Process for preparing stable bacterial extracts and their use as pharmaceuticals

Abstract

To provide stable bacterial extract preparations with substantially increased stability with time, methods for preparation thereof, pharmaceutical formulations based on these stable bacterial extracts, and administration routes and delivery devices to treat and / or preventing immunological disorders due to infection and / or inflammation and / or neoplasms and / or dysbiosis.SOLUTION: Provided is a pharmaceutical composition comprising a bacterial extract from one or more Gram-positive or Gram-negative bacterial species and a pharmaceutically acceptable excipient or vehicle, where the bacterial extract is prepared as follows: subjecting one or more Gram-positive or Gram-negative bacterial species to alkaline lysis at a pH higher than 10; adjusting the pH of the lysate to 5-8 with one or more organic acids; conducting filtration using a filter of 1.2 micron to 0.1 micron followed by filtration using a filter of a cut-off value of 10 kDa to 100 kDa for purification, and finally adjusting the final physiological pH to from 5 to 8.SELECTED DRAWING: Figure 18

Description

[Technical Field]

[0001]

[0001] The present invention relates to novel stable bacterial extract preparations with substantially increased stability over time, novel methods for their preparation, pharmaceutical formulations based on these novel stabilized bacterial extracts, and novel administration routes and delivery devices for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation. [Background technology]

[0002]

[0002] There is ever-increasing interest in developing pharmaceutical formulations, liquid or solid, that are stable over time and can be administered to human subjects to treat infection and inflammation, and that can be easily adapted to a wider range of administration routes, e.g., oral, intranasal, intratracheal, intrapulmonary, transmucosal, topical and buccal, and to the patient's disease state and stage (acute stage, exacerbation, etc.).

[0003] Bacterial infections are frequently involved in many respiratory conditions, and antibiotic treatment is common. The effectiveness of antibiotics in treating such disease states and exacerbations is controversial. Their excessive use is associated with increased costs and the potential for increased antibiotic resistance in microorganisms.

[0004] Bacterial extract lysate preparations containing antigens from several bacterial strains have been shown to increase resistance to infection by these organisms. The manner in which these bacterial extract lysates may exert their effects is diverse and not yet fully understood. Numerous bacterial extracts have been used as immunostimulants and antitumor agents. Examples include Bacillus Calmette-Guerin (BCG), polysaccharides, beta-1,3 glucans, Maruyama vaccine, and extracts of Bifidobacterium, L. lactis, L. fermentum, L. acidophilus, and S. lactis. Such extracts are thought to stimulate the immune system in several ways. One important way is by stimulating lymphocytes to grow and produce cytokines. The ability to induce the production of such cytokines has a powerful effect on the immune system.

[0005]

[0005] In particular, for the treatment and / or prevention of upper respiratory tract disorders, the applicant has already successfully developed several bacterial extract preparations. In this regard, mention may be made, inter alia, of the drug Broncho-Vaxom®, a bacterial lytic extract from several pathogens frequently causing respiratory tract infections, as described in U.S. Pat. No. 9,463,209 B2. Broncho-Vaxom® is an immunostimulant administered via the enteral-oral route as capsules, sachets, or drops for the prevention and prophylaxis of respiratory tract infections, recurrent respiratory tract infections, such as acute bronchitis, chronic bronchitis, asthma, chronic obstructive pulmonary disease, and emphysema. It has been shown to enhance the production of TNF-α and interferon-γ from cultured human peripheral blood mononuclear cells, leading to the activation of alveolar macrophages and stimulating polymorphonuclear leukocytes to kill bacteria. The applicant has also developed Lactobacillus bacterial extracts and shown that these bacterial lysed extracts were effective when administered as capsules via the enteral oral route for the treatment of infections, allergies, autoimmune diseases and inflammation (see International Application WO2010 / 027344).

[0006]

[0006] Constant exposure of mucosal surfaces, such as the respiratory tract and lungs, to inhaled viruses, bacteria, and toxins presents a challenge to the immune system. The situation is exacerbated when the host is exposed to a viral infection and subsequently superinfected with a microorganism that leads to increased mortality. Thus, secondary bacterial infections after viral infections with influenza (e.g., H1N1), human rhinovirus (HRV), rhinosyncitial virus (RV), and coronaviruses (CoV, SARS-CoV, MERS-CoV, COVID-19, etc.) are an urgent problem facing respiratory medicine.

[0007]

[0007] Experimentally, numerous in vivo studies have demonstrated that the bacterial extract Broncho-Vaxom® administered by the enteral oral route provided protection in respiratory tract infection models. Most studies have used solid dosage forms (lyophilized) and liquid bacterial extracts, which have limited stability. An example of such an in vivo mouse model using a human dose (7 mg dry weight bacterial extract, 40 mg lyophilized) showed that it was able to enhance protection from secondary bacterial and / or viral infections after influenza infection (Pasquali et al., Frontiers in Medicine 2014, 1, 41).

[0008]

[0008] Furthermore, functional dysbiosis, which is caused by "junk food" rich in lipids, sugars and proteins and leads to dysregulation of the microbiota (Clarence M. et al., Abstract, March 30, 2018 - St-John University, Queens), was normalized by supplementing mice with Broncho-Vaxom (registered trademark), reducing associated sequelae and comorbidities.

[0009] Thus, for several years, Broncho-Vaxom® has been administered via the enteral route in solid dosage forms (capsules or sachets) to stimulate immune defenses and prevent common airway disorders and associated exacerbations. The protective effect of these bacterial lysates, designed and developed for oral administration, initiates an immune response in the gut (gut-associated lymphoid tissue, GALT). These organs sense and send immune cells to the lungs, armed to prevent and cure pulmonary infections from the respiratory tract. Thus, the bacterial lysate is not directly exposed to the lungs. By contrast, by replacing the enteral oral route with alternative "perioral" administration routes, such as intranasal, nasal, inhalation, nebulization, and intratracheal routes, the anti-infective effect of the stabilized bacterial lysate is thought to occur directly on lung cells or nasal and surrounding mucous membranes where infection occurs. Other preferred "perioral" routes for reaching mucosal tissues are the sublingual and buccal areas, where infection frequently initiates. Summary of the Invention [Problem to be solved by the invention]

[0010]

[0010] In accordance with the present invention, novel bacterial extract formulations have been developed for these alternative perioral administration routes, thus better fitting the broader range of potential routes of infection and inflammation. The ability of the mucous membranes to distinguish between harmful and harmless antigens is important in defense against pathogens and protection against damage caused by the body's own inflammatory response.

[0011]

[0011] However, these bacterial lysate extracts have generally been administered via the enteral oral route in solid form, i.e., capsules and sachets. One of the main technical difficulties when moving to alternative administration routes and / or alternative pharmaceutical formulations is that these bacterial extracts exhibit some physical instability, particularly the appearance of sediment. This physical instability of these bacterial extracts poses substantial problems during manufacturing, preparation, and storage, and thus constitutes a limiting factor for the development of alternative pharmaceutical dosage forms and / or alternative routes of administration, such as intranasal, pulmonary, intratracheal, mucosal, transmucosal, topical, topical, buccal, sublingual, oral, pulmonary, intrabronchial, or intrapulmonary. Since this significantly impacts regulatory approval, suitable stable formulations for these alternative routes and pharmaceutical dosage forms are essential for the success of therapeutic bacterial lysate-based drug products.

[0012]

[0012] Therefore, addressing both the aggregation and precipitation problems of these bacterial extracts and providing improved, stable and soluble bacterial extracts was important to improve manufacturing process solutions and to enable a wider range of administration routes and alternative pharmaceutical dosage forms.

[0013]

[0013] Importantly, the novel stable bacterial extract formulation can now be administered at a more precise dosage using specific delivery devices suitable for oral or peroral administration, particularly user-friendly delivery devices such as aerosols, nasal sprays, nebulizers, and pens, thereby increasing treatment compliance and patient convenience. Another substantial advantage is that the novel stable bacterial extract can be administered in liquid form via the oral route more easily to patients who cannot swallow tablets or capsules, such as infants and children, particularly those between 3 months and 6 years of age, as well as adults, including some elderly people who have difficulty swallowing. Alternative formulations of bacterial extract, such as emulsions, microemulsions, dispersions, creams, etc., can be envisioned for topical or topical skin-type administration routes.

[0014]

[0014] Finally, the novel stable bacterial extract does not precipitate at low temperatures or room temperature. The bacterial extract drug can therefore be stored and preserved intact by patients or pharmacies under normal conditions, without the risk of clogging any drug delivery device, allowing for accurate and precise dosage of the bacterial extract. Such stable bacterial extracts are also a great advantage to the pharmaceutical industry for storing intermediate drug products, either during the manufacturing process or during the formulation of final drugs. [Means for solving the problem]

[0015] The present invention thus relates to bacterial extract formulations prepared from Gram-positive and / or Gram-negative bacterial species that have improved stability characteristics and are therefore suitable for a variety of pharmaceutical formulations and routes of administration. In particular, the novel stabilized bacterial extract pharmaceutical compositions can be formulated for nasal, intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, oral, pulmonary, intrabronchial and / or intrapulmonary administration.

[0016] The present invention also relates to suitable dosage forms and delivery systems for delivering the novel stabilized bacterial extracts according to the present invention.

[0017] The present invention further relates to a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasms and / or dysbiosis.

[0018]

[0018] Finally, the present invention relates to a novel process for preparing stable bacterial extracts in liquid, semi-solid or aerosol form either as a final drug before administration to a patient or during the manufacture and / or formulation of a pharmaceutical product. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 is a diagram of a tangential flow filtration (TFF) system for bacterial extracts after alkaline lysis. The diagram shows two different configurations for the filtration membranes: a parallel mode, in which all membranes work simultaneously, and a serpentine mode, in which the membranes are configured in series. [Figure 2] Figure 2 shows a diagram of a tangential flow filtration (TFF) system used for the purification of bacterial extracts after alkaline lysis. The diagram shows the different chambers for the crude bacterial extract, purified fraction, diafiltration medium, pure or concentrated organic acid solution, two pumps, a microfiltration membrane holder, a nanofiltration / ultrafiltration membrane holder, valves, a transmembrane pressure regulator (TMP), a propeller, a pH electrode, and connections to the waste stream. A series of membranes can be connected in parallel mode, where all membranes work simultaneously, or in serpentine mode, where the membranes are configured in series. [Figure 3] Figure 3 shows the study design of the viral lung infection followed by bacterial lung infection superinfection experiment. The administration schedule of OM bacterial extract by oral route (group 2) versus intranasal route (groups 3 and 4) and intranasal saline (group 1) is shown. [Figure 4] Figure 4 shows results obtained after superinfection showing that intranasal OM bacterial extract treatment (in dose A = 50 micrograms and in dose B = 5 micrograms) significantly increased survival compared to oral (po 7 milligrams) OM bacterial extract and saline control treated animals. [Figure 5]Figure 5 shows results obtained after coinfection demonstrating that prophylactic intranasal OM bacterial extract treatment significantly reduced morbidity and mortality following influenza B. infection, summarized here using clinical score measurements after administration of both intranasal (in) 5 and 50 micrograms of OM bacterial extract versus oral (po) 7 milligrams of OM bacterial extract, all compared to saline controls. [Figure 6] Figure 6 is a photograph showing the difference in comorbidity signs 1 day after bacterial infection (day 8 from the scheme in Figure 3) observed in mice treated with 7 milligrams of OM bacterial extract via the oral route (shown here as transient ragged fur) versus 50 micrograms of OM bacterial extract via the intranasal route (shown as healthy animals). [Figure 7] Figure 7 shows the study design of a viral lung infection followed by a bacterial lung infection superinfection experiment. The administration regimens of OM bacterial extract administered by the intranasal (intranasal) route versus the intratracheal (it) route versus saline control are shown in the figure. [Figure 8] Figure 8 is a graph showing viral titers in lung tissue after 5 days of OM bacterial extract at doses A (50 micrograms) and B (5 micrograms) via intranasal (in) and intratracheal (it) administration versus intranasal (in) and intratracheal (it) saline control. [Figure 9] FIG. 9 is a graph showing the survival rate of mice treated by the intranasal route with OM bacterial extract at 50 micrograms (dose AIN) and 5 micrograms (dose BIN) or saline (IN). [Figure 10] 10 is a graph showing that intranasal prophylactic OM bacterial extract treatment significantly reduced morbidity and mortality following influenza B. infection as summarized here using clinical scoring measures after administration of both intranasal 5 micrograms (Dose A) and 50 micrograms (Dose B) of OM bacterial extract versus a saline control group. Clinical efficacy was dose-proportional. [Figure 11]Figure 11 is a graph showing survival of mice after administration of OM bacterial extract or saline by the intratracheal route: 50 micrograms (Dose A IT) and 5 micrograms (Dose B IT) of OM bacterial extract. [Figure 12] Figure 12 is a graph showing that prophylactic intratracheal OM bacterial extract treatment significantly reduced morbidity and mortality as summarized here using clinical scoring measures after administration of doses of 50 micrograms (AIT dose) and 5 micrograms (BIT dose). [Figure 13] Figure 13 shows the study design of the coinfection experiment: analysis of influenza virus load in the lungs after different doses and regimens of OM bacterial extract administered via the intranasal (intranasal) and oral routes compared to the control group, followed by bacterial lung infection after viral lung infection. Groups 1 to 11 are listed in Table 6. [Figure 14] FIG. 14 is a graph showing viral titers in lung tissue 5 days after infection following prophylactic intranasal treatment with OM bacterial extract compared to oral treatment. [Figure 15]Figure 15: Human rhinovirus (RV16) infection rate of human primary bronchial epithelial cells (BEC) derived from lung biopsies of healthy donors pretreated with various OM314A stable bacterial extracts. (A) Pretreatment of BEC cells with OM314A containing organic acids or HCl was performed 1 day before infection with RV-16 at a multiplicity of infection (MOI) of 1. RV-16 mRNA expression was used as an indicator of viral replication and is expressed relative to RV16 (100%) (percentage, Figure 15A). Control 1 (0%) was uninfected cells. RV16-1 was BEC cells infected with RV16 for 24 hours. Bars represent the mean ± SD. Samples tested: Control 1, RV16; OM314A samples: HCl; 10% HCl centrifugal force; butyrate; butyrate centrifugal force; propionate; propionate centrifugal force; aspartate; aspartate centrifugal force. Centri. indicates the supernatant of the sample obtained after centrifugation compared to the non-centrifuged control. (B) RV16 protein-positive human primary BECs (n=3) 24 hours after infection with three different virus concentrations. (C) Effect of preincubation (24 hours) with various OM314A preparations (20 μL / mL) on RV16 protein staining in BECs (n=3) 24 hours after infection with 0.1 MOI. Bars represent mean ± SEM. [Figure 16] Figure 16: Experimental scheme describing the protocol used to monitor interferon release from human BECs. Cells were seeded on day -2, serum-starved on day -1, and stimulated with OM314A samples (OM) for 24 hours as shown in Figures 17 and 18. Cell supernatants were collected at the indicated times for dose determination of interferon beta and gamma using ELISA. [Figure 17]Figure 17: (A) Dose response of type 1 interferon beta (IFN-beta) secretion by human BECs after 24 hours of incubation with HCl-neutralized OM bacterial extract (from 0.1 to 50 micrograms / ml). Bars represent the mean ± SD of n=5 donors. *=p<0.01 compared to 24 hours. (B) IFN-beta secretion by human BECs (n=3) over 24 hours after RV16 infection. (C) Concentration-dependent effect of various OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) preparations on IFN-beta secretion by uninfected (n=3) human BECs. Bars represent the mean ± SEM for each condition. [Figure 18] Figure 18: (A) Dose response of type 2 interferon gamma (IFN-gamma) secretion by human lung-derived primary epithelial cells (BECs) after 24 hours of incubation with HCl-neutralized OM bacterial extract (from 0.1 to 50 micrograms / ml). Bars represent the mean ± SD of n=5 donors. *=p<0.01 compared to 24-hour control. (B) IFNγ secretion by human BECs (n=3) over 24 hours after RV16 infection. (C) Concentration-dependent effect of various OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) preparations on IFNγ secretion by uninfected human BECs. Bars represent the mean ± SEM for each condition. [Figure 19] Figure 19 shows antiviral beta β-defensin-1 expression by human lung-derived primary epithelial cells (BECs). (A) Effect of infection (three different concentrations) on β-defensin-1 secretion by BECs (n=3) 24 hours after RV16 infection. (B) Concentration-dependent effect of OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5) stable bacterial extracts on β-defensin-1 secretion by uninfected BECs (n=3). Bars represent the mean ± SEM of triplicates for each condition. [Figure 20]Figure 20: ICAM-1 viral receptor expression. (A) ICAM-1 receptor expression by human lung-derived primary epithelial cells (n=3) 24 hours after RV16 infection. (B) Concentration-dependent effects of OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5) stable bacterial extracts on ICAM-1 expression by infected primary epithelial cells (n=3). Bars represent the mean ± SEM of triplicates of each condition. [Figure 21] Figure 21 shows toll-like receptor (TLR)-4-dependent TNFα release from murine bone marrow-derived dendritic cells (BMDCs) derived from wild-type (WT, black bars) or TLR-4 knockout (TLR4- / -, white bars) mice. Cells were stimulated with increasing dilutions of OM314A stable bacterial extract (P1, P2, P3), OM314B (P4), and OM-314C (P5), or with LPS (2 μg / mL) or an industrial batch (IB#1619057) as a control using the same dilution set. The level of TNFα in the supernatant was measured by ELISA 16 hours after induction according to the manufacturer's protocol. [Figure 22] Figure 22 shows linear discriminant analysis (LDA) scores in saline solution, normal chow-fed control mice (NCD (NCD-Sham), normal chow-fed mice treated with bacterial extracts from 21 strain lysates (NCD-BE), high-fat-fed control mice (HFD-Sham) given saline solution, and high-fat-fed mice treated with bacterial extracts from 21 strain lysates (HFD-BE). [Figure 23] Figure 23: (A) shows graphs of glucose concentrations in bacterial extracts from 21 strain lysates before feeding (pre-feed), (B) after normal chow feeding mice (post-NCD), and (C) after a high-fat feeding (post-HFD). Mice were weighed weekly for weight gain. Food consumption measures were assessed weekly by weighing pellets at the beginning and end of the week. Large black and small star significance symbols indicate HFD-Sham compared to HFD-L. Plantarum and HFD-Sham compared to HFD-BE, as noted in the graph with their respective significance values. [Figure 24]Figure 24 shows body weight and food consumption. (A) shows body weight in grams for high-fat diet sham controls fed saline solution (HFD-Sham), high-fat diet treated with bacterial extracts from 21 strain lysates (HFD-BE), and high-fat diet treated with L. plantarum (NCD-L plantarum). (B) shows food consumption in grams between high-fat diet sham controls fed saline solution (HFD-Sham) and high-fat diet treated with bacterial extracts from 21 strain lysates (HFD-BE). Mice were weighed weekly for weight gain. Food consumption was assessed weekly by weighing pellets at the beginning and end of the week. Circled star-shaped significance symbols indicate HFD-Sham compared to HFD-BE, as indicated with the respective significance values ​​in the graph. Plain star-shaped significance symbols indicate HFD-Sham compared to HFD-L. plantarum. [Figure 25] Figure 25 shows the results of insulin tolerance tests in high-fat diet (HFD) mice fed either saline solution (HFD-Sham), bacterial extract from 21 strain lysates (HFD-BE), or L. plantarum (NCD-L plant) in all 42 mice before treatment (pre-diet) and at the end of the 8-week treatment period (post-diet). Significance is indicated by a simple black star for HFD-Sham compared to HFD-BE (p<0.0001), as shown on the graph. [Figure 26]Figure 26 shows the effect of bacterial extracts from 21 strain lysates on various intestinal species. 16S ribosomal RNA sequencing and analysis was performed as described in Example 10. A. Clostridiales Lachnospiraceae Blautia; B. Clostridiales Ruminococcaceae GCA-900066225; C. Clostridiales Ruminococcaceae UCG-0101; D. Bacteroidales Muribaculaceae uncultured bacteria; E. Lachnospiraceae [Eubacterium] fissicantena group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 27] FIG. 27 shows a noisy spectrum indicating precipitate in solution (shown as typical of unstable bacterial extracts). [Figure 28] FIG. 28 shows a smooth spectrum indicating a clear solution (typical of a stable bacterial extract). [Figure 29] Figure 29: Process 1 during stability - Bioassay results show that Process 1-E3-neutralized filtrate (OM314A) exhibited comparable bioactivity by secreting MIP-3α on THP-1 cells for at least 4 months at room temperature (20°C ± 5°C) or 4°C. Process 1 TO was compared to T4 samples stored for 4 months at 4°C and room temperature (RT). [Figure 30] Figure 30: Process 2 - Bioassay results during stability show that Process 2-E3-neutralized filtrate (OM314A) exhibited comparable bioactivity by secreting MIP-3α on THP-1 cells for at least 4 months at room temperature (20°C ± 5°C) or 4°C. Process 2 T0 was compared to T4 samples stored for 4 months at 4°C and room temperature (RT). [Figure 31]Figure 31: Process 3 - Bioassay results during stability show that Process 3 - E3-neutralized filtrate (OM314A) exhibited comparable bioactivity through MIP-3α secretion on THP-1 cells for at least 5 months at room temperature (20°C ± 5°C) or 4°C. Process 3 T0 was compared to T5 samples stored for 4 months at 4°C and room temperature (RT). [Figure 32] Figure 32: Process 5 during stability - Bioassay results show that Process 5-E3-neutralized filtrate (OM314A) exhibited comparable bioactivity through MIP-3α secretion on THP-1 cells for at least 5 months at room temperature (20°C ± 5°C) or 4°C. Process 5 T0 was compared to T5 samples stored for 4 months at 4°C and room temperature (RT). DETAILED DESCRIPTION OF THE INVENTION

[0020]

[0051] The present invention thus relates to a stable purified bacterial extract obtainable by alkaline lysis of Gram-positive and / or Gram-negative bacterial species and neutralization with one or more specific organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof or pharmaceutically acceptable salts and esters thereof, followed by purification by filtration of the neutralized bacterial extract and adjustment to a final physiological pH by addition of the organic acid used for said neutralization, combinations thereof or salts and esters thereof.

[0021]

[0052] Applicants have discovered that contacting a bacterial extract with a selection of specific organic acids after alkaline lysis, where the pH of the lysate is greater than 10 (with a pH variation of ±0.1), surprisingly results in a bacterial extract that exhibits excellent stability. More specifically, bacterial extract formulations according to the invention retained physical stability in liquid form for several months. Such novel bacterial extract formulations with improved physical stability can therefore be stably stored in liquid, semi-solid, or aerosol form as the final pharmaceutical for administration to a patient or during the manufacture or formulation of a pharmaceutical product.

[0022]

[0053] A "stable" formulation is intended to mean a bacterial extract that essentially retains its physical stability in liquid form during pharmaceutical storage or as an intermediate drug product during manufacturing or formulation. A bacterial extract formulation retains its physical stability in a pharmaceutical formulation if there is no significant increase in aggregation and / or precipitation, as measured by visual inspection for clarity or by light scattering, size exclusion chromatography (SEC), and dynamic light scattering. The absence of significant precipitation or physical change at room temperature is observed for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months at 4°C, -20°C, or -80°C. Preferably, no more than 10%, preferably no more than 5%, of aggregation and precipitation forms.

[0023]

[0054] The term "organic acid" refers to an organic compound characterized by weak acidity and that does not completely dissociate in the presence of water.

[0024]

[0055] The term "alternative routes of administration" generally refers to perioral and oral routes, and may include intranasal, intratracheal, mucosal, transmucosal, topical, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary routes of administration, among others.

[0025]

[0056] The term "OM314A bacterial extract" refers to a multivalent immunomodulatory agent comprising a purified bacterial extract or lysate extracted by alkaline lysis from one or more of the most frequent bacterial pathogens of the upper respiratory tract, including Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and / or Streptococcus sanguinis. Preferably, the OM314A bacterial extract is obtainable by alkaline lysis of a combination of one or more of the above bacterial pathogens, and most preferably, includes eight of the above pathogens. This OM314A bacterial extract, prepared in a stable formulation according to the present invention, corresponds to the second generation of bacterial extract previously described in several scientific publications and International Publication WO 2008 / 109669. The first generation of bacterial extract, hereinafter referred to as "OM bacterial extract," is administered orally to patients as a solid, swallowable formulation, such as a capsule or tablet, and has been shown to be effective in preventing respiratory tract infections in adults and children. Furthermore, several clinical trials have demonstrated that enteral administration (oral) of this first-generation OM bacterial extract has been shown to prevent allergic asthma and wheezing attacks caused by acute respiratory tract illness in children when administered orally. The first generation of OM bacterial extract medicine is commercialized under the trade name Broncho-Vaxom® in a solid dosage form, typically a capsule or sachet, administered orally to patients at a dosage regimen of one capsule of 7 mg of lyophilized bacterial extract per day for adult treatment and one capsule of 3.5 mg of lyophilized bacterial extract per day for children.

[0026]

[0057] Thus, OM314A bacterial extract, as opposed to first generation OM bacterial extracts, refers to a second generation of drugs that contain OM bacterial extracts but that are stabilized so that they can be formulated in any possible pharmaceutical dosage form, either in liquid, gaseous or solid form, and are suitable for a wider range of possible routes of administration, including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary.

[0027]

[0058] The term "OM314B bacterial extract" refers to a multivalent vaccine obtainable by alkaline lysis of one or more bacterial species selected from Lactobacillus bacterial strains as described in International Publication WO2010 / 027344. In particular, the stable bacterial extracts include Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus casei ssp. casei ... The OM314B bacterial extract may comprise one or more Lactobacillus bacterial strains selected from Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and / or Lactobacillus delbrueckii. The OM314B bacterial extract is prepared in a stable formulation according to the present invention and corresponds to the second generation of the Lactobacillus bacterial extract previously described in International Publication WO 2010 / 027344. Because the OM314B bacterial extract is stabilized, it can be formulated into any possible pharmaceutical dosage form, whether in liquid, gaseous, or solid form, suitable for various possible routes of administration, including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial, and / or intrapulmonary routes.

[0028]

[0059] Thus, the term "OM314C bacterial extract" refers to a stable bacterial extract obtainable by alkaline lysis of one or more Escherichia coli bacterial strains as described in International Publication WO 2008 / 109667. The OM314C bacterial extract is prepared in a stable formulation according to the present invention and corresponds to the second generation of the E. coli bacterial extract previously described in International Publication WO 2008 / 109667. Because the second generation OM314C bacterial extract is stabilized, it can be formulated in any possible pharmaceutical dosage form, either in liquid, gaseous, or solid form, suitable for various possible routes of administration, including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial, and / or intrapulmonary.

[0029]

[0060] The term "stable bacterial extract formulation" refers to a stabilized form of any bacterial extract drug that may be obtained by alkaline lysis extract as described in International Publications WO2008 / 109669, WO2010 / 027344 or WO2008 / 109667, but that has been adapted into a pharmaceutical dosage form suitable for alternative routes, including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary, as described above.

[0030]

[0061] Thus, the stable purified bacterial extract according to the present invention may comprise bacterial alkaline lysates of any combination of Gram-positive and / or Gram-negative bacterial species having therapeutic immunomodulatory properties that, because they are stabilized, can be formulated in any pharmaceutical dosage form for a wider variety of routes of administration, including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary, as described above.

[0031]

[0062] According to a first embodiment, the novel stable bacterial extract formulation may comprise a bacterial lysate of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and / or Streptococcus sanguinis, as described in the applicant's International Publication No. WO 2008 / 109669. Preferably, the novel stable bacterial extract formulation according to this embodiment, hereinafter referred to as the stable OM314A bacterial extract formulation, is obtainable by alkaline lysis of the following bacterial species: Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis.

[0032]

[0063] According to this embodiment, there is thus provided a stable purified bacterial extract derived from one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, obtainable by alkaline lysis of the bacterial strain and neutralization with one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof and / or pharmaceutically acceptable salts and esters thereof, followed by purification by filtration of the neutralized extract and adjustment to a final physiological pH by addition of the same organic acids or the same combinations thereof used for said neutralization.

[0033]

[0064] According to a second embodiment, the stable bacterial extract formulation is prepared from one or more bacterial species selected from Lactobacillus bacterial strains. In particular, the stable bacterial extract may comprise one or more Lactobacillus bacterial strains selected from Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and / or Lactobacillus delbrueckii, as described in the applicant's International Publication WO 2010 / 027344.

[0034]

[0065] Thus, according to a second embodiment, there is provided a stable purified bacterial extract derived from one or more Lactobacillus bacterial strains, obtainable by alkaline lysis of the bacterial strains and neutralization with one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof and / or pharmaceutically acceptable salts and esters thereof, purification of the neutralized extract by filtration and adjustment to a final physiological pH by addition of the same organic acids or the same combinations thereof used for said neutralization.

[0035]

[0066] In a third embodiment, a stable bacterial extract derived from one or more E. coli bacterial strains is obtainable by alkaline lysis of the bacterial strains, neutralization with one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof, and / or pharmaceutically acceptable salts and esters thereof, purification of the neutralized extract by filtration, and adjustment to a final physiological pH by addition of the same organic acid or combinations thereof used for neutralization. In particular, the E. coli bacterial extract may comprise one or more E. coli bacterial strains as described in Applicant's International Publication WO 2008 / 109667.

[0036]

[0067] According to this third embodiment, there is thus provided a stable purified bacterial extract from one or more E. coli bacterial strains, obtainable by alkaline lysis and neutralization with one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof or pharmaceutically acceptable salts and esters thereof, followed by purification by filtration of the neutralized extract and adjustment to a final physiological pH by addition of the same organic acids or the same combinations thereof used for said neutralization.

[0037]

[0068] In the above embodiments, the bacterial extract comprises at least one strain from each of the above bacterial species, although in other embodiments, one or more specific strains from the above list may be omitted or replaced with one or more different strains.

[0038]

[0069] As mentioned above, grams + and / or grams - None of these specific embodiments are intended to limit the preparation of stable bacterial extract formulations, as any other combination of bacteria can be prepared and formulated in accordance with the present invention and may exhibit improved stability characteristics.

[0039]

[0070] Typically, bacterial extracts are prepared by fermentation followed by heat inactivation and alkaline lysis and filtration, which are already known in the art and are disclosed, inter alia, in International Publications WO2008 / 109667, WO2010 / 027344 and WO2008 / 109669.

[0040]

[0071] Fermentation is typically performed by growing each bacterial strain in a culture medium to an appropriate optical density. To obtain sufficient material for each strain, fermentation cultures can be initiated from available seed lots, which can then be used to inoculate larger fermentation vessels. For example, fermentation can be initiated with a small culture, e.g., 0.1 to 1.0 liters, and incubated at 30-40°C, e.g., 37°C, for approximately 3-6 hours to obtain an optical density (OD) at 700 nm of 3.0-5.0. Following the small-scale cultivation step, additional cultures in one or a series of larger fermenters can be performed at 30-40°C for a period of 3-20 hours, e.g., 3-10 hours or 8 hours.

[0041]

[0072] The culture medium preferably 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, such as serum or meat extracts from animals, such as cattle or sheep, or any other animals that can transmit prion-based diseases. For example, non-animal media, such as plant-based media, e.g., soy-based media, or synthetic or semi-synthetic media, can be used. Alternatively, media using horse serum or media containing materials from animal species that do not transmit prion diseases can be used. The culture medium can also contain biological extracts, such as yeast extract and horse serum, which also do not pose a risk of such diseases. Supplementary growth factors can also be introduced to enhance the growth of some bacterial species.

[0042]

[0073] After fermentation, the biomass from each bacterial strain or combination of bacterial strains is usually inactivated by heat treatment, concentrated, and frozen.

[0043]

[0074] Alkaline lysis is used to lyse bacterial cells under basic conditions and is usually carried out using organic or inorganic bases. Alkaline lysis can be carried out on a single bacterial biomass or a mixture of bacterial biomass or fermentation batches under basic conditions, typically using concentrated solutions of hydroxide ions, such as NaOH.

[0044]

[0075] Alkaline lysis is preferably carried out at a pH greater than about 10, with a pH variation of ±0.1. The duration of lysis can be estimated by one skilled in the art and depends on the amount of initial bacterial biomass. Lysis can be carried out at temperatures ranging from 30 to 60°C, e.g., 30-40°C or 35-40°C, e.g., 37°C. Lysis is generally stopped when all bacterial cells appear to be destroyed based on visual observation, as known to those skilled in the art. When multiple strains of bacteria of the same genus are used, the strains can be lysed together or separately. The strains can thus be mixed before or after lysis.

[0045]

[0076] During lysis, bacterial cells are destroyed and their components are degraded and chemically modified. In particular, amino acid racemization creates D-amino acids from naturally occurring L-amino acids found in natural proteins. D-amino acids can be beneficial in increasing the bioavailability of the extract because proteins composed primarily or partially of D-amino acids are not efficiently digested in the mammalian intestine. Therefore, antigenic molecules in the extract that have been chemically modified to contain D-amino acids during lysis can remain in the patient's body for a longer period of time, allowing for stronger immunostimulatory activity.

[0046]

[0077] After lysis, according to the present invention, the bacterial lysate is neutralized, i.e. the pH of the lysate may be adjusted to a final pH between 5 and 8, between 6 and 8, between 6.3 and 7.8 or between 6.5 and 7.8 by the addition of one or more specific organic acids, which according to the present invention may be selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof and / or pharmaceutically acceptable salts and esters thereof.

[0047]

[0078] The lysate is then purified by centrifugation and / or filtration to remove large cell debris or any insufficiently digested components, and any insoluble or particulate material, to obtain a soluble bacterial extract. Purification methods, including centrifugation and filtration, are known in the art for removing particulate matter from extracts. For example, the lysate can be centrifuged at 9000 g followed by one or more rounds of filtration. Typically, filtration can involve passing the extract or combined extract through one or more filtration membranes, such as microfiltration membranes (i.e., microfiltration) or ultrafiltration membranes (i.e., ultrafiltration), which can be repeated for several passes or cycles. For example, successive rounds of filtration through larger pore membranes can be followed by microfiltration using smaller pore membranes, such as 0.2 micron filtration membranes. Ultrafiltration can also be employed to aid in the extraction of soluble materials from the extract. For example, the ultrafiltration permeate can be recycled for further microfiltration.

[0048]

[0079] Tangential flow filtration (TFF) can be used to filter the extract and separate solubilized 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). An example of a filtration loop process is shown in Figure 1 below. At the beginning of such a process, diluted bacterial lysate can be stored in a first tank. A microfiltration (MF) loop can be initiated, and the product is pumped out. The resulting MF retentate can be recycled, while the MF permeate can be transferred to a second tank. After reaching an appropriate concentration, an ultrafiltration (UF) loop can be initiated. The UF permeate can be recycled back to the first tank for continuous extraction of solubilized compounds from the lysate, while the UF retentate can be stored in the second tank. During continuous extraction, the volumes of Tanks 1 and 2 can be adjusted by adjusting the flow rates of the microfiltration and ultrafiltration permeates. Several such extraction cycles can be performed using either TFF or another filtration method. In embodiments using TFF, at the end of the last cycle, the ultrafiltration loop can be closed and the microfiltration loop can be run alone, with the MF permeate being transferred to Tank 2. The microfiltration loop can be fitted with 1.2 micron to 0.1 micron filtration membranes, e.g., 0.65 to 0.2 micron or 0.45 micron filtration membranes. The cross-flow is at a transmembrane pressure (TMP) of 0.6 to 2 bar, e.g., between 0.8 and 1.5 bar or 1.0 bar, and a flow rate of 1000 liters / hour m 2(LHM) and 3000 LHM, for example, between 1500 and 2500 LHM or 2000 LHM. The ultrafiltration loop can be fitted with a filtration membrane of from 10 KDa to 1000 KDa, for example from 10 KDa to 100 KDa, or from 10 KDa to 30 KDa, or from 30 KDa to 100 KDa, or from 30 kDa to 300 kDa, or from 100 kDa to 300 kDa, or from 30 kDa to 1000 kDa, or from 100 kDa to 1000 kDa, or from 300 kDa to 1000 kDa. The cross flow may be between 30 and 1000 LHM, for example between 20 and 500 LHM, at a TMP of 0.2 to 1.5 bar, for example between 0.4 and 1.2 bar or 0.5 bar.

[0049]

[0080] Between 5 and 20 diafiltration volumes can be used to extract solubilized compounds from bacterial cell walls. The diafiltration medium can be water adjusted to a pH value between 7 and 11. In some embodiments, between 8 and 15 volumes are used. Thus, for example, in some embodiments, between 5 and 15 cycles of filtration can be used, and in some cases between 8 and 15 cycles, e.g., 8, 9, 10, 11, 12, 13, 14, or 15 cycles.

[0050]

[0081] In addition to removing any insoluble particles, such filtration also aims to remove any nucleic acids. As a result of filtration, the amount of nucleic acids present in the bacterial extract may remain below 100 micrograms / mL. However, filtration can preserve sugar components, including monosaccharides, disaccharides, as well as larger sugars, such as linear and branched polysaccharides, particularly lipopolysaccharide (LPS) components. Indeed, during the lysis process, sugars (including LPS components) are cleaved into smaller structures or substituted with different functional groups. In this regard, although it was previously thought that sugar components, including potentially toxic LPS components, should be removed from bacterial extracts for safety reasons (see U.S. Pat. No. 5,424,287), the present applicant has shown that sugar components, including LPS, can be safely retained, since these components actually contribute additional antigens to the extract, thereby improving therapeutic efficacy. The extract may be further diluted, concentrated, or centrifuged, if desired.

[0051]

[0082] After diafiltration, the alkali-purified soluble bacterial extract is further adjusted according to the present invention by the addition of one or more specific organic acids to neutralize the lysate. The organic acids according to the present invention can be selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof, and / or pharmaceutically acceptable salts and esters thereof. Preferably, the final pH of the bacterial extract preparation can be adjusted to between 5 and 8, between 6 and 8, between 6.3 and 7.8, or between 6.5 and 7.8.

[0052]

[0083] Such purified soluble bacterial extracts can therefore be advantageously preserved and stored as liquids in accordance with the present invention and remain clear without any settling or sedimentation, thus maintaining excellent physical stability. Alternatively, the purified bacterial extracts can be lyophilized if necessary, after which they can be reformulated under liquid, gaseous or solid forms for therapeutic use or further galenic processing.

[0053]

[0084] When bacterial extract formulations are stored as liquid formulations, they can be stored at room temperature for extended periods of time with excellent physical stability over time while maintaining biological activity. Advantageously, bacterial extracts can be stored at room temperature, 4°C, -20°C, or -80°C during the formulation process or storage without forming aggregates or precipitates. According to the present invention, the formation of both insoluble and soluble aggregates in bacterial extract formulations can be significantly reduced. Furthermore, in addition to improved physical stability, the formulations also maintained excellent biological activity of polysaccharides, lipopolysaccharides, proteins, racemized amino acids, and other biological components during manufacturing and storage without substantial chemical degradation or modification of the biological components.

[0054]

[0085] Bacterial extract formulations according to the present invention can thus be stored at RT or 4°C, -20°C, or -80°C for periods of at least 1 month, or at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, or at least 24 months, and the formulations do not exhibit substantial physical changes that occur during production and / or storage of the bacterial extract, e.g., due to the effects of light, temperature, pH, water, or due to reaction with excipients and / or the immediate container closure. Furthermore, the same bacterial extract formulation with known initial biological activity when subjected to the same storage conditions retains at least the initial biological activity. In related embodiments, the bacterial extract has not reached its labeled expiration date during the storage period.

[0055]

[0086] The composition and chemical properties of the major components of the therapeutically active soluble bacterial extract thus obtained were precisely determined and maintained over time in the liquid formulation. In particular, as described above, the amount of nucleic acids present in the bacterial extract is less than 100 μg / mL. The bacterial extract also contains more than 0.1 mg / mL of polysaccharides, or polysaccharides in a range starting or ending at 0.1 to 4.5 mg / mL, or 0.1 to 4 mg / mL, or 0.1 to 4 mg / mL, or 0.1 to 3.5 mg / mL, or 0.6 to 3 mg / mL, or 0.3 to 1 mg / mL, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mg / mL, e.g., 0.4 to 0.5 mg / mL. Furthermore, the yield of solubilized protein can be measured by Lowry in the soluble purified bacterial extract and can be, for example, greater than 50%, or greater than 60%, or 50 to 90%, or 60-90%. Thus, the bacterial extract may contain 5-75 mg / mL protein, or 10-65 mg / mL, or 20-45 mg / mL, or 5-40 mg / mL, or 5-20 mg / mL, or 5-10 mg / mL, or 6-8 mg / mL protein, or a range starting or ending at 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg / mL; 1.5 to 2.5 mg / mL free amino acids (AA), calculated from glutamic acid (147.1 g / mol), or 1.5 to 2 mg / mL, or 2 to 2.5 mg / mL free AA; or 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.5, 2.6, 2.7, 2.8, 2.9 ... and free AA in the range starting or ending at 0.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 mg / mL; and polysaccharides and monosaccharides from 0.3 to 4.5 mg / mL, or 0.3 to 4 mg / mL, or 0.4 to 4 mg / mL, or 0.5 to 3.5 mg / mL, or 0.6 to 3 mg / mL, or 0.3 to 1 mg / mL, or a range starting or ending at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5 mg / mL, for example, 0.4 to 0.5 mg / mL polysaccharides and monosaccharides.

[0056]

[0087] Furthermore, such dissolution results in partial hydrolysis of proteins, as well as deamination, deamidation, and partial racemization of L-to-D amino acids. Racemization of amino acids during the dissolution process creates D-amino acids from naturally occurring L-amino acids found in native proteins. Analytical studies of bacterial extracts determined the percentage of racemization. Peaks representing D-aspartic acid, D-glutamic acid, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, and D-lysine were observed. The percentage of these types of D-amino acids ranged from 3% to 40%. Thus, racemization of one or more of serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine, and / or lysine was evident. At least 10% of one or more of the above amino acids may be racemized from D to L. D-amino acids can be beneficial for increasing the bioavailability of the extract because proteins composed primarily or partially of D-amino acids are not efficiently digested in the mammalian intestine. Thus, antigenic molecules in the extract that are chemically modified during dissolution to contain D-amino acids may remain in the patient's body for a longer period of time, allowing for stronger immunostimulatory activity.

[0057]

[0088] Finally, lysis of bacteria according to the present invention may result in a reduction in the molecular weight of the component molecules from 0-300 kDa to 0-100 kDa, or 0-60 kDa due to hydrolysis.

[0058]

[0089] For example, bacterial extracts may contain approximately 6 to 8 mg / mL protein, 1.5 to 2.5 mg / mL amino acids (AA) (measured after HCl hydrolysis), and / or approximately 0.4 to 0.5 mg / mL polysaccharides and monosaccharides. Protein concentration is measured by the Lowry assay according to Method 2 of the European Pharmacopoeia 2.5.33. Sugar concentration is assayed after acid hydrolysis and derivatization according to D. Herbert et al., Meth. Microbiol. 5B:266 et seq. (1971). Glutamate (glutamic acid) concentration is measured by converting amino acids to isoindole derivatives and measuring absorbance at 340 nm according to Roth M., Fluorescence reaction for amino acids, Anal. Chem., 43, 880-882, (1971).

[0059]

[0090] As mentioned above, it was experimentally demonstrated that OM bacterial extract administered orally in solid form at a human dose (7 mg dry weight bacterial extract) in mice was able to enhance protection from secondary bacterial infections after influenza infection (Pasquali et al., Frontiers in Medicine 2014, 1, 41).

[0060]

[0091] The route of administration of the OM bacterial extract has always been oral (e.g., enteral), with a dosage regimen for adult treatment of one capsule of 7 mg of lyophilized bacterial extract per day, and one capsule of 3.5 mg of lyophilized bacterial extract per day for children. Applicants have found, however, that administration of the OM bacterial extract via alternative peroral parenteral routes, such as intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial, and / or intrapulmonary routes, results in a strong immune response and is more effective in combating potential infection and / or inflammation.

[0061]

[0092] Importantly, the observed superior therapeutic efficacy of these novel perioral administration routes allowed for a substantial reduction in the dosage of the bacterial extract drug. The dosage regimen could be divided into two, with a daily perioral dosage regimen of 3.5 mg of lyophilized bacterial extract for adult treatment and 1.75 mg of lyophilized bacterial extract for children. Thus, Applicant has demonstrated that these novel perioral administration routes resulted in higher therapeutic efficacy at lower doses.

[0062]

[0093] Furthermore, Applicants have shown that these novel perioral routes of administration of bacterial extracts (either the stabilized bacterial extract formulations described herein or not) induce stronger immune responses in the airways and lungs, but also in distal locations such as the gut.

[0063]

[0094] In particular, perioral administration of bacterial extracts (either in a stable formulation or not) makes it possible to protect patients against acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis. The perioral administration route is therefore particularly useful and effective in methods for treating and / or preventing these pathologies and disorders that usually worsen medical conditions and increase the risk of developing into chronic pathologies.

[0064]

[0095] According to the present invention, an infection can include upper or lower respiratory tract infections and / or associated sequelae, including allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosincitanal virus (RV), coronavirus (CoV, SARS-CoV, MERS-CoV, COVID-19, etc.), croup, pneumonia, hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory tract infection, obstructive pulmonary disease with acute upper respiratory tract infection, or diseases associated with impaired epithelial ciliary motility and / or impaired mucus clearance. Infections can also include secondary infections, non-respiratory viral infections, non-respiratory bacterial infections, and systemic infections, such as sepsis, septic shock, and virus-induced complications.

[0065]

[0096] According to the present invention, inflammation may include allergic / atopic respiratory and non-respiratory manifestations atopic dermatitis, acute and / or chronic associated dermatitis, anaphylaxis and food allergies. The inflammation can include skin disorders, skin inflammation such as eczema, rosacea, atopic dermatitis, psoriasis, photodamage (e.g., sun-induced skin inflammation and redness), atrophy of the skin, skin dyspigmentation (patches / spots), photodermatitis (erythema: inflammation and redness of the skin), telangiectasia, couperose, or actinic keratosis, inflammation including T helper 2-dominant autoimmune indications selected from Graves' disease, Hashimoto's disease, scleroderma, Ig4-related disease, or pemphigus, and inflammation including eosinophilic indications selected from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma, or eosinophilic pneumonia.

[0066]

[0097] According to the present invention, dysbiosis-associated disorders may include asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber regimens, atopic dermatitis, acute and / or chronic associated dermatitis, psoriasis, inflammatory bowel disease, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, renal failure, diseases associated with low fiber regimens.

[0067]

[0098] Finally, neoplasia may include neoplastic manifestations associated with immune disorders, such as mastocytosis, mast cell leukemia, T helper 2 biased and / or immunosuppressive tumors.

[0068]

[0099] Thus, according to a first aspect, the present invention relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, said bacterial extract comprising less than 100 micrograms / ml of nucleic acids, for use in a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis in a subject, said purified soluble bacterial extract being either a stabilized formulation or not, and being administered to the subject via a perioral route, in particular via intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration, at a dosage regimen lower than that used for enteral oral administration.

[0069]

[0100] The present invention also relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, said bacterial extract containing less than 100 micrograms / ml of nucleic acids and effective in preventing allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngolaryngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosinciar virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19) in a subject. 2. The purified soluble bacterial extract of claim 1, wherein the extract is a soluble or non-soluble bacterial extract, and the soluble or non-soluble bacterial extract is administered to a subject via a perioral route, in particular via intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration.

[0070]

[0101] The present invention further relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, said bacterial extract containing less than 100 micrograms / ml of nucleic acids, and which is useful for treating allergic / atopic respiratory and non-respiratory symptoms in a subject, including atopic dermatitis, acute and / or chronic associated dermatitis, anaphylaxis and food allergies, skin disorders, skin inflammation, e.g., eczema, rosacea, atopic dermatitis, psoriasis, photodamage (e.g., sun-induced skin inflammation and redness), atrophy of the skin, depigmentation of the skin (macula), etc. , and a T helper 2 dominant autoimmune indication selected from Graves' disease, Hashimoto's disease, scleroderma, Ig4-related disease or pemphigus, and an eosinophilic indication selected from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia, wherein the purified soluble bacterial extract is either a stable formulation or not and is administered to the subject via perioral routes, particularly intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration.

[0071]

[0102] The present invention further relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, said bacterial extract containing less than 100 micrograms / ml of nucleic acids, and for use in the treatment of asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber regimens, atopic dermatitis, or the like in a subject. the purified soluble bacterial extract is for use in a method for treating and / or preventing a microbiome-related disorder selected from: inflammatory bowel disease, acute and / or chronic associated dermatitis, psoriasis, inflammatory bowel disease, colitis, ulcerative colitis, Crohn's disease, obesity, a metabolic disease or disorder, liver failure, NASH, NAFLD, liver fibrosis, renal failure or a disease associated with a low fiber regimen, wherein said purified soluble bacterial extract is either a stable formulation or not and is administered to the subject via perioral routes, in particular intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration.

[0072]

[0103] The present invention finally relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, said bacterial extract containing less than 100 micrograms / ml of nucleic acid, for use in a method for treating and / or preventing a neoplastic manifestation associated with immune impairment in a subject, such as a neoplasm selected from mastocytosis, mast cell leukemia, T helper 2-biased and / or immunosuppressive tumors, said purified soluble bacterial extract being either a stable formulation or not, and being administered to the subject via perioral route, in particular intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration.

[0073]

[0104] According to a first aspect, the extract comprises at least one strain from each of the bacterial species listed above. Alternatively, one or more specific strains from the list above can be omitted or substituted with one or more different strains. In the case of a preferred perioral OM bacterial extract, the extract is derived from eight bacterial pathogens of the upper respiratory tract: Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis. The bacterial extract according to this aspect can be a perioral OM314A bacterial extract, i.e., a stabilized form of the OM bacterial extract administered via the perioral route.

[0074]

[0105] OM bacterial extracts are prepared from one or more bacterial species, namely Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis, by alkaline lysis at a pH preferably greater than 10 and containing less than 100 micrograms / ml of nucleic acids, at least 0.3 mg / mL of sugars, between 6 and 8 mg / mL of proteins with a molecular weight of less than 30 kDa, After subsequent purification, extracts containing amino acids equivalent to between 1.5 and 2.5 mg / mL of glutamic acid (147 g / mol) (measured after HCl hydrolysis), with 1 to 80% of the L-to-D racemized amino acids (wherein one or more racemized amino acids are selected from aspartic acid, asparagine, glutamic acid, glutamine, serine, methionine, histidine, alanine, arginine, phenylalanine, tyrosine, leucine, lysine, valine, and threonine) can be obtained. Further disclosure regarding the characteristics of the extracts and suitable methods for their preparation is provided below and in International Publication WO 2008 / 109669, the entire contents of which are incorporated herein by reference.

[0075]

[0106] According to this first aspect, the present invention thus also relates to a method for treating and / or preventing upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and / or dysbiosis-related disorders, wherein the perioral OM bacterial extract, stabilized or not, is administered to a subject via intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial, or intrapulmonary administration at a dosage regimen of 0.005 mg to 1 mg per day, i.e., at a dosage lower than that administered orally by enteral administration, and is effective in providing optimal protection. Preferably, the bacterial extract according to this aspect is an OM bacterial extract or a stabilized form thereof, OM314A bacterial extract. Furthermore, according to this aspect, the secondary infection to be treated and / or prevented may be a non-respiratory viral infection.

[0107] In the following examples, the applicant clearly demonstrated that intranasal administration of the OM bacterial extract according to the first aspect substantially reduced the viral titer in lung tissue after influenza virus infection and reduced the morbidity and mortality of co-infected animals at a lower dose compared with oral administration. Indeed, its intranasal or intratracheal administration as a preventive treatment was shown to be more effective than enteral administration. Intranasal administration constituted an effective preventive treatment against influenza in this mouse model, and this protective effect was shown to be dose-dependent. The applicant further demonstrated that direct intratracheal or intranasal administration of the purified bacterial extract according to the first aspect upregulated the long-chain isoforms of these two glycosaminoglycans, thereby demonstrating that the bacterial extract was involved in novel beneficial mechanisms, such as reduced inflammation and increased antigen presentation.

[0076]

[0108] According to a first aspect, the present invention therefore relates to a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis, comprising administering a therapeutically effective amount of an OM bacterial extract or a stabilized form OM314A via the perioral route.

[0077]

[0109] The present invention relates to, inter alia, the treatment of allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngolaryngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosynthetic virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19, etc.), croup, pneumonia, hypersensitivity reactions, and the like, comprising administering a therapeutically effective amount of OM bacterial extract or stabilized form OM314A via the perioral route. The present invention relates to a method for treating and / or preventing an upper or lower respiratory tract infection and / or associated sequelae, secondary infection, non-respiratory viral infection, non-respiratory bacterial infection, systemic infection, such as sepsis, septic shock and virus-induced complications, including pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory tract infection, obstructive pulmonary disease with acute upper respiratory tract infection, or diseases with impaired epithelial ciliary motility and / or impaired mucus clearance.

[0078]

[0110] The present invention also relates to methods for treating and / or preventing immune disorders including, but not limited to, an imbalance between T helper 1, T helper 17 and T helper 2 immune responses, Treg imbalance, type 2 hypersensitivity, immunosuppression, eosinophilia, allergy and atopy, comprising administering a therapeutically effective amount of OM bacterial extract or stabilized form OM314A by the perioral route.

[0079]

[0111] The present invention further provides a method for the treatment of allergic / atopic respiratory and non-respiratory symptoms including atopic dermatitis, acute and / or chronic related dermatitis, anaphylaxis and food allergies, skin disorders, skin inflammation such as eczema, rosacea, atopic dermatitis, psoriasis, photodamage (e.g. sun-induced skin inflammation and redness), atrophy of the skin, skin depigmentation (plaques), ultraviolet dermatitis (erythema: inflammation and redness), and the like, comprising administering a therapeutically effective amount of OM bacterial extract or stabilized form OM314A via the perioral route. The present invention also relates to methods for treating and / or preventing inflammation, including those involving skin redness), telangiectasia, couperose, actinic keratosis, or inflammation comprising a T helper 2 dominant autoimmune indication selected from Graves' disease, Hashimoto's disease, scleroderma, Ig4-related disease, or pemphigus, or inflammation comprising an eosinophilic indication selected from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma, or eosinophilic pneumonia.

[0080]

[0112] The present invention further relates to a method for treating and / or preventing a dysbiosis-associated disorder selected from asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber regimens, atopic dermatitis, acute and / or chronic associated dermatitis, psoriasis, inflammatory bowel disease, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, renal failure, diseases associated with low fiber regimens, comprising administering a therapeutically effective amount of OM bacterial extract or stabilized form OM314A via the perioral route.

[0081]

[0113] The present invention further relates to a method for treating and / or preventing immune disorders including, but not limited to, an imbalance between T helper 1, T helper 17 and T helper 2 immune responses, Treg imbalance, type 2 hypersensitivity, immunosuppression, eosinophilia, allergy or atopy, comprising administering a therapeutically effective amount of OM bacterial extract or stabilized form OM314A via the perioral route.

[0082]

[0114] The present invention finally relates to a method for treating and / or preventing neoplastic indications associated with immune disorders, such as neoplasms selected from mastocytosis, mast cell leukemia, T helper 2-biased and / or immunosuppressive tumors, comprising administering a therapeutically effective amount of an OM bacterial extract or stabilized form OM314A via the perioral route.

[0083]

[0115] According to a second aspect, the present invention also relates to a bacterial extract obtainable by alkaline lysis from one or more bacterial species selected from Lactobacillus bacterial strains for use in a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis in a subject as described above, wherein the Lactobacillus bacterial extract is either a stable formulation or not, which is administered to a subject by the perioral route, in particular via intratracheal inhalation, intranasal, mucosal, transmucosal, topical skin, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary administration, in a dosage regimen lower than that used for enteral oral administration. The bacterial extract may preferably comprise one or more Lactobacillus species selected from one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and Lactobacillus delbrueckii. Preferably, the perioral bacterial extract according to this aspect is a bacterial extract obtainable by alkaline lysis from one or more bacterial species selected from the above-mentioned Lactobacillus strains, or a stabilized form thereof, i.e., OM314B bacterial extract.

[0084]

[0116] According to this second aspect, the present invention therefore relates to a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis, comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract via the perioral route.

[0085]

[0117] The present invention thus particularly relates to a method for treating and / or preventing an upper or lower respiratory tract infection and / or associated sequelae, secondary infection, non-respiratory viral infection, non-respiratory bacterial infection, systemic infection, such as sepsis, septic shock or virus-induced complications, including allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngolaryngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosincitan virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19), croup, pneumonia, hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory tract infection, obstructive pulmonary disease with acute upper respiratory tract infection, or diseases associated with impaired epithelial ciliary motility and / or impaired mucus clearance, comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract via the perioral route.

[0086]

[0118] The present invention also relates to a method for treating and / or preventing immune disorders including, but not limited to, an imbalance between T helper 1, T helper 17 and T helper 2 immune responses, Treg imbalance, type 2 hypersensitivity, immunosuppression, eosinophilia, allergy and atopy, comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract via the perioral route.

[0087]

[0119] The present invention further provides a method for treating allergic / atopic respiratory or non-respiratory symptoms, including atopic dermatitis, acute and / or chronic related dermatitis, anaphylaxis or food allergies, skin disorders, skin inflammation such as eczema, rosacea, atopic dermatitis, psoriasis, photodamage (e.g. sun-induced skin inflammation and redness), atrophy of the skin, depigmentation of the skin (plaques), and photodermatitis (erythema: inflammation and redness of the skin), comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract via the perioral route. or inflammation comprising a T helper 2 dominant autoimmune indication selected from Graves' disease, Hashimoto's disease, scleroderma, Ig4-related disease or pemphigus, or inflammation comprising an eosinophilic indication selected from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia.

[0088]

[0120] The present invention further relates to a method for treating and / or preventing a dysbiosis-associated disorder selected from asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber regimens, atopic dermatitis, acute and / or chronic associated dermatitis, psoriasis, inflammatory bowel disease, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, renal failure or diseases associated with low fiber regimens, comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract via the perioral route.

[0089]

[0121] The present invention further relates to a method for treating and / or preventing neoplastic manifestations associated with immune disorders, such as mastocytosis, mast cell leukemia, T helper 2-biased and / or immunosuppressive tumors, comprising administering a therapeutically effective amount of a Lactobacillus bacterial extract by the perioral route.

[0090]

[0122] Stable bacterial extracts obtainable by alkaline lysis from one or more bacterial species selected from Lactobacillus bacterial strains are particularly useful for the treatment and / or prevention of rhinitis and / or allergic rhinitis, commonly known as cold symptoms accompanied by nasal congestion or runny nose.

[0091]

[0123] According to a preferred embodiment of the second aspect, the present invention therefore relates to a method for treating and / or preventing acute and chronic immune disorders resulting from infection and / or inflammation and / or neoplasia and / or dysbiosis as described above, comprising administering via the perioral route a therapeutically effective amount of a Lactobacillus bacterial extract obtainable by alkaline lysis from one or more bacterial species selected from Lactobacillus bacterial strains. Preferred Lactobacillus bacterial extracts include one or more Lactobacillus bacterial species selected from one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and / or Lactobacillus delbrueckii. Most preferably, the Lactobacillus bacterial extract is the stabilized OM314B bacterial extract described above.

[0092]

[0124] The stabilized Lactobacillus bacterial extract according to the second aspect is therefore administered via the perioral route, in particular via intratracheal inhalation or intranasal administration, at a dosage regimen lower than that used for enteral oral administration.

[0093]

[0125] Considering the direct and indirect antiviral nonspecific activity of OM and the new stable OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) bacterial extracts on epithelial cell surfaces, as evidenced by in vivo reduction of viral cellular titers and the initiation of antiviral antibodies against, but not limited to, H1N1, RSV, and in vitro efficacy against human RV (exemplified by the induction of IFN, β-defensin accompanied by a reduction in the viral receptor ICAM-1, thereby confirming the induction by OM and the new stable OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) of loss of attachment (to) viral attachment to epithelial cells), such oral It is expected that the peripheral administration route will be particularly useful and effective in methods of treating and / or preventing upper and lower respiratory tract infections, such as rhinitis, allergic rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngolaryngitis, influenza, respiratory syncytial virus, secondary bacterial infections following viral infection with influenza (such as H1N1), human rhinovirus (HRV), rhinosynthetic virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19, etc.), croup, pneumonia, bronchopneumonia, bronchitis, bronchiolitis, obstructive pulmonary disease associated with acute lower respiratory tract infection, obstructive pulmonary disease associated with acute upper respiratory tract infection, and diseases associated with impaired epithelial ciliary dyskinesia and / or impaired mucus clearance.

[0094]

[0126] These purified bacterial extracts can be stabilized as described above and thus administered as stable bacterial extract formulations in either solid, semi-solid, liquid or aerosol form.

[0095]

[0127] Pharmaceutical compositions comprising the stabilized bacterial extract and a pharmaceutically acceptable excipient are also provided. These pharmaceutical compositions can be stabilized and stored in a liquid formulation for several months and administered to patients in liquid or vapor form. Alternatively, they can be lyophilized and / or stored and then reformulated as liquid or aerosol medications.

[0096]

[0128] The stable pharmaceutical composition according to the present invention is particularly useful in a method for treating and / or preventing acute and chronic immune disorders resulting from the above-mentioned infections and / or inflammations and / or neoplasias and / or dysbiosis.

[0097]

[0129] These pharmaceutical compositions remain stable for several months in any form, liquid, gas or aerosol, semi-solid or solid, and can be formulated for administration via intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, oral, pulmonary, intrabronchial and / or intrapulmonary routes. Preferably, they can be administered to a subject by intratracheal inhalation or intranasal transmucosal routes.

[0098]

[0130] Pharmaceutical compositions that are liquid or aerosol and formulated as sprays, droplets, colloids, mist, nebulae, or atomized vapor are particularly preferred. Preferred pharmaceutical compositions may also be liquid or semisolid formulations, such as emulsions, microemulsions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, solutions, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, serums, ointments, mousses, pastes, or transdermal patches. In certain other preferred embodiments, the compositions are solid and formulated as powders or crushable tablets.

[0099]

[0131] In preparing liquid, semi-solid, solid and spray medicines, the above-mentioned materials can be used appropriately together with any additives, if necessary, such as vehicles, binders, perfumes, flavoring agents, sweeteners, colorants, preservatives, antioxidants, stabilizers and surfactants.

[0100]

[0132] In a preferred embodiment, the bacterial extract pharmaceutical composition is administered via the intranasal route, and the composition may be in a form selected from an emulsion, suspension, colloid, mist, cloud, atomized smoke or spray, nasal tampon, powder, ointment, cream, lotion, gel, paste, salve, liquid, tincture, patch, or strip.

[0101]

[0133] In another preferred embodiment, the bacterial extract pharmaceutical composition is administered orally and can be in the form of a single dose container, such as a monodose plastic bottle similar to the plastic bottles of physiological fluids used as eye drops for dry eyes, or in the form of a single dose ampoule. The oral bacterial extract liquid formulation according to this preferred embodiment is therefore a stable formulation and can be kept in the single dose container as a neutralized (pH close to 7) liquid formulation for an extended period of time.

[0102]

[0134] To prepare the pharmaceutical composition, the bacterial extract can be mixed with pharmaceutically acceptable carriers, adjuvants, and / or excipients according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers include any standard pharmaceutical carrier, such as phosphate-buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The bacterial extract may 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, and nonfat dry milk. Liquid and semisolid 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, and sesame oil. Liquid carriers, particularly those for injectable solutions, include water, saline, aqueous glucose, and glycols. For examples of carriers, stabilizers and adjuvants, see Remington's Pharmaceutical Sciences, edited by EW Martin, Mack Publishing Company, 18th ed., 1990. Pharmaceutical compositions may also include stabilizers and preservatives.

[0103]

[0135] In certain preferred embodiments, the pharmaceutical composition can be formulated as a crushable tablet. The tablet can be administered whole or lightly crushed, for example, with finger pressure, and sprinkled onto a suitable vehicle. Crushable tablets can be prepared using direct compression and excipients, taking care not to damage the coating of the individual subunits in the process. Suitable excipients for preparing crushable tablets include those typically used for chewable tablets, including monosaccharides and disaccharides, sugar polyols, etc., or combinations thereof. Exemplary excipients include mannitol, sorbitol, xylitol, maltitol, lactose, sucrose, maltose, or combinations thereof. Any pharmaceutical excipient, such as a diluent, lubricant, glidant, flavor, colorant, etc., or a combination comprising at least one of the above, can also be included in the tablet matrix. Crushable tablets can be prepared using tablet manufacturing methods known in the pharmaceutical industry.

[0104]

[0136] The bacterial extract formulation may be in the form of a colloid, e.g., containing a metal halide, most preferably a silver halide. One or more bacterial extracts and adjuvants can be incorporated into or encapsulated within the colloid particles. Alternatively, or in addition, one or more bacterial extracts and adjuvants can be attached to the surface of the colloid particles. The means by which the active agent and adjuvants are attached to the particles depends on the characteristics of the extract, adjuvants, and colloid particles. For example, proteins readily adsorb or adhere to hydrophobic particles through hydrophobic interactions with the particle surface, displacing some neutral emulsifiers.

[0105]

[0137] The present invention is based, in part, on the surprising discovery that the use of the above-described periocular delivery system of bacterial extracts results in significantly higher antibody titers, enhanced immune responses, and a safe and effective approach to enhancing the immunogenicity of a variety of antigens used in both prophylactic and therapeutic pharmaceutical compositions.

[0106]

[0138] The appropriate dosage according to the present invention and described herein in various embodiments varies depending on the condition, age, and species of the subject, and can be easily determined by one skilled in the art. However, according to the present invention, the total daily dosage can be significantly reduced to a range of 0.005 to 1 mg, preferably 0.05 to 0.5 mg, and most preferably 0.1 to 0.3 mg, which can be administered as a single or divided dose, and the upper limit can be exceeded if necessary. Advantageously, the dosage administered via the perioral route is lower (e.g., half the dosage) than that administered via the oral enteral route.

[0107]

[0139] Another aspect relates to a delivery device for the bacterial extract formulation according to the invention. Also provided is a delivery device for use in a method for treating and / or preventing upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and / or dysbiosis-related disorders.

[0108]

[0140] According to the present invention, bacterial extract formulations can be administered via the intranasal or intratracheal route by nasal insufflation devices, nasal inhalers, nasal 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 and nasal capsules.

[0109]

[0141] The nasal spray can be a liquid or solid nasal spray. The bacterial extract formulation can be administered in aerosol or non-aerosol form. The nasal delivery device can be metered to administer the precise effective dose to the nasal cavity. The nasal delivery device can be for single unit delivery or multiple unit delivery.

[0110]

[0142] When bacterial extract formulations are administered as aerosols, they can be prepared using standard procedures. For example, aerosol sprays can be generated from pressurized containers using suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen, carbon dioxide, or other suitable gases. Dosage units can be determined by providing a valve to deliver a metered amount. Pump spray dispensers can deliver metered doses or doses having a specific particle or droplet size. Aerosols can be suspensions or dispersions of either liquid droplets or solid powders in air (or gas). Liquid droplets can be formed from solutions, suspensions, and dispersions of drugs in liquids, such as water or non-aqueous solvents. Aerosols can be generated in any suitable device, such as MDIs, nebulizers, or mist sprayers.

[0111]

[0143] The aerosol according to the present invention can be insufflated or inhaled using a suitable mechanical device. The device can, for example, comprise a reservoir and a sprayer, which is a device adapted to expel a pharmaceutical dose in the form of a spray. The doses to be administered can be contained in the reservoir, optionally in a solution or suspension, or in a solid particle formulation, e.g., a solid particulate mixture.

[0112]

[0144] Nebulizer devices generate a stream of high velocity air that allows a therapeutic agent in liquid form to be sprayed as a mist. The therapeutic agent is formulated in liquid form, for example, as a solution or suspension of appropriately sized particles. The particles are micronized. The term "micronized" is defined as having about 90% or more of the particles having a diameter of less than about 10 μm. Suitable nebulizer devices are commercially available, for example, from PARI GmbH (Sternberg, Germany). Other nebulizer devices include Respimat (Boehringer Ingelheim) and those disclosed, for example, in U.S. Pat. Nos. 7,568,480 and 6,123,068 and WO 97 / 12687.

[0113]

[0145] The bacterial extract can be administered using a DPI device in the form of a free-flowing powder that can be dispersed in the patient's airstream during inspiration. DPI devices with an external energy source can also be used. To achieve a free-flowing powder, the bacterial extract formulation can be combined with a suitable excipient (e.g., lactose). A dry powder combination can be made, for example, by combining dry lactose having a particle size between about 1 μm and 100 μm with micronized particles of a benzodiazepine and dry blending. Alternatively, the benzodiazepine can be formulated without excipients. The formulation is loaded into a dry powder dispenser or into inhalation cartridges or capsules for use in a dry powder delivery device. Examples of commercially available DPI devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKline) (U.S. Patent No. 6,378,519); Turbuhaler (AstraZeneca, Wilmington, Del.) (U.S. Patent No. 4,524,769); and Rotahaler (GlaxoSmithKline) (U.S. Patent No. 4,353,365).

[0114]

[0146] Using an MDI device, a measured amount of the bacterial extract formulation can be delivered using a compressed propellant. Formulations for MDI administration include a solution or suspension of the bacterial extract formulation in a liquefied propellant. Examples of propellants include hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA227), and chlorofluorocarbons, such as CCl3F. Additional components of HFA formulations for MDI administration include cosolvents, such as ethanol, pentane, and water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. The bacterial extract formulation can be loaded into an aerosol canister, which forms part of the MDI device.

[0115]

[0147] The bacterial extract preparation can be delivered to the nasal cavity as a powder, e.g., microspheres, via a nasal insufflator. The bacterial extract preparation can be absorbed onto a solid surface, e.g., a carrier. The powder or microspheres can be administered in a dry, air-dispersible form. The powder or microspheres can be stored in the insufflator container. Alternatively, the powder or microspheres can be filled into a capsule, e.g., a gelatin capsule, or other single-dose unit suitable for nasal administration.

[0116]

[0148] The bacterial extract formulation is delivered via a nasal spray applicator. The composition can be administered by placing it in a nasal spray administration device or atomizer and spraying it into the subject's nostrils for delivery to the nasal mucosa. A sufficient amount is administered to achieve the desired systemic or localized level for therapeutic effect.

[0117]

[0149] Bacterial extract formulations can also be administered by oral inhalation to the respiratory tract, i.e., the lungs, via the intratracheal route. Such intratracheal administration requires aerosolization of a solid or liquid and delivery of the aerosol to the lungs via the mouth and throat. Medicament particles can be administered to the lungs as dry powder aerosols or liquid aerosols. Dry powder aerosols are typically administered to the lungs using dry powder inhaler (DPI) inhalation devices. Dry powder inhalers can include breath-actuated dry powder inhalers, such as those described in U.S. Pat. No. 7,434,579. Metered-dose inhalers contain medication suspended in a propellant, propellant mixture or solvent mixture, propellant, and / or other excipients in a compact pressurized aerosol dispenser. MDI products can dispense up to several hundred metered doses of medication. Each actuation can contain from a few micrograms (mcg) to milligrams (mg) of active ingredient in a volume typically between 25 and 100 microliters.

[0118]

[0150] As noted above, another type of liquid aerosol dispersion device is a nebulizer, which uses a jet, vibrating mesh, or other means to aerosolize a suspension containing particles of medication.

[0119]

[0151] Bacterial extract formulations according to the present invention may further comprise adjuvants, penetration enhancers, and / or solvents. For example, for intranasal delivery, penetration enhancers can be used to enhance penetration of the composition through the nasal mucosa. Compounds containing one or more hydroxy groups can be used as penetration enhancers. Some of these hydroxy group-containing compounds also function as solvents in the composition. Non-limiting examples of hydroxy group-containing compounds that can be used as penetration 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-menthane-3 ,8-diol; 2-methyl-2,4-pentanediol; triols (e.g., glycerin), polyols (e.g., suitable polymers containing multiple hydroxy groups, including polyethylene glycol or PEG, polypropylene glycol, polysorbates, and sorbitan esters; and suitable sugar alcohols), cyclitols (e.g., pinitol, inositol), cyclic diols (e.g., cyclohexanediol), aromatic diols (e.g., hydroquinone, bisphenol A, resorcinol, and catechol).

[0120]

[0152] Those skilled in the art will recognize that the present teachings can also be applied to other penetration enhancers. Non-limiting examples of other penetration enhancers useful in the present invention include simple long-chain esters that are generally recognized as safe (GRAS) in various pharmacopoeial compendia. 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 penetration enhancers include alcohols (e.g., short-chain and long-chain alcohols), polyhydric alcohols, amines and amides, urea, amino acids and their esters, amides, pyrrolidone and its derivatives, terpenes, fatty acids and their esters, macrocycles, sulfoxides, surfactants, benzyldimethylammonium chloride, cetyltrimethylammonium bromide, cineole, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dodecylpyridinium chloride, dodecylamine, hexadecyltrimethylammoniopropanesulfonate, limonene, linoleic acid ... Examples of suitable enhancers include oleic acid (OA), linoleic acid (LA), menthol, 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. Enhancers should be appropriate. Those skilled in the art will also recognize that substances that are incompatible or irritating to mucous membranes should be avoided.

[0121]

[0153] Examples of pharmaceutically acceptable solvents or excipients that can be used in the present compositions 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 pharmaceutically acceptable solvents that can be used in the present compositions include, but are not limited to, propylene glycol (also known 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.

[0122]

[0154] Particularly preferred is a bacterial extract preparation or pharmaceutical composition comprising said preparation for use in a method for the treatment and / or prevention of upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and / or dysbiosis-related disorders, which is administered to a subject by intratracheal inhalation or by the intranasal transmucosal route.

[0123]

[0155] It appears that intranasally administered bacterial extracts affect the nasopharyngeal-associated lymphoid tissue (NALT) or gut-associated lymphoid tissue (GALT), and subsequently induce gut-derived B and T cells and macrophages to traffic to the bronchus-associated lymphoid tissue, which may lead to an immune response against these pathogens in the respiratory tract.

[0124]

[0156] The present invention also relates to a method for treating, preventing, or attenuating viral infection and / or allergic diseases or disorders, such as asthma, chronic obstructive pulmonary disease, and viral-induced exacerbations of allergies or autoimmunity, comprising administering a therapeutically effective amount of a stable bacterial extract of the present invention to a subject via the perioral route.

[0125]

[0157] The asthmatic condition can be steroid-resistant asthma, neutrophilic asthma, or non-allergic asthma. The allergic disease or disorder can 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).

[0126]

[0158] The present invention relates to a method for treating and / or preventing an allergic disease or disorder, or ameliorating the condition of a subject suffering from an allergic disease or disorder, including, but not limited to, an allergic disease or disorder selected from the group consisting of asthma, rhinitis, dermatitis, drug reactions, eosinophilic diseases or disorders, esophageal and gastrointestinal allergies, comprising administering a therapeutically effective amount of the stable bacterial extract of the present invention to the subject via the perioral route.

[0127]

[0159] The present invention finally provides a novel extraction process for the preparation of bacterial extracts with enhanced stability. The process for preparing bacterial extracts with improved stability comprises the following steps: a. culturing each bacterial strain species in an appropriate medium; b. Lysing each strain at an initial pH preferably greater than 10 with a pH variation of ±0.1; c. Decreasing the pH of the extract obtained in step (b) by 1 or 2 units by adding one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof, or pharmaceutically acceptable salts and esters thereof; d. passing the product of step (c) through a microfiltration membrane at least once and retaining the product on an ultrafiltration membrane to obtain a purified soluble extract; e. adjusting the final pH to about 7 (±1.0) by adding the organic acid or combination thereof used in step (b); f. Adding a pharmaceutically acceptable excipient or vehicle.

[0128]

[0160] According to a first aspect of the present invention, the bacterial extract is obtained from one or more bacterial species selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and / or Streptococcus sanguinis. Preferably, the bacterial extract according to this aspect, like the first generation OM bacterial extract drugs, is derived from eight bacterial pathogens of the upper respiratory tract, namely Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis.

[0129]

[0161] According to a second aspect of the invention, the bacterial extract is obtained from one or more bacterial species selected from Lactobacillus bacterial strains, such as Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii.

[0130]

[0162] According to a third aspect of the invention, the bacterial extract is obtained from one or more strains of Escherichia coli bacteria, as described in International Publication No. WO2008 / 109667.

[0131]

[0163] The dissolution can be carried out at a temperature of 60° C. for a period of 40 hours to 10 days. Also, the microfiltration membrane that can be used in the process is 0.45 micron, and the ultrafiltration membrane is 30 KDa. Furthermore, part (c) of the process includes tangential flow filtration, where the tangential flow filtration can be carried out for 5 to 15 cycles.

[0132]

[0164] Furthermore, the present invention relates to bacterial extract formulation products and / or pharmaceutical compositions obtainable by the above process. [Example]

[0133] Example 1: Process for stabilizing alkaline extract Example 1.1: Addition of organic acids for the preparation of a stable bacterial extract (OM314A) The amounts of acid needed to acidify the alkaline OM314A bacterial extract to pH 5.0 and NaOH 1N to adjust to pH 7.0 were first determined in advance.

[0134] 25 mL of OM314A alkaline concentrate was placed in a 50 mL Falcon tube. The measured neat pH was typically approximately pH 10.5. After measuring the neat pH, a small magnetic stirrer was placed in the tube and stirring at 600 rpm was initiated. The required amount of each selected organic acid was added in small increments until a pH of 5.0 (5.0±0.2) was reached. The volume and weight of each selected organic acid were recorded. The acidic extract was then centrifuged at 5000 g for 5 minutes, and the supernatant was filtered through a 0.2 μm filter membrane. The pH of the stabilized bacterial extract was then adjusted to pH 7.0±0.2 using NaOH 1N, and the volume of NaOH 1N used was recorded.

[0135] In a second series of preparations, 35 mL of OM314A alkaline concentrate was adjusted from pH 10.5 to pH 5.0 (5.0±0.2) using the required amount of each selected organic acid, added in small increments until a pH of 5.0 (5.0±0.2) was reached. The volume and weight of the acid were recorded. The acidic extract was then centrifuged at 5000 g for 5 minutes, and the supernatant was filtered through a 0.2 μm filter membrane. The pH of the stabilized bacterial extract was then adjusted to pH 7.5±0.2 using NaOH 1N, and the volume of NaOH 1N used was recorded. A similar procedure was performed in a sterile laminar flow cabinet to prepare sterile samples of the stabilized bacterial extract.

[0136] 25 mL or 35 mL of alkaline OM314A bacterial extract concentrate was placed in a 50 mL Falcon tube. Each acid was added based on a predetermined volume or by weighing, as determined in a preliminary assay. Acidification was initially performed to pH 5.0, but precipitates formed. In a second step, the pH was adjusted to 7.0 ± 0.2 and 7.5 ± 0.2, respectively. Some solutions adjusted to pH 5 with organic acids and further adjusted to pH 7.0 and 7.5, respectively, remained clear and were stable at room temperature throughout the entire observation period, but precipitates formed after several weeks of storage at 4–8°C.

[0137] Table 1 below summarizes the organic acids and volumes used to adjust the pH to 7.5 for a 35 mL solution of OM314A bacterial extract.

[0138] [Table 1]

[0139] Example 1.2. Removal of inorganic cations To remove inorganic divalent cations present in the organic acid-stabilized OM314A extract previously described in Example 7.1, 25 mg of ammonium oxalate (1 mg / mL) was added. A strong milky precipitate formed. The precipitate was centrifuged at 5000 g for 5 minutes, and the clear supernatant was filtered through a 0.2 μm membrane filter. The clear sample was adjusted to pH 7.0±0.1 with NaOH 1N. A difficulty with this process is scale-up, which would require centrifugation. Furthermore, oxalate would not be suitable for intranasal or perioral formulations.

[0140] Example 1.3. Simultaneous removal of small molecules and inorganic salts and enrichment of the macromolecular fraction of organic acid-stabilized bacterial extracts An alkaline bacterial extract with a pH between 10.0 and 11.0 was added to the purification unit shown in the schematic diagram in Figure 2, which illustrates the connections between four vessels, two pumps, two filtration membranes, two transmembrane pressures (TMP1 and TMP2 pressure regulating valves) and four rotary valves. Transmembrane pressure (TMP): average applied pressure from the feed to the filtrate side of the filtration membrane. TMP [bar] = [(pressure at retentate + pressure at filtrate) / 2] - pressure at filtrate. The TMP1 and TMP2 valves generate pressure on the filtrate side of the filtration membrane, which adjusts the transmembrane pressure (TMP) to an appropriate pressure level. Microfiltration: In this setting, membrane filters, usually 0.45 to 0.2 μm in size, are used to remove particles, allowing soluble (non-particulate) material to pass through the pores of the filter membrane. Ultrafiltration or nanofiltration: These filtration membranes have very small pores with a range of cutoffs (cutoff value: pore size expressed as molecular weight in Daltons (1 Da = mass unit, 1 kDa = 1000 mass units). A 10 kDa filtration membrane will retain substances larger than the pores or molecules larger than 10 kDa. (See http: / / www.merckmillipore.com / CH / de / ps-learning-centers / ultrafiltration-learning-center / optimization-process-simulation / d#eb.qB.ZWQAAAFAUV8ENHoL,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F#tmp).

[0141] Example 1.4: Example of a five-step process leading to an organic acid-embedded stabilized bacterial extract according to the present invention (Figure 2) Step 1: An alkaline bacterial extract (pH 10-11) containing bacterial cell walls, cell wall fragments, and soluble materials was added to Tank 2 (Figure 2) loaded with a microfiltration 0.45 μm membrane and a 0.45 μm permeate connected to Pump 1 and Tank 3 (Figure 2). Tank 3 is connected to Pump 2 (Figure 2) and an ultrafiltration 10 kD (kDalton) nanofiltration membrane which returns the 10 kD permeate to Tank 2. The permeate containing small molecules and diluted sodium hydroxide is used for the continuous extraction of the active water-soluble components of the bacterial extract in Tank 2 (Figure 2). One half of the initial volume of bacterial extract in vessel 2 was microfiltered and the 0.45 μm cut-through was passed to vessel 3.

[0142] Step 2: The second filtration unit was then powered on (Pump 2) and the 10 kDa permeate leaving Tank 3 was returned to Tank 2 using Valve 3. A continuous extraction of the bacterial soluble components of the crude bacterial extract in Tank 2 was performed by extracting the 10 kDa permeate with a total of 10 times the initial volume of the bacterial extract.

[0143] Step 3: This step was performed to remove low molecular weight components present in Tank 3. The diafiltration process was initiated by connecting Tank 1 to Pump 1 with fine adjustment of TMP1 regulation to maintain the volume of Tank 2 at a constant level. Tank 1 contained water as the diafiltration medium, adjusted to a pH of 10.8 to 11.0 with sodium hydroxide. During the diafiltration process, the ultrafiltration permeate of Tank 3 was connected to waste using Valve 3 and TMP2 adjusted to an optimal flow rate. A total of five volumes of diafiltration medium were required to remove the undesired low molecular weight components present in the purified bacterial extract in Tank 3.

[0144] Step 4: The step of concentrating the purified extract present in tank 3 was carried out after disconnecting tank 1 from pump 1. The purified extract fraction in tank 3 was concentrated to half of its initial volume, with the permeate going to waste via valve 3.

[0145] Step 5: The concentrated purified bacterial extract was stabilized by the addition of organic acids to form embedded organic acid salts. This process was carried out after closing pump 2 and valve 2. A predetermined volume of pure liquid or solid organic acid, respectively, and a predetermined volume of a concentrated solution of organic acid were added from tank 4 to tank 3 through valve 4 to reach a pH value of 7.5±0.2. For pH adjustment, the propeller was turned on during the addition of the organic acid, and the pH was adjusted via a pH electrode installed in tank 3.

[0146] Step 6: Tank 3, containing the concentrated purified bacterial fraction in the form of organic acid salts at pH 7.5, was sterilized in-line using a sterile 0.2 μm membrane filtration unit loaded into a sterilization line connected to sterilization tank 5 (sterilization bag or heat-sterilized stainless steel tank). Depending on the final use and route of the liquid formulation (intranasal, inhalation as aerosol or solid), further concentration was performed using ultrafiltration connected to waste via pump 2 and valve 3, reaching 25% of the initial volume. The presence of organic acid salts allowed for a highly concentrated purified fraction to be achieved, allowing for direct use in the spray drying process, as droplets, and as an aerosol. Products of similar pH (7.5±0.5) were obtained using different organic acids that formed embedding salts.

[0147] The same process was repeated using hollow fibers with 10 kD, 30 kD, 100 kD, and 300 kD cutoffs as alternative tangential flow filtration membrane designs, along with 3 kD, 10 kD, 30 kD, 100 kD, and 300 kD polysulfone tangential flow filtration membranes, respectively. The process was initially carried out at a 1 L laboratory scale using a concentration step (2x, reaching 500 mL, then 5x, reaching 200 mL volume). Unwanted small molecules were washed away by a diafiltration step using a pH 10.5 to pH 11.0 NaOH-water solution, and the desired retentate of the ultrafiltration was further concentrated 2x. Organic acids were then added to stabilize the preparation by forming salts with positively charged groups present in the extract at pH 7.5 ± 0.5. Similarly, the high molecular weight fraction was concentrated using a concentration step with ultrafiltration membranes with different cutoffs to remove the low molecular weight fraction: 3 kD, 10 kD, 30 kD, 100 kD, 300 kD, and 1000 kD, respectively.

[0148] The organic acids listed in the table below were added at a target pH value of 7.5±0.5. The embedded organic acid salts of the positively charged soluble high molecular weight fraction of the bacterial extract were kept within the high molecular weight fraction during the process. A similar procedure was performed at 10 L, 100 L and finally 400 L to prepare pilot and industrial batch sizes with a 10 kD cutoff. Preferably, low-molecular-weight salts and molecules can be removed by ultrafiltration to yield a concentrated high-molecular-weight fraction with enhanced antiviral properties of the bacterial extract. 500 mL of bacterial extract at pH 10.8 was added to a laboratory-scale ultrafiltration system equipped with a 10 kDa polysulfone ultrafiltration membrane. The initial volume of 500 mL was concentrated four-fold to approximately 120 to 140 mL, followed by diafiltration using NaOH-water at pH 11 ± 0.2. After five volumes of diafiltration, the retentate was concentrated down to 100 mL, adjusted to a final volume of 125 mL with water, and then 10 mL aliquots were added with different organic acids.

[0149] The process was carried out using different bacterial extracts, such as purified fractions of OM alkaline extracts, Gram-positive bacterial purified fractions from alkaline extracts of Staphylococcus, Streptococcus, Bifidobacterium, and Lactobacillus, as well as Gram-negative bacterial purified fractions from alkaline extracts of Escherichia coli, Klebsiella, Branhamella, and Hemophilus strains. Table 2 below summarizes the organic acids used to adjust the pH to 7.5. The purified fractions of bacterial alkaline extracts after removal of low molecular weight components were adjusted to pH 7.5 using the following organic acids, and their stability was evaluated.

[0150] [Table 2]

[0151] Example 1.5: Simultaneous removal of small molecules and inorganic salts and enrichment of the high molecular weight fraction of an organic acid-stabilized bacterial extract of Lactobacillus fermentum (OM314B). A 1 L volume of alkaline bacterial extract of Lactobacillus fermentum at pH 10.5 ± 0.3 was added to an ultrafiltration system equipped with a 0.45 μm microfiltration membrane and a second ultrafiltration membrane with a 10 kDalton cutoff. The bacterial extract was purified using both filtration membranes as described in Example 7.3 and Figure 1. The first step was a concentration step, followed by sequential extractions using 10 volumes of the 10 kDalton flow-through as the extraction medium. A diafiltration process using 5 volumes of aqueous NaOH solution was then performed to adjust the pH to 10. A final 4-fold concentration step was added followed by the addition of different organic acids that form embedding salts.

[0152] The process was carried out at a 1 L scale using an initial concentration step (5-fold, down to approximately 200 mL), followed by a diafiltration step using 5 volumes of a pH 10.5 NaOH-water solution to wash out unwanted small molecules. The organic acids listed in Table 3 below were then added to stabilize the preparation by forming salts with the positively charged groups present in the extract. Similarly, the high molecular weight fraction was concentrated using a concentration step with polysulfone membranes with ultrafiltration cutoffs of 3 kD, 10 kD, 30 kD, 100 kD, and 300 kD, respectively, to remove the low molecular weight fraction.

[0153] The organic acids listed in the table below were added at a target pH value of 7.5±0.2. The embedded organic acid salts of the positively charged soluble high molecular weight fraction of the bacterial extract were kept within the high molecular weight fraction during the process. In other examples, the process was repeated using the following polysulfone ultrafiltration membrane cutoffs: 3 kD, 30 kD, 100 kD, 300 kD, respectively. In another example, removal of low-molecular-weight salts and molecules by ultrafiltration led to a concentrated high-molecular-weight fraction of Lactobacillus fermentum extract with enhanced antiviral properties. 500 mL of Lactobacillus fermentum alkaline extract at pH 10.8 was added to a laboratory-scale ultrafiltration system equipped with a 10 kDalton polysulfone ultrafiltration membrane. The initial volume of 500 mL was concentrated four-fold to approximately 120 to 140 mL, followed by diafiltration using NaOH-water at pH 10.8 to 11.0. After five volumes of diafiltration, the high-molecular-weight fraction (retentate) was concentrated down to 100 mL and adjusted to a final volume of 125 mL with water. 10 mL aliquots were then spiked with different organic acids to evaluate their physical stability and antiviral activity. Table 3 below lists the organic acids used to adjust the pH of Lactobacillus fermentum extract fractions >10 kDalton to 7.5.

[0154] [Table 3]

[0155] In another example, the process was repeated using microfiltration hollow fibers 0.45 μm and 0.2 μm and ultrafiltration hollow fibers with cutoffs of 30 kD and 100 kD as alternatives to tangential flow filtration membrane designs with better flow rates and shorter process times. A similar procedure was carried out at 10 L, 100 L and finally 400 L to prepare pilot and industrial batch sizes using a 10 kD cutoff.

[0156] Example 1.6: Process and analytical characterization for Lactobacillus fermentum I3929p (OM314B) Example 1.6.1 Process 4 Lactobacillus fermentum I3929p Dissolution: Lactobacillus fermentum I3929p biomass was thawed overnight at room temperature. The dissolution was performed using a total mass of 2 kg, with a total dry weight (RS) of 25 g per kg of lysate. The required amount of biomass (depending on the RS result) was placed in a 2500 mL mini-keg (reference: Semadeni No. 6863), and a 2 kg qsp was prepared using preheated purified water at 40°C ± 5°C. The pH of this solution was then adjusted to 10.0 ± 0.1 with NaOH 10N (pH: 9.98, adjusted with 2.8 mL of NaOH 10N). The alkaline lysis was transferred under stirring (with a 1 cm product vortex) to a warm room at 40° C.±1° C. After 4 hours±5 minutes of lysis, the pH was adjusted (end of lysis pH: 9.28). The sample corresponding to the lysate at the end of the lysis (called "Process 4-E1-Lysate") was subjected to this step.

[0157] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 30 kDa (ultrafiltration membrane in Figure 2). Filtration was carried out on a laboratory scale with a working volume of 2000 mL. Before the start of the process, the filtration system was checked for reproducibility over several batches. A normalized water permeability (NWP) was performed on the filtration system to verify the correct filtration capacity of the product.

[0158] The lysate used for production was stirred to create a 1 cm vortex of the product. The product was cooled to room temperature while waiting for the filtration process to begin. In this process, there was no initial pH adjustment, so the filtration process began immediately.

[0159] Initial concentration step: The product used in the first step of filtration had the following parameters: no pH adjustment after dissolution (pH: 9.28), temperature 38°C, and stirring to have a 1 cm vortex of the product. The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0160] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product with a concentration factor of 0.5 (pressure force: 270 mbar, permeate flow rate: 75 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0161] Diafiltration: When a concentration factor of 1 / 2 (0.5x) was reached, the UF permeate was opened and diafiltration of the product started (pressure force: 793 mbar, permeate flow rate: 53 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 1060 mbar, permeate flow rate: 47 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0162] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0163] Filtration 2: The collected product of interest (mass: 996.0 g) then underwent the second stage of purification for five cycles in a 30 kDa cutoff UF loop (tank 3, pump 2, and ultrafiltration membrane in Figure 2). The 30 kDa permeate was discarded with the valve open to waste (Figure 2). The volume of the 30 kDa retentate was kept constant during this second filtration step of the purification by adding a 0.001 N NaOH solution at pH 10.0 for diafiltration. The UF loop pump (pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system.

[0164] Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 640 mbar, permeate flow rate: 108 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 900 mbar, permeate flow rate: 134 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH 0.001 M solution was added as the level in the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH 0.001 M diafiltration solution added equal to five times the volume of the product of interest collected (5 cycles of diafiltration).

[0165] At the end of the second filtration step, the final product was collected (mass: 951.4 g) and then divided into two equal portions. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 μm and larger than 30 kDa in size. A sample corresponding to the filtrate before neutralization (referred to as "Process 4-E2-filtrate") was subjected to this step.

[0166] The first portion of the filtrate was then neutralized with 1% propionic acid at pH 7.0±0.2 (adjusted to pH 7.13 with 0.5 mL of 1% propionic acid) and then sterilized under a biosafety cabinet using filtration with a 0.2 μm polyethersulfone (PES 0.2 μm) sterilizing membrane. A sample corresponding to the neutralized filtrate at the end of the process (designated "Process 4-E3-Neutralized Filtrate (Propionic Acid)" OM314B) was run through this step. Meanwhile, the second portion of the filtrate was further divided into nine equal portions, each of which was then neutralized to 7.0 ± 0.2 with 2.5% hydrochloric acid (pH: 7.07) or one of the following organic acids (OM314B): formic acid 1 / 100 (pH: 7.16), acetic acid 1 / 100 (pH: 7.16), 3-hydroxybutanoic acid 1 / 100 (pH: 7.14), aspartic acid 0.1% (pH: 7.18), lactic acid 1 / 50 (pH: 7.09), glutamic acid 0.1% (pH: 7.14), pyruvic acid 1 / 100 (pH: 7.16), ascorbic acid 0.1% (pH: 7.18). Finally, the different products were sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to a different neutralized filtrate at the end of the process (called "Process 4-E4-Neutralized Filtrate (Name of Acid)") was run at this step.

[0167] [Table 4]

[0168] Example 1.6.2 Analytical Characterization a) Dry weight method (dissolved material) The dry residue of the melt was determined by halogen desiccation following the principle of thermogravimetric analysis. At the beginning of the measurement procedure, the weight of the sample was determined, and then the sample was rapidly heated using a built-in halogen heater to evaporate the moisture. During drying, the instrument continuously weighed the sample. Once drying was completed, the weight of the dry residue was displayed. Approximately 2 to 5 g of lysate (M) was accurately weighed. The following parameters were used: heating mode: progressive; stopping mode: constant weight; final temperature: 105°C. Once the analysis was finished, a weighing ticket was automatically printed showing the final mass of the residue obtained (m). Dry residue (expressed in mg / g) = (m / M) x 1,000. Measurements were performed directly on the sample (total) and after centrifugation at 5,000 x g for 5 minutes (supernatant).

[0169] b) Dry weight method (filtrate) The dry weight of the filtrate was determined according to European Pharmacopoeia 2.2.32 using approximately 5 g of filtrate dried at 105° C. (oven) for 16 hours. The results are expressed in mg / g.

[0170] c)Lowry method The assay was based on the reaction of proteins with alkaline copper tartrate solution and Folin's reagent (based on European Pharmacopoeia 2.5.33). Two steps led to a colorimetric reaction: the reaction between the protein and copper in alkaline medium, and the subsequent reduction of Folin's reagent by the copper-treated protein. The protein resulted in the reduction of Folin's reagent, yielding a reduced species with a characteristic blue color with an absorbance maximum of 750 nm. Results were expressed against a bovine serum albumin (BSA) standard curve.

[0171] Sample preparation: 1.9 mL of phosphate buffer pH 11 (3.55 g NaHPO and 41 mL of 0.1 M NaOH per L of water) was added to 100 μL of each sample and vortexed. BSA standard preparation: BSA solution was diluted with phosphate buffer to prepare a 6-point standard curve between 0 and 420 μg / mL. Procedure: 20 μL of sample and standard solutions were placed in a 96-well microplate. 25 μL of A reagent (Bio-Rad® Lowry kit) was immediately added to each well and incubated at room temperature for 10 minutes. 200 μL of B reagent (Bio-Rad® Lowry kit) was added, mixed, and incubated at room temperature for 20 minutes. After mixing, the absorbance was read at 750 nm. Results: Protein concentrations were calculated from the standard curve: protein concentration (mg / mL) = [(yb) / a] * sample dilution, y = absorbance of the sample, a = slope of the calibration curve; b = ordinate at the origin of the calibration curve. Results are expressed as mg protein / mL.

[0172] d) Total sugar method When heated in anthrone and sulfuric acid medium, the carbohydrate forms a chromophore that absorbs at 625 nm. Preparation of glucose standard: D-glucose was dissolved and diluted in purified water to prepare a standard curve with 5 points between 0 and 100 μg / mL. Procedure: 0.1 mL of the solution to be tested (i.e., concentrate) and 0.9 mL of purified water were placed in a tube in an ice bath. 5.0 mL of anthrone reagent (160 mg of anthrone in 100 mL of 85% sulfuric acid) was added and vigorously shaken. The solution was heated in a boiling water bath for 15 minutes, then cooled in an ice bath and shaken occasionally. The solution was left at room temperature for 15 minutes. The tube was vortexed, and the solution was transferred to a measuring cell. After leaving it at room temperature for 30 minutes, the absorbance was measured at 625 nm. Results: Carbohydrate concentrations were calculated from the standard curve: carbohydrate content (mg / mL) = ([(yb) / a]*10) / (1,000), where y = absorbance of the sample; a = slope of the calibration curve; b = ordinate at the origin of the calibration curve. Results are expressed as mg carbohydrate / mL.

[0173] e) Total RNA assay Total RNA purification and assay were performed according to the supplier's recommendations, based on the RNeasy® Mini Kit data sheet. Briefly, 600 μL of bacterial extract was transferred to an RNeasy® spin column and centrifuged at 8,000 × g for 15 seconds. Next, 700 μL of RW1 buffer was added to the RNeasy® column and centrifuged at 8,000 × g for 15 seconds to wash the spin column membrane. The same process was performed twice with 500 μL of RPE buffer, followed by centrifugation at 8,000 × g for 15 seconds and 2 minutes. To eliminate any possible carryover of RPE buffer, the spin column was centrifuged at full speed for 1 minute. To elute the RNA, 30 μL of RNase-free water was added directly to the spin column membrane, and the column was centrifuged at 8,000 × g for 1 minute. Purified total RNA was detected at 260 nm using a NanoDrop (ThermoFischer) spectrophotometer.

[0174] f) Total DNA assay Total DNA purification and assay were performed according to the supplier's recommendations and based on the DNeasy® Blood and Tissue Kit data sheet. Briefly, 600 μL of bacterial extract was transferred to a DNeasy® mini spin column and centrifuged at 6,000 × g for 1 minute. 500 μL of AW1 buffer was then added to the DNeasy® mini column and centrifuged at 6,000 × g for 1 minute. 500 μL of AW2 buffer was added, and the spin column was centrifuged at 20,000 × g for 3 minutes to dry the DNA membrane. 100 μL of AE buffer was added to the DNeasy® membrane, incubated for 1 minute, and centrifuged at 6,000 × g for 1 minute to elute the DNA. The elution was repeated once for maximum DNA yield. Purified total DNA was detected at 260 nm using a NanoDrop spectrophotometer.

[0175] g) Limulus amoebocyte lysate LAL assay The endotoxin assay was performed according to the supplier's recommendations using the Pierce™ Chromogenic Endotoxin Quantitation (Quant) Kit data sheet. All samples were diluted 1:10 to prevent the sample's inherent color from altering the absorbance reading. Briefly, endotoxin standard solutions (range 0.1-1.0 EU / mL) were prepared from endotoxin stock solution (10 EU / mL). 50 μL of endotoxin standard, blank (endotoxin-free water), and sample were added per well. 50 μL of reconstituted amebocyte lysate reagent was added, and the plate was incubated at 37°C for 15 minutes. 100 μL of chromogenic substrate solution was added per well and incubated at 37°C for 6 minutes. At exactly 6 minutes, 50 μL of stop solution (25% acetic acid) was added. Optical density was measured at 405 nm immediately after assay completion.

[0176] h) Amino acid method: D- and L-amino acids were determined by reversed-phase high-performance liquid chromatography (HPLC). After hydrolysis of the samples in a microwave oven, the amino acids were derivatized with o-phthalaldehyde and the chiral thiol N-isobutyryl-L-cysteine. Detection was performed by UV detection at 338 nm. (Bruckner, H., T. Westhauser, H. Godel. Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and N-isobutyryl-L-cysteine. J. Chromatography A, 1995, 711, 201-21). Standard solutions were prepared using different amino acids at 2.5 μmol / mL in 0.01 N hydrochloric acid (HCl): aspartic acid (Asp), serine (Ser), glutamic acid (Glu), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), lysine (Lys), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), glycine (Gly), and cystine (Cys). These solutions were diluted to 0.5 μmol / mL with 0.1 M sodium tetraborate decahydrate buffer at pH 9.2. In a tube, 2.0 mL of sample solution was added to 2.0 mL of water, 240 μl of 1-dodecanethiol, and 8 mL of 25% HCl. These were hydrolyzed in a microwave oven at 180°C and 1320 watts for 15 minutes. The solution was left at room temperature and filtered through a 5 μm filter. 50 μl was evaporated to dryness and reconstituted with 100 μl of 0.01N HCl. Sample and standard solutions were kept in an HPLC system at 10°C and injected using the following injection mode: 5 μl of 0.1 M sodium tetraborate decahydrate buffer, pH 9.2; 2 μl of derivatization solution (23 mg of phthalaldehyde and 50 mg of N-isobutyryl-L-cysteine ​​solubilized in 1 ml of methanol); 2 μl of sample or standard solution was withdrawn by autosampler, mixed, and injected five times. The HPLC column was a Supelcosil C18, 5 μm, 4.6 × 250 mm column with a Supelco LC18, 5 μm, 4.6 × 20 mm precolumn. For elution, a gradient was formed from 23 mM sodium acetate (eluent A) adjusted to pH 5.9 and a methanol-acetonitrile mixture (12:1, v / v) (eluent B). The gradient was formed from 4% B to 33% B in 45 min, then to 56% B in 30 min (with a rinse step to 85% B) at a flow rate of 1 ml / min. Free amino acids were also tested separately for each sample without the HCl hydrolysis step using the E2-filtrate solution directly in HPLC vials; no free amino acids were detected without hydrolysis.

[0177] i) Spectroscopic results obtained during stability The neutralized filtrate solutions were subjected to stability at room temperature (20°C ± 5°C) or 4°C. At each time point, the absorbance of the solutions was recorded between 300 and 700 nm. The spectral profiles were visually assessed as noisy (e.g., Figure 27, showing precipitate in the solution) or smooth (e.g., Figure 27). The absorbance was extracted at 320 nm for quantitative assessment.

[0178] j) Results at release All E2-filtrate solutions were frozen after processing and thawed overnight at 4°C before analysis.

[0179] Example 1.6.3. Analytical Characterization of Process 4 Lactobacillus fermentum I3929p Final Sample at Time of Release (T0) [Table 5]

[0180] [Table 6]

[0181] [Table 7]

[0182] The technical batch (OM-13-BV) described in WO2008 / 109669 (HCL technical batch 1619064) neutralized with hydrochloric acid showed precipitation starting already at T0 immediately after neutralization. Process 4-E4-neutralized filtrates were physically stable for at least 6 months at 4° C. or room temperature. They are expected to be stable for 12 months under these conditions.

[0183] Mip3-alpha (CCL20) results obtained during stability: Chemokine (CC motif) ligand 20 (CCL20), also known as liver activation-regulated chemokine (LARC) or macrophage inflammatory protein-3 (MIP-3-alpha, CCL20), is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and weakly attracts neutrophils. CCL20 is involved in the formation and function of mucosal lymphoid tissues through the chemoattraction of lymphocytes and dendritic cells toward the epithelial cells surrounding these tissues. CCL20 induces its effects on target cells by binding to and activating the chemokine receptor CCR6.

[0184] The THP-1 cell line was purchased from ATCC Collection #TIB-202. The THP-1 cell line was derived from the peripheral blood of a 1-year-old boy with acute monocytic leukemia. A vial from the THP-1 working cell bank was used as differentiated macrophage-like cells in this bioassay.

[0185] The neutralized filtrate solutions were subjected to stability at room temperature (20°C ± 5°C) or 4°C. Samples were taken at different time points and frozen for further bioassays. All solutions (TO and stability time points) were thawed overnight at 4°C before analysis.

[0186] Differentiation: THP-1 cells were cultured in cell suspension (1 × 10) using phorbol 12-myristate 13-acetate (PMA). 6 The cells were differentiated to a final PMA concentration of 100 ng / mL (cells / mL). 100.0 μL / well of the PMA-cell suspension was dispensed into each well of a 96-well cell culture plate. The cells were incubated at 37°C for 72 hours.

[0187] Stimulation: 10-point serial dilutions (3.16-fold serial dilutions) were performed with medium in deep-well plates: PAM3CSK4 (reference positive control for MIP-3α secretion) from 2 μg / mL to 0.06 μg / mL. 6-point serial dilutions (3.16-fold serial dilutions) were performed with medium in deep-well plates: 200 μL of test sample + 400 μL of medium into the first well of the deep well, then 190 μL was diluted with 410 μL of medium (3.16-fold). 100 μL / w of supernatant was removed from the plate with cells. 100.0 μL / w of medium was distributed into each well of the plate with cells. The plate was incubated at 37°C for 24 hours.

[0188] Supernatant collection: 75 μL of supernatant from each well was collected and distributed into a 96-well PP microplate. The plate was sealed and stored in an ultra-low temperature freezer until the ELISA assay was performed. ELISA test: Microplate wells were coated with 100.0 μL / well of anti-human MIP-3α (capture antibody). The plate was covered with sealing foil and appropriately incubated. After incubation was completed, a washing step was performed using an automated microplate washer. On the day of the saturation step, the supernatant plate was thawed at +4°C. Saturation step: 250.0 μL / well of reagent diluent (1% BSA in PBS) was added, and the plate was appropriately incubated. During the saturation step, serial dilutions of the supernatant and standard MIP-3α were prepared. A standard curve was performed for each ELISA plate. After incubation was completed, a washing step was performed, samples were distributed into wells, and the plate was appropriately incubated. After incubation was complete, a washing step was performed and biotinylated goat anti-human MIP-3α (detection antibody step) was dispensed. The plate was incubated appropriately. After incubation was complete, a washing step was performed and streptavidin-HRP conjugate was dispensed. The plate was incubated appropriately. After incubation was complete, a washing step was performed, enzyme substrate was added, and the OD was read at 450 nm. Blank subtraction and wavelength correction at 540 nm were performed according to the recommendations of the ELISA kit supplier.

[0189] Process 4-Bioassay results during stability indicate that Process 4-E3-Neutralized Filtrate was stable for at least one month at room temperature (20°C ± 5°C) or 4°C.

[0190] Example 1.7 Polyvalent Alkaline Lysate Mixture of 18 E. coli Strains Example 1.7.1. Process No. 5 Polyvalent Alkaline Lysate Mixture of 18 E. coli Strains (OM314C) Alkaline lysis: A portion of the alkaline lysed E. coli mixture of 18 strains described in WO2008 / 109667 was collected during production and stored in 2500 mL mini-barrels (reference: Semadeni no. 6863). The sample corresponding to the lysate at the end of the lysis (called "Process 5-E1-Lysate") was subjected to this step.

[0191] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 30 kDa (ultrafiltration membrane in Figure 2). Filtration was performed on a laboratory scale with a working volume of 2000 mL. Before the process started, it had to be ensured that the filtration system was reproducible across multiple batches. NWP (Standardized Water Permeability) was performed on the filtration system to prove the correct filtration capacity of the product.

[0192] The lysate used for purification was first diluted 4-fold with purified water (500.8 g of lysate and 1502.7 g of purified water). The product was stirred to create a 1 cm vortex. The product was allowed to cool to room temperature while the filtration process began. During this process, the pH was adjusted to 10.5-10.8 (adjusted to pH 10.68 using 2.1 mL of pure pyruvic acid).

[0193] Initial concentration: The product used in the first step of filtration had the following parameters (pH: 10.68, temperature: 29°C, agitation to create a 1 cm product vortex). The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated, and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditioned the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0194] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product with a concentration factor of 0.5 (pressure force: 200 mbar, permeate flow rate: 89 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0195] Diafiltration: When a 0.5 concentration factor was reached, the UF permeate was opened and the diafiltration of the product started (pressure force: 786 mbar, permeate flow rate: 80 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 950 mbar, permeate flow rate: 60 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0196] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0197] Filtration 2: The collected product of interest (mass: 1146.3 g) then underwent a second stage of purification in a UF loop (tank 3, pump 2 and ultrafiltration membrane in Figure 2) with a 30 kDa cutoff point for 5 cycles. The flow-through was waste as described in Figure 2. During the second filtration step of this purification, a constant volume was maintained by adding a 0.001 N NaOH solution at pH 10.0.

[0198] The UF loop pump (Pump 2 in Figure 2) was started at 40% of process speed to load the product into the system. Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 650 mbar, permeate flow rate: 76 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 960 mbar, permeate flow rate: 105 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH solution was added as the level of the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH solution added equal to five times the volume of the product of interest collected.

[0199] At the end of the second filtration step, the final product was collected (mass: 1042.7 g) and then divided into two equal portions. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 μm and larger than 30 kDa in size. A sample corresponding to the filtrate before neutralization (referred to as "Process 5-E2-Filtrate") was run at this step.

[0200] The first part of the filtrate was then neutralized with pyruvic acid 1 / 100 at pH 7.0±0.2 (adjusted to pH 7.08 with 3 mL of pyruvic acid 1 / 100) and then sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to the neutralized filtrate at the end of the process (referred to as "Process 5-E3-Neutralized Filtrate (Pyruvic Acid)", OM314C) was run through this step. Meanwhile, the second portion of the filtrate was further divided into nine equal portions. Each of these portions was then neutralized to 7.0 ± 0.2 with 2.5% hydrochloric acid (pH: 7.09) or organic acids (OM314C): formic acid 1 / 100 (pH: 7.11), acetic acid 1 / 100 (pH: 6.97), 3-hydroxybutanoic acid 1 / 100 (pH: 7.03), aspartic acid 0.1% (pH: 7.09), lactic acid 1 / 100 (pH: 7.15), glutamic acid 0.1% (pH: 7.10), propionic acid 1 / 100 (pH: 7.10), and pure ascorbic acid (pH: 7.04). Finally, the different products were sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to a different neutralized filtrate at the end of the process (called "Process 5-E4-Neutralized Filtrate (Name of Acid)") was run at this step.

[0201] [Table 8]

[0202] Example 1.7.2. Analytical Characterization The analytical method is described in 1.6.2.

[0203] Analytical characterization of Process 5 final samples at time of release (T0) The E2-filtrate solution was frozen after processing and thawed overnight at 4°C before analysis.

[0204] [Table 9]

[0205] [Table 10]

[0206] a) Spectroscopic results obtained during stability [Table 11]

[0207] Industrial batch 1619064 (OM-13-BV) described in WO2008 / 109669, which was neutralized with hydrochloric acid, showed precipitation starting at TO. With the exception of ascorbic acid, the Process 5-E4-neutralized filtrates were physically stable for at least 6 months at 4° C. or room temperature and are expected to be stable for 12 months under these conditions.

[0208] Mip3-alpha (CCL20) results obtained during stability: Figure 32: Process 5 during stability - Bioassay results show that Process 5-E3-neutralized filtrate exhibited comparable bioactivity by MIP-3α secretion in THP-1 cells for at least 5 months at room temperature (20°C ± 5°C) or 4°C. Process 5 TO was compared to T5 samples stored for 4 months at 4°C and room temperature (RT).

[0209] Example 1.8 Process 3: Streptococcus pn. 7466 Alkaline Lysate (OM314A) Example 1.8.1 Process 3: Streptococcus pn.7466 Alkaline lysis: 2794 kg of Streptococcus pneumoniae 7466 biomass (batch 1418123 - boxes 34 and 35) was thawed overnight at room temperature in a lysis barrel. 240 g of NaOH 10N and 4293 g of 8 g / L NaCl solution were added to obtain a lysis with a total weight of 7327 g. The alkaline lysis was transferred to a warm room at 37°C ± 2.5°C with stirring at 150 rpm ± 5 rpm for 8 days. After 3 h 00 ± 30 min of lysis, the J0 OD was controlled. Samples were diluted 100-fold and read on a spectrophotometer at 700 nm (reading OD: 0.258 and final OD: 25.8). On each working day, stirring (150 rpm ± 5 rpm), warm room temperature (37.0 °C ± 2.5 °C) and pH were controlled (J1 pH: 12.63 / J2 pH: 12.62 / J5 pH: 12.59 / J6 pH: 12.65 / J7 pH: 12.69 / J8 pH: 12.71). If the pH was not within the process range, it had to be adjusted with NaOH 10N (J1: 10 mL NaOH 10N / J2: 10 mL NaOH 10N / J5: 10 mL NaOH 10N / J6: 10 mL NaOH 10N / J7: 10 mL NaOH 10N).

[0210] At the end of lysis, J8 OD was controlled. The sample was diluted 5 times and read at 700 nm in a spectrophotometer (read OD: 0.092 and final OD: 0.46). The delta OD between J0 and J8 had to be greater than 12.8 (delta OD: 25.34). A portion of this Streptococcus pneumoniae 7466 lysis was collected and stored in a 2500 mL mini-barrel (reference: Semadeni No. 6863). The sample corresponding to the final lysate (referred to as "Process 3-E1-Lysate") was obtained from this step.

[0211] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 10 kDa (ultrafiltration membrane in Figure 2). Filtration was performed on a laboratory scale with a working volume of 2000 mL. Before the process started, it had to be ensured that the filtration system was reproducible across multiple batches. NWP (Standardized Water Permeability) was performed on the filtration system to prove the correct filtration capacity of the product.

[0212] The lysate used for purification was first diluted 2-fold with purified water (1000.0 g of lysate and 1000.0 g of purified water). The product was stirred to create a 1 cm vortex. The product was allowed to cool to room temperature while the filtration process began. During this process, the pH was adjusted to 10.5-10.8 (adjusted to pH 10.68 using 15 mL of pure 3-hydroxybutanoic acid).

[0213] Initial concentration: The product used in the first step of filtration had the following parameters (pH: 10.68, temperature: 29°C, agitation to create a 1 cm product vortex). The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated, and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditioned the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0214] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product to a concentration factor of 0.5 (pressure force: 210 mbar, permeate flow rate: 50 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0215] Diafiltration: When a 0.5 concentration factor was reached, the UF permeate was opened and the diafiltration of the product started (pressure force: 680 mbar, permeate flow rate: 47 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 995 mbar, permeate flow rate: 46 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0216] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0217] Filtration 2: The collected product of interest (mass: 945.7 g) then underwent a second stage of purification in a UF loop (tank 3, pump 2 and ultrafiltration membrane in Figure 2) with a 10 kDa cutoff point for 5 cycles. The flow-through was waste as described in Figure 2. During the second filtration step of this purification, a constant volume was maintained by adding a 0.001 N NaOH solution at pH 10.0.

[0218] The UF loop pump (Pump 2 in Figure 2) was started at 40% of process speed to load the product into the system. Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 690 mbar, permeate flow rate: 37 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 945 mbar, permeate flow rate: 52 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH solution was added as the level of the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH solution added equal to five times the volume of the product of interest collected.

[0219] At the end of the second filtration step, the final product was collected (mass: 931.7 g) and then divided into two equal portions. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 μm and larger than 10 kDa in size. A sample corresponding to the filtrate before neutralization (referred to as "Process 3-E2-Filtrate") was run at this step.

[0220] The first part of the filtrate was then neutralized with 3-hydroxy-butanoic acid 1 / 100 at pH 7.2±0.2 (adjusted to pH 7.25 with 10 mL of 3-hydroxy-butanoic acid 1 / 100) and then sterilized under a biosafety cabinet using filtration with a PES 0.2 μm sterile membrane. A sample corresponding to the neutralized filtrate at the end of the process (referred to as "Process 3-E3-Neutralized Filtrate (3-Hydroxy-Butanoic Acid)") was run through this step. Meanwhile, the second portion of the filtrate was further divided into nine equal portions. Each of these portions was then neutralized to 7.2 ± 0.2 with 2.5% hydrochloric acid (pH: 7.19) or organic acids (OM314A): formic acid 1 / 100 (pH: 7.16), acetic acid 1 / 100 (pH: 7.16), pyruvic acid 1 / 100 (pH: 7.14), aspartic acid 1 / 100 (pH: 7.19), lactic acid 1 / 100 (pH: 7.06), glutamic acid 0.1% (pH: 7.13), propionic acid 1 / 100 (pH: 7.20), and pure ascorbic acid (pH: 7.20). Finally, the different products were sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to a different neutralized filtrate at the end of the process (called "Process 3-E4-Neutralized Filtrate (Name of Acid)") was run at this step.

[0221] [Table 12]

[0222] Example 1.8.2 Analytical Characterization The analytical method is described in 1.6.2.

[0223] Analytical characterization of Process 3 final samples at time of release (T0) The E2-filtrate solution was frozen after processing and thawed overnight at 4°C before analysis. Results at launch (T0):

[0224] [Table 13]

[0225] [Table 14]

[0226] a) Spectroscopic results obtained during stability [Table 15]

[0227] Industrial batch 1619064 described in WO2008 / 109669 was neutralized with hydrochloric acid and showed precipitation starting at TO. With the exception of ascorbic acid, the Process 3-E4-neutralized filtrate was physically stable for at least 3 months at 4°C or room temperature.

[0228] b) Mip3-alpha (CCL20) results obtained during stability: Figure 31: Process 3 during stability - Bioassay results show that Process 3-E3-neutralized filtrate exhibited comparable bioactivity in MIP-3α secretion in THP-1 cells for at least 5 months at room temperature (20°C ± 5°C) or 4°C. Process 3 TO was compared to T5 samples stored at 4°C and room temperature (RT) for 5 months.

[0229] Example 1.9, 21-Strain Polyvalent Lysate Stable Bacterial Extract (OM314A) Example 1.9.1 Process 1 for Stabilization of a 21-Strain Polyvalent Lysate Bacterial Extract Preparation Lysis: A portion of the 21 strain polyvalent lysate was collected at the time of production and stored in 2500 mL mini-barrels (reference: Semadeni no. 6863). The sample corresponding to the lysate at the end of the lysis (called "Process 1-E1-lysate") was subjected to this step.

[0230] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 10 kDa (ultrafiltration membrane in Figure 2). Filtration was performed on a laboratory scale with a working volume of 2000 mL. Before the process started, it had to be ensured that the filtration system was reproducible across multiple batches. NWP (Standardized Water Permeability) was performed on the filtration system to prove the correct filtration capacity of the product.

[0231] The lysate used for production was first diluted 2-fold with purified water (1000.6 g of lysate and 999.9 g of purified water). The product was stirred to create a 1 cm vortex. The product was allowed to cool to room temperature while the filtration process began. During this process, the pH was adjusted to 10.5-10.8 (adjusted to pH 10.77 using pure aspartic acid).

[0232] Initial concentration: The product used in the first step of filtration had the following parameters (pH: 10.77, temperature: 25°C, agitation to create a 1 cm product vortex). The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated, and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditioned the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0233] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product to a concentration factor of 0.5 (pressure force: 300 mbar, permeate flow rate: 43 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0234] Diafiltration: When a 0.5 concentration factor was reached, the UF permeate was opened and the diafiltration of the product started (pressure force: 730 mbar, permeate flow rate: 58 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 1010 mbar, permeate flow rate: 58 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0235] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0236] Filtration 2: The collected product of interest (mass: 1232.7 g) then underwent a second stage of purification in a 10 kDa cutoff point UF loop (tank 3, pump 2 and ultrafiltration membrane in Figure 2) for 5 cycles. The flow-through was waste as described in Figure 2. During the second filtration step of this purification, a constant volume was maintained by adding a 0.001 N NaOH solution at pH 10.0.

[0237] The UF loop pump (Pump 2 in Figure 2) was started at 40% of process speed to load the product into the system. Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 655 mbar, permeate flow rate: 37 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 917 mbar, permeate flow rate: 57 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH solution was added as the level of the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH solution added equal to five times the volume of the product of interest collected.

[0238] At the end of the second filtration step, the final product was collected (mass: 1237.0 g) and then divided into two equal portions. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 μm and larger than 10 kDa in size. A sample corresponding to the filtrate before neutralization (referred to as "Process 1-E2-Filtrate") was run at this step.

[0239] The first portion of the filtrate was then neutralized with aspartic acid 0.1% at pH 7.2±0.2 (adjusted to pH 7.20 with 65 mL of aspartic acid 0.1%) and sterilized under a biosafety cabinet using filtration with a PES 0.2 μm sterile membrane. A sample corresponding to the neutralized filtrate at the end of the process (designated "Process 1-E3-Neutralized Filtrate (Aspartic Acid)" OM314A) was run through this step. Meanwhile, the second portion of the filtrate was further divided into nine equal portions. Each of these portions was then neutralized to a pH of 7.2 ± 0.2 with 0.25% hydrochloric acid (pH: 7.19) or organic acids (OM314A): formic acid 1 / 100 (pH: 7.16), acetic acid 1 / 100 (pH: 7.20), pyruvic acid 1 / 100 (pH: 7.12), 3-hydroxybutanoic acid 1 / 100 (pH: 7.17), lactic acid 1 / 100 (pH: 7.19), glutamic acid 0.1% (pH: 7.09), propionic acid 1 / 100 (pH: 7.11), and pure ascorbic acid (pH: 7.20). Finally, the different products were sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to a different neutralized filtrate at the end of the process (referred to as "Process 1-E4-Neutralized Filtrate (Name of Acid)", OM314A) was run through this step.

[0240] [Table 16]

[0241] Example 1.9.2. Analytical Characterization The analytical method is described in 1.6.2. a) Release Results: The E2-filtrate solution was frozen after processing and thawed overnight at 4°C before analysis.

[0242] [Table 17]

[0243] [Table 18]

[0244] Spectroscopic results obtained during stability [Table 19]

[0245] Industrial batch 1619064, described in WO2008 / 109669 as a 21 strain lysate, was neutralized with hydrochloric acid and showed a precipitate starting at TO. With the exception of ascorbic acid, the Process 1-E4-neutralized filtrate was physically stable for at least 3 months at 4°C or room temperature.

[0246] b) Mip3-alpha (CCL20) results obtained during stability: Figure 29: Process 1 during stability - Bioassay results show that Process 1-E3-neutralized filtrate exhibited comparable bioactivity by MIP-3α secretion in THP-1 cells for at least 4 months at room temperature (20°C ± 5°C) or 4°C. Process 1 TO was compared to T4 samples stored at 4°C and room temperature (RT) for 4 months.

[0247] Example 1.9 Haemophilus influenzae 8467 (OM314A) Example 1.10.1 Process 2: Haemophilus influenzae 8467 Lysis: 13,396 kg of Haemophilus influenzae 8467 biomass (batch 1419110 - boxes 10, 11, 12, and 13) was thawed overnight at room temperature in a lysing barrel. 692 g of NaOH 10N and 12,920 g of 8 g / L NaCl solution were added to obtain a lysate with a total weight of 27,008 g. The alkaline lysate was transferred to a warm room at 37°C ± 2.5°C with stirring at 150 rpm ± 5 rpm for 5 days. After 3 h 00 ± 30 min of lysis, the J0 OD was controlled. Samples were diluted 200-fold and read on a spectrophotometer at 700 nm (reading OD: 0.273 and final OD: 54.6). On each working day, stirring (150 rpm ± 5 rpm), warm room temperature (37.0 °C ± 2.5 °C), and pH were controlled (J1 pH: 11.87 / J2 pH: 11.74 / J5 pH: 11.45). If the pH was not within the process range, it had to be adjusted using NaOH 10N (J1: 20 mL NaOH 10N / J2: 20 mL NaOH 10N). At the end of the lysis, the OD of J8 was controlled. The sample was diluted 100-fold and read at 700 nm in a spectrophotometer (reading OD: 0.169 and final OD: 16.9). The delta OD between J0 and J5 had to exceed 13.1 (delta OD: 37.7). A portion of this Haemophilus influenzae 8467 lysis was collected and stored in a 2500 mL mini-keg (reference: Semadeni No. 6863). The sample corresponding to the lysate at the end of the lysis (called "Process 2-E1-lysate") was subjected to this step.

[0248] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 10 kDa (ultrafiltration membrane in Figure 2). Filtration was performed on a laboratory scale with a working volume of 2000 mL. Before the process started, it had to be ensured that the filtration system was reproducible across multiple batches. NWP (Standardized Water Permeability) was performed on the filtration system to prove the correct filtration capacity of the product.

[0249] The lysate used for production was first diluted 4 times with purified water (499.9 g of lysate and 1500.2 g of purified water). The product was stirred to create a 1 cm vortex. The product was cooled to room temperature while waiting for the filtration process to begin. In this process, there was no pH adjustment, so the filtration process began immediately.

[0250] Initial concentration: The product used in the first step of filtration had the following parameters (pH: 11.23, temperature: 32°C, agitation to create a 1 cm product vortex). The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated, and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditioned the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0251] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product with a concentration factor of 0.5 (pressure force: 325 mbar, permeate flow rate: 45 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0252] Diafiltration: When a 0.5 concentration factor was reached, the UF permeate was opened and the diafiltration of the product started (pressure force: 880 mbar, permeate flow rate: 63 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 1163 mbar, permeate flow rate: 68 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0253] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0254] Filtration 2: The collected product of interest (mass: 1039.1 g) then underwent a second stage of purification in a 10 kDa cutoff UF loop (tank 3, pump 2 and ultrafiltration membrane in Figure 2) for 5 cycles. The flow-through was waste as described in Figure 2. During the second filtration step of this purification, a constant volume was maintained by adding a 0.001 N NaOH solution at pH 10.0.

[0255] The UF loop pump (Pump 2 in Figure 2) was started at 40% of process speed to load the product into the system. Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 650 mbar, permeate flow rate: 44 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 960 mbar, permeate flow rate: 72 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH solution was added as the level of the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH solution added equal to five times the volume of the product of interest collected.

[0256] At the end of the second filtration step, the final product was collected (mass: 1033.9 g) and then divided into two equal portions. At the end of this second filtration step, the product of interest was smaller than 0.45 μm, It contained elements with sizes greater than 10 kDa. A sample corresponding to the filtrate before neutralization (referred to as "Process 2-E2-filtrate") was subjected to this step.

[0257] The first part of the filtrate was then neutralized with propionic acid 1 / 100 at pH 7.2±0.2 (adjusted to pH: 7.18 with 4.2 mL of propionic acid 1 / 100) and then sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to the neutralized filtrate at the end of the process (designated "Process 2-E3-Neutralized Filtrate (Propionic Acid)" OM314A) was run through this step. Meanwhile, the second portion of the filtrate was further divided into nine equal portions. Each of these portions was then neutralized to 7.2 ± 0.2 with 0.25% hydrochloric acid (pH: 7.09) or organic acids (OM314A): formic acid 1 / 100 (pH: 7.13), acetic acid 1 / 100 (pH: 7.20), pyruvic acid 1 / 100 (pH: 7.15), aspartic acid 0.1% (pH: 7.12), lactic acid 1 / 100 (pH: 7.19), glutamic acid 0.1% (pH: 7.21), 3-hydroxybutanoic acid 1 / 100 (pH: 7.16), and pure ascorbic acid (pH 7.25). Finally, the different products were sterilized under a biosafety cabinet by filtration using a PES 0.2 μm sterile membrane. A sample corresponding to a different neutralized filtrate at the end of the process (called "Process 2-E4-Neutralized Filtrate (Name of Acid)") was run at this step.

[0258] [Table 20]

[0259] Example 1.10.2. Analytical Characterization The analytical method is described in 1.6.2.

[0260] Results at launch (T0): The E2-filtrate solution was frozen after processing and thawed overnight at 4°C before analysis.

[0261] [Table 21]

[0262] [Table 22]

[0263] a) Mip3-alpha (CCL20) results obtained during stability: Figure 30: Process 2 during stability - Bioassay results show that Process 2-E3-neutralized filtrate exhibited comparable bioactivity by MIP-3α secretion in THP-1 cells for at least 4 months at room temperature (20°C ± 5°C) or 4°C. Process 2 TO was compared to T4 samples stored at 4°C and room temperature (RT) for 4 months.

[0264] Example 1.11-21 strain bacterial lysis extract 30 kDa (BE30 kD, OM314A) Example 1.11.1 Process 6: 21 strain bacterial lysis extract 30 kDa (BE30 kD, OM 314A) Lysis: A portion of the 21-strain bacterial polyvalent lysate (industrial batch 1619064 described as 21-strain lysate in WO2008 / 109669) was collected during production and stored in 2500 mL mini-kegs (reference: Semadeni no. 6863).

[0265] Filtration 1: The equipment for product filtration was prepared according to the diagram (Figure 2). The filtration system consisted of two filtration loops. The first microfiltration (referred to as MF) consisted of a tank (tank 2 in Figure 2), a pump (pump 1 in Figure 2), and a filtration system with a cutoff point of 0.45 μm (microfiltration membrane in Figure 2). The second loop, ultrafiltration (referred to as UF), consisted of a tank (tank 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cutoff point of 30 kDa (ultrafiltration membrane in Figure 2). Filtration was performed on a laboratory scale with a working volume of 2000 mL. Before the process started, it had to be ensured that the filtration system was reproducible across multiple batches. NWP (Standardized Water Permeability) was performed on the filtration system to prove the correct filtration capacity of the product.

[0266] The lysate used for production was first diluted 2-fold with purified water (1000.2 g of lysate and 1000.1 g of purified water). The product was stirred to create a 1 cm vortex. The product was allowed to cool to room temperature while the filtration process began. During this process, the pH was adjusted to 10.5-10.8 (adjusted to pH 10.73 using pure aspartic acid).

[0267] Initial concentration: The product used in the first step of filtration had the following parameters (pH: 10.73, temperature: 25°C, agitation to create a 1 cm product vortex). The MF loop pump (Pump 1 in Figure 2) was started at 40% process speed to load the product into the system. Once this step was completed, product recirculation in the MF loop was initiated, and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditioned the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0268] The filtration system was considered conditioned when the flow rate and pressure were stabilized. The permeate valve of the MF loop was then opened to perform an initial concentration of the product with a concentration factor of 0.5 (pressure force: 235 mbar, permeate flow rate: 52 mL / min). During the initial concentration, the pump (Pump 1 in Figure 2) speed was gradually increased to 100% of the process speed (100 rpm, equivalent to 600 mL / min). In parallel with this process, the UF loop was conditioned. The UF loop pump (Pump 2 in Figure 2) was started at 40% of the process speed to load the product into the system. Once this process was complete, product recirculation to the UF loop was initiated and the pump speed was periodically increased to reach 75% of the process speed. Recirculation conditions the hydrophilic filtration system by eliminating any air bubbles present in the system.

[0269] Diafiltration: When a 0.5 concentration factor was reached, the UF permeate was opened and the diafiltration of the product started (pressure force: 734 mbar, permeate flow rate: 52 mL / min). To obtain optimal extraction of the product, the MF TMP (transmembrane pressure), controlled by valve 1 in Figure 2, had to be set to 850 mbar (pressure force: 1010 mbar, permeate flow rate: 48 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was set to reach a UF permeate flow equal to the permeate flow MF. In fact, the volume in the MF tank (tank 2 in Figure 2) had to be kept as stable as possible during the diafiltration process. Diafiltration was performed in cycles. At the end of the initial concentration, a certain volume was present in the MF tank (tank 2 in Figure 2). Once this volume passed through the MF filtration system, it constituted one cycle. In this process, 5 diafiltration cycles were required.

[0270] Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. The MF pump was terminated when the MF input pressure began to increase. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 μm in size.

[0271] Filtration 2: The collected product of interest (mass: 1179.6 g) then underwent a second stage of purification in a UF loop (tank 3, pump 2 and ultrafiltration membrane in Figure 2) with a 30 kDa cutoff point for 5 cycles. The flow-through was waste as described in Figure 2. During the second filtration step of this purification, a constant volume was maintained by adding a 0.001 N NaOH solution at pH 10.3.

[0272] The UF loop pump (Pump 2 in Figure 2) was started at 40% of process speed to load the product into the system. Once this step was completed, product recirculation through the UF loop was initiated and the pump speed was periodically increased to reach 75% of process speed. Recirculation allows the hydrophilic filtration system to condition by eliminating any air bubbles present in the system. Once the filtration system was conditioned, the UF permeate was opened and product filtration 2 started (pressure force: 670 mbar, permeate flow rate: 67 mL / min). To obtain optimal extraction of the product, the UF TMP (transmembrane pressure), controlled by valves 2 and 3 in Figure 2, had to be set to 850 mbar (pressure force: 920 mbar, permeate flow rate: 88 mL / min). During the Filtration 2 step, the volume of the UF tank (Tank 3 in Figure 2) had to be kept as stable as possible. Therefore, NaOH solution was added as the level of the UF tank (Tank 3 in Figure 2) decreased. Five cycles correspond to a volume of NaOH solution added equal to five times the volume of the product of interest collected.

[0273] At the end of the second filtration step, the final product was collected (mass: 1070.5 g). At the end of this second filtration step, the product of interest contained elements smaller than 0.45 μm and larger than 30 kDa in size. The filtrate was then neutralized with aspartic acid 0.1% at pH 7.2±0.2 (adjusted to pH 7.19 with 180 mL of aspartic acid 0.1%) and then sterilized under a biosafety cabinet using filtration with a PES 0.2 μm sterile membrane. A sample corresponding to the neutralized filtrate at the end of the process (referred to as "Process 6-E3-Neutralized Filtrate (Aspartic Acid)", OM314A) was run through this step.

[0274] Example 2: Stable formulations for intranasal, intratracheal, inhalation and perioral use Example 2.1: Intranasal Formulation of OM314A-Stabilized Bacterial Extract The high molecular weight fraction (>10 kD) of organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. The final solution was added to nasal spray medical device vials (10 mL, range 1 to 25 mL) at a typical dose of 0.05 mL containing 0.25 mg of organic acid-stabilized OM bacterial extract (range 0.025 to 0.1 mL per dose).

[0275] Therapeutic Dosage: With these formulations, once-daily and twice-daily doses ranging from 0.025 to 0.1 mL per administration, including 0.05 to 1 mg, can be achieved.

[0276] Example 2.2: Intranasal Formulation of Lactobacillus fermentum Stabilized Bacterial Extract 20 (OM314B) The high molecular weight fraction (>10 kD) of organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. The final solution was added to nasal spray medical device vials (10 mL, range 1 to 25 mL) at a typical dose of 0.05 mL containing 0.25 mg of organic acid-stabilized Lactobacillus fermentum purified bacterial extract (range 0.025 to 0.1 mL per dose).

[0277] Therapeutic Dosage: With these formulations, once-daily and twice-daily doses ranging from 0.025 to 0.1 mL per administration, including 0.05 to 1 mg, can be achieved.

[0278] Example 2.3: Nasal Formulation of E. coli Stabilized Bacterial Extract (OM314C) The high molecular weight fraction (>10 kD) of organic acid-stabilized E. coli bacterial extract was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. The final solution was added to nasal spray medical device vials (10 mL, range 1 to 25 mL) at a typical dose of 0.05 mL containing 0.25 mg of organic acid-stabilized E. coli purified bacterial extract (range 0.025 to 0.1 mL per dose).

[0279] Therapeutic Dosage: With these formulations, once-daily and twice-daily doses ranging from 0.025 to 0.1 mL per administration, including 0.05 to 1 mg, can be achieved.

[0280] Example 2.4: Inhalation formulation of OM314A bacterial stabilized extract as a droplet spray The high molecular weight fraction (>10 kD) of organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. The final solution was added to spray inhaler medical device vials (10 mL, range 1 to 25 mL) with a typical dose of 0.1 mL containing 0.5 mg of organic acid-stabilized OM bacterial extract (range 0.05 to 0.4 mL per dose).

[0281] Therapeutic Dosage: With these formulations, once-daily and twice-daily doses ranging from 0.05 to 0.4 mL per administration, including 0.05 to 8 mg, can be achieved.

[0282] Example 2.5: Inhalation formulation of Lactobacillus fermentum bacterial stabilized extract (OM314B) as a droplet spray The high molecular weight fraction (>10 kD) of organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. The final solution was added to spray inhaler medical device vials (10 mL, range 1 to 25 mL) with a typical dose of 0.1 mL containing 0.5 mg of organic acid-stabilized Lactobacillus fermentum purified bacterial extract (range 0.05 to 0.4 mL per dose).

[0283] Therapeutic Dosage: With these formulations, once-daily and twice-daily doses ranging from 0.05 to 0.4 mL per administration, including 0.05 to 8 mg, can be achieved.

[0284] Example 2.6: Inhalation Formulation of OM314A Stabilized Bacterial Extract as Solid Particles The high molecular weight fraction (>10 kD) of organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 with sterile saline (0.9% NaCl in water for injection) and one or more excipients from the list* at a final concentration of 10 mg dry weight / mL (range 1 to 20 mg / mL) and sterilized by 0.2 μm filtration. In one example, a 10 mg / mL solution of bacterial extract was mixed with mannitol (25 mg / mL), lactose (25 mg / mL), and Mg stearate (1 mg / mL). After spray drying, the powder was compressed into tablets (12 mg tablets). The crushable tablets were dispensed into medical devices (particle size range 1 to 7 μm) and the inhaled dose from a 12 mg tablet was 2 mg bacterial extract. Typical excipients for inhalation include, but are not limited to, lactose, glucose, mannitol, trehalose, Mg stearate, DPPC, DSPC, DMPC, cholesterol, leucine, trileucine, poloxamer, bile salts, chitosan, trimethylchitosan, PLGA (for a review see G. Pilcer, K. Amighi, International Journal of Pharmaceutics, 2010, 392, 1-19).

[0285] Example 2.7: Inhalation Formulation of Lactobacillus fermentum Stabilized Bacterial Extract (OM314B) as Solid Particles The high molecular weight fraction (>10 kD) of organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was prepared at a final concentration of 10 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline (0.9% NaCl in water for injection) and one or more excipients from the list*, adjusted to pH 7.5, and sterilized by 0.2 μm filtration. In one example, a 10 mg / mL solution of bacterial extract was mixed with mannitol (25 mg / mL), lactose (25 mg / mL), and magnesium stearate (1 mg / mL). After spray drying, the powder was compressed into tablets (12 mg tablets). The crushable tablets were dispensed into medical devices (particle size range 1 to 7 μm) and the inhaled dose from a 12 mg tablet was 2 mg bacterial extract. Typical excipients for inhalation include, but are not limited to, lactose, glucose, mannitol, trehalose, Mg stearate, DPPC, DSPC, DMPC, cholesterol, leucine, trileucine, poloxamer, bile salts, chitosan, trimethylchitosan, PLGA (for a review see G. Pilcer, K. Amighi, International Journal of Pharmaceutics, 2010, 392, 1-19).

[0286] Example 3: Evidence of greater stability of novel bacterial extract formulations The extracts prepared according to Examples 1.1, 1.2, 1.3, 1.4 and the formulations according to Examples 2.1, 2.2, 2.3, 2.4, 2.5 are adjusted to pH 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 with different organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid and combinations thereof. The stability of the bacterial extracts stored at 5°C ± 3°C and at room temperature of 20°C to 25°C is visually observed for the presence of precipitates at different time points from day 0 (between 15 and 120 minutes after pH adjustment) and at 30, 60, 90, 180, and 360 days.

[0287] Quantification is performed using visible spectrophotometry by measuring the absorbance at 550, 600, 650, and 700 nm, determined from day 0 (between 15 and 120 minutes after pH adjustment) and at different time points after 30, 60, 90, 180, and 360 days. Spectrophotometry is performed on water samples. Stability is expressed as a change in absorbance and depends on the process, the organic acid or combination of organic acids used, and the final pH value.

[0288] Example 4: Prophylactic and curative efficacy of intranasal versus oral OM administration in an animal model of sublethal bacterial infection following primary influenza infection A stable perioral form of OM bacterial extract (bacterial extract from 21 strain lysates) was extemporaneously prepared for the purpose of experimentally testing perioral administration of OM bacterial extract in animal models. While this extemporaneous perioral form is stable over time, the results provide insight and demonstrate the substantial therapeutic benefits of perioral administration. The effectiveness of intranasal administration of extemporaneous perioral OM bacterial extract in reducing viral titers in lung tissue after influenza virus infection and (2) reducing morbidity and mortality in superinfected animals (animals treated with sublethal influenza virus infection followed by sublethal bacterial infection) was compared with that of oral administration of OM bacterial extract (Figure 3). Female BALB / c mice (8 weeks old, Charles River Laboratories) were anesthetized with an intraperitoneal injection of ketamine and xylasol and inoculated intranasally with 100 PFU of influenza A / Puerto Rico 8 / 34 strain in a volume of 50 μl PBS. Bacterial starter cultures were initiated on day 6 post-infection and subsequently expanded to logarithmic phase growth on day 7 post-infection.

[0289] For oral administration, 320 μL of OM concentrate was administered by gavage to achieve 360 ​​mg / kg of the active ingredient of OM lyophilizate per animal per day, resulting in a daily dose of 7.2 mg of active ingredient per mouse (Figure 3). For intranasal administration of LPS (within the scope of the COPD model), the dose used was 7 micrograms of LPS per nose. In the literature, administration of 1 microgram of LPS per nose has been reported to protect against allergic inflammation. The test schedule is summarized in Table 23 below.

[0290] [Table 23]

[0291] Statistics were performed using GraphPad Prism version 5.0d. Student's t-test was performed for viral load. Two-tailed ANOVA was performed for body weight, body temperature, and clinical score. Comparison of the curves was performed for survival. Overall, OM bacterial extract treatment protected mice against morbidity and mortality in the superinfection model. This protective effect was most pronounced with intranasal treatment, suggesting that mucosal administration of OM bacterial extract could significantly improve its efficacy (Figures 4 and 5) and comorbidities following secondary bacterial infection (Figure 6).

[0292] Example 5: Prophylactic efficacy of intranasal and intratracheal OM bacterial extract administration in an animal model of sublethal bacterial infection following primary influenza infection. This study, outlined in Figure 7, demonstrated the effectiveness of intranasal and intratracheal administration of OM bacterial extract (bacterial extract from 21 strain lysates) as a prophylactic treatment regimen for (1) reducing viral titers in lung tissue after influenza virus infection (Figure 8) and (2) reducing morbidity and mortality in superinfected animals (animals treated with sublethal influenza virus infection followed by sublethal bacterial infection) as evidenced by clinical scores (Figures 10 and 12). Two different doses of OM bacterial extract were tested: Dose A (50 micrograms of active ingredient per dose) and Dose B (5 micrograms of active ingredient per dose).

[0293] Female BALB / c mice (8 weeks old, Charles River Laboratories) were anesthetized with an intraperitoneal injection of ketamine and xylazole and inoculated intranasally with 100 PFU of influenza A / Puerto Rico 8 / 34 strain in a volume of PBS. Mice were divided into six groups, with 15 mice per group. Group 1 received saline drops via the intranasal (in) route on days d7, d5, and d3 (prophylactic control). Group 2 received prophylactic dose A of OM bacterial extract via the intranasal route on days d7, d5, and d3. Group 3 received prophylactic dose B of OM bacterial extract via the intranasal (in) route on days -7, -5, and -3. Group 4 received saline spray intratracheally (it) on days -7, -5, and -3. Group 5 received prophylactic dose A of OM bacterial extract via the intratracheal route on days -7, -5, and -3. Group 6 received prophylactic dose B of OM bacterial extract via the intratracheal route on days -7, -5, and -3. OM bacterial extract administration using 50 micrograms active ingredient (Dose A resulting in 2.2 microliters of OM bacterial extract concentrate) and 5 micrograms active ingredient (Dose B resulting in 0.22 microliters of OM bacterial extract) per mouse per time point. Bacterial starter cultures were initiated on day 6 post-influenza infection and then grown to log phase growth on day 7 post-influenza infection. The study design and dosing schedule is shown in Figure 7 along with Table 24 below.

[0294] [Table 24]

[0295] Animals were anesthetized with an intraperitoneal injection of ketamine and xylazole and administered OM bacterial extract intranasally or intratracheally using either 50 micrograms of active ingredient (Dose A resulting in 2.2 microliters of OM bacterial extract concentrate) or 5 micrograms of active ingredient (Dose B resulting in 0.22 microliters of OM bacterial extract concentrate) diluted in saline in a total volume of 50 microliters. Animals were anesthetized with an intraperitoneal injection of ketamine and xylazole and administered OM bacterial extract intranasally or intratracheally using either 50 micrograms of active ingredient (Dose A resulting in 2.2 microliters of OM bacterial extract concentrate) or 5 micrograms of active ingredient (Dose B resulting in 0.22 microliters of OM bacterial extract concentrate) diluted in saline in a total volume of 50 microliters.

[0296] Statistics were performed using GraphPad Prism version 5.0d. Student's t-test was performed for viral load. Two-tailed ANOVA was performed for body weight, body temperature, and clinical score. Comparison of the curves was performed for survival (Figures 8 and 10).

[0297] Prophylactic treatment of animals via intranasal administration of OM bacterial extract (5 micrograms OM dose B IN and 50 micrograms OM dose A IN) resulted in a reduction in virus titers in lung tissue measured 5 days after PR8 infection (Figure 8). This reduction was more prevalent with the 50 microgram dose of OM bacterial extract administered via the intranasal route, whereas 5 micrograms was sufficient to clear virus particles via the intratracheal route. Similar to the viral titer results, prophylactic intranasal OM bacterial extract treatment resulted in a significant reduction in morbidity and mortality following influenza bacterial infection. A 50 microgram dose of OM bacterial extract per nasal treatment resulted in 90% survival (OM dose A IN), and a 5 microgram dose of OM bacterial extract per nasal treatment resulted in 40% survival (OM dose B IN), whereas the intranasal saline control (saline IN) did not protect animals that died on day 6 post-infection (Figure 9). Consistent with the survival results, clinical scores and weight loss measurements were significantly reduced in animals treated with the 50 microgram dose of OM bacterial extract compared to saline-treated animals (Figure 9). Consistent with the 50 microgram dose of OM bacterial extract-treated animals, the 5 microgram dose-treated animals also had reduced clinical scores and weight loss compared to saline control animals. However, efficacy was lower for the 5 microgram intranasal dose.

[0298] Prophylactic intratracheal treatment of animals with 5 microgram doses of OM bacterial extract resulted in the best reduction in viral titers in lung tissue measured 5 days after PR8 infection compared to viral titers found in control saline intratracheally treated animals (Figure 8). With regard to morbidity and mortality, both the intratracheal 50 microgram dose (OM Dose A IT) and the 5 microgram dose (OM Dose B IT) of OM bacterial extract treatment increased survival (70%) compared to saline control treated animals (30%), with a dose-proportional effect through day 10 (Figure 11). This is summarized with comparable reductions in clinical score measurements for both the intratracheal 50 microgram and 5 microgram doses of OM bacterial extract treated animals compared to the saline control group (Figure 12). In summary, prophylactic intranasal OM bacterial extract administration resulted in a significant reduction in morbidity and mortality in co-infected animals and in reduced viral titers in lung tissue following influenza infection. This result was particularly evident after a 50 microgram dose of OM bacterial extract administered via the intranasal route. Surprisingly, prophylactic intratracheal OM bacterial extract treatment resulted in the best reduction in morbidity and mortality, with greater efficacy using the 5 microgram dose, which can be explained by deeper superficial lung exposure. These results clearly demonstrated that both intranasal and intratracheal administration are highly effective routes of administration for OM bacterial extract therapeutic treatment of respiratory diseases such as asthma, COPD and other pathogens.

[0299] Example 6: Novel treatment regimen for intranasal administration of bacterial extracts according to the present invention in an animal model of sublethal influenza infection This study (Figure 13) demonstrated the efficacy of intranasal administration of extemporaneously prepared OM bacterial extract (bacterial extract from lysates of 21 strains) in reducing viral titers in lung tissue after influenza virus infection. This study also demonstrated a dose-response relationship for OM bacterial extract. This study further provided a comparison of two different multiple-dose regimens (six doses and three doses) and a comparison between intranasal treatment regimens and oral administration. Female 7-week-old BALB / c mice (specific pathogen-free; SPF) were purchased from Charles River Laboratories and randomly assigned to cages with a total of five mice per cage. Mice were monitored weekly and allowed to acclimate to the facility for seven days prior to the start of the study (Study Day 0). Animals were 8 weeks old on Study Day 0. Drinking water and food were available ad libitum. Mice were divided into 13 groups: Groups 1 to 11 received the active ingredient, OM bacterial extract (OM), intranasally. Group 12 was the water control (320 μL water control, orally, daily from day -10 to -1) and Group 13 was the negative control (sublethal influenza virus infection only). Tables 25 and 26 show the different groups and treatment protocols as shown schematically in Figure 13.

[0300] [Table 25]

[0301] [Table 26]

[0302] On the day specified in the study protocol above, mice were anesthetized using a calibrated vaporizer system (VIP300, Provet, Vet.Med Center, Lyssach, CH) that delivered the anesthetic isoflurane (Provet AG, Cat. No. 2222) to a plexiglass chamber containing the mice. Anesthetized animals were then administered a total volume of 50 microliters of OM bacterial extract test article, which was dispensed into both nostrils using a 100 ul micropipette. Viral material (influenza virus PR8 strain (A / Puerto Rico / 8 / 34, H1N1) obtained from Virapur, San Diego) was stored at -75°C ± 10°C and thawed prior to administration. Once thawed, the material was diluted with cold PBS (4°C) equivalent to 100 PFU / 50 μl for A / PR / 8 / 34. The diluted virus was kept on ice until administered to mice. Animals were anesthetized with an intraperitoneal injection of 9.75 mg xylazole and 48.75 mg ketasol per kg body weight, and each animal received 50 μl of virus solution via intranasal inoculation. On day 5, animals were sacrificed by lethal intraperitoneal injection of pentobarbital (Streuli Pharma AG, Uznach, Cat: 1170139A), immediately followed by tissue isolation (lungs). Isolated lung lobes were prepared for quantification of viral load in lung tissue by quantitative PCR. Lung lobes were isolated, and RNA was prepared using TRI Reagent (Molecular Research Center) and then treated with DNase (Invitrogen) to avoid genomic DNA contamination. RNA was then converted to cDNA by reverse transcription using SuperScript III (Invitrogen). cDNA was quantified by real-time PCR (iCycler; Bio-Rad) using SYBR Green (Stratagene), and samples were normalized using GAPDH expression levels. All graphs were generated using Graphpad Prism version 6, and one-way ANOVA was applied. Error bars represent the standard error of the mean (SEM).

[0303] The results of this study clearly demonstrated that intranasal administration of OM bacterial extract effectively protected mice against infection with influenza virus at all doses used in the study, with the intranasal route being more effective than the oral route (Figure 14). Compared to untreated controls, there was a clear improvement in virus management in both the water and OM bacterial extract oral groups. Furthermore, this positive effect was significantly dose-dependent, from 5 micrograms to 100 micrograms, the most recent and highest dose evaluated in this study. The group receiving six intranasal doses of OM bacterial extract showed the best efficacy with the least variation, but mice receiving only three doses of OM bacterial extract were also significantly protected against the virus. These data clearly confirmed previous studies after intranasal administration, demonstrating that OM bacterial extract provides the most effective route of administration. Because a clear dose response was demonstrated using three and six treatments of OM bacterial extract, it can be concluded that this dose and regimen are important in this highly effective prophylactic treatment against influenza in this mouse model. Thus, longer total treatment duration, more frequent perioral, eg intranasal, administration and higher doses can be expected to be more effective.

[0304] Example 7: Effect of bacterial extracts according to the invention on the expression of rhinovirus docking proteins and type 1 and type 2 interferon responses in primary human epithelia from healthy donors

[0305] Previous data have been published on the antiviral activity of extemporaneously prepared perioral OM bacterial extracts (bacterial extracts from lysates of 21 strains) in human lung epithelial cells from healthy donors and COPD and asthma patients (Roth M et al., PLoS ONE 2017, 12(11), e0188010). To follow up on the antiviral efficacy demonstrated in animals by OM bacterial extract via the intratracheal route (direct lung exposure, Examples 4, 5, and 6), Applicant evaluated this direct lung exposure using primary bronchial epithelial cells (hBECs) from human pulmonary origin. To mimic the results obtained in mice via the intratracheal route, human bronchopulmonary epithelial cells were directly exposed to new stable OM bacterial extract formulations from 21 strain lysates (OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5)) to evaluate the antiviral-primed efficacy response from the lung cells. Given the direct antiviral effect on primary epithelial lung cells, these studies confirmed that the lung is the primary target organ for OM bacterial extract, as previously suggested by intranasal and intratracheal administration in animals (Examples 4, 5, and 6).

[0306] In this study, the applicant demonstrated on a molecular basis the consequences of cell exposure to OM bacterial extracts and their protective effect against rhinovirus infection. For this purpose, experiments on protein, mRNA expression, and immunofluorescence were performed. Detection was performed using ELISA and immunofluorescence techniques (direct cell counting with trypan blue exclusion staining), as well as RT-PCR on human lung epithelial cell cultures originating from the lungs of several healthy donors, COPD patients, and asthma patients. BEC isolation and characterization: Small pieces of bronchial tissue (1 x 1 x 1 mm up to 2 x 2 x 2 mm) were placed in cell culture vessels pre-wetted with BEC-specific medium Cnt-PR-A (CellnTech, Bern, Switzerland). Medium was changed every two days, and cells were passaged by mechanical shaking of dividing cells. Cells were characterized by positive staining for E-cadherin and pankeratin and negative staining for fibronectin (Roth M et al., PLoS One. 2017;12:e0188010).

[0307] In this study, Applicant demonstrated the superior results of a selected set of novel stable OM bacterial extract formulations and their preventative antiviral effects against rhinovirus infection on a molecular basis. Experimental readouts were quantitative viral load changes and anti-infective and anti-inflammatory mediators produced by hBECs, including, but not limited to, soluble mediators such as type 1 and type 2 interferons. These biological effects were measured by the following methods: mRNA detection by RT-PCR for viral load and soluble mediator detection by ELISA for type 1 interferons (IFN-beta and type II interferon-gamma). RV16 mRNA determination was performed for the different formulations listed in Table 27, with the antiviral effects expressed as percentage mRNA for RV16 and RV16 mRNA expression rates.

[0308] Example 7.1: Antiviral Results Results obtained with the various new stable OM bacterial extract formulations tested (OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5)) show similar or improved antiviral efficacy compared to the standard OM bacterial extract HCl formulation (Figure 15A). In this series, antiviral efficacy was tested with new formulations incorporating several acids into the manufacturing and purification process (Figure 15C). Compared to the unstable liquid formulation labeled "HCl," the new stable formulations demonstrated similar or better antiviral efficacy. With one exception, efficacy was similar or better than that of the HCl formulation, depending on the OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) bacterial extract used (compare the percentage of RV16 mRNA for HCl in Figure 15A and IB in Figure 15C with other sample preparations).

[0309] [Table 27]

[0310] Example 7.2: Type 1 and Type 2 Interferons The induction of type 1 interferon-beta production by first-generation OM bacterial extracts has previously been described in a mouse experimental cell model for primary bone marrow-derived dendritic cells (DCs) (Dang et al., Sci Rep. 2017 Mar 6;7:43844). Briefly, human BEC cells collected from healthy donors, asthma, and COPD patients were seeded on day -2, serum-starved on day -1, and stimulated with OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) (at a concentration of 10 micrograms / mL) at 0, 24, and 48 hours as described in the diagram (Figure 16). Cell supernatants were then collected at the indicated times for the amount of interferon-beta and gamma expressed using ELISA. Compared to the original IFN beta and gamma secretion by BECs using the previous OM bacterial extract (Figures 17A and 18A), the new OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) stabilized products were able to induce type 1 interferon beta and type 2 interferon gamma to a similar or better extent in Figures 17C and 18C (compare IB with other products). Depending on the process used, some significant differences were observed. In this study, dose-range induced interferon release of all OM314 stabilized bacterial extracts was based on five donors. The mean interferon values ​​for the five donors are shown in Figures 17B and 18B. Notably, and in contrast to Figures 17A and 18A, where IFN-dependent release was obtained from OM bacterial extract concentrates, volumes up to 20 microliters were used in Figures 17C and 18C, which corresponds to the lower amount of standard OM bacterial extract. Interferon alpha was not induced in human BEC cells in vitro by either the OM bacterial extracts or the new stable OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) (data not shown).

[0311] Example 8: Protective antiviral effect of bacterial extracts according to the invention on the alteration of beta β-defensin-1 and ICAM-1 expression on primary human epithelial cells (BEC) originating from human lung biopsies. Example 8.1: β-Defensin-1 and ICAM-1 For Example 8, and to further evaluate the new stable OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) preparations after systematic comparison with previous data obtained using the original bacterial extract OM, we aimed to confirm previous published data on human BECs but using other antiviral features previously induced by OM bacterial extracts (Roth et al., 2017). This is demonstrated in Figure 19, which shows the ability of the new stable OM314A (P1, P2, P3), OM314B (P4), and OM-314C (P5) preparations to induce antiviral beta-defensin-1 expression by human lung-derived primary epithelial cells (BECs) to a degree comparable to or superior to that of the industrial batch (IB#1619057). Similarly, this antiviral efficacy is also demonstrated using the reduction of rhinovirus ICAM-1 on the surface of these cells. Figure 20B shows the ability of the new stable OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5) preparations to reduce ICAM-1 viral receptor expression by human BECs to the same extent as the industrial batch (IB#1619057), thus confirming in both cases the ability of this newly prepared stable bacterial extract OM314 to maintain the potency and efficacy of the previous OM bacterial standard, with similar or superior results depending on the process and bacterial extract content.

[0312] Example 9: Ability of bacterial extracts according to the invention to activate the adaptor and TLR-dependent effector protein MyD88, demonstrated and monitored by the release of TNFα from murine bone marrow-derived dendritic cells. Example 9.1: BMDC preparation To further expand our investigation into the ability of the newly prepared stable bacterial extracts OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5) to maintain the same or superior levels of efficacy against innate immune activation, we performed the same studies that originally demonstrated the anti-inflammatory and regulatory effects of OM bacterial standards, as extensively illustrated in Dang et al. (Sci Rep. 2017 Mar 6;7:43844). For this purpose, we performed a single, yet intensive, type of study because it employs all necessary cellular components during activation: surface receptors (TLRs), an effector protein (MyD88) essential for OM bacterial extracts to induce protection, and a transcription factor (NFkB) that induces cytokine release upon cleavage and translocation from the cytoplasm to the nucleus, where transcription and cytokine release in the culture medium (TNFα) are measured. For this purpose, we used primary bone marrow-derived dendritic cells derived from mouse bone marrow. Bone marrow (BM) cells were extracted from the femurs and tibias of 6- to 10-week-old wild-type C57 / BL6 or various TLR knockout mice (here, only TLR4 knockout (TLR4- / -) and WT mice are shown) by flushing the bones with ice-cold PBS. Subsequent cell processing of BMDMs and their maturation and differentiation into dendritic cells (BMDCs) was performed according to Dang et al. (2017). The purity of the cultures was determined by staining the cells with anti-mouse CD11c and anti-mouse MHCII antibodies, and the percentage of CD11c+MHCIIhigh cells was analyzed by flow cytometry.

[0313] Example 9.2: BMDC stimulation and measurement of cytokine release BMDCs were cultured at 2 x 10 5Cells were seeded at a density of 1000 cells / well and stimulated for 16 h with LPS (4 μg / mL) or different concentrations of freshly prepared stable bacterial extracts OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5) from Enzo Life Science. Different concentrations (50–1600 μg / mL) of bacterial extracts from an industrial batch (IB#1619056OM) were used (top left corner), as shown in Figure 21. The concentration of TNF-α cytokine was measured in cell-free supernatants by ELISA using the TNF-α kit from eBioscience according to the manufacturer's instructions. As illustrated in Figure 21, all but one of the newly prepared stable bacterial extracts, OM314A (P1, P2, P3), OM314B (P4), and OM314C (P5), were able to induce TNF-α secretion from BMDCs to the same extent as controls (IB) from wild-type normal mice. Interestingly, this secretion was comparable or even better (concentration-dependent) in some cases, demonstrating the importance of the selective response of these different bacterial extract sources. Concomitantly, the new stable bacterial extracts, OM314A P3, P4, and P5, did not require the TLR4 receptor to induce TNF-α release from BMDCs, because this cytokine was secreted even in the absence of TLR4 (TLR4- / -). LDA revealed that different organisms dominated distinct populations, and that i) BE (bacterial extracts from 21 strain lysates) protected against harmful bacterial populations in HFD mice and ii) increased population diversity.

[0314] Example 10: Ability of perioral administration of bacterial extracts according to the invention to reverse gut dysbiosis in animals maintained on a high-fat diet. Example 10.1: Importance of bacterial flora balance, adverse consequences of dysbiosis, and taxonomic microbiome analysis. Specific microbiota patterns fluctuate and depend on many external factors, such as diet, age, genetics, and drug therapy (Dieterich et al., Med Sci (Basel). 2018;6(4):116. Published December 14, 2018, doi:10.3390 / medsci6040116). While research is still in its infancy in demonstrating how the microbiome can contribute to homeostasis, elucidating the precise mechanisms that lead to several medical conditions in which microbiome dysbiosis is present is crucial, and products to restore such imbalances are urgently needed. Therefore, restoring unfavorable populations of gut microbiota to a favorable microbial ecosystem may prevent human disease (Young VB et al., BMJ 2017;356: j831. In a study aimed at assessing the lipotoxic effects of consuming a high-fat diet (HFD) during pregnancy, we recently showed that 8 weeks of HFD consumption leads to increased gut dysbiosis, oxidative stress, inflammation, and an increased risk of inflammation-driven preterm birth (PTB). Therefore, in this example, we sought to determine the ability of the bacterial extract of the present invention to reverse HFD-induced gut dysbiosis and the associated deleterious effects on metabolism and immune status. Results from taxonomy allowed us to determine linear discriminant analysis (LDA) scores, as shown in Figure 22. This figure illustrates the specific effects of bacterial lysates on specific microorganisms. HFD control mice ( Undesirable Clostridiales, Firmicutes, Clostridiaceae, and Blautia species present in the HFD-Sham group were restored in mice fed the bacterial extract (HFD-BE) and, to a lesser extent, in mice fed the normal chow control diet (NCD-Sham). Furthermore, increased levels of desirable organisms depleted by HFD consumption were also identified in the microbiota contents of mice fed the bacterial lysate (HFD-BE and NCD-BE). Unlike standard taxonomic analysis, which shows the increase and decrease of a selected set of species, the LDA scores shown in Figure 22 indicate unique species in each of the four groups (NCD-Sham, NCD-BE, HFD-Sham, HFD-BE).LDA revealed that different organisms dominated different populations and that i) BE (bacterial extract from 21 strain lysates) protected against harmful bacterial populations in HFD mice and ii) increased population diversity, thus confirming the positive effect of the bacterial lysates from the present invention.

[0315] Example 10.2: Methods, Animals, Food Consumption, Glucose Tolerance, Insulin Resistance and Diet Two different mouse strains were used throughout this study: C57BL / 6 and CD1. C57BL / 6 mice are an inbred strain with the advantage of being known to become obese, hyperglycemic, and insulin-resistant when fed a high-fat diet (HFD). On the other hand, CD1 mice have mild metabolic dysfunction after consuming an HFD, but have the advantage of being outbred, thereby avoiding any idiosyncratic reactions to the HFD. Mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Equal numbers of male and female mice of each strain were housed in individually ventilated cages at 24°C in the Animal Care Center under a 12-hour light / dark cycle and provided with food and water ad libitum. HFD mice were fed a diet containing 60% lipids, a high saturated lipid content, and lipids derived primarily from lard and soybean oil, while NCD mice were fed an NCD containing 13.3% lipids. Food and water were provided ad libitum. OM bacterial extract was administered by perioral route, intranasal route (0.05 mL) by pipette for 14 days, or by direct oral route (≦0.15 mL) into the mouse's mouth daily for 4 and 8 weeks. Mice receiving "sham treatment" (negative control) received 0.05 mL of water (intranasal) once daily for 14 days by pipette or by direct oral route (≦0.15 mL) into the mouse's mouth daily for 4 and 8 weeks. Positive control: Lactobacillus plantarum, a probiotic that reverses gut dysbiosis, was administered to positive control mice by adding 2×108 CFU / mL to their drinking water for 6 days. Analysis of the effect of OM bacterial extract on the gut microbiome: Fecal samples were collected and analyzed by 16S rRNA sequencing. After sequencing, functional genes in the samples were characterized, and differences between functional genes in the microbial community were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The protein function classification of these genes was predicted using Cluster of Orthologous Group (COG) family information. Analysis of the effects of OM bacterial extracts on metabolic status: Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed on all mice at the beginning and end of the 8-week period.Mice are fasted for 8 hours and then challenged with an intraperitoneal injection of 2.0 g / kg glucose with or without insulin, and glucose levels are measured at 0, 15, 30 and 60 minutes.

[0316] Example 10.3: Effect of bacterial extract (BE) from 21 bacterial lysates on various genera To add to and further expand on the data summarized in Figure 22, some examples of changes in genera induced by BE are shown in Figure 26. In this example, BV showed a positive effect (reduced growth) on harmful genera while simultaneously favoring growth of authentic genera. Five examples are shown in this figure: (A) BE reduces the genus Blautia of the order Clostridiales, Lachnospiraceae, and Blautia caused by HFD. (B) BE reduces the genus GCA-900066225 of the class Clostridiales, Ruminococcaceae, and GCA-900066225 caused by HFD. (C) BE reduces the genus UCG-0101 of the order Clostridiales, Ruminococcaceae, and UCG-0101 in both NCD and HFD mice. (D) BE restores uncultured bacteria of the order Bacteroidales, Muribacraceae, and Bacteroidales, depleted by HDF. (E) BE reduces the Physicanthenae group of the family Lachnospiraceae [Eubacterium].

[0317] Example 11: Ability of Perioral Administration of Bacterial Extract to Improve Glucose Tolerance Example 11.1: Examination of Bacterial Extracts from 21 Strain Lysates for Glucose Concentrations in Pre-Feed (Pre-Feed), Normal Chow-Fed Mice (Post-NCD), and High-Fat Diet (Post-HFD) In addition to correcting gut dysbiosis with bacterial extracts, glucose tolerance was also examined in these diabetic mice. This parameter is clinically important. Figure 23B shows that normal chow-fed control mice (NCD-BE) fed bacterial extracts from lysates of 21 strains other than L. plantarum (usually used as a positive control to improve gut dysbiosis) showed significantly increased glucose tolerance, as indicated by lower glucose concentrations. Similarly, this effect was also observed, to a lesser extent, in high-fat-fed mice (HFD-BE) fed bacterial extracts from lysates of 21 strains, as shown in Figure 23C.

[0318] Example 11.2: Assessment of body weight and food consumption In Figure 24, both parameters were tested as controls. The results show that high-fat-fed mice (HFD-BE) fed bacterial extracts from strain 21 lysates exhibited significantly reduced weight gain. This is exemplified in these high-fat-fed mice (Figure 24A). Concomitantly, this absence of weight gain occurred without affecting food consumption, which was measured and represented here as a control (Figure 24B). Interestingly, this effect was more effective than L. plantarum, which is commonly used in such measures.

[0319] Example 11.3: Assessment of insulin tolerance Given the protective glucose tolerance results obtained from treatment with bacterial extract (BE) from strain 21 lysate in diabetic mice exposed to a high-fat diet (HDF) regimen, we further investigated whether insulin levels were altered for this protective purpose. Figure 25 demonstrates insulin tolerance in all mice (42) before treatment (pre-diet), which served as a reference, and after the final 8-week treatment period (post-diet) in high-fat diet (HFD) mice. As expected for normal chow mice fed NCD-L-plantarum (NCD-L-plant.), which exhibit reduced insulin resistance (Figure 25B), mice fed HFD-BE bacterial extract from strain 21 lysate exhibited insulin resistance at all time points, significantly more so than the high-fat mice. This protective effect of bacterial extract from strain 21 lysate is consistent with the glucose data and the absence of weight gain from previous figures, all of which demonstrate a positive reorganization of standard parameters measured in diabetic patients. Thus, it was confirmed that bacterial extract (BE) from 21 strain lysates not only induced protective gut dysbiosis in chronic HFD mice undergoing such a regimen, but also its associated sequelae exemplified by body weight, glucose, and insulin, which all positively influenced towards protection by bacterial lysates from 21 strains.

Claims

1. 1. A pharmaceutical composition comprising a bacterial extract from one or more Gram-positive or Gram-negative bacterial species and a pharmaceutically acceptable excipient or vehicle, The bacterial extract, during its preparation, is one in which one or more Gram-positive or Gram-negative bacterial species are subjected to alkaline lysis at a pH greater than 10, the pH is adjusted to 5-8 with one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof and / or pharmaceutically acceptable salts and esters thereof, and purified by filtration through a 1.2 micron to 0.1 micron filter membrane followed by filtration through a 10 kD to 100 kDa cut-off filter membrane, and the final physiological pH is adjusted to 5-8 by the addition of one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof and / or pharmaceutically acceptable salts and esters thereof. Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the purification comprises filtration using a 0.65 to 0.2 micron filtration membrane, or a 0.45 micron filtration membrane.

3. 3. The pharmaceutical composition of claim 1, wherein the alkaline dissolution is carried out at a pH greater than 10.

0.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the final pH is adjusted to 6 to 8, 6.3 to 7.8, or 6.5 to 7.

8.

5. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the one or more bacterial species are selected from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the one or more bacterial species are selected from Lactobacillus bacterial strains.

7. 7. The pharmaceutical composition of claim 6, wherein the Lactobacillus bacterial strain comprises one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and / or Lactobacillus delbrueckii.

8. The pharmaceutical composition according to any one of claims 1 to 4, wherein the one or more bacterial species are selected from Escherichia coli bacterial strains.

9. 9. The pharmaceutical composition of claim 1, wherein the bacterial extract contains less than 100 micrograms / ml of nucleic acids and at least 0.1 mg / mL of sugars.

10. The pharmaceutical composition according to any one of claims 1 to 9, which is stable in liquid form at room temperature, 4°C, -20°C or -80°C.

11. The pharmaceutical composition according to any one of claims 1 to 10, in the form of a solid, semi-solid, liquid or aerosol formulation.

12. 12. The pharmaceutical composition according to any one of claims 1 to 11, formulated for intranasal, intratracheal, mucosal, transmucosal, topical, oral mucosal, sublingual, oral, pulmonary, intrabronchial and / or intrapulmonary administration routes.

13. 13. The pharmaceutical composition according to any one of claims 1 to 12, which is a liquid or an aerosol and is formulated into a spray, droplets, colloid, mist, cloud and / or atomized smoke.

14. 13. The pharmaceutical composition according to any one of claims 1 to 12, which is liquid or semi-solid and formulated in the form of an emulsion, microemulsion, aqueous dispersion, oil, milk, balsam, foam, aqueous or oily lotion, aqueous or oily gel, cream, solution, hydroalcoholic solution, hydroglycolic solution, hydrogel, serum, ointment, mousse, paste or transdermal patch.

15. The pharmaceutical composition according to any one of claims 1 to 12, which is solid and formulated into a powder and / or a crushable tablet.

16. 6. A pharmaceutical composition comprising the bacterial extract of claim 5 for use in a method for treating and / or preventing acute and chronic immune disorders caused by infection and / or inflammation and / or neoplasia and / or dysbiosis in a subject.

17. 17. The pharmaceutical composition for use in the method of claim 16, wherein the immune disorder is selected from an imbalance between T helper 1, T helper 17 and T helper 2 immune responses, a Treg imbalance, type 2 hypersensitivity, immunosuppression, eosinophilia, allergy and atopy.

18. 17. A pharmaceutical composition for use in the method of claim 16, wherein the infection is an upper or lower respiratory tract infection and / or associated sequelae.

19. The upper or lower respiratory tract infection and / or the associated sequelae are allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngolaryngitis, influenza, respiratory syncytial virus, rhinovirus, coronavirus, croup, pneumonia, hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory tract infection, obstructive pulmonary disease with acute upper respiratory tract infection, or obstructive pulmonary disease with impaired epithelial ciliary motility and / or impaired mucus clearance. The pharmaceutical composition according to claim 18, wherein the treatment is selected from caries diseases.

20. 17. The pharmaceutical composition for use in the method of claim 16, wherein the infection comprises a secondary infection, a secondary bacterial infection after a viral infection such as influenza, a non-respiratory viral infection, a non-respiratory bacterial infection, or a systemic infection.

21. 21. The pharmaceutical composition of claim 20, wherein the systemic infection is sepsis, septic shock or a virus-induced complication.

22. 17. A pharmaceutical composition for use in the method of claim 16, wherein the inflammation is selected from allergic / atopic respiratory and non-respiratory manifestations atopic dermatitis, acute and / or chronic associated dermatitis, anaphylaxis or food allergy.

23. 17. A pharmaceutical composition for use in the method of claim 16, wherein the dysbiosis-associated disorder is selected from obesity, asthma, diabetes, autoimmune diseases, diseases associated with low fiber diets, atopic dermatitis, acute and / or chronic associated dermatitis, psoriasis, metabolic diseases and / or liver fibrosis.

24. The pharmaceutical composition according to claim 23, wherein the metabolic disease is NASH or NAFLD.

25. 17. The pharmaceutical composition for use in the method of claim 16, wherein the inflammation is a skin inflammatory disorder selected from the group consisting of eczema, rosacea, atopic dermatitis, psoriasis, photodamage including sun-induced skin inflammation and redness, atrophy of the skin, depigmentation of the skin, ultraviolet dermatitis, erythema, telangiectasia, couperose, or actinic keratosis.

26. 17. The pharmaceutical composition for use in the method of claim 16, wherein the inflammation is a T helper 2-dominant autoimmune syndrome.

27. 27. The pharmaceutical composition of claim 26, wherein the T helper 2 dominant autoimmune indication is selected from Graves' disease, Hashimoto's disease, scleroderma, Ig4-related disease, or pemphigus.

28. 17. The pharmaceutical composition for use in the method of claim 16, wherein the inflammation is an eosinophilic sign.

29. 29. The pharmaceutical composition of claim 28, wherein the eosinophilic indication is selected from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma, or eosinophilic pneumonia.

30. 17. A pharmaceutical composition for use in the method of claim 16, wherein the neoplasm is selected from neoplastic indications associated with immune disorders selected from mastocytosis, mast cell leukemia, T helper 2 biased and / or immunosuppressive tumors.

31. 17. The pharmaceutical composition for use in the method of claim 16, wherein the disorder associated with dysbiosis is selected from inflammatory bowel diseases including ulcerative colitis, Crohn's disease, colitis, metabolic disorders, obesity, type 2 diabetes, liver failure including NASH and / or NAFLD, liver fibrosis, renal failure, autoimmune diseases or diseases associated with a low fiber diet.

32. 32. The pharmaceutical composition for use in the method of any one of claims 16 to 31, wherein the pharmaceutical composition is administered to a subject by intratracheal, intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, oral, pulmonary, intrabronchial and / or intrapulmonary route.

33. A pharmaceutical composition for use in the method according to any one of claims 16 to 32, wherein the subject is a human or non-human animal subject.

34. 16. A delivery device comprising the pharmaceutical composition of any one of claims 1 to 15, wherein the delivery device is selected from the group comprising a nasal insufflation device, a nasal inhaler, a nasal 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 blowtorch, a two-way device, a dose ampoule, a nasal pad, a nasal sponge and a nasal capsule.

35. 35. A delivery device according to claim 34 for use in a method for treating and / or preventing upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and dysbiosis-related disorders in a human subject.

36. A method for producing the pharmaceutical composition according to any one of claims 1 to 15, comprising the steps of: a. culturing each bacterial strain species in an appropriate culture medium; b. Lysing each strain at an initial pH greater than 10; c) adjusting the pH of the extract obtained in step (b) to 5-8 by adding one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof, or pharmaceutically acceptable salts and esters thereof; d. passing the product of step (c) through a 1.2 micron to 0.1 micron microfiltration membrane at least once and retaining the product on an ultrafiltration membrane with a cutoff of 10 kD to 100 kDa to obtain a purified soluble extract; e. adjusting the final pH to 6-8 by adding one or more organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, combinations thereof, or pharmaceutically acceptable salts and esters thereof; f) adding a pharmaceutically acceptable excipient or vehicle.

37. The pharmaceutical composition according to any one of claims 1 to 15, wherein the cutoff value is 10 kDa to 30 kDa.

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