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    • 专利摘要:
    The present invention relates to a method for preparing a homogeneous mixture of fecal microbiota from at least two preselected donors. The homogeneous mixture of fecal microbiota thus obtained has a high bacterial diversity and a high viability. The homogeneous mixture of fecal microbiota can be used for the treatment of intestinal dysbiosis, for the treatment of pathologies associated with such dysbiosis.
    公开号:FR3078627A1
    申请号:FR1852084
    申请日:2018-03-09
    公开日:2019-09-13
    发明作者:Carole SCHWINTNER；Alice Leroux；Clemence Mader；Herve Affagard
    申请人:Maat Pharma SA；
    IPC主号:
    专利说明:

    The present invention relates to a stool collection procedure from multiple donors and a method of preparing a fecal microbiota sample.
    The invention also relates to the use of said sample in the transplantation of fecal microbiota (TMF, also called fecal microbiota transfer), preferably for treating intestinal dysbiosis.
    The human intestinal microbiota, commonly called “intestinal flora”, is the set of microorganisms (bacteria, yeasts and fungi) that are found in the human gastrointestinal system (stomach, intestine and colon). Bacterial diversity is currently estimated at around 10 3 bacterial species making up the dominant intestinal microbiota of an adult individual, with an abundance of 10 14 bacteria, representing a bacterial metagenome of 200,000 to 800,000 genes in each individual, or 10 to 50 times the number of genes in the human genome. Sterile in utero, the intestine colonizes from the first days of life until it evolves into a unique individual microbiota. Each person has relatively close bacteria in terms of species, but the exact composition of their microbiota (species, proportions) is largely (more than 2/3 of the species) specific to the host. Thus, the human intestinal microbiota is a very diverse, complex and specific ecosystem of each individual.
    Maintaining a great diversity of the microbiota promotes its stability. However, certain pathologies or treatments unbalance the microbiota: antibiotics for example, as well as diseases with an inflammatory component, such as inflammatory bowel disease (IBD), can limit the diversity of the microbiota in the intestine. Antibiotic treatments (or antibiotic therapy), in particular, result in an alteration of the microbiota, which can promote the proliferation of pathogenic organisms such as Clostridium difficile.
    A certain number of pathologies are associated with intestinal dysbiosis, for example graft versus host disease (Graft-Versus-Host Disease - GvHD). Allogeneic hematopoietic stem cell transplantation or transplantation (allo-HSCT) is an effective treatment for hematopoietic malignancies and hereditary hematopoietic disorders, and it is considered to be the most effective tumor immunotherapy to date [Sung, AD and Chao, NJ. (2013), Concise Review: Acute Graft-Versus-Host Disease: Immunobiology, Prévention, and Treatment, Stem Cells Transi. Med. 2: 25-32]. However, T lymphocytes derived from transplanted stem cells can attack the host receptor tissues causing GvHD, a major complication of wing-HSCT associated with significant mortality (15 to 25% of deaths after allo-HSCT). Patients undergoing allo-HSCT may be simultaneously exposed to cytotoxic chemotherapy, total body irradiation, immunosuppressants and broad-spectrum antibiotics that can cause significant damage to the gut microbiota. Indeed, a significant enrichment of Enterococcaceae, as well as an increase in Lactobacillales and a decrease in Clostridiales, is often observed in patients after an allo-HSCT. As a result, commonly encountered dominant bacteria such as Enterococcus resistant to vancomycin, Streptococcus from the group of viridans and various proteobacteria, can enter the blood and cause sepsis. It is interesting to note that this change is particularly important in patients who then develop refractory graft versus host disease (GvHD) [Holler et al. (2014), Biol Blood MarrowTransplant. 20 (5): 640-645, and Peled, J. (2017) 59 th Annual Meeting of the American Society of Hematology, Atlanta, USA, Dec 9-11].
    In order to restore the intestinal microbiota and thus restore homeostasis (/.e. Symbiosis), transplantation of fecal microbiota (TMF) is considered and tested. It consists of the introduction of the stool of a healthy donor into the digestive tract of a recipient patient, in order to rebalance the altered intestinal microbiota of the host. This transplantation of fecal microbiota can be allogenic (that is to say from a healthy donor individual to a patient) or autologous (that is, from an individual to himself). The results obtained on Clostridium difficile infections are encouraging, and some patients have been treated successfully (Tauxe et al, Lab Medicine, Winter 2015, volume 46, Number 1). Recent studies have also shown that GvHD patients treated with TMF had an improvement in gastrointestinal symptoms and had a reduction or disappearance of diarrhea, associated with a reconstruction of the microbiota [Kakihana et al. (2017) Transplantation 128: 2083-2089, and Spindelboeck et al. (2017) Hematologica 102: e210].
    In the case of allogeneic transplantation, it is important that the transplanted sample has an acceptable profile in terms of viability and diversity of bacteria, since the suspension of microbiota in its diluent represents the active principle (active substance) of the drug. Current transplantation methods are often empirical and do not take special precautions to ensure the diversity of bacteria present in the samples of fecal microbiota used, or to best preserve the viability of anaerobic bacteria, the major components of the intestinal microbiota.
    American patent application US 2017/239303 describes compositions for the restoration of the intestinal microbiota, as well as their manufacturing processes and their administration. This document discloses that the compositions comprising samples have a Shannon diversity index of approximately 0.4-2.5 where the calculations are performed at the family level. It is of the order of 1-8 depending on the level of taxonomy (phyla, species etc.) according to which diversity is calculated.
    There is therefore a need to provide a method for transplanting fecal microbiota which is secure, efficient and easy to implement, in particular on an industrial scale. In addition, there is a need for a method of transplanting a fecal microbiota in which the bacterial diversity of the transplanted products is as high as possible. The viability of the bacteria should be preserved as much as possible during the process and during storage.
    There is a need to provide samples of fecal microbiota with optimal diversity and bacterial viability, to be administered for the treatment and prevention of bacterial intestinal dysbiosis (iatrogenic or non-iatrogenic) and / or associated pathologies. There is a need to provide a process for the preparation of such samples. The pathologies concerned can be refractory graft versus host disease (GvHD), Clostridium difficile infection, ulcerative colitis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, type II diabetes. , food allergies, cancer, including leukemia, obesity and morbid obesity. Other pathologies associated with dysbiosis include autism, sclerosis, traveler's diarrhea, chronic vaginal infection (cystitis, yeast infections), bone and joint infections, dysbiosis associated with intensive care in the hospital, Parkinson's disease, Alzheimer's disease, schizophrenia, intestinal dysbiosis associated with anticancer chemotherapy or immunotherapy, dysbiosis linked to alcoholic and non-alcoholic liver diseases.
    It is necessary to provide samples of fecal microbiota for use in the treatment or prevention of iatrogenic intestinal dysbiosis and / or associated conditions and complications including, but not limited to, sepsis, septic shock and gastrointestinal disorders -intestinal, including but not limited to diarrhea, mucositis, abdominal pain and gastrointestinal bleeding.
    The present invention meets the needs described above.
    It is therefore an object of the invention to provide samples of faecal microbiota, having optimal diversity and sufficient bacterial viability, for use in TMF (Transfer of Fecal Microbiota), and which can be easily produced from a reliably and reproducibly.
    The invention relates to a process for the preparation of a homogeneous mixture of fecal microbiota from at least two preselected donors, comprising the following steps:
    at. taking at least one fecal microbiota sample from said preselected donors,
    b. within less than 5 minutes of taking the sample, placing the sample obtained in a) in an oxygen-tight collection device,
    vs. the qualitative control of the samples taken and the exclusion of samples that do not meet the qualitative criteria,
    d. adding to each of the samples retained after the control step c) of an aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent,
    e. filtration of the samples obtained at the end of step d) to form a series of inocula,
    f. grouping said inocula to form a mixture of inocula,
    g. the homogenization of said mixture obtained in step f), in particular by manual stirring or using a stirring device, steps b) and d) to g) being carried out anaerobically.
    According to one embodiment of the invention, the donors are preselected according to the following preselection criteria:
    i. between the ages of 18 and 60, ii. having a Body Mass Index (BMI) between 18 and 30, iii. absence of a personal history of serious infectious diseases, such as AIDS, hepatitis etc., metabolic and neurological disorders, or depression, iv. lack of recent medication that may deteriorate the composition of the intestinal microbiota,
    v. absence of recent appearance of symptoms associated with gastrointestinal illness, such as fever, diarrhea, nausea, vomiting, abdominal pain, jaundice, absence of history of serious infectious diseases, in particular AIDS, hepatitis etc.
    vi. absence of recent travel to tropical countries, vii. absence of risky sexual behavior, viii. absence of injury, piercing and / or recent tattoo (s) (typically, in the last three months), ix. absence of recent chronic fatigue (typically, in the last three months),
    x. absence of recent allergic reaction (typically, within the last three months), xi. optionally, having a varied diet.
    According to one embodiment of the invention, the qualitative criteria of the samples of step c) include:
    consistency of the sample between 1 and 6 according to the Bristol scale, absence of blood and urine in the sample.
    According to one embodiment of the invention, the qualitative criteria of the samples of step c) include:
    absence of the following bacteria: Campylobacter, Clostridium difficile (toxin
    A / B), Salmonella Yersinia enterocolitica, Vibrio sp., Shiga-like toxinproducing E. coli (STEC) stxl / stx2, multi-resistant bacteria, Broad spectrum beta-lactamase (ESBL) - Enterococci resistant to vancomycin and glycopeptides (VRE , GRE) and Listeria monocytogenes, bacteria resistant to carbapenemases, absence of the following parasites: Cryptosporidium parvum, Cyclospora sp., Entamoeba histolytica, Giardia lamblia, Blastocystis hominis, Helminths, Strongyloides stercoralis, Isospora sp., Microsporidias and Dientamoe Adenovirus F40 / 41, Astrovirus, Norovirus, Rotavirus A, Sapovirus and Picornavirus (Aichi Virus and enterovirus), absence of the following bacteria: E.coli enteroaggregative (EAEC), E.coli enteropathogenic (EPEC), E.coli enterotoxigenic ( ETEC) It / st, Shigella / Enteroinvasive E.coli (EIEC) and Plesiomonas shigelloides.
    According to one embodiment of the invention, the at least one cryoprotective agent and / or bulking agent of step d) is a polyol, a di-, tri- or polysaccaride or their mixture and a bulking agent.
    According to one embodiment of the invention, the aqueous saline solution from step
    d) includes maltodextrin and trehalose.
    According to one embodiment of the invention, the filtration in step e) is carried out with a filter comprising pores of diameter less than or equal to 0.5 mm, preferably less than or equal to 265 μιτι.
    According to one embodiment of the invention, the time between the collection of the sample and the end of step g) is less than 76 hours.
    According to one embodiment of the invention, step g) of homogenization is carried out at a temperature between 2 ° C and 25 ° C, preferably between about 2 and 6 ° C, more preferably at about 4 ° C.
    According to one embodiment of the invention, the method comprises the following transfer step:
    h. the transfer of the homogenized mixture obtained in step g):
    i. in bags for storage at a temperature of about -50 ° C to -80 ° C, preferably at -80 ° C, or for storage at a temperature between about 2 to 6 ° C for use of the mixture within 16 hours approximately, or for storage at a temperature between 10 to 25 ° C for use of the mixture within approximately 4 hours, ii. in a lyophilization device for lyophilization.
    According to one embodiment of the invention, the homogeneous mixture of fecal microbiota comes from at least four donors, preferably from at least five donors.
    According to another aspect, the invention relates to the use of the homogeneous mixture of faecal microbiota capable of being obtained according to the method of the invention in the transplantation of allogenic faecal microbiota (TMF).
    According to one embodiment of the invention, said homogeneous mixture of fecal microbiota has a high diversity, having a Simpson index greater than 15, preferably greater than 20.
    The invention relates to the use of the homogeneous mixture of fecal microbiota capable of being obtained according to the method of the invention of the invention in the treatment of intestinal dysbiosis.
    The invention relates to the use of the homogeneous mixture of fecal microbiota obtainable according to the method of the invention of the invention in the treatment of graft versus host disease (GvHD).
    The invention relates to the use of the homogeneous mixture of fecal microbiota capable of being obtained according to the method of the invention in the treatment of iatrogenic intestinal dysbiosis and / or associated pathologies and complications including sepsis, septic shock and gastrointestinal disorders, including diarrhea, mucositis, abdominal pain, and gastrointestinal bleeding.
    The invention relates to the use of the homogeneous mixture of fecal microbiota capable of being obtained according to the method of the invention of the invention in the treatment of Clostridium difficile infection and associated diarrhea (CDI), intestinal disease inflammatory (IBD), irritable bowel syndrome (IBS), idiopathic constipation, celiac disease, Crohn's disease, obesity and morbid obesity, autism, multiple sclerosis, traveler's diarrhea, chronic vaginal infection (including cystitis and yeast infection), bone and joint infections, Parkinson's disease, type II diabetes, food allergies, cancer, refractory leukemia, Alzheimer's disease , schizophrenia and bipolar disorder, intestinal dysbiosis associated with cancer chemotherapy or immunotherapy, and alcoholic and non-alcoholic liver disease.
    Description of Figures
    Figure 1 is a schematic representation of the process for preparing a homogeneous mixture of fecal microbiota from samples of fecal microbiota from multiple donors for use in allogenic TMF.
    Figure 2 is a histogram representing the percentage of viability of the inoculum microbiota (inoculum 1, 4, 5, 6 and 2 + 8) and the mixture of inoculum (active substance) in inoculum bags 1 to 15 for storage.
    Figure 3 is a histogram showing the percentage viability of the microbiota of individual inocula and inocula mixtures (pooled inocula) for four batches of product. Lot No. 4 is the same lot as that used for Example 1 (and illustrated in Figure 2).
    Figures 4a and 4b show the richness and bacterial diversity of the inocula.
    Figure 4a: The richness (number of bacterial species) measured for the individual inocula and also for the mixture of inocula for lots, lot N ° 1 to lot N ° 4. The operational taxonomic units (OTUs) were evaluated in 16S ribosomal RNA (16S rRNA). For individual donors, the wealth is between 100 and 350 species.
    Figure 4b: The Bray Curtis similarity of individual inocula per batch (“Donors”), inocula compared between batches (“Inter-batch”) and inocula grouped in the bags in the same batch (“Intra-batch”).
    Detailed description
    The present invention relates to a process for collecting and preparing a homogeneous mixture of fecal microbiota from several donors. The invention also relates to the use of said homogeneous mixture in the transplantation of allogenic faecal microbiota, in particular for treating intestinal dysbiosis, and in particular the diseases associated with such dysbiosis.
    In general, according to the invention, the donors are healthy human subjects. By “healthy” subject is meant a subject not suffering from an intestinal microbiota imbalance or from a pathology diagnosed / recognized by the medical profession.
    To select potential donors, a number of criteria were defined. These criteria are as follows:
    i. between the ages of 18 and 60, ii. having a Body Mass Index (BMI) between 18 and 30, iii. absence of a personal history of serious infectious diseases such as AIDS or viral hepatitis, metabolic and neurological disorders, or depression, iv. absence of recent intake (during the approximately 3 months preceding the donation) of drugs which may deteriorate the composition of the intestinal microbiota, such as antibiotics,
    v. no recent onset (within about 3 months before donation) of symptoms associated with gastrointestinal illness, such as fever, diarrhea, nausea, vomiting, abdominal pain, jaundice, no history of serious infectious diseases, including AIDS, hepatitis etc.
    vi. absence of recent trips (in the 3 months or so preceding the donation) to tropical countries, vii. absence of risky sexual behavior, viii. absence of recent contact (during the 3 months approximately preceding the donation) with human blood, for example, via an injury, piercing and / or tattoo, ix. absence of recent chronic fatigue (during the 3 months preceding the donation),
    x. no recent allergic reaction (within 3 months or so prior to donation), xi. optionally, having a varied diet.
    Donor selection criteria are based on those commonly used in Europe for blood donation, but with additional criteria specific to stool donation and the context of transplantation of fecal microbiota. Thus, criteria (i) to (xi) were defined to select the donors.
    The optional criterion relating to the diverse diet aims to improve the possibility of having significant bacterial diversity in the sample of fecal microbiota. It is therefore preferable that the donor has a diverse diet.
    By "varied diet" is meant a diet composed of various vegetables and different cereals (which will allow the regular intake of fiber), but also fruits and meats.
    By “bacterial diversity” is meant the diversity or variability measured at the level of the genus or the species. Bacterial diversity can be expressed with terms such as "richness" (number of species observed in a sample), "Shannon index" and "Simpson index". The Shannon index gives an idea of the specific diversity, that is to say the number of species in the sample (specific richness) and the distribution of individuals within these species (specific fairness). The Simpson's index is derived from wealth and takes into account the relative abundance of each species. It is between 0 (low diversity) and 1 (high diversity).
    The method according to the invention, typically, comprises a step a) of taking at least one stool sample, comprising the fecal microbiota, from the donor subject. Step a) of taking at least one stool sample can thus be carried out by the donor himself, for example, at home, or by a healthcare professional.
    The collection of at least one stool sample is preferably carried out with a collection device designed for this function so that the stool sample is enclosed in an anaerobic environment. We can thus cite the collection device described in patent application WO2016 / 170290. Thus, typically, a device for collecting faeces at home is given to selected donors with a user guide. Preferably, a stool sample has a mass of at least 20g.
    Following this sampling step, and within a rapid time, for example, less than 5 minutes following the sampling, preferably less than 3 minutes, more preferably, less than 1 minute, the sample is placed in a sealed collection device. with oxygen: this is step b).
    According to one embodiment of the invention, the step of taking at least one fecal microbiota sample is carried out by directly depositing a stool sample in a collection device, such as that described in the application for Patent W02016 / 170290.
    Thereafter, generally the process is now carried out anaerobically (in an anaerobic atmosphere) or in confinement where exposure to air is limited. Control step c) can be carried out anaerobically or aerobically or in confinement where exposure to air is limited. According to one embodiment of the invention, the sampling of the sample for carrying out the control tests is done aerobically. According to one embodiment of the invention, steps b) and d) to g) of the process are carried out anaerobically or in confinement where exposure to air is limited. By limiting exposure to air, the viability of the bacteria constituting the fecal microbiota and present in the sample is thus preserved.
    Preferably, the airtight collection device is in the form of the type comprising:
    a container comprising a body which has an interior space adapted to receive the sample of faecal microbiota from the donor subject, and a neck which delimits an access opening to the interior space of the body, and
    - A cover adapted to be removably and tightly mounted on the neck of the container so as to close the access opening of the neck and to close the interior space of the body, in which the body of the container consists of a flexible pouch, and in which at least one of the container and the lid is provided with an evacuation member adapted to evacuate at least a portion of the gases contained in the interior space of the body of the container.
    Preferably, the evacuation member of the device comprises a passage formed through one of the container and the cover, and a member for closing the passage to prevent external fluids from entering the interior space of the body. of the container. Preferably, the device discharge member further comprises a microporous filtration membrane arranged in the passage.
    Alternatively, the airtight collection device is in the form of the type comprising:
    a container comprising a body which has an interior space adapted to receive the sample of faecal microbiota from the donor subject, and a neck which delimits an access opening to the interior space of the body, and
    a cover adapted to be removably and tightly mounted on the neck of the container so as to close the access opening of the neck and to close the interior space of the body,
    - in which the interior space of the container body possibly includes a chemical device which neutralizes oxygen.
    For example, to carry out steps a) to d) (or even e)) of the method, according to one embodiment of the invention, it is possible to use a collection device provided with at least one additional device, for example, an additional device supply device adapted to supply the interior space with fluid (for example, the solution added in step d)), as described in document WO 2016/170290.
    Furthermore, the annex device can also be an analysis tube, used to take out a sample for analysis (for example, a quality control according to step c)).
    Preferably, the airtight collection device is used for steps a) and b): the sample collection from step a) is carried out directly in said device, in particular in the container, and the closing the device, in particular thanks to the cover, places the sample in an oxygen-free atmosphere (step b)).
    In particular, the device mentioned above, used in step b), makes it possible to carry out steps b), d) and e) anaerobically.
    Optionally, a transport step can thus take place. This transport step allows the sample to be brought back from the place of collection to the laboratory, for further processing and analysis.
    After step b), preferably, in less than 24 hours after step b), step c) of quality control is carried out on the samples taken. The purpose of this quality control is to eliminate samples of fecal microbiota that do not meet the predefined qualitative criteria. Thus the samples retained after the control are considered acceptable to form the homogeneous mixture of desired fecal microbiota.
    In general, the quality criteria which are checked include: consistency of the sample between 1 and 6 according to the Bristol scale, by visual inspection, absence of blood and urine in the sample,
    Criteria may also include:
    absence of the following bacteria: Campylobacter, Clostridium difficile (toxin A / B), Salmonella, Yersinia enterocolitica, Vibrio sp., Shiga-like toxinproducing, E.coli (STEC) stxl / stx2, multi-resistant bacteria: Broad spectrum beta-lactamase ( ESBL) - Enterococci resistant to vancomycin and glycopeptides (VRE, GRE) and Listeria monocytogenes, bacteria resistant to carbapenemases, absence of the following parasites: Cryptosporidium parvum, Cyclosporasp, Entamoeba histolytica, Giardia lamblia, Blastocystis hominis, Helminthora, Strongminids sp., Microsporidia and Dientamoeba fragilis, absence of the following viruses: Adenovirus F40 / 41, Astrovirus, Norovirus, Rotavirus A, Sapovirus and Picornavirus (Aichi Virus and enterovirus), absence of the following bacteria: E. coli enteroaggregative (EAEC), E. enteropathogenic coli (EPEC), E.coli enterotoxigenic (ETEC) It / st, Shigella / Enteroinvasive E.coli (EIEC) and Plesiomonas shigelloides.
    The consistency check of the sample is generally carried out by visual inspection. If the sample has an appearance between 1 and 6, preferably 5, according to the Bristol scale [Lewis, SJ. ; Heaton, K.W. (September 1997). Stool form scale as a useful guide to intestinal transit time. Scand. J. Gastroenterol.], It is acceptable and will be accepted.
    The absence of blood and urine in the sample can be checked by visual inspection or by other means. For example, rapid immunoassays can be used. For example, the OC Sensor® test (available from MAST Diagnostic in France), a quantitative immunological test which detects hemoglobin using antibodies specific for human globin.
    If blood and / or urine is found, the sample is discarded.
    According to one embodiment of the invention, a control of the absence of certain parasites, viruses and bacteria from the sample can also be carried out. Typically, a check for the absence of certain parasites, viruses and bacteria in the sample is carried out once a week, by donor. This means that, typically, for a donor who provides, for example, five samples in the week, one in five samples will be checked. According to one embodiment of the invention, the step of controlling the absence of certain parasites, viruses and bacteria from the sample is carried out on each sample.
    The checks for the absence of certain bacteria, certain parasites and certain viruses from the samples are carried out according to methods known to those skilled in the art.
    Preferably, the following bacteria should be absent from the sample: Campylobacter, Clostridium difficile (toxin A / B), Salmonella Yersinia enterocolitica, Vibrio sp., Shiga-like toxin-producing E.coli (STEC) stxl / stx2, Listeria monocytogenes and multi-resistant bacteria, such as Gram-negative bacteria producing extended spectrum beta-lactamase (ESBL) and Enterococci resistant to vancomycin and glycopeptides (ERV, GRE), Listeria monocytogenes, bacteria resistant to carbapenemase, E. coli enteroaggregative (EAEC), E.coli enteropathogenic (EPEC), E.coli enterotoxigenic (ETEC) It / st, Shigella / Enteroinvasive E.coli (EIEC) and Plesiomonas shigelloides.
    Preferably, the bacteria mentioned above are pathogens whose presence in the collected fecal microbiota sample will exclude the donor of said sample from the selection.
    Similarly, preferably, the following parasites must be absent from the fecal microbiota sample: Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica and Giardia lamblia, Blastocystis hominis, Helminths, Strongyloides stercoralis, Isospora sp., Microsporidies and Dientamoeba.
    Similarly, preferably, the following viruses should be absent from the fecal microbiota sample: Adenovirus F40 / 41, Astrovirus, Norovirus, Rotavirus A, Sapovirus and Picornavirus (Aichi Virus and enterovirus).
    Checks for the presence of bacteria, parasites and viruses are carried out according to methods known to those skilled in the art. The following methods may be cited as examples: culture under selective conditions, detection of bacteria, parasites or viruses with antibodies, amplification (with, for example, PCR) and analysis of the DNA sequences present in samples. As a system for analyzing DNA sequences, mention may be made of FilmArray® from BioMerieux (France) an automated system which can be used for the detection of bacteria, parasites and viruses. For example, the following parasites can be detected using this system: Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica and Giardia lamblia. We can also cite the series of PCR tests "Ailplex-GI" available from Eurobio (France).
    Other parasites such as Blastocystis hominis, Isopora sp, Microsporidia and Dientamoeba fragilis can be detected by microscopic examination with concentration of the sample if necessary. Strongyloides stercoralis can be detected with, for example elective staining, after concentration of the sample if necessary.
    Multi-resistant bacteria, such as ESBL, ERV and GRE, can be detected by cultures under specific conditions, for example the ESBL, VRE or ALOA medium available on the market, for example from BioMerieux.
    For some species of bacteria and viruses, their absence from the sample is not required. According to one embodiment of the invention, the presence of the following bacteria is not a criterion for excluding the sample: E.coli enteroaggregative (EAEC), E.coli enteropothogenic (EPEC), E.coli enterotoxigenic (ETEC) It / st, Shigella / Enteroinvasive E.coli (EIEC) and Plesiomonas shigelloides. According to one embodiment of the invention, the presence of the EBV virus is not an exclusion criterion since this virus is prevalent today in the general human population.
    Generally, the samples retained after the control step c) which includes a control of the absence of blood and urine in the sample, and, possibly, a control of the absence of certain parasites, viruses and bacteria in the sample, go to step d). Step d) includes the addition of a cryoprotective diluent.
    Generally, for step d), the samples are separately transformed into liquid inocula by adding cryoprotective diluent. In general, an aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent is added to each of the samples retained after the control step c).
    Any suitable diluent / cryoprotective can be used for the preparation of inocula. Preferably, polyols or di-, tri- or polysaccharides, or a mixture thereof can be used. As polyol, there may be mentioned glycerol, mannitol, sorbitol, propylene glycol or ethylene glycol. As di-, tri- or polysaccharides, mention may be made of dimers, trimers, tetramers and pentamers of different or identical units, said units being chosen from glucose, fructose, galactose, fucose and N acetylneuraminic acid. Among the disaccharides which can be used, mention may be made of trehalose or one of its analogues or sucrose. Preferably, the cryoprotectant is chosen from glycerol, mannitol, sorbitol, propylene glycol, ethylene glycol, trehalose and its analogs, sucrose, galactose-lactose and their mixtures. More preferably, the cryoprotective is galactose-lactose or trehalose.
    Typically, the amount of cryoprotective present in the aqueous saline solution is between 3 and 30% by weight relative to the total volume of the final inoculum (w / v), preferably between 4 and 20% (w / v).
    As bulking agents, mention may, for example, be made of partial hydrolysates of starch, in particular of wheat or of corn, as well as partial hydrolysates of starchy foods, for example, of potato, containing large amounts of maltodextrin. Preferably, the bulking agent is a mixture of maltodextrins, in which the maltodextrin is present between 3 and 30%, preferably between 4 and 20% (relative to the total volume of the final weight / volume inoculum).
    According to one embodiment of the invention, the diluent / cryoprotective is an aqueous saline solution comprising at least one cryoprotective and a bulking agent.
    Typically, the solution contains water and physiologically acceptable salts. Typically, the solution will contain calcium, sodium, potassium or magnesium salts with chloride, gluconate, acetate or hydrogen carbonate irons. The aqueous saline solution can optionally also contain at least one antioxidant. The antioxidant can be chosen from ascorbic acid and its salts, tocopherols, cysteine and its salts, in particular the hydrochloride, and their mixtures. Preferably, the aqueous saline solution comprises at least one salt chosen from sodium chloride, calcium chloride, magnesium chloride, potassium fluoride, sodium gluconate and sodium acetate and optionally at least one antioxidant chosen among sodium L-ascorbate, tocopherol, cysteine hydrochloride monohydrate and mixtures thereof. Typically, the salt is present in the aqueous saline solution at a concentration of between approximately 5 and 20 g / L, preferably between 7 and 10 g / L (relative to the total volume of the final inoculum). Typically, the antioxidant is present in the aqueous saline solution in an amount between 0.3 and 1% by weight / volume, preferably between 0.4 and 0.6% by weight / volume (relative to the total volume of the final inoculum).
    In general, the aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent is added to the sample of fecal microbiota with the weight (g) / volume (ml) ratio of between 1: 0.5 and 1: 10, preferably between 1: 2 and 1: 8, more preferably 1: 4. A sample weight / volume ratio: solution equal to 0.5 weight: 10 volumes means that the sample is mixed at 0.5 weight (for example 0.5g) for 10 volumes of solution (for example 10ml).
    Preferably, step d) of adding an aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent is carried out anaerobically, or in confinement where exposure to air is limited. According to one embodiment of the invention, the saline solution comprising at least one cryoprotective agent and / or a bulking agent is added to the device annexed to the collection device mentioned below, and the solution is added to the sample. stool through a closed line. The mixing of the sample with at least one aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent can in particular be carried out by kneading, in order to obtain a homogeneous mixture.
    The samples obtained after this dilution step are then filtered, in the step
    e). Preferably, the filtration step is carried out with one (or more, having pores increasingly reduced in size) filter (s) comprising pores of diameter less than or equal to 0.5 mm, preferably less than or equal to 265 pm. In the case where several filters are used, the size of the pores gradually decreases. Preferably, the first filters used comprise pores of diameter less than or equal to 2mm, preferably less than or equal to 1mm. Preferably, the last filter used comprises pores of diameter less than or equal to 0.5 mm, preferably less than or equal to 265 μΐΐΐ. Thus, the individual inocula are obtained. Preferably, this filtration step e) is carried out anaerobically, or in confinement where exposure to air is limited. During filtration, the sample from step d) can be pushed through the filters manually, by mechanical action, by gravity, by vacuum or by other appropriate means.
    According to one embodiment of the invention, the same quantity (in terms of weight) of stools per donor is used to form the inoculum, that is to say to carry out steps d) and e). Mention may be made, as suitable quantity, for example, 25-80 g, preferably 40 g. Thus, each individual inoculum is produced using the same amount of fecal microbiota sample.
    Step f) consists in grouping the inocula from the filtration step e). In particular, typically, the individual inocula are transferred to a container, preferably a flexible pouch. Typically, the volume of this container which groups inocula is from IL to 5L, preferably 3L or 5L. This volume may be greater depending on the industrialization scale of the process. The transfer can be carried out manually or by using mechanical means. Preferably, the transfer of the inocula is carried out using mechanical means, for example, a syringe, more preferably, with a peristaltic pump. Suitable peristaltic pumps are commercially available, for example, from Interscience (France).
    Once combined, the inocula are mixed so as to form a homogeneous mixture (homogenization step g)). The mixing of the inocula can be achieved by any means. The mixing of the inocula can be carried out manually or by mechanical means known to those skilled in the art. For example, there may be mentioned a stirring plate, available, for example, from Stuart (England).
    In general, a stirring rate of 80-200rpm, preferably between 90 and 150 rpm, more preferably 100-135 rpm, can be used. In general, the homogenization can take place for a period of time between 10 minutes and 2 hours, preferably 20 minutes and 1 hour, more preferably for 30 minutes. The duration depends on the stirring speed. A person skilled in the art knows how to determine the homogenization time required as a function of the homogenization method that he uses.
    A colorimetric test can be used to check if the mixture is homogeneous. Visual inspection can also be used. Preferably, a colorimetric test followed by a visual inspection are carried out to determine if the mixture is homogeneous.
    The homogenization step can be carried out at a temperature between 2 and 25 ° C, preferably between 2 ° C and 8 ° C, more preferably at around 4 ° C.
    According to one embodiment of the invention, an analysis step can be carried out on the homogeneous mixture obtained after step g), before the mixture is stored or lyophilized (see details below). PH and pO2 can be measured. The methods known to those skilled in the art are used to carry out these measurements. Typically, the pO2 of the mixture is less than 10%, preferably less than 5%; typically the pH is between 4 and 7, preferably between 4.5 and 6.5.
    Preferably, less than 76 hours pass between the start of step a) and the end of step g).
    In general, the final product (the homogeneous mixture) meets the following specifications:
    The aspect: colored, homogeneous suspension, which has a yellow / brown color.
    Viability: greater than 20%, preferably, greater than 40% for lyophilization.
    The number of events (of bacteria): greater than 10 9 bacteria / ml.
    Bacterial diversity: an inverse Simpson index greater than or equal to 4. Preferably, the Simpson index of the mixture of inocula is greater than or equal to 10, more preferably, greater than 15, even more preferably, greater than 20.
    Generally, the homogeneous mixture is transferred (step h) of transfer) for storage or lyophilization. Thus the mixture can be transferred into bags:
    - for storage at a temperature of about -50 ° C to -80 ° C, preferably at about -80 ° C, for use of the mixture beyond 16 hours, or
    - for storage at a temperature between 2 to 6 ° C for use within approximately 16 hours, or
    - for storage at a temperature between 10 to 25 ° C for use of the mixture in the next four hours. Beyond four hours at room temperature. The number of bacteria increases and the homogeneity of the inoculum can be reduced.
    Otherwise, the homogeneous mixture can be transferred to a lyophilization device for subsequent or immediate lyophilization.
    According to one embodiment of the invention, an analysis step i) can be carried out on the homogeneous mixture obtained after the transfer step step h) of the homogeneous mixture in its place of storage (typically a bag) or in its place of lyophilization (typically in a lyophilization device). Specifically, this analysis step includes a visual inspection, viability measurements, taxonomic analysis and measurements of number of events / ml. The purpose of this analysis step is to determine if the sample meets the qualitative criteria for its use in TMF therapy.
    Typically, the mixture has a yellow / brown color. Typically, the viability should be> 20%. According to one embodiment of the invention, the viability is preferably> 40%. If the mixture is to be lyophilized, it is preferable that the viability is> 40%. In general, the cell concentration measured by flow cytometry is greater than 10 6 , preferably, greater than 10 7 bacteria / ml, and more preferably greater than 10 9 bacteria / ml. In terms of taxonomic analysis, in general, it is preferable that the Shannon index is greater than 3.5, preferably greater than 4.
    The good quality of the mixture resulting from the process according to an embodiment of the invention has been demonstrated by the applicants. The results of a qualitative evaluation of the process, according to an embodiment of the invention, carried out with six fresh stools of healthy donors, are shown in Example 1. Tests of viability of the microbiota were carried out on each inoculum before step f) of grouping and after step g) of homogenization.
    The applicants have found that the homogeneous mixture of the pooled inocula has its own bacterial viability, which is higher than can be expected.
    Figure 2 is a histogram representing the percentage of viability of the microbiota of the inocula of Example 1 (inoculum 1, 4, 5, 6 and 2 + 8) and of the inoculum mixture (homogeneous mixture) in bags 1 to 15 inoculum to be stored. Figure 2 shows that the individual inocula (inoculum 1,4, 5, 6, 2 + 8) do not have the same individual viability, but that the inoculum mixture has its own viability, different from the individual inocula. In addition, the method according to the invention ensures good reproducibility of the bags of final mixture, since the viability of the microbiota is approximately the same for each bag (greater than 40% °.
    A statistical analysis of the homogeneous mixture bags indicates that there is no significant difference according to a t-test and according to a rank test, either for the viability of the microbiota or for the number of events / pL. Thus, the applicants have shown that all of the prepared mixing bags are homogeneous with one another.
    The results of a qualitative evaluation of the process, according to an embodiment of the invention, carried out with four batches (Lot No. 1 to Lot No. 4) different from fresh stools from healthy donors, are shown in Example 2 Lot # 4 is the same as that used for Example 1 (and shown in Figure 2). In Example 2, the number of donors is 2, 7, 4 and 6 for Lot # 1, Lot # 2, Lot # 3 and Lot # 4, respectively. The applicants have measured the viability and the bacterial diversity of the individual inocula (at the end of step e)), of the homogenized grouped inocula (at the end of step g)) and of bags filled before storage.
    Figure 3 further demonstrates that individual inocula vary, but that the inoculum mixture has its own viability, different from the individual inoculum. The viability of lots 1 to 4 is between 46.1% and 60.1%, which is excellent.
    Figure 4a shows the richness of the samples. The coefficient of variation (CV) was calculated for each inoculum. The CV (coefficient of variation or relative deviation) is a measure of the dispersion of the data around the mean. This is the calculation of the ratio between the standard deviation and the mean. The CV allows you to calculate the degree of variation from one sample to another. The smaller the CV value, the more homogeneous or stable the values. The CV of the individual inocula of the four lots varies between 16% and 81%. This difference is normal because it is known that individual microbiota are very different. For each grouped inoculum (at the end of step g)), the coefficient of variation, per batch, is between 0.3% and 2%. This slight variation indicates that the mixture of inocula for each batch is homogeneous and stable.
    The richness measured at the level of the species or genus is clearly higher in the grouped inocula, in comparison with the individual inocula. On average, the richness increases by 64% in lot N ° 4 and by 147% in lots N ° 1 and 3 in comparison with the individual inocula.
    On average, the richness measured at the genus level of the combined inoculum increases by 25% for lot N ° 4 and by 61% for lots N ° 1 and 3 in comparison with the individual inocula. The richness of lots 1 to 4 is surprising, and cannot be deduced from the individual wealth of the individual inoculas; that is, this richness of mixtures of inocula (the grouped inocula) is not the average, neither direct nor weighted by the mass of the individual inocula, nor even by fraction of the individual wealth measured. This result is unexpected.
    The percentage of Proteobacteria present in the four combined inocula was measured (see Table 5). The values are 5.5%, 3.7%, 3.1% and 3.4% for lots N ° 1, 2, 3 and 4 respectively.
    In conclusion, the grouping of inocula results in an unexpected increase in the richness of the microbiota. Thus, the inocula mixture is a sample of fecal microbiota with its own characteristics.
    Figure 4b shows that the process involves standardization of the products (homogeneous mixture) obtained for a batch, and intended for use in the clinic. The Figure shows that the Bray Curtis similarity between the bags (containing the pooled inoculum) for each batch is greater than 96%. This result shows that in terms of taxonomic profile, the pockets of inoculum (the homogeneous mixture) are homogeneous.
    Bacterial diversity was measured using Simpson Inverse and Shannon indices (Table 5).
    The results indicate that the Simpson Inverse Index for donors varies between 5 and 30. This difference is normal since each microbiota is different. From the data presented in Table 5, we can observe that the index of
    Simpson Reverse for the pooled inoculum is between 20.2 and 27.2. The bacterial diversity of the pooled inoculum (after step g)) is higher than that of the individual donors (with the exception of lot No. 3 where the diversity of a donor is greater than that for the inoculum regroup).
    The Shannon index is between 2.4 and 4.2 for donors. This difference is normal since each microbiota is different. The Shannon index for the pooled inoculum is between 4 and 4.50 (see Table 5).
    We can also observe that for each batch the diversity of the pooled inoculum is higher than that of the individual donors. The values obtained, as for the wealth, could not be predicted and constitute for each one a characteristic characteristic of the homogeneous mixture.
    The mixture of inocula thus obtained has a taxonomic profile and a bacterial viability superior to those of certain individual inocula which imply that it is perfectly suited for use in the allogenic TMF (Transfer of Fecal Microbiota). The inocula mixture obtained according to the process of the invention is superior in quality for use in TMF because it is characterized by a high and stable viability, as well as a very high diversity. These characteristics allow a higher potential for recolonization of adapted microbiota compared to what would be obtained using an individual inoculum.
    The homogeneous mixture can be easily produced in a reliable and reproducible manner. The results of Example 1 show a homogeneity of viability and taxonomic profile between all the pockets of product mixture. The quality of the microbiota sample (the homogeneous mixture) administered is reproducible, and is identical between bags from a group of donors. An almost identical product can be administered with each bag. The patient can thus receive the same product during several treatments, if more than one treatment is necessary.
    In general, n donors can give enough homogeneous mixture to fill approximately 3n, preferably 3.2n bags, each bag being sufficient for a treatment of TMF.
    The homogeneous mixture of microbiota (or pooled inocula) can be used in the treatment of intestinal dysbiosis and associated pathologies. It indeed represents the active principle. The bacterial diversity of the pooled inoculum produced with the process of the invention is high, possibly having a Shannon index between 4 and 4.50, and a Simpson index located between 20.2 and 27.2. The applicants have also demonstrated that the individual inocula have very varied viabilities, but that the inoculum mixture (pooled inoculum) has excellent viability (from 41% to 60% approximately). According to one embodiment of the invention, the homogeneous mixture of inoculum has a viability greater than 40%, preferably greater than 45% and, more preferably, greater than 50%, more preferably still, greater than 55%. The bacterial diversity and viability criteria are very important in the evaluation of the quality of a product for TMF.
    It is also important that the products are homogeneous, according to regulatory criteria for pharmaceutical products. The data in the examples below demonstrate that the products according to the invention are homogeneous.
    According to one embodiment of the invention, the homogeneous mixture of microbiota (or grouped inocula) can be administered rectally. According to an embodiment of the invention, the homogeneous mixture of microbiota (or grouped inocula) can be formulated for oral administration. As an oral formulation, mention may be made of the formulations mentioned in patent application EP 17306602.8.
    As mentioned above, studies show that TMF can be effective in the treatment of graft versus host disease (GvHD). Thus, the homogeneous mixture of microbiota of the invention can be used in the treatment of GvHD. Thus, according to an embodiment of the invention, a patient who suffers from GvHD can receive an allogenic TMF comprising the homogeneous mixture of the invention. For example, the patient may receive an allogenic TMF rectally. He can also receive it orally.
    The homogeneous mixture of microbiota can be used in the treatment of iatrogenic intestinal dysbiosis and / or pathologies and associated complications including sepsis, septic shock and gastrointestinal disorders, including diarrhea, mucositis, abdominal pain and gastrointestinal bleeding.
    Homogeneous mixture of microbiota can be used in the treatment of Clostridium difficile infection and associated diarrhea (CDI), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), idiopathic constipation, celiac disease , Crohn's disease, obesity and morbid obesity, autism, multiple sclerosis, traveler's diarrhea, chronic vaginal infection (including cystitis and yeast infection), bone and bone infections joints, Parkinson's disease, type II diabetes, food allergies, cancer, refractory leukemia, Alzheimer's disease, schizophrenia and bipolar disorder, intestinal dysbiosis associated with cancer chemotherapy or immunotherapy and alcoholic and non-alcoholic liver disease.
    The homogeneous mixture of microbiota can be used in the treatment of complications due to hospitalization in intensive care.
    The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.
    EXAMPLES
    Example 1
    Steps a) and b): Eight fresh stools from six healthy donors were collected in the medical device described in W02016 / 170290, then stored at + 2 ° C / + 8 ° C for a period of 72 hours, during which controls qualitative (step c)) have been carried out. Based on the results of these checks, the stools of donors 3 and 7 were rejected.
    Stools 1,4, 5, 6 and 2+ 8 were separately transformed into a liquid inoculum by adding cryoprotective diluent (15% maltodextrin, 5% trehalose, 0.9% NaCl, 0.5% ascorbic acid, 0.05% cysteine, in a ratio of 4: 1 and clarified through a filter (265 µm) Stool 2 and 8 were combined to form an inoculum due to the small amounts of stool collected.
    The viability of inocula 1, 4, 5, 6 and 2+ 8 (five inocula) was tested. The individual inocula were then transferred to a flexible 5L bag using a peristaltic pump. The bag was then placed on a stirring plate in a refrigerated incubator set at 4 ° C and the inoculum mixture was made at 125 rpm +/- 5% for 30 minutes.
    Once the homogenization was complete, the inoculum mixture was transferred to a series of 15 bags suitable for lyophilization and stored at -80 ° C. The viability and impact of keeping the homogeneous mixture under different conditions were tested for each of the 15 bags before storage.
    Viability analysis:
    The viability tests were carried out using flow cytometry technology using an Accuri 6 cytometer (BD Science). The samples were diluted in 0.9% aqueous saline, with serial dilution 1:10 to 1:10 ' 3 . The samples were labeled with propidium iodide (PI) fluorides (10 | aL / mL) and SYTO9® 9 (3μί / ι · ηί). PI also targets DNA but only penetrates cells with damaged membranes; it emits at 635nm (red) after excitation at 470nm. SYTO9® penetrates all cells with or without integrity, attaches to DNA and emits at 540nm (green) after excitation at 470nm (blue laser). The stool, inoculum or homogeneous mixture samples are marked with the mixture of the two fluorophores before being analyzed by flow cytometry. The percentage of live bacteria compared to the number of total bacteria (live and dead) allows the bacterial viability of the sample to be obtained. Each batch of the analysis was validated with a positive control (reference inoculum sample kept at -80 ° C) and a negative control (reference sample kept at -80 ° C and treated by incubation for 10 minutes in 70 % isopropanol (1/9 ratio), centrifuged, resuspended in 0.9% NaCl and diluted in dilution series 10 times to 10 “ 3 ).
    Results:
    The assessment of the viability of individual inocula ranges from 30.5% to 67.6%. The 15 bags prepared with the same inocula mixtures show an average viability of 47.1% with a standard deviation of 1.98. The viability percentages of the microbiota of the individual inocula (inoculum 1, 4, 5, 6 and 2 + 8, colored in light gray) and of the inoculum mixture in storage bag 1 to 15 (colored in black) are presented on Figure 2.
    The summarized results are presented in Table 1.
    Analyzes Viability (%) Average 47.1 Standard deviation 1.98 Low 44.3 Max 51.4
    Table 1: Summary of the viability of the microbiota of the allogenic inoculum analyzed
    Statistical analysis :
    The viability and the number of events were measured on the 15 bags of homogeneous mixture. In order to assess whether these two measurements were homogeneous between the pockets, a random assignment of measurements to Group A for 7 pockets and to Group B for the remaining pockets was carried out 500 times.
    For each iteration, a t-test and a rank test were used to compare Group A to Group B. The final results presented below correspond to the proportion of iterations where the statistical test considered was not significant ( > 0.1). The conclusion is that the pockets are homogeneous in terms of viability and in terms of number of events / pL.
    Test Viability Number of events / pL Proportion of iterations where the t-test is not significant 93.4% 95.4% Proportion of iterations where the rank test is not significant 95.4% 96.8%
    Table 2: Statistical results
    The results show that there is no significant difference for the t-test and the rank test, either for the viability of the microbiota or for the number of events / pL.
    It can be concluded that the 15 mixing bags prepared are homogeneous with one another.
    Storage:
    During the mixture preparation process, intermediate storage must be carried out while the quality controls are carried out. In order to determine the impact of this intermediate storage on the viability of the microbiota, an evaluation was carried out:
    - control condition: once prepared, the inoculum is instantly stored at -80 ° C.
    - storage condition at room temperature: once prepared, the inoculum is left at room temperature for 16 hours, a sampling is carried out to determine the viability of the microbiota, the microbiota / pL events and the measurement of pH and pO2 , then the inoculum is stored at -80 ° C.
    - storage condition at 4 ° C: once prepared, the inoculum is stored at 4 ° C for 16 hours, sampling is carried out to determine the viability of the microbiota, the microbiota / pL events and the measurement of pH and pO2, then the inoculum is stored at -80 ° C.
    Then, the three inocula are thawed and the viability of the microbiota is measured.
    The data show that the median viability of the microbiota is the same for the inoculum stored for 16 hours at room temperature or at 4 ° C. After thawing, the median viability of the microbiota decreases both for the inoculum stored at 4 ° C and stored at room temperature. Viability is expected to decline after freezing. Here, we see that in both cases (4 ° C and room temperature), we observe the same phenomenon.
    It can be seen that the number of events / pL is the same for the inoculum and for the inoculum stored at 4 ° C, but, as might be expected, it increases for the inoculum stored at room temperature. The same observation is made after thawing the inoculum.
    All these results show that storage for 16 hours has no impact on the viability of the microbiota when stored at 4 ° C or at room temperature; the number of events / pL increases at room temperature indicating that the microbiota evolves, while at 4 ° C the microbiota is in a latent state. The results of measurement of pH and pO2 are shown in Table 3.
    Sample PH PO 2 (%) inoculum 6.33 1.4 Storage for 16h at room temperature 5.34 4.3 Storage for 16h at 4 ° C 6.28 1.6 Defrost control 6.55 1.5 Thawing of the inoculum stored at room temperature 16h 5.29 3.7 Thawing of the inoculum stored at 4 ° C for 16h 6.23 1.3
    Table 3: Results of pH and pO 2 measurement
    These results show that the storage for 16 hours has no impact on the pH and on the pO2 of the inoculum stored at 4 ° C.
    Example 2:
    Using almost the same conditions as for Γ Example 1, the process was carried out on four lots (lot N ° 1 to lot N ° 4) of fecal microbiota samples, with 2, 8, 4 and 6 donors for the lot N ° l, lot N ° 2, lot N ° 3 and lot N ° 4 respectively. Lot No. 4 corresponds to the batch of Example 1. Thus, for batch No. 4, two of the six stools were combined to form an inoculum given the small amounts of stool collected.
    The stools were separately transformed into a liquid inoculum by adding cryoprotective diluent (15% maltodextrin, 5% trehalose, 0.9% NaCl, 0.5% ascorbic acid, 0.05% cysteine, 4: 1), and clarified through a filter (265 µm).
    The viability and taxonomic profile of the inocula were tested. The individual inocula were then transferred to a flexible 3L or 5L bag using a peristaltic pump. The bag was then placed on a stirring plate in a refrigerated incubator set at 4 ° C and the inoculum mixture was made at 130 rpm + / 5% for 30 minutes in an incubator set at 4 ° C .
    Once homogenization was complete, for each batch, the inoculum mixture was transferred to a series of bags suitable for freezing and stored at -80 ° C. The viability and taxonomic profile of the homogeneous mixtures were tested before storage. For lot N ° 1, 2 saddles were collected and 5 pockets were filled. For lot N ° 2, 8 saddles were collected and 29 pockets were filled. For lot N ° 3, 5 saddles were collected and 21 bags were filled. For lot No. 4, 6 saddles were collected, 2 saddles were combined to have enough material to form an inoculum and 15 bags were filled.
    Viability analysis:
    The viability tests were carried out as for Example 1.
    Results:
    Viability:
    Table 4 below shows the viability of the batches. The assessment of the viability of individual inocula ranges from 16.8% to 67.6%. The viability of the batches of the inocula combined is given in the table below, and is between 46.1 and 60.2%.
    Analyzes Lot N ° 1 Lot N ° 2 Lot N ° 3 Lot N ° 4 Number of donors 2 7 4 6 Average (%) 60.2 46.1 53.2 47.1 Standard deviation 1/2 1.1 2.1 2.0 Low 59.4 45.0 50.6 44.3 Max 61.6 48.3 58.2 51.4
    Table 4
    Table 5 below shows the results.
    Analyzes Simpson Reverse Shannon proteobacteria Lot N ° 1 Donor 1 17.68 3.8Donor 2 8.23 2.6Inocula mixture 20.2 4 5.6 Lot N ° 2 Donor 1 17.4 3.9Donor 2 7.5 3Donor 3 16.4 3.5Donor 4 16.3 3.48Donor 5 26 3.8Donor 6 12.4 3.4Donor 7 22.07 4Inocula mixture 26.2 4.3 3.5 Lot N ° 3 Donor 1 21,0629,15 3.98Donor 2 4.1Donor 3 20.28 3.7Donor 4 6.5 2.5Inocula mixture 27.2 4.3 3.7 Lot N ° 4 Donor 1 12.3 3.59Donor 4 16.37 4.1Donor 5 21.37 3.48Donor 6 16.71 4Donor 2+ Donor 8 25,12 3.52Inocula mixture 26.4 3.27 3.1
    Table 5
    权利要求:
    Claims (17)
    [1" id="c-fr-0001]
    1. Process for the preparation of a homogeneous mixture of fecal microbiota from at least two preselected donors comprising the following steps:
    at. taking at least one fecal microbiota sample from said preselected donors,
    b. within less than 5 minutes of taking the sample, placing the sample obtained in a) in an oxygen-tight collection device,
    vs. the qualitative control of the samples taken and the exclusion of samples that do not meet the qualitative criteria,
    d. adding to each of the samples retained after the control step c) of an aqueous saline solution comprising at least one cryoprotective agent and / or a bulking agent,
    e. filtration of the samples obtained at the end of step d) to form a series of inocula,
    f. grouping said inocula to form a mixture of inocula,
    g. the homogenization of said mixture obtained in step f), in particular by manual stirring or using a stirring device, steps b) and d) to g) being carried out anaerobically.
    [2" id="c-fr-0002]
    2. Method according to claim 1, characterized in that the donors are preselected according to the following preselection criteria:
    i. between the ages of 18 and 60, ii. having a Body Mass Index (BMI) between 18 and 30, iii. absence of a personal history of serious infectious diseases, metabolic and neurological disorders, or depression, iv. lack of recent medication that may deteriorate the composition of the intestinal microbiota,
    v. absence of recent appearance of symptoms associated with a gastrointestinal illness, such as fever, diarrhea, nausea, vomiting, abdominal pain, jaundice, absence of history of serious infectious diseases, in particular AIDS, hepatitis, etc.
    vi. absence of recent travel to tropical countries vii. absence of risky sexual behavior, viii. absence of injury, piercing and / or recent tattoo (s), ix. absence of recent chronic fatigue,
    x. no recent allergic reaction, xi. optionally having a diverse diet.
    [3" id="c-fr-0003]
    3. Method according to claim 1 or 2, characterized in that the qualitative criteria of the samples of step c) include:
    consistency of the sample between 1 and 6 according to the Bristol scale, absence of blood and urine in the sample,
    [4" id="c-fr-0004]
    4. Method according to claim 3, characterized in that the qualitative criteria of the samples of step c) include:
    absence of the following bacteria: Campylobacter, Clostridium difficile (toxin
    A / B), Salmonella Yersinia enterocolitica, Vibrio sp., Shiga-like toxinproducing E. coli (STEC) stxl / stx2, multi-resistant bacteria, Broad spectrum beta-lactamase (ESBL) - Enterococci resistant to vancomycin and glycopeptides (VRE , GRE) and Listeria monocytogenes, bacteria resistant to carbapenemases, absence of the following parasites: Cryptosporidium parvum, Cyclospora sp., Entamoeba histolytica, Giardia lamblia, Blastocystis hominis, Helminths, Strongyloides stercoralis, Isospora sp., Microsporidias and Dientamoe Adenovirus F40 / 41, Astrovirus, Norovirus, Rotavirus A, Sapovirus and Picornavirus (Aichi Virus and enterovirus), absence of the following bacteria: E.coli enteroaggregative (EAEC), E.coli enteropathogenic (EPEC), E.coli enterotoxigenic ( ETEC) It / st, Shigella / Enteroinvasive E.coli (EIEC) and Plesiomonas shigelloides.
    [5" id="c-fr-0005]
    5. Method according to one of claims 1 to 4, characterized in that the at least one cryoprotective agent and / or bulking agent is a polyol, a di-, tri- or polysaccaride or their mixture and a bulking agent .
    [6" id="c-fr-0006]
    6. Method according to one of claims 1 to 5, characterized in that the aqueous saline solution comprises maltodextrin and trehalose.
    [7" id="c-fr-0007]
    7. Method according to one of claims 1 to 6, characterized in that the filtration in step e) is carried out with a filter comprising pores of diameter less than or equal to 0.5 mm, preferably less than or equal to 265 pm.
    [8" id="c-fr-0008]
    8. Method according to one of claims 1 to 7, characterized in that the time between the collection of the sample and the end of step g) is less than 76 hours.
    [9" id="c-fr-0009]
    9. Method according to one of claims 1 to 8, characterized in that step g) of homogenization is carried out at a temperature between 2 ° C and 25 ° C, preferably between about 2 and 6 ° C, more preferably around 4 ° C.
    [10" id="c-fr-0010]
    10. Method according to one of claims 1 to 9, characterized in that it comprises the following transfer step:
    h. the transfer of the homogenized mixture obtained in step g):
    i. in inoculum bags for storage at a temperature of about -50 ° C to -80 ° C, preferably at -80 ° C, or for storage at a temperature between about 2 to 6 ° C for use of the mixture within approximately 16 hours, or for storage at a temperature between 10 to 25 ° C for use of the mixture within approximately 4 hours, ii. in a lyophilization device for lyophilization.
    [11" id="c-fr-0011]
    11. Method according to one of claims 1 to 10, characterized in that the homogeneous mixture of fecal microbiota comes from at least four donors, preferably from at least five donors.
    [12" id="c-fr-0012]
    12. Homogeneous mixture of fecal microbiota capable of being obtained according to one of claims 1 to 11 for its use in the transplantation of allogenic fecal microbiota.
    [13" id="c-fr-0013]
    13. Homogeneous mixture of fecal microbiota capable of being obtained according to one of claims 1 to 11 characterized in that the mixture has a Simpson index greater than 15, preferably 20.
    [14" id="c-fr-0014]
    14. Homogeneous mixture of microbiota according to claim 12 or 13 for its use in the treatment of intestinal dysbiosis.
    [15" id="c-fr-0015]
    15. A homogeneous mixture of microbiota according to claim 12 or 13 for its use in the treatment of graft versus host disease (GvHD).
    [16" id="c-fr-0016]
    16. A homogeneous mixture of microbiota according to claim 12 or 13 for its use in the treatment of iatrogenic intestinal dysbiosis and / or pathologies and associated complications including sepsis, septic shock and gastrointestinal disorders, including diarrhea, mucositis, abdominal pain and gastrointestinal bleeding.
    [17" id="c-fr-0017]
    17. Homogeneous mixture of microbiota according to claim 12 or 13 for its use for the treatment of Clostridium difficile infection and associated diarrhea (CDI), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), idiopathic constipation, celiac disease, Crohn's disease, obesity and morbid obesity, autism, multiple sclerosis, traveler's diarrhea, chronic vaginal infection (including cystitis and yeast infections) , bone and joint infections, Parkinson's disease, type II diabetes, food allergies, cancer, refractory leukemia, Alzheimer's disease, schizophrenia and bipolar disorder, intestinal dysbiosis associated with anticancer chemotherapy or immunotherapy; and alcoholic and non-alcoholic liver disease.
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    同族专利:
    公开号 | 公开日
    US20200405776A1|2020-12-31|
    IL276969D0|2020-10-29|
    KR20200130367A|2020-11-18|
    EP3762001A1|2021-01-13|
    CN111836631A|2020-10-27|
    CA3091626A1|2019-09-12|
    AU2019229721A2|2020-10-22|
    FR3078627B1|2020-11-13|
    WO2019171012A1|2019-09-12|
    JP2021517131A|2021-07-15|
    AU2019229721A1|2020-10-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2016170285A1|2015-04-24|2016-10-27|Maat Pharma|Method for preparing a fecal microbiota sample|
    WO2018026913A1|2016-08-03|2018-02-08|Crestovo Llc|Methods for treating ulcerative colitis|
    ES2673581T3|2013-06-05|2018-06-22|Rebiotix, Inc.|Microbiota Restoration Therapy , compounds and manufacturing methods|
    FR3035317B1|2015-04-24|2019-06-14|Maat Pharma|MICROORGANISM SAMPLING DEVICE, SAMPLING KIT COMPRISING SUCH A DEVICE AND SAMPLING METHOD USING SUCH A DEVICE|EP3895716A1|2020-04-17|2021-10-20|Maat Pharma|Fmt performance prediction test to guide and optimize therapeutic management of gvhd patients|
    NL2025863B1|2020-06-19|2022-02-17|Acad Medisch Ct|Fecal matter for treatment of cachexia|
    法律状态:
    2019-01-11| PLFP| Fee payment|Year of fee payment: 2 |
    2019-09-13| PLSC| Search report ready|Effective date: 20190913 |
    2020-01-27| PLFP| Fee payment|Year of fee payment: 3 |
    2021-07-28| PLFP| Fee payment|Year of fee payment: 4 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1852084A|FR3078627B1|2018-03-09|2018-03-09|PROCEDURE FOR COLLECTING STOOL AND PROCEDURE FOR PREPARING A SAMPLE FOR TRANSPLANTATION OF FECAL MICROBIOTE|
    FR1852084|2018-03-09|FR1852084A| FR3078627B1|2018-03-09|2018-03-09|PROCEDURE FOR COLLECTING STOOL AND PROCEDURE FOR PREPARING A SAMPLE FOR TRANSPLANTATION OF FECAL MICROBIOTE|
    JP2020546950A| JP2021517131A|2018-03-09|2019-03-08|Fecal collection method and sample preparation method for transplanting fecal microflora|
    US16/979,077| US20200405776A1|2018-03-09|2019-03-08|Stool collection method and sample preparation method for transplanting fecal microbiota|
    CN201980018083.8A| CN111836631A|2018-03-09|2019-03-08|Fecal collection procedure and method of preparing samples for fecal microbiota transplantation|
    PCT/FR2019/050522| WO2019171012A1|2018-03-09|2019-03-08|Stool collection method and sample preparation method for transplanting fecal microbiota|
    CA3091626A| CA3091626A1|2018-03-09|2019-03-08|Stool collection method and sample preparation method for transplanting fecal microbiota|
    AU2019229721A| AU2019229721A1|2018-03-09|2019-03-08|Stool collection method and sample preparation method for transplanting fecal microbiota|
    KR1020207028567A| KR20200130367A|2018-03-09|2019-03-08|Fecal collection method and sample preparation method for fecal microbiota transplantation|
    EP19713110.5A| EP3762001A1|2018-03-09|2019-03-08|Stool collection method and sample preparation method for transplanting fecal microbiota|
    IL276969A| IL276969D0|2018-03-09|2020-08-27|Stool collection method and sample preparation method for transplanting fecal microbiota|
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