BRPI0808351B1 - BACTERIAL EXTRACT FOR DISEASES OF THE DIGESTIVE OR URINARY TRACT AND PROCESS FOR ITS PREPARATION - Google Patents

Abstract

BACTERIAL EXTRACT FOR DISEASES OF THE DIGESTIVE OR URINARY TRACT AND PROCESS FOR ITS PREPARATION. The present invention relates to an extract of bacterial strains useful as a treatment for diseases of the digestive or urinary tract, to compositions comprising the extract, and to processes for producing the extract using means that do not pose a risk of prion-related diseases.

Description

DESCRIPTION OF THE INVENTION

[0001] This application claims priority to U.S. Provisional Application No. 60/904,787, filed March 5, 2007, which is incorporated herein in its entirety by reference.

FIELD OF INVENTION

[0002] The present invention relates to extracts of bacterial strains useful as a treatment for symptoms such as diseases of the digestive or urinary tract, to compositions comprising the extracts, and to processes for producing the extracts using means that do not pose a risk of prion-related diseases.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] The present invention relates to compositions comprising bacterial extracts useful for treating symptoms such as urinary or digestive tract diseases. The extracts may comprise bacterial lysates from cultures chosen from one or more species of Escherichia coli. In some embodiments, the extracts may comprise one or more species chosen from the following E. coli strains: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707, and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088, and 089. These strains are deposited under the Budapest Treaty. The strains listed under the number I have been indexed by the Collection Nationale de Culture des Microorganismes at the Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France. All other strains have been indexed by the National Collection of Type Cultures, London.

[0004] In some embodiments, an extract is prepared from all of these strains. In other embodiments, only some of the strains are chosen. In some embodiments, for example, one or more strains are chosen from the "I" group and one or more strains are chosen from the "NCTC" group.

[0005] In some embodiments, one or more of the specific strains listed above may be omitted, or replaced with a different strain of E. coli, or from a different species of bacteria.

[0006] The extracts may be obtained by an alkaline lysis process after the cells are grown to a suitable optical density in a culture medium. In some embodiments, each of the bacteria is grown in a medium that does not pose a risk of prion-related diseases or a risk of other diseases that can be transmitted through ingestion of products obtained by animal-based means. For example, in some embodiments, a plant-based medium is used to grow the cells, such as a soy-based medium. A synthetic medium may be used for cell growth in some embodiments, or a medium including biological extracts, such as yeast extract and equine serum, that also do not pose such disease risks.

[0007] Lysates may also be filtered to remove nucleic acids and larger cellular debris. As a result of filtration, in certain embodiments, the amount of nucleic acid present in the extracts is less than 100 μg/mL. In certain embodiments, insolubilized compounds, such as cell wall debris and insufficiently degraded liposaccharides (LPS), are also removed by filtration. Thus, in some embodiments, the resulting extract comprises soluble molecular components and does not contain significant amounts of insoluble or particulate material.

[0008] Saccharide components may be preserved in the extracts, including liposaccharide (LPS) components. During the lysis process, saccharides may become chemically modified, e.g., cleaved into smaller structures or replaced by other functional groups.

[0009] The racemization of amino acids during the lysis process also creates D-amino acids from the naturally occurring L-amino acids found in natural proteins. D-amino acids may be beneficial in increasing the effectiveness time of extracts, since they are not sufficiently digested in the mammalian intestine. In this way, 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, possibly allowing for a stronger immunostimulatory action.

[00010] Although bacterial extracts have been used in the prior art to stimulate the immune system against diseases of the digestive and urinary tract, there has been a need to better standardize and control these extracts in order to make them safer, more effective, and longer lasting. For example, it was thought that saccharide components, including potentially toxic lipopolysaccharide (LPS) components, should be removed from bacterial extracts for safety reasons. (See, e.g., U.S. Patent 5,424,287.) However, the present invention proposes a process that results in sufficient chemical modification of the LPS components so that the saccharides can be safely retained. Retention of these components can improve efficacy in addition to providing additional antigens.

[00011] For example, the inventors have found that monitoring the pH and time of lysis allows sufficient degradation of potentially allergenic or toxic cell wall components. In contrast, prior lysis conditions at lower pHs or shorter times have produced extracts in which the cell wall components and LPS have been chemically modified to an insufficient extent. (See, for example, GB 2 054 374 A). The resulting extracts were too allergenic to be safely administered to patients. In general, the inventors have found that products lysed at too low a pH and/or too short a time have increased toxicity, decreased protein extraction, and decreased filtration capacity.

[00012] Furthermore, the present invention cultivates bacterial strains in culture media that do not pose disease risks, such as prion-related diseases.

[00013] The filtration process can also influence the properties of the resulting extract in some cases, such as the pore size of the filter, and sometimes the chemical properties of the filter surface, by altering the type of materials that are removed and retained. For example, the present invention uses a filtration process that retains certain saccharides but removes other molecular components, such as nucleic acids.

[00014] Thus, the present invention provides parameters that standardize bacterial extracts to help maintain consistent safety and efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

[00015] Figure 1: Diagram of a slow tangential filtration (TFF) system for preparing bacterial extracts after lysis of the bacteria. The diagram shows two different configurations for the filters: a parallel mode, in which all filters operate simultaneously, and a "serpentine" mode, in which the filters are configured in series.

[00016] Figure 2: Activity of the extracts in a peripheral blood mononuclear cell (PBMC) assay at an initial biomass concentration for lysis of 12.5 g/L (part A) and 25 g/L (part B). (See, for example, Example 5A for further details).

[00017] Figure 3: Activity of the extracts in a PBMS test at an initial biomass concentration for lysis of 25 g/l (part A) and 100 g/l (part B).

[00018] Figure 4: Activity of the extracts in a peripheral blood mononuclear cell (PBMC) test at a lysis time of 24 hours (part A) and 72 hours (part B).

[00019] Figure 5: Effect of NaOH concentration during lysis on macrophage nitrous oxide (NO) activity.

[00020] Figure 6: Mean CFU (colony forming unit) values in bladder (part A) and kidney (part B) tissues for different experimental groups.

[00021] Figure 7: Death records in the experimental groups during the 21-day period after infection with 104 CFU of Salmonella typhimurium.

DETAILED DESCRIPTION OF THE INVENTION

[00022] Definitions

[00023] Extract: An extract, as defined herein, refers to a material obtained after the lysis of one or more bacterial strains. In some cases, the extract is obtained from only one strain, while in others, the extract is a mixture of extracts from several different strains.

[00024] Alkaline lysis: This is a method of subjecting bacterial cells to lysis under basic conditions, such as by using an organic or inorganic base.

[00025] Lysate: An extract of bacteria obtained from a cell lysis procedure.

[00026] Filtration: The filtration process, as defined herein, refers to passing an extract, or mixture of extracts, through one or more filters, such as microfilters (i.e., microfiltration) or ultrafilters (i.e., ultrafiltration). Such filtration may not necessarily remove 100% of the components it is intended to remove. In some cases, filtration is repeated in multiple steps or cycles.

[00027] Initial pH: This term means the pH measured at the beginning of a procedure, such as bacterial lysis or filtration.

[00028] Saccharides: A saccharide, as defined herein, includes monosaccharides, disaccharides, as well as larger saccharides such as linear and branched polysaccharides. Saccharides also include chemically modified or substituted saccharides such as lipopolysaccharides (LPS) and its chemically modified variants.

[00029] D-amino acids: This term refers to amino acids that exist in dextrorotatory isometric forms, as opposed to biosynthetically produced L-amino acids, which exist in levorotatory isomeric forms.

[00030] Racemization: This term indicates the at least partial chemical modification of L-amino acids into D-amino acids.

[00031] Media that avoids the risk of prion-related diseases means a culture medium used at any stage of the preparation of extracts, which does not comprise materials such as serum or meat extracts obtained from animals, such as cows or sheep, or from any other animal that can transmit prion-related diseases. Examples of such media include synthetically chemically defined or plant-based media, and also media using horse serum or media comprising materials obtained from animal species that do not transmit prion-related diseases. Examples of prion-related diseases include, for example, mad cow disease, scrapie, and Creutzfeld-Jakob disease.

[00032] A non-animal medium is a medium that does not include components derived from animals. Examples include a plant-based (i.e., vegetable) medium, such as a soybean medium, and a synthetic medium.

[00033] Treatment, as used herein, refers to both the treatment of current infections and other conditions, for example, as well as the prevention or protection from the development of new infections, for example.

[00034] The term patient, as used herein, refers to any animal patient, including mammalian patients such as humans and domestic mammals.

[00035] It is understood that the specific bacterial strains identified in the present invention and used in the invention may include the strain obtained from the original deposit cited in the present invention or from a genetic clone thereof, including a strain that was redeposited at a later time under a different deposit code name but which is considered genetically to be the same strain as the originally deposited version.

[00036] All numbers used in the present invention are approximations, taking into account intrinsic errors in their measurement, rounding and significant figures.

[00037] Preparation of Extracts

[00038] The bacterial extracts of the present invention may be prepared by fermentation, followed by thermal inactivation, concentration and collection of biomass, alkaline lysis of single bacterial biomass or alkaline lysis of mixtures of bacterial biomass under defined conditions. The alkaline lysates under different conditions may be mixed prior to purification by filtration. The obtained filtrate may be further purified, such as to remove particulate matter, and may also be lyophilized and/or formulated.

[00039] For each strain, to obtain a sufficient quantity of material, fermentation cultures can be started from a working seed batch, followed by inoculation into larger fermentation vessels.

[00040] The media used may be the same for each species. However, supplemental growth factors may be introduced to improve the growth of some species. In some embodiments, a medium that avoids the risk of prion-related diseases is used for the cultivation of at least some or all of the strains. Examples include non-animal media, such as a plant-based medium and synthetic media. Other examples include a medium that includes equine serum or other animal extract, which is obtained from an animal species that does not pose a risk of prion-related diseases, as opposed to strains grown in the presence of bovine serum or meat extracts that may pose such risks.

[00041] In some embodiments, fermentation may begin with a small culture, such as 0.1 to 1.0 liters, incubated for about 3 to 6 hours at 30-40°C, such as 37°C, to obtain an optical density (OD) at 700 nm of 3.0 to 5.0. After a small-scale culture step, additional cultures in one or a series of larger fermenters may be performed at 30°C to 40°C for a duration of 3 hours to 20 hours, such as for 3-10 hours, or 8 hours.

[00042] Following fermentation, the biomass of each strain or set of strains may be inactivated by heat treatment, concentrated, and frozen. After thawing the frozen biomasses, the bacterial suspension may then be diluted and alkalized to cause lysis of the bacterial cells with a concentrated solution of hydroxide ions, such as NaOH. In some embodiments, about 10 to about 120 g/L of bacterial dry weight of one or a mixture of strains is lysed, such as about 15 to about 80 g/L, or about 15 to about 35 g/L, such as 15, 20, 25, 30, or 35 g/L. In some embodiments, about 40 to about 80 g/L is lysed, such as 40, 50, 60, 70, or 80 g/L (the "bacterial dry weight concentration" is defined as the amount of dry biomass per liter of lysis. The dry weight concentration is measured by drying 5 mL of material in a small porcelain dish at 105°C until a constant mass is reached, then recording the mass in grams per liter). In some embodiments, a strong base concentration of 0.01 N to 1.2 N is used, such as from 0.10 N to 1.1 N, or from 0.10 N to 0.65 N, or from 0.10 N to 0.4 N, or a range starting or ending from 0.1, 0.2, 0.3, or 0.4 N, or from 0.6 N to 1.1 N, or a range starting or ending of 0.6, 0.7, 0.8, 0.9, 1.0 or 1.1 N, or a base concentration is used to achieve an initial pH of 12 or higher, or a pH greater than 12, a pH greater than 12 and less than 13.5, such as greater than 12.5, greater than 12.6, greater than 12.8, or from pH 12.6 to pH 13.4. The pH during lysis may decrease when extracting the solubilized compounds. Therefore, the pH can be adjusted during the procedure. The lysis temperature may vary from 30 to 60oC, such as from 35 to 40oC, such as 37oC. The lysis time may vary from 20 hours to several days, such as 5, 6, 7, 8, 9 or 10 days, or from 30 to 120 hours, or from 30 to 50 hours, such as 30, 35, 40, 45 or 50 hours, or from 60 to 120 hours, such as 60, 72, 84, 96, 108 or 120 hours. The extracts may then be returned to a lower pH, mixed together as desired and filtered.

[00043] After lysis, the solubilized dry weight may be 20 to 180 mg/mL. (The solubilized dry weight is determined by the same method as the bacterial dry weight described above and may alternatively be reported in units of mg/g, assuming the density of the soluble lysate is 1 g/mL.) The remaining protein concentration measured by the Lowry method may be 8 to 75 mg/mL, such as 10 to 70 mg/mL, 20 to 60 mg/mL, or a range starting or ending from 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mg/mL. The saccharide concentration measured by the anthrone method for total reducing sugar, after lysis of a mixture of strains, may be from 0.8 to 4 mg/mL, such as from 1 to 3.5 mg/mL, 1.2 to 3 mg/mL, or a range starting or ending from 1, 1.5, 2, 2.5, 3, 3.5 or 4 mg/mL.

[00044] Lysis may be performed on only one strain at a time, or on a mixture of all desired strains. For example, a mixed extract may be obtained by mixing, for example, two or more bacterial lysates. Each lysate may contain 10 g/L dry weight of biomass at 90 g/L, such as 15-85 g/L, or 20-80 g/L, or 25-75 g/L, or 30-70 g/L, or 35-65 g/L, or 40-60 g/L, or 15-35 g/L, or 40-80 g/L. Each lysate may comprise, for example, from about 20% to 80% of the total extract by volume. The volume proportions of the lysate mixture may be, for example, 25%-75%, or 75%-25%, 70%-30%, or 30%-70%, or 60%-40%, or 40%-60%, or 50-50%.

[00045] The lysates may then be purified by centrifugation and/or filtration. For example, the lysates may be centrifuged at 900 x gravity, followed by one or more filtration sessions through a 0.2 micron filter, which may be used to purify the extract. In some cases, successive filtration sessions through larger pore filters may be used by filtration through a 0.2 micron filter. Ultrafiltration methods may also be employed to help extract soluble materials from the extract, for example, by recirculating the ultrafiltration permeate for further microfiltration.

[00046] In some embodiments, a tangential flow filtration (TFF) method can be used to filter the extracts and extract solubilized molecules from larger cellular debris. (See Figure 1). (See, e.g., Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson, Editor, Interpharm Press, Inc., Buffalo Grove, IL, U.S.A., pp. 126-135 - ISBN:0-935184-72-4.) At the beginning of such a TFF process, a diluted bacterial lysate can be stored in a first tank. A microfiltration (MF) cycle is initiated, and the product is pumped and the resulting MF retentate is recycled, while the MF permeate is transferred to a second tank.

[00047] After reaching a suitable concentration level, an ultrafiltration (UF) cycle is initiated. The UF permeate can be recirculated back to the first tank for continued extraction of the solubilized compounds from the lysate, while the UF retentate 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 permeates.

[00048] Several such extraction cycles may be performed, either with TTF or with another filtration method. In embodiments using TTF, at the end of the last cycle, the ultrafiltration cycle may be stopped and the microfiltration cycle may be performed alone and the MF permeate transferred to tank 2.

[00049] The microfiltration loop may be equipped with filters from 1.2 micron to 0.1 micron, such as 0.65 to 0.2 micron or 0.45 micron filters. The cross flow may 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 bar, such as between 0.8 and 1.5 bar, or 1.0 bar. The ultrafiltration loop may be provided with filters from 10 KDa to 1000 KDa, such as from 10 KDa to 100 KDa, or from 10 KDa to 30 KDa, or from 30 KDa to 100 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.

[00050] Between 5 and 20 diafiltration volumes may be used to extract solubilized compounds from the bacterial cell walls. In some embodiments, between 8 and 15 volumes may be used. Therefore, 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.

[00051] After filtration, the extract may be further concentrated or centrifuged if desired. For example, additional microfiltration may be performed using a smaller pore size filter, such as a 0.2 micron filter. After filtration, the yield of solubilized proteins as measured by Lowry may be 50 to 90% or greater than 60%. After filtration, the extract may be lyophilized prior to formulating it for use.

[00052] In some embodiments of the invention, a selection of lysis conditions may be performed so as to obtain "strong" or "moderate" lysis. For example, in some embodiments, strong lysis can be achieved by lysing from about 15 to about 35 g/L bacterial dry weight, such as 15, 20, 25, 30, and 35 g/L, or smaller ranges within such concentrations (e.g., 15-20, 20-25, 20-30, etc.), with 0.6 to 1.1 N hydroxide ion, such as 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 N, or smaller ranges within such concentrations for 60 to 120 hours, such as 60, 70, 80, 90, 100, 110, and 120 hours, or smaller ranges within such times, at 30-40°C, such as 35-40°C, 30-35°C, or 37°C. Thus, as used herein, a "strong" lysis involves the use of bacterial dry weight concentration, hydroxide ion concentration, time, and temperature, all within the broader ranges above. In other embodiments, a moderate lysis can be achieved by lysing from about 40 to about 80 g/L bacterial dry weight (e.g., 40, 45, 50, 55, 60, 65, 70, 75, or 80) with 0.1 to 0.4 N hydroxide ion (i.e., 0.1, 0.2, 0.3, or 0.4) for 30 to 50 hours (e.g., 30, 35, 40, 45, or 50 hours) at 30-40°C, such as 35-40°C, 30-35°C, or 37°C. Therefore, as used herein, a "moderate" lysis involves the use of bacterial dry weight concentration, hydroxide ion concentration, time, and temperature, all within the broader ranges above. In some embodiments, a mixture of two bacterial lysates, such as a strong and moderate lysate as described above, can be prepared, such as a 10% to 90% mixture, a 20/80, 25/75, 35/65, 50/50 mixture by volume. (See, for example, Examples 8 and 9 below.) In some embodiments, all strains to be used in the extract can be subjected to both a strong and a moderate lysis, followed by pooling of the resulting lysates. Or, alternatively, some strains can be subjected to a strong lysis, while others are subjected to a moderate lysis. Filtration of these lysates can occur before or after mixing, for example, by the TFF method through a MF cycle with a 0.65 to 0.2 micron filter, such as a 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3 or 0.25 micron filter and UF cycle with a 10 or 30 KDa filter, for a total of 8 to 15 cycles, such as 8, 9, 10, 11, 12, 13, 14, or 15 cycles. Once prepared, the extracts of the present invention can be purified to remove particulate matter.

[00053] Chemical Properties of Bacterial Extracts

[00054] Some embodiments according to the present invention may contain, for example, 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, of proteins, or a range starting or ending at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg/mL; 1.5 to 2.5 mg/mL free amino acids (A.A.), or 1.5 to 2 mg/mL, or 2 to 2.5 mg/mL free A.A., or a range starting or ending at 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 free A.A., calculated from glutamic acid (147.1 g/mol); and 0.3 to 4.5 mg/mL of polysaccharides and monosaccharides, or 0.3 to 4 mg/mL, or 0.4 to 4 mg/mL, or 0.5 to 3.5 mg/mL, or 0.6 to 3 mg/mL, or 0.3 to 1 mg/mL, or a range starting or ending at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mg/mL of polysaccharides and monosaccharides. For example, some embodiments contain 5 to 9 mg/mL of protein, 2 mg/mL of free amino acids (A.A.) calculated from glutamic acid (147.1 g/mol), and/or about 0.3 to 0.6 mg/mL of polysaccharides and monosaccharides.

[00055] In some embodiments, the concentration of LPS equivalents based on a horseshoe crab amebotic lysate (LAL) chromogenic assay is less than 5000 ng/mL, or less than 2000 ng/mL, or less than 1000 ng/mL, or less than 750 ng/mL, or less than 500 ng/mL, or less than 200 ng/mL, or less than 100 ng/mL.

[00056] Lysis of bacteria according to the present invention can result in partial hydrolysis of amphiphilic compounds such as lipopolysaccharides and lipopeptides. Lysis of bacteria according to the present invention can also result in partial hydrolysis of proteins as well as in deamination, deamidation and partial racemization of amino acids from L to D. In an analytical study of an extract according to the invention, after total HCl hydrolysis of the extract and derivatization of the amino acids, gas chromatography peaks representing D-aspartic acid and D-asparagine, D-glutamic acid and D-glutamine, D-serine, D-methionine, D-histidine, D-acyanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, D-lysine, D-valine, D-threonine were observed. The percentage of D-amino acids of these species in this study ranged from 3% to 80%. Therefore, some embodiments of the invention allow for the racemization of one or more of serine, methionine, aspartic acid and asparagine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine and lysine, such as all of the foregoing amino acids, or any selection of more than one but less than all of the foregoing amino acids, such as, for example, serine, aspartic acid, asparagine, alanine, phenylalanine, tyrosine and lysine, or a selection of these amino acids. In some embodiments, at least 10% of one or more of the foregoing amino acids can become racemized from L to D. In other embodiments, at least 30% of one or more of the foregoing amino acids can become racemized.

[00057] Biological Activities of Extracts

Bacterial

[00058] The extracts according to the invention may be effective in the treatment of patients suffering from or at risk of developing diseases of the urinary or digestive tract, such as infections of the digestive and urinary tract. The extracts according to the invention may be effective in preventing recurrent urinary tract infections. Examples of diseases that may be treated by the extracts of the invention include E. coli and other bacterial infections, urethritis, tubulointerstitial nephritis, obstructive pyelonephritis, urinary tract infections due to obstructive and reflux uropathy, cystitis, including chronic cystitis, prostatitis, including chronic prostatitis, prostatocystitis, pelvic inflammatory diseases in women, Crohn's disease and irritable bowel syndrome.

[00059] The biological activity of extracts can be determined by a variety of assays. For example, a peripheral blood mononuclear cell (PBMC) assay tests for the production of the cytokine IL-6 from PBMCs and can examine the ability of an extract to stimulate the immune system. For example, in some embodiments, the in vitro concentration of IL-6 measured in supernatants of PBMCs stimulated with the extracts of the invention ranged from 2000 pg/mL to 70,000 pg/mL, 2000 pg/mL to 50,000 pg/mL, 2000 pg/mL to 30,000 pg/mL, 2000 pg/mL to 20,000 pg/mL, 2000 pg/mL to 10,000 pg/mL, or 5000 pg/mL to 70,000 pg/mL, 5000 pg/mL to 50,000 pg/mL, 5000 pg/mL to 30,000 pg/mL, 5000 pg/mL to 25,000 pg/mL, or 5000 pg/mL to 10,000 pg/mL, or 15,000 pg/mL to 25,000 pg/mL. When LPS was used as an agonist control (at 0.01 μg/mL), the values obtained ranged, depending on the donors, from 5,000 pg/mL to 70,000 pg/mL.

[00060] A murine nitric oxide (NO) test measures the production of NO by murine macrophages, which also indicates immune stimulation. For example, macrophages produce NO in order to kill invading bacteria. In some embodiments, in vitro nitrous oxide (NO) activity for embodiments of the present invention tested at concentrations ranging from 0.001 mg/mL to 10 mg/mL soluble dry weight provided maximal responses ranging from 3 μM to 100 μM nitric oxide, or 3 μM to 90 μM, 3μM to 80 μM, 3 μM to 70 μM, 3 μM to 60 μM, 3 μM to 50 μM, 3 μM to 40 μM, 3 μM to 30 μM, 3 μM to 20 μM, 3 μM to 10 μM, or 5 μM to 80 μM, 5 μM to 60 μM, 5 μM to 40 μM, 5 μM to 20 μM, or 10 μM to 80 μM, 10 μM to 70 μM, 10 μM to 50 μM, 10 to 30 μM, or 10 μM to 15 μM, or bands starting or ending at 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM.

[00061] The activities observed in human peripheral blood mononuclear cells and murine macrophages in vitro may depend on variables such as the amount of bacterial dry weight to be lysed, i.e., the "starting material" for lysis, the duration of alkaline lysis, and the initial percentage of NaOH or the initial pH used in lysis.

[00062] Combining in vitro activity tests, such as PBMC and NO, with determination of LPS concentration, such as by LAL, can also provide information regarding the balance of activity vs. toxicity risk for a given bacterial extract.

[00063] The activities observed in vitro in animals in infection models show that some embodiments of the present invention have a protective effect. See, for example, Example 9, illustrating the repeated treatment of animals with exemplary extracts according to the invention. In an in vivo model of urinary tract infection with E. coli (Example 8), animals that received repeated oral administrations for prevention of exemplary extracts according to the invention exhibit a lower amount of bacteria in the urinary tract (bladder and kidneys).

[00064] For example, the survival rate 13 days after challenge of at least 8 LPS-insensitive mice with the uropathogenic E. coli strain 1677 is at least 60% when such mice are first treated for 10 days with effective amounts of some embodiments of the present invention. The dose of uropathogenic E. coli for challenge may be chosen such that untreated mice, or mice treated with a water control or blank formulation containing excipients but no extract, have a survival rate of 60% or less, such as 50% or less. In some cases, the survival rate of mice treated with embodiments of the present invention in such a model is at least 70%, at least 80%, at least 90%, or at least 95%.

[00065] As another example, the survival rate 13 days after challenge of at least 8 mice having wild-type LPS sensitivity with Salmonella thyphimurium is at least 60% when such mice are first treated for 10 days with effective amounts of some embodiments of the present invention. The dose of Salmonella thyphimurium for challenge may be chosen so that untreated mice, or mice treated with a water control or blank formulation containing excipients but no extract, have a survival rate of 60% or less, such as 50% or less. In some cases, the survival rate for the extract-treated mice is at least 70%, at least 80%, at least 80%, at least 90%, or at least 95%.

[00066] Compositions Comprising Bacterial Extracts

[00067] An extract according to the invention may be formulated in a variety of different forms for eventual administration. For example, oral tablets, capsules and pills may be prepared, as well as liquid or aerosol formulations. Formulations for infusion or injection may also be prepared.

[00068] Embodiments of the present invention may be formulated, for example, as solid dosage forms or liquid dosage forms. Examples of solid dosage forms include, for example, a tablet (e.g., a coated tablet, chewable tablet, effervescent tablet, sublingual tablet, powder or a capsule) containing the extract, and, as an option, one or more nutritional and/or dietary supplements. Solid dosage forms may also contain diluents, fillers and/or other excipients. Other excipient components may be additional, such as preservatives, colorants, flavorings and sweeteners. Powder or granulated formulations may also be prepared. Liquid dosage forms, such as solutions, syrups, suspensions or drops may also be used for the oral route.

FUNCTIONAL EXAMPLES

[00069] Example 1. Preparation of an E. coli Strain Culture

[00070] The medium for E. coli I-081 (E81), E. coli I-082 (E82), E. coli I-083 (E83), E. coli I-084 (E84), E. coli I -085 (E85), E. coli I-086 (E86), E. coli I-087 (E87), E. coli I-088 (E88), E. coli I-089 (E89), E. coli NCTC 8603 (E8603), E. coli NCTC 8621 (E8621), E. coli NCTC 8622 (E8622), E. coli NCTC 8623 (E8623), E. coli NCTC 9026 (E9026), E. coli NCTC 9111 (E9111), E. coli NCTC 9119 (E9119), E. coli NCTC 9707 (E9707), and E. coli NCTC 9708 (E9708) was prepared by dissolving the following components in purified water: Sodium chloride 3.75 g/L; Sodium monohydrogen phosphate 2.5 g/L; Sodium acetate 0.625 g/L; Vegetable peptone (soybean papain digest) 50 g/L; Inosine 0.125 g/L; Calcium chloride 0.025 g/L; Potassium chloride 0.125 g/L; Hydrogen carbonate sodium 0.75 g/L; Arginine 1 g/L; Sodium pyruvate 0.35 g/L; Glucose 3 g/L; and a "concentrated element solution" 0.625 mL/L (containing Copper Sulfate 3 mg/L; Iron Chloride 830 mg/L; Zinc Sulfate 860 mg/L; and Sulfuric Acid 1.1 mL/L).

[00071] 0.1 L of medium was inoculated with 1.5 mL of frozen bacteria and incubated in a 300 mL Erlenmeyer flask at 37°C for 3 hours with shaking. Then, 1.0 L of medium in a 3000 mL Erlenmeyer flask was inoculated with 30 mL of the previous 300 mL culture and incubated again at 37°C for 4 hours with shaking. TABLE 1.1: OPTICAL DENSITY FOR SUCCESSIVE CULTURE STEPS

[00072] The same medium used above was prepared for pre-fermenters, but with the addition of 0.08 mL/L of polypropylene glycol. One liter of the culture from the previous incubation step was transferred to the pre-fermenter. The incubation temperature was set at 30°C, and the pre-fermenter was shaken, and the pH was not regulated. The sterile air flow rate was adjusted to 3.3 L/min. After 6 hours, two 25-liter pre-fermenters were transferred to a larger fermenter. TABLE 1.2: OPTICAL DENSITY FOR PRE-FERMENTER CULTURES:

[00073] The same medium described above was prepared for the next incubation in larger-scale fermenters, but with the addition of 0.05 mL/L polypropylene glycol and 6 g/L glucose before sterilization. The incubation temperature was regulated at 37°C, with shaking and aeration during incubation. The pH was regulated at 6.8. Twelve kg of glucose were added during the culture. After approximately 10.5 hours, the cultures were inactivated by heating to between 90 and 100°C and transferred to a collection tank. The biomass was then separated into the culture medium and concentrated by centrifugation.

[00074] According to the medium and culture conditions described, cultures were prepared as illustrated in the following tables and descriptions. TABLE 1.3

[00075] Example 1.20

[00076] NCTC9111 was cultured with a synthetic medium under the same conditions described in example 1.2. The medium was prepared by dissolving in 540 L of purified water: 0.2220 Kg of inosine, 0.3330 Kg of citric acid monohydrate, 1.4430 Kg of glutamic acid, 1.1655 Kg of ammonium chloride, 0.7825 Kg of magnesium sulfate-2 H2O, 1.5096 Kg of potassium phosphate (KH2PO4), 0.3330 Kg of arginine, 0.1110 Kg of uracil, 0.0189 Kg of calcium chloride, 11.8200 Kg of sodium chloride, 0.5435 Kg of L-leucine, 0.5435 Kg of L-lysine HCL, 0.5435 Kg L-serine, 0.5435 Kg L-methionine, 0.5435 Kg L-valine, 0.5435 Kg L-alanine, 0.5435 Kg L-asparagine, 14.0000 L ammonium hydroxide, 0.3420 L potassium hydroxide, 40.5000 Kg glucose, 4.1667 g iron chloride, 4.1667 g cobalt chloride, 4.1667 g sodium molybdate, 4.1667 g manganese sulfate, 4.1667 g zinc sulfate, 4.1667 g nickel sulfate, 0.0833 g boric acid, 0.1667 g copper sulfate, 1.8333 mL sodium acid sulfuric (95-97%).

[00077] Example 2: Alkaline Lysis of Cells

[00078] Example 2.1

[00079] An aliquot of each of the following strains, E81, E82, E83, E084, E085, E086, E087, E088, E89, E9111, E8603, E8621, E8622, E8623, E026, E9119, E9707 and E9708, containing 1810 g of bacterial dry weight was thawed at room temperature, and then diluted with purified water to reach 26 g/L of bacterial biomass (dry weight). Alkalization to 0.8 M sodium hydroxide was performed, the pH was measured after 2 hours of lysis and was 13.1. Then, the lysis was incubated for 120 hours at 35-40oC under continuous shaking. After incubation, the pH was adjusted to 11.3 with concentrated HCI. (Soluble Dry Weight); SDW: 59.4 mg/mL; Prot: 17.4 mg/mL. The soluble dry weight was determined by obtaining 5 mL of the soluble fraction resulting from the lysis and drying it to a constant mass in a porcelain dish at 105oC.

[00080] Example 2.2

[00081] According to examples 1.1 to 1.19, 3 aliquots of E81, E82, E084, E086, E89, 2 aliquots of E83 and E085, 4 aliquots of E088, 6 aliquots of E9111, 9 aliquots of E8603, E9119, 7 aliquots of E8621, E9707 and 8 aliquots of E8622, E8623, E9026 and E9708 containing a total of 9264 g of bacterial dry weight were frozen at room temperature, and then diluted with purified water, to reach 50.1 g/L of bacterial biomass concentration (dry weight). The alkalization at 0.2 N hydroxide ion was 12.3. The lysis was incubated for 32 hours at 35-40oC under continuous agitation. During the lysis the pH was monitored so that it did not decrease more than 1.3 pH units. The pH was adjusted to 11.3 with the addition of concentrated HCI. (Soluble Dry Weight); SDW: 60.7 mg/mL; Prot: 32.0 mg/mL.

[00082] Example 2.3

[00083] According to Table 2, the biomasses were diluted to 58 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 1 day at 35-40oC under continuous agitation. (Soluble Dry Weight); SDW: 96.8 mg/mL; Prot: 35.3 mg/mL.

[00084] Example 2.4

[00085] According to Table 2, the biomasses were diluted to 92 g/L bacterial biomass concentration. Alkalization was performed to 0.4 M with 10N NaOH. The lysis was incubated for 1 day at 35-40oC under continuous agitation. SDW: 146.6 mg/mL; Prot: 62.9 mg/mL.

[00086] Example 2.5

[00087] According to Table 2, the biomasses were diluted to 58 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 7 days at 35-40oC under continuous agitation. SDW: 97.7mg/mL; Prot: 42.4 mg/mL.

[00088] Example 2.6

[00089] According to Table 2, the biomasses were diluted to 92 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 7 days at 35-40oC under continuous agitation. SDW: 153.0 mg/mL; Prot: 78.9 mg/mL.

[00090] Example 2.7

[00091] According to Table 2, the biomasses were diluted to 58 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 98.6 mg/mL; Prot: 29.6 mg/mL.

[00092] Example 2.8

[00093] According to Table 2, the biomasses were diluted to 92 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 127.4 mg/mL; Prot: 60 mg/mL.

[00094] Example 2.9

[00095] According to Table 2, the biomasses were diluted to 43 g/L bacterial biomass concentration. Alkalinization was performed at 0.2 M NaOH. The lysis was incubated for 168 hours at 35-40oC under continuous agitation. SDW: 43.2 mg/mL; Prot: 8.2 mg/mL.

[00096] Example 2.10

[00097] According to Table 2, the biomasses were diluted to 57 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 69.5 mg/mL; Prot: 20.2 mg/mL.

[00098] Example 2.11

[00099] According to Table 2, the biomasses were diluted to 30 g/L bacterial biomass concentration. Alkalinization was performed with 1 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 86.9 mg/mL; Prot: 13 mg/mL.

[000100] Example 2.12

[000101] According to Table 2, the biomasses were diluted to 27 g/L bacterial biomass concentration. Alkalinization was performed with 1 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 91.3 mg/mL; Prot: 11.8 mg/mL.

[000102] Example 2.13

[000103] According to Table 2, the biomasses were diluted to 14 g/L bacterial biomass concentration. Alkalinization was performed at 0.1 M NaOH. The lysis was incubated for 1 day at 35-40oC under continuous agitation. SDW: 25.2 mg/mL; Prot: 5.8 mg/mL.

[000104] Example 2.14

[000105] According to Table 2, the biomasses were diluted to 14 g/L bacterial biomass concentration. Alkalinization was performed at 0.2 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 26.6 mg/mL; Prot: 5.7 mg/mL.

[000106] Example 2.15

[000107] According to Table 2, the biomasses were diluted to 14 g/L bacterial biomass concentration. Alkalinization was performed at 0.1 M NaOH. The lysis was incubated for 10 days at 35-40oC under continuous agitation. SDW: 20.0 mg/mL; Prot: 2.1 mg/mL.

[000108] Example 2.16

[000109] According to Table 2, the biomasses were diluted to 114 g/L bacterial biomass concentration. Alkalinization was performed with 1 M NaOH. The lysis was incubated for 1 day at 35-40oC under continuous agitation. SDW: 163.3 mg/mL; Prot: 68.4 mg/mL.

[000110] Example 2.17

[000111] According to Table 2, the biomasses were diluted to 114 g/L bacterial biomass concentration. Alkalinization was performed at 0.4 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 163.0 mg/mL; Prot: 60.3 mg/mL.

[000112] Example 2.18

[000113] According to Table 2, the biomasses were diluted to 114 g/L bacterial biomass concentration. Alkalinization was performed with 1 M NaOH. The lysis was incubated for 10 days at 35-40oC under continuous agitation. SDW: 171.2 mg/mL; Prot: 71.0 mg/mL.

[000114] Example 2.19

[000115] According to Table 2, the biomasses were diluted to 25 g/L bacterial biomass concentration. Alkalinization was performed at 0.45 M NaOH. The lysis was incubated for 4 days at 35-40oC under continuous agitation. SDW: 48.5 mg/mL; Prot: 15.2 mg/mL; A.A: 4.0 mg/mL; Sugar: 0.86 mg/mL.

[000116] Example 2.20

[000117] According to Table 2, the biomasses were diluted to 28 g/L bacterial biomass concentration. Alkalinization was performed at 0.6 M NaOH. The lysis was incubated for 4 days at 35-40oC under continuous agitation. SDW: 59.4 mg/mL; Prot: 16.8 mg/mL; A.A: 5.4 mg/mL; Sugar: 1.22 mg/mL.

[000118] Example 2.21

[000119] According to Table 2, the biomasses were diluted to 58 g/L bacterial biomass concentration. Alkalinization was performed at 0.2 M NaOH. The lysis was incubated for 4 days at 35-40oC under continuous agitation. SDW: 64.4 mg/mL; Prot: 32.4 mg/mL; A.A: 5.6 mg/mL; Sugar: 1.84 mg/mL.

[000120] Example 2.22

[000121] According to Table 2, the biomasses were diluted to 56 g/L bacterial biomass concentration. Alkalinization was performed at 0.2 M NaOH. The lysis was incubated for 3 days at 35-40oC under continuous agitation. SDW: 61.0 mg/mL; Prot: 30.0 mg/mL; A.A: 5.6 mg/mL; Sugar: 1.88 mg/mL.

[000122] Example 2.23

[000123] According to Table 2, the biomasses were diluted to 39 g/L bacterial biomass concentration. Alkalinization was performed at 0.7 M NaOH. The lysis was incubated for 4 days at 35-40oC under continuous agitation. SDW: 78.32 mg/mL; Prot: 20.60 mg/mL; A.A: 7.8 mg/mL; Sugar: 1.54 mg/mL.

[000124] Example 2.24

[000125] An aliquot of each of the following strains, E82, E83, E084, E086, E087, E088, E9111, E 8603, E 8621, E 8622, E8623, E 026, E9119, E9707 and E9708, containing 1810 g of bacterial dry weight was thawed at room temperature, and then diluted with purified water to reach 25 g/L of bacterial biomass (dry weight). Alkalization to 1.0 M sodium hydroxide was performed, the pH was measured after 2 hours of lysis and was 13.5. Then, the lysis was incubated for 72 hours at 35-40oC under continuous stirring. After incubation, the pH was adjusted to 11.4 with concentrated HCl.

[000126] (Soluble dry weight); SDW: 75.4 mg/mL; Prot: 14.8 mg/mL; AA: 5.4 mg/mL; Sugar: 0.9 mg/mL. TABLE 2: COMPOSITION OF E.COLI LYSES

[000127] Example 3: Lysate Mixtures

[000128] Various bacterial extracts were mixed and had their pH adjusted, with the results below:

[000129] Example 3.1

[000130] 0.845 kg of E. coli Example 2.4 was mixed with 1.4 kg of E. coli Example 2.3. The mixture was diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to a microfiltration tank. The analytical results were: Soluble Dry Weight (SDW): 29.7 mg/mL; Prot: 10.0 mg/mL.

[000131] Example 3.2

[000132] 1.4 kg of E. coli Example 2.7 was mixed with 0.85 kg of E. coli Example 2.8. The mixture was diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to a microfiltration tank. The analytical results were: Soluble Dry Weight (SDW): 32.3 mg/mL; Prot: 10.3 mg/mL. SWD was measured as described in Example 2. Protein concentration was measured by a Lowry assay (see European Pharmacopoeia 2.5.33, under "total protein - method 2").

[000133] Example 3.3

[000134] 1.4 kg of E. coli Example 2.5 was mixed with 0.86 kg of E. coli Example 2.6. The mixture was diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to a microfiltration tank. The analytical results were: SDW: 26.8 mg/mL; Prot: 9.6 mg/mL.

[000135] Example 3.4

[000136] 2.4 kg of E. coli Example 2.7 was mixed with 1.5 kg of E. coli Example 2.8. The mixture was diluted with purified water to 20 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 22.0 mg/mL; Prot: 9.5 mg/mL.

[000137] Example 3.5

[000138] 9.0 kg of E. coli Example 2.9 was diluted with purified water to 9.3 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 45.7 mg/mL; Prot: 20.6 mg/mL.

[000139] Example 3.6

[000140] 8.1 kg of E. coli Example 2.10 were diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 39.7 mg/mL; Prot: 17.3 mg/mL.

[000141] Example 3.7

[000142] 3.2 kg of E. coli Example 2.11 was mixed with 3.2 kg of E. coli Example 2.12. The mixture was diluted with purified water to 12.8 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 39.9 mg/mL; Prot: 8.0 mg/mL.

[000143] Example 3.8

[000144] 248 kg of E. coli Example 2.19 were diluted with purified water to 400 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results are: SDW: 31.6 mg/mL; Prot: 10.2 mg/mL; free amino acids (A.A.): 2.6 mg/mL; Sugar: 0.59 mg/mL. The sugar concentration was analyzed after acid hydrolysis and derivatization according to D. Herbert et al., Meth. Microbiol. 5B: 266 et seq. (1971). The free amino acid concentration was measured by converting the free amino acids to isoindole derivatives and measuring the absorbance at 340 nm according to Roth M., Fluorescence "reaction for amino acids", Anal.Chem., 43, 880-882, (1971). The results are expressed in glutamic acid equivalents.

[000145] Example 3.9

[000146] 248 Kg of E. coli Example 2.20 were diluted with purified water to 400 Kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results are: SDW: 38.9 mg/mL; Prot: 10.8 mg/mL; A.A.: 3.6 mg/mL; Sugar: 0.74 mg/mL.

[000147] Example 3.10

[000148] 247 Kg of E. coli Example 2.21 were diluted with purified water to 400 Kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 40.08 mg/mL; Prot: 20.0 mg/mL; A.A.: 3.6 mg/mL; Sugar: 1.17 mg/mL.

[000149] Example 3.11

[000150] 247 kg of E. coli Example 2.22 were diluted with purified water to 400 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 43.9 mg/mL; Prot: 19.1 mg/mL; amino acids (A.A.): 3.9 mg/mL; Sugars: 1.27 mg/mL.

[000151] Example 3.12

[000152] 1.9 kg of E. coli Example 2.21 was mixed with 6 kg of E. coli Example 2.23. The mixture was diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 45.4 mg/mL; Prot: 12.6 mg/mL; A.A.: 3.8 mg/mL; Sugar: 0.82 mg/mL.

[000153] Example 3.13

[000154] 4 kg of E. coli Example 2.21 was mixed with 4 kg of E. coli Example 2.23. The mixture was diluted with purified water to 12 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 47.0 mg/mL; Prot: 15.0 mg/mL; A.A.: 3.9 mg/mL; Sugars: 1.14 mg/mL.

[000155] Example 3.14

[000156] 72.4 kg of E. coli Example 2.1 was mixed with 185.2 kg of E. coli Example 2.2. The mixture was diluted with purified water to 400 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 40.8 mg/mL; Prot: 17.7 mg/mL; A.A.: 3.7 mg/mL; Sugars: 1.24 mg/mL.

[000157] Example 3.15

[000158] 75.4 kg of E. coli Example 2.24 was mixed with 185.2 kg of E. coli Example 2.2. The mixture was diluted with purified water to 400 kg. The diluted bacterial lysate mixture was transferred to the microfiltration tank. The analytical results were: SDW: 43.6 mg/mL; Prot: 17.0 mg/mL; A.A.: 3.3 mg/mL; Sugars: 1.16 mg/mL.

[000159] Example 4: Purification of Lysates

[000160] Example 4.1

[000161] The bacterial lysate mixture from Example 3.1 was transferred to a microfiltration (MF) tank. The microfiltration (MF) unit utilized either a 0.45 micron tangential flow filtration (TFF) filter (PALL Procette) in a "coil" mode or a Schleicher & Schuell filter in parallel mode. (See Figure 1 for a diagrammatic example of a TFF configuration.) The crossflow, for the coil mode, was set at 2000 L/h m2 (LHM) and the Transmembrane Pressure (TMP) at 1.3 bar. The permeate was transferred to an ultrafiltration (UF) tank.

[000162] Once the volume of lysate in the microfiltration tank had reached half of the initial volume, the UF unit was started. The crossflow was set to 1500 LHM and the TMP to 0.3 bar. The volumes of both the MF tank and the UF tank (the Initial Volume of the MF is called ViMF) were kept at the same level. At each diafiltration volume (corresponding to the ViMF), protein extraction was followed by a protein measurement by the Bradford microplate assay. By this measurement, the number of extraction cycles was defined to extract all solubilized compounds.

[000163] After 10 volumes of diafiltration, the UF was stopped, and the bacterial lysate was concentrated in the MF tanks. The volume recovered was 9.55 kg. This product was then diluted to 7.0 mg/mL and then filtered through a 0.2 micron sterile filter. Soluble Dry Weight (SDW): 24.2 mg/mL; Prot: 6.4 mg/mL. A.A: 1.3 mg/mL, sugars: 0.5 mg/mL. Percentage of D-amino acids: 29% D-Ala, 4% D-Thr, 11% D-Leu, 45% D-Ser, 36% D-Asx, 29% D-Met, 26% D-Phe, 21% D-Glx, 25% D-Tyr, 9% D-Lys.

[000164] NOx (macrophage nitric oxide) production was measured after a 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution with results as follows: C1: 6.3 μM, C2: 7.4 μM and C3: 13.1 μM.

[000165] Several additional mixtures were filtered by Tangential Flow Filtration (TFF) as described below.

[000166] Example 4.2

[000167] The lysate from example 3.2 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for serpentine mode, was set at 2500 LHM and the TMP at 1.30 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 38.2 LHM. The dry weight extraction yield was 65%, the protein extraction yield was 80% and the volume yield was 80%. The concentrate obtained comprised: SDW: 26.1 mg/mL; Prot: 8.1 mg/mL; A.A.: 1.4 mg/mL; Sugars: 0.6 mg/mL; DNA: 4.8 μg/mL. Percentage of D-amino acids: 29% D-Ala, 4% D-Thr, 11% D-Leu, 45% D-Ser, 36% D-Asx, 29% D-Met, 26% D-Phe, 21% D-Glx, 25% D-Tyr, 9% D-Lys. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 5.5 μM, C2: 7.5 μM, C3: 5.5 μM.

[000168] Example 4.3

[000169] The lysate from example 3.3 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2450 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.3 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 26.2 L/h.m2. The dry weight extraction yield was 65%, the protein extraction yield was 75% and the volume yield was 88%. The concentrate obtained comprised: SDW: 24.5 mg/mL; Prot: 7.0 mg/mL; A.A.: 2.1 mg/mL; Sugars: 0.5 mg/mL; DNA: 8.2 μg/mL. Percentage of D-amino acids: 45% D-Ala, 11% D-Leu, 48% D-Ser, 44% D-Asx, 41% D-Met, 26% D-Phe, 25% D-Glx, 38% D-Tyr, 27% D-Lys. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 5.1 μM, C2: 6.8 μM, C3: 13.7 μM.

[000170] Example 4.4

[000171] The lysate from example 3.4 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2000 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.8 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 22.5 L/h.m2. The dry weight extraction yield was 67%, the protein extraction yield was 84% and the volume yield was 92%. The concentrate obtained comprised: SDW: 21.5 mg/mL; Prot: 6.1 mg/mL; A.A.: 1.5 mg/mL; Sugars: 0.3 mg/mL; DNA: 5.6 μg/mL. Percentage of D-amino acids: 44% D-Ala, 15% D-Thr, 12% D-Leu, 48% D-Ser, 40% D-Asx, 40% D-Met, 30% D-Phe, 26% D-Glx, 31% D-Tyr, 18% D-Lys.

[000172] Example 4.5

[000173] The lysate from example 3.5 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2000 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.3 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 27.7 L/h.m2. The dry weight extraction yield was 72%, the protein extraction yield was 69% and the volume yield was 86%. The concentrate obtained comprised: SDW: 36.9 mg/mL; Prot: 16.9 mg/mL; A.A.: 2.8 mg/mL; Sugars: 0.815 mg/mL; DNA: 46.7 μg/mL. Percentage of D-amino acids: 27% D-Ala, 16% D-Thr, 11% D-Leu, 48% D-Ser, 40% D-Asx, 39% D-Met, 36% D-Phe, 32% D-Glx, 31% D-Tyr. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 6.2 μM, C2: 10.9 μM, C3: 18.3 μM.

[000174] Example 4.6

[000175] The lysate from example 3.6 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2000 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.3 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 15.7 L/h.m2. The dry weight extraction yield was 53%, the protein extraction yield was 51% and the volume yield was 81%. The concentrate obtained comprised: SDW: 30.1 mg/mL; Prot: 12.7 mg/mL; A.A.: 2.6 mg/mL; Sugars: 0.364 mg/mL; DNA: 4.8 μg/mL. Percentage of D-amino acids: 40% D-Ala, 16% D-Thr, 12% D-Leu, 49% D-Ser, 43% D-Asx, 44% D-Met, 40% D-Phe, 38% D-Glx, 36% D-Tyr, 24% D-Lys. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 5.8 μM, C2: 7.1 μM, C3: 15.1 μM.

[000176] Example 4.7

[000177] The lysate from example 3.7 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2000 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.35 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 30.0 L/h.m2. The dry weight extraction yield was 82%, the protein extraction yield was 83% and the volume yield was 92%. The concentrate obtained comprised: SDW: 38.2 mg/mL; Prot: 5.4 mg/mL. NOx production after dilution 20,000 times (C1), 2000 times (C2) and 200 times (C3): C1: 0.4 μM, C2: 2.1 μM, C3: 16.3 μM.

[000178] Example 4.8

[000179] 500 mL of the lysate from example 2.13 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 45%. The concentrate obtained comprised: SDW: 25.21 mg/mL; Prot: 5.85 mg/mL; DNA: 7.5 μg/mL. Percentage of D-amino acids: 24% D-Ala, 16% D-Thr, 9% D-Leu, 43% D-Ser, 20% D-Asx, 16% D-Met, 10% D-Phe, 8% D-Glx, 7% D-Tyr.

[000180] Example 4.9

[000181] 500 mL of the lysate from example 2.14 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 53%. The concentrate obtained comprised: SDW: 26.62 mg/mL; Prot: 5.75 mg/mL; DNA: 6.5 μg/mL. Percentage of D-amino acids: 35% D-Ala, 22% D-Thr, 10% D-Leu, 45% D-Ser, 35% D-Asx, 35% D-Met, 31% D-Phe, 24% D-Glx, 22% D-Tyr, 12% D-Lys. PBMC at 1 mg active dry weight/mL: IL-6:9232 pg/mL.

[000182] Example 4.10

[000183] 500 mL of the lysate from example 2.15 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 53%. The concentrate obtained comprised: SDW: 20 mg/mL; Prot: 2.13 mg/mL; DNA: 3.5 μg/mL. Percentage of D-amino acids: 35% D-Ala, 4% D-Thr, 11% D-Leu, 46% D-Ser, 34% D-Asx, 32% D-Met, 24% D-Phe, 24% D-Glx, 15% D-Tyr. NOx production in mg active dry weight/mL: 0.01 mg/mL (C1), 0.1 mg/mL (C2) and 1.0 mg/mL (C3); C1: 3.6 μM, C2: 4.4 μM, C3: 7.9 μM.

[000184] Example 4.11

[000185] 500 mL of the lysate from example 2.16 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 33%. The concentrate obtained comprised: SDW: 163.33 mg/mL; Prot: 68.42 mg/mL; DNA: 14.1 μg/mL. Percentage of D-amino acids: 38% D-Thr, 1% D-Leu, 49% D-Ser, 43% D-Asx, 64% D-Met, 30% D-Phe, 31% D-Glx, 4% D-Tyr.

[000186] Example 4.12

[000187] 500 mL of the lysate from example 2.17 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 17%. The concentrate obtained comprised: SDW: 162.78 mg/mL; Prot: 60.26 mg/mL; DNA: 14.8 μg/mL. Percentage of D-amino acids: 10% D-Leu, 40% D-Ser, 34% D-Asx, 62% D-Met, 27% D-Phe, 23% D-Glx, 5% D-Tyr.

[000188] Example 4.13

[000189] 500 mL of the lysate from example 2.18 was centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The volume yield was 13%. The concentrate obtained comprised: SDW: 171.16 mg/mL; Prot: 71.02 mg/mL; DNA: 16.9 μg/mL.

[000190] Example 4.14

[000191] The lysate from example 3.8 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2300 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.5 bar. The UF was stopped after 11 diafiltration volumes and the permeate flow in the first cycle was 32.5 L/h.m2. The dry weight extraction yield was 86%, the protein extraction yield was 87% and the volume yield was 90%. The concentrate obtained comprised: SDW: 26.6 mg/mL; Prot: 8.1 mg/mL; A.A.: 2.3 mg/mL; Sugars: 0.42 mg/mL; DNA: 68.5 μg/mL. PBMC at 1 mg active dry weight/mL: IL-6: 29898 pg/mL, IL-10: 446 pg/mL, TNF-α: 3429 pg/mL. NOx production at mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2) and 2.0 mg/mL (C3); C1: 2.3 μM, C2: 16.6 μM, C3: 4.0 μM.

[000192] Example 4.15

[000193] The lysate from example 3.9 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2375 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.3 bar. The UF was stopped after 10 volumes of diafiltration and the permeate flow in the first cycle was 32.5 L/h.m2. The dry weight extraction yield was 83%, the protein extraction yield was 79% and the volume yield was 92%. The concentrate obtained comprised: SDW: 24.9 mg/mL; Prot: 7.2 mg/mL; A.A.: 2.3 mg/mL; Sugars: 0.49 mg/mL. PBMC at 1 mg active dry weight/mL: IL-6: 23709 pg/mL, IL-10: 385 pg/mL, TNF-α: 2917 pg/mL. NOx production in mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2) and 2.0 mg/mL (C3); C1: 1.1 μM, C2: 13.8 μM, C3: 4.2 μM.

[000194] Example 4.16

[000195] The lysate from example 3.10 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2500 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.0 bar. The UF was stopped after 10 diafiltration volumes and the permeate flow in the first cycle was 15.0 L/h.m2. The dry weight extraction yield was 58%, the protein extraction yield was 59% and the volume yield was 79%. The concentrate obtained comprised: SDW: 22.0 mg/mL; Prot: 8.1 mg/mL; A.A.: 1.6 mg/mL; Sugars: 0.46 mg/mL; DNA: 23.9 μg/mL. PBMC at 1 mg active dry weight/mL: IL-6: 21458 pg/mL, IL-10: 281 pg/mL, TNF-α: 123 pg/mL. NOx production in mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2) and 2.0 mg/mL (C3); C1: 14.6 μM, C2: 23.8 μM, C3: 16.9 μM. Percentage of D-amino acids: 27% D-Ala, 22% D-Thr, 11% D-Leu, 44% D-Ser, 27% D-Asx, 26% D-Met, 22% D-Phe, 16% D-Glx, 15% D-Tyr, 8% D-Lys.

[000196] Example 4.17

[000197] The lysate from example 3.11 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2500 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.0 bar. The UF was stopped after 10 volumes of diafiltration and the permeate flow in the first cycle was 17.5 L/h.m2. The dry weight extraction yield was 69%, the protein extraction yield was 69% and the volume yield was 78%. The concentrate obtained comprised: SDW: 22.2 mg/mL; Prot: 8.3 mg/mL; A.A.: 1.8 mg/mL; Sugars: 0.5 mg/mL; DNA: 33.3 μg/mL. PBMC at 1 mg active dry weight/mL: IL-6: 19304 pg/mL, IL-10: 343 pg/mL, TNF-α: 220 pg/mL. NOx production in mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2) and 2.0 mg/mL (C3); C1: 14.4 μM, C2: 20.5 μM, C3: 18.9 μM. Percentage of D-amino acids: 30% D-Ala, 18% D-Thr, 10% D-Leu, 44% D-Ser, 29% D-Asx, 28% D-Met, 23% D-Phe, 18% D-Glx, 18% D-Tyr, 11% D-Lys.

[000198] Seventy liters of the liquid form were neutralized with 196 mL of 25% HCI and then mixed with 10.44 kg of mannitol. The mixture was then lyophilized. During the lyophilization cycle, the product was frozen at -50°C and then heated to -25°C. The pressure in the lyophilizer was maintained between 0.2 and 0.5 mbar and controlled by means of injections of filtered air. Then, in the secondary desiccation, the product was heated to its final lyophilization temperature (+30°C) while maintaining a vacuum level of less than 0.2 mbar. When the lyophilization cycle was complete, normal pressure was reestablished in the lyophilizer with filtered air. The lyophilized product was sieved through an attached vibrating sieve. 1.95 kg of lyophilized product were recovered.

[000199] Example 4.18

[000200] 500 mL of the lysate from example 3.13 were centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCI. The concentrate obtained comprised: SDW: 42.8 mg/mL; Prot: 12.5 mg/mL; A.A.: 3.5 mg/mL; Sugars: 1.13 mg/mL; DNA: 14.1 μg/mL. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 0.4 μM C2: 5.7 μM C3: 25.1 μM. PBMC per mL of diluted concentrate volume: Dilution 100: IL-6: 2101 pg/mL. Percentage of D-amino acids: 25% D-Ala, 12% D-Thr, 10% D-Leu, 45% D-Ser, 35% D-Asx, 35% D-Met, 29% D-Phe, 26% D-Glx.

[000201] Example 4.19

[000202] 500 mL of the lysate from example 3.12 were centrifuged for 30 minutes at 9000 g. The supernatant was filtered through successive filters with porosities of 0.8 μm, 0.45 μm and 0.2 μm. The pH was adjusted to 10.5 with HCL. The concentrate obtained comprised: SDW: 38 mg/mL; Prot: 9.7 mg/mL; A.A.: 3.1 mg/mL; Sugars: 0.82 mg/mL; DNA: 80.1 μg/mL. NOx production after 20,000-fold (C1), 2000-fold (C2) and 200-fold (C3) dilution: C1: 0.3 μM, C2: 2.6 μM, C3: 19.2 μM. PBMC per mL of diluted concentrate volume: Dilution 100: IL-6: 2802 pg/mL. Percentage of D-amino acids: 35% D-Ala, 25% D-Thr, 10% D-Leu, 46% D-Ser, 40% D-Asx, 38% D-Met, 34% D-Phe, 32% D-Glx, 32% D-Tyr, 24% D-Lys.

[000203] Example 4.20

[000204] The lysate from example 3.14 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2500 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.2 bar. The UF was stopped after 13 diafiltration volumes and the permeate flow in the first cycle was 16.0 L/h.m2. The dry weight extraction yield was 64%, the protein extraction yield was 62% and the volume yield was 78%. The concentrate obtained comprised: SDW: 22.4 mg/mL; Prot: 8.7 mg/mL; A.A.: 1.8 mg/mL; Sugars: 0.54 mg/mL; DNA: 39.1 μg/mL. Percentage of D-amino acids: 32% D-Ala, 18% D-Thr, PBMC at 1 mg active dry weight/mL, PBMC at 1 mg active dry weight/mL: IL-6: 22303 pg/mL, IL-10: 561 pg/mL, TNF-α: 336 pg/mL. NOx production in mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2), and 2.0 mg/mL (C3); C1: 11.3 μM, C2: 15.3 μM, C3: 13.5 μM. Percentage of D-amino acids: 32% D-Ala, 18% D-Thr, 10% D-Leu, 44% D-Ser, 26% D-Asx, 23% D-Met, 17% D-Phe, 15% D-Glx, 14% D-Tyr, 9% D-Lys.

[000205] Example 4.21

[000206] The lysate from example 3.15 was transferred to an MF tank. The TFF installation was similar to example 4.1. The cross flow, for the serpentine mode, was adjusted to 2500 (L/h m2) (LHM) and the (Transmembrane Pressure) TMP to 1.0 bar. The UF was stopped after 13 diafiltration volumes and the permeate flow in the first cycle was 15.0 L/h.m2. The dry weight extraction yield was 69%, the protein extraction yield was 66% and the volume yield was 77%. The concentrate obtained comprised: SDW: 21.4 mg/mL; Prot: 7.6 mg/mL; A.A.: 1.6 mg/mL; Sugars: 0.5 mg/mL; DNA: 25.9 μg/mL. Percentage of D-amino acids: 12.5% D-Ala, 3.4% Val, 16.6%, 65% D-Ser, 26.1% D-Asx, 18.8% D-Met, 15.6% D-Glx, 9.1% D-Lys. NOx production in mg active dry weight/mL: 0.02 mg/mL (C1), 0.2 mg/mL (C2) and 2.0 mg/mL (C3): C1: 10.3 μM, C2: 15.2 μM, C3: 12.3 μM.

[000207] The liquid form was lyophilized as described in Example 4.17. The protein content (Lowey) was: 44 mg protein per gram of lyophilized product. NOx production in mg active dry weight/mL of lyophilized product: 0.01 mg/mL (C1), 0.1 mg/mL (C2) and 1.0 mg/mL (C3); C1: 6.4 μM, C2: 12.9 μM, C3: 19.4 μM.

[000208] The capsules were produced by mixing 50.4 kg of lyophilized product with 64.68 kg of starch 1500 PT, 2.52 kg of magnesium stearate and 50.4 kg of mannitol. NOx production in mg of active dry weight/mL of capsules: 0.01 mg/mL (C1), 0.1 mg/mL (C2) and 1.0 mg/mL (C3); C1: 6.8 μM, C2: 12.1 μM, C3: 19.1 μM.

[000209] Example 5

[000210] Example 5A: Effect of the Amount of "Starting Material" on the Activity of Bacterial Lysates Measuring IL-6 Secretion by Human PBMC.

[000211] Preparation of human PBMC and cell culture:

[000212] Peripheral blood from healthy donors (Centre de transfusion, Hopital Universitaire, Geneva) was centrifuged to obtain a buffy coat. The buffy coat was mixed with Hanks' balanced salt solution (HBSS, Sigma, Buchs, Switzerland), layered on Ficoll Paque Plus (Amersham Pharmacia) to 1.077 g/mL and centrifuged (2800 rpm, 20°C, 25 min). Cells collected from the interphase were washed twice in HBSS at 800 rpm for 15 min at room temperature and the precipitated cells were resuspended in HBSS. Cell counts were performed using a Neubauer cell. All cell cultures were performed in RPMI-1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mmol/L), and fetal bovine serum (FCS), all obtained from Sigma. For in vitro stimulation, cells were cultured at a concentration of 1 × 106 viable cells/well.

[000213] Stimulation and measurement of IL-6 in culture supernatants:

[000214] Peripheral blood mononuclear cells (PBMC) were incubated at 37 °C and under a 5% CO2 atmosphere with the products of the invention were tested at 0.25, 0.5, 1 and 2 mg/mL of "soluble dry weight, SDW" in RPMI-1640 culture medium.

[000215] Culture supernatants were collected after 24 h and the IL-6 concentration was measured by an enzyme-linked immunosorbent assay (ELISA) (Human UL-6 ELISA Kit, BD OptEIA, San Diego, USA) according to the manufacturer's instructions. The limit of detection was 10 pg IL-6/mL. Activity was first tested in the PBMC assay. (Refer to Figure 2a and 2b, showing the effect of increasing the amount of "starting material" (bacterial biomass concentration expressed in grams of dry weight per liter of lysate) from 12.5 g/L (Figure 2a) to 25 g/L (Figure 2b). The activity of the extracts was dependent on the amount of "starting material" that undergoes alkaline lysis (12.5 g/L vs 25 g/L). The percentage of NaOH (2% (v/v) and the time of alkaline lysis (72 h) were constant. Figures 3a and 3b show the effect of increasing the starting material from 25 g/L to 100 g/L. The time of alkaline lysis was fixed at 168 h and the percentage of NaOH was fixed at 1%.

[000216] According to Figures 2 and 3, the higher the level of "Starting Material", the higher the resulting activity.

[000217] Example 5B: Effect of duration of lysis on the activity of the products of the invention in the human PBMC test.

[000218] The "Starting Material" was 12.5 g/L and was treated with 2% NaOH for 24 h (Figure 4a) or 72 h (Figure 4b). According to these figures, the longer lysis time resulted in lower activity.

[000219] Example 6: Effect of the initial percentage of NaOH (v/v) on the activity of the bacterial lysates of the Invention on the murine nitric oxide test.

[000220] Six-week-old male C57/BL6 mice (6-week-old males, SPF quality, Charles Rivier, FR) were killed by CO2 inhalation. The hip, femur and tibia were removed from the posterior appendage. Bone marrow was extracted from the lumen by injecting Dulbecco's modified Eagle's Medium (DH) through the bone after cutting both ends. After washing, stem cells were resuspended (40,000 cells/mL) in DH medium supplemented with 20% horse serum and 30% L929 cell supernatant. The cell suspension was incubated for 8 days in a 37°C incubator under 8% CO2 and a humidity-saturated atmosphere. Macrophages were then removed with ice-cold PBS, washed and resuspended in DH medium supplemented with 5% fetal bovine serum (FCS), amino acids and antibiotics (DHE medium). Cell density was adjusted to 700,000 cells/mL. Aqueous solutions of the products were serially diluted in DHE medium directly into microtiter plates. The products were tested in triplicates and each microtiter plate comprised a negative control composed of the medium. The final volume in each well was 100 μL. 100 μL of the cell suspensions were added to the diluted products and the cells were incubated for 22 h in an incubator at 37°C under 8% CO2 and a humidity-saturated atmosphere. At the end of the incubation period, 100 μL of supernatant was transferred to another microtiter plate and the nitrite concentration produced in each supernatant was determined by performing a Griess reaction. 100 μL of Griess reagent (5 mg/mL sulfanilamide + 0.5 mg/mL N-(1-naphthyl)ethylenediamine hydrochloride) in 2.5% aqueous phosphoric acid was added to each well. The microtiter plates were read with a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm compared to a reference at 690 nm. The nitrite concentration was proportioned to the nitric oxide content being formed. The nitrite content was determined based on a standard curve. The results were given in nitric oxide (NO) as mean value ± standard deviation and plotted as a dose-response curve.

[000221] In the tests presented in Figure 5, the amount of starting material (25 g/L) and the lysis time (168 h) were similar, the variable tested was the NaOH concentration (1% in Part A vs 4% Part B) used to obtain both lysates. Lower activity was observed when 4% NaOH (B in Figure 5) vs 1% NaOH (A in Figure 5) was used, suggesting a likely alkaline dose-dependent degradation of the endotoxin-like molecules.

[000222] Depending on the starting material, NaOH concentration, and lysis time, different immunological activities in the NO macrophage assay can be generated.

[000223] Example 7: Limulus Amebocyte Lysate (LAL) Chromogenic Test

[000224] To determine the presence of endotoxin-like molecules, a LAL test was performed using the Bio-Whittaker Chromogenic Kit - LAL.

[000225] This test is based on the activation by lipopolysaccharides (LPS) or products of similar structure, by an enzymatic cascade present in LAL. This enzymatic activation is demonstrated by the cleavage of a chromogen linked to a peptide by a protease. The enzymatic reaction is carried out at 37°C and the formation of the chromogen over time is measured at 405 nm. The time required to reach 0.2 O.D. unit is recorded and the endotoxic activity is calculated in relation to an LPS standard (standard curve).

[000226] The results of such an exemplary experiment on the extracts according to the invention are expressed in the table below in EU (Endotoxin Unit) relative to a standardized preparation of E. coli LPS (1 EU corresponds to 0.09 ng of LPS equivalent).

[000227] Example 8: Effect of embodiments of the Invention on a model of urinary tract infection by Escherichia coli in a mouse strain insensitive to LPS

[000228] Embodiments according to the invention were tested in an E. coli urinary tract infection model in LPS-insensitive mice. (See Hopkins et al., "Inheritance of susceptibility to induced Escherichia coli bladder and kidney infections in female C3H/HeJ mice.", J Infect Dis. 2003 Feb 1;187(3):418-23). These groups of 10- to 12-week-old female C3H/HeJ mice (8 mice in each group) were treated orally with one of three different extracts (see below) for 10 days prior to E. coli infection. The C3H/HeJ mice contain a mutation in the toll-like receptor TLR4 gene, and are insensitive to TLR4 agonists, such as lipopolysaccharide (LPS). Therefore, this model is suitable for detecting the effects of drugs acting through pathways other than TLR4.

[000229] Mice were maintained during the treatment period under normal conditions at room temperature of 1826 oC and a relative humidity of 30 to 70%. The lighting program was set to a 12:12 hour light-dark cycle. Animals were fed a standard diet supplied by Harlan Sprague Dawley Laboratories (Indianapolis, IN). Tap water was used ad libitum unless otherwise noted in the text.

[000230] The three extracts tested were:

[000231] a) An extract obtained by strong lysis of 18 strains of E. coli

[000232] b) An extract obtained by moderate lysis of 18 strains of E. coli

[000233] c) An extract obtained by a mixture of a) and b) (1/4 and 3/4, respectively)

[000234] Twenty-five mg of bacterial extract was administered orally to each mouse once a day for 10 consecutive days. The mice were inoculated intravesically with phosphate buffered solution (PBS) or with the uropathogenic E. coli strain 1677 according to a minimum inoculum protocol that reduces the likelihood of inoculation associated with kidney reflux and induces infections in all inoculated animals. The E. coli strain 1677, isolated from the urine of a woman with a febrile UTI, was used in these experiments. This strain is O6 and contains genes for, for example, hemolysin, aerobactin, P fimbriae, and fimbriae type 1, but not for, for example, afimbrial adhesin I, cytotoxic necrotizing factor 1, and S fimbriae. To prepare the inoculum, bacteria were grown from frozen stock for 2 passages in tryptose broth (Difco Laboratories), washed with PBS, and resuspended to a concentration of 2 × 1010 bacteria/mL. Mice were deprived of water for 1 h, and urine was removed from their bladders immediately before inoculation. Ten microliters of bacterial inoculum was instilled into the bladder by urethral catheterization under isoflurane anesthesia, resulting in a dose of 2 × 108 E. coli per mouse. Animals were allowed to recover from anesthesia, and water was re-fed 1 h later.

[000235] Mice were killed 10 days after inoculation to assess the intensities of bladder and kidney infections. The bladder and both kidneys from each animal were removed, weighed, and homogenized in sterile PBS, after which the homogenates were serially plated on Levine's methylene blue-eosin agar (Difco Laboratories). The number of E. coli colonies on each plate was counted after overnight incubation at 37°C and was used to calculate the total number of bacteria in each bladder or kidney pair.

[000236] Fisher's protected least significant difference test was used to determine statistically significant differences between the mean total colony forming unit (CFU) values for different groups of mice (PBS, untreated infected group, and infected and treated group). Kidney and bladder infection data were transformed using total CFU = log 10[(CFU + 100)/mg tissue], where CFU is the total number of colony forming units calculated per tissue sample.

[000237] The results obtained are illustrated in Figures 6a and 6b, for the bladder and kidneys, respectively. In summary, the 3 bacterial lysates tested decreased, by a factor > 3 (bladder) and > 2 (kidneys), the logarithmic values obtained, suggesting that the number of colonies cultured from the bladder and kidneys was decreased by at least a factor of 1000 and 100, respectively. Since the results were generated in C3H/HeJ mice, the observed effects are independent of TLR4.

[000238] Example 9: Effect of embodiments of the Invention in a murine model of intraperitoneal infection by Salmonella typhimurium in a strain of mice sensitive to LPS.

[000239] Three extracts of the invention, described in Example 8, were also tested in C57/bl mice, which have a normal sensitivity to LPS. The three extracts tested were:

[000240] a) An extract obtained by strong lysis of 18 strains of E. coli

[000241] b) An extract obtained by moderate lysis of 18 strains of E. coli

[000242] c) An extract obtained by a mixture of a) and b) (1/4 and 3/4, respectively)

[000243] C57BL/6 mice were maintained for 7 days before oral treatment with the above-mentioned substances. The experiment consisted of 4 experimental groups - 3 groups treated with the compounds of the invention, and a control group. Each experimental group involved 20 mice. The mice in the control group received a sham treatment using the oral administration of 0.5 mL of water daily for 10 days. For the other groups, the extracts were dissolved daily in distilled water so as to have a single dose in a final volume of 0.5 mL. This volume of 0.5 mL was administered orally to each mouse once a day for 10 consecutive days before all mice were challenged with Salmonella. 10 mg of bacterial extract was administered to each animal at each administration.

[000244] For the challenge, a suspension of Salmonella typhimurium strain 415 (I. Mechnokov Institute for Vaccines and Sera, Russian Academy of Medical Sciences) was injected intraperitoneally into each mouse.

[000245] A preliminary dose-finding challenge (not illustrated) ranged from 102 to 105 CFU of Salmonellae per mouse. A dose of 104 was selected for the main experiment because this dose yielded approximately 50% survival in untreated animals. Following challenge, mice were maintained under standard laboratory animal conditions. Daily observation and death recordings were performed for a period of 21 days post-challenge.

[000246] The anti-infection efficacy of the compounds (see tables below) was estimated according to the post-infection survival rate (SR), the mean post-infection life span (ADL), the defense factor (DF), and the preparation efficacy index (EI), which were calculated for each experimental group. The SR was obtained as a percentage of animals alive in the experimental group on day 21 after infection. The ADL, DF and EI were calculated using the following formulas:

[000247] ADL = (XI + X2 +...+ Xn): N,

[000248] where ADL is an average life span, X1 through Xn are post-infection life spans for experimental mice #1 through #n, and N is the total number of animals in the experimental group.

[000249] DF = CD:ED,

[000250] where DF is the defense factor, CD is a percentage of death in the control group, and ED is a percentage of death in the experimental group.

[000251] EI = [(DF - 1): DF] x 100%,

[000252] where EI is the preparation effectiveness index and DF is the defense factor.

[000253] Death records in the experimental groups during the 21-day period after infection with 104 CFU of Salmonella typhimurium. The same results are also represented in Figure 7.

[000254] _*: Number of dead mice found dead on the day presented.

[000255] Defense efficacy of extracts in the lethal infection model with Salmonella Thyphimurium in C57BL/6 mice.

[000256] Extracts (a) and (c) formed from a strong alkaline lysis (strong) and from a mixture of strong and moderate lysis (mixture) were well tolerated. Extract (b) formed from a moderate lysis (moderate) demonstrated greater toxicity than the others. A reduction in body size of the mice, and retardations in the growth of the mice were observed, although not measured, in the group treated with extract (b). Four of the 20 mice in the experimental (b) or "moderate" groups died during the course of treatment. Thus, only 16 mice in this group were challenged with Salmonellae.

[000257] In the control group of mice pretreated with water, the survival rate during the observation period (21 days) was 58%, and the ADL was 15.3 days. The extract (b) appeared to offer no defense against Salmonella infection, and the extract accelerated the death of the infected animals. Specifically, 90% of the mice died by the seventh day after infection, and the ADL was 3.3 days. The survival rate in this group at the twenty-first day after infection was low, reaching 6% (see the tables above and Figure 7).

[000258] In contrast, extracts (a) and (c), formed from a strong lysis (Strong) or from a mixture of strong and moderate lysis (Mixture) increased the resistance of mice to infection with Salmonella thyphimurium. Both substances showed satisfactory protective efficacy. Specifically, the survival rate after challenge with extract (c) (Mixture) was 90%, while with extract (a) (Strong) it was 85% (tables above and Figure 7).

[000259] Additional Examples

[000260] An extract of one or more bacterial strains of Escherichia coli, wherein during preparation of the extract, the one or more bacterial strains are subjected to lysis at a pH greater than 12, and the extract is treated to remove nucleic acids; and wherein the extract does not present a risk of prion-related disease upon administration to the patient.

[000261] The extract of the previous paragraph, obtained from at least one strain of E. coli chosen from: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707 and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088 and 089.

[000262] The extract of the previous paragraph, obtained from each of the following E. coli strains: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707 and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088 and 089.

[000263] The extract of any of the three preceding paragraphs, wherein the extract comprises less than 100 μg/mL nucleic acid.

[000264] The extract of any of the preceding paragraphs, wherein the extract comprises at least 0.3 mg/mL of saccharides.

[000265] The extract of any of the preceding paragraphs, wherein at least one saccharide is selected from the group consisting of monosaccharides, disaccharides, and polysaccharides.

[000266] The extract from the previous paragraph, wherein at least one polysaccharide is a branched polysaccharide.

[000267] The extract of any of the preceding paragraphs, wherein at least one saccharide is chemically modified.

[000268] The extract of any of the preceding paragraphs, wherein the extract comprises between 0.3 and 4.5 mg/mL of saccharides.

[000269] The extract of any of the above paragraphs, wherein lysis is carried out at a pH of 12.6 to 13.4.

[000270] The extract of any of the preceding paragraphs, wherein the extract is obtained by lysis over a period of 30 to 120 hours with a biomass of 15 to 80 g/L.

[000271] The extract of any of the preceding paragraphs, wherein the extract is treated to remove particulate and/or insoluble components.

[000272] The extract of any of the preceding paragraphs, wherein the extract comprises between 1.5 and 2.5 mg/mL of free amino acids.

[000273] The extract of any of the preceding paragraphs, wherein each bacterial strain is grown in a medium that does not present a risk of prion-related disease.

[000274] The extract of any of the preceding paragraphs, wherein each bacterial strain from which the extract is derived is grown in a plant or synthetic medium.

[000275] The extract of any of the preceding paragraphs, wherein at least one amino acid chosen from aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine and lysine is racemized by at least 10%.

[000276] The extract of any of the preceding paragraphs, wherein the free amino acids of the extract comprise between 1 and 80% D-amino acids.

[000277] The extract of any of the preceding paragraphs, wherein the free amino acids of the extract comprise between 10 and 45% D-amino acids.

[000278] The extract of the previous paragraph, with the free amino acids of the extract comprising between 25 and 35% D-amino acids.

[000279] The extract of any of the preceding paragraphs, wherein the extract comprises at least one D-amino acid chosen from D-aspartic acid and D-asparagine, D-glutamic acid and D-glutamine, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, D-lysine, D-valine and D-threonine.

[000280] The extract from the previous paragraph, where the concentration of any D-amino acid comprises between 1 and 50% of the concentration of free amino acids.

[000281] The extract from the previous paragraph, where the concentration of any D-amino acid comprises between 10 and 40% of the concentration of free amino acids.

[000282] The extract of the previous paragraph, wherein the concentration of any D-amino acid comprises between 15 and 35% of the concentration of free amino acids.

[000283] The extract of any of the preceding paragraphs, wherein the extract comprises less than 5000 ng of LPS equivalents by a chromogenic test of limbus amebocyte lysate (LAL).

[000284] The extract of any of the preceding paragraphs, wherein the extract comprises between 8 and 75 mg/mL of one or more proteins.

[000285] The extract of any of the preceding paragraphs, wherein the one or more proteins have molecular weights of less than 30 kDa.

[000286] The extract of any of the preceding paragraphs, wherein the one or more proteins have molecular weights of less than 10 kDa.

[000287] The extract from any of the preceding paragraphs, wherein the survival rate of at least 8 LPS-insensitive mice 13 days after challenge with uropathogenic E. coli strain 1677 is at least 70%, wherein the dose of uropathogenic E. coli strain 1677 is chosen so that the survival rate of at least 8 control mice is 60% or less.

[000288] The extract from the previous paragraph, with the survival rate being at least 80%.

[000289] The extract from the previous paragraph, with the survival rate being at least 90%.

[000290] The extract from any of the preceding paragraphs, wherein the survival rate of at least 8 wild-type LPS-sensitive mice 13 days after challenge with Salmonella thyphimurium is at least 70%, wherein the dose of Salmonella thyphimurium is chosen so that the survival rate of at least 8 control mice is 60% or less.

[000291] The extract from the previous paragraph, with the survival rate being at least 80%.

[000292] The extract from the previous paragraph, with the survival rate being at least 90%.

[000293] A pharmaceutical composition comprising the extract of any of the above paragraphs.

[000294] A method of treating an individual suffering from or at risk of developing a disease of the digestive or urinary tract, comprising administering an effective amount of any of the extracts of the preceding paragraphs to said individual. The method of any of the preceding paragraphs, wherein said individual is a human being.

[000295] The method of any of the preceding paragraphs, wherein the digestive or urinary tract disease is chosen from urethritis, tubulointerstitial nephritis, obstructive pyelonephritis, urinary tract infections due to obstructive and reflux uropathy, cystitis including chronic cystitis, prostatitis including chronic prostatitis, prostatocystitis, pelvic inflammatory diseases in women, Crohn's disease and irritable bowel syndrome.

[000296] A process for preparing an extract obtained from one or more strains of E. coli, comprising:

[000297] (a) culturing each strain in a medium that does not present a risk of prion-related diseases;

[000298] (b) subjecting each strain to lysis at a pH greater than 12; and

[000299] (c) passing the product of (b) at least once through a microfilter and at least once through an ultrafilter.

[000300] The process of the previous paragraph, wherein the extract is obtained from at least one strain of E. coli chosen from: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707 and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088 and 089.

[000301] The process of the previous paragraph, wherein the extract is obtained from each of the following strains of E. coli: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707 and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088 and 089.

[000302] The process of any of the preceding paragraphs, wherein the lysis is carried out at an initial pH greater than 12.5.

[000303] The process of any of the preceding paragraphs, wherein the lysis is carried out at an initial pH of 12.6 to 13.4.

[000304] The process of any of the preceding paragraphs, wherein the lysis is carried out at an initial hydroxide ion concentration of 0.1 N to 1.1 N.

[000305] The process of any of the preceding paragraphs, wherein the lysis is carried out at an initial hydroxide ion concentration of 0.2 N to 1 N.

[000306] The process of any of the preceding paragraphs, wherein the lysis is carried out for a period of 20 hours to 10 days at 30-60 oC.

[000307] The process of any of the preceding paragraphs, wherein the lysis is carried out for a period of 40 hours to 72 hours at 35-40 oC.

[000308] The process of any of the preceding paragraphs, wherein the microfilter is 0.45 microns and the ultrafilter is 30 KDa.

[000309] The process of any of the preceding paragraphs, further comprising passing the product of (c) through a second microfilter at 0.2 microns.

[000310] The process of any of the preceding paragraphs, wherein part (c) is carried out by tangential flow filtration.

[000311] The process of the previous paragraph, where tangential flow filtration is carried out for 5 to 15 cycles.

[000312] The process of any of the preceding paragraphs, wherein tangential flow filtration is performed as shown in Figure 1.

[000313] The process of any of the preceding paragraphs, wherein the tangential flow filtration is performed as shown in Figure 1, in serpentine mode.

[000314] The process of any of the preceding paragraphs, wherein part (b) is carried out with 10-120 g/L bacterial dry weight of material.

[000315] The process of any of the preceding paragraphs, wherein part (b) is carried out with 15-80 g/L bacterial dry weight of material.

[000316] A product obtained by any of the processes of the preceding paragraphs.

[000317] A method of treating an individual suffering from or at risk of developing a disease of the digestive or urinary tract, comprising administering an effective amount of the product of any of the processes of the preceding paragraphs to said individual.

[000318] The method of the previous paragraph, where the individual is a human being.

Claims (26)

1. Extract comprising a bacterial lysate from all bacterial strains of Escherichia coli, selected from NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707, 9708, 1: 081, 082, 083, 084, 085, 086, 087, 088 and 089, which preserves and/or retains the lipopolysaccharide components, characterized by being obtained by lysis of the bacterial strains at an initial pH greater than 12, containing at least 0.3 mg/mL of saccharides, less than 100 μg/mL of nucleic acids, between 1.5 and 2.5 mg/mL of free amino acids in glutamic acid equivalents (147.1 g/mol) chosen from aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine and lysine, which are racemized into D-amino acids. 2. Extract, according to claim 1, characterized by comprising between 0.3 and 4.5 mg/mL of saccharides. 3. Extract according to claim 1, characterized in that at least one saccharide is selected from the group consisting of monosaccharides, disaccharides and polysaccharides. 4. Extract according to claim 3, characterized in that at least one saccharide is chemically modified. 5. Extract according to claim 4, characterized in that each bacterial strain from which the extract is derived is grown in a vegetable or synthetic medium. 6. Extract according to claim 1, characterized in that the free amino acids in the extract comprise between 1 and 80% D-amino acids. 7. Extract according to claim 6, characterized in that the free amino acids in the extract comprise between 10 and 45% D-amino acids. 8. Extract according to claim 7, characterized in that the free amino acids in the extract comprise between 25 and 35% D-amino acids. 9. Extract according to any one of claims 1 to 8, characterized in that it comprises at least one D-amino acid chosen from D-aspartic acid and D-asparagine, D-glutamic acid and D-glutamine, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, D-lysine, D-valine and D-threonine. 10. Extract according to claim 9, characterized in that the concentration of any D-amino acid is between 1 and 50% of the concentration of free amino acids. 11. Extract according to claim 10, characterized in that the concentration of any D-amino acid is between 10 and 40% of the concentration of free amino acids. 12. Extract according to claim 11, characterized in that the concentration of any D-amino acid is between 15 and 35% of the concentration of free amino acids. 13. Extract according to any one of claims 1 to 12, characterized in that it comprises less than 5000 ng of LPS equivalents by a chromogenic test of limbus amebocyte lysate (LAL). 14. Extract according to claim 13, characterized in that one or more proteins have molecular weights of less than 30 kDa. 15. Pharmaceutical composition, characterized by comprising the extract according to any one of claims 1 to 14. 16. A process for preparing an extract as defined in claim 1, comprising: (a) culturing each strain in a medium that does not present a risk of prion-related diseases; (b) subjecting each strain to lysis at a pH greater than 12; and (c) passing the product of (b) once through a microfilter and once through an ultrafilter, wherein the extract preserves and/or retains lipopolysaccharide components obtained by lysis of the bacterial strains at a pH higher than 12, and comprises at least 0.3 mg/mL of saccharides, 100 μg/mL nucleic acids, and between 1.5 to 2.5 mg/mL of free amino acids in glutamic acid equivalents (147.1 g/mol) chosen from aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine, which are racemized into D-amino acids. 17. Process according to claim 16, characterized in that the lysis is carried out at an initial pH of 12.6 to 13.4. 18. Process according to claim 16 or 17, characterized in that a portion of the lysis is carried out at an initial hydroxide ion concentration of 0.1N to 1.1N. 19. Process according to claim 17 or 18, characterized in that a portion of the lysis is carried out at an initial hydroxide ion concentration of 0.2N to 1N. 20. Process according to any one of the preceding claims, characterized in that part of the lysis is carried out for a period of 30 to 50 hours at 30 to 40°C. 21. Process according to any one of the preceding claims, characterized in that part of the lysis is carried out for a period of 60 to 120 hours at 30 to 40°C. 22. Process according to any one of the preceding claims, characterized in that the microfilter is 0.45 microns and the ultrafilter is 30 KDa. 23. The process of claim 16, further comprising passing the product of (c) through a second microfilter at 0.2 microns. 24. Process according to claim 16, characterized in that part (c) is carried out by tangential flow filtration. 25. Process according to claim 21, characterized in that the tangential flow filtration is carried out for 5 to 15 cycles. 26. The process of claim 16, wherein part (b) is carried out with 10-120 g/L bacterial dry weight of material.