STABLE BACTERIAL EXTRACTS AS PHARMACEUTICALS

Abstract

The present invention relates to novel stable bacterial extract preparations that have substantially increased stability over time, novel methods of preparing the same, pharmaceutical formulations based on the novel stabilized bacterial extracts, as well as novel routes of administration and delivery devices for treating and / or preventing acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis.

Description

STABLE BACTERIAL EXTRACTS AS DRUGS FIELD OF THE INVENTION

[001] The present invention relates to novel stable bacterial extract preparations that have substantially increased stability over time, novel methods of preparation thereof, pharmaceutical formulations based on the novel stabilized bacterial extracts, as well as novel administration routes and devices. release to treat and / or prevent acute and chronic immunological disorders resulting from infections and / or inflammation. BACKGROUND OF THE INVENTION

[002] There is increasing interest in the development of pharmaceutical formulations, either liquid or solid, stable over time, that can be administered to human subjects to treat infections and inflammations which can be easily adapted to broader routes of administration, such as oral, intranasal, intratracheal, intrapulmonary, transmucosal, topical, buccal, etc., as well as for conditions and stages of disease (acute stage, exacerbations, etc.) of the patients.

[003] Bacterial infections are frequently implicated in many respiratory conditions and antibiotic treatments are common. The effectiveness of antibiotics in treating such conditions and disease exacerbations has been debated. Its excessive use has been associated with increased costs and the potential for increased resistance of microbes to antibiotics.

[004] Bacterial extract lysate preparations, which contain antigens derived from various strains of bacteria, have been shown to increase resistance to infection by these organisms. The way in which these bacterial extract extracts can exert their effect could be multifactorial and is not yet completely known. Numerous bacterial extracts have been used as immune stimulants and antitumor agents. As examples we can mention Bacillus Calmette-Guerín (BCG), Polysaccharide, beta 1,3, glucan, the Maruyama vaccine and extracts of Bifídobacterium, L lactis, L fermentum, L acidophilus and S. lactis. These extracts are thought to stimulate the immune system in numerous ways. One important way is to stimulate lymphocytes to grow and produce cytokines. The ability to induce the production of these cytokines has a very powerful effect on the immune system.

[005] In particular, the applicant has already successfully developed several bacterial extract preparations for the treatment and / or prevention of upper respiratory disorders. In this regard, we can mention the drug Broncho-Vaxom® which is a bacterial extract of several pathogens frequently responsible for respiratory tract infections, as described inter alia in US Patent No. 9,463,209B2. Broncho-Vaxom® is an immunostimulant administered as a capsule, sachet or drops via the oral enteral route for the prevention and prophylaxis of respiratory tract infections, recurrent respiratory tract infections, such as acute bronchitis, chronic bronchitis, asthma, lung disease chronic obstructive and emphysema. It has been shown to increase the production of TNF-α and interferon-γ from cultured human peripheral blood mononuclear cells, to lead to the activation of alveolar macrophages and to stimulate bacterial clearance by Rn ι η / ι 7n7 / E / YL polymorphonuclear leukocytes. The applicant also developed extracts of Lactobacillus bacteria and showed that these bacterial lysate extracts were efficient when administered as a capsule via the oral-enteral route to treat infections, allergies, autoimmune disorders and inflammations (See international publication WO2010 / 027344).

[006] Constant exposure of mucosal surfaces such as the airways and lungs to viruses, bacteria, and inhaled toxins present a challenge to the immune system. The situation worsens when the host is exposed to a viral infection and is subsequently superinfected by microbes leading to an increased mortality rate. Consequently, secondary bacterial infections after viral infections with influenza (H1N1 and similar), human rhinovirus (HRV), syncytial rhinovirus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID19 and similar) are a problem. pressing challenge facing respiratory medicine.

[007] Experimentally, numerous in vivo studies have shown that BronchoVaxom® bacterial extract administered by the enteral oral route provides protection in models of respiratory tract infection. Most of the studies used the solid dosage form (lyophilized) as well as liquid bacterial extract, however, with limited stability. An example of that in vivo mouse model using the human dose (7 mg dry weight bacterial extract, 40 mg lyophilized) showed that it was able to increase protection against secondary bacterial and / or viral infections after influenza infection. (Pasquali et al, Frontiers in Medicine 2014,1,41).

[008] Additionally, functional dysbiosis that originates from “junk” rich in fat, sugars and protein that leads to dysregulation of the microbiota (Clarence M. et al., Abstract, March 30 9, 2018 - St-John University , Queens) was normalized by implementing the Broncho-Vaxom® in accompanied mice with decreased sequences and associated comorbidities.

[009] For several years, Broncho-Vaxom® has been administered in this way via enteral routes in solid form (capsules or sachets) to stimulate immune defenses and for the prevention of common respiratory tract disorders and associated exacerbations. The protective effects of these bacterial lysate extracts, which have been created and designed for oral administration, initiate an immune response in the gut (gut-associated lymphoid tissue, GALT). These organs detect and send armed immune cells to the lung to prevent and cure lung infections from the airways. Consequently, there is no direct exposure of the bacterial lysate to the lung. In contrast, moving from the enteral oral route to using alternative "perioral" routes of administration, such as the intranasal, nasal, inhalation, nebulization and intratracheal routes, it is assumed that the anti-infective effect of the stabilized bacterial used takes place directly on cells of the lung or on the nasal and surrounding mucosa, where infections take place. Other favorable “perioral” routes to reach the mucosal tissue are the sublingual and buccal routes where infections often start.

[0010] In accordance with the present invention, novel bacterial Usado extract formulations were developed for these alternative prior routes of administration and thus to better fit broader routes of possible infections and inflammations. The ability of the mucosa to distinguish between dangerous and non-dangerous antigens is important in defense against pathogens and in protecting against damage caused by the response. Rn ι η / ι 7n7 / E / YL body's own inflammatory.

[0011] Those Bacterial Usado extracts, however, have until now commonly been administered via the oral-enteral route in solid forms: capsules or sachets. One of the major technical difficulties when switching to alternative routes of administration and / or alternative pharmaceutical formulations is that these bacterial extracts present some physical instability, particularly with the appearance of sedimentation. This physical instability of these bacterial extracts is a substantial problem during the manufacturing process, preparation and storage and is thus a limiting factor for the development of alternative pharmaceutical forms and / or alternative routes of administration such as intranasal, pulmonary, intratracheal. , mucosal, transmucosal, topical, external skin topical, buccal, sublingual, oral, pulmonary, intrabronchial or intrapulmonary. Regulatory approval of significant impact such as, a stable formulation suitable for those routes and alternative dosage forms is essential for a successful therapeutic bacterial drug product.

[0012] Therefore, it was important to address any aggregation and precipitation issues of those bacterial extracts and provide improved stabilized soluble bacterial extracts, to improve on the one hand manufacturing process solutions and, on the other hand, allow broader administration routes and alternative pharmaceutical forms.

[0013] Importantly, novel stable bacterial extract formulations can now be administered in more precise doses with specific delivery devices suitable for perioral and oral administrations and, in particular user-friendly delivery devices, such as aerosols, nasal sprays, nebulizers, pens, therefore increasing adherence to treatment and patient convenience. Another substantial advantage is that the novel stable bacterial extract can be more easily administered in liquid forms, via the oral route, to patients who are unable to ingest a tablet or capsule such as infants, young children, particularly between 3 months and 6 years of age. age, as well as adults, for example, some elderly people, who have difficulty eating. Alternative formulations of the bacterial extracts such as emulsions, microemulsions, dispersions, creams etc. may be contemplated for topical or external skin topical types of administration routes.

[0014] Finally, the novel stable bacterial extracts do not precipitate either at low temperature as well as at room temperature. Bacterial extract drugs can in this way be preserved and stored as such, under normal conditions by patients or pharmacies and do not present any risk of obstruction of any of the drug delivery devices, therefore allowing precise administration of doses. exact details of the bacterial extracts. Such stable bacterial extracts also provide a major advantage for pharmaceutical industries to store the pharmaceutical intermediate either during the manufacturing process or during the formulation of the final pharmaceutical products. BRIEF DESCRIPTION OF THE INVENTION

[0015] The present invention thus relates to bacterial extract formulations prepared from Gram-positive and / or Gram-negative bacterial species which have improved stability properties and are thus 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 intrabronchial administration. intrapulmonary.

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

[0017] The present invention further relates to the method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis.

[0018] The present invention finally relates to a novel process for preparing stable bacterial extracts in liquid, semisolid or aerosol forms, either as final pharmaceutical products before administration to patients or during the manufacturing and / or formulation of pharmaceutical products. . BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1: is a diagram of a tangential flow filtration (TFF) system for the preparation of bacterial extracts after alkaline lysis. The diagram shows two different configurations for the filters: a parallel mode where all the filters work simultaneously and a coil mode where the filters are configured in a series mode.

[0020] Figure 2: is a diagram of tangential flow filtration (TFF) used for purification of bacterial extracts after alkaline lysis. The diagram shows the different vessels for the pure bacterial extract, the purified fraction, the diafiltration media, the pure organic acid or the concentrated organic acid solution, the two pumps, the microfilter holder, the nanofilter / ultrafilter holder, valves, regulators transmembrane pressure (TMP), propellant, pH electrode and connection to waste. The series of filters can be connected in parallel mode where all filters work simultaneously or in coil mode where the filters are configured in a series mode.

[0021] Figure 3: shows the study design of a superinfection experiment: viral lung infection followed by bacterial lung infection. The administration dosage schedule of the OM bacterial extract by the oral route (groups 2) versus intranasal route (groups 3 and 4) and intranasal saline solution (group 1) is shown.

[0022] Figure 4: shows the results obtained after superinfection and shows that treatment with intranasal OM bacterial extract (i.n. dose A=50 micrograms and dose B=5 micrograms) produced a significant increase in survival rate compared to the oral OM bacterial extract (p.o. 7 milligrams) and control animals treated with saline solution.

[0023] Figure 5: shows the results obtained after superinfection and shows that treatment with prophylactic intranasal OM bacterial extract produced significant alleviation of morbidity and mortality after the post-influenza bacterial infection summarized here with the measurement of Clinical score after administration of both intranasal (i.n.) OM bacterial extract 5 and 50 micrograms against oral OM bacterial extract (p.o.) 7 milligrams all compared to saline control group.

[0024] Figure 6: is a photograph illustrating the difference in signs of comorbidity one day after bacterial infection (day 8 of the scheme of Figure 3) observed in mice treated with 7 milligrams of OM bacterial extract via the oral route (here illustrated as transient rough hair coating) against 50 micrograms of OM bacterial extract via intranasal route (shown in healthy animals). z i «n i n / ι znz / E / Yl·

[0025] Figure 7: shows the study design of a superinfection experiment: viral lung infection followed by bacterial lung infection. Shown in the Figure is the dose regimen of administration of the OM bacterial extract by the intranasal (nasally) route versus the intratracheal (i.t.) route against saline controls.

[0026] Figure 8: is a graph showing the viral titer in lung tissue after day 5 after the OM bacterial extract at doses A (50 micrograms) and B (5 micrograms) via administrations (i.n.) and intratracheal ( i.t.) against controls with intranasal (i.n.) and intratracheal (i.t.) saline solution.

[0027] Figure 9: is a graph showing the survival rate of mice treated by the intranasal route with OM bacterial extract with 50 micrograms (A.I.N. dose) and 5 micrograms (B.I.N. dose) or with saline solution (I.N.).

[0028] Figure 10 is a graph showing that intranasal prophylactic OM bacterial extract treatment produced significant relief of morbidity and mortality after post-influenza bacterial infection summarized here with clinical score measurement after administration of both intranasal OM bacterial extracts of 5 micrograms (dose A) and 50 micrograms (dose B) against the saline control group. Clinical efficacy was proportional to dose.

[0029] Figure 11: is a graph showing the survival of mice after administration of OM bacterial extract or saline following the intratracheal route. 50 micrograms (AI. T. dose) and 5 micrograms (B I. T. dose) of OM bacterial extract.

[0030] Figure 12 is a graph showing that treatment with prophylactic intratracheal OM bacterial extract produced significant relief of morbidity and mortality summarized here with clinical score measurement after administration of 50 microgram doses (A.I.T. dose). and 5 micrograms (B.I.T. dose).

[0031] Figure 13: shows the study design of a superinfection experiment: viral lung infection followed by bacterial lung infection using different doses and regimens after intranasal (nasal) and oral administration of the OM bacterial extract compared with the control group followed by lung influenza virus load analysis. Groups 1 to 11 are described in Table 6.

[0032] Figure 14: is a graph showing viral titer in lung tissue on day 5 post-infection after intranasal preventive treatments with OM bacterial extracts and compared to oral treatment.

[0033] Figure 15A-15C: shows the rate of infection with Human Rhinovirus (RV16) in human primary bronchial epithelial cells (BECs) originating from lung biopsies of healthy donors pretreated with various stable OM314A bacterial extracts. (Fig. 15A) Pretreatment of BEC cells with OM314A containing organic acids or HCl was performed 1 day before infection with 1 Multiplicity of Infection (MOI) RV-16. Expression of RV-mRNA was used as an indicator of virus replication and expressed as the relative value (percent, Figure 15A) against RV16 (100%). Control 1 (0%) represented uninfected cells. RV16-1 represented BEC cells infected with RV16 for 24 hours. Bars represent mean ± S.D. Treated samples: control 1, RV16; samples with OM314A: HCL; 10 centrifuged HCL; butyric acid; centri. butyric acid; propionic acid; centri. propionic acid; Aspartic acid; centri aspartic acid Centri. Indicates supernatant of the pair of samples obtained after zLRnLn / Lznz / B / Yi centrifugation and compared in a non-centrifuged counterpart. (Fig.15B) Primary human BEC positive for RV16 protein (n=3) 24 hours after infection using 3 different viral concentrations. (Fig. 15C) Effect of preincubation (24 hours) with various preparations of OM314A (20 pL / mL) on RV16 protein staining in BEC (n=3) 24 hours after infection with 0.1 MCI. Bars represent mean ± S.D.

[0034] Figure 16: shows the 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 for 24 h with OM314A (OM) samples as indicated in Figures 17A-17C and 18A-18C. Cell supernatants were collected at the indicated times for interferon beta and gamma dosing using ELISA.

[0035] Figure 17A-17C: (Fig. 17A) The dose response of interferon beta (IFN-beta) type 1 secretion by human BECs after 24 hours of incubation with HCL-neutralized OM bacterial extract (0.1 up to 50 micrograms / ml). Bars represent the mean of n=5 donors ± S.D. * = p < 0.01 compared to 24 hrs. (Fig.17B) secretion of ΙΕΝβ by human BEC (n=3) for 24 hours after infection with RV16. (Fig. 17C) Concentration-dependent effect of various OM314A (Ρ1, P2, P3), OM314B (P4) and OM-314C (P5) preparations on ΙΕΝβ secretion by uninfected human BECs (n=3). Bars represent the mean ± S.D. of each condition.

[0036] Figure 18A-18C: (Fig. 18A) The dose response of interferon gamma (IFN-gamma) type 2 secretion by primary human lung-derived epithelial cells (BEC) after 24 hours of incubation with OM bacterial extract neutralized with HCL (0.1 to 50 micrograms / ml). Bars represent the mean of n=5 donors ± S.D. * = p < 0.01 compared to 24-hr controls. ((Fig. 18B) Secretion of IFNy by human BECs (n=3) for 24 hours after infection with RV16. ((Fig. 18C) Concentration-dependent effect of various OM314A preparations (P1, P2, P3) , OM314B (P4) and OM-314C (P5) on IFNy secretion by uninfected human BECs. Bars represent the mean ± S.D. of each condition.

[0037] Figure 19A-19B: Shows the expression of antiviral beta β-defensin-l by primary human lung-derived epithelial cells (BEC). (Fig. 19A) Shows the effect of infection (3 different concentrations) on the secretion of beta β-defensin-l by BEC (n = 3) for 24 hours after infection with RV16. (Fig.19B) Shows the concentration-dependent effect of the stable bacterial extract OM314A (P1, P2, P3), OM314B (P4) and OM314C (P5) on the secretion of β-defensin-l by uninfected BEC (n= 3). Bars represent the mean ± S.D. of triplicates of each condition.

[0038] Figure 20A-20B: shows the expression of the viral receptor ICAM-1. (Fig.20A) Shows the expression of ICAM-1 receptor by primary human lung-derived epithelial cells (n=3) for 24 hours after infection with RV16. (Fig.20B) Shows the concentration-dependent effect of the stable bacterial extract OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) on the expression of ICAM-1 by infected primary epithelial cells (n =3). Bars represent the mean ± S.D. of triplicates of each condition.

[0039] Figure 21: Shows TNFo (TLR)-4-dependent release of TNFo from Bone Marrow-derived Dendritic Cells (BMDCs) from wild-type murine (WT, black bars) or TLR-4-modified mouse (TLR4- / -, white bars). Cells were stimulated with increasing dilutions of stable bacterial extract OM314A (Ρ1, P2, zLRnLn / Lznz / E / Yi Ρ3), ΟΜ314Β (Ρ4) and OM-314C (Ρ5) or with either LPS (2 pg / mL) or industrial batch (I. B. #1619057) for controls using the same set of dilutions. The level of TNFo concentrations was measured in the supernatants by ELISA after 16 h of induction and according to the manufacturer's protocol.

[0040] Figure 22: shows the Linear Discriminant Analysis (LDA) scores on the normal diet of saline-fed control mice, normal chow diet (NCD (NCD-Sham), normal diet mice treated with bacterial extract of Usados ​​strain 21 (NCD-BE), saline-fed high-fat diet control mice (HFD-Sham), and high-fat diet mice treated with bacterial extract of Usados ​​strain 21 (HFD-BE ).

[0041] Figure 23A-23C: (Fig.23A) shows graphs of glucose concentration before the bacterial extract diet of Usados ​​strain 21 (Prediet), (Fig.23B) mice after the chow diet normal (Post-NCD), (Fig.23C) after high fat diet (Post-HFD). For weight gain, mice were weighed once a week. Feed consumption measurements were evaluated once a week by weighing the kibbles at the beginning and end of the week. The meanings of the large and small asterisk marks are HFDSimulated compared to HFD-L Plantarum and HFD-Simulated compared to HFD-BE according to what is indicated in the graphs and with the respective significant values.

[0042] Figure 24A-24B: shows weight and feed consumption. (Fig.24A) Shows the weight in the sham control with high-fat diet fed with saline solution (HFD-Sham), high-fat diet treated with bacterial extract of Usados ​​strain 21 (HFD-BE) and high-fat diet fat treated with L. Plantarum (NCD-L, plant) expressed in grams. (Fig.24B) Shows the comparison of food consumption between the simulated control with high-fat diet fed with saline solution (HFD-Simulated) and with high-fat diet treated with bacterial extract of Usados ​​strain 21 (HFD-BE ) expressed in grams. For weight gain, mice were weighed once a week. Feed consumption measurements were evaluated once a week by weighing the kibbles at the beginning and end of the week. The meanings of the circled asterisk marks are HFD-Simulated compared to HFD-BE, the meanings of the normal * marks are HFD-Simulated compared to HFD-L Plantarum as indicated in the graphs and with the respective significant values.

[0043] Figure 25A-25B: shows the results of insulin tolerance tests in the 42 mice before treatment (PRE-DIET) and at the end of the eight-week treatment period (POST-DIET) in mice with diet high-fat (HFD) fed either saline (HFD-Sham), bacterial extract of Usados ​​strain 21 (HFD-BE) or L. Plantarum (NCD-L, plant). The meanings of the black asterisk marks are HFDsimulated compared to HFD-BE as indicated in the graphs (p<0.0001).

[0044] Figure 26A-26E: shows the effect of the bacterial extract of Bacterial Usages 21 on various intestinal species. 16S ribosomal RNA sequencing and analysis were performed as described in EXAMPLE 10. Fig.26A. Clostridiales Lachnospiraceae blautía', Fig.26B. Clostridia Clostridiales ruminococcaceae GCA900066225; Fig.26C. Clostridiales Ruminococcaceae ruminococcaceae UCG-0101; Fig.26D. Bacteroídales Muríbaculaceae uncultured bacterium; Fig.26E. Lachnospiraceae [Eubacteríum] físsícantena\ *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0045] Figure 27: represents a noisy spectrum indicating precipitate in solution (given as a typical example of unstable bacterial extract).

[0046] Figure 28: represents a smooth spectrum indicating clear solution (given as a typical example of stable bacterial extract).

[0047] Figure 29: shows Process 1 - bioassay results during stability show that the E3 neutralized filtrate from Process 1 (OM314A) exhibited comparable bioactivity through MIP-3a secretion onto THP-1 for at least 4 months at room temperature (20°C +1- 5°C) or 4°C. Process 1 T0 was compared with T4 samples stored at 4°C and at room temperature (RT) for 4 months.

[0048] Figure 30: Shows Process 2 - bioassay results during stability show that the E3 neutralized filtrate from Process 2 (OM314A) exhibited comparable bioactivity through the secretion of MIP3o onto THP-1 for at least 4 months at room temperature (20°C + / - 5°C) or 4°C. Process 2 T0 was compared with T4 samples stored at 4°C and at room temperature (RT) for 4 months.

[0049] Figure 31: Shows Process 3 - bioassay results during stability show that the E3 neutralized filtrate from process 3 (OM314A) exhibited comparable bioactivity through the secretion of MIP-3a on THP-1 for at least 5 months at room temperature (20°C + / - 5°C) or 4°C. Process 3 T0 was compared with T5 samples stored at 4°C and at room temperature (RT) for 5 months.

[0050] Figure 32: Shows Process 5 - bioassay results during stability show that Process 5 - E3 - the neutralized filtrate (OM314A) exhibited comparable bioactivity through the secretion of MIP-3a on THP-1 during at least 5 months at room temperature (20°C + / - 5°C) or 4°C. Process 5 T0 was compared with T5 samples stored at 4°C and room temperature (RT) for 4 months. DETAILED DESCRIPTION OF THE INVENTION

[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, acid 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, a combination thereof or pharmaceutically acceptable salts and esters thereof, followed by purification by filtration of the neutralized bacterial extract and adjusting to a final physiological pH by adding the organic acid, a combination of salts and esters thereof used for neutralization.

[0052] The applicant has discovered that the contact of the bacterial extract, after alkaline lysis by which the lysate pH is greater than 10 (with variations of ± 0.1 of the pH) with a selection of specific organic acid, leads to a bacterial extract exhibiting surprisingly superior stability properties. More precisely, the bacterial extract formulation according to the present invention retained physical stability for a few months under liquid form. Such novel bacterial extract formulations having improved physical stability could then be stably stored under liquid, semi-solid or aerosol forms, as final pharmaceutical formulations for administration to patients or during manufacturing or formulation of the pharmaceutical product. Rn ι η / ι 7n7 / E / YL

[0053] “Stable” formulation is intended to mean a bacterial extract which essentially retains its physical stability in liquid form either during storage of the drug or in liquid form as a pharmaceutical intermediate during manufacturing or formulation. The bacterial extract formulation retains its physical stability in a pharmaceutical formulation if it shows no significant increase in aggregation and / or precipitation after visual examination for clarity or, as measured by light diffraction, size exclusion chromatography (SEO). and dynamic light diffraction. Non-significant precipitation or physical changes at ambient temperature are observed at 4°C, -20°C or -80°C for at least 3 months or 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. Preferably, no more than 10%, preferably 5% aggregation and precipitation is formed.

[0054] The terms "organic acid" refer to an organic compound that is characterized by weak acidic properties and does not completely dissociate in the presence of water.

[0055] The terms “alternative routes of administration” generally refer to perioral and oral routes and may include inter alia intranasal, intratracheal, mucosal, transmucosal, topical, external skin topical, buccal, oral, sublingual routes of administration. , pulmonary, intrabronchial and / or intrapulmonary.

[0056] The terms "OM314A bacterial extract" refer to a polyvalent immunomodulator that comprises purified bacterial extract or extract which is extracted by alkaline lysis of one or more of the most frequent bacterial pathogens of the upper respiratory tract, which comprises Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis. Preferably, the OM314A bacterial extract can be obtained by alkaline lysis of the combination of one or more of the above bacterial pathogens and, more preferably, comprises the eight pathogens mentioned above. This OM314A bacterial extract has been prepared in a stable formulation according to the present invention and corresponds to the second generation of the bacterial extract previously described in several scientific publications and in the international publication WO2008 / 109669. The first generation of bacterial extract, referred to herein below as “OM bacterial extract”, is administered orally to patients as solid ingested formulations such as capsules and tablets and has been shown to be efficient in preventing respiratory tract infections in adults and children. In addition, several clinical trials have been carried out and demonstrated that enteral (oral) administration of this first-generation OM bacterial extract was shown to prevent allergic asthma and wheezing attacks caused by acute respiratory tract diseases in children when administered orally. orally. The first generation of OM bacterial extract drugs have been marketed under the trade name Broncho-Vaxom®, in solid form, usually a capsule or sachet, which was administered to patients orally and at single-capsule dosing regimens. per day of 7 mg of lyophilized bacterial extract for the treatment of adults and one capsule per day of 3.5 mg of lyophilized bacterial extract for children.

[0057] Therefore, OM314A bacterial extract refers - as opposed to the first generation OM bacterial extract - to a second generation drug that comprises the OM bacterial extract, but which has been stabilized so that it can be formulated in any possible pharmaceutical form. , whether liquid, gaseous or Solid Rn ι η / ι 7n7 / E / YL, which are suitable for the widest possible routes of administration including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary.

[0058] The terms “OM314B bacterial extract” refer to a polyvalent vaccine obtainable by alkaline lysis of one or more bacterial species chosen from the Lactobacillus bacterial strains as described in international publication No. WO2010 / 027344. In particular, the stable bacterial extract may comprise one or more Lactobacillus bacterial strains chosen from Lactobacillus fermentum. Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, 15 Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuterí, Lactobacillus salivarius, Lactobacillus lactis and / or Lactobacillus delbrueckii. The OM314B bacterial extract has been prepared in a stable formulation according to the present invention and corresponds to the second generation of the Lactobacillus bacterial extract previously described in the international publication WO2010 / 027344. The OM314B bacterial extract has been stabilized so that it can be formulated into any possible pharmaceutical 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.

[0059] Therefore, the terms “OM314C bacterial extract” refer to a stable bacterial extract obtainable by alkaline lysis of one or more bacterial strains of Escherichia coli as described in international publication No. WO2008 / 109667. The OM314C bacterial extract has been prepared in a stable formulation according to the present invention and corresponds to the second generation of the Escherichia coli bacterial extract previously described in the international publication WO2008 / 109667. The second generation of the OM314C bacterial extract has been stabilized so that it can be formulated in any possible pharmaceutical form, whether in liquid, gaseous or solid form and is thus suitable for a variety of routes of administration including intranasal, intratracheal, mucosal , transmucosal, topical of the external skin, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary.

[0060] The terms "stable bacterial extract formulation" refers to a stabilized form of any bacterial extract drug obtainable by alkaline lysis extraction as described in international publications WO2008 / 109669, WO2010 / 027344, or WO2008 / 109667, but which has been adapted into dosage forms suitable for alternative routes including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary as described above.

[0061] Therefore, the stable purified bacterial extracts according to the present invention may comprise the alkaline bacterial lysate of any combination of Gram-positive and / or Gram-negative bacterial species that have therapeutic immunomodulatory properties that have been stabilized and can thus be formulated. in any pharmaceutical form for a wide variety of routes of administration including intranasal, intratracheal, mucosal, transmucosal, topical, buccal, oral, sublingual, pulmonary, intrabronchial and / or intrapulmonary as described above.

[0062] According to a first embodiment, the novel stable bacterial extract formulations Rn ι η / ι 7n7 / E / YL may comprise bacterial lysate of one or more species of bacteria chosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, as described in Applicant's international publication No. WO2008 / 109669. Preferably, the novel stable bacterial extract formulation according to this embodiment is designated herein below as stable OM314A bacterial extract formulation and 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.

[0063] According to this embodiment, there is thus provided a stable purified bacterial extract derived from one or more species of bacteria chosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, where the bacterial extract is 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, a combination thereof and / or pharmaceutically acceptable salts and asters thereof, followed by purification by filtration of the neutralized extract and, adjusting to a final physiological pH by adding the same organic acid or the same combination thereof used for neutralization.

[0064] According to a second embodiment, the stable bacterial extract formulation is prepared from one or more bacterial species chosen from Lactobacillus bacterial strains. In particular, the stable bacterial extract may comprise one or more Lactobacillus bacterial strains chosen 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 delbrueckií, as described in the applicant's International Publication No. WO2010 / 027344.

[0065] Therefore, according to this second embodiment, a stable purified bacterial extract derived from one or more bacterial strains of Lactobacillus is provided, wherein the bacterial extract is 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, a combination thereof and / or salts and pharmaceutically acceptable asters thereof, purification by filtration of the neutralized extract and, adjusting to a final physiological pH by adding the same organic acid or the same combination thereof used for neutralization.

[0066] In a third embodiment, the stable bacterial extract derived from one or more bacterial strains of Escherichia cotí, where the bacterial extract is 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, a combination thereof and / or pharmaceutically acceptable salts and esters thereof, zLRnLn / Lznz / E / Yi purification by filtration of the neutralized extract and, adjusting to a final physiological pH by adding the same organic acid or the same combination thereof used for neutralization. In particular, the Escheríchia coli bacterial extract may comprise one or more Escheríchia coli bacterial strains as described in Applicant's International Publication WO2008 / 109667.

[0067] According to this third embodiment, a stable purified bacterial extract of one or more bacterial strains of Escheríchia coli is thus provided, wherein the bacterial extract is 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, a combination thereof or pharmaceutically acceptable salts and esters thereof , followed by purification by filtration of the neutralized extract and adjusting to a final physiological pH by adding the same organic acid or the same combination thereof used for neutralization.

[0068] In the above embodiments, the bacterial extracts comprise at least one strain of each of the above bacteria species, while in other embodiments, one or more specific strains from the list above may be removed or replaced with one or more strains different.

[0069] As indicated above, it is not intended to limit the preparation of the stable bacterial extract formulations to any of those specific modalities since any other combinations of Gram+ and / or Gram bacteria can be prepared and formulated in accordance with the present invention. and would also exhibit the property of improved stability.

[0070] Typically, bacterial extracts are prepared by fermentation followed by heat inactivation and alkaline lysis and filtration. Fermentation, alkaline lysis and filtration are now well known in the state of the art and have been described inter alia in international publications WO2008 / 109667, WO2010 / 027344 and WO2008 / 109669.

[0071] Fermentation is generally carried out by growing each bacterial strain to a suitable optical density in a culture medium. For each strain, to obtain a sufficient amount of material, fermentation cultures can be started from a seed batch followed by inoculation into larger fermentation containers. For example, fermentation can begin with a small culture such as 0.1 to 1.0 liter, incubated for approximately 3 to 6 hours at 30 to 40°C, such as 37°C, to obtain an optical density (OD) at 700 nm of 3.0. to 5.0. After the small scale culture step, additional cultures can be carried out in one or a series of larger thermometers at 30°C to 40°C for a duration of 3 hours to 20 hours, such as for 3-10 hours or 8 hours.

[0072] The culture medium is preferably a medium that does not possess a risk of prion-related diseases (i.e., mad cow disease, scrapie and Creutzfeld-Jacob disease) or other diseases and thus does not comprise materials animal-based such as whey or meat extracts taken from animals such as cows or sheep or any other animal that can transmit prion-based diseases. For example, a non-animal medium may be used, such as a vegetable-based medium, such as a soy-based medium, a synthetic or semi-synthetic medium. Alternatively, media using horse serum or media comprising materials taken from animal species that do not transmit prion diseases may be used. The zLRnLn / Lznz / B / Yi culture medium may also include biological extracts such as yeast extract and horse serum, which also do not pose these disease risks. Supplemental growth factors can also be introduced to enhance the growth of some bacterial species.

[0073] After fermentation, the biomass of each bacterial strain or combined bacterial strains is generally activated by heat treatment, concentrated and frozen.

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

[0075] Alkaline lysis can preferably be carried out at a pH greater than about 10, with variations of ± 0.1 of the pH. The duration of lysis can be evaluated by the person 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, such as 3040°C or 35-40°C, such as 37°C. In general, lysis stops when all bacterial cells appear to have been ruptured based on visual observation as is well known to those skilled in the art. When more than one strain of the same bacterial genus is used, the strains can be used together or separately. The strains can thus be mixed before or after lysis.

[0076] During lysis, bacterial cells are broken and their components are degraded and chemically modified. In particular, the racemization of amino acids creates D-amino acids from the natural Lamino acids found in natural proteins. D-amino acids may be beneficial to increase the bioavailability of extracts, since proteins made up mainly partially of D-amino acids are not efficiently digested in the mammalian intestine. Thus, antigenic molecules in extracts that are chemically modified during lysis to contain D-amino acids remain in the patient's body for a longer time, potentially allowing a stronger immunostimulatory action.

[0077] After lysis, according to the present invention, the Bacterial Used was neutralized, i.e. that the pH of the Used was 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 adding one or more specific organic acids. One or more of the specific organic acids can be selected according to the invention from acetic acid, propionic acid, lactic acid, 3-hydroxypropanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, glutamic acid, aspartic acid, a combination thereof and / or pharmaceutically acceptable salts and esters thereof.

[0078] The Used are then purified by centrifugation and / or filtration to remove large cellular debris or any component that is insufficiently degraded, any insoluble or particulate material to obtain a soluble bacterial extract. Purification, including centrifugation and filtration, are well known in the state of the art to remove particulate matter from extracts. For example, Used can be centrifuged at 9000 g, followed by one or more rounds of filtration. Typically, filtration may comprise passing an extract, or a mixture of extracts, through one or more filters such as microfilters (i.e., microfiltration) or ultrafilters (i.e. ΖίβΠίΠ / ίΖηΖ / Ε / ΥΓ ultrafiltration) which can be repeated in several passes or cycles. For example, successive rounds of filtration can be performed on larger pore filters followed by microfiltration using a smaller pore filter, such as a 0.2 micrometer filter. Ultrafiltration can also be employed to help extract soluble materials from the extract, for example, by recirculating the ultrafiltration infiltrate for additional microfiltration.

[0079] The tangential flow filtration (TFF) method can be used to filter the extract and to extract solubilized molecules from larger cell debris. This is well known in the art and is described inter alia by Wayne P. Olson (Separations Technology, Pharmaceutical and Biotechnology Applications, Interpharm Press, Inc., Buffalo Grave, IL, U. S. A., pp. 126-135). An example of a filtration loop process is shown in Figure 1 below. At the beginning of this process, a diluted bacterial waste can be stored in a first tank. A microfiltration (MF) loop can be started and the product is pumped. The resulting MF retentate is recycled, while the MF infiltrate is transferred to a second tank. After reaching an adequate degree of concentration, an ultrafiltration (UF) loop can be started. The UF infiltrate can be recirculated back to the first tank for continuous extraction of solubilized compounds from the lysate while the retentate UF fraction can be stored in the second tank. During continuous extraction, the volumes in tanks 1 and 2 can be adjusted by regulating the flow rates of the microfiltration and ultrafiltration infiltrates. Several such extraction cycles can be carried out, either with TFF or another filtration method. In embodiments using TFF, at the end of the last cycle, the ultrafiltration loop can be stopped and the microfiltration loop can be performed alone and the MF infiltrate transferred to tank 2. The microfiltration loop can be equipped with filters from 1.2 micrometers to 0.1 micrometers, such as 0.65 to 0.2 micrometer or 0.45 micrometer filters. The cross flow can be between 1000 Liters / hours m2 (LHM) and 3000 LHM, such as between 1500 and 2500 LHM or 2000 LHM with a transmembrane pressure (TMP) of 0.6 to 2 bars, such as between 0.8 and 1.5 bars or 1.0 bar . The ultrafiltration loop can be equipped with filters from 10 KDa to 1000 KDa, such as 10 KDa to 100 KDa or 10 KDa to 30 KDa or 30 KDa to 100 KDa or 30 kDa to 300 kDa or 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 can be between 30 LHM and 1000 LHM, such as between 20 and 500 LHM with a TMP of 0.2 to 1.5 bar, such as between 0.4 and 1.2 bar or 0.5 bar.

[0080] Between 5 and 20 diafiltration volumes can be used to extract solubilized compounds from bacterial cell walls. The diafiltration media could be water adjusted to pH values ​​between 7 and 11. In some embodiments, between 8 and 15 volumes are used. Accordingly, for example, in some embodiments, between 5 and 15 filtration cycles may be used, in some cases between 8 and 15 cycles, such as 8,9,10,11,12,13,14 or 15 cycles.

[0081] In addition to removing any insoluble particles, this filtration also has the purpose of removing any nucleic acid. As a consequence of filtration, the amount of nucleic acid present in the bacterial extract may remain less than 100 micrograms / mL. However, saccharide components, including monosaccharides, disaccharides, as well as larger saccharides such as linear and branched polysaccharides and, particularly lipopolysaccharide (LPS) components, can be preserved by filtration. In fact, during the lysis process, saccharides (including LPS components) were cleaved into smaller structures or replaced by different functional groups. In this regard, although it was previously thought that κη ι η / ι 7n7 / E / YL saccharide components, including potentially toxic LPS components, should be removed from bacterial extracts for safety reasons (See, US Patent No. 5,424,287). , the applicant showed that saccharide components, including LPS, can be safely retained since these components in effect provide additional antigens to the extracts and thus improve therapeutic efficacy. The extracts can be further diluted, concentrated or centrifuged, if desired.

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

[0083] Such purified soluble bacterial extracts can thus be advantageously preserved and stored as a liquid in accordance with the present invention and remain clear without any sedimentation or precipitation and thereby retaining excellent physical stability. Alternatively, purified bacterial extracts can be lyophilized if necessary, before reformulating them into liquid, gaseous or solid forms for therapeutic uses or additional galenic processes.

[0084] When bacterial extract formulations are maintained as liquid formulations, they can be stored at room temperature for an extended period of time with excellent physical stability over time while still maintaining biological activity. Advantageously, the bacterial extracts can be stored at room temperature or at 4°C, -20°C or -80°C without formation of aggregates and precipitates during the formulation or storage process. According to the present invention, it was possible to greatly reduce the formation of any aggregates, whether insoluble or soluble in the bacteria extract formulations. Furthermore, not only was improvement in physical stability observed, but the formulation contained the superior biological activity of polysaccharides, lipopolysaccharides, proteins, racemized amino acids, and other biological components during production or storage, without substantial chemical degradation or modification of the biological components.

[0085] The bacterial extract formulations according to the present invention could thus be stored at RT or at 4°C, -20°C or at -80°C for a period of at least 1 month or at least 2 months, at 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 At least 18 months, at least 24 months, the formulations do not show substantial physical change caused during the manufacture and / or storage of the bacterial extract due to the effect of, for example, light, temperature, pH, water or by reaction with excipient and / or or the immediate container closing system. Furthermore, the same bacterial extract formulation of a known initial biological activity when placed under the same storage conditions could retain the initial biological activity. In a related embodiment, the bacterial extract has not reached the labeled expiration date during the period Storage ΖίβΠίΠ / ίΖηΖ / Ε / ΥΙ.

[0086] The compositions and chemical properties of the main components of the therapeutically active soluble bacterial extracts thus obtained have been precisely determined and maintained over time in liquid formulations. In particular, as indicated here above, the amount of nucleic acid present in the bacterial extract is less than 100 mg / mL. Also, bacterial extracts comprise more than 0.1 mg / mL of polysaccharides or between 0.1 to 4.5 mg / mL or from 0.1 to 4 mg / mL or from 0.1 to 4 mg / mL or from 0.1 to 3.5 mg / mL or from 0.6 to 3 mg / mL or 0.3 to 1 mg / mL or a starting or ending range of 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 of polysaccharides, such as 0.4 to 0.5 mg / mL. Furthermore, the yields of solubilized proteins measured by Lowry in the soluble purified bacterial extract may be greater than 50% or may be greater than 60% or may be 50 to 90% or may be 60-90%, for example. Therefore, the bacteria extract may comprise 5-75 mg / mL of proteins 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 start or end range of 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 mg / mL; from 1.5 to 2.5 mg / mL of free amino acids (A. A.) or from 1.5 to 2 mg / mL or from 2 to 2.5 mg / mL of free A. A. or an initial or final range of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.1, 2.2, 2.3, 2.4 or 2.5 mg / mL of free A. A., calculated from glutamic acid (147.1 g / mol); and from 0.3 to 4.5 mg / mL of polysaccharides and monosaccharides or from 0.3 to 4 mg / mL or from 0.4 to 4 mg / mL or from 0.5 to 3.5 mg / mL or from 0.6 to 3 mg / mL or from 0.3 to 1 mg / mL or a starting or ending range of 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 of polysaccharides and monosaccharides, such as from 0.4 to 0.5 mg / mL.

[0087] Furthermore, such lysis results in the partial hydrolysis of proteins as well as deamination, deamidation and partial racemization of amino acids from L to D. The racemization of amino acids during the lysis process creates Damino acids from natural L-amino acids found in proteins natural. Analytical studies of the bacterial extracts have determined the racemization percentages. Peaks representing Daspartic 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 D-amino acids of those species fluctuated from 3% to 40%. Accordingly, racemization of one or more of serine, threonine, histidine, alanine, arginine, tyrosine, phenylamine, leucine and / or usine has been revealed. At least 10% of one or more of the above amino acids can be slowed from D to L. D-amino acids may be beneficial in increasing the bioavailability of extracts, since proteins made up primarily or partially of D-amino acids are not digested efficiently. in the intestine of mammals. Thus, the antigenic molecules in the extracts that are chemically modified during lysis to contain D-amino acids remain in the patient's body for a longer time, allowing for a potentially stronger immunostimulant action.

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

[0089] As an example, the bacterial extract may thus contain approximately 6 to 8 mg / mL of proteins, 1.5 to 2.5 mg / mL of amino acids (A.A.) (measured after hydrolysis with HCl) and / or or from zLRnLn / Lznz / B / Yi approximately 0.4 to 0.5 mg / mL of polysaccharides and monosaccharides. The protein concentration is measured by the Lowry test 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). The concentration of glutamate (glutamic acid) is measured by converting isoindole-derived amino acids and measuring the absorbance at 340 nm, according to Roth M., Fluorescence reaction for amino acids, Anal. Chem., 43, 880-882, (1971).

[0090] According to what was indicated above, it was experimentally demonstrated that the OM bacterial extract administered orally in solid form at the human dose (7 mg of bacterial extract by dry weight) to the mouse was able to improve protection against bacterial infection. secondary after influenza infection (Pasquali et al, Frontiers in Medicine 2014,1,41).

[0091] The routes of administration of the OM bacterial extract have always been by the oral route (for example, the enteral route) and the dose regimen for the treatment of adults was one capsule per day of 7 mg of lyophilized bacterial extract and one capsule per day of 3.5 mg of lyophilized bacterial extract for children. However, the Applicant has found that administration of the OM bacterial extract via non-enteral routes of administration by alternative oral routes such as intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary routes provided a Strong and more efficient immune response to counteract possible infections and / or inflammation.

[0092] Importantly, the observed superior therapeutic effects of these novel perioral routes of administration allowed for a substantial decrease in the bacterial extract drug dose. The dose regimens could be divided in two, with daily perioral dose regimens for treatment of adults of 3.5 mg of lyophilized bacterial extract and 1.75 mg of lyophilized bacterial extract for children. Thus, it has been demonstrated by the Applicant that these novel perioral administration routes provided greater therapeutic efficacy at lower doses.

[0093] In addition, the Applicant showed that these novel perioral routes of administration of the bacterial extract - either stabilized bacterial extract formulations as described herein above or not - produced a stronger immune response in the respiratory tract and lungs, also producing a at the same time strong immune responses in distal sites, such as the intestine, etc...

[0094] In particular, the perioral administrations of the bacterial extracts (whether in stable formulation or not) allowed the protection of patients against acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis. Perioral routes of administration are particularly useful and effective in methods of treatment and / or prevention of those pathologies and disorders, which generally worsen medical conditions and increase the risk of developing chronic pathologies.

[0095] In accordance with the present invention, infections may comprise upper and lower respiratory tract infections and / or associated sequelae comprising allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, viruses respiratory syncytial virus, human rhinovirus (HRV), rhinosyncytial virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19 and the like), couperosis, pneumonia, Rn ι η / ι 7n7 / E / YL Hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive lung disease with acute lower respiratory infection, obstructive lung disease with acute upper respiratory infections or diseases with epithelial cilia movement disorders and / or mucus clearance disorders. Infections may also comprise secondary infections, non-respiratory viral infections, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock, and viro-induced complications.

[0096] According to the present invention, inflammations may comprise atopic dermatitis with allergic / atopic and non-respiratory respiratory indications, associated acute and / or chronic dermatitis, anaphylaxis and food allergy. Inflammations may also comprise skin disorders, inflamed skin, such as eczema, rosacea, atopic dermatitis, psoriasis, including photodamage (such as sun-induced redness or inflamed skin) skin atrophy, skin depigmentation (patches / dots) , photodermatitis (erythema: inflammation and reddened skin), telangiectasia, couperosis or actinic keratosis, as well as inflammations comprising predominant autoimmune indications of T helper 2 chosen from Grave's disease, Hashimoto's disease, scleroderma, diseases related to Ig 4 or pemphigus e inflammations that include eosinophilic indications chosen from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia.

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

[0098] Finally, the neoplasm may comprise neoplastic indications with immunological disorders such as mastocytosis, mast cell leukemia, tumors diverted by T helper 2 and / or immunosuppressed.

[0099] Therefore, according to a first aspect, the present invention relates to a purified bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from Moraxella catairhalis, Haemophilus influenzae, Klebsíella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae , Streptococcus pyogenes and / or Streptococcus sanguinis, where the bacterial extract comprises less than 100 micrograms / ml of nucleic acids, for use in a method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammations and / or neoplasms and / or dysbiosis, in a subject, where the purified soluble bacterial extract is, whether in a stable formulation or not, is administered to the subject by perioral routes, particularly via intratracheal inhalation, intranasal, mucosal, transmucosal, topical administrations of the external skin, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary and, at a dose regimen lower than the dose for oral, enteral administrations.

[00100] The present invention also relates to the purified bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from Moraxella catarrhalís, Haemophilus influenzae, Klebsíella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis, z ι "η ι η / ι znz / E / YL where the bacterial extract comprises less than 100 micrograms / ml of nucleic acids, for use in a method of treatment and / or prevention of infections chosen from infections of the upper and lower respiratory tract and / or associated sequelae that include allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosyncytial virus (RV), coronavirus (CoV, SARS- CoV, MERS-Cov, COVID-19 and similar), couperose, pneumonia, Hypersensitivity Pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute upper respiratory infections or diseases with disorders of epithelial cilia movement and / or mucus clearance disorders, secondary infections, non-respiratory viral infections, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock and viro-induced complications, in a subject, where the purified soluble bacterial extract is , whether in a stable formulation or not, is administered to the subject by perioral routes, particularly via intratracheal, intranasal, mucosal, transmucosal, external skin topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary inhalation administrations.

[00101] The present invention also relates to purified bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumonias, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, where the bacterial extract comprises less than 100 micrograms / ml of nucleic acids, for use in a method of treatment and / or prevention of inflammations chosen from atopic dermatitis with allergic / atopic and non-respiratory respiratory indications, associated acute and / or chronic dermatitis, anaphylaxis and food allergy, skin disorders, inflamed skin, such as eczema, rosacea, atopic dermatitis, psoriasis, including photodamage (such as sun-induced redness or inflamed skin) skin atrophy, skin depigmentation (patches / spots) , photodermatitis (erythema: inflammation and reddened skin), telangiectasia, couperosis or actinic keratosis, as well as inflammations chosen from predominant autoimmune indications of T helper 2 chosen from Grave's disease, Hashimoto's disease, scleroderma, diseases related to Ig 4 or pemphigus e inflammations comprising eosinophilic indications chosen from eosinophilic cystitis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia in a subject, wherein the purified soluble bacterial extract is, either, in a formulation stable or not, it is administered to the subject by perioral routes, particularly via intratracheal, intranasal, mucosal, transmucosal, topical external skin, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary inhalation administrations.

[00102] The present invention further relates to the purified bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, where The bacterial extract comprises less than 100 micrograms / ml of nucleic acids, for use in a method of treatment and / or prevention of dysbiosis-related disorders selected from asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low-carbohydrate regimens. fiber, atopic dermatitis, associated acute and / or chronic dermatitis, psoriasis, inflammatory bowel diseases, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, renal failures or diseases associated with low fiber regimens, in a subject, where the purified soluble bacterial extract is, whether in a stable formulation or not, is administered to the subject by perioral routes, particularly via administrations inhalation intratracheal, intranasal, mucosal, transmucosa, topical external skin, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary.

[00103] The present invention finally relates to the purified bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphyiococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and / or Streptococcus sanguinis, where the bacterial extract comprises less than 100 micrograms / ml of nucleic acids, for use in a method of treatment and / or prevention of neoplasm chosen from neoplastic indications with immunological disorders such as mastocytosis, mast cell leukemia, tumors diverted by T helper 2 and / or immunosuppressed, in a subject, where the purified soluble bacterial extract is, whether in a stable formulation or not, is administered to the subject by perioral routes, particularly via inhalation, intratracheal, intranasal, mucosal, transmucosal, external skin topical administrations , buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary.

[00104] According to this first aspect, the extract comprises at least one strain of each of the above bacteria species. Alternatively, one or more specific strains from the list above may be removed or substituted with one or more different strains. In the case of the preferred perioral OM bacterial extract, the extract is obtained from the eight bacterial pathogens of the upper respiratory tract, i.e. Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphyiococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis. The bacterial extract according to this aspect may also be the perioral OM314A bacterial extract, that is, the OM bacterial extract in stabilized form administered via the perioral routes.

[00105] The OM bacterial extract is obtainable by alkaline lysis, preferably at a pH greater than 10, from one or more bacterial species i.e. Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphyiococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis and subsequent purification to contain less than 100 micrograms / mL of nucleic acids, at least 0.3 mg / mL of saccharides, between 6 and 8 mg / mL of proteins having a molecular weight of less than 30 kDa, between 1.5 and 2.5 mg / mL of amino acids (measured after hydrolysis with HCl) in glutamic acid equivalents (147 g / mol), from 1 to 80% of the racemized amino acids from L to D, from one or more racemized amino acids that are chosen of aspartic acid, asparagine, glutamic acid, glutamine, serine, methionine, histidine, alanine, arginine, phenylalanine, tyrosine, leucine, lysine, valine and threonine. Additional descriptions of the properties of the extract and its suitable preparation methods have been provided below and are also provided in international publication WO2008 / 109669, the entire contents of which are incorporated herein by reference.

[00106] According to this first aspect, the present invention thus also relates to a method of treatment and / or prevention of infections of the upper and lower respiratory tract, associated sequelae and / or secondary infections, dysbiosis and / or disorders related to dysbiosis, where the bacterial extract OM perioral ΑΠI 0 / 1 7Λ7 / Ε / ΥΙ1 whether stabilized or not, is administered to the subject via intratracheal inhalation, intranasal, mucosal, transmucosal, topical, buccal, sublingual, pulmonary, intrabronchial or intrapulmonary administration and at a dose regimen of 0.005 mg to 1 mg per day, that is, at doses lower than oral enteral administration and was efficient in conferring optimal protection. Preferably, the bacterial extract according to this aspect is the OM bacterial extract or its stabilized form, the OM314A bacterial extract. Furthermore, according to this aspect, the secondary infections treated and / or prevented may be non-respiratory viral infections.

[00107] The Applicant clearly demonstrates in the following Examples that the intranasal administration of the OM bacterial extract according to this first aspect substantially reduced the viral titer in the lung tissue after influenza viral infection and reduced the morbidity and mortality of animals superinfected when compared to oral administration and at a much lower dose. In fact, intranasal or intratracheal administration of it as a prophylactic treatment was shown to be more effective than the enteral route of administration. Intranasal administration constituted an effective prophylactic 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 this first aspect upregulated the long chain isoforms of those two glycosaminoglycans, therefore evidencing that this bacterial extract was involved in the novel beneficial mechanism, such as reduction of inflammation and increase of antigen presentation.

[00108] According to this first aspect, the present invention thus relates to a method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis, which It comprises administering via the perioral route a therapeutically effective amount of the bacterial extract OM or the stabilized form OM314A.

[00109] The present invention relates in particular to a method of treatment and / or prevention of infections chosen from infections of the upper and lower respiratory tract and / or associated sequelae comprising allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis , laryngitis, tracheitis, laryngopharyngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosyncytial virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19), couperose, pneumonia, Hypersensitivity Pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive lung disease with acute lower respiratory infection, obstructive lung disease with acute upper respiratory infections or diseases with epithelial cilia movement disorders and / or mucus clearance disorders, secondary infections, non-viral infections respiratory, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock or viro-induced complications, which comprises administering via the perioral route a therapeutically effective amount of the OM bacterial extract or the stabilized form OM314A.

[00110] The present invention also relates to a method of treatment and / or prevention of immunological disorders including but not limited to imbalance between the immune response of T helper 1, T helper 17 and T helper 2, T reg imbalance, hypersensitivity type 2, immunosuppression, eosinophilia, allergy and atopy that comprises administering via the perioral route a therapeutically effective amount of the OM bacterial extract or the Rn ι η / ι 7n7 / E / YL stabilized form OM314A.

[00111] The present invention also relates to a method of treatment and / or prevention of inflammations comprising atopic dermatitis with allergic / atopic and non-respiratory respiratory indications, associated acute and / or chronic dermatitis, anaphylaxis and food allergy, disorders of the skin, inflamed skin, such as eczema, rosacea, atopic dermatitis, psoriasis, including photodamage (such as sun-induced redness or inflamed skin) skin atrophy, skin depigmentation (patches / dots), photodermatitis (erythema: inflammation and red skin), telangiectasia, couperosis, actinic keratosis or inflammations comprising predominant autoimmune indications of T helper 2 chosen from Grave's disease, Hashimoto's disease, scleroderma, diseases related to Ig 4 or pemphigus or inflammations comprising eosinophilic indications chosen from cystitis eosinophilic, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia that comprises administering via the perioral route a therapeutically effective amount of the bacterial extract OM or the stabilized form OM314A.

[00112] The present invention further relates to a method of treatment and / or prevention of dysbiosis-related disorders selected from asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber diets, atopic dermatitis, associated acute and / or chronic dermatitis, psoriasis, inflammatory bowel diseases, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, kidney failure or diseases associated with low-in regimens fiber comprising administering via the perioral route a therapeutically effective amount of the bacterial extract OM or the stabilized form OM314A.

[00113] The present invention still further relates to a method of treatment and / or prevention of immunological disorders including but not limited to the imbalance between the immune response of T helper 1, T helper 17 and T helper 2, imbalance of T reg, type 2 hypersensitivity, immunosuppression, eosinophilia, allergy or atopy, which comprises administering via the perioral route a therapeutically effective amount of the OM bacterial extract or the stabilized form OM314A.

[00114] The present invention finally relates to a method of treatment and / or prevention of neoplasms chosen from neoplastic indications with immunological disorders such as mastocytosis, mast cell leukemia, tumors diverted by T helper 2 and / or immunosuppressed, which comprises administering via route periorally a therapeutically effective amount of the OM bacterial extract or the stabilized form OM314A.

[00115] According to a second aspect, the present invention also relates to a bacterial extract obtainable by alkaline lysis of one or more bacterial species chosen from bacterial strains of Lactobacillus, for use in a method of treatment and / or prevention of disorders Acute and chronic immunological infections resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis as described above in a subject, where the Lactobacillus bacterial extract is, whether in a stable formulation or not and where it is administered to the subject. subject by perioral routes, particularly via intratracheal, intranasal, mucosal, transmucosal, external skin topical, buccal, sublingual, pulmonary, intrabronchial and / or intrapulmonary inhalation administrations and at a dose regimen lower than the dose used for oral enteral administration. The bacterial extract may comprise ΑΓ>I n / Ι 7O7 / E / Yl· preferably one or more Lactobacillus bacterial strains chosen comprising one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus caseí defensis, Lactobacillus casei ssp. case!, Lactobacillus paracasei, Lactobacillus bulgarícus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii. Preferably, the perioral bacterial extract according to this aspect is the bacterial extract obtainable by alkaline lysis from one or more bacterial species chosen from Lactobacillus bacterial strains as described above or its stabilized form, i.e. the OM314B bacterial extract.

[00116] According to this second aspect, the present invention is thus related to the method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis, which It comprises administering via the perioral route a therapeutically effective amount of Lactobacillus bacterial extract.

[00117] The present invention thus relates in particular to a method of treatment and / or prevention of infections chosen from infections of the upper and lower respiratory tract and / or associated sequelae comprising allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, pharyngitis , tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, respiratory syncytial virus, human rhinovirus (HRV), rhinosyncytial virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19), couperose, pneumonia, Pneumonitis Hypersensitivity, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive lung disease with acute lower respiratory infection, obstructive lung disease with acute upper respiratory infections or diseases with epithelial cilia movement disorders and / or mucus clearance disorders, secondary infections, infections non-respiratory viral infections, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock or viro-induced complications, which comprises administering via the perioral route a therapeutically effective amount of Lactobacillus bacterial extract.

[00118] The present invention also relates to a method of treatment and / or prevention of immunological disorders including but not limited to imbalance between the immune response of T helper 1, T helper 17 and T helper 2, T reg imbalance, hypersensitivity type 2, immunosuppression, eosinophilia, allergy and atopy, which includes administering a therapeutically effective amount of the Lactobacillus bacterial extract via the perioral route.

[00119] The present invention also relates to a method of treatment and / or prevention of inflammation chosen from allergic / atopic and non-respiratory respiratory indications, atopic dermatitis, associated acute and / or chronic dermatitis, anaphylaxis and food allergy, disorders of the skin, inflamed skin, such as eczema, rosacea, atopic dermatitis, psoriasis, including photodamage (such as sun-induced redness or inflamed skin) skin atrophy, skin depigmentation (patches / dots), photodermatitis (erythema: inflammation and reddened skin), telangiectasia, couperosis or actinic keratosis or inflammations comprising predominant autoimmune indications of T helper 2 chosen from Grave's disease, Hashimoto's disease, scleroderma, diseases related to Ig 4 or pemphigus or inflammations comprising eosinophilic indications chosen from cystitis eosinophilic, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic asthma or eosinophilic pneumonia, comprising Rn ι η / ι 7n7 / E / YL administer via the perioral route a therapeutically effective amount of the Lactobacillus bacterial extract.

[00120] The present invention further relates to a method of treatment and / or prevention of selected dysbiosis-related disorders comprising asthma, diabetes, type 2 diabetes, autoimmune diseases, diseases associated with low fiber regimens, atopic dermatitis, dermatitis associated acute and / or chronic, psoriasis, inflammatory bowel diseases, colitis, ulcerative colitis, Crohn's disease, obesity, metabolic diseases or disorders, liver failure, NASH, NAFLD, liver fibrosis, kidney failure or diseases associated with low fiber regimens which comprises administering via the perioral route a therapeutically effective amount of the Lactobacillus bacterial extract.

[00121] The present invention is also related to a method of treatment and / or prevention of neoplasms that include neoplastic indications with immunological disorders such as mastocytosis, mast cell leukemia, tumors diverted by T helper 2 and / or immunosuppressed, which comprises administering via route periorally a therapeutically effective amount of the Lactobacillus bacterial extract.

[00122] the stable bacterial extract obtainable by alkaline lysis from one or more bacterial species chosen from Lactobacillus bacterial strains is particularly useful for treating and / or preventing rhinitis and / or allergic rhinitis, which is trivially called common cold symptoms with nasal congestion or runny nose.

[00123] According to a preferred embodiment of the second aspect, the present invention thus relates to a method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms 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 chosen from Lactobacillus bacterial strains. The preferred Lactobacillus bacterial extract comprises one or more bacterial strains chosen comprising one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus case! defensis, Lactobacillus case! ssp. case!, Lactobacillus paracasei, Lactobacillus bulgarícus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii. More preferably, the Lactobacillus bacterial extract is a stabilized OM314B bacterial extract as described hereinbefore.

[00124] The stable Lactobacillus bacterial extract according to this second aspect is thus administered via perioral routes, particularly via intratracheal inhalation or intranasal administration and at a dose regimen lower than the dose used for oral enteral administration.

[00125] Considering the non-specific direct and indirect antiviral activity of the bacterial extracts OM and newly stable OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) on the surface of epithelial cells demonstrated in vivo with reduction of viral cell titers and the assembly of antiviral antibodies against, but not limited to, H1N1, RSV as well as in vitro efficacy against human RV (exemplified by the induction of IFNs, β-defensins accompanied by a decrease in the receptor viral ICAM-1, thus confirming the induction by OM- and newly stable OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) of the loss of binding (until loss) of viral binding to epithelial cells, it can be anticipated that those perioral administration routes are particularly useful and effective in the methods of treatment and / or prevention of upper and lower respiratory tract infections such as rhinitis, allergic rhinitis, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, respiratory syncytial virus, secondary bacterial infections after viral infections with influenza (H1N1 and similar), human rhinovirus (HRV), rhinosyncytial virus (RV), coronavirus (CoV, SARS-CoV, MERS-Cov, COVID-19 and similar), couperose, pneumonia, bronchopneumonia, bronchitis, bronchiolitis, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute upper respiratory infections, diseases with movement disorders of the epithelial cilia and / or mucus clearance disorders.

[00126] Such purified bacterial extracts can be stabilized as described herein above and thus administered as stable bacterial extract formulations either as solid, semi-solid, liquid aerosol forms.

[00127] Pharmaceutical compositions comprising the stabilized bacterial extracts and a pharmaceutically acceptable excipient are also provided. Such pharmaceutical compositions can be stabilized and stored in liquid formulations for a few months and administered to patients in liquid or vaporized forms. Alternatively, they can be lyophilized and / or stored before being reformulated as liquid or aerosol drugs.

[00128] The stable pharmaceutical compositions according to the present invention are particularly useful in a method of treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammation and / or neoplasms and / or dysbiosis as described above .

[00129] Since these pharmaceutical compositions remained stable for several months in any form, liquid, gaseous or aerosol, semisolid or solid, they can be formulated for administration via the intranasal, intratracheal, mucosal, transmucosal, topical, buccal, sublingual, oral routes. , pulmonary, intrabronchial and / or intrapulmonary. Preferably, they may be administered to the subject by intratracheal inhalation or by the intranasal transmucosal route.

[00130] Particularly preferred are pharmaceutical compositions where the composition is liquid or aerosol and is formulated in a mist, drops, colloidal, mist, nebulizer or in atomized vapor. Also preferred pharmaceutical compositions can be liquid or semi-solid formulations such as emulsions, microemulsions, aqueous dispersions, oils, milks, balms, foams, aqueous or oily lotions, aqueous or oily gels, creams, solutions, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, serums. , ointments, foams, pastes or transdermal patches. In certain more preferred embodiments, the composition is solid and is formulated into a powder or a disintegratable tablet.

[00131] In the preparation of liquid, semi-solid, solid and spray medicines, the above-mentioned materials can be appropriately used with any additives such as carriers, binding agents, perfumes, flavoring agents, sweeteners, colorants, antiseptics, antioxidants, stabilizing agents and surfactants. , if desired.

[00132] In a preferred embodiment, the bacterial extract pharmaceutical composition is administered via the intranasal route and the composition may be in a form chosen from an emulsion, suspension, colloidal form, mist, nebulizer, atomized vapor or mist, a nasal plug. , powder, ointment, cream, lotion, gel, paste, balm, solution, ΖίβΠίΠ / ίΖηΖ / Ε / ΥΙ tincture, patch or strip.

[00133] In another preferred embodiment, the bacterial extract pharmaceutical composition can be administered orally and be present in the form of a single-dose container or single-dose container such as a single-dose plastic bottle similar to those plastic bottles of physiological liquid used as drops. for dry eyes or in the form of single-dose vials. The liquid formulations of oral bacterial extract according to this preferred embodiment are thus a stable formulation and can be maintained in single-dose containers as a neutralized liquid formulation (close to pH 7) for prolonged periods of time.

[00134] To prepare the pharmaceutical compositions herein, the bacterial extracts can be mixed with a pharmaceutically acceptable carrier, adjuvant and / or excipient, according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers encompass any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water and emulsions, such as oil / water or water / oil emulsion, and various types of wetting agents. The bacterial extracts may additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, skimmed milk powder and the like. The liquid and semi-solid excipients can be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. . Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose and glycols. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). Pharmaceutical compositions may also include stabilizers and preservatives.

[00135] In one of the preferred embodiments, the pharmaceutical composition can be formulated with a disintegrating tablet. The tablet may be administered whole or slightly disintegrated, such as by finger pressure and sprinkled on the appropriate vehicle. The disintegrating tablet can be prepared using direct compression processes and excipients taking care in the process to avoid damaging the coating of the individual subunits. Suitable excipients for preparing the disintegrating tablet include those typically used for chewable tablets including mono- and disaccharides, sugar polyols and the like or a combination thereof. Exemplary excipients include mannitol, sorbitol, xylitol, maltitol, lactose, sucrose, maltose or a combination thereof. Optional pharmaceutical excipients such as diluents, lubricants, glidants, flavorings, colorants, etc., ... or a combination comprising at least one of the above may also be included in the compression matrix. Disintegratable tablets can be prepared using tablet manufacturing methods known in the pharmaceutical art.

[00136] The bacterial extract formulation may also be presented in colloidal form, comprising for example, a metal halide, more preferably silver halide. One or more bacterial extracts and the adjuvant may be incorporated into or encapsulated by means of the colloidal particle. Alternatively or in addition, one or more bacterial extracts and adjuvant may be attached to the surface of the colloidal particle. The means by zLRnLn / Lznz / B / Yi which active agent and adjuvant bind to the particle depends on the characteristics of the extracts, adjuvants and the colloidal particles. For example, proteins readily adsorb or bind to hydrophobic particles via hydrophobic interactions with the particle surface and displace some of the neutral emulsifier.

[00137] The present invention is based, in part, on the surprising discovery that the use of a perioral delivery system of the bacterial extracts described above provides significantly higher antibody titers and improved immune response and a safe and effective approach to increase the immunogenicity of a variety of antigens for use in prophylactic and therapeutic pharmaceutical compositions.

[00138] Suitable doses according to the invention and as described herein in the different embodiments will vary depending on the condition, age and species of the subject and can be easily determined by those skilled in the art. However, according to the present invention, the total daily doses are greatly reduced and may be in the range of 0.005 to 1 mg, preferably 0.05 to 0.5 mg, more preferably 0.1 to 0.3 mg and those can be administered as a single or divided dose and, in addition, the upper limit may also be exceeded when this is found to be indicated. Advantageously, doses administered via perioral routes are lower (e.g., half the doses) than doses administered via oral enteral routes.

[00139] Another aspect relates to a device for releasing the bacterial extract formulations according to the invention. Also provided is a delivery device for use in a method of treating and / or preventing upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and / or dysbiosis-related disorders.

[00140] According to the present invention, the bacterial extract formulations can be administered via the intranasal or intratracheal route, by means of a nasal insufflator device, intranasal inhaler, intranasal mist device, atomizer, nasal spray bottle, dose container unit, pump, dropper, squeeze bottle, nebulizer, metered dose inhaler (MDI), pressurized dose inhalers, insufflators, bidirectional devices, dose vials, nasal pillows, nasal sponges and nasal capsules.

[00141] The nasal sprays can be liquid or solid nasal sprays. Bacterial extract formulations can be administered as aerosols or in non-aerosol forms. The nasal delivery device can be dosed to deliver an exact effective dose amount to the nasal cavity. The nasal delivery device may be to release a single unit or to release multiple units.

[0142] When bacterial extract formulations are administered as aerosols, they can be prepared using standard procedures. For example, an aerosol mist may be generated from the pressurized container with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen, carbon dioxide or other suitable gas. The dosage unit can be determined by providing a valve to release a measured amount. Pump mist dispensers can dispense a metered dose or a dose that has a specific particle or droplet size. The aerosol may be a suspension or dispersion of liquid droplets or solid powder in air (or in a gas). Liquid droplets can be formulated from Rn ι η / ι 7n7 / E / YL solutions, suspensions, and dispersions of drug in a liquid, such as water or a nonaqueous solvent. Aerosols can be produced in any suitable device, such as an MDI, nebulizer, or mist sprayer.

[00143] An aerosol according to the invention can be inflated or inhaled using a suitable mechanical apparatus. The apparatus may include, for example, a reservoir and sprayer, which is a device adapted to expel the pharmaceutical dose in the form of a mist. A number of doses to be administered may be contained within the reservoir, optionally in a liquid solution or suspension or in a solid particulate formulation, such as a solid particulate mixture.

[00144] Nebulizer devices produce a high-velocity airflow that causes a therapeutic agent in liquid form to be sprayed as a mist. The therapeutic agent is formulated in a liquid form as a solution or a liquid suspension of particles of suitable size. The particles are micronized. The term "micronized" is defined as having approximately 90% or more of the particles with a diameter less than approximately 10 pm. Suitable nebulizing devices are commercially available, for example, from PARI GmbH (Sternberg, Germany). Other nebulizer devices include the Respimat (Boehringer Ingelheim) and those described in, for example, US Patent Nos. 7,568,480 and 6,123,068 and WO 97 / 12687.

[00145] DPI devices can be used to deliver the bacterial extract formulation in the form of a free-flowing powder that can be dispersed into a patient's airstream during inspiration. DPI devices having an external power 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 blend can be produced, for example, by combining dry lactose having a particle size between about 1 pm and 100 pm with micronized benzodiazepine particles and dry mixing. Alternatively, the benzodiazepine can be formulated without excipients. The formulation is loaded into a dry powder dispenser or inhalation cartridges or capsules for use with a dry powder delivery device. Examples of commercially provided DPI devices include Diskhaler (GlaxoSmith line, Research Triangle Park, N.C.) (U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKIine) (U.S. Patent No. 6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (U.S. Patent No. 4,524,769); and Rotahaler (GlaxoSmithKIine) (U.S. Patent No. 4,353,365).

[00146] MDI devices can be used to discharge a measured amount of the bacterial extract formulation using compressed propellant gas. MDI delivery formulations include a solution or suspension of bacterial extract formulation in a liquefied propellant. Examples of propellants include hydrofluoroalkanes (HFA), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227) and chlorofluorocarbons, such as CCI3F. Additional components of HFA formulations for MDI administration include cosolvents, such as ethanol, pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin and glycerin. The bacterial extract formulation is loaded into an aerosol bottle, which forms a portion of an MDI device.

[00147] The bacterial extract formulation can be released into the nasal cavity in powder form as microspheres via a nasal insufflator. The bacterial extract formulation can be absorbed to a solid surface, Rn 1 η / ι 7n7 / E / YL for example, a carrier. The powder or microspheres may be administered in a dry form, dispersed in air. The powder or microspheres can be stored in an insufflator container. Alternatively, the powder or microspheres may be filled into a capsule, such as a gelatin capsule or other individual dosage unit adapted for nasal administration.

[00148] The bacterial extract formulation is delivered through a nasal spray applicator. The composition may be placed in an intranasal mist dosing device or may be applied by spray to the nasal passages of a subject to be released to the mucous membrane of the nasal passages. A sufficient amount is applied to achieve the systemic or localized levels desired for the therapeutic effect.

[00149] The bacterial extract formulation may further be administered via the intratracheal route, by oral inhalation into the respiratory tract, i.e., the lungs. Such intratracheal administration requires aerosolization of a solid or liquid and release of the aerosol to the lungs via the mouth and throat. The drug particles can be delivered to the lungs as dry powder aerosols or liquid aerosols. Dry powder aerosols are usually administered to the lungs with the dry powder inhaler (DPI) inhalation device. Dry powder inhalers may include breath-actuated dry powder inhalers, as described in US Patent No. 7,434,579. Metered-dose inhalers contain medication suspended in a propellant, such as a mixture of propellants or a mixture of solvents, propellants and / or other excipients in compact, pressurized aerosol dispensers. An MDI product can deliver up to several hundred metered doses of medication. Each actuation can contain from a few micrograms (mcg) to milligrams (mg) of the active ingredients released in a volume typically between 25 and 100 microliters.

[00150] As described 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 drug particles.

[00151] Bacterial extract formulations according to the present invention may further comprise adjuvants, infiltration improvers and / or solvents. For example, for intranasal delivery, the filtration improver can be used to improve or increase infiltration of the composition through the nasal mucosa. Compounds containing one or more than one hydroxyl group can be used as infiltration enhancers or enhancers. Some of those compounds containing hydroxyl group may also serve as solvents in the composition. Non-limiting examples of hydroxyl group-containing compounds that can be used as infiltration enhancers or enhancers include alcohols (such as ethanol), diols (such as 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; etohexadiol; pentane-3,8diol ; 2-metll-2,4-pentanediol); triols (such as glycerin), polyols (such as suitable polymers containing multiple hydroxyl groups, including polyethylene glycols or PEG, polypropylene glycols, polysorbates and sorbitan esters; and suitable sugar alcohols), cyclitols (such as pinitol, inositol), cyclic diols (such as cyclohexane diol), aromatic diols (such as hydroquinone, bisphenol A, resorcinol and catechol).

[00152] One skilled in the art would recognize that the teachings herein would also be ΑΠI 0 / 1 7Λ7 / Ε / ΥΙ1 applicable to other infiltration improvers. Non-limiting examples of other infiltration enhancers useful in the present invention are the simple long chain esters Generally Recognized as Safe (GRAS) in the different compendia of the pharmacopoeia. These may include aliphatic, unsaturated or simple saturated esters. Non-limiting examples of such esters include isopropyl myristate, myristyl myristate, octyl palmitate and the like. Non-limiting examples of other infiltration enhancers include alcohols (e.g., short and long chain alcohols), polyalcohols, amines and amides, urea, amino acids and their esters, amides, pyrrolidone and its derivatives, terpenes, fatty acids and their esters, macrocyclic compounds, sulfonic compounds, surfactants, benzyldimethylammonium chloride, cetyl trimethyl ammonium bromide, cineole, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dodecyl pyridinium chloride, dodecylamine, hexadecyl trimethylaminopropane sulfonate, limonene, linoleic acid (OA), linolenic acid (LA), menthol, methyl laurate, methylpyrrolidone, N-decyl-2-pyrrolidone, NLS, nicotine sulfate, nonyl-1,3dioxolane, octyl trimethylammonium 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. Improvers will be suitable. The person skilled in the art will also appreciate that those materials that are not compatible or irritating to mucous membranes should be avoided.

[00153] Examples of pharmaceutically acceptable solvents or excipients that can be used in the present composition can be found in reference books such as the Handbook of Pharmaceutical Excipients (Fifth Edition, Pharmaceutical Press, London and American Pharmacists Association, Washington, 2006). Non-limiting examples of pharmaceutically acceptable solvents that may be used in the present composition include, but are not limited to, propylene glycol (also known as 1,2-dihydroxypropane, 2-hydroxypropanol, methyl ethylene 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 sosorbide, propylene carbonate, Nmethylpyrrolidone, glycofurol, tetraethylene glycol , propylene glycol fatty acid esters and mixtures thereof.

[00154] Particularly preferred are bacterial extract formulations or pharmaceutical compositions comprising the formulations for use in a method of treatment and / or prevention of upper and lower respiratory tract infections, associated sequelae and / or secondary infections, dysbiosis and / or disorders related to dysbiosis, which are administered to the subject by intratracheal inhalation or by the transmucosal-intranasal route.

[00155] The intranasally administered bacterial extract is likely to have an effect on nasal associated lymphoid tissue (NALT) or gut associated lymphoid tissue (GALT), followed by trafficking of intestinally derived B and T cells and macrophages to the associated lymphoid tissue with the bronchi and, this can lead to an immune response against those pathogens in the respiratory tract.

[00156] The present invention also relates to a method of treating, preventing or attenuating viral infections and / or virus-induced exacerbations of allergic diseases or disorders such as asthma, chronic obstructive pulmonary disease and allergy or autoimmunity comprising administering to a subject via the perioral route a therapeutically effective amount of the stable bacterial extract of the present invention.

[00157] The asthmatic condition may be steroid-resistant asthma, neutrophilic asthma or non-allergic asthma. κη ι η / ι 7n7 / E / YL The allergic disease or disorder may be an eosinophilic disease or disorder, particularly a disease or disorder selected from the group consisting of nodules, eosinophilia, eosinophilic rheumatism, dermatitis and swelling (NERDS).

[00158] The present invention relates to a method of treatment and / or prevention of an allergic disease or disorder or for alleviating the condition of a subject suffering from an allergic disease or disorder, including, but not limited to, a allergic disease or disorder selected from the group consisting of asthma, rhinitis, dermatitis, drug reactions, eosinophilic diseases or disorders, esophageal and gastrointestinal allergy, comprising administering to a subject via the perioral route a therapeutically effective amount of the stable bacterial extract of the present invention.

[00159] The present invention finally provides a novel extraction process for the preparation of bacterial extracts with improved stability. The process to prepare the bacterial extract with improved stability includes the following steps: to. cultivate each species of bacterial strain in a suitable culture medium, b. Use each strain at an initial pH, preferably greater than 10, with variations of ± 0.1 of the pH, c. decrease the pH of the extracts 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-hydroxy butanoic acid, glutamic acid, aspartic acid, a combination thereof or pharmaceutically acceptable salts and esters thereof, d. passing the product from step (c) at least once through a microfilter and retaining the product on an ultrafilter to obtain a purified soluble extract, and. adjust a final pH around 7 (+ / - 1.0) by adding the organic acid or the combination thereof used in step (b) and F. add a pharmaceutically acceptable excipient or vehicle.

[00160] According to a first aspect of the present invention, the bacterial extract is obtained from one or more bacterial species chosen 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 is derived from the eight bacterial pathogens of the upper respiratory tract, namely Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis in a similar manner to the first generation of drug from bacterial extract OM.

[00161] According to a second aspect of the present invention, the bacterial extract is obtained from one or more bacterial species chosen from Lactobacillus bacterial strains, such as, for example, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii , Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. case!, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii. ZLRnLn / LZnZ / E / Yl·

[00162] According to a third aspect of the present invention, the bacterial extract is obtained from one or more bacterial strains of Escherichia coli as described in international publication No. WO2008 / 109667.

[00163] Lysis can be carried out over a period of 40 hours to 10 days at a temperature of 60°C. Also, the microfilter that can be used in the process is 0.45 micrometers and the ultrafilter is 30 KDa. Additionally, part (c) of the process comprises tangential flow filtration, where the tangential flow filtration can be filled out for 5 to 15 cycles.

[00164] Furthermore, the present invention relates to bacterial extract formulation products and / or pharmaceutical compositions obtainable by the above processes. EXAMPLES Example 1: Process to stabilize the alkaline extract Example 1.1: Addition of organic acids for the preparation of stable bacterial extracts (OM314A) First, preliminary measurements of the amounts of acid required to acidify the alkaline OM314A bacterial extract and 1 N NaOH to adjust the pH to pH 5.0 were made. 25 mL of OM314A alkaline concentrate was placed in 50 mL Falcon tubes. The native pH as measured is generally around pH 10.5. After native pH measurement, a small magnetic stirrer was introduced into the tube and stirring began at 600 rpm. The required amount of each selected organic acid was introduced in small amounts stepwise 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 for 5 min at 5000 g and the supernatant was filtered through a 0.2 pm filter. The pH of the stabilized bacterial extract was then adjusted to pH 7.0 ± 0.2 using 1 N NaOH and the volume of 1 N NaOH used was recorded. 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) with the required amount of each selected organic acid added in small amounts in steps until pH 5.0 (5.0) was reached. ±0.2). The volume and weight of acid were recorded. The acid extract was then centrifuged for 5 min at 5000 g and the supernatant was filtered on a 0.2 pm filter. The pH of the stabilized bacterial extract was then adjusted to pH 7.5 ± 0.2 using 1 N NaOH and the volume of 1 N NaOH used was recorded. A similar procedure was performed in a sterile laminar flow hood to prepare sterile samples of the stabilized bacterial extract. 25 mL or 35 mL of alkaline OM314A bacterial extract concentrate was introduced into 50 mL Falcon tubes. Each acid was added based on a predetermined volume or by weighing as determined by preliminary measurements. Acidification was initially carried out at pH 5.0 but a precipitate formed. In a second step, the pH was adjusted to 7.0 ± 0.2 and respectively 7.5 ± 0.2. Some dilutions were adjusted to pH 5 with the organic acids and further adjusted to pH 7.0 and respectively 7.5 remained clear and stable throughout the observation period at room temperature but not at 4 to 8°C where a precipitate formed after several weeks of storage. The following Table 1 summarizes the organic acids and volumes used to adjust the pH to 7.5 for a 35 mL solution of OM314A bacterial extract. Rn ι η / ι 7n7 / E / YL Table 1: Acid Liquid [% purity] Solid [% purity] Volume / weight added to adjust pH to 7.5 Turbidity Stability from 20°C to 25°C Stability from 4°C to 8°C HCI (acid control) Liquid, 25% 120 μΙ high Not stable, precipitates Not stable, precipitates Formic acid Liquid, 98-100% 30 μΙ slightly turbid Not stable, precipitates Not stable, precipitates Propanoic acid Liquid, 99% 70 μΙ slightly turbid Relatively stable, very small amounts of precipitate Relatively stable , Very small amounts of precipitate Aspartic acid Solid > 98% 118 mg clear solution Relatively stable, Very small amounts of precipitate Relatively stable, Very small amounts of precipitate Acid Liquid [% purity] Solid [% purity] Volume / weight added for adjust to pH 7.5 turbidity Stability 20°C to 25°C Stability 4°C to 8°C Lactic Acid Liquid, 90% 120 μΙ slightly turbid Relatively stable, very low amounts of precipitate Relatively stable, very low amounts of precipitate 3-Hydroxy-propanoic acid Liquid, 30% is water solution 250 μΙ slightly turbid Relatively stable, very small amounts of precipitate Relatively stable, very small amounts of precipitate Butanoic acid Liquid, 99% 70 μΙ slightly turbid Relatively stable, very small amounts of precipitate Relatively stable, Very small amounts of precipitate Glutamic acid Solid 150 mg slightly turbid Relatively stable, Small amount of precipitate Relatively stable, Small amount of precipitate Example 1.2. Removal of inorganic cations To remove inorganic divalent cations present in the OM314A extract stabilized as described above in Example 7.1, 25 mg of ammonium oxalate (1 mg / mL) was added. A precipitate with strong opalescence formed. The precipitate was centrifuged for 5 min at 5000 g and the clear supernatant was filtered through a 0.2 pm filter. The clear sample was adjusted to pH 7.0 ± 0.1 using 1 N NaOH. The difficulty of the process is scaling which would require centrifugation. Additionally, oxalate may not be suitable for intranasal or perioral formulations. Example 1.3. Simultaneous removal of small molecules and inorganic salts and concentration of the high molecular z i «n i η / ι ζηζ / Ε / γ fraction of the bacterial extract stabilized with organic acid. Alkaline bacterial extracts between pH 10.0 to 11.0 were added to the purification unit, schematized in Figure 2 which shows a diagram with the concentrations between 4 vessels, 2 pumps, two filters, two transmembrane pressure valves (TMP1 pressure regulation valves and TMP2) and four rotary valves. Transmembrane pressure (TMP): The average pressure applied from the feed to the filtrate side of the membrane. TMP [bar] = [(Pressure in the retained fraction + Pressure in the filtrate) / 2] - Pressure in the filtrate. Valves TMP1 and TMP2 generate pressure on the filter side of the filter and this regulates the transmembrane pressure (TMP) to the appropriate pressure value. Microfiltration: This filter usually 0.45 to 0.2 pm in this configuration is used to remove particles and allow soluble (non-particulate) material to pass through the filter pores. Ultrafiltration or nanofiltration: These filters have very small holes with a cut-off range (cut-off value: pore size expressed in molecular weight in Dalton units (1 Da = 1 mass unit, 1 kDa = 1000 mass units). 10 kDa filter retains material larger than the pore or molecules larger than 10 kDa (See http: / / www.merckmillipore.com / CH / de / ps-learninq-centers / ultrafiltration-learning-center / optimization-processsimulation / d eb.qB.ZWQAAAFAUV8ENHoL,nav?ReferrerURL=https%3A%2F%2Fwww.qooqle.com%2F#tmp). Example 1.4: Example of a five-step process leading to the stabilized bacterial extract containing included organic acids according to the invention (Figure 2) Step 1: The alkaline bacterial extract (pH 10 to 11) containing bacterial cell walls, cell wall fragments and soluble material was added to vessel 2 (Figure 2) assembled with pump 1 and microfiltration with a 0.45 pm filter and the 0.45 pm infiltrate connected to container 3 (Figure 2). Vessel 3 was connected to pump 2 (Figure 2) and a 10 KD (kDalton) ultrafiltration nanofilter which sends the 10 KD infiltrate back to vessel 2. The infiltrate containing small molecules and dilute sodium hydroxide was used for continuous extraction in vessel 2 (Figure 2) of the active water-soluble components of the bacterial extract. Half of the initial volume of the bacterial extract in container 2 was microfiltered and the 0.45 μm infiltrate was passed to container 3. Step 2: The second filtration unit (pump 2) was then turned on and the 10 kDa infiltrate 5 from vessel 3 was returned to vessel 2 using valve 3. The continuous extraction of the bacterial soluble components from the pure bacterial extract present in Vessel 2 was made using 10 KD infiltrate extraction for a total of 10 initial volumes of bacterial extract. Step 3: This step was carried out to remove the low molecular weight components present in container 3, a diafiltration process was started by connecting container 1 to pump 1 with fine adjustment of the TMP1 regulation to maintain the volume in container 2 at a constant level. Vessel 1 contained water adjusted to pH 10.8 to 11.0 with sodium hydroxide as the diafiltration media. During the diafiltration process, the ultrafiltration infiltrates from vessel 3 were connected to the waste using valve 3 and the TMP2 was adjusted to the optimal flow rate. A total of five volumes of diafiltration media were required to remove undesirable small molecular weight components present in the purified bacterial extract in vessel 3. zLRnLn / Lznz / B / Yi Step 4: A concentration step of the purified extract present in vessel 3 was carried out after disconnecting vessel 1 and pump 1. The purified extract fraction in vessel 3 was concentrated to half the initial volume with the infiltrate passing to waste via valve 3. Step 5: The concentrated purified bacterial fraction was stabilized by the addition of an organic acid to form included organic acid salts. This process was carried out after closing pump 2 and valve 2. A predefined volume of pure liquid organic acid or respectively for solid organic acids, a predefined volume of a concentrated solution of the organic acid, was added from container 4 to container 3 via valve 4 to reach a pH value of 7.5 ± 0.2. For pH adjustment, the propellant was turned on during the addition of the organic acid and the pH was adjusted via a pH electrode mounted in vessel 3. Step 6: Vessel 3 containing the concentrated purified bacterial fraction in the form of the organic acid salt at pH 7.5 was sterilized online using a 0.2 μm sterile filter mounted on sterile lines connected to a sterilized vessel 5 (sterile bag or container heat sterilized stainless steel). Depending on the final use of the liquid formulation and the route (intranasal, inhalation or aerosol or solid) an additional concentration was applied using pump 2 and ultrafiltration connected to the waste via valve 3, to reach 25% of the initial volume. The presence of the organic acid salt allowed to achieve a highly concentrated purified fraction that allows the spray drying process, direct use as liquid droplets and aerosols. Products with similar pH (7.5 ± 0.5) were obtained with different organic acids forming included salts. The same process was repeated using respectively 3 kD, 10 kD, 30 kD, 100 kD, 300 kD polysulfone tangential flow filter as well as hollow filters using 10 kD, 30 kD, 100 kD, 300 kD cutoffs as filter design. alternative tangential flow. The process was initially carried out on a 1 L Laboratory scale using first a concentration step (2-fold to reach 500 mL and up to a 5-fold concentration to reach a volume of 200 mL), undesirable small molecules were washed for a diafiltration step using a NaOH-water solution at pH 10.5 to pH 11.0 and the desired retentate from the ultrafiltration was further concentrated 2 times. Organic acids were then added to form salts at pH 7.5 ± 0.5 with the positively charged groups present in the extract to stabilize the preparation. Similarly, the concentration of the high molecular weight fractions was carried out using a concentration step on the ultrafiltration filters removing the low molecular fraction using different cutoffs, respectively 3 kD, 10 kD, 30 kD, 100 kD, 300 kD, 1000 kD of the respective ultrafiltration filters. The organic acids listed in the following table were added with a target pH value of 7.5 ± 0.5. The organic acid salts included from the positively charged soluble high molecular fraction of the bacterial extract were maintained within the high molecular fraction during the process. A similar procedure to 10 L, 100 L and finally 400 L was carried out to prepare pilot and industrial batch sizes using a 10 kD cut-off. Preferably, salts and low molecular weight molecules can be removed by zLRnLn / Lznz / E / Yi ultrafiltration leading to a high molecular weight fraction thereby improving the antiviral properties of the bacterial extract. 500 mL of bacterial extract at pH 10.8 was added to a laboratory scale ultrafiltration system fitted with a 10 kDa polysulfone ultrafiltration filter. After a 4-fold concentration of the initial 500 mL volume to approximately 120 to 140 mL, diafiltration was applied using NaOH-water at pH 11 ± 0.2. After 5 volumes of diafiltration, the retentate was concentrated to 100 mL and the final volume adjusted to 125 mL with water before the addition of the different organic acids in 10 mL aliquots. The process was applied with different bacterial extracts such as purified fractions of OM alkaline extract, purified fractions of Gram-positive bacteria from alkaline extracts of Staphylococcus, Streptococcus, Bifidobacterium, Lactobacillus as well as purified fractions of Gram-negative bacteria from alkaline extracts of Escheríchia coli, Klebsíella strains. , Branhamella, Hemophilus. Table 2 below recapitulates the organic acids that were used to adjust the pH to 7.5. Purified fractions of alkaline bacterial extracts after removal of low molecular weight components were adjusted to pH 7.5 using the following organic acids and their stability evaluated. Rn ι η / ι 7n7 / E / YL Table 2: Acid / organic acid turbidity Stability at 20°C to 25°C Stability at 4°C to 8°C Formic acid slightly cloudy Not stable, cloudy, precipitates Not stable, cloudy, precipitates Propanoic acid slightly cloudy Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Slightly cloudy lactic acid Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Slightly turbid 3-hydroxypropanoic acid Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Butanoic acid clear solution Stable , clear solution, does not precipitate Stable, clear solution, does not precipitate 2-Hydroxybutanoic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate 3-Hydroxybutanoic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution , does not precipitate Aspartic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Glutamic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Example 1.5: Simultaneous removal of small molecules and inorganic salts and concentration of the high molecular fraction of the organic acid-stabilized bacterial extract of Lactobacillus fermentum (OM314BY 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 pm microfilter and a second ultrafiltration filter with a 10 kDalton cutoff. The bacterial extract was purified using both filters as described in example 7.3 and in Figure 1. The first step was a concentration step which was followed by continuous extraction using 10 10 kD infiltrate volumes as the extraction media. Then, a diafiltration process was carried out with 5 volumes of a solution of NaOH in water adjusted to pH 10. A 4-fold final concentration step was added before the addition of the different organic acids forming included salts. The process was carried out on a 1 L scale using a first concentration step (5 times, up to approximately 200 mL), undesirable small molecules were washed out by means of a diafiltration step using 5 volumes of NaOH-water solution at pH 10.5. . So. The organic acids listed in Table 3 to form salts with the positively charged groups present in the extract were added to stabilize the preparation. Similarly, concentration of the high molecular fractions was performed using a concentration step on the ultrafiltration filters by removing the low molecular fractions using other cutoffs, respectively 3 kD, 10 kD, 30 kD, 100 kD, 300 kD from the filters. of respective polysulfone. The organic acids listed in the table below were added with a target pH value of 7.5 ± 0.2. The organic acid salts included from the positively charged soluble high molecular fraction of the bacterial extract were maintained within the high molecular fraction during the process. In another example the process was repeated using the following cuts of the polysulfone ultrafiltration filter, respectively 3 kD, 30 kD, 100 kD, 300 kD. In another example, the removal of salts and low molecular weight molecules was carried out by ultrafiltration leading to a concentrated high molecular weight fraction which improves the antiviral properties of the Lactobacillus fermentum extract. 500 mL of alkaline Lactobacillus fermentum extract at pH 10.8 was added to a laboratory-scale ultrafiltration system mounted with a 10 kDalton polysulfone ultrafiltration filter. After a 4-fold concentration of the initial volume of 500 mL to approximately 120 to 140 mL, diafiltration was applied using NaOH-water at pH 10.8 to 11.0. After 5 volumes of diafiltration, the high molecular weight fraction (retentate fraction) was concentrated to 100 mL and the final volume was adjusted to 125 mL with water before the addition of the different organic acids in 10 mL aliquots to evaluate the physical stability and antiviral activity. The following Table 3 lists the organic acids used to adjust the pH to 7.5 for Lactobacillus fermentum extract fractions > 10 kDalton. z i «n i n / ι znz / E / YL Table 3: Acid / organic acid turbidity Stability from 20°C to 25°C Stability from 4°C to 8°C Formic acid slightly cloudy Not stable, cloudy, precipitates Not stable, cloudy, precipitates Propanoic acid slightly cloudy Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Lactic acid slightly cloudy Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate 3-hydroxy-propanoic acid slightly cloudy Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Butanoic acid clear solution Stable , clear solution, does not precipitate Stable, clear solution, does not precipitate 2-hydroxybutanoic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate 3-hydroxybutanoic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution , does not precipitate Aspartic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate Glutamic acid clear solution Stable, clear solution, does not precipitate Stable, clear solution, does not precipitate In other examples, the process was repeated using microfiltration hollow fibers of 0.45 pm and 0.2 pm and ultrafiltration hollow fibers with cuts of 30 kD and 100 kD as an alternative to the tangential flow filter design with a better flow rate and process time. shorter. A similar process was carried out at 10 L, 100 L and finally at 400 L to prepare pilot and industrial batch sizes with a cut-off of 10 kD. Example 1.6: Process for Lactobacillus fermentum I 3929p (OM314B) and analytical characterization Example 1.6.1 Process 4 of Lactobacillus fermentum I 3929p Lysis: Lactobacillus fermentum 13929p biomass was thawed overnight at room temperature. The lysis carried out was a lysis with a total mass of 2 kg including 25 g of total dry weight after desiccation (RS) per kg of lysis. The necessary amount of biomass (depending on the result of the biomass RS) was deposited in a 2500 mL mini barrel (reference: Semadeni no. 6863) and the 2 kg qsp was made with purified water preheated to 40°C ± 5 °C. The pH of this solution was then adjusted to 10.0 ± 0.1 using 10 N NaOH (pH: 9.98 adjusted with 2.8 mL of 10 N NaOH). The alkaline lysis was transferred in a warm room at 40°C ± 1°C under agitation (to have a 1 cm product vortex). After 4 h 00 ± 5 min of lysis, the pH was controlled (final lysis pH: 9.28). In this step, a corresponding sampling of lysate was carried out at the end of lysis (called Process Use 4E1-I). Filtration 1: The facility for product filtration was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiltration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2 ). The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cut-off point of 30 kDa (ultrafiltration filter in Figure 2). The filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before starting the process, the filtration system was checked for reproducibility over several batches. To verify the correct filtration capacity of the product, a Normalized Water Permeability (NWP) was carried out on the filtration system. The Used used for production was agitated to have a 1 cm product vortex. The product temperature 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. Initial concentration step: The product used for the first filtration step had the following parameters: no pH adjustment after lysis (pH: 9.28), temperature 38°C, stirred to have a 1 cm product vortex. The MF loop pump (pump 1 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF loop was opened to carry out an initial concentration of the product with a concentration factor of 0.5 (inlet pressure: 270 mbar, infiltrate flow rate: 75 mL / min). During the initial concentration, the pump speed (pump 1 in Figure 2) was gradually increased up to 100% (100 Rn ι η / ι 7n7 / E / YL rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation over the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Filtration When the concentration factor of half (0.5x) was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 793 mbar, infiltrate flow rate: 53 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in Figure 2, should be set to 850 mbar (inlet pressure: 1060 mbar, infiltrate flow rate: 47 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted to achieve a UF infiltrate flow rate equal to the MF infiltrate flow. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was carried out per cycle. At the end of the initial concentration, a volume was present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. When the MF inlet pressure began to increase, the MF pump was shut down. At the end of this first filtration step, the product of interest contained elements smaller than 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 996.0 g) was then subjected to a second stage of 5 purification cycles in the UF loop with a cutoff of 30 kDa (vessel 3, pump 2 and ultrafiltration filter in Figure 2). The 30 kDa infiltrate was discarded with the valve open toward the waste (Figure 2). The volume of the 30 kDa retentate was kept constant during this second filtration purification step, adding 0.001 N NaOH solution at pH 10.0 for diafiltration. The UF loop pump (pump 2 in Figure 2) started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 640 mbar, infiltrate flow rate: 108 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, must be set to 850 mbar (inlet pressure: 900 mbar, infiltrate flow rate: 134 mL / min). During filtration step 2, the volume of the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. So, when the level of the UF tank (vessel 3 in Figure 2) decreased, there was an addition of 0.001 M NaOH solution. The 5 cycles corresponding to a volume of diafiltration solution «η ι η / ι 7n7 / E / YL of 0.001 M NaOH added the equivalent of 5 times the volume of product of interest harvested (5 diafiltration cycles). At the end of filtration step 2, the final product was harvested (mass: 951.4 g) and then separated into 2 equal parts. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 pm and larger than 30 kDa in size. In this step, a sample corresponding to neutralization was carried out before filtration (called filtrate E2 of process 4). The first part of the filtrate was then neutralized with 1% propionic acid to pH 7.0 ± 0.2 (pH: 7.13 adjusted with 0.5 mL of 1% propionic acid), then sterilized under a biosafety cabinet using a filter with a sterilizing polyethersulfone membrane. from 0.2 pm (PES 0.2 pm). In this step, a sample was carried out corresponding to the neutralized filtrate at the end of the process (called neutralized filtrate (propionic acid) E3 from the 4” OM314B process). At the same time, the second part of the filtering was subdivided into 9 equal parts. Each of those parts was then neutralized to 7.0 ± 0.2 with 2.5% hydrochloric acid (pH: 7.07) or with organic acids (OM314B): formic 1 / 100 (pH: 7.16), acetic 1 / 100 (pH: 7.16) , 3-hydroxy-butanoic 1 / 100 (pH: 7.14), aspartic 0.1% (pH: 7.18), lactic 1 / 50 (pH: 7.09), glutamic 0.1% (pH: 7.14), pyruvic 1 / 100 ( pH: 7.16), ascorbic 0.1% (pH: 7.18). Finally, the different products were sterilized under a biosafety cabinet using filtration with a 0.2 pm PES sterilizing membrane. In this step, a sample was carried out corresponding to the different neutralized filtrates at the end of the process (called neutralized filtrate (acid name) E4 from Process 4). Table 4: Summary of sample codes Rn ι η / ι 7n7 / E / YL Used Used E1 of Process 4 Lysed Filtration E2 of Process 4 Standard Neutralized Filtration Neutralized Filtration (propionic acid) E3 of Process 4 Neutralized Filtration with Acid 1 Neutralized Filtration (hydrochloric acid) E4 of Process 4 Neutralized Filtration with Acid 2 Neutralized Filtration (formic acid ) E4 of Process 4 Neutralized Filtrate with Acid 3 Neutralized Filtrate (acetic acid) E4 of Process 4 Neutralized Filtrate with Acid 4 Neutralized Filtrate (3-hydroxy-butanoic acid) E4 of Process 4 Neutralized Filtrate with Acid 5 Neutralized Filtrate (aspartic acid) E4 of Process 4 Neutralized Filtrate with Acid 6 Neutralized Filtrate (lactic acid) E4 of Process 4 Neutralized Filtrate with Acid 7 Neutralized Filtrate (glutamic acid) E4 of Process 4 Neutralized Filtrate with Acid 8 Neutralized Filtrate (pyruvic acid) E4 of Process 4 Filtrate Neutralized with Acid 9 Neutralized filtrate (ascorbic acid) E4 from Process 4 Example 1.6.2 Analytical Characterization Method a) Dry weight method (Used) The determination of the dry lysate residue was carried out by halogen desiccation following the principles of thermogravimetry: at the beginning of the measurement operation, the weight of the sample was defined, ours was then heated rapidly with an integrated halogen heater and evaporated humidity. During drying, the device weighed the sample continuously. Once drying was completed, the weight of the dry residue was indicated. Approximately 2 to 5 g of Used (M) were accurately weighed. The following parameters were used: Heating mode: progressive; Interrupt mode: constant weight; Final temperature: 105°C. Once the analysis was completed, the weight ticket indicating the final mass of the residue obtained (m) was automatically printed. Dry residue (expressed in mg / g) = (m / M) x 1,000. Measurements were carried out on the sample directly (total) and after centrifugation for 5 min at 5,000 x g (supernatant). b) Dry weight method (filtered) The dry weight of the filtrate was carried out according to Ph. Eur. 2.2.32 using approximately 5 g of filtrate dried for 16 hours at 105°C (oven). The results were expressed in mg / g. c) Lowry method This assay was based on the reaction of proteins with an alkaline solution of copper tartrate and Folin's reagent (based on Ph. Eur. 2.5.33). There were two steps that led to a colorimetric reaction: the reaction between proteins and copper in an alkaline medium and the subsequent reduction of the Folin reagent by the protein treated with copper. The proteins carried out a reduction of the Folin reagent producing reduced species which had a characteristic blue color with a maximum absorbance at 750 nm. The results were expressed relative to a Bovine Serum Albumin (BSA) protein standard curve. Sample preparation: 1.9 mL of pH 11 phosphate buffer (3.55 g of Na2HPO4 and 41 mL of 0.1 M NaOH per 1 L of water) was added to 100 pL of each sample and shaken. Preparation of BSA standard: The BSA solution was diluted with phosphate buffer to prepare the standard curve with 6 points between 0 and 420 pg / mL. Procedure: 20 pL of samples and standard solutions were loaded into a 96-well microplate. 25 pL of reagent A (Lowry Bio-Rad® kit) was immediately added to each well and incubated for 10 min at room temperature. 200 pL of reagent B (Lowry Bio-Rad® kit) was added, mixed and incubated for 20 min at room temperature. Absorbances were read at 750 nm after mixing. Results: Protein concentrations were calculated from the standard curve: Protein Concentration (mg / mL) = [(y - b) / a] * sample dilution, with y = sample absorbance; a = slope calibration curve; b = ordered to the origin of the calibration curve and the results were expressed as mg of protein / mL. d) Total sugar method Carbohydrates, when heated with anthrone in a sulfuric medium, form a chromophore that absorbs at 625 nm. Preparation of Glucose Standard: D-Glucose was solubilized and diluted with purified water to prepare the standard curve with 5 points between 0 and 100 (pg / mL). Procedure: 0.1 mL of the solution (i.e. the concentrate) was examined and 0.9 mL of purified water was added to a tube placed in an ice bath. 5.0 mL zLRnLn / Lznz / B / Yi of Antrone reagent (160 mg of Antrone in 100 mL of 85% sulfuric acid) was added and shaken vigorously. The solutions were heated for 15 min in a boiling water bath, then cooled in an ice bath and stirred occasionally. The solutions were allowed to stand at room temperature for 15 min. The tubes were shaken and the solutions were transferred to the measurement cell and allowed to stand for 30 min at room temperature before absorbance measurement at 625 nm. Results: Carbohydrate concentrations were calculated from the standard curve: Carbohydrate content (mg / mL) = ([(y - b) / a] * 10) / (1,000), with y = sample absorbance; a = slope of the calibration curve; b = ordered to the origin of the calibration curve and the results were expressed as mg carbohydrates / mL. e) Total RNA assay Total RNA purification and assay were carried out based on the RNeasy® Mini kit data sheet according to the supplier's recommendations. Briefly, 600 pL of bacterial extract was transferred to the spin column of the RNeasy® and centrifuged for 15 s at 8,000 x g. Next, 700 μl of RW1 buffer was added to the RNeasy® column and a wash and spin column membrane was centrifuged for 15 s at 8,000 x g. The step was carried out twice with 500 pL of RPE buffer and concentrated for 15 s at 8,000 x g and for 2 min at 8,000 x g, consecutively. To eliminate any possible RPE buffer carryover, the spin column was centrifuged at full speed for 1 min. To elute the RNA, 30 μl of RNase-free water was added directly to the spin column membrane and the column was centrifuged for 1 min at 8,000 × g. Purified total RNA was detected at 260 nm with the NanoDrop spectrophotometer (ThermoFischer). f) Total DNA Assay Total DNA purification and assay were carried out based on the DNeasy® Blood and Tissue kit data sheet according to the supplier's recommendations. Briefly, 600 pL of bacterial extract was transferred to the DNeasy® mini spin column and centrifuged for 1 min at 6,000 x g. Next, 500 µl of AW1 buffer was added to the DNeasy® mini column and centrifuged for 1 min at 6,000 x g. 500 µL of AW2 buffer was added and the spin column was centrifuged for 3 min at 20,000 x g to dry the DNA membrane. 100 µl of AE buffer were added on the DNeasy® membrane, incubated for 1 min and centrifuged for 1 min at 6,000 x g to elute the DNA. For the maximum yield of DNA, the elution was repeated once. Purified total DNA was detected at 260 nm with a NanoDrop spectrophotometer. g) Limulus amebocyte Used LAL Assay The endotoxin assay was carried out based on the Pierce™ Chromogenic Endotoxin Quant Kit data sheet according to the supplier's recommendations. All samples were diluted 1:10 to avoid altered sample intrinsic color of absorbance readings. Briefly, endotoxin standard solutions (range 0.1-1.0 EU / mL) were prepared from the endotoxin standard solution (10 EU / mL). 50 pL of endotoxin standard, blank (water without endotoxin) and samples were placed per well. 50 pL of reconstructed Amebocyte Used Reagent were added and the plate was incubated for 15 min at 37°C. 100 pL of Chromogenic Substrate Solution were added per well and incubated at 37°C for 6 min. At exactly 6 min, 50 pL of stop solution (25% acetic acid) was added. The optical density was measured at 405 nm immediately after completion of the ΑΓ>I n / Ι 7O7 / E / Yl· test. h) Amino acid method: The determination of D- and L-amino acid was carried out by reversed-phase high-performance liquid chromatography (HPLC). After hydrolysis of the samples in a microwave oven. Amino acids were derived using o-phthaldialdehyde together with chiral N-isobutyryl-L-cysteine ​​thiol. Detection was carried out by UV detection at 338 nm. (Brückner, Η,, 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 -twenty-one). Standard solutions were prepared with the different amino acids: Aspartic acid (Asp), Serine (Ser), Glutamic acid (Glu), Histidine (His), Arginine (Arg), Threonine (Thr), Alanine (Ala), Tyrosine (Tyr) , Valine (Val), Methionine (Met), Usine (Lys), Isoleucine (lie), Leucine (Leu), Phenylalanine (Phe), Glycine (Gly), cystine (Cys)) at 2.5 pmol / mL in hydrochloric acid ( HCl) 0.01 N. Those solutions were diluted to 0.5 pmol / mL using 0.1 M sodium tetraborate decahydrate buffer at pH 9.2. In a tube, 2.0 mL of sample solutions were added to 2.0 ml of water, 240 μΙ of 1-dodecanethiol and 8 mL of 25% HCl. They were hydrolyzed in a microwave oven at 180°C, 1320 Watts for 15 min. The solutions were allowed to stand at room temperature and filtered at 5 pm. 50.0 μL was evaporated to dryness and reconstituted with 100 μL of 0.01 N HCl. The sample and standard solutions were kept in the HPLC system at 10°C and injected with the following injection mode: 5.0 μL of tetraborate buffer of sodium decahydrate at 0.1 M at pH 9.2, 2.0 pl of derivatization solution (23 mg of phthaldialdehyde and 50 mg of N-isobutyryl-Lcysteine ​​solubilized in 1.0 ml of methanol), 2.0 pl of sample or standard solution were removed by means of an automatic sampler, they were mixed 5 times and the injected column was Supelcosil C18 HPLC, 5 pm, 4.6 x 250 mm, with the Supelco LC 18 guard column, 5 pm, 4.6 x 20 mm. For elution, a gradient of 23 mM sodium acetate adjusted to pH 5.9 (eluent A) and a mixture of methanol-acetonitrile (12:1, v / v) (eluent B) was formed. The gradient was formed from 4% B to 33% B in 45 min, then to 56% B for 30 min (and 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 Filter Solution E2 directly into HPLC flasks and no free amino acids were detected without hydrolysis. i) Spectrophotometric results obtained during stability The neutralized filtered solutions were placed in stability at room temperature (20°C + / - 5°C) or at 4°C. At each time point, the absorbance of solutions between 300 and 700 nm was recorded. The spectral profiles were visually evaluated as noisy (e.g., Figure 27 - indicating precipitation in solution) or smooth (e.g., Figure 27). Absorbances were extracted at 320 nm for quantitative evaluation. j) Results in release: All E2 Filtration Solutions were frozen after processing and thawed at 4°C overnight before analysis. Example 1.6.3. Analytical Characterization of the final samples of Lactobacillus fermentum I 3929p from Process 4 in the release (T0) Table 5: Results of Process 4 zLRnLn / Lznz / B / Yi samples Test Sample Result Unit Dry weight (total) Used E1 from Process 4 27.7 [mg / g] Dry weight (supernatant) Used E1 from Process 4 5.5 [mg / g] Dry weight (filtered) Filtrate E2 from Process 4 1.5 [mg / g / g] Total proteins Filtrate E2 from Process 4 0.44 [mg / mL] Total sugar Filtrate E2 from Process 4 0.21 [mg / mL] Endotoxin LAL* Neutralized filtrate (propionic acid) E3 from Process 4 ND [EU / mL] DNA** Neutralized filtrate (propionic acid) E3 from Process 4 ND [pg / mL] RNA*** Neutralized filtrate (propionic acid) E3 from Process 4 ND [pg / mL] ΖίβΠίΠ / ίΖηΖ / Ε / ΥΙ ND = not detected; *LAL endotoxin: detection limit = 0.1 EU / mL; **DNA: detection limit = 3.60 pg / mL, quantification limit = 12.01 pg / mL; ***RNA: detection limit = 4.29 pg / mL, quantification limit = 14.32 pg / mL Table 6: Process 4 - Total amino acids Amino Acid Concentration (pmol / mL) % / AA (D vs. L) L-Asp 0.00 NA D-Asp 0.00 NA L-Glu 0.17 100 D-Glu 0.00 0 L-Ser 0.00 NA D-Ser 0.00 NA L-Thr 0.00 0 D-Thr 0.31 100 L-His 0.00 NA Gly 0.00 NA D-His 0.00 NA L-Ala 0.11 NA L-Arg 0.04 NA Amino Acid Concentration (pmol / mL) % / AA (D vs. L) D-Arg + D-Ala 0.00 NA L-Tyr 0.00 NA D-Tyr 0.00 NA L-Val 0.00 NA L-Met 0.00 NA D-Met 0.00 NA L -Cys 0.00 NA D-Val 0.00 NA L-lle 0.00 NA L-Phe 0.00 NA D-Phe 0.00 NA L-Leu 0.32 100 D-lle 0.00 NA D-Leu 0.00 0 L-Lys 0.08 100 D-Lys 0.00 0 ΖίβΠίΠ / ίΖΓΙΖ / Ε / ΥΙ Table 7: Process 4 - solution stability through absorbance measurement Time Points (Months) Process 4 Propionic Acid O o o o Aspartic Acid Formic Acid Lactic Acid 3-Hydroxybutanoic Acid Ascorbic Acid Acetic Acid Pyruvic Acid Glutamic Acid HCL Industrial Lot 1619064 T0 Abs [AU] 0.837 0.833 0.809 0.841 0.846 0. 835 0.815 0.84 0.844 0.794 2.865 Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth T1 4°C Abs [AU] 0.852 0.847 0.829 0.857 0.857 0.851 0.836 0.869 0.866 0.807 3.17 R(%) 102% 102% 102% 102% 101% 102% 103% 103 % 103% 102% 111% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy TA Abs [AU] 0.821 0.848 0.793 0.84 0.82 0.833 0.811 0.821 0.839 0.796 3.17 R[%) 98% 102% 98% 100% 97 % 100 % 100% 98% 99% 100% 111% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy T3 4°C Abs [AU] 0.838 0.844 0.817 0.832 0.862 0.84 0.835 0.851 0.86 0.803 3.148 R[%) 100% 1 01% 101% 99% 102% 101% 102% 101% 102% 101% 110% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy TA Abs [AU] 0.816 0.853 0.808 0.848 0.823 0.85 0.823 0.825 0.851 0.806 3,159 R(%) 97% 102% 100% 101% 97% 102% 101% 98% 101% 102% 110% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy T6 4°C Abs [AU] 0.884 0.866 0.804 0.837 0.834 0.819 0.816 0.8 39 0.836 0.778 3.046 R(%) 106% 104% 99% 100% 99% 98% 100% 100% 99% 98% 106% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy TA Abs [AU] 0.81 0.914 0.837 0.879 0.863 0.878 0.857 0.859 0.906 0.835 2.985 R(%) 97% 110% 103% 105% 102% 105% 105% 102% 107% 105% 104% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy Rn i n / ι 7r>7 / E / Yl· Abs: absorbance at 320 nm; RT: room temperature (20°C + / - 5°C); Evaluation: visual spectrum evaluation Industrial batch described in WO 2008 / 109669 (OM-13-BV) neutralized with hydrochloric acid (HCL industrial batch 1619064) presented a precipitate starting at T0 immediately after neutralization. Neutralized E4 filtrates from Process 4 were physically stable at 4°C or room temperature for at least 6 months. They were considered stable in those conditions for 12 months. Mip3-alpha (CCL20) results obtained during stability: Chemokine (C-C motif) ligand 20 (CCL20) or liver activation-regulated chemokine (LARC) or Macrophage Inflammatory Protein-3 (ΜΙΡ-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 via chemoattraction of lymphocytes and dendritic cells to the epithelial cells surrounding these tissues. CCL20 produces its effect on its target cells by binding to and activating the CCR6 chemokine receptor. The THP-1 cell line was purchased from the ATCC collection, #TIB-202. The THP-1 cell line was derived from the peripheral blood of a 1-year-old male with acute monocytic leukemia. A THP-1 Working Cell Bank flasks were used as macrophage-like cells after differentiation in this bioassay. The neutralized filtered solutions were placed in stability at room temperature (20°C + / - 5°C) or at 4°C. Samples were taken at different time points and frozen for further bioassay. All solutions (TO and stability time points) were thawed at 4°C overnight before analysis. Differentiation: THP-1 cells were differentiated using Phorbol 12-Myristate 13-Acetate (PMA) to have a final concentration of 100 ng / mL of PMA in the cell suspension (1 x 106 cells / mL). 100.0 pL / well of PMA cell suspension was distributed in each well of the 96-well cell culture plate. The cells were incubated 72 hours at 37°C. Stimulation: A 10-point serial dilution (3.16-fold serial dilution) was performed with culture medium in a deep-well plate: from 2 μg / mL to 0.06 pg / mL of PAM3CSK4 (reference positive control for secretion). of MIP-3a). A 6-point serial dilution (3.16-fold serial dilution) was performed with culture medium in a deep-well plate: 200 pL of Test Sample in the first well of deep-well culture medium +400 pL, then 190 pL were diluted with 410 pL of culture medium (3.16 times). 100 pL / p of plate supernatants with cells were removed. 100.0 pL / p of culture medium were distributed in each well of the plate with cells. The plate was incubated 24 hours at 37°C. Harvest of supernatant: 75 pL of supernatant was harvested from each well and distributed in a 96 PP microplate. The plate was sealed and stored in a deep freezer until the ELISA assay was performed. ELISA test: Microplate wells were coated with 100.0 pL / well anti-human MIP-3a (capture antibody). The plate was covered with a sealing film and appropriately incubated. After completion of the incubation, the washing steps were performed with an automatic microplate washer. On the day of the saturation step, the supernatant plate was thawed at +4°C. Saturation step: 250.0 pL / well of reagent diluent (1% BSA in PBS) was added and plates were incubated as deemed appropriate. During the saturation step, serial dilutions for MIP-3a supernatants and standards were prepared. A standard curve was run on each ELISA plate. After completing the incubation, washing steps were carried out and the samples were distributed in the wells, the plates were incubated as considered appropriate. After completing the incubation, washing steps were performed and biotinylated goat anti-human MIP-3o was dispensed (Detection Antibody Step). Plates were incubated as deemed appropriate. After completion of the incubation, washing steps were performed and the streptavidin-HRP conjugate dispensed. Plates were incubated as deemed appropriate. After completion of the incubation, washing steps were performed, enzyme substrate addition was performed, and Do was read at 450 nm. Blank reduction and wavelength correction to 540 nm were performed according to the recommendations of the ELISA equipment supplier. Process 4 - Bioassay results during stability show that the E3 neutralized filtrates from Process 4 were stable for at least 1 month at room temperature (20°C + / - 5°C) or 4°C. Example 1.7 Mixture of polyalkaline waste from 18 strains of E. coli Example 1.7.1. Used polyalkaline mixture of 18 strains of E. cotí from Process No. 5 (OM314C) Alkaline lysis: One part of the 18-strain E. coli mixture used alkaline as described in the Rn ι η / ι 7n7 / E / YL WO2008 / 109667 was recovered in production and stored in a 2500 mL mini barrel (reference: Semadeni no. 6863). In this step, a sample corresponding to the Used at the end of the lysis was carried out (called “Used E1 of process 5”). Filtration 1: The facility for product filtration was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiltration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2) . The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cut-off point of 30 kDa (ultrafiltration filter in Figure 2). The filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before starting the process, we had made sure that the filtration system was reproducible over batches. To verify the correct filtration capacity of the product, a NWP (Normalized Water Permeability) was carried out on the filtration system. The Used used for production was first diluted 4 times with purified water (500.8 g of Used and 1502.7 g of purified water). The product was stirred to have a 1 cm product vortex. The product temperature was cooled to room temperature while waiting for the filtration process to begin. In this process there was a pH adjustment to pH 10.5 -10.8 (pH: 10.68 adjusted with 2.1 mL of pure pyruvic acid). Initial concentration: The product used for the first filtration step had the following parameters (pH: 10.68, temperature 29°C, stirred to have a product vortex of 1 cm). The MF loop pump (pump 1 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, product recirculation over the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF in loop was opened to carry out an initial concentration of the product with a concentration factor up to 0.5 (inlet pressure: 200 mbar, infiltrate flow rate: 89 mL / min). During the initial concentration, the pump speed (pump 1 in Figure 2) was gradually increased to 100% (100 rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Diafiltration: When the concentration factor of 0.5 was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 786 mbar, infiltrate flow rate: 80 mL / min). To have optimal extraction of the product, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in the zLRnLn / Lznz / E / Yi Figure 2, should be set to 850 mbar (inlet pressure: 950 mbar, infiltrate flow rate: 60 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted until a UF infiltrate flow rate equal to the MF infiltrate flow was reached. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was carried out per cycle. At the end of the initial concentration, there was a volume present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. when the MF inlet pressure began to increase, the MF pump was turned off. At the end of this first step, the product of interest contained smaller elements of 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 1146.3 g) was then subjected to a second stage of 5 purification cycles in the UF loop with a cut-off point of 30 kDa (vessel 3, pump 2, the ultrafiltration filter in the Figure 2). The infiltrate was discarded as mentioned in Figure 2. The volume was kept constant during this second purification step by filtration, by adding 0.001 N NaOH solution at pH 10.0. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 650 mbar, infiltrate flow rate: 76 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, should be set to 850 mbar (inlet pressure: 960 mbar, infiltrate flow rate: 150 mL / min). During filtration step 2, the volume in the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. So, when the UF tank level (vessel 3 in Figure 2) decreased, there was an addition of NaOH solution. The 5 cycles corresponding to one volume of NaOH solution added the equivalent of 5 times the volume of product of interest harvested. At the end of filtration step 2, the final product (mass: 1042.7 g) was harvested and then separated into 2 equal parts. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 pm and larger than 30 kDa in size. In this step, a sample corresponding to the filtrate before neutralization was carried out (called “filtrate E2 from process 5”). The first part of the filtrate was then neutralized with 1 / 100 pyruvic acid to pH 7.0 ± 0.2 (pH: 7.8 adjusted with 3 mL of 1 / 100 pyruvic acid), then sterilized under a safety cabinet using PES sterilizing membrane filtration. from 0.2 p.m. In this step, a sample was carried out corresponding to the neutralized filtrate at the end of the process (called “neutralized filtrate (pyruvic acid) E3 from process 5”, OM314C). Rn ι η / ι 7n7 / E / YL At the same time, the second part of the filtering was subdivided into 9 equal parts. Each of these parts was then neutralized to 7.0 ± 0.2 with 0.25% hydrochloric acid (pH: 7.09) or with organic acids (OM314C): formic 1 / 100 (pH: 7.11), acetic 1 / 100 (pH: 6.97), 3-hydroxy-butanoic 1 / 100 (pH: 7.03), aspartic 0.1% (pH: 7.09), lactic 1 / 100 (pH: 7.15), glutamic 0.1% (pH: 7.10), propionic 1 / 100 (pH: 7.10 ), pure ascorbic (pH: 7.04)). Finally, the different products were sterilized under a safety cabinet using filtration with a 0.2 pm PES sterilizing membrane. In this step, a sample was carried out corresponding to the neutralized filtrates at the end of the process (called neutralized filtrate (name of the acid) E4 of Process 5”). Table 8: Summary of sample codes: Rn ι η / ι 7n7 / E / YL Used Used E1 from process 5 Filtrate E2 filtrate from process 5 Neutralized filtrate Standard Neutralized filtrate (pyruvic acid) E3 from Process 5 Neutralized filtrate with acid 1 Neutralized filtrate (hydrochloric acid) E4 from Process 5 Neutralized filtrate with acid 2 Neutralized filtrate (formic acid ) E4 from Process 5 Acid neutralized filtrate 3 Neutralized filtrate (acetic acid) E4 from Process 5 Acid neutralized filtrate 4 Neutralized filtrate (3-hydroxy-butanoic acid) E4 from Process 5 Acid neutralized filtrate 5 Neutralized filtrate (aspartic acid) E4 from Process 5 Acid neutralized filtrate 6 Neutralized filtrate (lactic acid) E4 from Process 5 Acid neutralized filtrate 7 Neutralized filtrate (glutamic acid) E4 from Process 5 Acid neutralized filtrate 8 Neutralized filtrate (propionic acid) E4 from Process 5 Filtration neutralized with acid 9 Neutralized filtrate (ascorbic acid) E4 from Process 5 Example 1.7.2 Analytical Characterization Analytical methods are described in 1.6.2 Analytical Characterization of the final samples of process 5 in the release (T0) E2 filtrate solutions were frozen after processing and thawed at 4°C overnight before analysis. Table 9: Process 5 - Analytical Results Test Sample Result Unit Dry weight (total) Used E1 from process 5 11.6 [mg / g] Dry weight (supernatant) Used E1 from process 5 12.0 [mg / g] Dry weight (filtered) Filtrate E2 from process 5 8.0 [mg / g / g] Total proteins Filtrate E2 from process 5 5.9 [mg / mL] Total sugar Filtrate E2 from process 5 0.19 [mg / mL] Endotoxins LAL* Neutralized filtrate (pyruvic acid) E3 from process 5 ND [EU / mL] DNA** Neutralized filtrate (pyruvic acid) E3 from process 5 13.2 [pg / mL] RNA*** Neutralized filtrate (pyruvic acid) E3 from process 5 6.9 [pg / mL] zLRnLn / Lznz / B / Yi ND = not detected; *LAL endotoxin: detection limit = 0.1 EU / mL; **DNA: detection limit = 3.60 pg / mL, quantification limit = 12.01 pg / mL; ***RNA: detection limit = 4.29 pg / mL, quantification limit = 14.32 pg / mL. Table 10: Process 5 - Total amino acids after hydrolysis with HCl Amino Acid Concentration (pmol / mL) % / AA (D vs. L) L-Asp 0.74 62 D-Asp 0.46 38 L-Glu 1.17 69 D-Glu 0.52 31 L-Ser 0.29 51 D-Ser 0.28 49 L-Thr 0.44 14 D-Thr 2.69 86 L-His 0.00 0 Gly 0.21 NA D-His 0.19 100 L-Ala 1.26 NA L-Arg 0.94 NA D-Arg + D-Ala 0.43 NA L-Tyr 0.51 76 D-Tyr 0.16 24 L-Val 0.83 84 L-Met 0.31 100 D-Met 0.00 0 L-Cys 0.11 NA D-Val 0.16 16 L-lle 0.44 100 L -Phe 0.70 66 D-Phe 0.36 34 L-Leu 1.53 91 D-lle 0.00 0 a) Spectrophotometric results obtained during stability Table 11: Process 5 - solution stability through absorbance measurement Time points (Months) Process 5 Pyruvic Acid Hydrochloric Acid Aspartic Acid Formic Acid Lactic Acid 3hydroxybutanoic Acid Ascorbic Acid Acetic Acid Propanoic Acid Glutamic Acid HCL Industrial Batch 1619064 T0 Abs [AU] 1,668 1,559 1,273 1,634 1,567 1,591 1,602 1.59 1,847 1,394 2,865 Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy T1 4°C Abs [AU] 1.687 1.592 1.259 1.622 1.589 1.589 2.262 1.599 1.605 1.405 3.17 R(%) 101% 102% 99% 99% 101% 100% 14 1% 101% 87% 101% 111% Evaluation smooth smooth smooth smooth smooth smooth noisy smooth smooth smooth noisy TA Abs [AU] 1,679 1,565 1,265 1,631 1,602 1,598 2,868 1,595 1,597 1,397 3.17 R(%) 101% 100% 99% 100% 102% 100% 179% 100% 86% 100% 111% Evaluation smooth smooth smooth smooth smooth smooth rriidOSO smooth smooth smooth noisy T3 4°C Abs [AU] 1.694 1.64 1.295 1.643 1.61 1.601 2.466 1.592 1.649 1.408 3.148 R(%) 10 2% 105% 102 % 101% 103% 101% 154% 100% 89% 101% 110% Evaluation smooth smooth smooth smooth smooth smooth knot» smooth smooth smooth noisy TA Abs [AU] 1,693 1,601 1,285 1,655 1.62 1,622 3,094 1,657 1.63 1,433 3.1 59 R(%) 101% 103% 101% 101% 103% 102% 193% 104% 88% 103% 110% Evaluation smooth smooth smooth smooth smooth smooth noisy smooth smooth smooth rudcso' T6 4°C Abs [AU] 1,665 1,559 1,243 1,607 1,569 1,563 2.6 42 1,576 1,565 1,376 3,046 R(%) 100% 100% 98% 98% 100% 98% 165% 99% 85% 99% 106% Evaluation smooth smooth smooth smooth smooth smooth noise smooth smooth smooth noise» TA Abs [AU] 1,707 1,657 1,337 1,728 1,672 1,691 2,964 1,683 1,686 1,427 2,985 R(%) 102% 106% 105% 106% 107% 106% 185% 106% 91% 102% 104% Evaluation smooth smooth smooth smooth smooth iíWO smooth smooth smooth noisy Abs: absorbance at 320 nm; RT: room temperature (20°C + / - 5°C); Evaluation: visual Spectrum evaluation Industrial Batch 1619064 described in WO 2008 / 109669 OM-13-BV was neutralized with hydrochloric acid and presented a precipitate starting at T0. Except for ascorbic acid, the E4 neutralized filtrates from Process 5 were physically stable at 4°C or room temperature for at least 6 months. They are considered stable in these conditions for 12 months. Mip3-alpha (CCL20) results obtained during stability: Figure 32: Process 5 - bioassay results during stability show that the neutralized filtrate E3 Process 5 exhibited comparable bioactivity through secretion of MIP-3o into THP-1 for at least 5 months at room temperature (20°C + / - 5°C) or 4°C. The T0 of process 5 was compared to T5 samples stored at 4°C and at room temperature (RT) for 4 months. 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 Streptococcuspneumoniae 7 4QQ biomass (lot 1418123 - boxes 34 and 35) was thawed overnight at room temperature in a lysis barrel. 240 g of 10 N NaOH and 4293 g of 8 g / L NaCl solution were added to have a total lysis weight of 7327 g. The alkaline lysis was transferred to a warm room at 37°C ± 2.5°C with shaking at 150 rpm ± 5 rpm for 8 days. After 3 h 00 ± 30 min of lysis, the OD of JO was monitored. The sample was diluted 100 times and read with a spectrophotometer at 700 nm (read OD: 0.258 and final OD: 25.8). Each working day, agitation (150 rpm ± 5 rpm), hot ambient temperature (37.0°C ± 2.5°C) and pH (J1 pH: 12.63 / J2 pH: 12.62 / J5 pH: 12.59 / J6 pH) were controlled. : 12.65 / J7 pH: 12.69 / J8 pH: 12.71). If the pH did not fall within the process range, an adjustment with 10 N NaOH had to be performed (J1: 10 mL of 10 N NaOH / J2: 10 mL of 10 N NaOH / J5: 10 mL of 10 N NaOH / J6: 10 ml of 10 N NaOH / J7:10 ml of 10 N NaOH). At the end of lysis the OD of JO was monitored. The sample was diluted 5 times and read with a spectrophotometer at 700 nm (read OD: 0.092 and final OD: 0.46). The DO delta between JO and J8 had to be greater than 12.8 (Delta DO: 25.34). A part of this Streptococcus pneumoniae 7466 lysis was recovered and stored in a 2500 mL mini-barrel (reference: Semadeni no. 6863). In this step, a sample corresponding to the lysate at the end of the lysis was carried out (called “lysate E1 of process 3”). Filtration 1: The facility for product filtration was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiltration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2) . The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cut-off point of 10 kDa (ultrafiltration filter in Figure 2). The filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before starting the process, we had to ensure that the filtration system was reproducible over batches. To verify the correct filtration capacity of the product, a NWP (Normalized Water Permeability) was carried out on the filtration system. Rn ι η / ι 7n7 / E / YL The Used used for the product was first diluted 2 times with purified water (1000.0 g of lysate and 1000.0 g of purified water). The product was stirred to have a 1 cm product vortex. The product temperature was cooled to room temperature while waiting for the filtration process to begin. In this process, a pH adjustment was carried out to pH 10.5 -10.8 (pH: 10.68 adjusted with 15 mL of pure 3-hydroxy-butanoic acid). Initial concentration: The product used for the first filtration step had the following parameters (pH: 10.68, temperature 29°C, stirred to have a product vortex of 1 cm). The MF loop pump (pump 1 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, product recirculation over the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF in loop was opened to carry out an initial concentration of the product with a concentration factor up to 0.5 (inlet pressure: 210 mbar, infiltrate flow rate: 50 mL / min). During the initial concentration, the pump speed (pump 1 in Figure 2) was gradually increased to 100% (100 rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Diafiltration: When the concentration factor of 0.5 was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 680 mbar, infiltrate flow rate: 47 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in Figure 2, should be set to 850 mbar (inlet pressure: 995 mbar, infiltrate flow rate: 46 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted until a UF infiltrate flow rate equal to the MF infiltrate flow was reached. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was carried out per cycle. At the end of the initial concentration, there was a volume present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Final concentration: at the end of the 5 diafiltration cycles, the UF pump was stopped. when the MF inlet pressure began to increase, the MF pump was turned off. At the end of this first step, the product of interest contained smaller elements of 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 945.7 g) was then subjected to a second stage of 5 purification cycles in the UF loop with a cut-off point of 10 kDa (vessel 3, pump 2, the ultrafiltration filter in the Figure 2). The infiltrate was discarded as mentioned in Figure 2. The volume was kept constant during Rn ι η / ι 7n7 / E / YL this second purification step by filtration, by adding 0.001 N NaOH solution at pH 10.0. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 690 mbar, infiltrate flow rate: 37 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, should be set to 850 mbar (inlet pressure: 945 mbar, infiltrate flow rate: 52 mL / min). During filtration step 2, the volume in the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. So, when the UF tank level (vessel 3 in Figure 2) decreased, there was an addition of NaOH solution. The 5 cycles corresponding to one volume of NaOH solution added the equivalent of 5 times the volume of product of interest harvested. At the end of filtration step 2, the final product (mass: 931.7 g) was harvested and then separated into 2 equal parts. At the end of this second filtration step, the product of interest contained elements smaller than 0.45 pm and larger ones of 10 kDa in size. In this step, a sample corresponding to the filtrate before neutralization (called (“filtrate E2 of process 3”) was carried out. The first part of the filtrate was then neutralized with 1 / 100 3-hydroxy-butanoic acid to pH 7.2 ± 0.2 (pH: 7.25 adjusted with 10 mL of 1 / 100 3-hydroxy-butanoic acid), then sterilized under a cabinet. safety using 0.2 pm PES sterilizing membrane filtration. In this step, a sample corresponding to the neutralized filtrate at the end of the process was carried out (called “neutralized filtrate (3-hydroxy-butanoic acid) E3 from process 3”). At the same time, the second part of the filtering was subdivided into 9 equal parts. Each of these parts was then neutralized to 7.2 ± 0.2 with 2.5% hydrochloric acid (pH: 7.19) or with organic acids (OM314A): formic 1 / 100 (pH: 7.16), acetic 1 / 100 (pH: 7.16), pyruvic 1 / 100 (pH: 7.14), aspartic 1 / 100 (pH: 7.19), lactic 1 / 100 (pH: 7.06), glutamic 0.1% (pH: 7.13), propionic 1 / 100 (pH: 7.20), ascorbic pure (pH: 7.20)). Finally, the different products were sterilized under a safety cabinet using filtration with a 0.2 pm PES sterilizing membrane. In this step, a sample was carried out corresponding to the neutralized filtrates at the end of the process (called “neutralized filtrate (name of the acid) E4 from Process 3”). Rn ι η / ι 7n7 / E / YL Table 12: Sample with codes, summary: Used Used E1 from process 3 Filtrate E1 filtrate from process 3 Filtrate Neutralized Standard Neutralized filtrate (3-hydroxy-butanoic acid) E3 from Process 3 Filtrate neutralized with acid 1 Neutralized filtrate (hydrochloric acid) E4 from Process 3 Filtrate neutralized with acid 2 Filtrate neutralized (formic acid) E4 from Process 3 Acid neutralized filtrate 3 Neutralized filtrate (acetic acid) E4 from Process 3 Acid neutralized filtrate 4 Neutralized filtrate (pyruvic acid) E4 from Process 3 Acid neutralized filtrate 5 Neutralized filtrate (aspartic acid) E4 from Process 3 Acid neutralized filtrate 6 Neutralized filtrate (lactic acid) E4 from Process 3 Acid neutralized filtrate 7 Neutralized filtrate (glutamic acid) E4 from Process 3 Acid neutralized filtrate 8 Neutralized filtrate (propionic acid) E4 from Process 3 Filtration neutralized with acid 9 Neutralized filtrate (ascorbic acid) E4 from Process 3 ΑΠI 0 / 1 7Λ7 / Ε / ΥΙ1 Example 1.8.2 Analytical Characterization Analytical methods are described in 1.6.2 Analytical Characterization of the final samples of process 3 in the release (T0) E2 filtrate solutions were frozen after processing and thawed at 4°C overnight before analysis. Release results (T0): Table 13: Process 3 - Analytical Results Test Sample Result Unit Dry weight (total) Used E1 from process 3 38.0 [mg / g] Dry weight (supernatant) Used E1 from process 3 38.8 [mg / g] Dry weight (filtrate) Filtrate E2 from process 3 12.1 [mg / g / g] Total proteins Filtrate E2 from process 3 9.9 [mg / mL] Total sugar Filtrate E2 from process 3 0.67 [mg / mL] Endotoxins LAL* Neutralized filtrate (3-hydroxybutanoic acid) E3 from process 3 ND [EU / mL] DNA ** Neutralized filtrate (3-hydroxybutanoic acid) E3 from process 3 9.5 [Mg / mL] RNA*** Neutralized filtrate (3-hydroxybutanoic acid) E3 from process 3 ND [pg / mL] ND = not detected; *LAL endotoxin: detection limit = 0.1 EU / mL; **DNA: detection limit = 3.60 pg / mL, quantification limit = 12.01 pg / mL; ***RNA: detection limit = 4.29 pg / mL, quantification limit = 14.32 pg / mL. Table 14: Process 3 - Total amino acids after hydrolysis with HCl «η ι η / ι 7n7 / E / YL Amino Acid Concentration (pmol / mL) % / AA (D vs. L) L-Asp 1.39 53 D-Asp 1.25 47 L-Glu 2.11 59 D-Glu 1.44 41 L-Ser 0.33 48 D-Ser 0.36 52 L-Thr 0.56 13 D-Thr 3.71 87 L-His 0.00 0 Gly 0.46 NA D-His 0.24 100 L-Ala 2.00 NA L-Arg 1.00 NA D-Arg + D-Ala 0.80 NA L-Tyr 0.66 68 D-Tyr 0.31 32 L-Val 1.74 89 L-Met 0.42 100 D-Met 0.00 0 L-Cys 0.21 NA D-Val 0.22 11 L-lle 0.80 76 L-Phe 1.38 69 D-Phe 0.62 31 L-Leu 2.30 81 D-lle 0.25 24 D-Leu 0.53 19 L-Lys 1.68 72 D-Lys 0.65 28 a) Spectrophotometric results obtained during stability Table 15: Process 3 - solution stability through zLRnLn / Lznz / B / Yii absorbance measurement Time points (Months) Process 3 3-Hydroxybutanoic acid Hydrochloric acid Aspartic acid Formic acid Lactic acid Propionic acid Ascorbic acid Acetic acid Pyruvic acid Glutamic acid Industrial batch of HCL 1619013 TO Abs [AU] 1.105 1.056 0.74 1.125 1.087 1,099 1,267 1,142 1,258 0.888 3,038 Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy T1 4°C Abs [AU] 1.087 1.064 1.151 1.114 1.117 1.119 2.049 1.128 1.294 0.898 3.196 R(%) 98% 101% 156% 99% 103% 102% 162% 99 % 103% 101% 105% Evaluation smooth smooth smooth smooth smooth smooth iwtoso smooth smooth smooth noisy TA Abs [AU] 1.134 1.121 0.757 1.17 1.143 1.165 3.488 1.243 1.372 0.951 3.033 R(%) 103% 106% 102% 104% 105% 106 % 275% 109% 109% 107% 100% Evaluation smooth smooth smooth smooth smooth smooth noisy smooth smooth smooth noisy Abs [AU] 1.104 1.112 0.738 1.166 1.124 1.141 1.155 1.316 0.891 R(%) 100% 105% 100% 104% 103% 104% 101% 105% 100% Si®l Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy Abs [AU] 1.165 1.162 1.235 1.223 1.179 1.219 1.229 1.388 0.948 i® R(%) 105% 110% 167% 109% 10 8 % 111% 272% 108% 110% 107% 103% Evaluation smooth smooth smooth smooth smooth smooth noisy smooth smooth smooth noisy Abs: absorbance at 320 nm; RT: room temperature (20°C + / - 5°C); Evaluation: visual Spectrum evaluation Industrial batch 1619064 described in WO 2008 / 109669 was neutralized with hydrochloric acid and presented a precipitate starting at T0. Except for ascorbic acid, the E4 neutralized filtrates from Process 3 were physically stable at 4°C or room temperature for at least 3 months. b) Mip3-alpha (CCL20) results obtained during stability: Figure 31: Process 3 - bioassay results during stability show that the E3 neutralized filtrate from Process 3 exhibited comparable bioactivity through secretion of MIP-3a into THP-1 for at least 5 months at room temperature (20° C + / -5°C) or 4°C. The T0 of Process 3 was compared with samples of T5 stored at 4°C and at room temperature (RT) for 5 months. Example 1.9 Stable Bacterial Extract of Strain 21 OM314A Polylysate Example 1.9.1 Process 1 to stabilize a polylysate bacterial extract formulation of strain 21 Lysis: A part of the polylysate of strain 21 was recovered in production and stored in a 2500 mL mini-barrel (reference: Semadeni no. 6863). In this step, a sample corresponding to the lysate at the end of the lysis (called "Used E1 from Process 1) was carried out." Filtration 1: The installation for the filtration of the product was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiItration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2). The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cut-off point of 10 kDa (ultrafiltration filter in Figure 2). Filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before beginning the process, we had to make sure that the filtration system was reproducible on batches. To verify the correct filtration capacity of the product, a NWP (Normalized Water Permeability) was carried out on the filtration system. 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 have a 1 cm product vortex. The product temperature was cooled to room temperature while waiting for the filtration process to begin. In this process there was a pH adjustment to pH 10.5 -10.8 (pH: 10.77 adjusted with pure aspartic acid). Initial concentration'. The product used for the first filtration step had the following parameters (pH: 10.77, temperature 25°C, stirred to have a product vortex of 1 cm). The MF loop pump (pump 1 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, product recirculation over the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF in loop was opened to carry out an initial concentration of the product with a concentration factor up to 0.5 (inlet pressure: 300 mbar, infiltrate flow rate: 43 mL / min). During the initial concentration, the pump speed (pump 1 in Figure 2) was gradually increased to 100% (100 rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was regularly increased until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Diafiltration: When the concentration factor of 0.5 was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 730 mbar, infiltrate flow rate: 58 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in Figure 2, should be set to 850 mbar (inlet pressure: 1010 mbar, infiltrate flow rate: 58 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted until a UF infiltrate flow rate equal to the MF infiltrate flow was reached. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was carried out per cycle. At the end of the initial concentration, there was a volume present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Rn ι η / ι 7n7 / E / YL Final concentration: At the end of the 5 diafiltration cycles, the UF pump was stopped. When the MF inlet pressure began to increase, the MF pump was turned off. At the end of this first step, the product of interest contained smaller elements of 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 1232.7 g) was then subjected to a second stage of 5 purification cycles in the UF loop with a cut-off point of 10 kDa (vessel 3, pump 2, the ultrafiltration filter in the Figure 2). The infiltrate was discarded as mentioned in Figure 2. The volume was kept constant during this second purification step by filtration, by adding 0.001 N NaOH solution at pH 10.0. The UF loop pump (pump 2 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 655 mbar, infiltrate flow rate: 37 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, should be set to 850 mbar (inlet pressure: 917 mbar, infiltrate flow rate: 57 mL / min). During filtration step 2, the volume in the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. So, when the UF tank level (vessel 3 in Figure 2) decreased, there was an addition of NaOH solution. The 5 cycles corresponding to one volume of NaOH solution added the equivalent of 5 times the volume of product of interest harvested. At the end of filtration step 2, the final product (mass: 1237.0 g) was harvested and then separated into 2 equal parts. At the end of this second filtration step, the product of interest contained smaller elements of 0.45 pm and larger elements of 10 kDa in size. In this step, a sample corresponding to the filtrate before neutralization was carried out (called “filtrate E2 from process 1”). The first part of the filtrate was then neutralized with 0.1% aspartic acid to pH 7.2 ± 0.2 (pH: 7.20 adjusted with 65 mL of 0.1% aspartic acid), then sterilized under a safety cabinet using 0.2 PES sterilizing membrane filtration. p.m. In this step, a sample was carried out corresponding to the neutralized filtrate at the end of the process (called “neutralized filtrate (aspartic acid) E3 from Process Τ' OM314A). At the same time, the second part of the filtering was subdivided into 9 equal parts. Each of these parts was then neutralized to 7.2 ± 0.2 with 0.25% hydrochloric acid (pH: 7.19) or with organic acids (OM314A): formic 1 / 100 (pH: 7.16), acetic 1 / 100 (pH: 7.20), pyruvic 1 / 100 (pH: 7.12), 3-hydroxy-butanoic 1 / 100 (pH: 7.17), lactic 1 / 100 (pH: 7.19), glutamic 0.1% (pH: 7.09), propionic 1 / 100 (pH: 7.11), pure ascorbic (pH: 7.20)). Finally, the different products were sterilized under a safety cabinet using filtration with a 0.2 pm PES sterilizing membrane. Rn ι η / ι 7n7 / E / YL In this step, a sample was carried out corresponding to the different neutralized filtrates at the end of the process (called “neutralized filtrate (name of the acid) E4 from Process 1”, OM314A). Table 16: Sample Summary: Rn ι η / ι 7n7 / E / YL Used Used E1 from Process 1 Filtrate E2 Filtrate from Process 1 Neutralized Filtrate Standard Neutralized filtrate (aspartic acid) E3 from Process 1 Acid neutralized filtrate 1 Neutralized filtrate (hydrochloric acid) E4 from Process 1 Acid neutralized filtrate 2 Neutralized filtrate (formic acid) ) E4 from Process 1 Acid neutralized filtrate 3 Neutralized filtrate (acetic acid) E4 from Process 1 Acid neutralized filtrate 4 Neutralized filtrate (pyruvic acid) E4 from Process 1 Acid neutralized filtrate 5 Neutralized filtrate (3-hydroxy-butanoic acid) E4 from Process 1 Acid neutralized filtrate 6 Neutralized filtrate (lactic acid) E4 from Process 1 Acid neutralized filtrate 7 Neutralized filtrate (glutamic acid) E4 from Process 1 Acid neutralized filtrate 8 Neutralized filtrate (propionic acid) E4 from Process 1 Neutralized filtrate with acid 9 Neutralized filtrate (ascorbic acid) E4 from Process 1 Example 1.9.2. Analytical Characterization Analytical methods are described in 1.6.2 a) Results in release: E2 filtrate solutions were frozen after processing and thawed at 4°C overnight before analysis. Table 17: Process 1 -Analytical Results Test Sample Result Unit Dry weight (total) Lysate E1 from Process 1 31.3 [mg / g] Dry weight (supernatant) Used E1 from Process 1 30.0 [mg / g] Dry weight (filtered) Filtrate E2 from Process 1 9.1 [mg / g / g] Total proteins Filtrate E2 from Process 1 7.3 [mg / mL] Total sugar Filtrate E2 from Process 1 0.10 [mg / mL] Endotoxin LAL* Neutralized filtrate (aspartic acid) E3 from Process 1 ND [EU / mL] DNA** Neutralized filtrate (aspartic acid) E3 from Process 1 10.4 [pg / mL] RNA*** Neutralized filtrate (aspartic acid) E3 from Process 1 ND [pg / mL] ND = not detected; *LAL endotoxin: detection limit = 0.1 EU / mL; **DNA: detection limit = 3.60 pg / mL, quantification limit = 12.01 pg / mL; ***RNA: detection limit = 4.29 pg / mL, quantification limit = 14.32 pg / mL. Table 18: Process 1 - Total amino acids after acid hydrolysis κη ι η / ι 7n7 / E / YL Amino Acid Concentration (pmol / mL) % / AA (D vs. L) L-Asp 1.17 60 D-Asp 0.77 40 L-Glu 1.59 68 D-Glu 0.75 32 L-Ser 0.39 55 D-Ser 0.32 45 L-Thr 0.46 11 D-Thr 3.56 89 L-His 0.00 0 Gly 0.32 NA D-His 0.35 100 L-Ala 1.71 NA L-Arg 0.83 NA D-Arg + D-Ala 0.50 NA L-Tyr 0.61 77 D-Tyr 0.19 23 L-Val 1.23 90 L-Met 0.28 100 D-Met 0.00 0 L-Cys 0.11 NA D-Val 0.13 10 L-lle 0.61 77 L-Phe 1.03 73 D-Phe 0.38 27 L-Leu 1.83 88 D-lle 0.19 23 D-Leu 0.26 12 L-Lys 1.82 81 D-Lys 0.42 19 Spectrophotometric results obtained during stability Table 19: Process 1 – solution stability through absorbance measurement Time points (Months) Process 1 Aspartic Acid O o o o ·< Propionic Acid Formic Acid Lactic Acid 3-hydroxybutanoic Acid Ascorbic Acid O o •Φ o o Pyruvic Acid Glutamic Acid •Industrial Batch 1619064 T0 Abs [AU] 1,317 1,496 1.54 1,553 1,519 1,509 1.646 1.504 1.638 1.294 2.73 Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth T1 4°C Abs [AU] 1.329 1.496 1.552 1.574 1.53 1.528 2.144 1.543 1.66 1.325 3.032 R(%) 101 % 100% 101% 101% 101% 101 % 130% 103% 101% 102% 111% Evaluation smooth smooth smooth smooth smooth smooth noise» smooth smooth smooth noise» TA Abs [AU] 1,397 1,579 1,589 1,634 1,591 1,602 3,043 1,602 1,727 1,365 3,152 R(%) 106% 1 06% 103 % 105% 105% 106% 185% 107% 105% 105% 115% Evaluation smooth smooth smooth smooth smooth smooth noisy smooth smooth smooth noisy T3 4°C Abs [AU] 1,397 1,587 1,574 1,599 1,545 1,532 2,457 1,603 1,692 1 .347 3.441 R( %) 106% 106% 102% 103% 102% 102% 149% 107% 103% 104% 126% Evaluation smooth smooth smooth smooth smooth smooth smooth smooth smooth noisy TA Abs [AU] 1,438 1.66 1,679 1,705 1,687 1,693 2,831 1,688 1,769 1,423 3.232 R(%) 109% 111% 109% 110% 111% 112% 172% 112% 108% 110% 118% Evaluation smooth smooth smooth smooth smooth smooth wfosso smooth smooth smooth noisy Abs: absorbance at 320 nm; RT: room temperature (20°C + / - 5°C); Evaluation: visual spectrum evaluation Industrial batch 1619064 described in WO 2008 / 109669 was neutralized with hydrochloric acid and presented a precipitate starting at T0. Except for ascorbic acid, the E4 neutralized filtrates from Process 1 were physically stable at 4°C or room temperature for at least 3 months. b) Mip3-alpha (CCL20) results obtained during stability: Figure 29: Process 1 - bioassay results during stability show that the E3 neutralized filtrate from Process 1 exhibited comparable bioactivity through secretion of MIP-3a into THP-1 for at least 4 months at room temperature (20° C + / -5°C) or 4°C. The T0 of Process 1 was compared with T5 samples stored at 4°C and at room temperature (RT) for 4 months. Example 1.9 Haemophilus influenzae 8467 (OM314A) Example 1.10.1 Process 2: Haemophilus influenzae 8467 Lysis: 13396 kg of biomass of Haemophilus influenza 8467 (lot 1419110 - boxes 10, 11, 12 and 13) were thawed overnight at room temperature in a lysis barrel. 692 g of 10 N NaOH and 12920 g of 8 g / L NaCI solution were added to give a total lysis weight of 27008 g. The alkaline lysis was transferred in a warm room at 37°C ± 2.5°C under shaking of 150 rpm ± 5 rpm for 5 days. After 3 h 00 ± 30 min of lysis, the OD of JO was monitored. The sample was diluted 200 times and read with a spectrophotometer at 700 nm (read OD: 0.273 and final OD: 54.6). Every 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, an adjustment had to be made with 10 N NaOH (J1: 20 mL 10 N NaOH / J2: 20 mL 10 N NaOH). At the end of lysis, the OD of J8 was monitored. The sample was diluted 100 times and read with a spectrophotometer at 700 nm (read OD: 0.169 and final OD: 16.9). The delta DO between JO and J5 had to be greater than 13.1 (Delta DO: 37.7). A part of this lysis of Haemophilus influenza 8467 was recovered and stored in a 2500 mL minibarrel (reference: Semadeni n° 6863). In this step, a sample corresponding to the one used at the end of the lysis (called "used E1 of process 2") was carried out. Filtration 1: The installation for the filtration of the product was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiltration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2). . The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a 10 kDa cutoff (ultrafiltration filter in Figure 2). Filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before starting the process, we had to make sure the filtration system was reproducible across batches. To verify the correct filtration capacity of the product, an NWP (Normalized Water Permeability) was performed on the filtration system. The used used for the production was first diluted 4 times with purified water (499.9 g of used and 1500.2 g of purified water). The product was stirred to have a 1 cm product vortex. The product temperature 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. Initial concentration'. The product used for the first filtration step had the following parameters (pH: 11.23), temperature 32°C, stirred to have a 1 cm product vortex). The MF loop pump (pump 1 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, the recirculation of the product over the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF loop was opened to carry out an initial concentration of the product with a concentration factor up to 0.5 (inlet pressure: 325 mbar, infiltrate flow rate: 45 mL / min). During the initial concentration, the speed of the pump (pump 1 in Figure 2) was gradually increased up to 100% (100 rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Diafiltration: When the concentration factor of 0.5 was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 880 mbar, infiltrate flow rate: 63 mL / min). to have a ΑΓ>I n / Ι 7O7 / E / Yl· optimal product extraction, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in Figure 2, should be set at 850 mbar (inlet pressure: 1163 mbar , infiltrate flow rate: 68 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted until a UF infiltrate flow rate equal to the MF infiltrate flow was reached. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was performed per cycle. At the end of the initial concentration, there was a volume present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Final concentration: at the end of the 5 diafiltration cycles, the UF pump was stopped. When the MF inlet pressure began to increase, the MF pump was turned off. At the end of this first step, the product of interest contained smaller elements of 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 1039.1 g) was then subjected to a second stage 5 cycles of purification in the UF loop with a cut-off point of 10 kDa (vessel 3, pump 2 the ultrafiltration filter in the Figure 2). The infiltrate was discarded as mentioned in Figure 2. The volume was kept constant during this second purification step by filtration, by adding 0.001 N NaOH solution at pH 10.0. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 650 mbar, infiltrate flow rate: 44 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, should be set to 850 mbar (inlet pressure: 960 mbar, infiltrate flow rate: 72 mL / min). During filtration step 2, the volume in the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. So, when the level of the UF tank (vessel 3 in Figure 2) decreased, there was an addition of NaOH solution. The 5 cycles corresponding to one volume of NaOH solution added the equivalent of 5 times the volume of product of interest harvested. At the end of filtration step 2, the final product (mass: 1033.9 g) was harvested and then separated into 2 equal parts. At the end of this second filtration step, the product of interest contained smaller elements of 0.45 pm and larger elements of 10 kDa in size. In this step, a sample corresponding to the filtrate before neutralization was carried out (called E2 filtrate from process 2”). The first part of the filtrate was then neutralized with 1 / 100 propionic acid to pH 7.2 ± 0.2 (pH: 7.18 adjusted with 4.2 mL of 1 / 100 propionic acid), then sterilized under a biosafety cabinet using PES sterilizing membrane filtration. from 0.2 pm. zLRnLn / Lznz / E / Yi In this step, a sample was carried out corresponding to the neutralized filtrate at the end of the process (called “neutralized filtrate (propionic acid) E3 from process 2” OM314A). At the same time, the second part of the filtering was subdivided into 9 equal parts. Each of these parts was then neutralized at 7.2 ± 0.2 0.25% hydrochloric acid (pH: 7.09) or with organic acids (OM314A): formic 1 / 100 (pH: 7.13), acetic 1 / 100 (pH: 7.20), pyruvic 1 / 100 (pH: 7.15), aspartic 0.1% (pH: 7.12), lactic 1 / 100 (pH: 7.19), glutamic 0.1% (pH: 7.21), 3-hydroxy-butanoic 1 / 100 (pH: 7.16) , pure ascorbic (pH: 7.25)). Finally, the different products were sterilized under a safety cabinet using filtration with a 0.2 pm PES sterilizing membrane. In this step, a sample was carried out corresponding to the different neutralized filtrates at the end of the process (called “neutralized filtrate (acid name) E4 from process 2”). Table 20: Summary of sample codes: Rn ι η / ι 7n7 / E / YL Used Used E1 from Process 2 Filtrate E2 Filtrate from Process 2 Neutralized Filtrate Standard Neutralized filtrate (propionic acid) E3 from Process 2 Neutralized filtrate with acid 1 Neutralized filtrate (hydrochloric acid) E4 from Process 2 Neutralized filtrate with acid 2 Neutralized filtrate (formic acid ) E4 from Process 2 Acid neutralized filtrate 3 Neutralized filtrate (acetic acid) E4 from Process 2 Acid neutralized filtrate 4 Neutralized filtrate (pyruvic acid) E4 from Process 2 Acid neutralized filtrate 5 Neutralized filtrate (aspartic acid) E4 from Process 2 Acid neutralized filtrate 6 Neutralized filtrate (lactic acid) E4 from Process 2 Acid neutralized filtrate 7 Neutralized filtrate (glutamic acid) E4 from Process 2 Acid neutralized filtrate 8 Neutralized filtrate (3-hydroxy-butanoic acid) E4 from Process 2 Neutralized filtrate with acid 9 Neutralized filtrate (ascorbic acid) E4 from Process 2 Example 1.10.2. Analytical Characterization Analytical methods are described in 1.6.2 Release results (T0): E2 filtrate solutions were frozen after processing and thawed at 4°C overnight before analysis. Table 21: Process 2 - Analytical results Test Sample Result Unit Dry weight (total) Used E1 from Process 2 24.5 [mg / g] Dry weight (supernatant) Used E1 from Process 2 21.2 [mg / g] Dry weight (filtered) Filtrate E2 from Process 2 8.4 [mg / g / g] Total proteins Filtrate E2 from Process 2 7.7 [mg / mL] Total sugar Filtrate E2 from Process 2 0.20 [mg / mL] Endotoxin LAL* Neutralized filtrate (propionic acid) E3 from Process 2 16.2 [EU / mL] DNA** Neutralized filtrate (propionic acid) E3 from Process 2 ND [pg / mL] RNA*** Neutralized filtrate (propionic acid) E3 from Process 2 ND [pg / mL] zLRnLn / Lznz / B / Yi ND = not detected; *LAL endotoxin: detection limit = 0.1 EU / mL; **DNA: detection limit = 3.60 pg / mL, quantification limit = 12.01 pg / mL; ***RNA: detection limit = 4.29 pg / mL, quantification limit = 14.32 pg / mL Table 22: Process 2 - Total amino acids after acid hydrolysis Amino Acid Concentration (pmol / mL) % / AA (D vs. L) L-Asp 0.87 77 D-Asp 0.26 23 L-Glu 1.25 87 D-Glu 0.18 13 L-Ser 0.40 54 D-Ser 0.34 46 L-Thr 0.47 17 D-Thr 2.31 83 L-His 0.00 NA Gly 0.21 NA D-His 0.00 NA L-Ala 1.18 NA L-Arg 1.07 NA P-Arg + P-Ala 0.11 NA L-Tyr 0.51 85 P-Tyr 0.09 15 L-Val 0.68 100 L-Met 0.41 100 P-Met 0.00 0 L-Cys 0.08 NA P-Val 0.00 0 L-lle 0.58 100 L -Phe 0.63 84 P-Phe 0.12 16 L-Leu 1.31 100 P-lle 0.00 0 P-Leu 0.00 0 L-Lys 1.67 94 P-Lys 0.12 6 a) Mip3-alpha (CCL20) results obtained during stability: Figure 30: Process 2 - bioassay results during stability show that the E3 neutralized filtrate from Process 2 exhibited comparable bioactivity through secretion of MIP-3o onto THP-1 for at least 4 months at room temperature (20° C + / - 5°C) or 4°C. The TO of Process 2 was compared with T4 samples stored at 4°C and at room temperature (RT) for 4 months. Example 1.11 - Bacterial Usado extract of strain 21 of 30 kDa (BE30kP, OM314A) Example 1.11.1 Process 6: Bacterial Usado extract of strain 21 of 30 kDa (BE30kD, OM314A) Lysis: A part of bacterial polylysate of strain 21 (Industrial Lot 1619064 described as Used strain 21 in WO 2008 / 109669) was recovered in production and stored in a 2500 mL mini barrel (reference: Semadeni no. 6863 ). Filtration 1: The installation for the filtration of the product was prepared according to the diagram (Figure 2). The filtration system consists of 2 filtration loops. A first microfiltration (called MF) consisting of a tank (vessel 2 in Figure 2), a pump (pump 1 in Figure 2) and a filtration system with a cut-off point of 0.45 pm (microfilter in Figure 2). . The second loop, ultrafiltration (called UF), consists of a tank (vessel 3 in Figure 2), a pump (pump 2 in Figure 2), and a filtration system with a cut-off point of 30 kPa (ultrafiltration filter in Figure 2). Filtration took place on a laboratory scale with an implemented volume of 2000 mL. Before starting the process, we had to ensure that the filtration system was reproducible over the batches. To verify the correct filtration capacity of the product, an NWP (Natural Permeability) was carried out. Normalized Water) on the filtration system. The Used used for production was first diluted 2 times with purified water (1000.2 g of Used and 1000.1 g of purified water). The product was stirred to have a 1 cm product vortex. The product temperature was cooled to room temperature while waiting for the filtration process to begin. In this process there was a pH adjustment to pH 10.5 -10.8 (pH: 10.73 adjusted with pure aspartic acid). Initial concentration: The product used for the first filtration step had the following parameters (pH: 10.73, temperature 25°C, stirred to have a product vortex of 1 cm). The MF loop pump (pump 1 in Figure 2) was started at 40% of process speed to charge the system with product. Once this step was completed, the recirculation of the product over the MF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. When the flow rate and pressure were stable, the filtration system was considered conditioned. Then, the infiltrate valve of the MF loop was opened to carry out an initial concentration of the product with a concentration factor up to 0.5 (inlet pressure: 235 mbar, infiltrate flow rate: 52 mL / min). During the initial concentration, the speed of the pump (pump 1 in Figure 2) was gradually increased up to 100% (100 rpm corresponding to 600 mL / min) of the process speed. Parallel to this step, the UF loop was conditioned. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Diafiltration: When the concentration factor of 0.5 was reached, the UF infiltrate was opened to begin diafiltration of the product (inlet pressure: 734 mbar, infiltrate flow rate: 52 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of MF which was controlled by valve 1 in Figure 2, should be set to 850 mbar (inlet pressure: 1010 mbar, infiltrate flow rate: 48 mL / min). During diafiltration, the speed of the UF pump (pump 2 in Figure 2) was adjusted until a UF infiltrate flow rate equal to the MF infiltrate flow was reached. In reality, the volume in the MF tank (vessel 2 in Figure 2) had to remain as stable as possible during the diafiltration step. Diafiltration was performed per cycle. At the end of the initial concentration, there was a volume present in the MF tank (vessel 2 in Figure 2). Once this volume passed through the MF filtration system, a cycle was performed. In this process, diafiltration required 5 cycles. Final concentration: at the end of the 5 diafiltration cycles, the UF pump was stopped. When the MF inlet pressure began to increase, the MF pump was turned off. At the end of this first step, the product of interest contained smaller elements of 0.45 pm in size. Filtration 2: The harvested product of interest (mass: 1179.6 g) was then subjected to a second stage of purification cycles in the UF loop with a cut-off point of 30 kDa (vessel 3, pump 2 and ultrafiltration filter zLRnLn / Lznz / B / Yi in Figure 2). The infiltrate was discarded as mentioned in Figure 2. The volume was kept constant during this second purification step by filtration, by adding 0.001 N NaOH solution at pH 10.3. The UF loop pump (pump 2 in Figure 2) was started at 40% process speed to charge the system with product. Once this step was completed, product recirculation in the UF loop began and the pump speed was increased regularly until reaching 75% of the process speed. Recirculation allows the hydrophilic filtration system to be conditioned by eliminating air bubbles present in the system. Once the filtration system was conditioned, the UF infiltrate was opened to begin filtration 2 of the product (inlet pressure: 670 mbar, infiltrate flow rate: 67 mL / min). To have optimal product extraction, the TMP (Transmembrane Pressure) of UF which was controlled by valves 2 and 3 in Figure 2, should be set to 850 mbar (inlet pressure: 920 mbar, infiltrate flow rate: 88 mL / min). During filtration step 2, the volume in the UF tank (vessel 3 in Figure 2) had to remain as stable as possible. Thus, when the level of the UF tank (vessel 3 in Figure 2) decreased, there was an addition of NaOH solution. The 5 cycles corresponding to one volume of NaOH solution added the equivalent of 5 times the volume of product of interest harvested. At the end of filtration step 2, the final product (mass: 1070.5 g) was harvested. At the end of this second filtration step, the product of interest contained smaller elements of 0.45 pm and larger elements of 30 kDa in size. The filtrate was then neutralized with 0.1% aspartic acid to pH 7.2 ± 0.2 (pH: 7.19 adjusted with 180 mL of 0.1% aspartic acid), then sterilized under a biosafety cabinet using 0.2 pm PES sterilizing membrane filtration. In this step, a sample was carried out corresponding to the neutralized filtrate at the end of the process (called neutralized filtrate (aspartic acid) E3 from process 6”, OM314A). Example 2: Stable formulations for intranasal, intratracheal, inhalation and perioral use Example 2.1: Intranasal formulation of stabilized bacterial extract OM314A The high molecular fraction (>10 kD) of the organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline (NaCl 0.9% in water for injection) and sterilized by 0.2 pm filtration. The final solution was added to the vial of the nasal spray medical device (10 mL, range 1 to 25 mL) with a typical dose of 0.05 mL containing 0.25 mg of the organic acid-stabilized OM bacterial extract (range 0.025 to 0.1 mL per dose). Therapeutic dose: A single daily dose as well as a twice daily dose range of 0.025 to 0.1 mL per administration with a content of 0.05 to 1 mg can be achieved with these formulations. Example 2.2: intranasal formulation of stabilized bacterial extract of Lactobacillus fermentum 20 (OM314B) The high molecular fraction (>10 kD) of organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was adjusted to pH 7.5 at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline solution (0.9% NaCl in water for injection) and sterilized by 0.2 pm filtration. The solution Final Rn ι η / ι 7n7 / E / YL was added to the nasal spray medical device vial (10 mL, range 1 to 25 mL) with a typical dose of 0.05 mL containing 0.25 mg of the purified bacterial extract of Lactobacillus fermentum stabilized with organic acid (range 0.025 to 0.1 mL per dose). Therapeutic dose: A single daily dose as well as a twice daily dose range of 0.025 to 0.1 mL per administration with a content of 0.05 to 1 mg can be achieved with these formulations. Example 2.3: Intranasal formulation of stabilized bacterial extract of Escherichia cotí (OM314C) The high molecular fraction (>10 kD) of the organic acid-stabilized Escherichia coli bacterial extract was adjusted to pH 7.5 at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline. (0.9% NaCl in water for injection) and sterilized by 0.2 pm filtration. The final solution was added to the nasal spray medical device bottle (10 mL, range 1 to 25 mL) with a typical dose of 0.05 mL containing 0.25 mg of the purified Escherichia coli bacterial extract stabilized with organic acid (range 0.025 to 0.1 mL per dose). Therapeutic dose: A single daily dose as well as a twice daily dose range of 0.025 to 0.1 mL per administration with a content of 0.05 to 1 mg can be achieved with these formulations. Example 2.4: Inhalation formulation as a spray of liquid droplets of stabilized bacterial extract OM314A The high molecular fraction (>10 kD) of the organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline (NaCl 0.9% in water for injection) and sterilized by 0.2 pm filtration. The final solution was added to a mist inhaler medical device vial (10 mL, range 1 to 25 mL) with a typical dose of 0.1 mL containing 0.5 mg of the organic acid-stabilized OM bacterial extract (range 0.05 to 0.4 mL per dose). Therapeutic dose: A single daily dose as well as a twice daily dose range of 0.05 to 0.4 mL per administration with a content of 0.05 to 8 mg can be achieved with these formulations. Example 2.5: Inhalation formulation as a spray of liquid droplets of stabilized bacterial extract of Lactobacillus fermentum (OM314B) The high molecular fraction (>10 kD) of the organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was adjusted to pH 7.5 at a final concentration of 5 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline solution (0.9% NaCl in water for injection) and sterilized by 0.2 pm filtration. The final solution was added to a mist inhaler medical device vial (10 mL, range 1 to 25 mL) with a typical dose of 0.1 mL containing 0.5 mg of the purified bacterial extract of Lactobacillus fermentum stabilized with organic acid (range 0.05 to 0.4 mL per dose). Therapeutic dose: A single daily dose as well as a twice daily dose range of 0.05 to 0.4 mL per administration with a content of 0.05 to 8 mg can be achieved with these formulations. Example 2.6: Inhalation formulation as solid particles of stabilized bacterial extract OM314A The high molecular fraction (>10 kD) of the organic acid-stabilized OM314A bacterial extract was adjusted to pH 7.5 at a final concentration of 10 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline «η ι η / ι 7n7 / E / YL (0.9% NaCl in water for injection), one or more excipients from the list* and sterilized by 0.2 pm filtration. In one example, a 10 mg / mL solution of the 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 disintegrating tablets were dispensed in a medical device (particle size range 1 to 7 pm) and the inhalation dose of a 12 mg tablet was 2 mg of bacterial extract. Typical excipients for inhalation are, but are not limited to: lactose, glucose, mannitol, trehalose, Mg stearate, DPPC, DSPC, DMPC, cholesterol, leucine, trileucine, Poloxamer, bile salts, chitosan, trimethylchitosan, PLGA (see G Pilcer, K. Amighi, International Journal of Pharmaceutics, 2010, 392,1-19 for a review) Example 2.7: Inhalation formulation as solid particles of stabilized bacterial extract of Lactobacillus fermentum (OM314B) The high molecular fraction (>10 kD) of the organic acid-stabilized Lactobacillus fermentum bacterial extract (OM314B) was adjusted to pH 7.5 at a final concentration of 10 mg dry weight / mL (range 1 to 20 mg / mL) using sterile saline solution (0.9% NaCl in water for injection), one or more excipients from the list* and sterilized by 0.2 pm filtration. In one example, a 10 mg / mL solution of the 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 disintegrating tablets were dispensed in a medical device (particle size range 1 to 7 pm) and the inhalation dose of a 12 mg tablet was 2 mg of bacterial extract. Typical excipients for inhalation are, but are not limited to: lactose, glucose, mannitol, trehalose, Mg stearate, DPPC, DSPC, DMPC, cholesterol, leucine, trileucine, Poloxamer, bile salts, chitosan, trimethylchitosan, PLGA (see G. Pilcer, K. Amighi, International Journal of Pharmaceutics, 2010, 392, 1-19 for a review). EXAMPLE 3: Evidence of increased 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 were 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 using the different organic acids selected from acetic acid, propionic acid, lactic acid, 3-hydroxy-propanoic acid, pyruvic acid, butanoic acid, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, acid glutamic acid, aspartic acid and a combination thereof. The stability of the 10 bacterial extracts stored at 5°C ± 3°C and at room temperature of 20°C to 25°C was visually observed by the presence of a precipitate at different time points from day 0 (between 15 min and 120 min after pH adjustment) and after 30,60, 90,180, 360 days. Quantification was carried out using visible spectrophotometry by measuring its absorbance at 550, 600, 650, 700 nm determined at different time points starting from day 0 (between 15 min and 120 min after pH adjustment) and after 30, 60, 90,180, 360 days. Spectrophotometry was carried out against a water sample. ΑΠI Π / 1 7Λ7 / Ε / ΥΙ1 Stability was expressed as a change in absorbance depending on the process, on the organic acid or combination of organic acid as well as on the final pH value. EXAMPLE 4: Prophylactic and curative efficacy of intranasal OM versus oral administration in an animal model of sublethal bacterial infection after primary influenza infection. A stable perioral form of the OM bacterial extract (Usados ​​bacterial extract of strain 21) has been prepared extemporaneously for the purpose of experimentally testing the perioral administration of the OM bacterial extract in animal models. This extemporaneous perioral form was stable over time, but the results provided insight and evidence of substantial therapeutic benefits from perioral administrations. The efficacy of intranasal administration of extemporaneous perioral OM bacterial extract in reducing viral titer in lung tissue after influenza viral infection and (2) reducing morbidity and mortality of superinfected animals (animals treated with sublethal influenza viral infection followed by sublethal bacterial infection) was compared with that of oral administration of the OM bacterial extract (Figure 3). Female BALB / c mice (8 weeks old, Charles River Laboratories) were anesthetized by intraperitoneal injection of ketamine and xilasol and inoculated intranasally with 100 PFU of Influenza A / Puerto Rico8 / 34 strain in a volume of 50 µl PBS. On day 6 after influenza infection the bacteria started bacterial cultures, followed by expansion until the growth phase on day 7 after influenza infection. For oral administration, 320 pL of concentrated OM was administered by gavage yielding 360 mg / kg of active ingredient of lyophilized OM per animal / day, which produced a daily administration of 7.2 mg of active ingredient per mouse (Figure 3). For intranasal administration of LPS (within the scope to perform a COPD model), the dose used was 7 micrograms of LPS per dose via the nasal route. In the literature, administration of 1 microgram of LPS nasally has been reported to protect against allergic inflammation. The test program is recapitulated in the following Table 23: I η / l 7O7 / E / Yl· Table 23: Day-10 to-1 Oral water control administration (Group 1). Day-10 to-1 Administration of oral OM bacterial extract (Group 2) Day-7, -5 and -3 Extemporaneous intranasal administration of OM bacterial extract (Groups 3 and 4) DayO Sublethal infection with influenza A virus (100 PFU nasally, H1N1 influenza strain PR / 8 / 34) Day 5 Sacrifice of 5 animals per group for testing (Groups 1-4) Day 5 Interim bleeding (12 animals per group, Groups 1-4) Day 7 Sublethal infection with Streptococcus pneumoniae (15 animals per group, Groups 1 to 4) Day 0, 7-12 Measurement of weight, temperature, clinical score and lethality (12 animals per group, Groups 1 to 4) Day 12-16 Measurement of weight, temperature, clinical score and lethality every second day until animals fully recover (all remaining animals) κη ι η / ι 7n7 / E / YL Statistics were performed using GraphPad Prism version 5.0d. A Student's t test was performed on the viral load. A two-way ANOVA was performed on weight, temperature, and clinical score. Comparisons of survival curves were made. In total, treatment with OM bacterial extract protected mice against morbidity and mortality in the superinfection model. This protective effect was more notable with intranasal treatment suggesting that mucosal administration of OM bacterial extract could further improve its efficiency (Figures 4, 5) and comorbidity after secondary bacterial infection (Figure 6). EXAMPLE 5: Prophylactic efficacy of the intranasal and intratracheal administration of the bacterial extract OM in an animal model of sublethal bacterial infection after primary infection by influenza. This study mentioned in Figure 7 evidences the efficacy of intranasal and intratracheal administration of the bacterial extract OM (Usados ​​bacterial extract of strain 21) as a prophylactic treatment regimen to (1) reduce the viral titer in the lung tissue after influenza viral infection (Figure 8) and (2) reduced morbidity and mortality of superinfected animals (animals treated with sublethal influenza viral 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 administration) and DOSE B (5 micrograms of active ingredient per administration). Female BALB / c mice (8 weeks old, Charles River Laboratories) were anesthetized by intraperitoneal injection of ketamine and xilasol and inoculated intranasally with 100 PFU of Influenza A / Puerto Rico8 / 34 strain in a volume of PBS. The mice were divided into 6 groups of 15 each. Group 1 was administered saline drops via the intranasal route (I.N.) on day d7, d5 and d3 (prophylactic control). Group 2 was administered a prophylactic dose of OM bacterial extract via the intranasal route, on day d7, d5 and d3. Group 2 was administered a prophylactic dose B of OM bacterial extract via the intranasal (I.N.) route, on day -7, -5 and -3. Group 4 was administered a saline spray intratracheally (I.T.) on day -7, -5 and -3. Group 5 was administered a prophylactic dose A of OM bacterial extract via the intratracheal route, on day -7, -5 and -3. Group 6 was administered a prophylactic dose B of OM bacterial extract via the intratracheal route, on day -7, -5 and -3. Administration of OM bacterial extract with 50 micrograms of active ingredient (DOSE A, producing 2.2 microliters of OM bacterial extract concentrate) and with 5 micrograms of active ingredient (DOSE B producing 0.22 microliters of OM bacterial extract concentrate) per mouse / per time point. On day 6 after influenza infection, bacterial cultures were started, followed by expansion to the log growth phase. on day 7 after influenza infection. The study design and dosing schedule are shown in Figure 7 as well as in the following Table 24. Table 24: Rn ι η / ι 7n7 / E / YL Day-7, -5, -3 Administration of intranasal (Group 1) or intratracheal (Group 4) saline solution Day-7, -5, -3 Administration of intranasal (Groups 2 and 3) or intratracheal (Groups 5) OM bacterial extract and 6). DayO Sublethal infection with influenza A virus (100 PFU nasally, influenza strain H1N1 PR / 8 / 34). Day 5 Sacrifice of 5 animals per group for analysis (Groups 1-6). Day 7 Sublethal infection with Streptococcus pneumoniae (10 animals per group, Groups 1-6) Day 7 Sublethal infection with Streptococcus pneumoniae (12 animals per group, Groups 1-3). Days 0, 7-12 Daily measurement of weight, temperature, clinical score and lethality (10 animals per group, Groups 1-6). Day >12 Measurement of weight, temperature, clinical score and lethality every second or third day until animals fully recovered (all remaining animals) The animals were anesthetized by intraperitoneal injection of ketamine and xyllasol and the OM bacterial extract was administered intranasally or intratracheally diluted in saline with 50 micrograms of active ingredient (DOSE A, producing 2.2 microliters of concentrated OM bacterial extract) or with 5 micrograms of ingredient active (DOSE B producing 0.22 microliters of OM bacterial extract concentrate) in a total volume of 50 microliters. The animals were anesthetized by intraperitoneal injection of ketamine and xyllasol and the OM bacterial extract was administered intranasally or intratracheally diluted in saline with 50 micrograms of active ingredient (DOSE A, producing 2.2 microliters of OM bacterial extract concentrate) or with 5 micrograms of active ingredient (DOSE B producing 0.22 microliters of OM bacterial extract concentrate) in a total volume of 50 microliters. Statistics were performed using GraphPad Prism version 5.0d. A Student's t test was performed on the viral load. A two-way ANOVA was performed on weight, temperature, and clinical score. Comparisons of survival curves were made (Figures 8 and 10). Prophylactic treatment of animals via intranasal administration of OM bacterial extract (OM I.N. DOSE B: 5 micrograms and OM AI.N. DOSE: 50 microgram doses) produced a reduction in viral titer in lung tissue measured 5 days later. of infection with PR8 (Figure 8). This reduction was more predominant in the dose of 50 micrograms of OM bacterial extract by the intranasal route whereas 5 micrograms was sufficient to eliminate viral particles by the intratracheal route. Similar to the viral titer results, treatment with prophylactic intranasal OM bacterial extract produced significant alleviation of morbidity and mortality after influenza bacterial infection. OM bacterial extract 50 microgram dose by nasal treatment produced 90% survival (OM A I.N. dose), OM bacterial extract 5 microgram dose by nasal treatment produced 40% survival (OM B I.N. dose), while control treatment with nasal saline solution (I.N. saline) did not protect animals that died day 6 after infection (Figure 9). Comparable to the survival results, clinical score and weight loss measurements were significantly reduced in animals with a dose of 50 micrograms of OM bacterial extract compared to animals treated with saline (Figure 9). Consistent with animals treated with the 50 microgram dose of OM bacterial extract, animals treated with the 5 microgram dose also had reduced clinical score and weight loss compared to saline control animals; however, with less efficacy for the intranasal dose of 5 micrograms. Prophylactic treatment of animals with the 5 microgram dose of OM bacterial extract produced the best reduction in viral titer in lung tissue measured at 5 days after infection with PR8 compared to viral titer measurements found in animals of control treated with intratracheal saline (Figure 8). With respect to morbidity and mortality, more than the 50 microgram intratracheal dose (OM A I. T. dose) and 5 microgram dose (OM B I. T. dose) of OM bacterial extract treatment produced an increased survival rate (70% ) compared to control animals treated with saline (30%) with a dose-proportional effect until day 10. (Figure 11). This is summarized by the clinical score measurement equally reduced for both 50 microgram intratracheal and 5 microgram doses of OM bacterial extract treated animals, compared to the saline control group (Figure 12). In summary, prophylactic administration of OM bacterial extract via the nasal route resulted in a significant reduction in morbidity and mortality of superinfected animals and reduced the viral titer in lung tissue after influenza infection. This result was particularly clear after a treatment regimen with bacterial extract at a dose of 50 micrograms administered via the intranasal route. Surprisingly, prophylactic treatment with intratracheal OM bacterial extract resulted in the best relief of morbidity and mortality with higher efficacy using the 5 microgram dose, which could be explained by the deeper surface exposure of the lungs. Rn ι η / ι 7n7 / E / YL Those results clearly demonstrate that both intranasal and intratracheal administrations were highly effective administration routes for the therapeutic treatment of OM bacterial extract for respiratory diseases, such as asthma, COPD, and other pathogens. EXAMPLE 6: Novel treatment regimen for intranasal administration of bacterial extract according to the present invention in an animal model of sublethal influenza infection This study (Figure 13) showed the efficacy of intranasal administration of extemporaneously prepared OM bacterial extract (strain 21 lysate bacterial extract) in reducing the viral titer in lung tissue after influenza viral infection. This study also showed a dose-response relationship of the OM bacterial extract. This study further provided a comparison of two different multiple-dose regimens (a dose 6 and a dose 3) with a comparison between the intranasal treatment regimen and oral administration. Seven-week-old female BALB / c mice (Specified Pathogen Free; SPF) were purchased from Charles River Laboratories and randomly assigned to cages, totaling 5 mice per cage. Mice were monitored weekly and acclimated to the facility for 7 days prior to the start of the study (Study Day 0). Animals were 8 weeks old on Study Day 0. Drinking water and feed were available ad libitum. Mice were divided into 13 groups: Groups 1 to 11 received the active substance (OM) OM bacterial extract intranasally. Group 12 was the water control (control with 320 pL of water, orally, daily, from day -10 to day -1) and Group 13 was the negative control (influenza viral infection, sublethal, only). Tables 25 and 26 show the different groups and treatment protocols schematized in Figure 13. Table 25: Rn ι η / ι 7n7 / E / YL Group Dose / schedule / route of treatment groups 1 2.5 micrograms, d-14, -12, -10, -7, -5, -3, intranasal 2 5 micrograms, d-14, -12, -10, - 7, -5, -3, intranasal 3 25 micrograms, d-14, -12, -10, -7, -5, -3, intranasal 4 50 micrograms, d-14, -12, -10, -7, -5, -3, intranasal 5 100 micrograms, d-14, -12, -10, -7, -5, -3, intranasal 6 2.5 micrograms, d-7, -5, -3, intranasal 7 5 micrograms, d-7, -5, -3, intranasal 8 25 micrograms, d-7, -5, -3, intranasal 9 50 micrograms, d-7, -5, -3, intranasal T^aJ g1.00 micrograms, d -7, -5, -3, intranasal 11 7.2 mg orally, daily, from day -10 to day -1 Drug Group Route Day of administration before viral infection Volume per administration (microliters) Active ingredient (micrograms) Volume of concentrated drug solution used to produce the target amount of active ingredients per administration Saline (microliters) 1 OM i.n. -14,-12,-10, -7,-5, -3 50 2.5 0.11 49.89 2 OM i.n. -14,-12,-10, -7,-5, -3 50 5 0.22 49.78 3 OM i.n. -14,-12,-10, -7,-5, -3 50 25 1.1 48.9 4 OM i.n. -14,-12,-10, -7,-5, -3 50 50 2.2 47.8 5 OM i.n. 14,-12,-10, -7, -5, -3 50 100 4.4 45.6 6 OM i.n. -7, -5,-3 50 2.5 0.11 49.89 7 OM i.n. -7, -5, -3 50 5 0.22 49.78 8 OM i.n. -7, -5, -3 50 25 1.1 48.9 9 OM i.n. -7, -5, -3 50 50 2.2 47.8 10 OM i.n. -7, -5,-3 50 100 4.4 45.6 11 OM p.o. daily, from -10 to -1 320 7200 320 N / A 12 water p.o. daily, from -10 to -1 320 N / A N / A N / A 13 N / A N / A N / A N / A N / A N / A N / A Rn ι η / ι 7n7 / E / YL Yo. η. = intranasal; p.o. = orally On the days specified in the Study Protocol, above, mice were anesthetized using a calibrated vaporizer system (VIP300, Provet, Vet.Med Center, Lyssach, CH) that releases the anesthetic agent, isoflurane Provet AG, Catalog Number: 2222 ) in a plexiglass chamber containing the mice. The anesthetized animals were then administered with a total volume of 50 microliters of OM bacterial extract test material, which was lightly rubbed onto both nostrils using a 100 μΙ micropipette. The virus material (Influenza virus strain PR8 (A / Puerto Rico / 8 / 34, Η1N1) from Virapur (San Diego)) was stored at -75°C ± 10°C and thawed before administration . Once thawed, the material was diluted with cold PBS (4°C) corresponding to 100 PFU / 50 μΙ of A / PR / 8 / 34. The diluted virus was kept on ice until administration to the mice. The animals were anesthetized by intraperitoneal injection with 9.75 mg of Xilasol and 48.75 mg of Ketasol per kg of body weight and each animal received 50 μΙ of virus solution per inoculation. On days 5, the animals were sacrificed by lethal intraperitoneal injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A) immediately by tissue isolation (lung). Isolated lung lobes were prepared for quantification of viral load in lung tissue by quantitative PCR. Isolated lung lobes and RNA were prepared with TRI Reagent (Molecular Research Center) and then treated with DNase (Invitrogen) to avoid contamination with genomic DNA beforehand. RNA was converted to cDNA by reverse transcription using SuperScript III (Invitrogen). cDNA was quantified by real-time PCR (Cycler; Bio-Rad) using Green SYBR (Stratagene) and samples were normalized to GAPDH expression levels. All graphs were generated with Graphpad Prism Version 6 and a one-way ANOVA was applied. Error bars represent the Standard Error of the Mean (SDE). The results of this study clearly showed that intranasal administration of the OM bacterial extract effectively protected mice against infection with influenza virus at any dose used in the study with greater efficacy by the intranasal route when compared by the oral route. (Figure 14). Compared to the untreated control, there was an apparent improvement in virus control in both the water and the OM bacterial extract for the oral groups. Furthermore, this protective effect was significantly dose dependent, from 5 micrograms up to 100 micrograms, the latter being the highest dose evaluated in this experiment. The groups that received six rounds of intranasal administration of OM bacterial extract showed the best efficacy with the least variation, although mice that were administered the OM bacterial extract only three times were also significantly protected against the virus. Those data clearly confirmed previous experiments after intranasal administration demonstrating that OM bacterial extract provides the most effective route of administration. Since a clear dose response was evident with both treatments 3 and 6 of OM bacterial extract, it can be concluded that this dose and regimen is important in this very effective prophylactic treatment against influenza in this mouse model. Thus it can be anticipated that longer total treatment duration, higher frequency of perioral administration as well as intranasal administration and higher doses are more effective. EXAMPLE 7: The effects of the bacterial extract according to the present invention on the expression of Rhinovirus coupling proteins and on type 1 and type 2 interferon responses on primary human epithelium originating from healthy donors. Previous data on the antiviral activity of extemporaneously prepared perioral OM bacterial extract (strain 21 lysate bacterial extract) on human lung epithelial cells derived from healthy donors as well as patient with COPD and asthma were published (Roth M et al , PLoS ONE 2017, 12(11), e0188010). Monitoring demonstrated antiviral efficacy in animals by the OM bacterial extract via the intratracheal route (direct pulmonary exposure, examples 4, 5 and 6). The Applicant evaluated this direct pulmonary exposure using human lung-derived bronchial epithelial cells (hBEC). To mimic the results obtained in mice by the intratracheal route, human bronchial lung epithelial cells were directly exposed to the new formulations of stable OM bacterial extract from Usados ​​strain 21 (OM314A (P1, P2, P3), OM314B (P4) and OM314C (P5)) to evaluate the mounted antiviral efficacy response of lung cells. Considering the antiviral effect on primary lung epithelial cells, those studies confirmed that the lung is the main target organ of the OM bacterial extract according to what was previously suggested by intranasal and intratracheal administrations in animals (Examples 4, 5 and 6). In this study, the Applicant demonstrated on a molecular basis the results of cellular exposure to Rn ι η / ι 7n7 / E / YL OM bacterial extract and its protective effects on Rhinovirus infections. Up to this point, experiments on protein, mRNA expression and immunofluorescence were performed. Detection was performed using ELISA and immunofluorescence (direct cell counting with Trypan blue exclusion stain) techniques, as well as TA-PCR on human lung epithelial cell cultures from lungs of several healthy donors, patients with COPD, and patients with asthma. Isolation and characterization of BEC: small pieces of bronchial tissues (1 x 1 x 1 mm up to 2x2x2 mm) were placed in cell culture vessels, which were pre-moistened with BEC specific medium Cnt-PR-A (CeilnTech, Bern, Switzerland). . The medium was replaced every second day and the cells were passed through mechanical agitation to divide the cells. Cells were characterized by positive staining for E-Cadherin and Pan-Keratin and negative staining for fibronectin (Roth M et al, PLoS One. 2017;12:e0188010). In this study, the applicant evidenced on a molecular basis the superior results of a selected set of new stable OM bacterial extract formulations and their preventive antiviral effects on Rhinovirus infection. The experimental readouts were quantitative viral load changes, as well as anti-infective and anti-inflammatory mediators produced by hBEC, including, but not limited to, soluble mediators such as type 1 and type 2 interferon. Those biological effects were measured by the following methods: detection of mRNA by TA-PCR for viral load and detection of soluble mediator release by ELISA for type 1 interferon (IFN-beta and type II gamma interferon). The determination of RV16 mRNA was carried out for the different formulations listed in Table 27 with antiviral effect expressed as a percentage of mRNA for RV16 and RV16 mRNA expression rate. Example 7.1: Antiviral results The results obtained with the different stable OM bacterial extract formulations tested (OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5)) show improved antiviral effect over the standard OM HCl bacterial extract formulation (Figure 15A). In this series, the antiviral effects were tested with the new formulations where various acids were integrated in the purification process in manufacturing (Figure 15C). Compared to unstable liquid formulations labeled “HCI,” the new stable formulations demonstrated equivalent or better antiviral efficacy. Depending on the bacterial extract OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) used and except in one case, an equivalent and better efficacy than the HCl formulation was demonstrated (compare the percentage of mRNA of RV16 for HCL in Figure 15A and I.B. in Figure Figure 15C with the other sample preparations). zLRnLn / Lznz / E / Yi Table 27: Percentage of RV16 mRNA and RV16 mRNA expression rate corresponding to Figure 15A Rn ι η / ι 7n7 / E / YL Controls and OM314A Percentage of RV16 mRNA Expression of RV16 mRNA Negative control 0.18 0.0000016 Positive control of RV16- 100 0.0008809 HCl 61.7 0.0005436 HCl-centri. 51.0 0.0004492 butyric acid 61.0 0.0005377 butyric acid-centri. 51.6 0.0004546 propionic acid 53.2 0.0004687 propionic acid-centri. 66.1 0.0005820 aspartic acid 39.4 0.0003467 aspartic acid -centri. 35.6 0.0003136 Example 7.2: Type 1 and Type 2 Interferons The induction of type 1 interferon beta production by the first generation of the OM bacterial extract has been previously described in experimental mouse cell models in bone marrow-derived primary dendritic cells (DCs) (Dang et al, Sel Rep. 6 Mar 2017;7:43844). Briefly, human EBC cells taken from healthy donors, asthma and COPD patients were seeded on Day 2, serum starved on Day 1 and stimulated for 0, 24 and 48 h with OM314A (Ρ1, P2, P3 ), OM314B (P4) and OM-314C (P5) (concentration of 10 micrograms / mL) according to what is indicated in the scheme (Figure 16). Cell supernatants were then collected at the indicated times for beta and gamma interferon dosing using ELISA. Compared with the original INF beta and gamma secretion by BECs using the previous OM bacterial extract (Figures 17A and 18A, the stabilized product OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) was capable of inducing interferon beta type 1 and interferon gamma type 2 to a greater or similar degree in Figures 17C and 18C (compare I.B. with the other product). Depending on the process used, some marked differences were observed. In this study, A dosage range-induced interferon release of the entire stable bacterial extract OM314 was performed based on 5 donors. The mean interferon values ​​of the 5 donors are shown in Figures 17B and 18B. Denote and contrast With Figures 17A and 18A where IFN-dependent release of OM bacterial extract concentrations was obtained, a maximum volume of 20 microliters was used in Figures 17C and 18C, a volume corresponding to the lowest amount of the standard OM bacterial extract . Alpha interferon was not induced by the OM bacterial extracts nor was it induced by the new stable OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) in human BEC cells in vitro (not shown). EXAMPLE 8: The protective, antiviral effects of the bacterial extract according to the present invention on the expression changes of beta β-defensin-1 and ICAM-1 on primary human epithelial cells (BEC) from human lung biopsies. Example 8.1: β-defensin-l and ICAM-1 As for EXAMPLE 8 and to further pursue the evaluation of the newly stable preparations OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) after systematic comparison with previous data obtained with the OM bacterial extract original, we aimed to confirm previous data also published on human BECs, but using other antiviral marks previously induced by the OM bacterial extract (Roth et al. 2017). This is demonstrated in Figure 19A-19B which shows the ability of recent stable preparations OM314A (Ρ1, P2, P3), OM314B (P4) and OM-314C (P5) to induce expression of antiviral beta β-defensin-l by primary epithelial cells (BEC) derived from human lung in the same or higher grade than the industrial batch (IB # 1619057). Identically, this antiviral efficacy was also demonstrated with the decrease of Rhinovirus ICAM-1 on the surface of these cells. Figure 20B shows the ability of recent stable preparations OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) to decrease the expression of the viral receptor ICAM-1 by human BECs to the same extent as the industrial lot (IB # 1619057). Thus, in both cases the ability of these freshly prepared OM314 stable bacterial extracts to maintain the potency and efficacy of previous OM bacterial standards is confirmed with similar or greater differences depending on the process, bacterial extract content. EXAMPLE 9: The ability of the bacterial extract according to the present invention to activate the TLR-dependent adapter and effector protein MyD88 demonstrated and monitored by the release of TNFα from dendritic cells derived from mouse bone marrow. Example 9.1: Preparation of BMDC To extend research on the ability of freshly prepared stable bacterial extracts OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5) to maintain the same or higher level of efficacy on the activation of innate immunity , we performed identical studies originally demonstrating the anti-inflammatory and modulatory effect of bacterial OM standards exhaustively exemplified in Dang et al. (Sel Rep. Mar 6, 2017; 7: 43844). Up to this point, only one type of study was carried out, but summarized as using all the cellular components necessary after activation. A surface receptor (TLR), an obligatory effector protein for the bacterial extract OM, for inducer of protection (MyD88) and transcription factor to induce the release of cytokine after its cleavage and translocation from the cytoplasm to the nucleus (NFkB) where it is measured the transcription and release of cytokine in the medium (TNFa). Up to this point, primary dendritic cells derived from bone cells from mouse bones were used. Bone Marrow (BM) cells were extracted from the femur and tibiae of 6-10 week old C57 / BL6 mice or various TLR4 genetically modified mice (shown here only with TLR4 genetically modified mice (TLR4 - / -) and WT) washing the bones with ice-cold PBS. Subsequently, the cellular treatment of BMDM and its maturation and differentiation into dendritic cells (BMDC) were carried out according to Dang et al. 2017. Culture purity was determined by measuring cells with anti-mouse CD11c and anti-mouse MHCII antibodies and the percentage of CD11c+MHCIIhigh cells was analyzed by flow cytometry. zLRnLn / Lznz / B / Yi Example 9.2: Stimulation of BMDC and measurement of cytokine release BMDCs were plated on 96-well tissue culture plates at a density of 2x105 cells / well and stimulated for 16 h with LPS (4 pg / mL) from Enzo Life Sciences or different concentrations of freshly prepared bacterial extracts OM314A (P1, P2, P3), OM314B (P4) and OM-314C (P5). As a reference in Figure 21, different concentrations (50 - 1600 pg / mL) of the Industrial Batch (I. B. #1619056OM) of the bacterial extract were used (upper left corner). TNF-α cytokine concentrations were measured in cell-free supernatants by ELISA kits according to the manufacturer's instructions using eBioscience TNF-α kits, as exemplified in Figure 21, all freshly stable bacterial extracts. Preparations OM314A (P1, P2), OM314B (P4) and OM-314C (P5) except one (P3 3-hydroxy-butanoic acid) were able to induce TNF-α secretion from BMDC to the same extent as the control ( I.B.) from normal and wild mice. Interestingly, this secretion was equivalent or better (depending on the dose) in some cases, demonstrating the importance of the selective response to the origin of the different bacterial extracts. Concomitantly, the fresh stable bacterial extract OM314A P3, P4 and P5 did not require TLR4 receptor to induce TNF-α release from BMDCs, since this cytokine was also secreted in the absence of this TLR4 (TLR4 - / -). The LDA reveals that different organisms dominate different groups and that i) BE (used bacterial extract of strain 21) prevents harmful bacterial groups in HFD mice and ii) increases group diversity EXAMPLE 10: The ability of perioral administration of bacterial extract according to the present invention to reverse gut dysbiosis in animals maintained on a High Fat Diet. Example 10.1: The importance of the balance of a microbiota and harmful consequences of dysbiosis and analysis of the microbiome by taxonomy. Specific microbiota patterns are available and depend on many external factors such as diet, age, genetics and medications. (Dieterich et al. Med Sci (Basel). 2018;6(4):116. Published 2018 Dec 14, doi: 10.3390 / medsc¡6040116). Although invention is still beginning to show that the microbiome can contribute to homeostasis, producing precise mechanisms in which microbiome dysbiosis leads to certain medical conditions, the importance of maintaining a balanced microbiome is paramount and products are urgently needed to reestablish that imbalance. Consequently, restoring an unfavorable gut flora population to a favorable microbial ecosystem can prevent human diseases (Young VB et al. BMJ 2017;356:j831). In a study conducted to evaluate the lipotoxic effects of consuming a high-fat diet (HFD) in pregnancy, we recently showed that consuming a HFD for eight weeks leads to gut dysbiosis, oxidative stress, increased inflammation and inflammation-induced increased risk of preterm birth (PTB). Consequently and in the present EXAMPLE, we set out to determine the capacity of the bacterial extract of the present invention to reverse HFD-induced intestinal dysbiosis and the associated dangerous effects on metabolism and immune status. The taxonomy results allowed the Linear Discriminant Analysis (LDA) scores to be determined as indicated in Figure 22. This Figure illustrates the specific effect of Bacterial Use on the specific microorganisms. The undesirable Clostridiales, Firmucutes, Clostridia and Blautia species present in control mice with HFD (Sham HFD) were re-established ΑΓ>I n / Ι 7O7 / E / Yl· in mice fed bacterial extract (HFD-BE) and to a lesser extent, also in mice fed a normal chow control diet (NCD-Sham). Furthermore, increased levels of desirable organisms depleted by consumption of an HFD were also identified in the microbiota content of mice fed bacterial lysate (HFDBE and NCD-BE). Unlike standard taxonomic analysis that demonstrates the rise and fall of a selected set of species, the LDA score demonstrated in Figure 22 illustrates distinct species from each of the 4 groups (NCD-Simulated, NCD-BE, HFD-Simulated , HFD-BE). LDA reveals that different organisms dominate different groups and that i) BE (bacterial extract lysate of strains 21) prevents harmful bacterial groups in HFD mice and i) increases group diversity, thus confirming the positive effect of the bacterial lysate of the present invention. Example 10.2: Methods, animal, feed 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 that they are known to become obese, hyperglycemic, and insulin resistant when fed a high-fat diet (HFD). CD1 mice, on the other hand, have more moderate metabolic dysfunction after consuming a HFD, but have the advantage that they reproduce more quickly, so any idiosyncratic response to the HFD will be avoided. Mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). An equal number of male and female mice of each strain were housed in individually ventilated cages in the Animal Care Center at 24°C, on a day and night cycle of 12 h each, and had ad libitum access to food and water. . A diet containing 60% fat, high saturated fat, fat derived mainly from lard and soybean oil was provided to HFD mice and NCD containing 13.3% fat was provided to NCD mice. Food and water were provided ad libitum. The OM bacterial extracts were administered periorally by pipetting via intranasal route (0.05 ml) for 14 days or orally route directly into the mouths (< 0.15 mL) of the mice daily for four and eight weeks. Mice receiving “substitute treatment” (negative controls) were administered by pipetting 0.05 mL of water (intranasal) once a day for 14 days or via the oral route directly into the mouths (<0.15 mL) of the mice daily for four and eight weeks. Positive control. Lactobacillus plantarum, a probiotic that reverses intestinal dysbiosis, is administered to positive control mice by adding 2 x 108CFU / ml to their drinking water for six days. Extract analysis of OM bacterial extracts on gut microbiome: Fecal samples were collected and 16S rRNAs were analyzed by sequencing. After sequencing functional genes in samples were characterized and differences between functional genes of microbial communities were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The protein function classification of those genes was predicted using family information from the Set of Orthologous Groups (COG). Analysis of the effects of OM bacterial extract on metabolic status: Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed on all mice at the beginning and at the end of the eight-week period. Mice were fasted for 8 h and then challenged with an intraperitoneal injection of 2.0 g / kg glucose with or without insulin and glucose levels were measured at 0.15, 30, and 60 min. Rn ι η / ι 7n7 / E / YL Example 10.3: Effect of bacterial extract of bacterial lysates 21 (BE) on various genera As an addition and to further populate the data summarized in Table 22, an example of several BE-induced changes in gender is demonstrated in Figure 26A-26E. In this example, BV demonstrated a positive effect on harmful genes (reducing growth) and simultaneously, favored growth on good genera. Five examples are shown in this Figure. (Fig.26A) BE reduces clostridial genera lachnospiraceae blautia activated by HFD; (Fig.26B) BE reduces clostridial clostridial ruminococcaceae genera GCA-900066225 induced by HFD; (Fig.26C) BE reduces clostridial Ruminococcaceae ruminococcaceae UCG-0101 in both NCD and HFD mice; (Fig.26D) BE restores uncultured bacteria Bacteroidales Muríbaculaceae depleted by HDF; (Fig.26E) BE reduces Lachnospiraceae of the group [Eubacterium] físsicantena. EXAMPLE 11: The ability of perioral administration of the bacterial extract to improve glucose tolerance. Example 11.1: Test of pre-diet glucose concentration of bacterial extract of strain 21 lysates (Pre-diet), from mice after normal chow diet (Post-NCD), after high-fat diet fat (After HFD). Furthermore, for the correction of intestinal dysbiosis by the bacterial extract, glucose tolerance was also investigated in those diabetic mice. This parameter is essential from a clinical point of view. Figure 23B, mice fed control diet with normal chow with bacterial extract of strain 21 (NCD-BE), but not L plantarum (normally used as a positive control to improve intestinal dysbiosis reorganization) increased significantly glucose tolerance as shown by lower glucose concentration. Identically, this effect was also demonstrated in high-fat diet mice fed the bacterial extract of strain 21 lysates (HFD-BE), shown in Figure 23C, also to a lesser extent. Example 11.2: Evaluation of weight and feed consumption In Figure 24A-24B, both parameters were tested as controls. The results show that high-fat diet mice fed strain 21 bacterial lysate extract (HFD-BE) significantly decreased weight gain. This is exemplified in those mice on a high-fat diet (Figure 24A). Concomitantly, this absence of weight takes place without affecting the food consumption measured and expressed here as a control (Figure 24B). Interestingly, this effect was even more effective than L. plantarum normally used for that measurement. Example 11.3: Insulin tolerance assessment Considering the above-cited protective results on glucose tolerance obtained from treatment with the bacterial extract of strain 21 lysates (BE) in diabetic mice exposed to the high-fat diet (HDF) regimen, we further investigated whether the insulin level It was also modified to its protective end. Figure 25A-25B demonstrates insulin tolerance in all mice (42) before baseline treatment (BEFORE DIET) and after the final eight-week treatment period (POST DIET) in diet mice. high fat mice (HFD). As expected for normal mice fed with L. Plantarum (NCD-L-plant.) showing decreased insulin resistance (Figure 25B), mice fed with zLRnLn / Lznz / E / Yi bacterial extract HFD-BE of Used 21 showed significantly, and for all time points, insulin resistance in the high-fat mice. This protective effect by the bacterial extract of Usados ​​21 in line with the glucose data and the absence of weight gain from previous Figures, all points towards a positive reorganization of the standard parameters measured in diabetic patients. This confirms that the bacterial extract of Used strain 21 (BE) not only induced a protective intestinal dysbiosis in mice with chronic HFD undergoing that regimen, but also its associated sequelae exemplified here with weight, glucose and insulin, all positively influenced towards protection by the Usados ​​bacterial strain 21. Rn ι η / ι 7n7 / E / YL

Claims

1. A bacterial extract of Gram-positive or Gram-negative bacterial species wherein the bacterial extract is obtainable by alkaline lysis of the bacterial species 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, a combination thereof and / or pharmaceutically acceptable salts and esters thereof, followed by purification by filtration of the neutralized extract and adjusting to a final physiological pH by adding the organic acid or combination thereof using neutralization.

2. The bacterial extract according to claim 1, comprising a reduced fraction of molecular weight components of 0 to 300 kDa, 0 to 100 kDa, or 0 to 60 kDa.

3. The bacterial extract according to claim 1 or 2, wherein the alkaline lysis is carried out at a pH greater than 10, with variations of ± 0.1 pH.

4. The bacterial extract according to any of the preceding claims, wherein the final pH is adjusted between 5 and 8, between 6 and 8, between 6.3 and 7.8 or between 6.5 and 7.

8.

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

6. The bacterial extract according to any of claims 1-4, wherein one or more bacterial species selected from Lactobacillus bacterial strains.

7. The bacterial extract according to claim 6, wherein the Lactobacillus strains comprise 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 reuterí, Lactobacillus salivarlas, Lactobacillus lactis and Lactobacillus delbrueckíi.

8. The bacterial extract according to any of claims 1-4, wherein one or more bacterial species selected from bacterial strains of Escherichia coli.

9. The bacterial extract according to any of the preceding claims, wherein the stable purified bacterial extract comprises less than 100 micrograms / ml of nucleic acids, at least 0.1 mg / mL of saccharides.

10. The bacterial extract according to any of the preceding claims, the bacterial extract being stable in liquid form at room temperature, 4°C, -20°C or -80°C.

11. The pharmaceutical composition comprising the bacterial extract according to any of the preceding claims and a pharmaceutically acceptable excipient or vehicle, wherein the pharmaceutical composition may be in a solid, semi-solid, liquid or aerosol formulation and wherein the composition may be formulated for intranasal, intratracheal, mucosal, transmucosal, external skin topical, buccal, sublingual, oral, pulmonary, intrabronchial and / or intrapulmonary routes of administration.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is liquid or aerosol and is formulated as a spray, drops, colloidal, mist, nebulizer and / or atomized vapor or wherein the composition is liquid or semi-solid and is formulated as emulsions, microemulsions, aqueous dispersions, oils, milks, balms, foams, aqueous or oily lotions, aqueous or oily gels, creams, solutions, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, serums, ointments, foams, pastes or transdermal patches or wherein the composition is solid and is formulated as a powder and / or disintegrating tablet.

13. The pharmaceutical composition according to claim 11 or 12, for use in the treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammations and / or neoplasms and / or dysbiosis.

14. The pharmaceutical composition for use according to claim 13, wherein the infections are selected from upper and lower respiratory tract infections and / or associated sequelae such as allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute upper respiratory infections, or diseases with disorders of epithelial ciliary movement and / or mucus clearance disorders, and wherein the infections may further comprise secondary infections, secondary bacterial infections followed by viral infections with influenza, non-respiratory viral infections, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock, or virus-induced complications.

15. The pharmaceutical composition for use according to claim 13, wherein the infections are selected from viral infections, influenza, respiratory syncytial virus, rhinovirus, coronavirus, CoV, SARS-CoV, and MERSCoV.

16. The pharmaceutical composition for use according to claim 13, wherein the infection is COVID-19, 17. The pharmaceutical composition for use according to claim 13, wherein the inflammations are selected from atopic dermatitis with allergic / atopic and non-respiratory respiratory indications, associated acute and / or chronic dermatitis, anaphylaxis and food allergy or wherein the inflammations comprise skin disorders, inflamed skin, eczema, rosacea, atopic dermatitis, psoriasis, actinic keratosis or wherein the inflammations are selected from eosinophilic indications such as eosinophilic asthma or eosinophilic pneumonia or wherein the inflammations are selected from predominantly T helper 2 autoimmune indications.

18. The pharmaceutical composition for use according to claim 13, wherein the dysbiosis-related disorders are selected from obesity, asthma, diabetes, autoimmune diseases, diseases associated with low-fiber regimens, atopic dermatitis, acute and / or chronic dermatitis associated with atopic dermatitis, or wherein the dysbiosis-related disorders are selected from inflammatory bowel diseases, comprising ulcerative colitis, Crohn's disease, colitis, type 2 diabetes, autoimmune diseases, or diseases associated with low-fiber regimens.

19. A delivery device comprising the pharmaceutical composition according to any one of claims 11 to 18, the delivery device being selected from the group comprising a nasal insufflator device, intranasal inhaler, intranasal spray device, atomizer, nasal spray bottle, unit dose container, pump, dropper, squeeze bottle, nebulizer, metered dose inhaler (MDI), pressurised dose inhalers, insufflators, bidirectional devices, dose ampoules, nasal pads, nasal sponges, and nasal capsules.

20. A process for preparing a stable bacterial extract according to any of claims 1-9, comprising the following steps: a. cultivating each species of bacterial strain in a suitable culture medium, b. thermally removing the bacteria, removing the culture medium, and harvesting the concentrated biomass, c. using each strain at an initial pH greater than 10, d. lowering the pH of the extracts obtained in step (c) 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, a combination thereof, or pharmaceutically acceptable salts and esters thereof, e. passing the product of step (d) at least once through a microfilter and retaining the product on an ultrafilter to obtain a purified soluble extract.

21. Use of the pharmaceutical composition according to claim 11 or 12, to prepare a medicament for the treatment and / or prevention of acute and chronic immunological disorders resulting from infections and / or inflammations and / or neoplasms and / or dysbiosis.

22. Use according to claim 21, wherein the infections are selected from upper and lower respiratory tract infections and / or associated sequelae such as allergic rhinitis, rhinitis, nasopharyngitis, sinusitis, hypersensitivity pneumonitis, bronchopneumonia, bronchitis, bronchiolitis, pneumonia, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute upper respiratory infections, or diseases with disorders of epithelial ciliary movement and / or mucus clearance disorders, and wherein the infections may further comprise secondary infections, secondary bacterial infections followed by viral infections with influenza, non-respiratory viral infections, non-respiratory bacterial infections, systemic infections such as sepsis, septic shock, or virus-induced complications.

23. Use according to claim 21, wherein the infections are selected from viral infections, influenza, respiratory syncytial virus, rhinovirus, coronavirus, CoV, SARS-CoV, and MERS-CoV.

24. Use according to claim 21, wherein the infection is COVID-19.

25. Use according to claim 21, wherein the inflammations are selected from atopic dermatitis with allergic / atopic and non-respiratory respiratory indications, associated acute and / or chronic dermatitis, anaphylaxis and food allergy or wherein the inflammations comprise skin disorders, inflamed skin, eczema, rosacea, atopic dermatitis, psoriasis, actinic keratosis or wherein the inflammations are selected from eosinophilic indications such as eosinophilic asthma or eosinophilic pneumonia or wherein the inflammations are selected from T helper 2 predominant autoimmune indications.

26. Use according to claim 21, wherein dysbiosis-related disorders are selected from obesity, asthma, diabetes, autoimmune diseases, diseases associated with low-fiber diets, atopic dermatitis, and associated acute and / or chronic dermatitis, or wherein dysbiosis-related disorders are selected from inflammatory bowel diseases, including ulcerative colitis, Crohn's disease, colitis, type 2 diabetes, autoimmune diseases, or diseases associated with low-fiber diets. 71 κη ι η / ι 7n7 / E / YL