% to 0.5% (v / v). β-propiolactone may be added to the preparation in one or more steps, for example in two successive steps, the two portions being preferably equal in volume, the second portion being added in about 15 to 45 minutes, for example about 30 minutes. The addition of β-propiolactone is preferably performed in liquid form. Addition in the form of a vapor or aerosol or other forms is also possible. A preferred embodiment of bacterial sterilization with β-propiolactone is an incubation time of 10 to 60 minutes, more preferably 15 to 45 minutes, even more preferably 25 to 35 minutes, as described in WO 2009 / 090093.

[0038] Furthermore, the preferred incubation temperature during the chemical sterilization process is 2°C to 55°C, more preferably 20°C to 40°C. The alcohol or beta-propiolactone is aseptically removed from the inactivated bacterial particles by extensive washing with purified water or a pharmacy-acceptable neutral buffer with a pH of 6 to 8, more preferably a pH of 6.5 to 7.5. The washing of the bacterial particles is performed with at least 10 times the incubation volume to efficiently remove the sterilizing agent.

[0039] The resulting chemically inactivated bacterial particles are of food grade origin and are non-viable, making them a safe tolerizing material. According to the present invention, an antigen is mixed with the chemically inactivated bacterial particles and forms part of a therapeutic formulation (e.g. a mucoadhesive patch, mucoadhesive gel, capsule, tablet, or liquid) that is delivered to humans through the mucosal layer of the oral cavity or other parts of the gastrointestinal tract without the need for injection. Preferably, the therapeutic formulation comprising the chemically inactivated bacterial particles and the antigen is delivered to humans through the mucosal layer of the oral cavity using a mucoadhesive patch, film, or hydrogel. Even more preferably, the therapeutic formulation comprising the chemically inactivated bacterial particles and the antigen comprises additional tolerizing compounds known in the art, such as cytotoxic T-lymphocyte antigen 4-immunoglobulin G (Maazi et al., 2013, Clin Exp Immunol 172:113-120), Zebularine (WO 2008 / 147283), retinoic acid (Coombes et al., 2007, J Exp Med 204:1757-1764), rapamycin (Maldonado, von Adrian 2010, Adv Immunol 108:111-165), vitamin D3 (Taher et al., 2008, J Immunol 180:5211-5221), and / or D-mannose (Zhang et al., 2017, Nature Med 23:1036-1047).

[0040] antigen The antigen-encoding sequence can be obtained from any natural source and / or prepared synthetically using known DNA synthesis techniques. The antigen-encoding sequence is then incorporated into (for example) a suitable expression vector, which is used to transform or transfect the intended host. The resulting recombinant host cell is then cultured, and the isolated antigen can then be used to formulate a therapeutic composition and form a powder, optionally after further purification and / or processing steps such as lyophilization. Optionally, recombinant host cell bacteria are used to produce BG, in which case the antigen forms part of the BG and no protein purification steps are required. However, instead, the BG containing the antigen of interest is used directly to formulate a therapeutic composition. Antigens can also be produced using known protein chemistry techniques. Additionally, antigens can be obtained as whole or partial original antigens from extracts such as pollen, gluten, house dust mites, cat saliva, birch, or peanuts.

[0041] The antigen may be any antigen known to those skilled in the art. An antigen as used herein throughout the application is any substance that preferably induces an immune response when introduced into an animal, said immune response may be a T cell mediated response and / or a B cell mediated response. The antigen may comprise a T cell epitope and / or a B cell epitope. There is no particular limit to the length of the antigen, provided that said antigen is produced. In a preferred embodiment of the invention, the antigen is the entire original antigen. The antigen may be a protein or a part thereof, such as a polypeptide or a peptide. Antigens of the invention include linear and / or conformational epitopes. T cell mediated responses include Th1, Th2, and / or Th17 responses. The antigen may be any antigen, such as but not limited to allergens (including food allergens), alloantigens, autoantigens, and therapeutic molecules or antigens that induce an immune response. Preferably, said antigen is involved in the induction of an immune response associated disease. Even more preferably, said antigen is involved in allergic rhinitis, allergic asthma, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, food allergy, celiac disease, pemphigus vulgaris, neuromyelitis optica, graft versus host disease, or inducing the development of anti-drug antibodies (ADA). Examples of antigens as part of the present invention include, but are not limited to, gliadin, hordein, Der p1, Der p2, Der p2.1, Fel d1, pINS, GAD65, InsB 9-23 , MBP, Dsg3, aquaporin 4, or IA2. A more extensive list of antigens as part of the present invention is given in Table 8.

[0042] The inventors have observed that delivery of immunodominant antigens in combination with the bacterial particles of the invention via mucoadhesive carriers suppresses systemic inflammatory T cell responses, and that formulation and delivery of such antigens is necessary and sufficient to induce a pronounced tolerogenic effect.

[0043] Regulatory T cells (Treg) play a key role in the induction and maintenance of oral tolerance. Induction of Treg is a major goal of immunotherapy of several autoimmune, allergic, and inflammatory diseases. Current strategies for the therapeutic induction of antigen-specific suppressor cells face significant challenges and usually require difficult procedures to isolate, handle, and transfer adequate numbers of regulatory cells. The bacterial particle-antigen mucoadhesive carrier delivery system of the present invention circumvents these problems and effectively induces antigen-specific Treg. In the present invention, it has been demonstrated that the induction of Treg can be achieved by exposing the mucosal immune system to a low dose of antigen. The exposure to the low dose of antigen is preferably continuous exposure. Thus, the present invention provides a method for the induction of Treg cells, preferably CD4 + CD25 + , CD4 + CD25 - , and CD8 + It relates to an antigen that induces and / or expands Treg cells.

[0044] In the present invention, it has been further demonstrated that Treg cells induced and / or expanded by the antigen according to the present invention function by a mechanism dependent on TGF-β and / or IL-10. Evidence has previously been presented that TGF-β plays an important role in oral tolerance and in the development of peripherally induced Tregs. Thus, the present invention provides immunodominant antigens that stimulate the expression of endogenous TGF-β and / or IL-10.

[0045] Furthermore, antigen-specific TGF-β-producing Th3 cells express antigen-specific Foxp3 in the periphery. + It has been shown that it accelerates the differentiation of regulatory cells. + CD25 - CD25 on T cells + , CD45RB - / low TGF-β-dependent conversion to suppressor cells has been reported. Oral tolerance induced by CTB-conjugated Ag is mediated by Foxp3 + CD25 + Both and Foxp3 + and Foxp3 - CD25- CD4 + These data suggest that Foxp3 is involved in the induction and maintenance of oral tolerance. + This suggests an important role for "adaptive" Tregs. It also shows significant mucosal Foxp3 induction. Moreover, bacterial particles alone are unable to induce this Foxp3 upregulation in the GALT, so the "mucosal" induced regulatory T cells tend to be antigen-specific. Therefore, the present invention preferably targets Foxp3 + Regarding Treg cells.

[0046] The present invention further demonstrated that Treg cells induced and / or expanded by the antigens of the present invention reduce inflammation, especially in spleen and secondary lymph node cells. Furthermore, the production of IFN-γ and IL-12 is reduced. Thus, the present invention provides immunodominant antigens that reduce the production of endogenous IFN-γ and / or IL-12 and / or stimulate the expression of endogenous TGF-β and / or IL-10. Furthermore, the present invention relates to antigens that reduce the proliferation of spleen and / or lymph node cells. The present invention also relates to antigens that suppress inflammatory antigen-specific T cell responses.

[0047] Immune response As used herein, an immune response-related disease is a disease caused by an unwanted immune response of the body to an antigen, which may be a xenoantigen or an autoantigen. Preferably, in the method of the present invention for inducing immune tolerance to an antigen, the antigen causes or is associated with an immune response-related disease. Immune response-related diseases include allergic reactions, including, but not limited to, food allergies, celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, type I diabetes, pemphigus vulgaris, and multiple sclerosis. Immune response-related diseases also include unwanted immune reactions, such as graft-versus-host disease or immune activation by medicines, such as antibody production against non-endogenous Factor VIII. Preferably, the disease is selected from the group consisting of allergic rhinitis, allergic asthma, food allergies, celiac disease, type I diabetes, and immune inactivation of therapeutic drugs.

[0048] In one embodiment, the present invention relates to a mucoadhesive carrier comprising a non-viable bacterial particle and an antigen as defined herein for use in the prevention and / or treatment of an immune response associated disease as defined herein, wherein the mucoadhesive carrier is administered mucosally to a subject as defined herein, preferably the antigen causes or is associated with an immune response associated disease.

[0049] According to the present invention, the term "immunodominance" refers to the principle of an antigen, including peptide fragments and specific epitopes thereof, inducing an immune response.

[0050] In view of the above, the present invention relates to the methods or uses as described herein, said methods or uses being therapeutic and / or prophylactic.

[0051] A further aspect of the invention relates to a method for inducing immune tolerance to an antigen comprising a peptide fragment and a specific epitope thereof, comprising the oral mucosal administration of said antigen in combination with a bacterial particle in a mucoadhesive patch, film or hydrogel. The antigen may be produced by the microorganism used to produce the bacterial particle. In a preferred embodiment of the invention, a purified antigen is produced from another source and mixed with the bacterial particle. The antigen may be physically associated with the tolerizing bacterial particle compound, but there may also be a mixing mode without physical interaction between the bacterial particle and the antigen.

[0052] Medication and Administration Compounds refer to any chemical or biological compound or complex, including simple or complex organic and inorganic molecules, peptides, peptidomimetics, proteins, protein complexes, antibodies, carbohydrates, nucleic acids, or derivatives thereof. Immunomodulatory compounds are compounds that modify the function of the immune system. As used herein, immunomodulatory compounds are tolerance-inducing compounds, and tolerance induction can be obtained directly, by inducing regulatory T cells, such as Treg, Tr1, or Th3, or by shifting the balance of Th1 / Th2 towards Th1 or Th2, or by inhibiting Th17, as non-limiting examples, or indirectly, by activating non-mature dendritic cells into tolerizing dendritic cells and / or inhibiting the Th2 immune response, which induces the expression of "co-stimulatory" factors on mature dendritic cells. Immunomodulatory and immunosuppressive compounds are known to those skilled in the art and include, but are not limited to, organic molecules such as vitamin D3 (or its precursors), D-mannose, zebularine, retinoic acid and phosphatidylserine, bacterial metabolites such as spergualin, fungal and actinomycete metabolites such as tacrolimus, rapamycin or cyclosporine, immunosuppressive cytokines such as IL-4, IL-10, IFN-α, TGF-β (as selective adjuvants for regulatory T cells), Flt3L, TSLP and Rank-L (as selective tolerogenic DC inducers), antibodies and / or antagonists such as anti-CD40L, anti-CD25, anti-CD20, anti-IgE, anti-CD3, anti-IL6 (or IL6R), and proteins, peptides or fusion proteins such as CTL-4Ig or CTLA-4 agonist fusion proteins.

[0053] Delivery or administration as used herein refers to any method of delivery or administration via mucoadhesive materials known to those skilled in the art, including but not limited to patches, biofilms, or hydrogels that contain or carry the antigen or bacterial particles combined with the antigen, optionally in the presence of compounds such as vitamin D3 and / or D-mannose to further enhance the tolerogenic effect and / or penetration enhancers such as surfactants or chelators to facilitate oral mucosal administration and / or mucosal uptake.

[0054] As used herein, hydrogel or film refers to a water-swellable cross-linked polymer that can be impregnated or loaded with antigens and bacterial particles. The bacterial particle-antigen combination loaded into the hydrogel is released in a controlled manner as the hydrogel hydrates in the oral cavity. In a preferred embodiment, the hydrogel matrix of the present invention comprises polysaccharides such as chitosan, alginate, and hyaluronic acid. In a more preferred embodiment of the present invention, the hydrogel matrix comprises chitosan. Chitosan is a bioactive, biocompatible, biodegradable, non-toxic compound with properties suitable for a range of industrial and biomedical applications such as drug delivery, wound healing, and biomedical implants. Chitosan is a polysaccharide that contains 1,4-linked residues of 2-amino-2-deoxy-β-D-glucose (glucosamine) and 2-acetamido-2-deoxy-β-D-glucose (N-acetylglucosamine). Chitosan is prepared by at least partial deacetylation of chitin (poly-N-acetylglucosamine or (1→4)-2-deoxy-β-D-glucan), a naturally occurring polysaccharide found naturally in the shells of crustaceans, insects, and fungi, where the acetyl groups are removed from at least some of the N-acetylglucosamine residues of chitin to form glucosamine residues.

[0055] In commercial preparations of chitosan, typically about 50% to about 100% of the N-acetylglucosamine residues of chitin are deacetylated to glucosamine residues. In the present invention, chitosan with a deacetylation rate of 70% to 95% is preferred. Chitosan dissolves in significant amounts in acidic solutions below pH 6.5. Soluble chitosan is therefore cationic and can bind to negatively charged surfaces and biological materials. Chitosan is a prominent example of a polysaccharide that can be ionically crosslinked. Chitosan hydrogels can be physically mixed into a stable network by introducing anionic ions or polymers to neutralize the positively charged chitosan and induce electrostatic attraction in the gelatinized network. Ionic crosslinking is a relatively safe technique used to produce biocompatible hydrogels without the use of toxic catalysts. In accordance with the present invention, chitosan-based hydrogels formulated from non-viable bacterial particles and antigens can be dried for long-term storage and easy application in the oral cavity.

[0056] In another preferred embodiment of the present invention, a thermosensitive chitosan solution is used. To obtain a thermosensitive hydrogel, the chitosan solution is neutralized with a counterion monosalt of a polyol. Under these conditions, chitosan remains liquid below 25°C and can be stored for long periods without losing its thermosensitive properties. The system has a physiologically acceptable neutral pH range (6.8-7.2), the liquid or gel state is determined only by the temperature of the environment, and gel formation is observed with increasing temperature. In a preferred embodiment of the present invention, the counterion monosalt of the polyol is β-glycerophosphate, and gel formation begins at temperatures above 32°C. The production and use of thermosensitive chitosan-based hydrogels are described in detail in literature such as "Biological Activities and Application of Marine Polysaccharides" (Arguelles-Monal et al., eds.).

[0057] Mucoadhesive patch as used herein refers to a patch consisting of multiple polymer layers with different functions. In such a multi-layer patch, the mucoadhesive layer covers and overlies the nanoscaffold reservoir layer described in Masek et al. (2017, J Control Rel 249:183-195). The mucoadhesive layer serves to attach / adhere to the oral mucosa and is optionally covered by a non-adhesive backing layer to prevent diffusion of the medicament into the oral cavity. The nanoscaffold reservoir layer is the actual carrier of the medicament and faces the oral mucosa. The manufacture of this kind of mucoadhesive patch, including electrospinning of nanofibers in the reservoir layer, is described in detail in WO 2016 / 051159. Thus, in a preferred embodiment, the mucoadhesive (e.g. particulate) carrier comprises a) a nanoscaffold (or matrix) carrying or comprising at least one substance or API (e.g. in the form of particles), and b) a mucoadhesive (layer) that is capable of adhering (to the mucosa) or overlapping the nanoscaffold on at least a part of its surface. The nanoscaffold preferably comprises or has pores with a size of 10 nm to 1000 μm and / or is a nanofiber layer with a thickness in the range of 0.1 to 1000 μm, or comprises a layer of a biocompatible polymer or a mixture thereof. The mucoadhesive carrier according to this embodiment may be further characterized in that a) the mucoadhesive layer (at least partially) overlaps the nanoscaffold, and the edges of the mucoadhesive layer overlap the edges of the nanoscaffold and / or the mucoadhesive layer surrounds the nanoscaffold along its edges, or b) the mucoadhesive carrier is configured to be applied to a target mucosa, the nanoscaffold facing the mucosa (e.g., in the same orientation as the mucoadhesive layer), and / or the portion of the mucoadhesive layer overlapping the nanoscaffold is configured to adhere and immobilize (adhere) the mucoadhesive carrier to the target mucosa.

[0058] In a preferred use of the invention, multiple layers of the mucoadhesive patch are preformed and then loaded onto the nanofiber reservoir layer with the bacterial particles and antigens. For this purpose, the bacterial particles and antigens are applied to the nanoscaffold reservoir layer as a solution, colloid, or suspension. Uptake by the reservoir layer occurs by diffusion and absorption. Excess liquid can be removed by conventional drying or freeze-drying techniques. Inclusion of a cryoprotectant may be necessary to adequately protect the bacterial particles and antigens.

[0059] As used herein, the terms "treatment", "treating" and the like include the amelioration or elimination of a psychological disease or condition once it has been established, or the alleviation of symptoms characteristic of such a disease or condition. As used herein, these terms also include preventing the onset of a disease or condition or the onset of symptoms associated with a disease or condition depending on the condition of the patient, including reducing the severity of the disease or condition or symptoms associated therewith prior to the onset of distress associated with said disease or condition. Such prevention or reduction prior to distress refers to administering a compound or composition of the invention to a patient who is not afflicted by the disease or condition at the time of administration. "Preventing" also includes preventing the recurrence or prevention of relapse of a disease or condition or symptoms associated therewith, for example after a period of improvement. It will be apparent that the psychological condition is the cause of the physical symptoms. In this regard, the term "treating" also includes the prevention of a physical disease or condition, or the amelioration or elimination of a physical disease or condition once it has been established, or the alleviation of symptoms characteristic of such a condition.

[0060] As used herein, the term "medicament" also encompasses the terms "drug," "therapeutic agent," or other terms used in the medical arts to refer to a preparation having a therapeutic effect.

[0061] It is recognized that the antigen present in the present invention is delivered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" is meant to refer to an amount of the compound or composition of the present invention that induces the desired therapeutic or prophylactic effect or response when administered according to a desired treatment regimen. It has been observed that when the immunodominant antigen is continuously present, the inflammatory antigen-specific cellular response is further reduced. This reduction is significantly greater than administration of the antigen or bacterial particle as is, or the antigen is non-continuously present.

[0062] Preferably, the compound or composition is provided in a unit dosage form, for example a mucoadhesive patch, biofilm, or hydrogel, which is administered to a subject, for example the oral cavity of a patient.

[0063] The active ingredient is administered 1 to 6 times per day, sufficient to provide the desired activity. These daily doses may be administered as a single dose once per day, or as two or more smaller doses administered at the same or different times throughout the day that add up to the specified daily dose. Preferably, the active ingredient is administered once or twice per day. For example, one dose may be taken in the morning and one dose later in the day.

[0064] In all aspects of the invention, the daily maintenance dose may be administered in the patient for a clinically desirable period, such as from one day to several years (e.g., for the entire life expectancy of the mammal), such as from about (2, 3, or 5 days, 1 or 2 weeks, or 1 month), and / or for example from about (5 years, 1 year, 6 months, 1 month, 1 week, or 3 or 5 days). Administration of a daily maintenance dose for about 3 days to about 5 days, or from about 1 week to about 1 year, is typical. Other components of the final formulation may include preservatives, inorganic salts, acids, bases, buffers, nutrients, vitamins, or other pharmaceuticals.

[0065] The bacterial particles in combination with the antigen may be delivered at a dose of at least 0.1 mg to 60 mg (dry weight) per day, preferably 0.5 to 20 mg per day, most preferably 1 to 10 mg per day.

[0066] The present invention further relates to the following numbered embodiments: 1. A mucoadhesive carrier comprising non-viable bacterial particles for use in oral mucosal administration to treat an immune response associated disease in a patient, wherein the non-viable bacterial particles are combined with at least one antigen that elicits said immune response, said immune response associated disease being selected from the group consisting of celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, or food allergies. 2. The mucoadhesive carrier according to embodiment 1, wherein the carrier is a mucoadhesive patch, a biofilm, or a hydrogel. 3. The mucoadhesive patch of embodiment 2, wherein said patch comprises electrospun fibers. 4. The mucoadhesive carrier according to any one of embodiments 1 to 3, wherein the antigen is α-gliadin or hordein. 5. The mucoadhesive carrier according to any one of embodiments 1 to 3, wherein said antigen is one or more immunodominant epitopes of the house dust mite, preferably Der p2.1. 6. The mucoadhesive carrier according to any one of embodiments 1 to 3, wherein said antigen is one or more immunodominant epitopes of peptides from feline saliva, skin or glands, preferably Fel d1. 7. The antigen is one or more immunodominant epitopes of peptides involved in type 1 diabetes, preferably pINS, GAD65, InsB 9-23 4. The mucoadhesive carrier according to any one of embodiments 1 to 3, wherein the mucoadhesive carrier is IA1 or IA2. 8. A non-viable bacterial particle for use in oral mucosal administration to treat an immune response associated disease in a patient, wherein the non-viable bacterial particle is combined with at least one antigen that elicits said immune response, and wherein said immune response associated disease is selected from the group consisting of celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, or food allergies. 9. A mucoadhesive carrier according to any one of embodiments 1 to 7, or a non-viable bacterial particle according to embodiment 8, wherein the non-viable bacterial particle is an acidic heat-treated Gram-positive bacterium, preferably L. lactis. 10. A mucoadhesive carrier according to any one of embodiments 1 to 7, or a non-viable bacterial particle according to embodiment 8, wherein the non-viable bacterial particle is an empty bacterial envelope obtained by lysing a gram-negative bacterium, preferably Escherichia coli Nissl. 11. A mucoadhesive carrier according to any one of embodiments 1 to 7, or a non-viable bacterial particle according to embodiment 8, wherein the non-viable bacterial particle is a chemically sterilized bacterium, preferably L. lactis, Lb. rhamnosus, or E. coli Nissul. 12. A mucoadhesive carrier or a non-viable bacterial particle according to any one of embodiments 1 to 11 for use as a medicament for treating humans and / or animals.

[0067] In this document and the claims thereto, the verb "to comprise" and its conjugations are used in an open-ended sense, meaning that the matter that follows the word is included, but not the matter that is not specifically mentioned is excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0068] The word "about" or "approximately" when used in connection with a numerical value (e.g., about 10) preferably means that the value may be within plus or minus 0.1% of the given value (10). All patents and references cited herein are hereby incorporated by reference in their entirety.

[0069] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0070] Example A1: Determination of Loading and In Vitro Release of Bacterial Particles from Nanoreservoir Layers Introduction The ability to efficiently load BLP into preformed nanoreservoir layers made from different types of polymer fibers was tested, as well as the in vitro release of BLP from these nanoreservoirs into buffer solution after drying.

[0071] Materials and Methods Bacterial particles: BLPs of Lactococcus lactis MG1363 were prepared essentially according to WO 02 / 101026.

[0072] Nanoreservoir layers: Nanoreservoir layers were prepared by electrospinning using polycaprolactone (PCL, Mw 80,000 g / mol, Sigma Aldrich) or a mixture of polycaprolactone and silk fibroin (1:1) (PSF) polymers. The procedure was as described by Masek et al. (2017, J Control Rel, 249:183-195). In this way, nanoreservoir layers of PCL and PSF with an average pore size of approximately 5 μm were obtained.

[0073] Experimental setup: A 1 cm drop of liquid was placed on the surface of the reservoir layer. 2The nanoreservoir layer was loaded with 10 μL of BLP solution (5 mg / mL BLP stock in demineralized water). The droplet was allowed to absorb into the layer and air-dried for approximately 20 min at room temperature. The dried reservoir layer containing BLP was examined under a scanning electron microscope (SEM, Hitachi 8010) or was first immersed in 1 mL of PBS for 10 min and then dried as above for SEM analysis.

[0074] result A drop of BLP solution placed on the surface of the PCL nanoreservoirs remained substantially on the surface and was not absorbed into the nanoreservoir layer. After 20 min of incubation, the droplet was removed and the PCL reservoir layer was dried. SEM analysis revealed that despite the apparent hydrophobicity of the PCL polymer, BLP was absorbed into the polymer fibers and had also penetrated into some pores in limited amounts. Immersion of the PCL nanoreservoirs in PBS with BLP resulted in the complete release of BLP from the nanoreservoirs, indicating that the absorption of BLP into the PCL nanofibers is reversible under these conditions.

[0075] A drop of BLP solution placed on the surface of the PSF nanoreservoir was immediately absorbed into the nanoreservoir layer. After drying, SEM analysis revealed that the BLP was densely loaded onto the PSF fibers and that the BLP had entered the pores of the reservoir layer. By immersing the PSF nanoreservoir in PBS with the BLP, more than 90% of the BLP was released from the nanoreservoir, indicating that the absorption of BLP into the PSF nanofibers was reversible under these conditions.

[0076] conclusion PCL and PSF nanofiber reservoir layers can be used to load and release BLP, with the PSF nanoreservoir layer being the most efficient.

[0077] Example A2: Determination of ex vivo release and penetration of bacterial particles combined with antigens from mucoadhesive patches or hydrogels into oral mucosal tissues Introduction The ability of the mucoadhesive patches and hydrogels to target and release relevant bacterial particles formulated with antigens under real conditions (on moist mucosal tissue surfaces with limited water content typical of mucosal layers, etc.) was investigated by incubating the mucoadhesive films and hydrogels with freeze-denatured and barrier-compromised mucosa at 37° C. In a separate experiment, the release of bacterial particles formulated with antigens from the mucoadhesive patches and hydrogels was investigated by determining the fluorescent signal by confocal microscopy of adjacent tissue cross-sections.

[0078] Materials and Methods Bacterial particles: BLP of Lactococcus lactis MG1363 and BG of E. coli Nisle 1917 were prepared essentially according to WO 02 / 101026 and WO 9906567, respectively. Lactococcus lactis MG1363, Lactobacillus rhamnosus GG, and E. coli Nisle were grown overnight in GM17, MRS, and TB growth media using standard growth conditions for these organisms. After overnight growth, cultures were harvested and washed with one volume of purified water. Chemically inactivated bacterial particles of Lactococcus lactis MG1363, Lactobacillus rhamnosus GG, and E. coli Nisle were obtained by incubating the overnight cell harvest with 70% ethanol at 20–23°C for 16–20 h. After chemical inactivation, the bacterial particle solution was centrifuged and the particles were washed three times with the same volume of initial medium. After washing the untreated bacterial cultures (viable bacteria) and chemically inactivated bacterial particles, the bacteria / particles were resuspended in PBS at a concentration of 30 mg / mL and stored at 2–8 °C until use.

[0079] Bacterial particles were labeled with fluorescein isothiocyanate (FITC) by incubating 3 mg of bacterial particles with 0.01 g of FITC (Sigma) in 0.1 M boric acid at pH 9.0 for 60 min at room temperature in a shaker. After incubation, the bacterial particles were harvested by centrifugation, washed extensively with PBS, and finally stored at 2–8°C at a concentration of 30 mg / mL.

[0080] Antigen: In all experiments, untreated LPS-free OVA grade V protein (Sigma Aldrich) was used as antigen. A stock solution of 1 mg / mL in PBS was prepared. OVA was labeled with AlexaFluor647 (ThermoFisher) according to the supplier's instructions.

[0081] Oral mucosa tissue: Immediately after the animals were killed, the sublingual gland and cheek tissue were removed using surgical instruments. The excised tissue was immediately transported to the laboratory in PBS buffer, cut into small cube-shaped pieces, and used for testing. The time between the killing of the animals and the start of the experiment was less than 20 minutes.

[0082] Mucoadhesive patches / hydrogels: Mucoadhesive patches and hydrogels were prepared using polycaprolactone-silk fibron (PSF), polylactide-co-glycolide-polyethylene glycol (PLGA-PEG) polymer, or deacetylated chitosan essentially as described in the referenced patents and above. Prior to the hydrogel polymerization process, bacterial particles and OVA were added to the polymer solution, and the resulting hydrogels were allowed to stand for 0.5 cm. 2 The final concentration contained 0.1 mg of labeled bacterial particles and / or 1 μg of labeled OVA per microwell. To obtain the mucoadhesive patch, the PSF nanoreservoir layer was overlaid with a mucoadhesive layer consisting of Carbopol 934P (Lubrizol Advanced Materials) and hydroxypropyl methylcellulose K4M (HPMC, Colorcon Ltd) in a 2:1 ratio, with the mucoadhesive layer overlying the PSF nanoreservoir layer. The side of the mucoadhesive layer not attached to the nanoreservoir layer was further covered with a non-adhesive backing layer consisting of Eudragit® L100-55 polymer. The procedure for preparing the preformed mucoadhesive PSF patch is described in Masek et al. (2017, supra).

[0083] A mixture of labeled bacterial particles and OVA was loaded onto a mucoadhesive PSF patch and placed in a 0.5 cm 2The compositions of each mucoadhesive patch / hydrogel tested are summarized in Table 1.

[0084] [Table 1]

[0085] Experimental setup: Ex vivo penetration of antigen-loaded bacterial particles into adjacent mucosa was tested in freshly excised porcine sublingual and buccal tissues essentially as described in Masek et al. (2017, supra). Nanofiber mucoadhesive films / patches containing fluorescently labeled bacterial particles and / or OVA were applied to the mucosa and incubated for 2 h at 37°C. During the experiment, the surface of each mucosa was wetted with PBS (flow rate 0.1 mL / min) using a linear pump and tubing.

[0086] The release of bacterial particles formulated with antigens from mucoadhesive patches and hydrogels was studied by determining the fluorescent signal in adjacent tissue cross-sections by confocal microscopy (Leica SP2) essentially as described in Masek et al. (2017, supra).

[0087] result The ex vivo penetration of bacterial particles formulated with antigen into the oral mucosa was tested in freshly excised porcine sublingual and buccal tissues. A given nanofiber mucoadhesive film / patch containing preloaded and labeled bacterial particles and / or OVA was applied to the excised mucosa and incubated at 37°C for 2 h. During the entire experiment, the mucosal surface was moistened with PBS buffer to reproduce the conditions in the oral mucosa. For control purposes, free bacterial particles were applied to the excised mucosa. The difference was clearly distinguishable by the intensity of the staining at the application site. In the nanofiber mucoadhesive patch / film and hydrogel, the antigen-loaded fluorescently labeled bacterial particles were maintained in the center of the mucoadhesive ring during the incubation, whereas the free bacterial particles were washed off from the mucosal surface after a short test and did not penetrate into the mucosa at all.

[0088] The penetration of bacterial particles formulated with OVA into the oral mucosa was confirmed by sectioning of adjacent mucosa and confocal microscopy. Freshly excised sublingual and buccal oral mucosa were incubated and tested with mucoadhesive patches and hydrogels. Penetration into the oral mucosa epithelium was observed in all tested samples containing bacterial particles. Fluorescence intensity reflects the concentration gradient of bacterial particles diffusing deep into the submucosa. Transport of bacterial particles in the epithelium is via a paracellular pathway, as clearly demonstrated by confocal microscopy of tissue sections.

[0089] conclusion Mucoadhesive patches and hydrogels allow bacterial particles formulated with antigens to have an extended exposure time in mucosal tissues, resulting in more efficient uptake of the particles and antigens by the mucosal tissues.

[0090] Example A3: Determination of in vivo release and penetration of BLP combined with antigen from mucoadhesive patches into oral mucosal tissues and draining lymph nodes Introduction The ability of the mucoadhesive patch to target and release relevant bacterial particles formulated with antigens was investigated under real conditions, i.e. in the piglet oral cavity model, by applying PSF mucoadhesive patches preloaded with labeled BLP and / or OVA to the sublingual gland or buccal oral mucosa. The porcine oral mucosa is generally recognized as a highly suitable model for this type of study, since the epithelium of the porcine oral mucosa is not keratinized and in that sense is similar to the human oral mucosal epithelium. The release of bacterial particles formulated with antigens from the mucoadhesive patch was studied by determining the fluorescent signal by confocal microscopy of cross sections of adjacent tissues and draining lymph nodes.

[0091] Materials and Methods Bacterial particles: BLPs of Lactococcus lactis MG1363 were prepared essentially according to WO 02 / 101026. Particles were labeled as described in Example A2. Antigen: Untreated LPS-free OVA grade V protein was used as antigen as described in Example A2.

[0092] Animals: Landrace-Yorkshire piglets, 2-3 months old

[0093] Mucoadhesive patch: PSF mucoadhesive patch was prepared as described in Example A2. Approximately 2 cm 2 A final concentration of 0.5 cm was obtained using a patch of 2 The patches were loaded with labeled BLP and / or OVA to give 0.1 mg BLP and / or 1 μg labeled OVA per patch. Some patches contained the permeation enhancer EDTA. The composition of each mucoadhesive patch tested is summarized in Table 2.

[0094] [Table 2]

[0095] Each type of patch was applied to the sublingual gland (anterior or lateral to the tongue) or cheek.

[0096] Experimental setup: Piglets were briefly anesthetized with Zoletil (Virbac) for application of the patches at the indicated sites in the oral cavity. After 3 or 24 h of incubation, animals were anesthetized and sacrificed by intravenous application of T61. Sublingual and buccal mucosa with attached mucoadhesive patches as well as draining lymph nodes were excised, frozen and stored at -80°C. Tissues were processed and analyzed by confocal microscopy essentially as described in Masek et al., 2017.

[0097] result Confocal microscopy of histochemically stained sublingual and buccal tissue sections demonstrated endocytosis of fluorescent BLP and OVA by antigen-presenting cells (APCs). BLP and OVA were internalized by APCs. Free diffusion of OVA was observed, but not BLP. Uptake by APCs was more pronounced 24 hours after application of the patch to the oral mucosal tissue. There was somewhat better uptake with patches placed in the buccal position of the oral cavity and / or with patches containing the penetration enhancer EDTA. APCs containing fluorescent BLP and OVA were also seen in the draining lymph nodes. This result was most pronounced with patches containing EDTA and applied to the buccal mucosa.

[0098] conclusion The mucoadhesive PSF patch allows efficient uptake of BLP and antigens in the oral mucosal tissue, with the particles and antigens draining to regional lymph nodes. Uptake is further stimulated by the presence of a penetration enhancer such as EDTA.

[0099] Example B: Ex vivo determination of bacterial particle-mediated antigen activation of antigen-presenting cells from the cheeks of autoimmune and allergic patients Introduction To demonstrate that oral mucosal tolerogenic APCs can be targeted and activated in an effective manner by extended exposure to a formulation of immunodominant antigens combined with bacterial particles, a human immune disorder-relevant ex vivo model was established by using fresh human oral mucosal tissues from patients with celiac disease and cat allergy, and the binding of antigen-loaded bacterial particles to human oral mucosal APCs was studied using a standardized protocol.

[0100] Materials and Methods Bacterial particles: Bacterial particles of Lactococcus lactis MG1363, Lactobacillus rhamnosus GG, and Escherichia coli Nissl 1917 were prepared essentially as described in Example A2.

[0101] Additional tolerizing compounds: Vitamin D3 (Sigma Aldrich) and D-mannose (Sigma Aldrich) were used to stimulate mucosal uptake and enhance mucosal responses. A single stock solution of vitD3-mannose (VM) containing 500 μg / mL of each compound was prepared.

[0102] Standardized ex vivo model of resorption of antigen-loaded particles: Oral mucosal tissue samples from the vestibular region were obtained from patients with celiac disease, house dust mite allergy, and cat allergy undergoing oral surgery. Intestinal biopsy samples were obtained from patients with celiac disease. Samples of peripheral blood mononuclear cells (PBMC) were also used. Patients with tumors were excluded and only clinically non-inflamed tissue was collected. Samples were used from individuals with a history of celiac disease based on genetic markers (HLA-DQ2+ and / or HLA-DQ8+) and serological markers (IgA anti-endomysial antibodies and / or IgA-tTG) combined with histological alterations of the duodenum assessed by duodenoscope. Samples from individuals with a history of allergy to house dust mites or cats were confirmed by skin prick tests and / or serum specific IgE.

[0103] All samples were obtained with the approval of the local and hospital ethical committees and after obtaining informed consent from the patients.

[0104] Antigens: Feline recombinant antigen Fel d1 was provided by ALK-Albello. Purified Der p1 and Der p2 house dust mite antigens were purchased from Citeq. Recombinant Der p2.1 (amino acids 1-53) house dust mite antigen was obtained as described by Chen et al. (Mol Immunol. 2008, 45:2486-2498). Vital wheat gluten (food grade, Gluvital) was purchased from Cargill, The Netherlands. Deamidated celiac disease antigen gliadin α1 peptide (amino acids PFPQPELPY), α2 peptide (PQPELPYPQ) and α2 peptide 33-mer (LQLQPFPQPELPYPQPELPYPQPQLPYPQPQPF) were purchased from Genscript.

[0105] Calculation of oLC transferred: Harvested human oral mucosal tissues were placed on sterile Petri dishes with the mucosal side up and incubated with PBS containing fluorescein isothiocyanate (FITC)-linked antigen-loaded particles or dextran at concentrations ranging from 10 to 1000 mg / mL at 37°C. For repeated antigen challenges, 10 mg / mL of antigen mixed with bacterial particles was administered to the oral mucosal tissues by removing the previous antigen solution and adding new antigen solution every 6 min without washing in between. As control conditions, oral mucosal tissues were incubated with PBS alone or cooled to 4°C before incubation with FITC-linked antigen-loaded particles or dextran to determine nonspecific antigen binding. After incubation, oral mucosal tissues were washed with PBS and placed in RPMI1640 medium (Invitrogen) containing 10% heat-inactivated FCS (Sigma), 1% antibiotic / antimycotic (Invitrogen), and 500 IU / mL GM-CSF (Berlex Laboratories) at 37°C. For time kinetics, cells that migrated out of the tissue were harvested 24 and 36 hours after antigen exposure and further processed for flow cytometry analysis. The medium was replaced and tissue incubation continued. Migrated cells were finally harvested after 48 hours, and dormant tissue was further processed and digested, followed by Ca2+ extraction as described elsewhere. 2+Single cell suspensions were prepared by trypsinization in 0.5% trypsin buffer without trypsin for 1 h at 37° C. Antigen binding to oLCs was calculated by gating on the FITC fluorescence of the major CD1a1 population that migrated out of the tissue at 4° C. Specific binding of antigen was calculated by subtracting the percentage of FITC-positive CD1a cells at 48° C. from the percentage of FITC-positive CD1a cells at 37° C.

[0106] Oral mucosal Langerhans cells were gated by their CD1a expression and location in forward / side scatter at 24, 36, and 48 hours after antigen / dextran exposure. The remaining tissue was treated with trypsin, and resting oLCs were identified by their CD1a expression. The gated cells, as well as cells in the remaining tissue, were counted at each migration time point. The percentage of oLCs that had migrated at each time point was calculated by dividing the sum of the number of oLCs at a particular time point and the previous time point by the total number of oLCs detected.

[0107] Proliferation assay: Irradiated APCs were pre-stimulated overnight with antigen mixed with bacterial particles (concentration range 10 per well 4 ~10 7 Optionally, vitamin D3 and D-mannose were also added to the mixture at a final concentration of 5 μg per well. The next day, allogeneic T cells were added, and 2 days later, 3 After a further 12-16 h incubation, plates were harvested and counted using a Betaplate Counter (Wallac, Turku) as described by Arentz-Hansen et al. (J. Exp. Med. 2000, 191:603-612). 3 H]thymidine incorporation was counted.

[0108] T cell proliferation assays using PBMCs were performed as described by Seyfert-Margolis et al. (Diabetes 2006, 55:2588-2594). Briefly, mononuclear cells were enriched on a Ficoll-Pacque gradient and plated at 1 × 10 per well in Hybrimax 2897 protein-free medium (Sigma) in culture-grade flat-bottom 96-well culture plates. 5 Plates were seeded at a concentration of 100 μCi / mL. To allow detection of preactivated and anergic T cells, 10 units / mL of recombinant IL-2 diluted in Hybrimax medium was added to the cultures. Responses measured in the presence of IL-2 supplement retain the absolute and dose-dependent requirement for antigen. Plates were incubated in a standard CO2 incubator for 1 week, with 1 μCi of [ 3 [H]thymidine was added and automated harvesting and scintillation counting was performed. PBMC cultures were stimulated with test antigen and test antigen-BLP combinations. To compare the proliferative responses in different samples, data were normalized as a stimulation index (cpm of antigen stimulation / cpm of unstimulated). A positive response was defined as 1.5 times greater than the mean stimulation index + 3SD in replicates of antigen-stimulated cultures. Background counts ranging from 500 to 1200 cpm were not significantly different from those in antigen-stimulated cultures (P > 0.3). Positive responses were summed for each sample to derive a T cell response score (range 0 to 15). A score of 4 was considered a positive response.

[0109] result The kinetics and uptake of antigen-formulated bacterial particles binding to oLCs and their translocation were investigated. To address this issue, freshly ex vivo isolated oral mucosal tissues were incubated with 100 mg / mL of antigen with or without bacterial particles for 1 h and placed in cell culture medium to allow translocation of allergen-loaded oLCs out of the tissue. Vitamin D3 and D-mannose were also added (see Table 3 for a complete list of all formulations tested).

[0110] [Table 3]

[0111] The antigen binding of the transferred oLCs was analyzed after 24, 36, and 48 hours. It was shown that LCs had a high dextran uptake. Therefore, oral mucosal tissues incubated with FITC-dextran were used as a reference for the binding of oLCs to antigens. In this regard, the total percentage of oLC-bound antigens was smaller than that of oLC-bound dextran after 24 and 36 hours of exposure. Interestingly, the percentage of oLC-bound antigens was similar after 24, 36, and 48 hours of exposure, whereas the percentage of oLC-bound dextran was maximum after 24 hours of exposure and continuously decreased after 36 and 48 hours of exposure.

[0112] Proliferation assays showed clear patient-specific T cell proliferation in the presence of antigen. Addition of bacterial particles caused a significant reduction in T cell proliferation, indicating a tolerizing effect. Addition of vitamin D3 and D-mannose also reduced T cell proliferation, but the effect was most pronounced when bacterial particles were combined with vitamin D3 and D-mannose.

[0113] conclusion We demonstrate that bacterial particles formulated with antigen target antigen uptake to tolerogenic oral APCs more efficiently than antigen alone, which is further enhanced by the addition of vitamin D3 and / or D-mannose. We further demonstrate that bacterial particles formulated with antigen have a tolerizing effect on T cells, which is further enhanced by the addition of vitamin D3 and / or D-mannose.

[0114] Example C1: In vivo induction of antigen-specific desensitization and disease-modifying effects by oral mucosal administration of bacterial particles combined with antigen to antigen-sensitized mice Introduction Having established that bacterial particles can be used to efficiently target antigen uptake to oral APCs and enhance tolerogenic responses in vitro, we tested the antigen-specific desensitization and disease-modifying potential of these formulations in an in vivo sublingual immunotherapy (SLIT) mouse model similar to that described by Hesse et al. (Allergy 2018, 73:862-874) for a subcutaneous immunotherapy (SCIT) mouse model.

[0115] Materials and Methods Bacterial particles: Lactococcus lactis BLP were prepared essentially as described in Example A1. Optionally, the BLP was formulated with vitamin D3 and / or D-mannose to a final concentration of 10 mg / mL BLP and 0.1 mg / mL vitamin D3 and D-mannose.

[0116] Antigen: Naturally purified Der p1 and Der p2 in a ratio of 50:1 were purchased from Citeq. In all experiments, untreated LPS-free OVA grade V protein (Sigma Aldrich) was used as antigen.

[0117] Animals: BALB / cByJ mice were purchased from Charles River Laboratories at 6-8 weeks of age and housed in individually ventilated cages (IVCs).

[0118] Sensitization of mice: All mice were injected intraperitoneally on days 1 and 15 with 5 μg crude extract HDM (Citeq) adsorbed to 2.25 mg Alum (Imject, Pierce) or 25 μg OVA in 100 μL PBS.

[0119] SLIT procedure: Groups of eight mice were treated sublingually with OVA-BLP (ratio 1:1 or 5:1 or 1:5) or Der p1 / 2-BLP (ratio 1:1 or 5:1 or 1:5) or PBS buffer, with a maximum corresponding dose of 100 μg free OVA or 100 μg free Der p1 / 2 (50:1), or a maximum corresponding dose of 100 μg free BLP, five times a week for 8 weeks (from day 29 to day 82). SLIT was performed by holding the mouse by the scruff of the neck and applying 2×5 μL of each treatment under the tongue. The mice were immobilized for an additional 20 s after each administration to prevent them from immediately swallowing the solution. The formulations used in this study are summarized in Table 4.

[0120] [Table 4]

[0121] Challenge: Two weeks after the SLIT procedure, mice were challenged by intranasal administration of antigen in PBS (total volume 25 μL) and by intranasal introduction of 25 μg of HDM or OVA on days 94, 96, and 98. Airway responsiveness was then determined and bronchoalveolar lavage fluid (BALF), lungs, and blood were collected and stored for analysis.

[0122] Ear Swelling Response (ESR): To test for allergic sensitization, ear swelling tests (EST) were performed before and after SCIT treatment to evaluate the early phase response to HDM.

[0123] Measurement of airway hyperresponsiveness to methacholine: Airway hyperresponsiveness (AHR) was assessed by measuring airway resistance (R, cmH2O.s / mL) and lung compliance (C, mL / H2O) to increasing doses of intravenous methacholine (Sigma-Aldrich). AHR was expressed as the effective dose (ED3) of methacholine required to induce an R of 3 cmH2O.s / mL.

[0124] Bronchoalveolar lavage fluid (BALF): The lungs were lavaged and cytocentrifugation preparations were made according to Hesse, Nawijn, Inflammation: Methods and Protocols (2017, New York, NY: Springer New York; 2017:137-168).

[0125] T cell response: restimulation of lung cells. Single cell suspensions of lung (5 × 10 5 Cells (1000 x 1000 cells / well) were stimulated with 0 or 10 μg of DerP1 / 2 per well in RPMI 1640 for 5 days, and supernatants were stored in triplicate (-80°C). IL-5, IL-10, IL-13, and IFN-γ concentrations were determined by ELISA according to the manufacturer's instructions (BD Pharmingen).

[0126] Analysis of cytokine levels in lung tissue: The right upper lobe was used for measurement of total protein, and concentrations of IL-4, IL-5, IL-10, IL-13, IL-17, IL-33, IFN-γ, TGF-β, eotaxin / CCL11, TARC / CCL17, and MIP3-α / CCL20 were measured using the MILLIPLEX Map Kit (Merck Millipore) and analyzed according to the manufacturer's protocol.

[0127] Antigen-specific immunoglobulins: Blood was collected at several time points during the experiment (pre- and post-serum). Mouse sera were analyzed by ELISA for levels of house dust mite (HDM)-spIgE, Der p1-spIgE and p2-spIgE, Der p1-spIgG1 and Der p1-spIgG2a, or OVA-spIgE, OVA-spIgG1, and OVA-spIgG2a. Briefly, polystyrene high-binding 96-well flat-bottom plates (Immulon2HB, VWR International) were coated with 100 μL / well of antigen (100 μg / mL) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) by overnight incubation at 4°C. The plates were washed three times with 200 μL / well of PBS containing 0.5% Tween 20 (PBST) (Automatic Plate Washer, ELX405, BioTek Instruments Inc.) and the wells were blocked with 200 μL / well of 3% Tween in PBS for 1.5 h at room temperature. The plates were washed three times with PBST and sera diluted 100-fold in PBST were added in triplicate at 100 μL / well and incubated at room temperature for 2 h. After washing three times with PBST, 100 μL of alkaline phosphatase-conjugated secondary antibody (whole molecule, Sigma) diluted 8000-fold was added to the plates and incubated at room temperature for 1 h. The plates were washed and a solution of polynitrophenol-phosphatase substrate (Sigma, 1 mg / mL) in diethanolamine buffer (pH 9.8) (100 μL / well) was added and incubated at room temperature in the dark. Controls included wells without serum, as well as wells with positive (pooled day 35 sera with high antibodies) and negative serum (pooled serum from day 10). The optical density (OD) of the wells was measured at 405 nm using an ELISA plate reader (EL808, BioTek Instruments Inc.) when the OD of the positive control reached 1. Net absorbance values ​​were calculated for all samples using the instrument software, which corrects for non-specific binding effects by subtracting the blank absorbance (no serum) value from the absorbance value of the wells with antigen.The mean OD of each triplicate test serum was expressed as a percentage of the positive control (% positive) as follows: % OD change = [sample OD / (positive control OD-negative control OD)]. Statistical analysis: All data are expressed as mean ± SEM. Mann-Whitney U test was used to analyze the results, and P < 0.05 was considered significant. Among AHR measures, generalized estimating equation (GEE) analysis was used using SPSS Statistics 20.0.0.2.

[0128] result Having established that bacterial particles can be used to efficiently target antigens to oral APCs and that they have a tolerizing effect on patient-specific T cells, these formulations were tested in a preclinical SLIT model using mice sensitized with OVA or HDM. These mice exhibit severe AHR, i.e., lung inflammation with cellular infiltration, and systemic OVA or HDM-specific Th2 immune responses. Mice sensitized with ovalbumin or house dust mite were treated sublingually with PBS, OVA or Der p1 / 2 alone, BLP alone, or OVA or Der p1 / 2 formulated with BLP, 5 days per week for 8 weeks. OVA or Der p1 / 2 formulated with BLP was in the presence and absence of vitamin D3 and D-mannose. Sublingual treatment with OVA-BLP or Der p1 / 2-BLP reduced AHR and eosinophil infiltration in all animals tested, which was most pronounced at a 1:1 antigen:BLP ratio. Furthermore, a trend towards more pronounced AHR and eosinophil infiltration was observed when vitamin D3 and D-mannose were present in the formulation. In contrast, treatment with soluble OVA or Der p1 / 2 alone had only a moderate effect on AHR. This hierarchy was similar to cytokine measurements in animals treated with OVA-BLP or Der p1 / 2-BLP, which showed a marked suppression of type 2 cytokine levels in the lung and BALF and an increase in antigen-spIgG responses. The levels of antigen-specific IgG1 after SLIT negatively correlated with the levels of IL-5, IL-13, and CCL20 after challenge, indicating a protective role for this neutralizing antibody response in the mouse model. Furthermore, a higher production of IL10 and TGF-β was seen in response to antigen compared to animals treated with PBS or particles alone, indicating an immune shift and induction of a regulatory tolerogenic response with the combination.

[0129] conclusion Successful SLIT using HDM-BLP and OVA-BLP shifted Th2 responses in lung tissue and BAL fluid toward a more protective Th1 / T regulatory profile that suppressed antigen-specific responses via the production of IL-10 and TGF-β, thereby suppressing AHR and eosinophil infiltration after challenge.

[0130] Example C2: In vivo induction of antigen-specific tolerance in antigen-sensitized wild-type piglets by bacterial particles combined with antigen Introduction Since it was demonstrated that bacterial particles can be used to efficiently desensitize antigen-sensitized mice and the piglet oral mucosal system is known to be highly similar to that of humans, the tolerance-inducing ability of these formulations delivered in mucoadhesive patches was tested in an in vivo piglet food allergy model.

[0131] Materials and Methods Bacterial particles: Lactococcus lactis BLP was prepared essentially as described in Example A1. Optionally, the BLP was formulated with vitamin D3 and / or D-mannose to a final concentration of 30 mg / mL BLP and 0.1 mg / mL vitamin D3 and D-mannose.

[0132] Antigen: In all experiments, untreated LPS-free OVA grade V protein or Ovm protein (Sigma Aldrich) was used as antigen. A stock solution of 1 mg / mL in PBS was prepared.

[0133] Mucoadhesive patch: A mucoadhesive patch containing PSF nanoreservoirs was prepared as described in Example A2. The resulting mucoadhesive PSF patch had a size of 0.5 cm 2 BLP and OVA or Ovm were added to a final concentration of 0.1 mg BLP and / or 5 μg vitD3-mannose and / or 1 μg OVA or Ovm per patch. The mucoadhesive patches used in the study are summarized in Table 5.

[0134] [Table 5]

[0135] Sensitization of pigs: Outbred, naive Landrace-Yorkshire piglets were used in a split-litter design to induce hypersensitivity to OVA or Ovm according to the protocols described by Rupa et al. (Int Arch Allergy Immunol. 2008, 146:11-18) and Rupa et al. (Vet Immunol Immunopathol. 2011, 140:23-29). Pigs were bled via the retro-orbital sinus at 10 days of age to obtain pre-sensitization serum and randomly assigned to four treatment groups (n=6 / group). Three groups were intraperitoneally injected with 100 or 200 μg OVA or 100 μg Ovm along with various doses (10, 25, or 50 μg) of cholera toxin (CT, List Biological Laboratories, Inc.) in 200 μL of phosphate buffered saline (PBS, 0.1 M, pH 7.4) on days 14, 21, and 35. Group 4 (control) received 50 μg CT alone. To detect the progression of immune responses, blood was collected for serum on days 21, 35, and 45. After clotting at room temperature for 1 hour, individual sera were collected and stored at −20° C. for detection of IgG (whole molecule)-associated antibodies against OVA or Ovm. For PCA reactions, individual sera from the same litter were pooled in equal amounts (separately sensitized controls were pooled). Animals were treated with an oromucosal PSF patch applied to the sublingual gland or buccal oral epithelium on days 45, 52, 59, 66, 73, and 80. After challenge, pigs were monitored (0–60 min) for clinical signs of allergy.

[0136] Challenge: On day 92, animals were orally challenged with 40 mL of yogurt slurry containing 3:2 (v / v) egg white:yogurt.

[0137] Serum analysis: Pig sera were analyzed for anti-OVA or anti-Ovm antibodies by ELISA. Briefly, polystyrene high-binding 96-well flat-bottom plates (Immulon2HB, VWR International) were coated with 100 μL / well of OVA or Ovm (100 μg / mL) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) by overnight incubation at 4°C. Plates were washed three times with 200 μL / well of PBS containing 0.5% Tween 20 (PBST) (Automatic Plate Washer, ELX405, Biotek Instruments Inc.) and wells were blocked with 200 μL / well of 3% Tween in PBS for 1.5 h at room temperature. Plates were washed three times with PBST and sera diluted 1:100 in PBST were added in triplicate at 100 μL / well and incubated at room temperature for 2 h. After washing three times with PBST, 100 μL of alkaline phosphatase-conjugated rabbit anti-pig IgG (whole molecule, Sigma) diluted 1:8000 was added to the plate and incubated for 1 h at room temperature. The plate was washed and a solution of polynitrophenol-phosphatase substrate (Sigma, 1 mg / mL) in diethanolamine buffer (pH 9.8) (100 μL / well) was added and incubated at room temperature in the dark. Controls included wells without serum, as well as wells with positive (pooled day 35 sera with high antibodies) and negative sera (pooled sera from day 10). When the optical density (OD) of the positive control reached 1, the OD of the wells was measured at 405 nm using an ELISA plate reader (EL808, BioTek Instruments Inc). Net absorbance values ​​were calculated for all samples using the instrument software, which corrects for non-specific binding effects by subtracting the blank absorbance (no serum) value from the absorbance value of wells with OVA or Ovm antigen. The mean OD of each triplicate test serum was expressed as a percentage of the positive control (positive rate) as follows: % OD change = [sample OD / (positive control OD-negative control OD)]. Skin hypersensitivity: To test for immediate active hypersensitivity reactions, intradermal skin tests were performed on all animals on day 10 pre-sensitization and days 21, 35, 46, 67, and 88 post-sensitization. Injection sites on the inner thighs were marked with an indelible ink pen. For each skin test, 100 μL (100 μg) of OVA or Ovm in PBS was injected intradermally using a tuberculin syringe and a 25-gauge needle. The negative control was PBS. All intradermal tests (serum and PBS) were performed and read simultaneously. One investigator performed all injections. 15 minutes after injection, at least two observers independently examined each site for swelling and redness responses, and consensus was reached for positive or negative results.

[0138] Clinical scores: Following induction by oral mucosal challenge on day 92, hypersensitivity responses were assessed using a scoring system. Responses were scored visually by at least three blinded observers at 0-60 min after oral challenge. Each pig was evaluated individually and scored as follows: 0 = no signs; 1 = immobility, lethargy, lethargy, scratching, rash; 2 = diarrhea, vomiting; 3 = increased respiratory rate, neck extension; 4 = forced expiration; 5 = other signs (variable). A total score was obtained by summing the individual scores assigned to each animal.

[0139] Passive cutaneous anaphylaxis: Serum was pooled from treatment groups (CT-treated control and sensitized) and aliquots were heated at 56°C for 4 hours to inactivate putative IgE-anti-OVA or anti-Ovm PCA-mediated antibodies. Serum was injected intradermally in duplicate at marked sites on the inner thigh of naive pigs in a volume of 100 μL. Twenty-four hours later, 5 mg of OVA or Ovm was injected in 1.0 mL of sterile PBS into the dorsal ear vein using a 23-gauge needle. The skin injection sites were examined by at least three observers for the development of a reaction of swelling or redness. Serum from sensitized pigs in each litter was also examined individually for PCA responses and each site was graded based on relative severity (+, ++, +++, ++++).

[0140] Analysis: Data were analyzed using InStat Package 4.0 (GraphPad Software Inc). Confidence intervals were set at at least 95% for significance assessment. Comparisons were made between serum IgG-anti-OVA or anti-Ovm on days 10 and 45, and between control (CT) and sensitized (OVA or Ovm) antibodies. Primary data for comparison of detected antibodies were analyzed by one-way ANOVA test with animals as experimental units followed by Tukey's multiple comparison test for significant differences between treatment groups. Relationships between PCA responses and clinical scores were evaluated by linear regression and Pearson correlation analysis (GraphPad Prism, version 4.0).

[0141] result Having established that bacterial particles can be used to efficiently target antigens to oral APCs and that they have a tolerizing effect on patient-specific T cells, these formulations were tested in a preclinical model using piglets sensitized with OVA or Ovm. These piglets exhibit severe AHR, i.e., lung inflammation with cellular infiltration, and a systemic OVA or Ovm-specific Th2 immune response. Ovalbumin or ovomucoid-sensitized piglets were treated orally with PBS, OVA or Ovm, BLP alone, or OVA or Ovm formulated with BLP, delivered via a mucoadhesive PSF patch once a week for 6 weeks.

[0142] Oral mucosal treatment with OVA or Ovm-loaded BLP administered with mucoadhesive PSF patches dramatically reduced AHR in all animals tested, whereas treatment with soluble OVA or Ovm alone had only a modest effect on AHR. OVA or Ovm-loaded BLP had a modest effect as measured by cellular responses assessed in mediastinal and cervical lymph nodes harvested from piglets after 6 weeks of oromucosal administration. This hierarchy was similar to cytokine measurements from cervical lymph nodes of animals treated with mucoadhesive OVA-BLP or Ovm-BLP, which showed lower IL-13 and IL-5 production and higher IL10 production in response to antigen compared to animals treated with PBS or particles alone, indicating immune shift and induction of a regulatory tolerogenic response with the combination.

[0143] conclusion In a human-like food allergy model, oral mucosal delivery of antigens by the BLP mucoadhesive PSF patch was superior to antigen alone in controlling immune responses in a short period of time and leading to a tolerogenic effect, and the tolerogenic effect was further enhanced in the presence of vitamin D3 and D-mannose.

[0144] Example D1: Induction of tolerance to insulin following oromucosal administration of BLP in combination with said autoantigens Introduction Autoimmunity is characterized by spontaneous inflammatory tissue damage and impaired physiological function due to loss of tolerance to self-antigens. It is partly associated with an overactive immune system, which is characterized by an excess of helper T (Th) cells. Predisposing factors such as susceptibility genes and environmental factors are difficult to govern, and therefore recent efforts to develop immunotherapy have been focused on re-establishing a functional balance between the two by reducing pathogenic effector cells and / or increasing immunoregulatory T cells. Autoimmune destruction of pancreatic islet β cells is the primary cause of type 1 diabetes (T1D). This destruction is associated with cellular and humoral immune responses against several β cell autoantigens, both of which may precede the clinical manifestations of the disease.

[0145] Here, oral mucosal delivery of autoantigens in combination with bacterial particles induces antigen-specific CD4 + We demonstrate that diabetes-specific immune responses are suppressed through the induction of regulatory T cells.

[0146] Materials and Methods Bacterial particles: Lactococcus lactis BLP were prepared essentially as described in Example A1. BLP were formulated with vitamin D3 (Sigma Aldrich) and / or D-mannose (Sigma Aldrich) to a final concentration of 10 mg / mL BLP, 0.1 mg / mL vitamin D3 and D-mannose, and / or 10 mg / mL antigen. The formulations used are summarized in Table 6. Antigen: insulin peptide fragment InsB 9-23

[0147] [Table 6]

[0148] mouse Non-obese female and male diabetic (NOD) mice and NOD-severe combined immunodeficiency (SCID) (Balb / c background) mice were purchased from Jackson Laboratory. Balb / c wild-type (WT) mice were purchased from Charles River Italy. Mice were maintained in a specific pathogen-free central animal facility. Mice were handled and used in accordance with institutional guidelines.

[0149] Experimental setup In a therapeutic setting, BLP-InsB 9-23 (or BLP as a control) was administered sublingually to diabetic NOD mice five times a week for eight consecutive weeks. One week after the end of the SLIT procedure, mice were sacrificed and analyses were performed.

[0150] SLIT procedure: Groups of eight NOD female mice exhibiting stable diabetes and hyperglycemia (12-23 weeks) were injected with BLP-InsB 9-23Solution (100 μg BLP and 100 μg InsB per dose) 9-23 ) or BLP (100 μg) and PBS control were administered sublingually 5 times per week. SLIT was performed by holding the mouse by the scruff of the neck and applying 2 × 5 μL of each treatment under the tongue. After each administration, the mouse was restrained for an additional 20 seconds to prevent the mouse from immediately swallowing the solution. As a positive (tolerization) control group, diabetic NOD mice were treated as described by Bresson et al. (J. Clin Invest. 2006, 116:1371-1381). Complete remission was defined as disappearance of glucose in urine and return to normoglycemia.

[0151] In the setting of syngeneic islet transplantation, female NOD mice that recently developed diabetes were treated with BLP-InsB 9-23 or BLP as a negative control for 4 weeks. After 4 weeks, 500 freshly isolated islets from nondiabetic NOD mice were transplanted into diabetic NOD mice. Blood glucose was then monitored three times a week until recurrence of diabetes or until 15 weeks after grafting. Animals with glucose levels ≥ 250 mg / dL on two consecutive occasions were considered diabetic and were sacrificed, serum collected, and grafts analyzed histologically.

[0152] The exact mechanism of tolerance induction was analyzed in vitro and in vivo by adoptive T cell transfer into NOD-SCID mice after rechallenge of NOD mice with specific self-antigens.

[0153] Diabetes detection Glucose monitoring: Urinary glucose was measured using Diastix (Miles) and confirmed by blood glucose measurement using a blood glucose monitoring system, One Touch Ultra (LifeScan Inc.). Diabetes was defined as two consecutive blood glucose readings of 250 mg / dL or higher.

[0154] Insulitis: Mice were sacrificed by CO2 asphyxiation, pancreases were fixed overnight in 10% formalin, embedded in paraffin, and serial 5 μm sections were stained with hematoxylin and eosin. Insulitis scores (mean ± SD) were determined by microscopic grading of the degree of cellular infiltration in 10-15 islets per mouse as follows: 0, no visible signs of islet infiltration; 1, peri-islet infiltration; 2, less than 50% infiltration; 3, more than 50% infiltration. Islet isolation and transplantation: After aseptic removal of islets from 14-21 day old donor NOD mice without insulitis or diabetes, islets were isolated by digesting the pancreatic glands with collagenase in Hanks' balanced salt solution with vigorous shaking. Isolation of islets was performed by direct manual picking under a stereomicroscope. Recipient diabetic NOD mice were anesthetized by intraperitoneal injection of Avertin (0.02 mL / g BWT), the left kidney was exposed through a lumbar incision, and 500 freshly isolated islets were administered under the renal capsule.

[0155] Immunohistochemistry: To detect insulin, CD4, and CD8 expression in pancreatic β-cells, primary antibodies (guinea pig anti-pig insulin (1:300 dilution) from Dako, anti-CD4 RM4.5 (BD Biosciences), and anti-CD8a IHC (1:50 dilution) from BD Biosciences) were applied to frozen tissue sections as described by Christen et al. (Diabetes 2004, 53:591-596).

[0156] In vitro proliferation assay Prepare single cell suspensions of spleen, mesenteric lymph nodes (MLN), and PLN. Proliferation assay of total spleen cell populations, 2 x 10 5 The cells were treated alone or with graded concentrations (1-100 μg / mL) of purified human insulin or a peptide specific for CD4 T cells (InsB 9-23 , H-2 d or g Restrictive) or CD8 T cell specific peptide (InsB 15-23 , K dThe cells were cultured in 96-well U-bottom plates in a total volume of 200 μL of complete medium with CD3+ (Sigma) or with or without anti-IL-10 or anti-TGF-β neutralizing monoclonal antibodies. Neutralizing antibodies were added at 1, 0.1, and 0.01 μg / mL. + T cells, CD8 + T cells, CD4 + T cells and CD4 + CD25 - For T cell population proliferation assays, 0.2 x 10 5 T cells were then subjected to insulin or GAD65 or CD4 + or CD8 + 1 × 10 of WT Balb / c mice loaded with specific peptides to T cells 5 Irradiated splenocytes were cultured in 96-well U-bottom plates with or without neutralizing antibodies in a total volume of 200 μL of complete medium. After 72 hours at 37°C in a humidified incubator with 5% CO2, 1 μCi / well of 3 Proliferation was assessed by adding [H]thymidine. After 16–18 h, DNA-bound radioactivity was harvested onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation was measured in a scintillation counter (Perkin Elmer). T cells were expressed as CD3 + , CD4 + , or CD8 + CD4 cells were purified from PLN or spleen by negative selection with magnetic bead separation using an isolation kit (MACS, Milteny Biotec, Auburn, CA). + T cells were used as whole cells or CD25 + CD25 was isolated by MACS using an isolation kit (Milteny Biotec). + and CD25 - The purity of the cell population (>90%) is determined by flow cytometry analysis.

[0157] For cytokine measurements, the supernatants of the cell cultures used in the various proliferation assays (antigen-specific stimulation) described above were harvested after 72 hours of culture and frozen at -80°C until cytokine analysis was performed. Cytokine production was quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, CA, USA). + T cells, CD4 + T cells, or CD8 + T cells were cultured and non-specifically stimulated in vitro with an anti-CD3 / anti-CD28 mix (1 μg / mL each) for 24 h or left unstimulated as a control. Supernatants were harvested and analyzed for IL-10, IL-4, IL-5, and IFN-γ production using a BD™ Cytometric Bead Array flex set on a BD FACSArray Bioanalyzer with FCAP array software (BD Biosciences). TGF-β1 was determined using a capture ELISA experiment using the Quantikine kit (R&D Systems).

[0158] In vitro T cell proliferation inhibition assay 2 × 10 isolated from a recently diabetic female NOD (8-12 weeks old) 4 Purified whole spleen CD4 + CD25 - T cells were cultured in WT Balb / c mice with 2 × 10 T cell-depleted and irradiated insulin or peptide-loaded 4 In the presence of 1000 splenocytes, various numbers of CD8 + T cells, CD4 + T cells and CD4 + CD25 - After 72 hours at 37°C in a humidified incubator with 5% CO2, 1 μCi / well of 3Proliferation was assessed by adding [H]thymidine. After 16–18 h, DNA-bound radioactivity was harvested onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation was measured in a scintillation counter (Perkin Elmer).

[0159] In vitro cytotoxicity assay The lymphoblast targets used were ConA-activated splenocytes from BALB / c mice. 6 100 μCi of target cells 51 The cells were labeled with Cr (Amersham International, Buckinghamshire, UK) at 37°C for 90 minutes, washed three times, and then incubated with 1 μg / ml of the peptide (InsB 15-23 The target cells were washed twice and plated at 10 per well. 4 CD8 cells isolated from spleen, MLN, and PLN were seeded. + T cells were added to each well in triplicate at various effector:target (E:T) ratios. Plates were centrifuged at 500 rpm for 2 minutes and incubated at 37°C for 4 hours. After incubation, 51 Supernatants were harvested to determine Cr release [% lysis = 100 × (test cpm - spontaneous cpm) / (total cpm - spontaneous cpm)]. For indirect killing assays, CD8 + T cells were incubated with 5 μg / mL of anti-CD3 antibody (clone 145-2C11, Pharmingen).

[0160] Adoptive transfer of diabetes Eight- to ten-week-old NOD-SCID mice were inoculated with graded numbers of bead-purified CD3 + T cells, CD8 + T cells, CD4 + T cells, CD4 + CD25 - , or CD4 + CD25 +2 × 10 splenocytes isolated from diabetic female NOD mice (6 weeks, 12 weeks, and 18 weeks) in combination with or without T cells. 7 or 5 × 10 6 The mice were intraperitoneally injected with 100 mg / kg of 1000 mg ...

[0161] result BLP-InsB 9-23 Delays relapse of diabetes after syngeneic islet transplantation. To assess whether BLP-InsB(9-23) induces oral tolerance, relapse of diabetes after syngeneic islet transplantation was studied. Therefore, mice were treated with oral mucosa as described above (experimental set-up) and transplanted with pancreatic islets as described above (isolation and transplantation). Relapse of diabetes was associated with increased expression of InsB 9-23 was delayed in the group compared with the controls.

[0162] BLP-InsB 9-23 free InsB in non-obese diabetic mice 9-23 To study the induction of oral tolerance, the oral mucosa of mice was treated as described above (experimental setup). 9-23 The autoantigen-specific proliferation response of splenocytes was significantly increased in the control and free InsB 9-23 The BLP-InsB 9-23 Sublingual administration of significantly enhanced the induction of tolerance to self-antigens.

[0163] BLP-InsB 9-23 enhanced oral tolerance with reduced insulitis, reduced rate of β-cell destruction, and increased IL-10 production by splenocytes. To study the induction of oral tolerance, mice were orally fed as described above (experimental setup). The presence of insulitis, rate of β-cell destruction, and cytokine production in response to the autoantigens were determined as described above. Histological analysis revealed that BLP-InsB 9-23 In the control group and the free InsB 9-23The results show a significant reduction in insulitis and beta cell destruction as well as an increase in IL-10 production compared to the control group.

[0164] BLP-InsB 9-23 CD4 + To assess whether CD4+ T cells mediate the induction of oral tolerance, we studied autoantigen-specific proliferative CD4+ T cell responses in pancreatic cells and lymph nodes. Thus, mice were orally fed as described above (experimental setup) and autoantigen-specific CD4+ T cell responses were proliferated as described above (in vitro amplification assay). + T cell proliferation was assessed. 9-23 BLP-InsB compared with the group 9-23 Autoantigen-specific CD4 T cell responses in the groups.

[0165] Autoaggressive CD8 in NOD mice + The response is BLP-InsB 9-23 Our combinatorial approach allows the suppression of CD4 T cells, which can control diabetes through bystander suppression mechanisms. + To examine whether T cells are induced, CD8 + The effect on autoaggressive T cells was analyzed. Antigen-specific autoaggressive CD8 + The percentage and / or activity of cells expressing BLP-InsB 9-23 This was significantly reduced after therapy.

[0166] BLP-InsB 9-23 After therapy, antigen-induced T regulatory cells can confer protection from autoimmune-like responses in vivo. To test active suppression of diabetes-like responses in mice treated with the oral tolerance protocol, splenocytes were adoptively transferred from the other treatment groups as described above (adoptive transfer of diabetes). Control and free InsB 9-23 Compared with the BLP-InsB group, diabetes-like responses were 9-23 The regulatory CD4 + It showed activation of T cells.

[0167] conclusion Oral mucosal delivery of autoantigens by bacterial particles enhances immune responses in autoimmune diseases by targeting antigen-specific CD4 + We demonstrate that it is suppressed in the oral mucosa via the induction of regulatory T cells.

[0168] Example D2: Induction of tolerance to gliadin following oral administration of BLP in combination with the autoantigen Introduction Celiac disease is caused by loss of tolerance to gliadin in the diet and is mediated by HLA-DQ2 or HLA-DQ8 restricted T cell responses. Effective treatment can only be achieved by a lifelong socially restricted diet that excludes foods containing gliadin, which is present in the proteins of wheat or related cereals such as rye or barley. Although a strict gluten-free diet heals the gut, intolerance to gluten is permanent and better treatment options are needed. For celiac disease, the trigger (gluten protein gliadin), genetic association (HLA-DQ2 or HLA-DQ8), and highly specific humoral responses have been well characterized. Because disease activity strongly correlates with the presence and amount of antigens, antigen-specific induction of oral tolerance (SLIT) using sublingual delivery of drugs is an attractive therapeutic approach. Oral tolerance is mediated by multiple mechanisms, including anergy, deletion of antigen-specific effector T cells, and / or active suppression by regulatory T cells (Tregs).

[0169] Here, we demonstrate that oral delivery of deaminated gliadin peptides in the presence of BLP inhibits NOD AB 0 We will investigate whether suppression of systemic DQ8-specific T cell responses in DQ8 transgenic mice can be induced, providing a method for inducing DQ8d antigen-specific tolerance.

[0170] Materials and Methods Bacterial particles: Lactococcus lactis BLP were prepared essentially as described in Example A1. Optionally, BLP were formulated in PBS with Vitamin D3 (Sigma Aldrich) and D-mannose (Sigma Aldrich) to a final concentration of 10 mg / mL BLP and 0.1 mg / mL Vitamin D3 and D-mannose, and / or 10 mg / mL antigen.

[0171] Antigen: Deaminated DQ8d peptide GAPVPYPDPLEPRQYPSGEGSFQPSQENPQA was purchased from Genscript.

[0172] The oral formulations used in the study are summarized in Table 7.

[0173] [Table 7]

[0174] mouse NOD AB 0 DQ8 transgenic mice and NOD AB 0 Mice were purchased from Mayo Clinic. Mice aged 7-16 weeks were used in the experiments. Mice were weaned, maintained on a gluten-free diet, and kept in a conventional animal facility until they reached the age of 8-12 weeks. Mice were treated and used in accordance with national guidelines.

[0175] Experimental setup NOD AB fed a gluten-free diet 0DQ8 mice were sensitized on day 1 by subcutaneous injection of 100 μg of DQ8d peptide at the base of the tail in 100 μL of 1:1 CFA (Complete Freund's Adjuvant, purchased from Difco, Becton, Dickinson and Company) saline. The peptide used for sensitization had the same sequence as the peptide used for treatment. Mice were used for the experiments at 7-16 weeks of age. Mice were weaned, maintained on a gluten-free diet, and kept in a conventional animal facility until they were 8-12 weeks of age. Groups of 8 mice per treatment group were used. Mice were administered PBS as a negative control, and the formulations shown in Table 7. Mice were administered sublingually 100 μg of DQ8d and / or 100 μg of BLP per dose, or PBS buffer, 5 times a week for 8 weeks. SLIT was performed by holding the mouse by the scruff of the neck and applying 2×5 μL of each treatment under the tongue. Mice were immobilized for an additional 20 seconds after each dose to prevent them from immediately swallowing the solution. Antigen-specific DTH responses were assessed four times at weekly intervals starting one week after SLIT treatment. DTH measurements were performed 24 hours thereafter. For antigen-specific DTH response measurements, baseline ear thickness was measured using an engineer's micrometer (Mitsutoyo). Mice were then injected into the ear with 10 μg of DQ8d in 10 μL of saline. Ear thickness was remeasured in a blinded manner 24 hours after challenge. DTH responses were expressed as the difference between baseline ear thickness and ear thickness 24 hours after injection of DQ8d. Mice were then sacrificed and spleens and lymph nodes were harvested to assess DQ8d-specific proliferation of cells and cytokine production.

[0176] Isolation of lamina propria cells: Mice were anesthetized with CO2 inhalation, and the small intestine was removed and cut into 2 cm long pieces longitudinally and transversely using a scalpel blade. The tissue was washed six times with CMF / HEPES solution (1x Hanks balanced salt solution, 15 mM HEPES, 2% FBS) and then incubated for 1 h in CMF / FBS / EDTA solution (1x Hanks balanced salt solution, 10% FBS, 15 mM HEPES, 5 mM EDTA, 100 μg penicillin / streptomycin) to remove epithelial cells. It was then digested with collagenase (100 U / mL) for 1 h to release lymphocytes from the lamina propria.

[0177] Cell culture, proliferation, and cytokine production assays: On day 11 of the experiment, spleen and lymph node cell suspensions were prepared by homogenizing the tissue with a tissue grinder (VWR International Inc.) in 1× PBS. Red blood cells were removed from the spleen cell suspension by incubation with ammonium chloride / potassium chloride lysis buffer. 5×10 cells were cultured at 37° C. in a volume of 0.2 mL in RPMI 1640 (1.5% HEPES, 1% Penstrep, and 10% FBS) supplemented with medium alone, 10 μg / mL ConA, 50 μg / mL irrelevant peptide, or 50 μg / mL DQ8d epitope-containing supplement. 5 Cells were incubated in 96-well microtiter plates at 10 cells / well. In separate experiments, IL-10, TGF-β, IL-10 and TGF-β, or LAP neutralizing antibody were added to splenocytes from treated mice. After 24 hours, 1 μCi / well of [ 3Proliferation was assessed by the addition of [H]thymidine. DNA-bound radioactivity was harvested onto glass fiber filter mats (Perkin Elmer) and thymidine incorporation was measured in a scintillation counter (Perkin Elmer). Results were expressed as mean counts per minute (CPM) of triplicate wells. For neutralizing antibody proliferation assays, results were expressed as the percentage of proliferation of the treated group compared to the PBS-treated group. For cytokine measurements, supernatants of cell cultures used in the various proliferation assays described above were harvested after 24 hours of culture and frozen at -20°C until cytokine analysis was performed. Cytokine production was quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences).

[0178] T cell proliferation assay using lamina propria cells: Lamina propria cells were isolated from SLIT treatment. These cells (5×10 6 The cells (cells / mL) were incubated with medium (RPMI, Sigma-Aldrich), DQ8d epitope (50 μg / mL) or irrelevant peptide (50 μg / mL) for 24 h and then incubated for an additional 24 h. 3 [H] thymidine was added, and the cells were then harvested in the same manner as the splenocytes.

[0179] Flow cytometry analysis: Spleen and cervical lymph node tissues from SLIT-treated mice were isolated, prepared as described above, and stained for CD4, CD25, and Foxp3. Intracellular staining for Foxp3 was performed according to the manufacturer's instructions (eBiosciences) and measured by flow cytometry on a Becton Dickinson FACSCaliburs. Cells were CD4 + CD25 + and CD4 + CD25 - Subpopulations were gated and within these populations the mean fluorescence intensity (MFI) was determined using Foxp3 histograms.

[0180] T cell phenotyping: CD4 and CD25 antibodies were purchased from BD Biosciences. APC anti-Foxp3 staining kit was purchased from eBiosciences. Anti-IL-10 neutralizing monoclonal antibody (1 μg / mL, clone JES052A5), TGF-β neutralizing monoclonal antibody (1 μg / mL, clone 1D11), and LAP (latency associated peptide) neutralizing antibody (1 μg / mL, clone 27235) were obtained from R&D Systems.

[0181] Statistical analysis: Results of cytokine measurements are expressed as mean ± SEM. DQ8d-specific proliferation, ear thickness, and cytokine measurements were tested for significance by one-way ANOVA followed by student's t-test comparison to determine differences between individual groups. A P value of less than 0.05 was used to indicate statistical significance for all tests.

[0182] result Inhibition of DTH and proliferation responses by DQ8d-BLP. NOD AB 0 DQ8 transgenic mice were treated with SLIT for 10 consecutive days with DQ8d, BLP, Dq8d-BLP, and DQ8d-BLP-VM (with additional vitamin D3 and D-mannose) and PBS (as a negative control). On day 10, mice were injected with 10 μg of DQ8d into the ear, and ear thickness measurements were performed 24 hours later. Control mice (PBS) were clearly sensitized to DQ8d. Daily SLIT treatment with DQ8d-BLP significantly reduced the DTH response throughout the study period (P<0.05). Ear swelling was also significantly reduced in DQ8d-BLP-VM treated mice compared to controls (P<0.01), but was not statistically different from the Dq8d-BLP group. Ear swelling in the DQ8d and BLP groups was slightly reduced, but did not reach statistical significance compared to the PBS group. These data indicate that orally administered DQ8d-BLP, in the presence or absence of vitamin D3 and D-mannose, inhibited the proliferation and proliferation of immunized NOD AB mice. 0 We show that both antigen and BLP are required to suppress systemic inflammatory T cell responses in DQ8 transgenic mice and to induce significant tolerogenic effects.

[0183] The peripheral immune response was further analyzed by examining DQ8d-specific proliferation of splenic and draining cervical lymph node cells. Splenocytes from mice treated with PBS, BLP, or DQ8-BLP or DQ8-BLP-VM were isolated 11 days after SLIT, and DQ8d-specific proliferative responses were evaluated by ex vivo stimulation with DQ8d peptide or an irrelevant peptide. Splenocytes from sensitized mice showed high DQ8d-specific proliferative responses, which were significantly suppressed by daily SLIT treatment with DQ8-BLP or DQ8-BLP-VM compared to PBS-treated control mice, with a trend toward better suppression in DQ8-BLP-VM. No significant suppression was observed in the BLP group. However, because cervical lymph nodes are the primary antigen recognition site in this SLIT protocol, the proliferative capacity of these lymphocytes was also examined. Proliferation of cervical lymph node cells was significantly reduced in the DQ8d-BLP or DQ8d-BLP-VM treated groups compared with the PBS treated group, and there was no significant proliferation in the BLP group. In all three treatment groups, splenocytes and cervical lymph node cells showed no proliferative response when an irrelevant control peptide was added.

[0184] To determine whether there was antigen-specific suppression in lamina propria cells, these cells were isolated from mice treated with SLIT with DQ8d-BLP, DQ8d-BLP-VM, and mice treated with PBS alone or BLP. These cells were then treated in vivo with medium, DQ8d, or an irrelevant control peptide. A high background of lamina propria cell proliferation was observed in all three groups of treated mice. However, proliferation was suppressed by addition of the DQ8d peptide alone in mice treated with DQ8d-BLP or DQ8d-BLP-VM. Proliferation in the lamina propria was not abolished by addition of the irrelevant control peptide. This demonstrates that the suppression induced by SLIT treatment with DQ8d-BLP(-VM) is specific to the DQ8d peptide in the lamina propria.

[0185] To investigate the mechanism involved in the reduction of antigen-induced T cell proliferation, the cytokine profile of ex vivo stimulated splenocytes or inguinal lymph node cells was determined. Splenocytes stimulated ex vivo with DQ8d showed a significant upregulation of IL-10 and downregulation of IL-12 production only in the groups treated with DQ8d-BLP and DQ8d-BLP-VM compared to the negative control (PBS). Furthermore, DQ8d-BLP and DQ8d-BLP-VM treatment significantly reduced DQ8d-induced IFN-γ production in inguinal lymph nodes compared to the negative control (PBS)-treated mice. When splenocytes and inguinal lymph node cells were stimulated with an irrelevant peptide, there were no differences in cytokine levels across treatment groups. The addition of an irrelevant peptide also did not change the levels of IL-10 and IL-12p70 in the culture medium of the four treatment groups. In summary, the proliferation and cytokine data suggest that DQ8d-BLP(-VM)SLIT treatment significantly increased the proliferation and cytokine levels of NOD AB 0 These results demonstrate that T cell responses in DQ8 transgenic mice can be systemically suppressed in an antigen-specific manner.

[0186] The essential role of TGF-β and IL-10 in DQ8d-BLP-mediated suppression. The functional significance of TGF-β, IL-10, and LAP (membrane-associated TGF-β) on the DQ8d-specific splenocyte proliferative response of splenocytes from mice treated with DQ8d-BLP and DQ8d-BLP-VM compared to the PBS control group was assessed using neutralizing antibodies. Although individual neutralization of IL-10, TGF-β, or LAP did not significantly interfere with the reduced splenocyte proliferative response of mice treated with DQ8d-BLP and DQ8d-BLP-VM, the addition of a combination of neutralizing monoclonal antibodies for TGF-β and IL-10 completely abolished the reduced Dq8d-specific proliferative ability of splenocytes. These data strongly suggest that the suppression of T cell activation mediated by DQ8d-BLP or DQ8d-BLP-VM treatment is due to the interaction of IL-10 and TGF-β.

[0187] CD4 + CD25 + and CD4 + CD25 -Increased Foxp3 expression by T cells. To analyze the role of regulatory T cells (Treg) in the induction of tolerance induced by DQ8d-BLP or DQ8d-BLP-VM, CD4 + The expression of Foxp3 in T cell populations was examined by FACS analysis. Foxp3 expression was significantly higher in the spleens of mice treated with DQ8d-BLP and DQ8d-BLP-VM than in the control group (PBS). + CD4 + CD25 + Cells and Foxp3 + CD4 + CD25 - There was a significant increase in the number of cells, whereas the BLP or DQ8d groups showed no significant increase in the number of Foxp3CD4 + There was also a significant increase in the number of Foxp3 T cells in the oral draining lymph node tissues of mice treated with DQ8d-BLP and DQ8d-BLP-VM compared to mice treated with PBS, BLP, or DQ8d. + CD4 + CD25 - An increase in the number of cells was detected, but CD4 + CD25 + These data suggest that SLIT administration of DQ8d-BLP(-VM) upregulates Foxp3 + This suggests a strong association between the increase in T cells and the suppression of the immune response to gliadin.

[0188] conclusion Here, sublingual intravenous delivery (SLIT) of deaminated gliadin peptides in the presence of BLP was shown to enhance the expression of NOD AB 0 We demonstrate that NOD AB induces suppression of systemic DQ8-specific T cell responses in DQ8 transgenic mice, providing a method to induce DQ8d antigen-specific tolerance. Results were most prominent when BLP was co-formulated with vitamin D3 and D-mannose. Furthermore, this approach, in conjunction with non-viable bacterial particles, provides a way to deliver the correct antigen in an appropriate manner to the oral mucosal immune system, potentially becoming an effective and non-toxic treatment for celiac disease. 0Although DQ8d-BLP was used to induce suppression of systemic DQ8-specific T cell responses in DQ8 transgenic mice, the same results would be obtained if DQ2.5 or DQ2.2 were used in place of these antigens in similar animal models.

[0189] [Table 8]

[0190] [Table 9]

[0191] Further embodiments are as follows. [Embodiment 1] 1. A mucoadhesive carrier comprising a non-viable bacterial particle and an antigen for use in inducing immune tolerance to an antigen in a subject, wherein said mucoadhesive carrier is administered transmucosally to said subject. [Embodiment 2] The mucoadhesive carrier of embodiment 1, wherein the antigen causes or is associated with an immune response associated disease. [Embodiment 3] 3. The mucoadhesive carrier according to embodiment 2, wherein the immune response associated disease is selected from the group consisting of celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, or food allergy. [Embodiment 4] A mucoadhesive carrier according to any one of embodiments 1 to 3, wherein the antigen is selected from the group consisting of immunodominant epitopes of α-gliadin, hordein, house dust mites, immunodominant epitopes of peptides from feline saliva, skin or glands, and immunodominant epitopes of peptides involved in type 1 diabetes. [Embodiment 5] A mucoadhesive carrier as described in embodiment 4, wherein the immunodominant epitope of the house dust mites is Der p2.1, the immunodominant epitope of the peptide from cat saliva, skin or glands is Feld1, and the immunodominant epitope of the peptide involved in type 1 diabetes is pINS, GAD65, InsB9-23, or IA2. [Embodiment 6] The mucoadhesive carrier according to any one of embodiments 1 to 5, wherein the mucoadhesive carrier is administered to the oral mucosa. [Embodiment 7] 7. The mucoadhesive carrier according to any one of embodiments 1 to 6, which is a mucoadhesive patch, a biofilm, or a hydrogel. [Embodiment 8] 8. The mucoadhesive carrier of embodiment 7, which is a mucoadhesive patch comprising electrospun fibers. [Embodiment 9] The mucoadhesive carrier according to any one of embodiments 1 to 8, wherein the non-viable bacterial particles are derived from intestinal beneficial bacteria. [Embodiment 10] The mucoadhesive carrier according to embodiment 9, wherein the intestinal beneficial bacteria is Lactobacillus, Lactococcus, Bifidobacterium, or Escherichia coli Nissul. [Embodiment 11] The non-viable bacterial particles a) an acidic heat treated Gram-positive bacterium, preferably L. lactis; b) empty bacterial envelopes obtained by lysis of Gram-negative bacteria, preferably E. coli Nissl, or c) Chemically sterilized bacteria, preferably L. lactis, L. rhamnosus, or E. coli Nissl. The mucoadhesive carrier according to embodiment 9 or 10, selected from: [Embodiment 12] A mucoadhesive carrier according to any one of embodiments 1 to 11 for use as a medicament for treating humans and / or animals. [Embodiment 13] A non-viable bacterial particle derived from intestinal beneficial bacteria for use in inducing immune tolerance to an antigen in a subject, said non-viable bacterial particle being administered transmucosally to said subject in combination with said antigen, preferably said antigen causing an immune response associated disease or associated with an immune response associated disease. [Embodiment 14] 14. The non-viable bacterial particle of embodiment 13, wherein the intestinal beneficial bacteria is Lactobacillus, Lactococcus, Bifidobacterium, or Escherichia coli Nissul. [Embodiment 15] The non-viable bacterial particles a) an acidic heat treated Gram-positive bacterium, preferably L. lactis; b) empty bacterial envelopes obtained by lysis of Gram-negative bacteria, preferably E. coli Nissl, or c) Chemically sterilized bacteria, preferably L. lactis, L. rhamnosus, or E. coli Nissl. 15. The non-viable bacterial particle according to embodiment 13 or 14, selected from: [Embodiment 16] 16. The non-viable bacterial particle according to any one of embodiments 13 to 15, wherein the immune response-related disease is selected from the group consisting of celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, or food allergy. [Embodiment 17] A non-viable bacterial particle according to any one of embodiments 13 to 16, wherein the antigen is selected from the group consisting of immunodominant epitopes of α-gliadin, hordein, house dust mites, immunodominant epitopes of peptides from cat saliva, skin or glands, and immunodominant epitopes of peptides involved in type 1 diabetes. [Embodiment 18] A non-viable bacterial particle as described in embodiment 17, wherein the immunodominant epitope of the house dust mites is Der p2.1, the immunodominant epitope of the peptide from cat saliva, skin or glands is Feld1, and the immunodominant epitope of the peptide involved in type 1 diabetes is pINS, GAD65, InsB9-23, or IA2.

Claims

1. 1. Use of a mucoadhesive carrier for the preparation of a medicine, medical food or nutritional food for inducing immune tolerance in a subject, comprising: The mucoadhesive carrier is a patch comprising mucoadhesive nanofibers incorporated or loaded with non-viable bacterial particles and antigen-containing material; or a hydrogel or film of a water-swellable cross-linked mucoadhesive polymer impregnated or loaded with said non-viable bacterial particles and antigen-containing material; the non-viable bacterial particles are derived from harmless bacteria used in probiotics, The harmless bacteria a) acid-heat treated Gram-positive bacteria, or b) Chemically sterilized bacteria That is, use.

2. The use according to claim 1 , wherein at least one antigen in the antigen-containing substance causes or is associated with an immune response-related disease.

3. 3. The use according to claim 2, wherein the immune response-related disease is selected from the group consisting of celiac disease, allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, pemphigus vulgaris, and food allergies.

4. 2. The use according to claim 1, wherein the antigen-containing material is obtained from pollen, gluten, house dust mites, cat saliva, birch or peanuts.

5. The antigen-containing substance, immunodominant epitopes of α-gliadin, Hordein immunodominant epitopes, Immunodominant epitopes of house dust mites, and Immunodominant epitopes of peptides from cat saliva, skin or glands The use according to claim 1, comprising at least one immunodominant epitope selected from the group consisting of:

6. The immunodominant epitope of the house dust mite is Der p2.1; The use according to claim 5, wherein the immunodominant epitope of said peptide from cat saliva, skin or glands is Feld1.

7. The use according to claim 1 , wherein the mucoadhesive carrier is a patch comprising electrospun fibers.

8. 8. The use according to claim 7, wherein the harmless bacterium is Lactobacillus, Lactococcus, Bifidobacterium or Escherichia coli Nissl.

9. a) the acid-heat-treated Gram-positive bacterium is L. lactis; 7. The method according to claim 6, wherein the chemically sterilized bacteria is L. lactis, Lb. rhamnosus, or E. coli Nissl.

10. The use according to claim 1 , wherein the subject is a human or an animal.

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