Method of treating and / or preventing asthma, asthma exacerbations, allergic asthma and / or associated conditions with microbiota related to respiratory disorders

Abstract

To provide bacterial extracts for treating and / or preventing microbiota-associated respiratory disorders selected from asthma, asthma exacerbation and / or allergic asthmatic conditions in human subjects.SOLUTION: Provided is a bacterial extract, comprising Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and / or Streptococcus sanguinis, the bacterial strains being dissolved at a pH greater than 10 during preparation of the extract, and the bacterial extract being administered to the subject via an intranasal or intratracheal route.SELECTED DRAWING: Figure 18A

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel methods and routes of administration and delivery devices of specific bacterial extracts obtainable by alkaline lysis of Gram-positive or Gram-negative bacterial species for the treatment and / or prevention of asthma, asthma exacerbations, allergic asthma and / or wheezing related symptoms and / or related disease states in human subjects with a microbiota associated with respiratory diseases, for the reorganization of a protective microbiota in asthmatic subjects and for the organization of the microbiota towards protective immunity in healthy subjects. [Background technology]

[0002] 2. Background of the Invention Chronic airway diseases such as asthma, allergic asthma and other wheezing disorders are one of the major health care problems, especially in children. Asthma is an inflammatory disease of the airways in the lungs and bronchi, which temporarily results in inflammation, constriction and narrowing of the airways. Asthma can be caused by a primary infection, an allergen or irritant that is aspirated into the lungs. Symptoms include difficulty breathing, excess mucus and sputum production, wheezing, coughing and chest tightness. The underlying cause of asthma is a complex product of genetic and environmental factors that result in a significant heterogeneity of the disease. The prevalence of asthma has increased dramatically in Westernized countries in recent decades. This is likely due to changes in environmental exposure.

[0003] Recent evidence also supports a role for the microbiota in the development of asthma and wheezing disorders, suggesting that alterations in the microbiota are associated with chronic airway disease states, particularly asthma and wheezing disorders. The hypothesis is that early-life exposures may perturb the composition of the microbiota, thereby promoting immune dysregulation in the form of hypersensitivity disorders. A healthy microbiome provides numerous benefits to the host, including resistance to colonization by a wide range of pathogens, biosynthesis and absorption of essential nutrients, and immune defense and stimulation that maintain a healthy gastrointestinal epithelium and well-regulated systemic immunity. Thus, the pulmonary airway and intestinal microbiota play prominent roles in the pathogenesis of many diseases and disorders, including various pathogen infections of the gastrointestinal tract. Summary of the Invention [Problem to be solved by the invention]

[0004] In situations of imbalance, the function of the microbiota may be lost or disturbed, resulting in increased pathogen susceptibility, altered metabolic profile, or induction of pro-inflammatory signals (which may result in local or systemic inflammation or autoimmunity). In particular, microbiota alterations are associated with several chronic airway diseases and conditions, such as asthma and wheezing. Thus, patients become more susceptible to pathogen infections when normal gut microbiota is disturbed by the use of broad-spectrum antibiotics. Many of these diseases and disorders are chronic conditions that significantly reduce the quality of life of patients and may ultimately be fatal. [Means for solving the problem]

[0005] Summary of the Invention The present invention is based on the surprising discovery that the administration of specific bacterial extracts, obtainable by alkaline lysis of Gram-positive or Gram-negative bacterial species, provides an effective and safe approach to prevent allergic inflammation in the airways whilst restoring a healthy microbiome in hosts suffering from asthma and allergy-induced exacerbations clearly associated with an imbalanced microbiota. The present invention relates to novel routes of administration and dosing of bacterial extracts for use in methods for treating and / or preventing asthma, asthma exacerbations, allergic asthmatic conditions, wheezing and related microbiota-associated disorders in subjects in need thereof. Different experiments using similar or different airway inflammation inducers are illustrated in the figures.

[0006] The present invention also relates to a method for monitoring the treatment or prevention of a disease or abnormal or imbalanced microbiome in a subject, comprising: (a) treating the subject with a therapeutically effective amount of a purified bacterial extract obtainable by alkaline lysis of Gram-positive and / or Gram-negative bacterial species; (b) analyzing the signature (characteristics) of the microbiome of the subject; and (c) comparing said microbiome signature or pattern of the subject with that of a healthy subject, where an increase in the similarity of the microbiome signature of the subject with that of a healthy subject after treatment with a therapeutically effective amount of the stable purified bacterial extract compared to the similarity of the microbiome signature of the subject with that of the healthy subject before treatment indicates the efficacy of the treatment. Also shown are various figures intended to illustrate the genetic correlations and changes as well as the discrimination, correlation and taxonomic changes with the subject. [Brief description of the drawings]

[0007] [Figure 1] Figure 1: Shows the protocol used in the first study to evaluate the effect of BV bacterial extract (BV) in a classical allergen-driven mouse model of experimental asthma using BALB / c mice. [Diagram 2] Figure 2: (A) shows the effect of BV on bronchoalveolar lavage (BAL) eosinophilia expressed as a percentage of cell volume in the BAL fluid obtained in the second study. The study was performed using the protocol in Figure 1, except for the BV dose. A significant inhibition of eosinophilia was obtained with 14 doses of 1 mg BV. (B) shows a representative example of efficacy with BV, demonstrating a dose-dependent reduction in BAL eosinophilia in an asthma model using various doses. [Diagram 3]Figure 3: (A) Effect of BV on bronchoalveolar lavage (BAL) eosinophilia in different experiments, as in Figure 2. In this figure, BAL eosinophil load is expressed as total cell count. (B) BV suppresses key molecular phenotypes of asthma-induced lung inflammation: BAL protein levels for IL-5 and IL-13 (pg / mL). [Figure 4] Figure 4: Representative examples of the function efficacy correlations shown in Figures 2 and 3 are shown with lung function as the readout. Airway hyperresponsiveness (AHR) was significantly prevented by BV at all test points, with the 1 mg dose being the most effective. Significant reductions in AHR were expressed relative to OVA. [Diagram 5] Figure 5: Additional examples demonstrating that BV suppresses key cellular and molecular phenotypes of asthma-induced pulmonary inflammation. This figure also shows a marked reduction in mucus-secreting goblet cells in BV-treated animals as detected by PAS* staining. *PAS = Periodic Acid-Schiff staining. [Figure 6] Figure 6: Shows the protocol used to evaluate the protective effect of BV in an allergic asthma model driven by Alternaria alternata. [Figure 7] Figure 7: Representative example of protective effect of BV in an Alternaria allergic asthma model after intranasal administration. Monitoring of efficacy with BV is based on the difference in BAL cell amount, demonstrating an approximately 40% inhibition of eosinophils, expressed in this figure as a percentage of cells in the BAL fluid obtained after the experimental scheme described in Example 5. [Figure 8] Figure 8: Effect of in (intranasal) BV in the Alternaria model: Respiratory system resistance (Rrs) - Maximal response (representing total airway resistance). Rrs responses were significantly inhibited by BV at all test points, with the 1 mg dose being the most potent. Significant reductions in AHR were expressed relative to Alternaria. [Figure 9] Figure 9: In vitro prevention by BV of stress- and Alternaria-induced barrier function impairment (measured as TEER (transepithelial electrical resistance)) in human bronchial epithelial cells (16HBE14o cell line). [Figure 10] Figure 10: A shorter protocol for OVA-induced asthma (19 days instead of 39 days, 8 treatments instead of 14 treatments, compare with Figure 1). [Figure 11] FIG. 11: Blockade of BAL eosinophilia by BV, expressed as percentage (left) and cell number (right), obtained with the modified OVA induction scheme shown in FIG. [Figure 12] Figure 12: Additional protocol for OVA-induced asthma in C57 BL6 mice. In this protocol, BV is tested at a dose of 1 mg / day / mouse delivered intranasally for 14 treatments as before. [Figure 13] FIG. 13: Protective decrease in eosinophils and increase in neutrophils in BAL induced by BV bacterial extract (expressed as percentage of cells). [Figure 14] Figure 14: WGCNA - Identification of gene clusters (modules) regulated by OVA and / or BV and associated with major asthma phenotypes in lungs of mice treated as in Figure 1 and analyzed by RNA-Seq. Each box shows the Pearson correlation coefficient (r) for the relationship of each module eigengene (first principal component of the normalized gene expression profile of that module, i.e. summary of normalized module gene expression data) with traits (percent BAL eosinophils, AHR Z-score) and / or comparisons of interest (OVA vs. PBS, OVA+BV vs. OVA). The significance level is shown as q-value in brackets in each box. The number of genes in each module is shown in brackets after each module name. The color intensity reflects the strength of the association. [Figure 15]Figure 15: Ingenuity Pathway Analysis (IPA) of the core set of genes of the WGCNA-identified module highly associated with eosinophilia, AHR and OVA / BV treatment demonstrates that the genes cluster not only in pathways related to AHR and eosinophil cell migration, as predicted, but also in pathways related to Th2 cell migration and differentiation and dendritic cell chemotaxis. The core gene set identification strategy is shown on the left. The right panel shows selected biological functions (boxes) that are enriched in members of this core gene set (symbols). Genes are labeled with log2 values ​​(fold change) between OVA+BV and OVA-treated mice from differential expression analysis. Enrichment (P-value) and predicted activation value (Z-score) for each pathway are listed in each box. Symbol shape and color correspond to module membership: cyan: grey diamonds, brown: grey circles, blue: open circles and yellow: open squares. [Figure 16] Figure 16: PCoA plot of unweighted UniFrac β diversity across 3 experiments (#100, 101, 113) showing clustering of gut microbiota samples from mice treated intranasally with BV (1 or 2.25 mg / mouse x 14 treatments) alone or with OVA, or with OVA alone or PBS. All treatments were significantly different from each other (q<0.05; PERMANOVA). [Figure 17] Figure 17: On the left, taxonomic profile at phylum level for all groups and samples shown in Figure 16 (demonstrating community differences); on the right, α-diversity as measured by observed ESVs and Shannon diversity index for all groups and samples shown in Figure 16 (boxes represent 25-75% quartiles, whiskers represent 95% confidence intervals). [Figure 18]Figure 18: (A) Linear discriminant analysis effect size (LEfSe) of the samples from Figures 16 and 17 divided into two groups: with BV (BV / OVA and BV alone) and without BV (OVA alone and PBS alone). In the figure, taxa enriched in each group are indicated by color. The analysis revealed discriminant features at multiple taxonomic levels, using cutoffs of q<0.05 and LDA score>2. (B) Phylogenetic position of each of the discriminant taxa (g, genus; f, family; o, order; c, class). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention Thus, the present invention provides a method for treating and / or preventing asthma, asthma exacerbations, allergic asthmatic conditions, wheezing-related symptoms and microbiota-related disorders in a subject in need thereof or at risk of developing said disorders, comprising administering a therapeutically effective amount of a BV bacterial extract obtainable by alkaline lysis of a Gram-positive or Gram-negative bacterial species. The bacterial extract may be administered by oral, intranasal or intratracheal route. Particularly preferred routes of administration include the intranasal and intratracheal routes. The asthmatic condition may be steroid-resistant asthma, neutrophilic asthma or non-allergic asthma. The allergic disease or disorder may be an eosinophilic disease or disorder, particularly a disease or disorder selected from the group consisting of nodules, eosinophilia, eosinophilic rheumatism, dermatitis and swelling (NERDS).

[0009] The intranasal and intratracheal routes of administration of the BV bacterial extract according to the present invention have been shown to be particularly advantageous for use in methods for treating and / or preventing and / or reducing asthma, asthma exacerbations, allergic asthmatic conditions, wheezing-related symptoms and microbiota-associated respiratory diseases in subjects in need thereof. In the following examples of this specification, the applicants have demonstrated that administration of BV bacterial extracts provides effective protection against allergic asthma at lower doses, and is efficient in providing complete protection against allergen-driven airway hyperresponsiveness (AHR) and airway inflammation, complete abolition of eosinophilia, and protective enhancement of neutrophilia, using various lung airway inflammation models. In addition, the applicants have shown that lower doses of BV bacterial extracts by intranasal or intratracheal routes, compared to oral administration, result in complete abolition of eosinophilia in lung BAL and significant reduction of several TH2 cytokines (e.g., IL13 and IL5) levels.

[0010] In particular, intranasal or intratracheal administration allows the use of lower doses of BV bacterial extract, and it has been demonstrated that even such lower doses induce a significant reorganization of the microbiome mass in the gastrointestinal tract, providing a protective effect against allergic asthma and a substantial reduction in allergic responses due to the alteration of the microbiome. "BV bacterial extract" refers to a bacterial extract obtainable by alkaline lysis of one or more pathogens selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, as described in International Publication No. WO2008 / 109669. A process for the production of a BV bacterial extract produced by alkaline lysis, preferably at a pH greater than 10, is also described in WO2008 / 109669. Preferably, said BV bacterial extract may comprise any combination of the above pathogens, such as those marketed under the trademark Broncho-Vaxom® for treating respiratory diseases.

[0011] Typically, BV bacterial extracts are produced by fermentation followed by heat inactivation and alkaline lysis and filtration. Fermentation, alkaline lysis and filtration are well known in the art and are described, inter alia, in International Publication WO 2008 / 109669. Fermentation is generally performed by growing each bacterial strain in a culture medium to an appropriate optical density. To obtain sufficient amounts of material for each strain, the fermentation culture may be started from a working seed lot, followed by inoculation of a larger fermentation vessel. For example, the fermentation may be started with a small culture (e.g., 0.1-1.0 liter) and incubated at 30-40°C (e.g., 37°C) for about 3-6 hours until an optical density (OD) at 700 nm of 3.0-5.0 is obtained. After the small-scale cultivation step, additional cultivation in one or a series of larger fermentation vessels may be performed at 30°C-40°C for 3-20 hours (e.g., 3-10 hours or 8 hours). The culture medium is preferably one that does not pose a risk of prion-related diseases (i.e., Mad Cow Disease, Scrapie and Creutzfeldt-Jakob Disease) or other diseases and therefore does not contain animal-based materials (e.g., serum or meat extracts from animals, such as cows or sheep or other animals that can transmit prion-based diseases). For example, non-animal media (e.g., plant-based media, such as soy-based media, or artificial or semi-artificial media) may be used. Alternatively, media using horse serum or media containing materials from animal species that do not transmit prion diseases may be used. The culture medium may also contain biological extracts that do not pose a risk of the above diseases, such as yeast extract and horse serum. Supplemental growth factors may be introduced to enhance the growth of some bacterial species.

[0012] After fermentation, the biomass from each bacterial strain or combination of bacterial strains is generally inactivated by heat treatment, concentrated and frozen. Alkaline lysis is used to lyse bacterial cells under basic conditions and is generally carried out using organic or inorganic bases. Alkaline lysis can be carried out on a single bacterial biomass or a mixture of bacterial biomasses or fermentation batches under basic conditions, typically using a concentrated solution of hydroxide ions (e.g. ions from NaOH). Alkaline lysis can be carried out at a pH preferably greater than about 10 (which can have a variation of ±0.1). The lysis time can be determined by the skilled artisan and depends on the initial amount of bacterial biomass. Lysis can be carried out at a temperature in the range of 30-60°C, for example 30-40°C or 35-40°C, for example 37°C. Generally, lysis is stopped when all the bacterial cells appear to be destroyed by visual observation, as is well known to the skilled artisan. When two or more strains of the same bacterial genus are used, the strains can be lysed together or separately. Thus, the strains can be mixed before lysis or after lysis.

[0013] The lysate is then purified by centrifugation and / or filtration to remove any components that are large cellular debris or poorly broken down insoluble or particulate material to obtain a soluble bacterial extract. Purification, including centrifugation and filtration, to remove particulate matter from the extract is well known in the art. For example, the lysate is centrifuged at 9000 g, followed by one or more filtrations. Typically, filtration may involve passing the extract or mixture of extracts through one or more filters, such as a microfilter (i.e., subjecting it to microfiltration) or an ultrafilter (i.e., subjecting it to ultrafiltration), which may be repeated several times or cycles. For example, several successive filtrations through a larger pore filter may be followed by microfiltration using a smaller pore filter (e.g., a 0.2 micron filter). Ultrafiltration may also be used to aid in the extraction of soluble materials from the extract, for example, the ultrafiltration permeate may be recycled for further microfiltration. Tangential flow filtration (TFF) methods may be used to filter the extract and extract soluble molecules from larger cellular debris. This is well known in the art and is described, inter alia, in Wayne P. Olson (Separations Technology, Pharmaceutical and Biotechnology Applications, Interpharm Press, Inc., Buffalo Grove, IL, USA, pp. 126-135). To date, BV bacterial extract has been orally administered to patients for the prevention of respiratory tract infections in adults and children. Several clinical trials have demonstrated that enteral administration (oral administration) of BV bacterial extract can prevent allergic asthma and wheezing caused by acute airway disease in children. The BV bacterial extract is commercially available under the trademark Broncho-Vaxom®. BV bacterial extract medicine is in solid form and is generally administered orally to patients, with one capsule per day of 7 mg of freeze-dried bacterial extract for the treatment of adults and 3.5 mg of freeze-dried bacterial extract for children. It is a capsule with a dosage regimen of one capsule per day.

[0014] According to a preferred embodiment, the present invention relates to a method for the treatment and / or prevention of asthma, asthma exacerbations, allergic asthmatic conditions, wheezing associated symptoms, microbiota associated disorders in a subject in need thereof, comprising administering a therapeutically effective amount of a BV bacterial extract by an intranasal or intratracheal route.The present invention also relates to a BV bacterial extract for use in a method for the treatment and / or prevention of asthma, asthma exacerbations, allergic asthmatic conditions, wheezing associated symptoms, microbiota associated disorders in a subject in need thereof, wherein the BV bacterial extract is administered to said subject by an intranasal or intratracheal route. Intranasal or intratracheal administration of BV bacterial extracts is particularly useful for the treatment and / or prevention of asthma, asthma exacerbations, allergic asthmatic conditions and / or wheezing related symptoms and / or microbiota-associated disorders in human subjects, reorganization of protective microbiota in asthmatic subjects and organization of the microbiota towards protective immunity in healthy subjects.

[0015] The term "microbiota" refers to a community of commensal, symbiotic and / or pathogenic microbes. Microbes or microorganisms actually live in every part of the biosphere and are found everywhere in or on the human body, including mucous membranes in the nasal cavity, oral cavity, vagina, skin, gastrointestinal tract and genitourinary tract. The term "microbiome" refers to the entire collection of microbes and genetic information in a particular body area ("habitat") of a host. Each type of microbe may produce different effects in light of the situation in which it invades for survival. The composition of the microbiota (which may, for example, survive in a mammal) may affect immune function, nutrient processing and other physiological aspects. The composition of the microbiota may change over time and may be affected by age, diet, exposure to antibiotics and other environmental influences. When different microbial species produce roughly the same effect, the microbiota is said to have some functional redundancy. The addition or loss of microbial species that produce similar effects is likely to have little effect on the overall effect that the microbiota has on a mammal's physiological system, but the addition or loss of some microbial species, even when present in low numbers, can produce significant effects on mammalian physiology.

[0016] The term "microbiome imbalance" or "dysbiosis" refers to a state in which a system has an imbalance of beneficial and harmful microbes. Dysbiosis can occur when the microbiome has a low diversity of beneficial microbial species and / or no functional redundancy of beneficial microbes. Microbiome imbalance occurs when the microbial species that produce what is considered to be beneficial effects on the system are at least equal to the microbial species that produce what is considered to be harmful effects on the system. Typically, in the case of dysbiosis, the microbiome signature shows an altered microbiome reorganization and an increase in lactobacillus taxa. The present invention also relates to methods for the treatment and prevention of said diseases and microbiota-associated disorders in human subjects, limited to the reorganization of a protective microbiota in asthmatic subjects and its organization towards protective immunity in healthy subjects. In the past few years, a major international effort by the National Institutes of Public Health (NIH) called the Human Microbiome Project (HMP) and more widely known as the Human Microbiome Consortium (IHMC) aims to characterize the microbes that inhabit the human body (see http: / / hmpdacc.org / data_browser.php). The large intestine, home to an estimated 100 trillion microbes, appears to play an essential role in the metabolism of foods, drugs, and dietary supplements that are not absorbed in the upper gastrointestinal (GI) tract. In addition, some of these microbes produce essential nutrients and vitamins required for health maintenance. This microbial interaction in the gut environment plays a key role in signaling the metabolic, behavioral, and immune regulatory systems of the human host. The microbiome includes commensal, symbiotic and pathogenic bacteria, fungi and viruses that form an ecological entity and interact with itself and with a particular host. For a long time, it was thought that the colonization of the microbiota was limited to body surfaces such as the skin and the gastrointestinal tract. However, in recent years, it has become clear that microbes live in the vicinity of all human tissues, including the mammary gland, ovaries, uterus, placenta and lungs. Thus, the human body is colonized by trillions of inhabitants. They constitute an ecological community that is diverse, individual and also changes with age.

[0017] Different microbiomes have been directly implicated in the etiopathogenesis of several pathological conditions as diverse as asthma, asthma exacerbations, allergic asthmatic states and wheezing-related conditions. Therefore, the BV bacterial extracts described in the above embodiments are beneficial in restoring the natural microbiome and are particularly useful in the treatment and / or prevention of asthma, asthma exacerbations, allergic asthmatic conditions, wheezing-related symptoms, and microbiota-related disorders. The therapeutically effective amount of BV bacterial extract provided is such that the signature or pattern of the subject's microbiome becomes more similar to that of a healthy subject, thereby treating and / or preventing pathological conditions associated with microbiome imbalance. What is considered a healthier system can be established or re-established by promoting the development of beneficial microbes while limiting harmful microbes. In this case, a state of eubiosis is promoted in which beneficial microbes in the system have a dominant effect because there is a high diversity and / or functional redundancy of beneficial microbial species in the system. The BV bacterial extract may be in any form suitable for oral administration, for example in solid or liquid form, and may be formulated as a pill, tablet, film tablet, coated tablet, capsule, syrup, powder and / or deposit. According to a preferred embodiment, the BV bacterial extract may be present in any form, either liquid, gaseous or solid form, that is suitable for intranasal and intratracheal administration. When present in liquid or aerosol form, the BV bacterial extract may be formulated into a spray, droplets, colloid, mist, nebula or atomized vapor. Alternatively, the BV bacterial extract may be present in solid form and formulated into a powder or crushable tablet.

[0018] When administered by the intranasal route, the BV bacterial extract preparation is preferably in a form selected from an emulsion, a suspension, a colloidal form, a mist, a cloud, a finely divided smoke or spray, liquid drops, a colloidal form, a mist, a cloud, a finely divided smoke, a nasal tampon, a nasal emulsion, a powder, an ointment, a cream, a lotion, a gel, a paste, a salve, a solution, a tincture, a patch, a bioadhesive strip. When the BV bacterial extract preparation is administered by the intratracheal route, delivery requires aerosolization of a solid or liquid and delivery of the aerosol through the mouth and throat to the lungs. Particles of the BV bacterial extract may be administered to the lungs as a dry powder aerosol or a liquid aerosol. Dry powder aerosols are typically administered to the lungs with dry powder inhaler (DPI) inhalation devices. Dry powder inhalers may include breath-actuated dry powder inhalers, such as those described in U.S. Pat. No. 7,434,579. Metered dose inhalers contain medicament suspended in a propellant, a mixture of propellants, or a mixture of solvents, propellants, and / or other excipients in a compact pressurized aerosol dispenser. MDI products can eject hundreds of metered doses of the BV bacterial extract. One actuation may contain a few micrograms (mcg) to milligrams (mg) of active ingredient delivered in a volume, typically 25-140 microliters. Another type of liquid aerosol dispersion device is a nebulizer, which utilizes a jet, vibrating mesh or other means to aerosolize a suspension containing particles of the BV bacterial extract. When preparing liquid, semi-solid, solid and spray medicaments, the BV bacterial extract may be formulated with any additives, such as vehicles, binders, flavors, fragrances, sweeteners, colorants, preservatives, antioxidants, stabilizers and surfactants, if desired.

[0019] To prepare the pharmaceutical preparation, the BV bacterial extract can be mixed with pharma- ceutically acceptable carriers, adjuvants and / or excipients according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers that can be used in the composition include any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The composition can additionally contain solid pharmaceutical excipients, such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, etc. Liquid and semi-solid excipients can be selected from glycerol, propylene glycol, water, ethanol, and various oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences (E. W. Martin, ed., Mack Publishing Company, 18th ed., 1990). The bacterial extract preparations may also contain stabilizers and preservatives. In certain embodiments, it may be desirable to extend the residence time in the nasal cavity, for example to enhance absorption. Thus, the BV bacterial extract may be formulated, if desired, with a bioadhesive polymer, gum, such as xanthan gum, chitosan (e.g., a high-purity cationic polysaccharide), pectin or any carbohydrate that thickens or emulsifies like a gel when applied to the nasal mucosa, microspheres, such as starch, albumin, dextran, cyclodextrin or derivatives thereof, gelatin, liposomes, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and / or cellulose (e.g., methyl or propyl; hydroxy or carboxy; carboxymethyl or hydroxypropyl).

[0020] Alternatively, the BV bacterial extract preparation may be formulated as a crushable tablet. The tablet may be crushed whole or lightly, for example, by finger pressure, and sprinkled into a suitable vehicle for administration. Crushable tablets may be manufactured using direct compression processes and excipients, with care taken to avoid damage to the coating of the individual subunits during the process. Suitable excipients for manufacturing crushable tablets include those typically used in chewable tablets, including mono- and disaccharides, sugar polyols, and the like, or combinations thereof. Exemplary excipients include mannitol, sorbitol, xylitol, maltitol, lactose, sucrose, maltose, or combinations thereof. Optional pharmaceutical excipients, such as diluents, lubricants, glidants, flavoring agents, coloring agents, and the like, or combinations comprising at least one of the above agents, may also be included in the compression base. Crushable tablets may be manufactured using tablet manufacturing methods known in the pharmaceutical arts. The BV bacterial extract formulation may further be in a colloidal form, for example, containing a metal halide, most preferably a silver halide. The BV bacterial extract and adjuvants may be incorporated or encapsulated within the colloidal particles. Alternatively or additionally, one or more of the bacterial extracts and adjuvants may be attached to the surface of the colloidal particles. For example, proteins are easily adsorbed or attached to hydrophobic particles by hydrophobic interactions with the particle surface, replacing a portion of the neutral emulsifier.

[0021] The appropriate dosage of BV bacterial extract will vary depending on the condition, age and species of the subject and can be readily determined by one of skill in the art, however, in accordance with the present invention, the total daily dosage may be in the range of 0.005-2 mg, preferably 0.05-1 mg, most preferably 0.5-1 mg, administered as a single or divided dose, and may exceed the upper limit of 2 mg if found to be effective. The BV bacterial extract may be administered intranasally by a nasal insufflation device, an intranasal inhaler, an intranasal spray device, an atomizer, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle, a nebulizer, a metered dose inhaler (MDI), a pressurized metered dose inhaler, a blower, a two-way device, a dose ampoule, a nasal pad, a nasal sponge, and a nasal capsule. Also provided is a delivery device for use in a method for treating and / or preventing asthma, asthma exacerbations, allergic asthmatic conditions, wheezing-related symptoms, microbiota-associated disorders in a subject in need thereof. The delivery device may be selected from the group including nasal insufflation devices, intranasal inhalers, intranasal spray devices, atomizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, nebulizers, metered dose inhalers (MDIs), pressurized metered dose inhalers, insufflators, two-way devices, dose ampoules, nasal pads, nasal sponges, nasal capsules, and the like. The nasal spray may be a liquid, solid nasal spray for administration as an aerosol or in a non-aerosol form. The nasal delivery device may be metered to administer a precise effective dosage of the BV bacterial extract to the nasal cavity. The nasal delivery device may be for single unit delivery or for multiple unit delivery. The therapeutically effective amount of the BV bacteria of the present invention may be delivered by tube, catheter, syringe, packtail, pledget, nasal tampon, or submucosal injection (as described in U.S. Patent Publications US 2009 / 0326275, 2009 / 0291894, 2009 / 0281522, and 2009 / 0317377).

[0022] When intranasal or intratracheal administration is performed using an aerosol, the spray can be generated, for example, from a pressurized container using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen, carbon dioxide, or other suitable gas. The dosage unit can be determined by providing a valve to deliver a metered amount. A pump spray dispenser can dispense a metered dose or a dose having a specific particle or droplet size. As used herein, the term "aerosol" can refer to a suspension or dispersion of liquid droplets or solid powder in air (or gas). Droplets can be formed from solutions, suspensions, and dispersions of the drug in liquids, such as water or non-aqueous solvents. The aerosol can be blown through the nose or inhaled. The aerosol can be produced in any suitable device, for example, MDI, nebulizer, atomizer. Typically, the aerosol may be insufflated using a suitable mechanical device. In some embodiments, the device may comprise a reservoir and a sprayer, which is a device that expels a pharmaceutical dose in the form of a spray. Doses of the BV bacterial extract to be administered may be contained within the reservoir, optionally in a liquid solution or suspension, or in a solid particulate formulation, e.g., a solid particulate mixture. Alternatively, nebulizer devices can be used which produce a stream of high velocity air that causes the BV bacterial extract to be administered in liquid form to spray as a mist, e.g., microparticles (wherein 90% or more of the particles have a diameter of less than about 10).

[0023] Another delivery device for intranasal or intratracheal administration of the BV bacterial extract according to the present invention can be a DPI device, which typically administers a therapeutically effective amount in the form of a free-flowing powder that is dispersible in the patient's inspiratory air-stream. DPI devices using an external energy source can also be used in the present invention. To achieve a free-flowing powder, the BV bacterial extract may be formulated with a suitable excipient, such as lactose. A dry powder formulation can be produced, for example, by combining and dry blending dry lactose having a particle size of about 1 μm to about 100 μm with microparticles of the BV bacterial extract. Alternatively, the BV bacterial extract may be formulated without excipients. The formulation is loaded into an inhalation cartridge or capsule or dry powder dispenser for use with a dry powder delivery device. Examples of commercially available DPI devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (U.S. Pat. No. 5,035,237); Diskus (U.S. Pat. No. 6,378,519); Turbuhaler (U.S. Pat. No. 4,524,769); and Rotahaler (U.S. Pat. No. 4,353,365). Further examples of suitable DPI devices are further described in U.S. Pat. Nos. 5,415,162; 5,239,993; and 5,715,810.

[0024] MDI devices typically use compressed propellant gas to expel a therapeutically effective amount of BV bacterial extract. Formulations for MDI administration include solutions or suspensions of active ingredients in a liquefied propellant. The formulations are loaded into an aerosol canister (forming part of the MDI device). Examples of propellants include hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), and chlorofluorocarbons, such as CC13F. Additional components of HFA formulations for MDI administration include cosolvents, such as ethanol, pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. MDI devices are described, inter alia, in U.S. Pat. No. 5,225,183 and International Publication WO 92 / 22286. When the BV bacterial extract is in the form of a powder, it may be administered by the intranasal or intratracheal route according to the present invention using a nasal insufflator. Typically, the BV bacterial extract may be adsorbed onto a solid surface, such as a carrier, and then delivered to the nasal cavity as a powder, such as a powder in the form of microspheres. The powder or microspheres may be stored in a reservoir in the insufflator and then administered in a dry, air-dispensable form. Alternatively, the powder or microspheres may be filled into capsules (e.g. gelatin capsules) or other single-dose units adapted for nasal administration. When delivered by a nasal spray applicator, the BV bacterial extract of the present invention may be placed in a nasal spray dispensing device or atomizer and applied by spraying into the subject's nostril for delivery to the mucous membrane of the nasal passage. For a nasal spray, up to about 200 microliters, about 50 to about 150 microliters, or about 75 to about 120 microliters may be applied. It may be administered to one or more nostrils, and application may be as frequent as desired or required. A first dose (in microliters) may be administered to a nostril. After a suitable period of time (during which time the liquid is absorbed), a second dose may be subsequently administered to the same or the other nostril. Separate metered sprays may be applied to alternating nostrils until the complete target therapeutic dose has been administered to the patient. The interval between applications of the benzodiazepine drug to the same nostril may be in time increments of several seconds to 5 minutes, preferably about 10 seconds to about 1 minute. This allows time for the drug to cross the nasal mucous membrane and enter the bloodstream. Multiple applications of metered sprays to each nostril (optionally separated by time intervals) allow for complete absorption of the composition into the bloodstream and allow for administration of a complete therapeutic dose in time increments short enough to avoid loss of drug down the back of the throat.

[0025] The BV bacterial extract may further comprise an adjuvant, a pharma- ceutically acceptable solvent, and a permeation enhancer. For example, for intranasal delivery, the permeation enhancer may enhance the permeation of the composition through the nasal mucosa. In some embodiments, a compound containing one or more hydroxyl groups may be used as a permeation enhancer. Some of these hydroxyl group-containing compounds may act as solvents in the composition. Non-limiting examples of hydroxyl group-containing compounds that may act as permeation enhancers include alcohols (e.g., ethanol), diols (e.g., propylene glycol, also known as 1,2-propanediol; 1,3-propanediol; butylene glycol, including 1,3-butanediol, 1,2-butanediol, 2,3-butanediol, and 1,4-butanediol; hexylene glycol; dipropylene glycol, 1,5-pentanediol, 1,2-pentanediol, 1,8-octanediol, ethohexadiol, p-methanol ... Examples of suitable diols include dimethylaminopropylamine, ...

[0026] Those skilled in the art will recognize that the present teachings are also applicable to other permeation enhancers. These may include simple aliphatic unsaturated or saturated esters. Non-limiting examples of such esters include isopropyl myristate, myristyl myristate, octyl palmitate, and the like. Non-limiting examples of other permeation enhancers include alcohols (e.g., short and long chain alcohols), polyalcohols, amines and amides, urea, amino acids and their esters, amides, azones or pyrrolidones and their derivatives, terpenes, fatty acids and their esters, macrocycles, sulfoxides, tensides, benzyldimethylammonium chloride, cetyltrimethylammonium bromide, cineole, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dodecylpyridinium chloride, dodecylamine, hexadecyltrimethylammoniopropanesulfonate, limonene, linoleic acid (OA), linolenic acid (LA), methanol, methyl laurate, methylpyrrolidone, N-decyl-2-pyrrolidone, NLS, nicotine sulfate, nonyl-1,3-dioxolane, octyltrimethylammonium bromide, oleyl betaine, PP, polyethylene glycol. Dodecyl ether, polyoxyethylene sorbitan monolaurate (Tween 20 or polysorbate 20), SLA, sodium oleate, sodium lauryl sulfate, sodium octyl sulfate (SOS), sorbitan monolaurate (S20), tetracaine, and Triton X-100.

[0027] Examples of pharma- ceutically acceptable solvents that can be used in the present invention can be found in references such as Handbook of Pharmaceutical Excipients (5th ed., Pharmaceutical Press, London and American Pharmacists Association, Washington, 2006).Non-limiting examples of pharma-ceutically acceptable solvents that can be used in the compositions of the present invention include, but are not limited to, propylene glycol, such as 1,2-dihydroxypropane, 2-hydroxypropanol, methylethylene glycol, methyl glycol or propane-1,2-diol), ethanol, methanol, propanol, isopropanol, butanol, glycerol, polyethylene glycol (PEG), glycol, Cremophor EL or any form of polyethoxylated castor oil, dipropylene glycol, dimethyl isosorbide, propylene carbonate, N-methylpyrrolidone, glycofurol, tetraethylene glycol, propylene glycol fatty acid esters and mixtures thereof. The present invention further provides a kit comprising the above-described BV bacterial extract or a delivery device for containing said BV bacterial extract and a package insert incorporating manuals / instructions for use. EXAMPLES

[0028] First series of experiments: Asthma protection Example 1 Effect of different doses of BV bacterial extract on OVA-induced asthma model Six doses of BV bacterial extract (hereafter also referred to as "BV") were tested in two separate experiments (Studies 1 and 2) using the intranasal route under anesthesia in four mice / group: - Study 1: 5-50-1000 micrograms / treatment x 14 treatments - Study 2: 14 treatments of BV 1-10-100-1000-2250 micrograms / treatment. The dosing schedule was selected from a previous study performed during the setup of an ovalbumin-induced (OVA) asthma model (Stein et al., N Engl J Med 2016;375:411-421) using different courses of Amish dust. The dose of BV was derived from a previous optimization study using a viral infection model and was extended to 1 and 2.2 mg / dose in this experimental scheme. For this purpose, in a phase 1 planned dose-response experiment, OVA-immunized mice were treated by intranasal / inhalation (in) route with increasing doses of BV bacterial extract (1, 5, 10, 50, 100 or 1000 micrograms / mouse / treatment) every 2-3 days from day 1 to day 32 (total of 14 times). A dose of 1 mg / mouse was proposed for exploratory feasibility. In phase 2, the same protocol was adapted to further test the BV concentration that showed optimal efficacy (1000 micrograms / mouse / treatment) and to add a higher dose (2250 micrograms / mouse / treatment) to assess whether a plateau of maximum protection was reached.

[0029] OVA-induced asthma animal model Male BALB / c mice aged 7-8 weeks were purchased from Envigo (USA) and maintained under specific pathogen-free conditions in the animal facility of the BIO5 Institute. The animals were fed a standard hypoallergen diet. In the example scheme of FIG. 1, BV bacterial extract concentrate was diluted in 0.9% saline. The OVA model was adapted from Stein M et al. Briefly, 7-8 week old BALB / c mice (Envigo) were sensitized intraperitoneally (ip) with ovalbumin (OVA: grade V, Sigma, 20 micrograms)-alum (Pierce) on days 0 and 14, and administered BV bacterial extract (1, 5, 10, 50, 100, 1000 micrograms / mouse / treatment) intranasally (in) every 2-3 days (total 14 times) from day 0 to day 32 under light isoflurane anesthesia, and the mice were challenged in with OVA (50 micrograms) on days 28 and 38. Group 1 of mice received saline at the time of treatment, sensitization, and challenge. Group of mice in experiment 3 was sham-treated. Final evaluation on day 39 included the use of non-exhaustive readouts: 1) invasive measurements of AHR; 2) cellularity with differentials in bronchoalveolar lavage (BAL) fluid; 3) pulmonary cytokine RNA and protein levels; 4) analysis of selected lung cell populations; and 5) measurements of serum OVA-specific IgE.

[0030] In the first exploratory experiments (Study 1 and Study 2), airway resistance in response to increasing concentrations of nebulized methacholine (0–40 and 0–60 mg / ml) was assessed in animals anesthetized with ketamine and xylazine (100 and 10 mg / kgy, respectively) and paralyzed with pancuronium bromide (4 μg / g). A 20-gauge cannula (BD) was inserted into the excised trachea and secured in place by suturing. Mice were then artificially ventilated on a mini-ventilator (FlexiVent FX, SCIREQ, Inc.) with a tidal volume of 10 mL / kg, an inspiratory / expiratory ratio of 66.67%, a ventilation rate of 150 / min, and a maximum pressure of 30 cmH2O. In the final experiment (Study 3) with the optimal BV bacterial extract concentration (1,000 micrograms / mouse / treatment), airway resistance in response to increasing doses of acetylcholine (0–1 micrograms / mouse) administered intravenously was measured. BAL fluid was obtained by delivering 1% cold BSA in PBS (2 mL) to the airways via a tracheal cannula and gently suctioning. Cells were counted using a Countess II FL automated cell counter (Thermo Fisher Scientific). Differentials were determined by an operator blinded to mouse ID / grouping by examining at least 400 cells / slide after Hema 3 (Fischer) staining. BAL fluid cytokines (IL-5, IL-13, IL-17) were measured by ELISA (R&D). Statistical analysis: Statistical differences for all parameters were evaluated using Student's t-test (two-tailed unpaired test). A p-value of <0.05 was considered statistically significant.

[0031] Example 2 Example of the effect of intranasal administration of BV bacterial extract in the OVA asthma model Efficacy monitoring based on BAL eosinophilia (expressed as a percentage) following the experimental scheme described in Example 1. To evaluate the efficacy of BV bacterial extract in reducing eosinophilia associated with protection from pulmonary airway hyperresponsiveness (AHR), two separate experiments were performed with different doses of BV. The best efficacy in reducing eosinophilia (Y-axis) was achieved with a dose of 1 mg BV, with significant inhibition. The complete absence of eosinophilic effect in the placebo group, regardless of the dose used, also confirms the safe use of BV in non-diseased animals. Intranasal / inhaled delivery of BV suppresses key cellular phenotypes of asthma-induced pulmonary inflammation.

[0032] Example 3 Data set from a different experiment, identical to Example 2, but expressing BAL eosinophilia as cell (FIG. 3A) and molecular content (FIG. 3B). As in Example 2, BV bacterial extract induced inhibition of BAL eosinophilia, with significant inhibition at the 1 mg dose. Given that OVA induced a high degree of eosinophilia (>800,000 cells) in terms of cell counts in the BAL, a near complete and highly significant (P=0.005) arrest of eosinophilia was predicted for this formulation class.

[0033] Example 4 A representative example of a two-group experiment is shown in Figure 4. This figure demonstrates the protection from AHR by BV bacterial extract. The best protective effect was obtained with the 1 mg dose. The arrest of eosinophilia and concomitant pulmonary AHR function shown in Figures 2 and 3 demonstrate the ability of intranasally administered BV bacterial extract to provide protection in a well-established model of allergic asthma, and indicates that a dose of 1 mg / day / mouse x 14 treatments is adequate to confer optimal protection against allergen-driven AHR and airway inflammation. The protection achieved with this dosing regimen is significant, even though BV is administered to adult mice rather than newborn or young mice, and dosing is initiated simultaneously with allergen exposure rather than prior to exposure. In addition to this, as shown in Figure 5A, the periodic acid-Schiff (PAS) +The apparent absence of lung tissue-derived airway inflammatory goblet cells in the BV-treated OVA group (OVA / BV), quantified in Fig. 5B as a highly significant reduction in mucus-secreting cells (P=0.00003), functionally corroborates the protection against AHR shown in Fig. 4 .

[0034] Example 5 The scheme (FIG. 6) represents the experimental setting of intranasal (in) BV bacterial extract in the Alternaria allergic asthma model, using a daily dose of 1 mg according to the dosing regimen consisting of 14 doses as indicated.

[0035] Example 6 The effect of in BV bacterial extract in the Alternaria allergic asthma model was monitored using BAL differences. In Figure 7, approximately 40% inhibition of eosinophilia in the BAL contents was achieved by BV bacterial extract using 1 mg doses (14 times) as shown in Figure 6. Given the high relevance of this asthma model translatable to humans (same in sensitization and challenge route, without adjuvant), these results clearly demonstrate the ability of BV bacterial extract to confer protection in allergy-induced asthma. BALB / c mice (4–6 / group) were sensitized with Alternaria in (Greer Laboratories: 50 μg dry weight, 10 μg protein in 50 μl PBS) on days 0 and 1 and challenged with Alternaria in (25 μg dry weight, 5 μg protein in 50 μl) on days 17, 18, and 19. BV (concentrate, 1 mg in 50 μl (25 μl in each nostril)) was administered every 2 days for 14 doses starting on day -10. Final evaluation was performed on day 20. BAL fluid was obtained by delivering cold 1% BSA in PBS (2 mL) to the airways via a tracheal cannula and gently aspirating. Cells were counted using a Countess II FL automated cell counter (Thermo Fisher Scientific) and differentials were determined by an operator blinded to mouse ID / grouping by examining at least 400 cells / slide after Hema 3 (Fischer) staining.

[0036] Example 7 A representative example of a functional efficacy correlation using pulmonary function as the readout is shown in Figure 8. Respiratory resistance (Rrs) responses were significantly inhibited by BV bacterial extract, with the 1 mg dose having the best efficacy at all test points. A significant reduction in AHR to Alternaria was noted. Airway resistance in response to increasing concentrations of acetylcholine (0-2 μg / g mouse) administered intravenously was assessed in animals anesthetized with ketamine and xylazine (100 and 10 mg / kg, respectively). Mice were tracheotomized and an 18-gauge metal cannula was inserted into the trachea and tightly tied with sutures to prevent air leakage. The tracheal cannula was connected to a computer-controlled animal ventilator (FlexiVent, Scireq Inc., Canada) and mice were mechanically ventilated at 150 breaths / min, tidal volume of 10 mL / kg, and positive end-expiratory pressure (PEEP) of 3 cmH2O. Animals were placed on a heating pad to maintain body temperature and paralyzed with pancuronium bromide (1 mg / kg, ip). To standardize lung volume history, lungs were inflated twice to a pressure of 30 cmH2O (recruitment maneuver). Acetylcholine chloride (ACh) diluted in room temperature saline was injected as a bolus into the right jugular vein at increasing doses of 0.25, 0.5, 1.0 and 2.0 μg / g body weight. Saline was used as a control. Measurements were started immediately after each ACh administration and were performed every 30 seconds for 5 minutes. The peak response after each ACh administration was measured. Respiratory mechanics were evaluated using a steady-phase model. This model has the ability to partition respiratory properties into central and peripheral airways and to distinguish between different tissue properties. The parameters evaluated were Newtonian resistance (Rn), an approximation of the resistance in the proximal conducting airways and tissue damping (G) (related to lung tissue resistance and reflecting energy dissipation in the alveoli). Only coefficients of determination (COD) of 0.9 or greater were used for this steady-phase model.

[0037] As shown in FIG. 7, the 30-40% reduction in eosinophilia in BAL correlated perfectly with lung function readouts, confirming that BV provides near complete protection against AHR in this extreme acute allergic asthma model. In addition to this, monitoring the protective effect of BV on the tightness of the epithelial cell layer expressed as TEER (transepithelial electrical resistance), a measure of barrier function, shows the prevention by BV of stress- and Alternaria-induced damage to human bronchial epithelial cells, enabling drug transport studies in Figure 9. - This is demonstrated in an in vitro airway cell model using cell lines (see Forbes B, Int J Pharm. 2003, 257(1-2), 161-167).

[0038] Example 8 Figure 10 shows a representative example of the protocol used in Figure 1, with a modification of OVA-induced asthma. This model again used a prophylactic regimen of BV, but with two consecutive OVA-induced sensitizations and challenges after a reduced treatment of eight doses (treatments T1-T8) and at a reduced time (final evaluation on day 19 instead of day 39). Even in this new setting with a reduced number of treatments, significant inhibition of eosinophilia by BV was confirmed.

[0039] Example 9 FIG. 11 shows the effect of BV on bronchoalveolar lavage (BAL) eosinophilia, expressed as percentage (left) and cell count (right), similar to FIG. 2 but obtained from a modified OVA induction scheme. FIG. 11 shows significant inhibition of eosinophilia by BV bacterial extract, along with a significant increase in neutrophil recruitment in the BAL. The number of mice was n=4 mice / group, and 1 mg / 50 μl was used.

[0040] Example 10 Evaluation of OVA-induced asthma in a different mouse strain (C57BL6). Figure 12 shows a representative example of the protocol used in Figure 1, with a modification of OVA-induced asthma. This model again used a prophylactic regimen of BV bacterial extract, but with two consecutive OVA-induced sensitizations and three OVA challenges after 14 doses of treatment (treatments T1 to T14). The final evaluation was on day 30.

[0041] Example 11 Example showing the effect of intranasal administration of BV bacterial extract in an OVA-induced asthma model using the scheme shown in Figure 12 adapted to C57 BL6 mice. Efficacy monitoring was based on BAL eosinophilia, expressed as a percentage of total BAL cell volume (Y-axis) in Figure 13. Results showed the efficacy of BV in reducing eosinophilia, which is associated with protection of airway hyperresponsiveness (AHR). The best efficacy in reducing eosinophilia was obtained with a dose of 1 mg BV.

[0042] Example 12 Core lung expressed genes strongly associated with major asthma phenotypes (AHR and BAL eosinophilia) in mice treated with OVA and / or BV clustered in pathways negatively associated with Th2 and dendritic cell migration, as shown in FIG. 14. Unfractionated lung tissue was collected from 27 Balb / c mice treated with PB, BV, OVA or OVA+BV as in Figure 1, and RNA was isolated and subjected to RNA sequencing (RNA-Seq). Expression data was evaluated for 24,538 genes and filtered to include only genes with at least one read in 20% of the samples (19,613 genes total). Weighted gene co-expression analysis (WGCNA) was performed for all 19,613 genes on the entire lung expression data to construct co-regulated gene networks (modules) using a signed network algorithm. Expression data within each module was summarized using the module eigengene vector (i.e., the first principal component of the module) and correlation with immune phenotypes related to airway and protection was assessed using Pearson correlation. Ingenuity pathway analysis was used to determine enrichment for key biological terms or functions among differentially expressed features, where negative values ​​correspond to predicted inhibition and positive values ​​correspond to predicted activation.

[0043] WGCNA, shown in Figure 15, identified a total of 16 co-regulated gene networks (modules), of which only four were strongly and significantly associated with both eosinophilia and AHR (|Pearson's r|>0.5 and P≦0.001). The cyan and brown modules were positively associated with AHR and eosinophilia and negatively associated with BV+OVA treatment (vs. OVA), while the blue and yellow modules showed a negative association with AHR and eosinophilia and a positive association with BV+OVA treatment. Ingenuity pathway analysis (IPA) of the core set of genes (n=333, Figure 15 left) from the brown, cyan, blue, green, and yellow modules highly associated with eosinophilia and AHR showed that genes affected by BV+OVA were not only clustered in pathways related to AHR and eosinophil cell migration as expected, but also in pathways related to Th2 cell and dendritic cell migration (Figure 15 right). Each of these pathways was predicted to be downregulated by IPA.

[0044] The DC-associated BV-induced transcriptional signature identified by RNA-Seq motivated us to investigate the effect of in vitro administration of BV extract on bone marrow-derived DC (BMDC). Expression of MHC class II and costimulatory molecules (CD40, CD80, and CD86) was strongly inhibited in BV-treated BMDC. We therefore asked whether reprogramming of DC was sufficient to explain the suppressive effect of BV bacterial extract on allergic asthma. To this end, BMDC pulsed with OVA in vitro for 2 days with or without 2-day pretreatment with BV extract were administered in naive Balb / c mice and challenged with OVA for 3 consecutive days 10 days later. OVA-pulsed BMDC effectively elicited experimental asthma in these animals, as evidenced by enhanced HR, BAL eosinophilia, and increased type 2 cytokine expression in the lungs and airway-draining lymph nodes. Strikingly, preincubation of BMDCs with BV bacterial extracts was sufficient to potently and significantly inhibit all of these allergen-driven responses, identifying DCs as the primary target for BV-induced asthma protection in this model.

[0045] Second series of experiments: Microbiota changes Example 13: Effects of intranasal or intratracheal BV bacterial extracts on the mouse gut microbiome In these experiments, a protective effect induced directly by BV bacterial extract was demonstrated. This protective effect was mediated by an alteration of the gastrointestinal microbiota of the tested mice. Intranasal and intratracheal administration of BV bacterial extract in an asthmatic mouse model was shown to induce an alteration of the gastrointestinal microbiome, which may contribute to a reduction in the allergic response. Mice were treated intranasally with BV bacterial extract (5, 50, 1000 or 2500 micrograms / treatment every 2 days) or PBS, and sensitized and challenged with OVA or PBS. Fecal samples were collected for microbiome analysis. Compared to PBS-only controls, significant changes in gut microbial community structure were observed in mice administered 1000 or 2500 micrograms / treatment of BV bacterial extract (i.e., any high dose of BV bacterial extract) with either OVA or PBS, as shown by beta diversity analysis (unweighted UniFrac) (Figure 16). Furthermore, there was an enrichment of Lactobacillus in any high dose of BV bacterial extract versus PBS-only treated mice. These data clearly demonstrated that BV bacterial extracts induced significant changes in the mouse gut microbiota.

[0046] Example 14: Effects of intranasal treatment with high and low doses of BV bacterial extract on the gut microbiome profile of mice Mice were administered 14 intranasal doses (1 mg or 2.25 mg) of BV bacterial extract. OVA sensitization was performed on days 0 and 15, and OVA challenge on days 28, 38, and 39. Control mice received PBS instead of OVA. At the end of the experiment (day 39), fecal pellets were collected. Mice challenged with PBS or OVA were treated with a 1 mg dose (BV1000) or a 2.25 mg dose (BV2250). DNA representative of the gut microbiome was extracted from the fecal pellets. Extracted DNA was sent to Argonne National Laboratory for 16S rRNA amplicon sequencing on an Illumina MiSeq. Sequence data were analyzed using QIIME2 and R. Linear discriminant analysis effect size (LEfSe) was performed to reveal differential features at multiple taxonomic levels. This analysis used cutoffs of q<0.05 and LDA score>2 to compare two groups of samples: any BV (BV / OVA and BV alone regardless of BV treatment dose) and no BV (OVA alone and PBS alone) within experiments 100, 101 and 113 (Figure 18). Phylogenetic relationships among differential traits were also characterized (g, genus; f, family; o, order; c, class) (Figure 17).

[0047] These data suggest that intranasal BV bacterial extracts can protect against eosinophilia and AHR, and that protected myeloma In mice, a molecule known to produce short-chain fatty acids and be associated with regulatory immunity / tolerance This clearly demonstrates that BV-treated mice were significantly reorganized in the gut microbiome with an increase in BV-treated asthma-protected mice, particularly Lactobacilli. Indeed, the expansion of these bacteria may contribute to the induction of T regulatory cells that we found in BV-treated asthma-protected mice. Furthermore, the results of 16s rRNA sequencing experiments clearly demonstrated the association between intranasal or intratracheal administration of BV bacterial extracts and immune modulation.

Claims

1. 1. An extract of bacteria comprising Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis for the treatment and / or prevention of a microbiota-associated respiratory disease selected from among asthma, asthma exacerbations and / or allergic asthmatic conditions in a human subject in need thereof, wherein during the preparation of said extract, said bacterial strains are dissolved at a pH greater than 10 and administered to said subject by intranasal or intratracheal route.

2. 2. The bacterial extract of claim 1, administered to the subject at a daily dose in the range of 0.05 to 2 mg, at a daily dose in the range of 0.1 to 1 mg, at a daily dose in the range of 0.5 to 1 mg, or at a daily dose of about 1 mg.

3. 3. The bacterial extract of claim 1 or 2, which is a liquid or aerosol, formulated into a spray, droplets, colloid, mist, cloud or finely divided smoke.

4. The bacterial extract of claim 3 which is administered in solid form and is formulated into a powder or crushable tablet.

5. 5. The bacterial extract of any one of claims 1 to 4, administered by a delivery device selected from the group comprising a nasal insufflation device, an intranasal inhaler, an intranasal spray device, an atomizer, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle, a nebulizer, a metered dose inhaler (MDI), a pressurized metered dose inhaler, an insufflator, a two-way device, a dose ampoule, a nasal pad, a nasal sponge and a nasal capsule.

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