Dead or inactivated spores for the treatment of allergy

Abstract

The invention relates to allergy, and to the treatment, amelioration or prevention of symptoms of allergy, such as rhinitis, allergic rhinitis and rhinosinusitis. The invention is especially concerned with the use of inactivated or killed bacterial spores in treating, preventing or ameliorating allergy or allergic reactions, and in particular, symptoms of allergy, including rhinitis, rhinosinusitis and allergic rhinitis. The invention extends to dead or inactivated spores, compositions (both pharmaceutical and non-pharmaceutical) comprising such spores and methods of making the compositions.

Description

[0001] DEAD OR INACTIVATED SPORES FOR THE TREATMENT OF ALLERGY

[0002] Field of the invention

[0003] The present invention relates to allergy, and particularly, although not exclusively, to the treatment, amelioration or prevention of symptoms of allergy, such as rhinitis, allergic rhinitis and rhinosinusitis. The invention is especially concerned with the use of inactivated or killed bacterial spores in treating, preventing or ameliorating allergy or allergic reactions, and in particular, symptoms of allergy, including rhinitis, rhinosinusitis and allergic rhinitis. The invention extends to dead or inactivated spores, compositions (both pharmaceutical and non-pharmaceutical) comprising such spores and methods of making the compositions.

[0004] Background of the invention

[0005] Allergies, also known as allergic diseases or allergic reactions, are a range of conditions caused by hypersensitivity of the immune system to typically harmless substances in the environment. Such diseases include hay fever, food allergies, atopic dermatitis, allergic asthma and anaphylaxis. Symptoms of such allergic reactions can include red or itchy eyes, and itchy rash, sneezing, coughing, a runny or itchy nose, shortness of breath, or swelling. Common allergens causing allergic reactions include pollen and certain foods, for example nuts. Food, insect stings and medications are common causes of severe allergic reactions, and their development is due to both genetic and environmental factors. The underlying mechanism involves immunoglobulin E antibodies (IgE) binding to an allergen and then to a receptor on mast cells or basophils where it triggers the release of inflammatory chemicals, such as histamine.

[0006] Rhinitis, a symptom of allergy, is a common nasal disease that is characterized by the presence of one or more of the following symptoms: nasal itching, sneezing, rhinorrhoea, and nasal congestion. Other symptoms sometimes encountered include headache, exaggerated pain response, cough, and fever (Baraniuk 2009). Rhinitis can be idiopathic or due to a variety of causes, including allergens, drugs, endocrine / metabolic, infections, inflammatory, and structural abnormalities of the nose (Bousquet, Khaltaev et al. 2008, Wallace, Dykewicz et al. 2008). The term "allergic rhinitis" is often used to describe inflammation of the inside of the nose that is specifically caused by an allergen, such as pollen, dust, mould, or flakes of skin from certain animals. Rhinosinusitis is an inflammation of the lining of the nose and paranasal sinuses, caused by many different phenomena such as bacteria, viruses, fungi, allergies, drugs or occupational factors. Rhinosinusitis is a common disease in children under 6 years of age and is also a common nasal disease in adults. Depending on the course of the disease, rhinosinusitis is divided into three types: acute rhinosinusitis lasting less than 4 weeks, subacute rhinosinusitis lasting from 4 to 8 weeks and chronic rhinosinusitis lasting at least 8 to 12 weeks regardless of treatment.

[0007] Rhinosinusitis in general is not a dangerous medical condition, but the frequency is relatively high with statistics showing that everyone has at least one episode in their life. Although most cases of acute rhinosinusitis are viral and resolve spontaneously, patients in many countries are prescribed antibiotics because it is clinically difficult to distinguish between viral and bacterial infections, and also to prevent serious infectious complications. Many pathogenic bacteria are resistant to previously used drugs and current concerns about antibiotic-resistant bacteria have led to the use of more selective antibiotics. The current first-line drugs used for patients with rhinosinusitis include antibiotics and a corticosteroid. Also, amoxicillin-clavulanate (875 / 125 mg) can be prescribed, which is taken orally every 12 hours, The duration of treatment for patients is about 10 days depending on the severity of the disease. During the treatment period, a change in the systemic antibiotic regimen is necessary if the observations after 3 to 5 days of treatment do not show signs of a satisfactory response. Additionally, anti-histamines are also commonly used to treat rhinosinusitis, especially among allergic rhinitis patients.

[0008] Allergen immunotherapy, aka allergen desensitisation or hypo-sensitization, involves exposing people to larger and larger amounts of allergens in an attempt to change the immune system response (Abramson, Puy et al. 2000, Abramson, Puy et al. 2003, Abramson, Puy et al. 2010). It is commonly believed that allergy results from the imbalance between Thl and Th2 subtypes of T-helper cells in favour of a Th2- mediated response. Insufficient induction of Thl cells leads to an exaggerated Th2 response. Allergic conditions (Th2) result from increases in IL-4, IL-13, IL-5 and IL-6 as well as production of IgE. In desensitization immunotherapy, the aim is to induce or restore tolerance to the allergen by reducing its tendency to induce IgE production. Treatment involves the administration of escalating doses of allergen that gradually decreases the IgE-dominated response. The objective of immunotherapy is to direct the immune response away from humoral immunity (Th2) and toward cellular immunity (Thl), thereby encouraging the body to produce fewer IgE antibodies and more CD4+ T regulatory cells that secrete IL-10 and TGF-0, which skews the response away from IgE production, There is good evidence that allergen immunotherapy works for allergic rhinitis in children and in asthma.

[0009] Many bacteria induce Thl responses (anti-inflammatory IFNy, IL-2, IL-10 and TNFo) and so down-regulate Th2 responses. Nasal dosing of live spores of Bacillus clausil has been shown to immunomodulate a number of cytokines, with decreases in IL-4 and increases in IFN-y, IL-10 and TGF-0 being observed in allergic children with recurrent respiratory tract infections (Ciprandi, Tosca et al. 2004, Ciprandi, Vizzaccaro et al. 2005). More recently, a human phase II study proposed using nasal dosing of a mixed formulation of live probiotic bacteria for treatment of allergic rhinitis (rhinosinusitis) in children (Meng, Li et al. 2019).

[0010] However, repeated exposure to "live" Bacillus has also been linked to illness. Inhalation of Bacillus, or more specifically, enzymes produced by Bacillus, has been linked to occupational asthma (Hole, Draper et al. 2000). This has mostly arisen in the detergent industry where inclusion of flac / 7 / us-produced enzymes notably, amylases, proteases and lipases in final products has led to high rates of asthma and associated IgE among factory workers (Pepys, Longbottom et al. 1969, Hole, Draper et al. 2000). In some cases, this has led to anaphylaxis and even death. The probable cause of allergy is most likely inhalation of enzymes associated with Bacillus cells and / or spores.

[0011] The present invention arises from the inventors' work in attempting to overcome the problems associated with the prior art.

[0012] Sterilization kills the bacterium without rupturing the cell or spore ensuring shape and physical integrity is maintained (Fonzi, Montomoli et al. 1999, Huesca-Espitia, Suvira et al. 2016). Spore proteins residing internally and surface exposed are presumably denatured; yet the inventors have previously found that this does not impair their ability to induce innate immunity (Song, Hong et al. 2012, James, Meyer et al. 2022). Sterilisation ensures that, first, the spore is unable to germinate and potentially proliferate, and second, that any enzyme produced by the bacterium and potentially adsorbed onto the spore surface is also inactivated.

[0013] Following on from their earlier work, the inventors then set out to test their hypothesis that dead or inactivated spores (e.g., of the species, Bacillus subtilis)' could be used to treat or prevent allergy and, in particular, various symptoms of allergic reactions, such as allergic rhinitis. As described in the Examples, the inventors have surprisingly shown that inert and non-viable bacterial spores are sufficient to clinically resolve symptoms of patients with acute rhinosinusitis, and therefore allergy per se. As described in the Examples, the inventors have surprisingly found that dead or inactivated spores reduce Th2 cytokine expression and increase Thl cytokine expression, as well as physically bind allergens to thereby block interactions between allergen and host.

[0014] Summary of the invention

[0015] Hence, in a first aspect of the invention, there is provided a dead or inactivated spore or a fragment thereof, for use in treating, preventing or ameliorating allergy.

[0016] In a second aspect of the invention, there is provided a method of treating, preventing or ameliorating allergy, the method comprising administering, or having administered, to a subject in need of such treatment, a therapeutically effective amount of a dead or inactivated spore or a fragment thereof.

[0017] Accordingly, the inventors believe that a dead or inactivated spore or a fragment thereof may therefore be used in allergen immunotherapy.

[0018] Hence, in a third aspect, there is provided a dead or inactivated spore or a fragment thereof, for use in allergen immunotherapy.

[0019] In a fourth aspect, there is provided a method of allergen immunotherapy, the method comprising administering, or having administered, to a subject in need of such treatment, a therapeutically effective amount of a dead or inactivated spore or a fragment thereof.

[0020] Detailed description of the invention

[0021] As described in Examples 1 and 2, the symptoms of acute sinusitis (Example 1) and allergic rhinitis (Example 2) in both live and dead (i.e., inactivate) spore treatment groups were significantly relieved relative to those in the Placebo group. The inventors have also shown that the use of bacterial spores significantly decreased levels of cytokines associated with a Th2-mediated response (IL-4 and IL-13), and significantly increased those associated with a Thl-mediated response (INF-y). Surprisingly, this shift from a Th2 to Thl response was much more pronounced when treating patients with inert and non-viable bacterial spores (as opposed to live spores). That is, the use of dead (i.e., inactivated) spores better promoted Thl-type cytokines (IFN-y) and diminished Th2-type cytokines (IL-4 and IL-13), demonstrating a better efficacy in ameliorating the symptoms of allergic rhinitis than live spores, and this was totally unexpected. Accordingly, a dead or inactivated spore or a fragment thereof may be used to dampen Th2-mediated immune responses for treating conditions or diseases associated with over-expressed Th2 cells. As described in Example 3 and as shown in Figure 5, the dead or inactivated spore is able to physically bind an allergen (e.g. Natural Ambrosia artemisiifolia allergen 1) to thereby block interactions between the allergen and host, and reduce allergic reactions therebetween.

[0022] As such, in an embodiment, the dead or inactivated spore or a fragment thereof may bind to an allergen and thereby block its interaction with a host cell. Thus, the dead or inactivated spore or a fragment thereof may reduce symptoms of allergy by binding and blocking interaction of allergen with host cells.

[0023] The dead or inactivated spore or a fragment thereof may be used to treat all types of allergies, allergic diseases or allergic reactions. For example, the dead or inactivated spore or a fragment thereof may be used to treat hay fever, allergic asthma, anaphylaxis, a food allergy, or atopic dermatitis. Typically, the dead or inactivated spore or fragment thereof is for use in treating, preventing or ameliorating hay fever.

[0024] Typically, the dead or inactivated spore or a fragment thereof is used to treat symptoms of allergy selected from a group consisting of: red or itchy eyes, and itchy rash, sneezing, coughing, a runny or itchy nose, shortness of breath, and swelling.

[0025] Typically, the dead or inactivated spore or a fragment thereof is for use in treating allergy in the respiratory tract.

[0026] Typically, the dead or inactivated spore or a fragment thereof is for use in treating rhinitis. The dead or inactivated spore or a fragment thereof may be used to treat all types of rhinitis, including allergic rhinitis and non-allergic rhinitis. Preferably, however, the dead or inactivated spore or a fragment thereof is for use in treating allergic rhinitis.

[0027] Typically, the allergen is selected from a group consisting of: pollen, dust or dust mites, mould, flakes of skin from certain animals, such as dogs and cats, wood dust, flour dust, and latex. In some embodiments, the pollen may be pollen from trees, grass and / or weeds. In other embodiments, the flakes of skin from certain animals may be from dogs and / or cats. Rhinitis can extend into and affect the sinuses. Rhinosinusitis describes inflammation of both the nose and the sinuses. Thus, in one embodiment, the dead or inactivated spore or a fragment thereof is used to treat Rhinosinusitis.

[0028] Rhinitis, or Rhinosinusitis, may be classified as acute (symptoms lasting less than 4 weeks), subacute (symptoms lasting 4-12 weeks), recurrent acute (four or more episodes per year), and chronic (symptoms lasting for greater than 12 weeks). In one embodiment, the dead or inactivated spore or a fragment thereof is used to treat acute rhinosinusitis, subacute rhinosinusitis, recurrent acute rhinosinusitis and / or chronic rhinosinusitis.

[0029] Typically, however, the dead or inactivated spore or a fragment thereof is used to treat acute rhinosinusitis.

[0030] The dead or inactivated spore or a fragment thereof may be derived from any sporeforming bacteria. In an embodiment, however, the dead or inactivated spore or a fragment thereof is derived from a bacterium belonging to the Bacillus species.

[0031] Typically, the Bacillus spp is selected from a group of Bacillus spp consisting of: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus clausii, Bacillus coagulans, Bacillus pumilus, Bacillus firmus, Bacillus flexus, Bacillus lichen! formis, Bacillis marisflvi, Bacillus polyfermenticus, Bacillus megaterium, Bacillus flexus, and Bacillus Indicus.

[0032] Most typically, the bacterium is Bacillus subtilis. Hence, it is preferred that the dead or inactivated spore or a fragment thereof comprises a Bacillus subtilis spore or a fragment thereof.

[0033] In one embodiment, the dead or inactivated spore or a fragment thereof comprises a Bacillus subtilis strain DSM 32444 spore or a fragment thereof. This strain has been deposited under the Budapest treaty at the DSMZ, InhoffenstraBe 7B, 38124 Braunschweig, Germany. In embodiments, the dead or inactivated spore or a fragment thereof comprises a Bacillus subtilis strain selected from HU58, PY79, CUI, or DE11.

[0034] The skilled person will appreciate that there are several ways in which the bacterial spore may be killed, inactivated or rendered non-viable, such as autoclaving, gamma irradiation, UV irradiation or formaldehyde treatment. Autoclaving may comprise exposing the bacterial spore to 121°C for about 10-45 minutes, ideally at a pressure of about 15 p.s.i, Typically, the exposure is for greater than 10 minutes, and ideally at least 20 minutes.

[0035] Gamma irradiation may comprise exposing the bacterial spore to about 1.5 x 106rads of gamma radiation. Such an exposure would be sufficient to inactivate >99% of spores (L.A. Dauphin et al., Applied and Environ. Microbiol. 2008, pp. 4427-4433).

[0036] UV irradiation may comprise exposing the bacterial spore to UV-C radiation (for example from a germicidal lamp) with a major UV emission of 254 nm. Irradiation times (minutes typically) would be first calibrated using spore samples in aqueous solution. Examples of UV irradiation of Bacillus spores are given in Khaneja R. et al. Carotenoids found in Bacillus. J Appl Microbiol. 2010; 108(6): 1889-902. doi: 10, 1111 / j.1365-2672.2009.04590.x, and Moeller, R. et al., (2005) Role of pigmentation in protecting Bacillus sp. endospores against environmental UV radiation. FEMS Microbiol Ecol 51, 231-236.

[0037] Formaldehyde treatment may comprise contacting the bacterial spore with formalin (i.e., formaldehyde) (e.g., 5%) for a defined period of time and then assessing viability of the spore. Conditions are first validated regarding % formalin and incubation time. Examples of formaldehyde inactivation of spores are given in Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard, Authors: Richard J. Manchee, Malcolm G. Broster, Anthony J. Stagg, Stephen E. Hibbs, https: / / doi.org / 10.1128 / aem.60. ll.4167-4171.1994.

[0038] The skilled person wili appreciate that a bacterial spore or "endospore" is a dormant, tough and non-reproductive structure produced by some bacteria in the phylum Bacillota. Either the whole spore or only a part or fragment or integument of the spore may be used in accordance with the invention, for treating, preventing or ameliorating rhinitis.

[0039] For example, the spore may be surrounded by a thin covering known as the exosporium, which overlies a spore coat. The spore coat, which acts like a sieve that excludes large toxic molecules such as lysozyme, is resistant to many toxic molecules and may also contain enzymes that are involved in spore germination. In Bacillus subtilis endospores, the spore coat is estimated to contain more than 70 coat proteins, which are organized into an inner and an outer coat layer. The cortex lies beneath the spore coat and consists of peptidoglycan. The core wall lies beneath the cortex and surrounds the protoplast or core of the endospore. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs (small acid-soluble spore proteins), that protect the spore DNA from UV radiation and heat. The core also contains normal cell structures, such as ribosomes and other enzymes, but is not metabolically active. Up to 20% of the dry weight of the endospore consists of calcium dipicolinate within the core, which is thought to stabilize the DNA. Dipicolinic acid could be responsible for the heat resistance of the spore, and calcium may aid in resistance to heat and oxidizing agents. However, mutants resistant to heat but lacking dipicolinic acid have been isolated, suggesting other mechanisms contributing to heat-resistance are also at work. Small acid-soluble proteins (SASPs) are found in endospores. These proteins tightly bind and condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals.

[0040] The arrangement of spore layers is as follows:

[0041] • Exosporium

[0042] • Spore coat

[0043] • Spore cortex

[0044] • Core wall

[0045] • Core

[0046] Accordingly, by "fragment of spore", any of the following components are envisaged, including : core, exosporium, spore coat, inner spore coat, outer spore coat, spore cortex, and core wall. Thus, it is preferred that the spore fragment comprises one or more of: core, exosporium, spore coat, inner spore coat, outer spore coat, spore cortex, and core wall. In addition, components secreted by the cell and adsorbed onto the spore may also exhibit therapeutic activity, such as HELMs, i.e., lipopeptide micelles and / or lipopeptides.

[0047] The inventors have shown in Example 2 that the use of bacterial spores significantly decreased levels of cytokines associated with a Th2-mediated response (IL-4 and IL- 13), and significantly increased those associated with a Thl-mediated response (IFN- y). Surprisingly, this shift from a Th2 to Thl response was more pronounced when treating patients with inert and non-viable bacterial spores (as opposed to live spores). Accordingly, a dead or inactivated spore or a fragment thereof may be used to dampen Th2-mediated immune responses for treating conditions or diseases associated with over-expressed Th2 cells.

[0048] Thus, typically the dead or inactivated spore or fragment thereof is configured to stimulate a shift from a Th2- to Thl-mediated response.

[0049] Typically, the dead or inactivated spore or fragment thereof is configured to reduce Th2 cytokine expression in a subject who has been administered with the dead or inactivated spore or fragment thereof. In other words, the level Th2 expression is typically reduced in the presence of the dead or inactivated spore or fragment thereof compared to the level of Th2 expression in the absence of the dead or inactivated spore or fragment thereof. Typically, the Th2 cytokine is selected from a group of Th2 cytokines consisting of: IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, IL-17E (IL-25), and IL-31. In one embodiment, the Th2 cytokine is IL-4 and / or IL-13. Typically, IL-4 and IL-13 expression is reduced.

[0050] Typically, the dead or inactivated spore or fragment thereof is configured to increase Thl cytokine expression in a subject who has been administered with the dead or inactivated spore or fragment thereof. In other words, the level of Thl expression is typically increased in the presence of the dead or inactivated spore or fragment thereof compared to the level of Thl expression in the absence of the dead or inactivated spore or fragment thereof. Typically, the Thl cytokine is selected from a group of Thl cytokines consisting of: IL-2, IFNy, TNFo, and TNFp / LTa. In one embodiment, the Thl cytokine is IFN-y.

[0051] Typically, the dead or inactivated spore or fragment thereof is not configured to induce tissue resident memory T cells.

[0052] Typically, the dead or inactivated spore or fragment thereof is not configured to modulate innate immunity.

[0053] Typically, the dead or inactivated spore or fragment thereof is not configured to induce an innate immune response.

[0054] Typically, the dead or inactivated spore or fragment thereof is not configured to induce secretory IgA and / or IgG production. Typically, the dead or inactivated spore or fragment thereof is not configured to act as an adjuvant.

[0055] The inventors have also surprisingly shown in Example 3 (Figure 5) that killed spores of B. subtilis bind to an exemplary allergen and, therefore, are capable of blocking interactions between allergen and host.

[0056] Thus, typically the dead or inactivated spore or fragment thereof is configured to bind an allergen. In one embodiment, the allergen is derived from ragweed pollen. In one embodiment, the allergen is derived from Ambrosia artemisiifolia. In one embodiment, the allergen is natural Ambrosia artemisiifolia allergen 1 (nAmb A 1).

[0057] Typically, the dead or inactivated spore or fragment thereof is configured to block interactions between the allergen and host.

[0058] It will be appreciated that the dead or inactivated spore or a fragment thereof used according to the invention may be used in a monotherapy (i.e., the sole use of the dead bacterial spore of fragment thereof), for treating, ameliorating or preventing allergy and, in particular, symptoms of allergy, such as rhinitis, allergic rhinitis or rhinosinusitis. Alternatively, such antiviral agents and formulations according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing allergy, such as allergic rhinitis.

[0059] The inventors also believe that the dead or inactivated spore or a fragment thereof according to the invention may advantageously be used in combination with other therapies for treating, preventing or ameliorating allergy and, in particular, symptoms of allergy, such as rhinitis, allergic rhinitis or rhinosinusitis. For example, the dead or inactivated spore or a fragment thereof may be used with an antibiotic, for example amoxicillin-clavulanate (amoxicillin 875 mg / day for 6 days and clavulanate 125 mg / day for 6 days).

[0060] Additionally, anti-histamines are also commonly used to treat rhinosinusitis, especially among allergic rhinitis patients, and so the dead or inactivated spore or a fragment thereof may be used with an anti-histamine. Histamines are usually divided into two main groups:

[0061] • antihistamines that make you feel drowsy - such as chlorphenamine

[0062] (Piriton), cinnarizine, diphenhydramine, hydroxyzine and promethazine; and • non-drowsy antihistamines that are less likely to make you feel sleepy - such as acrivastine, cetirizine, fexofenadine and loratadine.

[0063] Anti-histamines also come in several different forms - including tablets, capsules, liquids, syrups, creams, lotions, gels, eyedrops and nasal sprays.

[0064] Antihistamine nasal sprays - antihistamines treat many health problems, including allergies. An antihistamine nose spray may ease the symptoms of nonallergic rhinitis too. These sprays include azelastine (Astepro, Astepro Allergy) or olopatadine hydrochloride (Patanase).

[0065] Antihistamines taken by mouth often do not work as well for non-allergic rhinitis as they do for allergic rhinitis. These antihistamines include diphenhydramine (Benadryl), cetirizine (Zyrtec Allergy), fexofenadine (Allegra Allergy) and loratadine (Alavert, Claritin).

[0066] Ipratropium nose spray. This prescription spray can ease a runny, drippy nose.

[0067] Steroids. These medicines help prevent and treat swelling linked with some types of non-allergic rhinitis. Steroid sprays that you can buy off the shelf include a fluticasone (Flonase Allergy Relief) and triamcinolone (Nasacort Allergy 24 Hour). Stronger steroid sprays also can be prescribed.

[0068] Decongestant nasal sprays are a type of medicine that can provide short-term relief for a blocked or stuffy nose (nasal congestion). They can help ease the symptoms of conditions such as colds and flu, hay fever and the allergic reactions, catarrh and sinusitis. They work by reducing the swelling of the blood vessels in your nose, which helps to open the airways. Examples include pseudoephedrine (sometimes called by the brand name Sudafed).

[0069] Decongestants. These medicines help narrow the blood vessels in the nose and lessen congestion. Examples include drugs with pseudoephedrine (Sudafed 24 Hour) and phenylephrine.

[0070] Salt water nasal sprays - Saline is a mixture of salt and water. Saline nose spray helps moisturize the nose. It also helps thin mucus and soothes the tissue that lines the inside of the nose. A home remedy known as nose irrigation is possible, and involves using a large amount of saline or a saltwater mixture to help clean out irritants and mucus.

[0071] The dead or inactivated spore or a fragment thereof may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used, preferably nasally. Thus, for example, the composition may be in the form of an aerosol, liquid, spray, micellar solution, liposome suspension, liquid, ointment, cream, gel, hydrogel, powder, or any other suitable form that may be administered to a person or animal in need of treatment, preferably nasally. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

[0072] Compositions and formulations of the invention are preferably administered by mucosal delivery. Compositions and formulations of the invention may be preferably administered by inhalation (e;g., intranasally).

[0073] Therefore, preferably the dead or inactivated spore or a fragment thereof for use in treating rhinitis is applied nasally. Nasal application may comprise application by a spray, mist, droplet, cream, gel or injection. In one embodiment, it is preferred that a dead or inactivated bacterial spore is used to treat rhinitis, and is applied nasally, preferably by spray, mist, droplet, cream, gel or injection. The dosage used for nasal administration may be 102to 1015, preferably 106or 108to 1012, in particular 10sto 1010, spores. Preferably, the use comprises mucosal application, to a subject, of a dead or inactivated spore or a fragment thereof. Mucosal application may comprise nasal, rectal, ocular, oral or sub-lingual application of the dead or inactivated spore or a fragment thereof. Preferably, therefore, the composition is a mucosally or nasally administrable composition. The dead or inactivated spore or a fragment thereof may be applied sub-lingually. Typically, however, the dead or inactivated spore or a fragment thereof is applied nasally. Nasal application may comprise application by a liquid, spray or droplet.

[0074] In one embodiment, it is envisaged that a dead or inactivated spore or a fragment thereof is used to treat rhinitis, preferably allergic rhinitis or rhinosinusitis, and is applied nasally, typically by liquid, spray or droplet. It will be appreciated that the amount of the compositions and formulations of the invention that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the compositions and formulations, and whether they are being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the dead or inactivated spore or a fragment thereof within the subject being treated . Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular dead or inactivated spore or a fragment thereof in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of rhinitis. Additional factors depending on the particular- subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0075] Generally, a daily dose of between 0,001pg / kg of body weight and lOmg / kg of body weight of the compositions and formulations of the invention may be used for treating, ameliorating, or preventing rhinitis, depending upon which of the compositions and formulations of the invention is used. More preferably, the daily dose is between O.Olg / kg of body weight and lmg / kg of body weight, more preferably between O.lg / kg and lOOg / kg body weight, and most preferably between approximately O.lg / kg and lOg / kg body weight.

[0076] Compositions and formulations of the invention may for instance be characterized in that they contain 102to 1015, preferably 106or 108to 1012, in particular 10Bto IO10, dead or inactivated spores. In a preferred embodiment, the composition of the invention comprises at least 0.1, 0.5, 1, 2, 3, 4 or 5 billion dead or inactivated spores. The dead or inactivated spores may be suitably lyophilized or spray dried.

[0077] The compositions and formulations may be administered before, during or after the onset of rhinitis. Daily doses may be given as a single administration (e.g., a single daily injection, or oral dose). Alternatively, the compositions and formulations may require administration two or more times during a day or one or more times a week, or one or more times a month. As an example, the compositions and formulations may be administered as two (or more depending upon the severity of the viral infection being treated) daily doses of between 0.07 g and 700 mg (i.e., assuming a body weight of 70 kg). A. patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two-dose regime) or at 3- or 4- hourly intervals thereafter. The dead or inactivated spore or a fragment thereof as defined by the first aspect, or the composition as defined by the third aspect, may be administered before, during or after the onset of Rhinitis. Daily doses may be given as a single administration (e.g., a single daily injection, or oral dose). Alternatively, the agents and formulations may require administration twice or two or more times during a day or one or more times a week, or one or more times a month.

[0078] Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g., in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents and formulations according to the invention and precise therapeutic regimes (such as daily doses of the agents and formulations and the frequency of administration).

[0079] The invention also provides in a fifth aspect, a pharmaceutical or non-pharmaceutical composition for use in treating allergy, wherein the pharmaceutical composition comprises the dead or inactivated spore or a fragment thereof as defined in the first aspect, and a pharmaceutically acceptable vehicle or carrier.

[0080] Most typically, the composition is a nasally administrable composition.

[0081] Thus, in an embodiment, the composition of the invention comprises a nasal spray (e.g. 10 mL) comprising at least 0.1, 0.5, 1 2, 3, 4 or 5 billion heat-treated spores of Bacillus subtilis, most typically Bacillus subtilis DSM32444. Postbiotic DSM32444 is manufactured by HURO BIOTECH Company as a drug-grade active pharmaceutical ingredient.

[0082] The composition is ideally administered 2-3 times per day to each of the nostrils, each time 1-2 sprays corresponding to 0.2 ml / spray.

[0083] In a sixth aspect, there is also provided a process for making the pharmaceutical or non-pharmaceutical composition according to the fifth aspect, the process comprising combining a therapeutically effective amount of the dead or Inactivated spore or a fragment thereof as defined in the first aspect, with a pharmaceutically acceptable vehicle or carrier.

[0084] A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g., a horse), pets, or may be used in other veterinary applications. Most preferably, the subject is a human being.

[0085] A "therapeutically effective amount" of the dead or inactivated spore or a fragment thereof as defined in the first aspect, or the composition as defined in the third aspect, is any amount which, when administered to a subject, is the amount of drug that is needed to treat allergy or an allergic reaction, or produce the desired effect.

[0086] For example, the therapeutically effective amount may be from about 0.001 pg to about 1 mg, and preferably from about 0.01 pg to about 100 pg. It is preferred that the amount of agent is an amount from about 0.1 pg to about 10 pg, and most preferably from about 0.5 pg to about 5 pg. The therapeutically effective amount may comprise 102to 1015, preferably 106or 108to 1012, in particular 108to 1010, dead or inactivated spores. Preferably, the therapeutically effective amount comprises at least 0.1, 0.5, 1, 2, 3, 4 or 5 billion spores.

[0087] A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0088] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tabletdisintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0089] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0090] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0091] All features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0092] Brief description of figures

[0093] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0094] Figure 1 is a. graph showing cumulative incidence of clinical resolution following treatment with one embodiment of the dead or inactivated Bacillus subtilis strain DSM 32444 spore or a fragment thereof according to the invention (referred to herein as "SPEROVID") (i.e. treatment group = 1) vs. active control (i.e. treatment group = 0). Figure 2 is a graph showing cumulative incidence of clinical improvement following treatment with SPEROVID (treatment group = 1) vs. active control (treatment group = 0).

[0095] Figure 3 is a graph showing changes in total symptom score (TSS) score compared to the baseline following treatment with SPEROVID vs. active control.

[0096] Figure 4A-4C are graphs showing cytokine levels following 2-week treatment with live spores (grey) vs. dead spores (black) of Bacillus subtilis strain DSM 32444 compared to a placebo control (white). Figure 4A Is a graph showing changes in IFN-y levels. Figure 4B is a graph showing changes in IL-4 levels. Figure 4C is a graph showing changes in IL-13 levels. All graphs show data before treatment (baseline) and after 2-week treatment.

[0097] Figure 5 is a graph showing inactivated (killed) spores of B. subtilis DSM 32444 binding to a known ragweed allergen. Data shown is a representative experiment from six independent experiments. Examples

[0098] The inventors have investigated the efficacy of dead spores of Bacillus subtilis strain DSM 32444 to treat or prevent allergy. The inventors have surprisingly shown that inert and non-viable bacterial spores are sufficient to clinically resolve symptoms of patients with acute allergic rhinosinusitis. The inventors have also investigated the effect of dead spores of Bacillus subtilis strain DSM 32444 on the expression levels of Thl and Th2 cytokines. In doing so, the inventors have found that inert and non- viable bacterial spores produce a more pronounced shift from a Th2 to Thl-mediated response. when compared with live spores, which is totally unexpected. Example 1: Treatment of acute sinusitis

[0099] A Phase II open labelled, randomized, active-control study was conducted to compare the safety and efficacy of a formulation of killed or dead Bacillus spores of Bacillus subtilis strain DSM 32444, known as DSM 32444K(herein referred to as "SPEROVID spray") compared to Neomycin / Dexamethasone / Xylometazoiine nasal spray, which is a standard therapeutic for acute sinusitis. Materials & Methods

[0100] Study Cohort

[0101] A total of 60 patients with acute rhinosinusitis were identified and randomly selected to use either SPEROVID spray (active agent of the invention) or Neomycin / Dexamethasone / Xylometazoline nasal spray (active control). Patients were randomized on a 1: 1 ratio (30 patients in each treatment group) to use:

[0102] • SPEROVID Spray as an adjunct to Amoxicillin-Clavulanate Standard Treatment for 10 days, or

[0103] • Neomycin / Dexamethasone / Xylometazoline Nasal Spray as an adjunct to Amoxicillin-Clavulanate Standard Treatment for 10 days.

[0104] Selection of Study Participants Participants were assigned to study treatment only if they meet all the inclusion criteria and none of the exclusion criteria. Participants who withdraw from the study after randomization were not replaced. Deviations from the inclusion and exclusion criteria are not allowed because they can potentially jeopardize the scientific integrity of the study, regulatory acceptability, or participant safety. Therefore, adherence to the eligibility criteria as specified in the protocol is essential.

[0105] Inclusion Criteria

[0106] Each participant must meet all the following criteria to be enrolled in this study:

[0107] 1. Confirmed rhinosinusitis1patients, > 18 years of age, at the time of signing the informed consent.

[0108] 2. Must be in generally good health, except rhinosinusitis

[0109] 3. Patients who are able to use an e-Diary or Paper Diary during the study to report their health status

[0110] 4. Patients capable of giving a signed informed consent form (ICF)

[0111] Exclusion Criteria

[0112] Individuals meeting any of the following criteria were excluded from the study:

[0113] 1. Presence of any nasal mucosal erosion, nasal ulceration, or nasal septal perforation (Grade lb - 4) 2. Other nasal disease(s) likely to affect deposition of intranasal medication, such as sinusitis, rhinitis medicamentosa, clinically significant polyposis, or nasal structural abnormalities. 3. Nasal surgery or sinus surgery within the previous year

[0114] 4. Chronic sinusitis - more than 3 episodes per year

[0115] 5. Asthma (with the exception of mild, intermittent asthma). Subjects with mild, intermittent asthma who only require short-acting inhaled bronchodilators (not more often than twice per week) and who do not have nocturnal awakening as a result of asthma are eligible for enrolment.

[0116] 6. Planned travel outside of the study area during the study period.

[0117] 7. Use of any investigational drug within 30 days prior to Day screening. No investigational products are permitted for use during the conduct of this study.

[0118] 8. Existence of any medical condition, which in the opinion of the investigator, might significantly affect the subject's ability to complete this trial; or their safety in this trial.

[0119] Randomization strategies resulted in highly homogenous study groups.

[0120] Treatment

[0121] Patients received either SPEROVID spray or Neomycin / Dexarnethasone / Xylometazoline nasal spray (active control), depending on which treatment group they were randomly allocated.

[0122] The following treatment groups were as follows:

[0123] 1. SPEROVID spray consists of a formulation of inert and non-viable, bacterial spores (species: Bacillus subtil is) of strain DSM 32444, referred to as DSM 32444K. SPEROVID bio-nasal spray (10 mL) contains SPOR-COV® substance with 5 billion heat-treated spores of Bacillus subtilis DSM32444. Postbiotic DSM32444 is manufactured by HURO BIOTECH Company as a drug-grade active pharmaceutical ingredient.

[0124] 2. Neomycin / Dexamethasone / Xylometazoline nasal spray (active control - Dexamethasone 15mg, Xylometazoline 7.5mg, and Neomycin 52.500 HJ). Dexamethasone is a glucocorticoid commonly used to treat allergies and rhinitis.

[0125] Treatments were administered as nasal sprays to the nasal mucosa.

[0126] Both the SPEROVID spray and Neomycin / Dexamethasone / Xylometazoline nasal spray were used as an adjunct to Amoxicillin-Clavulanate (875 / 125 mg) standard treatment for 10 days. Amoxicillin-Clavulanate (875 / 125 mg) was taken orally every 12 hours. The SPEROVID Spray or the Neomycin / Dexamethasone / Xylometazoline Nasal Spray are recommended to be used 2-3 times per day to each of the nostrils, each time 1-2 sprays corresponding to 0.2 ml / spray were used.

[0127] Study endpoints

[0128] The safety and efficacy of the SPEROVID spray or Neomycin / Dexamethasone /

[0129] Xylometazoline nasal spray (Active Control) was assessed over the course of the today treatment period.

[0130] Safety assessments were performed on the safety analysis set (SAS) for adverse events of special interest and adverse events in general. Safety was assessed by the incident rate of serious adverse events, adverse events leading to discontinuation, withdrawing subjects from the study, adverse events requiring medical care, solicited adverse events and unsolicited adverse events.

[0131] Efficacy was assessed based on the severity of Acute sinusitis symptoms.

[0132] TSS includes the following symptoms, each will be scored from 0 to 6: • Nasal obstruction / blockage / congestion

[0133] • Discoloured nasal drainage: anterior / posterior .

[0134] • Headache

[0135] » Fatigue

[0136] • Decreased sense of smell » Ear pain / pressure / fullness

[0137] » Cough

[0138] • • Halitosis

[0139] • Dental pain

[0140] • Fever

[0141] Score Descriptions

[0142] 0 or 1 • None - to an occasional limited episode

[0143] 2 or 3 Mild - Steady symptoms but easily tolerable

[0144] 4 or 5 Moderately bothersome - Symptoms hard to tolerate, might interfere with activities of daily living, sleep, or both

[0145] 6 Very severe - Symptoms are so bad that person cannot function virtually all the time Efficacy was defined in terms of the following:

[0146] Clinical resolution: all the symptoms return to "None - to an occasional limited episode" or a score of 0-1 in the TSS scale.

[0147] Clinical improvement: all the symptoms return to "Mild - Steady symptoms but easily tolerable" or score of 2-3 in the TSS scale. In this outcome, only improvements of clinical symptoms are recorded.

[0148] Clinical failure: the persistence of 1 or more signs and symptoms of rhinosinusitis or patients who have received additional (or new) antibiotics. Results

[0149] Efficacy

[0150] Time to symptom resolution based on total symptom score (TSS)

[0151] The percentage of the patients with clinical resolution response at the end of treatment are shown in Table 1. The inventors have surprisingly shown that the number and percentage of patients achieving clinical resolution in the SPEROVID group is significantly higher than that of the active control group (p = 0.0242). There were 25 patients (83.3%) in SPEROVID group that achieved clinical resolution during the 10 days follow-up compared to only 17 patients (56.7%) in the active control group.

[0152] For the patients who met clinical resolution criteria, the time (number of days) to clinical resolution in the SPEROVID group and the active control group are shown in Table 2 and Figure 1.

[0153] The median time to clinical resolution between the active control group compared to the SPEROVID group was not significantly different (p = 0.0616).

[0154] Time to clinical improvement based on total symptom score CTSS)

[0155] The percentage of the patients with clinical improvement response at the end of treatment are shown in Table 3.

[0156] All 30 patients (100%) in each of the SPEROVID group and the active control group achieved clinical improvement during the 10 days follow-up. No patients needed to change their standard oral antibiotics at Day 5,

[0157] The time (number of days) to clinical improvement in the SPEROVID and the active control group are shown in Table 4 and Figure 2.

[0158] The time to clinical improvement was comparable between the SPEROVID group and the active control group (median of 5 days). The median time to clinical improvement between the two groups was not statistically significant (p = 0.0832).

[0159] Change From Baseline in Total Symptom Score (TSS)

[0160] Changes in Total Symptom Score (TSS) at days 3, 7 and 10 compared to baseline are shown in Table 5 and Figure 3.

[0161] The changes in TSS compared to the baseline are comparable between the SPEROVID group and the active control group. Conclusions

[0162] No adverse events were observed in both the SPEROVID group and the active group.

[0163] Clinical improvements were observed in all patients in both the SPEROVID group and the active group when used in combination with Amoxicillin / Clavulanate (875 / 125 mg), which is the standard treatment for acute rhinosinusitis patients. However, the use of SPEROVID spray unexpectedly resulted in a significantly higher number and percentage of patients achieving clinical resolution compared to using Neomycin / Dexamethasone / Xylometazoline nasal spray. Example 2: Treatment of allergic rhinitis with live and dead Spores of B. subtilis DSM

[0164] 32444

[0165] Individuals with acute allergic rhinitis showed the impact on Thl and Th2 cytokine production in subjects treated with nasal spray preparations that contain either live or dead spores of B. subtilis DSM 32444.

[0166] Materials & Methods

[0167] Study Cohort

[0168] A total of 9 adults with acute allergic rhinitis were identified and randomly allocated into three separate treatment groups: placebo, treatment with live spores of B. subtilis 32444 (Live spores) and treatment with dead spores of B. subtilis 32444 (Dead spores - an embodiment according to the invention). Each group consisted of three participants.

[0169] Treatment Participants were treated with a solution containing either live or dead spores of B. subtilis 32444.

[0170] Dead spores were produced by autoclaving at 121°C, 20-60 mins]. The inventors confirmed that autoclaved DSM 32444 spores exhibited no protease, amylase or biosurfactant activity following sterilisation (data not shown), and were therefore dead or inactivated.

[0171] Participants assigned to the placebo group received a saline solution (NaCI 0.9%). The saline solution, live spores and dead spores solutions were contained in identical packages, which is a closed aerosol spray (nasal spray).

[0172] The three groups were instructed to administer the solution into the nasal mucosa through the nasal spray by spraying three times per day over the course of 14 days. Each application involved one spray per nostril at a dosage of 0.5 ml / spray.

[0173] Study endpoints

[0174] All participants were tested and compared for the levels of IFN-y, IL-4 and IL-13 by ELISA analysis.

[0175] Results

[0176] The inventors assessed individuals with allergic rhinitis for any changes in the levels of cytokines expressed by Thl cells (IFN-y) and Th2 cells (IL-4 and IL-13) following treatment with live or dead spores of Bacillus strain DSM 32444, and the results are shown in Figure 4.

[0177] The changes in the cytokine levels of IFN-y (a Thl cytokine) are shown in Figure 4A.

[0178] As can be seen, the levels of IFN-y are significantly increased following treatment with both live and dead spores of Bacillus strain DSM 32444 compared to the placebo control. Treatment with the placebo does not affect expression of IFN-y. Furthermore, surprisingly, the levels of IFN-y are much higher for individuals treated with dead spores when compared to those treated with live spores. The changes in the cytokine levels of IL-4 (a Th2 cytokine) are shown in Figure 4B. As shown, the levels of IL4 are significantly decreased following treatment with both live and dead spores of Bacillus strain DSM 32444 compared to the placebo control.

[0179] Treatment with the placebo does not affect expression of IL4. Surprisingly, the levels of IL4 are lower for individuals treated with dead spores when compared to those treated with live spores.

[0180] Finally, the changes in the cytokine levels of IL-13 (another Th2 cytokine) are shown in Figure 4C. As can be seen, the levels of IL-13 are significantly decreased following treatment with both live and dead spores of Bacillus strain DSM 32444 compared to the control. Treatment with the placebo does not affect expression of IL-13. Moreover, as with IL-4, the levels of IL-13 are lower for individuals treated with dead spores when compared to those treated with live spores.

[0181] Conclusions

[0182] After 14 days, the overall symptoms of allergic rhinitis in both spore treatment groups were significantly relieved relative to those in the Placebo group. This correlated with significantly decreased levels of IL-4 and IL-13, and surprisingly increased levels of INF-y. Since IFN-y is known as a Thl-cytokine, and IL-4 and IL-13 are Th2-type cytokines, the data demonstrate that treatment with a solution containing B. subtilis DSM 32444 spores helps balance Thl and Th2 reactions in patients with Th2-dominant reactions to ameliorate the allergic symptoms.

[0183] Interestingly, the use of dead spores of B. subtilis DSM 32444 unexpectedly resulted in a more significant decrease of IL-4 and IL-13, and a greater rate of increase in IFN- y levels, when compared to treatment with live spores of the same. That is, the use of dead spores better promoted Thl-type cytokines (IFN-y) and diminished Th2-type cytokines (IL-4 and IL-13), demonstrating a better efficacy in ameliorating the symptoms of allergic rhinitis than live spores, and this was totally unexpected.

[0184] Example 3: Binding of Allergen (nAmb A 1 ) to Killed Spores of B. subtilis

[0185] The inventors set out to determine whether killed spores of B. subtilis can bind an allergen. The example allergen used was Natural Ambrosia artemisiifolia allergen 1 (nAmb A 1) (38-40 kDa) obtained from short ragweed pollen. The spores used were inactivated (killed) spores of B. subtilis DSM 32444 manufactured (WHO-GMP) by HURO Biotech in Vietnam. The spores were completely sterile and unable to proliferate. Binding between the allergen and killed spores of B. subtilis was tested under conditions that mimicked the pH found in the mucosa of the respiratory tract (pH 5.6-6.7).

[0186] Materials & Methods

[0187] Reagents

[0188] • Inactivated (killed) spores of B. subtilis DSM 32444. Spores were WHO-GMP manufactured at 5 X1010 / mL (Lot. HURO-S1 / 02-21).

[0189] • Natural Ambrosia artemisiifoiia allergen l(nAmb A 1), short ragweed pollen, 250 pg (Inbio NA-AAR1-1).

[0190] • Primary Ab-. Monoclonal antibody 7F11 (Inbio MA-7F11).

[0191] • Secondary Ab: Anti-Mouse IgG (whole molecule)-Peroxidase antibody produced in goat (Sigma A4416).

[0192] • TMB substrate (Biolegend 421101). • PBS pH 7.4 (Sigma P4417).

[0193] •< Diluent buffer (1% BSA in PBS pH 7.4).

[0194] • 2N H2SO4

[0195] Protocol Step 1 : Suspend spores (2 X 109spores of DSM 32444) in 50 pL of PBS using an Eppendorf tube and incubate for 18h at 4°C.

[0196] Step 2 : Add nAmb A 1 protein (27 mg).

[0197] Incubate, at room temperature for 20 min with gentle orbital shaking. Step 3 : Pellet spores (15,000xg for 1 minute) and wash 2X with PBS (0.01M, 500 uL; pH 7.4).

[0198] Step 4 : Add 50 pL of antibody against nAmb A 1 7F11 (diluted 1:50 in diluent buffer). Incubate for 90 min at room temperature with gentle orbital shaking.

[0199] Step 5 : Pellet spores (15,000xg for 1 minute) and wash 4X with PBS (0.01M, 500 11L; pH 7.4).

[0200] Step 6 : Add secondary antibody (50 mL; anti-mouse IgG; diluted 1:2000 in diluent buffer).

[0201] Step 7: Pellet spores (15,000xg for 1 minute) and wash 4X with PBS (0.01M, 500 uL; pH 7.4) Step 8 : Add TMB substrate (50 mL).

[0202] Incubate at room temperature for 5 min.

[0203] Step 9 : Stop reaction by adding H2SO4 (50 mL).

[0204] Read OD 450. nm. . . Results

[0205] The data shown in Figure 5 and Table 6 demonstrates that spores are able to bind a known ragweed allergen at pH 6, which is approximately the pH of the upper respiratory tract. This data has been repeated independently and binding was also observed at pH 4. Conclusions

[0206] The inventors have demonstrated that killed spores reduce symptoms of allergy by binding and blocking interaction of allergen with host cells.

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Claims

Claims1. A dead or inactivated spore or a fragment thereof, for use in treating, preventing or ameliorating allergy.

2. A dead or inactivated spore or a fragment thereof, for use in allergen immunotherapy.

3. A dead or inactivated spore or a fragment thereof, for use according to either claim 1 or claim 2, wherein the dead or inactivated spore or a fragment thereof is used to treat hay fever, allergic asthma, anaphylaxis, a food allergy, or atopic dermatitis.

4. A dead or inactivated spore or a fragment thereof, for use according to claim 3, wherein the dead or inactivated spore or fragment thereof is for use in treating, preventing or ameliorating hay fever.

5. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is used to treat symptoms of allergy selected from a group consisting of: red or itchy eyes, and itchy rash, sneezing, coughing, a runny or itchy nose, shortness of breath, and swelling.

6. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is for use in treating allergy in the respiratory tract.

7. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is for use in treating rhinitis.

8. A dead or inactivated spore or a fragment thereof, for use according to claim 7, wherein the dead or inactivated spore or a fragment thereof is for treating allergic rhinitis.

9. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the allergen is selected from a group consisting of: pollen, dust or dust mites, mould, flakes of skin from certain animals, optionally dogs and cats, wood dust, flour dust, optionally wherein the pollen is pollen from trees, grass and / or weeds.

10. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is used to treat Rhinosinusitis.

11. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is used to treat acute rhinosinusitis, subacute rhinosinusitis, recurrent acute rhinosinusitis and / or chronic rhinosinusitis.

12. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is used to treat acute rhinosinusitis.

13. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is derived from a bacterium belonging to the Bacillus species.

14. A dead or inactivated spore or a fragment thereof, for use according to claim 13, wherein the Bacillus spp is selected from a group of Bacillus spp consisting of:Bacillus. subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus clausii, Bacillus coagulans, Bacillus pumilus, Bacillus firmus, Bacillus flexus, Bacillus licheniformis, Bacillis marisflvi, Bacillus po / yfermenticus, Bacillus megaterium, Bacillus flexus, and Bacillus indicus.

15. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof comprises a Bacillus subtilis spore or a fragment thereof.

16. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof comprises a Bacillus subtilis strain DSM 32444, HU58, PY79, CUI, or DE11 spore, or a fragment thereof.

17. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the bacterial spore has been killed, inactivated or rendered non-viable by autoclaving, gamma irradiation, UV irradiation or formaldehyde treatment.

18. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the spore fragment comprises one or more of: core, exosporium, spore coat, inner spore coat, outer spore coat, spore cortex, and core wall,19. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof binds to am allergen and thereby block its interaction with a host cell, optionally wherein the dead or inactivated spore or a fragment thereof reduces symptoms of allergy by binding and blocking interaction of allergen with host cells.

20. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or fragment thereof is configured to stimulate a shift from a Th2- to Thl-mediated response.

21. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or fragment thereof is configured to reduce Th2 cytokine expression in a subject who has been administered with the dead or inactivated spore or fragment thereof.

22. A dead or inactivated spore or a fragment thereof, for use according to claim 20, wherein the Th2 cytokine is selected from a group of Th2 cytokines consisting of: IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, IL-17E (IL-25), and IL-31.

23. A dead or inactivated spore or a fragment thereof, for use according to claim 22, wherein the Th2 cytokine is IL-4 and / or IL-13, optionally wherein IL-4 and IL-13 expression is reduced.

24. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or fragment thereof is configured to increase Thl cytokine expression in a subject who has been administered with the dead or inactivated spore or fragment thereof.

25. A dead or inactivated spore or a fragment thereof, for use according to claim24. wherein the Thl cytokine is selected from a group of Thl cytokines consisting of: IL-2, IFNy, TNFo, and TNF|3 / LTa.

26. A dead or inactivated spore or a fragment thereof, for use according to claim25, wherein the Thl cytokine is IFN-y.

27. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dead or inactivated spore or a fragment thereof is mucosally or nasally administrable.

28. A dead or inactivated spore or a fragment thereof, for use according to any preceding claim, wherein the dosage is 102to 1015, 108to 1O1Z, or 108to 1010, spores.

29. A pharmaceutical or non-pharmaceutical composition for use in treating allergy, wherein the pharmaceutical composition comprises the dead or inactivated spore or a fragment thereof as defined in any one of claims 1-28, and a pharmaceutically acceptable vehicle or carrier.

30. The pharmaceutical or non-pharmaceutical composition according to claim 29, wherein the composition is a nasally administrable composition.

31. The pharmaceutical or non-pharmaceutical composition according to either claim 29 or claim 30, wherein the composition of the invention comprises a nasal spray comprising at least 0.1, 0.5, 1, 2, 3, 4 or 5 billion heat-treated spores of Bacillus subtilis.

32. The pharmaceutical or non-pharmaceutical composition according to any one of claims 2.9-31, wherein the composition is administered 2-3 times per day to each of the nostrils, each time 1-2 sprays, preferably corresponding to about 0.2 ml / spray.

33. A process for making the composition according to any one of claims 29-31, the process comprising combining a therapeutically effective amount of the dead or inactivated spore or a fragment thereof as defined in any one of claims 1-28, with a pharmaceutically acceptable vehicle or carrier.

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