Vaginal Microbiota Related Methods, Compositions, and Devices

Abstract

The present invention relates to a pharmaceutical composition for use in treating inflammation in the female urogenital tract of a human subject, the female subject exhibiting a dysbiotic microbiota in the urogenital tract. The pharmaceutical composition comprises a substantially complete vaginal microbiota preparation, the preparation comprising (i) one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) less than 5% of the genera Gardnerella, Atopobium, and Prevotella; the pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent. The invention further relates to methods of preparing said compositions, as well as devices comprising and / or using same.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 156,328, filed March 3, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] The vagina is a fibromuscular tube that leads from the female uterus to the outside of the body. A healthy vagina is colonized by a mutually symbiotic flora of microorganisms, especially the genus Lactobacillus, that protect its host from vaginal infections. The acidity (pH about 3.8-4.5) of the healthy vagina of women of childbearing age is thought to result from the breakdown of secreted glycogen / glucose into lactic acid and acetic acid by lactobacilli. Acidic conditions are unfavorable for the growth of many pathogenic microorganisms and pathogenic commensals, including bacteria, protozoa and viruses. However, an imbalance in the vaginal microbiota can result in the overgrowth of pathogenic microorganisms and pathogenic commensals, resulting in dysbiosis, inflammation and / or infection.

[0003] There are several types of vaginal infections (vaginitis) depending on the pathogenic microorganism involved, such as bacterial vaginosis, vaginal candidiasis, and trichomoniasis, as well as combinations thereof.

[0004] Lactobacillus-containing products (e.g., containing Lactobacillus acidophilus, Lactobacillus rhamnosus or Lactobacillus gasseri) for vaginal or oral use have been available for many years. These products include vaginal suppositories containing freeze-dried Lactobacillus acidophilus or other Lactobacillus species of human origin (e.g., Lactobacillus rhamnosus or Lactobacillus gasseri) and various nutritional supplements. These products have been largely ineffective due to the inability of the products to colonize the vagina with exogenous lactobacilli. This may be due to poor quality or the use of ecologically inappropriate strains.

[0005] The colonization and establishment of isolated bacteria, e.g., single strains, in the vaginal microbial niche is difficult to predict from in vitro experiments, e.g., it is common for different bacterial species in the vaginal microbial niche to interact, and these interactions are difficult to reproduce in the laboratory. It is well established and widely accepted that different women can be colonized with different compositions of lactobacilli species / strains, and that this composition of lactobacilli changes over time. Thus, even if a bacterial species shows promising in vitro results, it may not colonize or engraft in vivo and provide the desired therapeutic effect, or the effect is transient because the delivered strain(s) are unable to collectively adapt and maintain colonization over time. For example, specific values ​​for adherence to the vaginal mucosa (vaginal epithelial cells - VEC), ability to produce antimicrobial / antipathogenic substances such as hydrogen peroxide and / or bacteriocins, ability to produce lactic acid for acidification, and fast doubling times are often measured in vitro during culture, but these characteristics do not routinely lead to successful prediction of the ability of a particular strain to colonize the vaginal niche in vivo (ability to colonize the vagina).

[0006] Thus, there is a need for the development of additional therapeutic compositions for vaginal diseases and adverse health conditions in women. Summary of the Invention

[0007] The present invention addresses this unmet need by providing compositions, substantially complete vaginal microbiota preparations, methods of producing them, and methods of treatment that are effective in treating diseases, symptoms, and / or disorders of the female urogenital tract, including urogenital tract inflammation, and are well tolerated, have no adverse effects, and / or effectively colonize the vaginal niche.

[0008] For example, it is understood that the topics discussed in the first embodiment also apply to the second embodiment and any other embodiment, unless the technical context requires otherwise or the topics discussed are clearly related to only one embodiment. For example, in the context of the first embodiment, it is mentioned that the substantially complete vaginal microbiota preparation comprises four bacterial species of the genus Lactobacillus, the one to four bacterial species constituting about 80-99.9% of the preparation, and the one to four Lactobacillus species include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. The preparation may comprise about 80-99.9% Lactobacillus crispatus. The preparation may comprise about 80-99.9% Lactobacillus iners. The preparation may comprise about 80-99.9% Lactobacillus jensenii. The preparation may contain about 80-99.9% Lactobacillus gasseri.The preparation may contain about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0009] When in the context of the second or other embodiments a substantially complete vaginal microbiota preparation is mentioned, this also means that it is intended that the same Lactobacillus species, relative amounts and combinations as in the first embodiment may be used in the substantially complete vaginal microbiota preparation of the second or any other embodiment, even if not explicitly mentioned in the second embodiment.

[0010] As such, the disclosures of the first, second, and any other aspects should be evaluated in combination as reflecting embodiments of the same overarching principles, and not as unrelated separate chapters.

[0011] Described herein in a first aspect is a pharmaceutical composition for use in treating inflammation in the female urogenital tract of a human subject, wherein the female subject exhibits a dysbiotic microbiota in the urogenital tract, the method comprising administering to the subject an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, the preparation comprising (i) one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) less than 5% of the genera Gardnerella, Atopobium and Prevotella; the pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

[0012] Provided herein in a second aspect is a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, the preparation comprising (i) one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) <5% of the genera Gardnerella, Atopobium, and Prevotella; optionally comprising a pharmaceutical acceptable carrier or diluent for use in treating inflammation in the female urogenital tract of a human subject, the female subject exhibiting a dysbiotic microbiota in the urogenital tract.

[0013] Provided herein in a third aspect is a substantially complete vaginal microbiota preparation or a pharmaceutical composition comprising the same, the preparation comprising (i) one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) less than 5% of the genera Gardnerella, Atopobium, and Prevotella; the pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

[0014] Provided herein in a fourth aspect is a dosage form comprising a pharmaceutical composition or a substantially complete vaginal microbiota preparation as described herein formulated for vaginal administration, the dosage form being a solid, semi-solid, liquid formulation or a film-forming formulation.

[0015] In a fifth aspect, the present invention provides a vaginal delivery system comprising a dosage form as described herein, said dosage form being suitable for administration to the vaginal cavity, wherein the dosage form is an applicator, dispenser or suppository.

[0016] Provided herein in a sixth aspect is a method of vaginally administering a pharmaceutical composition or a substantially complete vaginal microbiota preparation as described herein to a human female subject, wherein the composition or preparation is administered using a device, the device comprising an open end (e.g. a tip) for insertion into the vaginal cavity and a dispensing end (e.g. a plunger or piston) for expelling the composition or preparation through the open end, the method comprising a) introducing the open end into the vaginal cavity; and b) expelling the composition or preparation into the vaginal cavity, thereby administering the composition or preparation to the vaginal cavity.

[0017] Provided herein in a seventh aspect is a method of producing a substantially complete vaginal microbiota preparation, the method comprising: A. providing a microbiota sample from the female urogenital tract of a donor; step A comprising one, two, or three of steps (1), (2), (3), or any combination thereof, and both of steps (4) and (5): (1) adding a diluent to the microbiota sample to create a diluted sample; (2) removing a portion of the diluted microbiota sample for testing (e.g., nucleic acid sequencing); (3) pre-cooling the remaining microbiota sample for refrigeration or freezing; (4) storing the refrigerated or frozen microbiota sample in isolation; and (5). providing, including one, two, or three of: (a) testing the donor to exclude substantial presence of one or more infectious pathogens (e.g., blood, and / or ectocervical secretions, and / or urine sample testing); (b) confirming microbiota composition and viability; or (c) holding the refrigerated or frozen microbiota sample in isolation until completion of any combination of: (a) testing the donor to exclude substantial presence of one or more infectious pathogens (e.g., blood, and / or ectocervical secretions, and / or urine sample testing); (b) confirming microbiota composition and viability; or (c) further confirming the health status of the female donor by multiple post-screening tests performed within a period of 30 to 90 days after donation; and B. releasing the refrigerated or frozen microbiota sample from isolation to define a substantially complete vaginal microbiota preparation.

[0018] A substantially complete vaginal microbiota preparation obtainable by the methods described herein is provided in an eighth aspect herein.

[0019] Provided herein in a ninth aspect is an isolated substantially complete vaginal microbiota preparation suitable for vaginal administration, said preparation comprising Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in said preparation, and said preparation comprising less than 5% of the genera Gardnerella, Atopobium, and Prevotella.

[0020] Provided herein in a tenth aspect is an isolated substantially complete vaginal microbiota preparation suitable for vaginal administration, said preparation comprising Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in said preparation, and said preparation comprising less than 5% of the genera Gardnerella, Atopobium, and Prevotella.

[0021] Provided herein in an eleventh aspect is an isolated substantially complete vaginal microbiota preparation suitable for vaginal administration, said preparation comprising Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in said preparation, and said preparation comprising less than 5% of the genera Gardnerella, Atopobium, and Prevotella.

[0022] Provided herein in a twelfth aspect is an isolated substantially complete vaginal microbiota preparation suitable for vaginal administration, said preparation comprising Lactobacillus crispatus, Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in said preparation, and said preparation comprising less than 5% of the genera Gardnerella, Atopobium and Prevotella.

[0023] Provided herein in a thirteenth aspect is an isolated substantially complete vaginal microbiota preparation suitable for vaginal administration, said preparation comprising Lactobacillus iners, Lactobacillus gasseri and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in said preparation, and further said preparation comprising less than 5% of the genera Gardnerella, Atopobium and Prevotella.

[0024] Provided herein in a further aspect is a vaginal delivery system comprising: a) a dosage form suitable for administering a composition to the vaginal cavity; and b) a composition comprising a substantially complete vaginal microbiota preparation, wherein (i) the preparation comprises one to five bacterial species from the genus Lactobacillus, and (ii) the one to five bacterial species constitute about 80-99.9% of the preparation, and the composition further comprises a pharmaceutically acceptable carrier or diluent. In some embodiments, the composition comprises one to four bacterial species from the genus Lactobacillus. In embodiments, the dosage form may be an applicator, dispenser, or suppository. The preparation may comprise about 80-99.9% Lactobacillus crispatus.

[0025] The preparation may comprise about 80-99.9% Lactobacillus iners. The preparation may comprise about 80-99.9% Lactobacillus jensenii. The preparation may comprise about 80-99.9% Lactobacillus gasseri. The preparation may comprise about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0026] The preparation may be free or substantially free of pathogens and pathogenic symbionts. The pathogens or pathogenic symbionts are one or more of the genera Gardnerella, Atopobium, and Prevotella. The preparation may contain less than 5% of the genera Gardnerella, Atopobium, and Prevotella.

[0027] The preparation may be free or substantially free of antimicrobial resistance (AMR) genes.

[0028] The preparation may be free or substantially free of human sperm (spermatozoa).

[0029] In addition to the vaginal microbiota preparation, the composition may further comprise a spermicide. The composition may further comprise an acidifying agent. The composition may further comprise lactic acid. The preparation may comprise a glycan. The glycan may be mucus.

[0030] The preparation may contain fewer than 30 or fewer than 20 species outside the genus Lactobacillus.

[0031] The composition may have an acidifying effect on the vaginal microflora.The composition may have a pH of 4.5 or less.

[0032] The dosage form is 10 2 ~10 11 Preferably, the dosage form contains at least 10 CFU / VCC of lactobacilli. 6 CFU / VCC, at least 10 7 CFU / VCC, or at least 10 8 The composition may be dispensed or formulated into a dosage form. The dosage form may be packaged as sterile.

[0033] The applicator or dispenser may have an open end (eg, a tip) for insertion into the vaginal cavity and a dispensing end (eg, a plunger or piston) for expelling the composition.

[0034] The preparation may be obtained by culture-independent methods.

[0035] The dosage form can be calibrated (e.g., have a volume indication or maximum volume indication or control). The dosage form can be a single-use or multi-use device. The dosage form can be designed from an inert polymeric material such as polystyrene or polypropylene.

[0036] Provided herein in a further aspect is a pharmaceutical composition formulated for vaginal administration comprising a substantially complete vaginal microbiota preparation, wherein (i) the preparation comprises one to five bacterial species from the genus Lactobacillus, and (ii) the one to five bacterial species constitute about 80-99.9% of the preparation, and the composition further comprises a pharma- ceutically acceptable carrier or diluent. In one embodiment, the pharmaceutical composition comprises one to four bacterial species from the genus Lactobacillus. The preparation may comprise 80-99.9% Lactobacillus crispatus. The preparation may comprise 80-99.9% Lactobacillus iners. The preparation may comprise about 80-99.9% Lactobacillus jensenii. The preparation may comprise about 80-99.9% Lactobacillus gasseri. The preparation may contain about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri.

[0037] The preparation may further comprise a further Lactobacillus species, where the further Lactobacillus species may be present in a low relative amount, for example 0.001%, 0.001-0.002%, 0.002-0.005%, 0.005-0.01%, 0.01-0.05%, 0.05-0.1% or 0.1-1%.

[0038] The preparation may be free or substantially free of pathogens and pathogenic commensals. The pathogens or pathogenic commensals may be one or more of the genera Gardnerella, Atopobium, and Prevotella. The preparation may contain less than 5% of the genera Gardnerella, Atopobium, and Prevotella. The preparation may be substantially free of antimicrobial resistance (AMR) genes. The preparation may be substantially free of human sperm (spermatozoa).

[0039] The composition may further comprise a spermicide. The composition may further comprise an acidifying agent. The composition may further comprise lactic acid. The preparation may comprise a glycan. The glycan may be mucus.

[0040] The preparation may include fewer than 20 species outside the genus Lactobacillus.

[0041] The composition may have a pH of 4.5 or less.

[0042] The preparation may be obtained by culture-independent methods.

[0043] In a further aspect, there is provided herein a method for vaginally administering a composition comprising a substantially complete vaginal microbiota preparation to a human female subject in need thereof using a device, the device comprising an open end (e.g., a tip) for insertion into the vaginal cavity and a dispensing end (e.g., a plunger or piston) for expelling the composition through the open end, the method comprising: a) introducing the open end into the vaginal cavity; b) expelling the composition into the vaginal cavity, thereby administering the composition to the vaginal cavity. The device may be an applicator or dispenser as described herein. The composition may be a pharmaceutical composition as described herein.

[0044] Releasing the composition may include contacting the vaginal mucosa or endometrial surface. The subject is in need of improved vaginal health. Administration to the subject may be performed while the subject is in a lithotomy position. The composition may be of suitable viscosity to allow a majority of the composition to remain in the vaginal cavity for at least 5, 10, 20, or 30 minutes when the subject is in an upright position.

[0045] The device can be calibrated (e.g., have a volume indication or a maximum volume indication or control). The device can be calibrated to about 10 per dose. 2 ~10 11 Preferably, the device is capable of administering at least 10 CFU of lactobacilli. 6 CFU / VCC, at least 10 7 CFU / VCC, or at least 10 8 CFU / VCC can be administered.

[0046] Administration may occur once, twice or three times within 3 to 21 days, or once per cycle for one, two, three or more cycles. Administration may occur once every 1 to 4 weeks for a period of 1 to 6 months. The device may be a single use device or a multi-use device.

[0047] The method may further comprise colonizing the vaginal mucosa with one or more Lactobacillus species. The one or more Lactobacillus species may be one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri. The preparation may comprise about 80-99.9% Lactobacillus crispatus. The preparation may comprise about 80-99.9% Lactobacillus iners. The preparation may comprise about 80-99.9% Lactobacillus jensenii. The preparation may comprise about 80-99.9% Lactobacillus gasseri. The preparation may contain about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0048] The preparation may be substantially free of pathogens and pathogenic commensals. The pathogens or pathogenic commensals may be one or more of the genera Gardnerella, Atopobium, and Prevotella. The preparation may contain less than 5% of the genera Gardnerella, Atopobium, and Prevotella. The preparation may be substantially free of antimicrobial resistance (AMR) genes. The preparation may be substantially free of human sperm (spermatozoa).

[0049] The composition may further comprise a spermicide. The composition may further comprise an acidifying agent. The composition may further comprise lactic acid. The preparation may comprise a glycan. The glycan may be mucus.

[0050] The preparation may include fewer than 20 species outside the genus Lactobacillus.

[0051] The composition may have a pH of up to 4.5. The composition may have a pH effective to achieve a pH in the vaginal canal of about pH 3.5 to 4.5.

[0052] The preparation may be obtained by culture-independent methods.

[0053] In another aspect, a kit is provided herein that includes the dosage form (a) and composition (b) described above. The dosage form (a) and composition (b) can be in separate sterile packages. The dosage form can be a multi-dose form. The dosage form (a) and / or composition (b) can include multiple doses. The dosage form can be a single-use or multi-use device. The composition can be freeze-dried or lyophilized. The kit can further include a resuspension medium for the freeze-dried or lyophilized composition.

[0054] Provided herein in another aspect is a substantially complete vaginal microbiota preparation comprising one to five, or one to four, bacterial species from the genus Lactobacillus derived from urogenital tract (e.g., vaginal microbial niche) secretions of a female donor, wherein the one to five bacterial species constitute about 80-99.9% of the preparation, and wherein one or more of said bacterial species are capable of engrafting (e.g., colonizing) the urogenital tract of a female recipient. In one embodiment, the substantially complete vaginal microbiota preparation comprises one to four bacterial species from the genus Lactobacillus.

[0055] The preparation may comprise 80-99.9% Lactobacillus species typically found in a healthy (e.g., non-dysbiotic) microbiome of the urogenital tract (e.g., the vaginal microbial niche). These Lactobacillus species may include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. The preparation may comprise about 80-99.9% Lactobacillus crispatus. The preparation may comprise about 80-99.9% Lactobacillus iners. The preparation may comprise about 80-99.9% Lactobacillus jensenii. The preparation may comprise about 80-99.9% Lactobacillus gasseri. The preparation may contain about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri.

[0056] The preparation may be formulated as a pharmaceutical composition as described herein. The pharmaceutical composition may further comprise an active agent selected from the group consisting of an antibiotic, an immunological agent, a hormonal agent, a metabolite, or a peptide. In some embodiments, the pharmaceutical composition may comprise at least one Lactobacillus strain added to the preparation contained in the pharmaceutical composition. The pharmaceutical composition may be in the form of a suspension, a spray, a gel, a cream, a powder, a capsule, a solution for douche, an egg, a vaginal insert, a tampon, a tablet, or a microencapsulated product.

[0057] Provided herein, in another aspect, is a method for producing a substantially complete vaginal microbiota preparation according to any one of the preceding claims, the method comprising: A. providing a microbiota sample from a donor female urogenital tract, wherein step A optionally comprises (1), (2), (3), (4), (5) or any combination thereof: (1) adding a diluent to the microbiota sample to create a diluted sample; (2) removing a portion of the diluted microbiota sample for testing (e.g., nucleic acid sequencing); (3) pre-cooling the remaining microbiota sample for refrigeration or freezing; and (4) storing the refrigerated or frozen microbiota sample in isolation. (5) including holding the refrigerated or frozen microbiota sample in isolation until completion of any combination of: (a) testing the donor to exclude the substantial presence of one or more infectious pathogens (e.g., blood, and / or ectocervical secretions, and / or urine sample testing), (b) confirming the composition and viability of the microbiota, or (c) further confirming the health of the female donor by multiple post-screening tests performed within a period of 30 to 90 days after donation; and B. releasing the refrigerated or frozen microbiota sample from isolation to define a substantially complete vaginal microbiota preparation.

[0058] The microbiota sample from the donor female may be ectocervical secretions. The microbiota sample may comprise glycans. The glycans may be mucus. The microbiota sample may weigh at least 100 mg, 150 mg, 200 mg or more. The microbiota sample may comprise 80-99.9% Lactobacillus species typically found in a healthy (e.g., non-dysbiotic) microbiome of the urogenital tract (e.g., vaginal microbial niche). These Lactobacillus species may include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri. The microbiota sample may comprise about 80-99.9% Lactobacillus crispatus. The microbiota sample may comprise about 80-99.9% Lactobacillus iners. The microbiota sample may comprise about 80-99.9% Lactobacillus jensenii. The microbiota sample may comprise about 80-99.9% Lactobacillus gasseri. The microbiota sample may comprise about 80-99.9% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0059] Step B of the method may include determining that the microbiota sample is substantially free of pathogens and pathogenic commensals. Step B may include determining that the microbiota sample is substantially free of Gardnerella, Atopobium, and Prevotella genera. Step B may include determining that the microbiota sample is substantially free of antimicrobial resistance (AMR) genes. Step B may include determining that the microbiota sample is substantially free of human sperm (spermatozoa). Step B may include determining that the female donor is substantially free of any one or more (two or more, three or more, or four or more) of: (i) bacteria involved in bacterial vaginosis (e.g., Gardnerella spp., and Prevotella spp.); (ii) yeasts (e.g., Candida, Cryptococcus, and Saccharomyces spp.); (iii) sexually transmitted pathogens (e.g., Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis); (iv) bacteria involved in urinary tract infections (e.g., E. coli, Staphylococcus, Chlamydia, and Mycoplasma); and (v) viruses (e.g., HIV, human papilloma virus (HPV), Hepatitis B virus, Hepatitis C virus, HSV-2), or any combination thereof.

[0060] Provided herein in another aspect is a method for restoring the balance of healthy human vaginal microbiota in the female urogenital tract of a human subject, comprising administering to a subject in need of such restoration an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system, thereby restoring the balance of healthy vaginal microbiota. The female subject may exhibit a dysbiotic microbiota in the urogenital tract, and by restoring the balance of healthy vaginal microbiota, treatment of dysbiosis is provided. The female subject may be treated with an effective amount of an antibacterial agent (e.g., an antibiotic) to substantially reduce the dysbiotic microbiota present prior to administration of the pharmaceutical composition comprising a substantially complete vaginal microbiota preparation.

[0061] In some embodiments, the female subject is subjected to vaginal irrigation prior to administration of the substantially complete vaginal microbiota preparation to reduce pathogen load. The vaginal irrigation can be performed with any suitable solution, including but not limited to saline, an acidic solution, or an antiseptic solution. In some embodiments, the acidic solution is lactic acid, and optionally the lactic acid solution has a pH of about 3.5-4.5, optionally about pH 3.8-4.3. The antiseptic solution can be chlorhexidine or providone-iodine, and the povidone-iodine solution comprises about 10% or 5-10% povidone-iodine. In certain embodiments, the vaginal irrigation is performed with chlorhexidine, and the solution comprises about 0.5% chlorhexidine.

[0062] Restoring the balance of a healthy vaginal microbiota may include the colonization and engraftment of one or more species provided by a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation. Restoring the balance of a healthy vaginal microbiota may include one or more of: (a) a decrease in the relative abundance of pathogens and / or pathogenic commensals present in the urogenital tract (e.g., a relative abundance of pathogens and / or pathogenic commensals of less than 10%); (b) an increase in the relative abundance of Lactobacillus of more than 50% (e.g., more than 60%, or more than 70%); (c) a decrease in one or more pro-inflammatory markers (e.g., locally or systemically); and / or (d) a decrease in vaginal pH (e.g., at least pH 0.3, 0.5, 1.0 or 1.5); and / or (e) a change in grade based on the Ison-Hay scoring system (e.g., grade 0 or grade I), when compared to values ​​of (a), (b), (c), (d) or (e) determined in the same subject prior to administration of a pharmaceutical composition comprising a substantially intact vaginal microbiota preparation. Restoring the balance of a healthy vaginal microbiota involves the engraftment of a microbial community dominated (e.g., at least 50%, 60%, 70% or at least 80%) by one or more lactic acid-producing bacteria.

[0063] The one or more species of lactic acid producing bacteria may be selected from one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0064] Restoring the balance of healthy vaginal microflora may include achieving a vaginal pH of about pH 3.5 to 4.5. Preferably, administration of one or more species of lactic acid-producing bacteria restores the vaginal pH to about 3.7 to about 4.2, more preferably about 3.8 to about 4.0. The microbial community may remain dominated by lactic acid-producing bacteria (e.g., lactobacilli) for at least 1 week, or at least 2, 3, 4 weeks. The microbial community may remain dominated by lactic acid-producing bacteria (e.g., lactobacilli) for at least 2, 3, 4, 5, or at least 6 months.

[0065] In some embodiments, administration of the preparation further comprises administration of an additional Lactobacillus species, which may be present in a low relative amount, for example 0.001%, 0.001-0.002%, 0.002-0.005%, 0.005-0.01%, 0.01-0.05%, 0.05-0.1% or 0.1-1%.

[0066] The method may include administering one or more additional active agents selected from the group consisting of antibiotics, immunological agents, and hormonal agents.

[0067] The subject may be Caucasian. The subject may be Asian. The subject may be Black / African. The subject may be Hispanic. The subject may be of childbearing age or pre-menopausal. The subject may be a post-menopausal subject.

[0068] Restoring the balance of healthy vaginal microbiota can provide an anti-pathogenic microbial niche / vaginal environment. Restoring the balance of healthy vaginal microbiota can provide an anti-inflammatory or low-inflammatory microbial niche / vaginal environment. A substantially complete vaginal microbiota preparation provides a Lactobacillus-dominant microbiota in the vaginal microenvironment, resulting in a lower vaginal pH, a lower amount of pathogenic bacteria, and a restored balance of the vaginal microbiota. Furthermore, inflammation of the vaginal niche of dysbiosis is treated, even if the subject does not show any vaginal symptoms.

[0069] Provided herein in another aspect is a method for treating dysbiosis in the female urogenital tract of a human subject, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system according to any one of the claims, thereby treating the dysbiosis. The dysbiosis may be associated with an infection of the urogenital tract. The infection may be one or more of (recurrent) bacterial vaginosis (BV), vaginal candidiasis and trichomoniasis.

[0070] Provided herein in another aspect is a method for treating (chronic) inflammation in the female genitourinary tract of a human subject, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system as described in any one of the claims, thereby treating the inflammation.

[0071] Provided herein in another aspect is a method for treating (recurrent) bacterial vaginosis (BV) in the female urogenital tract of a human subject, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system, thereby treating the (recurrent) BV.

[0072] Provided herein in another aspect is a method of treating vaginal candidiasis in the female urogenital tract of a human subject, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system, thereby treating vaginal candidiasis.

[0073] Provided herein in another aspect is a method for treating trichomoniasis in the female urogenital tract of a human subject, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation, optionally using a vaginal delivery system, thereby treating trichomoniasis.

[0074] The methods described herein may further comprise administering one or more additional active agents selected from the group consisting of antibiotics, antifungal agents, immunological agents, and hormonal agents. [Brief description of the drawings]

[0075] [Figure 1] Schematic diagram of the donor program for producing a substantially complete vaginal microbiota preparation. Donors were selected based on microbiome sequencing, medical testing, and the absence of specific diseases. Testing was performed both before and after the donor visit. All samples provided by donors were subjected to quality control (see, for example, FIG. 5). Samples were released only if the donor and sample met all requirements. [Diagram 2] Figure 1 shows the distribution of the vaginal microbiome from a cohort of 96 female subjects assessed by shotgun DNA sequencing to identify suitable donors for obtaining ectocervical secretions. The relative abundance of bacteria was measured and subjects were classified as "healthy", "dysbiotic" and "undefined" using the classification parameters shown. [Diagram 3]Stacked bar graphs of relative bacterial abundance in donors from a cohort of 96 female subjects (see FIG. 2) with healthy vaginal microbiomes (n=61) assessed by shotgun sequencing. All donor microbiomes in this graph contain at least 80% vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners) or mixtures thereof, and less than 5% selected pathogens (Atopobium spp., Prevotella spp., B. vaginale, and F. vaginae) or mixtures thereof. [Figure 4] Stacked bar graphs of relative bacterial abundance in donors from a cohort of 96 female subjects (see FIG. 2) with dysbiotic vaginal microbiomes (left, n=27) or undefined microbiomes (right, n=8) assessed by shotgun sequencing. The dysbiotic microbiome contains at least 20% species from selected vaginal pathogens (Atopobium spp., Prevotella spp., B. vaginale, and F. vaginae) or mixtures thereof, and less than 10% vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners) or mixtures thereof. [Figure 5A] A flow chart showing an exemplary procedure for obtaining cervicovaginal secretions and their processing is shown. Figure 5A provides a general overview, and Figure 5B provides a specific exemplary embodiment of the procedure. The secretions were divided into samples for characterization and quality control (including, for example, viability counts, DNA sequencing, and pathogen screening) and processing into substantially complete vaginal microbiota preparations. [Figure 5B]A flow chart showing an exemplary procedure for obtaining cervicovaginal secretions and their processing is shown. Figure 5A provides a general overview, and Figure 5B provides a specific exemplary embodiment of the procedure. The secretions were divided into samples for characterization and quality control (including, for example, viability counts, DNA sequencing, and pathogen screening) and processing into substantially complete vaginal microbiota preparations. [Figure 6A] Figures 6A-6C show stacked bar graphs of the four major Lactobacillus species present in substantially complete vaginal microbiota preparations from nine donors (A-I) assessed by shotgun sequencing. Results from eight representative donor donation visits are shown for each donor. If present, vertical lines indicate new visits as recurrent donors. [Figure 6B] Figures 6A-6C show stacked bar graphs of the four major Lactobacillus species present in substantially complete vaginal microbiota preparations from nine donors (A-I) assessed by shotgun sequencing. Results from eight representative donor donation visits are shown for each donor. If present, vertical lines indicate new visits as recurrent donors. [Figure 6C] Figures 6A-6C show stacked bar graphs of the four major Lactobacillus species present in substantially complete vaginal microbiota preparations from nine donors (A-I) assessed by shotgun sequencing. Results from eight representative donor donation visits are shown for each donor. If present, vertical lines indicate new visits as recurrent donors. [Figure 7] 1 shows a plot illustrating the stability of a substantially complete vaginal microbiota preparation after storage at -80°C for up to 11 months. Each data point represents one sample; each connecting line indicates samples from one donation visit of one donor. Similar symbols and shading indicate similar microbiomes. [Figure 8]Bar graphs of the viability of a substantially intact vaginal microbiota preparation after thawing from -80° C. A. Samples are slowly thawed and warmed to approximately 30° C. B. Effect of repeated freeze-thaw cycles or prolonged incubation at ambient temperature (RT = room temperature) on cell viability. [Figure 9A] 9A-9B show stacked bar graphs of relative bacterial abundance in two recipients (A-B) of substantially complete vaginal microbiota preparations and their respective donors, as measured by shotgun sequencing. All recipients were enrolled based on their microbiome at screening; baseline microbiome samples were taken immediately prior to dosing. An asterisk (*) indicates that one menstrual period occurred before the sample was taken. Two asterisks (**) indicate that two menstrual cycles had passed. [Figure 9B] 9A-9B show stacked bar graphs of relative bacterial abundance in two recipients (A-B) of substantially complete vaginal microbiota preparations and their respective donors, as measured by shotgun sequencing. All recipients were enrolled based on their microbiome at screening; baseline microbiome samples were taken immediately prior to dosing. An asterisk (*) indicates that one menstrual period occurred before the sample was taken. Two asterisks (**) indicate that two menstrual cycles had passed. [Figure 10] Shown are principal component analysis (PCA) plots for two recipients and their respective donors before and after administration of substantially complete vaginal microbiota preparations. Variables used as input values ​​are the relative abundance of bacterial species measured by shotgun sequencing. The same input values ​​were used as in the graph of Figure 9. [Figure 11A]Figure 1 shows plots of a panel of inflammatory markers in the screening cohort (n=60) comparing women with dysbiotic (n=19) and healthy (n=41) microbiomes. A. PCA plot (each dot corresponds to one woman; 92 inflammatory panel markers were used as variables). Asterisks (*) indicate women with a healthy microbiome who may have unrecognized infections, e.g. Candida. B. Box plots of selected inflammatory markers associated with bacterial vaginosis. [Figure 11B] Figure 1 shows plots of a panel of inflammatory markers in the screening cohort (n=60) comparing women with dysbiotic (n=19) and healthy (n=41) microbiomes. A. PCA plot (each dot corresponds to one woman; 92 inflammatory panel markers were used as variables). Asterisks (*) indicate women with a healthy microbiome who may have unrecognized infections, e.g. Candida. B. Box plots of selected inflammatory markers associated with bacterial vaginosis. [Figure 12A] Figure 11 shows plots of inflammatory markers from the inflammation panel described in Figure 11 in recipient 2 pre- and post-administration of a substantially complete vaginal microbiota preparation. A. PCA plot showing the shift in inflammatory status in recipients pre- and post-administration compared to donor samples and a subset of the screening cohort for comparison. 92 inflammation panel markers were used as variables. B. Dot plots of recipient 2 pre- and post-administration showing the same selected inflammatory markers as Figure 11B for the screening cohort. Horizontal lines represent medians; each dot represents a technical replicate: for each time point, two aliquots of the same sample were used and run in duplicate. [Figure 12B]Figure 11 shows plots of inflammatory markers from the inflammation panel described in Figure 11 in recipient 2 pre- and post-administration of a substantially complete vaginal microbiota preparation. A. PCA plot showing the shift in inflammatory status in recipients pre- and post-administration compared to donor samples and a subset of the screening cohort for comparison. 92 inflammation panel markers were used as variables. B. Dot plots of recipient 2 pre- and post-administration showing the same selected inflammatory markers as Figure 11B for the screening cohort. Horizontal lines represent medians; each dot represents a technical replicate: for each time point, two aliquots of the same sample were used and run in duplicate. [Figure 13]FIG. 13 shows an exemplary vaginal delivery system (100) in three different configurations (FIG. 13A, FIG. 13B, and FIG. 13C). FIG. 13A shows the vaginal delivery system in a pre-filled configuration. The basic dispensing device (102) includes a dispensing end (e.g., plunger or piston) (101), which may optionally be configured manually or electromechanically; and an open end (e.g., tip) (105) for insertion into the vaginal cavity, which may optionally be shaped into any suitable form to improve insertion into the vaginal cavity. Optionally, the device (102) includes some type of calibration portion (104), e.g., having a volume indication or maximum volume indication or control, e.g., to aid in administration. In the pre-filled configuration, a composition (103) (e.g., frozen or liquid form) comprising a substantially complete vaginal microbiota preparation is included in the dispensing device (102). To administer the composition (103), the dispensing end (101) is pushed into the dispensing device (102) with some force and the composition is expelled through the open end (105), e.g., into the vaginal cavity. If the contents are provided in frozen form, the user first thaws the frozen contents (103) before administration. FIG. 13B shows an alternative configuration of the vaginal delivery system (100). The dispensing device (102) is not pre-filled and is empty. The composition (103) comprising the substantially complete vaginal microbiota preparation is provided separately from the dispensing device (102) in a container (107) having a cap (106), e.g., a cryovial or other suitable storage container. The dispensing device and the container can be packaged, shipped, and / or stored separately. The user then typically dispenses the contents (103) of the container (107) immediately prior to use (e.g., administration to the urogenital tract), dispensing the contents into a dispensing device (102). If desired, the contents (103) can be frozen, in which case the user thaws the frozen contents (103) prior to administration, or the contents (103) can be provided in lyophilized form, in which case the user rehydrates the lyophilized contents (103) prior to administration.The dispensing tip (101) may be provided detached from the dispensing device (102) as shown, or alternatively attached thereto and then removed immediately prior to loading the dispensing device (102) with a composition (103) comprising a substantially complete vaginal microbiota preparation. FIG. 13C shows an alternative configuration of the vaginal delivery system (100). The dispensing device (108) includes a dispensing tip (101) and an open end (105), but unlike (102), the dispensing device (108) includes a docking platform (110) that allows connection with a container (109), such as a cartridge, for example, to allow for quick connection. Optionally, the container (109) can be loaded within the dispensing device (108), for example, in a suitable space between the docking platform (110) and the dispensing tip (101). The container (109) includes a composition (103) containing a substantially complete vaginal microbiota preparation, and optionally some type of calibration portion (104), and caps (106) on both ends. One end of the container (109) can be connected to the docking platform (110) of the dispensing device (108), and the other end can be connected to the dispensing end (101). The caps (106) of the container (109) may be configured such that their seals are broken upon contact with the docking platform (110) and the dispensing end (101), e.g., made from a soft material (e.g., rubber or other soft polymers or films, etc.) that can be pierced by both the docking platform (110) and the dispensing end (101) to release the contents (103). All configurations can be adapted for single or multiple use applications. For example, the vaginal delivery system (100) illustrated in FIG. 13C may be adapted for multiple use by leaving the dispensing end (101) of the vaginal delivery system permanently attached to the dispensing device (108, dashed line), and (105) and / or (110) may be configured to be discarded after each use or may be made of a washable / sterilizable, multi-use material and removable from the device (108). [Figure 14]The design of the first clinical trial is presented. The study will investigate and map the shift in the vaginal microbiome upon three consecutive administrations of a substantially complete vaginal microbiota preparation to recipients. Recipients will be screened for vaginal dysbiosis based on defined criteria from metagenomic sequencing of vaginal samples. [Figure 15] FIG. 1 is a schematic diagram of an exemplary design for a clinical trial administering antibiotics together with a substantially complete vaginal microbiota preparation. [Figure 16] The design of a second clinical trial is presented. The study will investigate and map shifts in the vaginal microbiome when recipients are administered up to three doses of a substantially complete vaginal microbiota preparation, isolated by follow-up visits and evaluation of treatment success. Recipients will be screened for vaginal dysbiosis based on defined criteria from metagenomic sequencing of vaginal samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The present invention relates to compositions, devices and kits comprising substantially complete vaginal microbiota preparations, as well as methods of making and using the same. Provided herein are substantially complete vaginal microbiota preparations and compositions for modulation of vaginal microbiota for the prevention or treatment of conditions and diseases in which the vaginal microbiota composition is implicated. The applicant has developed a safe and effective process for making a substantially complete vaginal microbiota preparation, and provides devices and methods for its administration and use to, for example, restore or maintain a balance of healthy human vaginal microbiota in the female urogenital tract and treat dysbiosis and / or inflammatory conditions.

[0077] Vaginal flora, vaginal microbial niche

[0078] A healthy vaginal flora is characterized by an acidic environment inhabited mainly by lactic acid bacteria, mainly species of Lactobacillus, which reside in the vaginal microbial niche. The microbial composition in healthy women can vary, but is typically dominated by one of four Lactobacillus species: L. crispatus, L. iners, L. gasseri, L. jensenii and mixtures thereof. Healthy vaginas of women of childbearing age contain approximately 10 7 ~10 9 It is estimated that the vaginal flora is dominated by lactic acid producing bacteria (e.g., Lactobacillus) in the colony forming units of the vagina. The distribution of species differs among women of different geographical backgrounds, different races (e.g., Asians, white women, blacks, Hispanics), age, lifestyle, etc. The composition of the vaginal flora is also influenced by which specific strains and / or species were inherited from the woman's mother and / or which strains and / or species were transferred from the female's gastrointestinal tract to the urogenital tract. Healthy fertile women exhibit a pH of approximately 3-5.5 (more specifically, pH 3.5-4.5) in the vagina, primarily as a result of lactic acid production. Vaginal pH varies physiologically from birth to menopause. An increase in vaginal pH above 4.0-4.5 is detrimental to the survival of Lactobacillus bacteria, but not to other microorganisms. Vaginal lactobacilli are believed to have a protective effect against colonization of the vagina by pathogenic microorganisms (e.g., yeasts (Candida albicans), Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis, as well as viruses such as HIV, HSV-2, and various anaerobic organisms) and to prevent colonization of the vagina by bacteria present in the colon, such as Gardnerella vaginalis, Mobiluncus, Bacteroides, Prevotella, and Escherichia coli.

[0079] Several factors can contribute to the disturbance of the vaginal flora. These factors can include: a) the use of antibiotics to kill pathogenic bacteria, which can result in a significant decrease in the level of lactobacilli in the vagina; b) hormonal changes, especially the change in estrogen levels observed at several stages of a woman's life (e.g., puberty, pregnancy, childbearing age, premenopause and postmenopause) (estrogen levels are thought to be related to the level (predominance) of Lactobacillus in the vagina); c) sexual intercourse, which can be associated with an increase in pH (semen is generally alkaline) that can disturb the vaginal flora (as an increase in vaginal pH can cause bacteria other than lactobacilli to start to grow); d) the use of medicines, such as chemotherapeutic or antifungal agents; e) the use of birth control products; f) during menstruation; g) poor hygiene (e.g., promoting the undesirable spread of microorganisms from the rectum to the urogenital area); h) general health conditions (e.g., suffering from diabetes).

[0080] Disturbance of the vaginal flora can result in vaginal dysbiosis and vaginal disorders, such as candidiasis and bacterial vaginosis, two common vaginal disorders that affect women worldwide. Bacterial vaginosis is believed to be the result of the replacement and substitution of vaginal lactic acid-producing bacteria by various undesirable species, such as Gardnerella vaginalis, Bacteroides, Mobiluncus, Prevotella, and Mycoplasma hominis. Vaginal infections are most often associated with one or more of Escherichia, Enterococcus, Pseudomonas, Proteus, Klebsiella, Streptococcus, Staphylococcus, Gardnerella, Ureaplasma, Bacteroides, Peptococcus, Neisseria, Serratia, Corynebacterium, Clostridium, and Candida.

[0081] The predominance of vaginal lactobacilli is thought to play an important role in the production of lactic acid and acidification of the vagina, as well as resistance to infections through the production of other antibacterial products, such as hydrogen peroxide. The presence of lactobacilli in the vagina is associated with a reduced frequency of bacterial vaginosis, yeast vaginosis, and sexually transmitted pathogens, including Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis. The predominance of Lactobacilli differs between ethnic groups (highly prevalent in Asian and white women, but thought to be less prevalent in black and Hispanic women, although they still constitute a majority).

[0082] Tests have shown that Lactobacillus-depleted communities are transient and may last only a few days, but in other cases, depleted communities may persist for weeks. Some women with Lactobacillus-depleted communities remain asymptomatic and healthy. However, such women may be at higher risk of infection and sexually transmitted diseases (STDs).

[0083] In some embodiments, the vaginal flora is restored by supplementing or adding to the disturbed vaginal flora a substantially complete vaginal microbiota preparation and compositions thereof described herein.

[0084] Substantially complete vaginal microbiota preparation and lactobacilli

[0085] Aspects of the present invention relate to a substantially complete vaginal microbiota preparation, methods of making and using the same, and pharmaceutical compositions comprising the same. There is great interest in developing a non-antibiotic, ecologically appropriate approach to replenish healthy vaginal flora to female subjects in need thereof. The substantially complete vaginal microbiota preparation described herein provides a solution to problems previously encountered by using isolated, culture-grown defined or single strain compositions, or compositions comprising multiple isolated, culture-grown strains. Problems include lack of vaginal colonization and engraftment of administered strains in the urogenital tract, including the vaginal cavity. The substantially complete vaginal microbiota preparation described herein can be administered to the vaginal cavity, for example, to regulate the vaginal microbial niche for the maintenance of a healthy vaginal microbiota, and to help restore an imbalanced vaginal microbiota. It has been found that the substantially complete vaginal microbiota preparation is preferred and advantageous to compositions containing a single culture-grown strain in that it can efficiently colonize the vaginal microbial niche upon administration.

[0086] The female urogenital tract (also known as the genitourinary tract) is composed of interconnected biogeographical niches. Bacteria from the vaginal microbial community can migrate through the cervix to distant sites of the urogenital tract. Dysbiosis in the vaginal microbial community can result in dysbiosis in distant sites. Dysbiosis in these sites is associated with various diseases and conditions, including urinary tract infections (UTIs) and pelvic inflammatory disease (PID). In some embodiments, administration of a substantially complete vaginal microbiota preparation to the vagina includes elimination of dysbiosis in distant sites of the urogenital tract.

[0087] In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three, four or five different bacterial species of the genus Lactobacillus. In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three or four different bacterial species of the genus Lactobacillus. In some embodiments, the bacterial species comprise about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.9%, 80-99%, 75%-95%, 85%-95%, 85%-99%, or 90%-99% of the preparation (the sum of all detectable bacterial taxa (e.g., species) of the preparation).

[0088] In some embodiments, the preparation comprises one of (a)-(o) Lactobacillus species and combinations of species: a) Lactobacillus crispatus; b) Lactobacillus iners; c) Lactobacillus jensenii; d) Lactobacillus gasseri; e) Lactobacillus crispatus and Lactobacillus iners; f) Lactobacillus crispatus and Lactobacillus jensenii; g) Lactobacillus crispatus and Lactobacillus gasseri; h) Lactobacillus iners and Lactobacillus jensenii; i) Lactobacillus iners and Lactobacillus gasseri; j) Lactobacillus jensenii and Lactobacillus gasseri; k) Lactobacillus crispatus, Lactobacillus iners and Lactobacillus jensenii; l) Lactobacillus crispatus, Lactobacillus iners and Lactobacillus gasseri; m) Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri; n) Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri; o) Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0089] In one aspect, the substantially complete vaginal microbiota preparation comprises (i) one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) less than 5% of the genera Gardnerella, Atopobium, and Prevotella; the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or diluent.

[0090] In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include one bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus crispatus. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus iners. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus jensenii. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus gasseri.

[0091] In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include two bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus crispatus and Lactobacillus iners. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus crispatus and Lactobacillus jensenii. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus crispatus and Lactobacillus gasseri. In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include Lactobacillus iners and Lactobacillus jensenii. In some embodiments, about 80-99.9% of all detectable bacterial species in the preparation include Lactobacillus iners and Lactobacillus gasseri.

[0092] In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include three bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri.

[0093] In some embodiments, about 80-99.9% of all detectable bacterial species in the preparation include Lactobacillus crispatus, Lactobacillus iners, and jensenii.In some embodiments, about 80-99.9% of all detectable bacterial species in the preparation include Lactobacillus crispatus, Lactobacillus iners, and gasseri.

[0094] In some embodiments, about 80-99.9% of all detectable bacterial species of the preparation include four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri.

[0095] In some embodiments, Lactobacillus crispatus or Lactobacillus iners is present in a greater relative amount than other bacterial species in the preparation, for example, 80-99.9% of all detectable bacterial species in a substantially complete vaginal microbiota preparation may be Lactobacillus crispatus and may further contain less than 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of other bacterial species.

[0096] Further Lactobacillus species

[0097] In some embodiments, one or more additional Lactobacillus species are present in the preparation in small amounts (e.g., less than 20%, 15%, 10%, 5%, 2%, 1% of the Lactobacillus species in the preparation). In some embodiments, these lactobacilli species are present at a concentration of about 0.01-1%, 0.02-0.5%, or 0.01-0.3% of all detectable bacterial species in a substantially complete vaginal microbiota preparation.

[0098] In a further embodiment, the substantially complete vaginal microbiota preparation comprises additional lactobacilli other than Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii or Lactobacillus gasseri, where the additional Lactobacillus species comprises, for example, Lactobacillus acidophilus, Limosilactobacillus fermentum (previously known as Lactobacillus fermentum), Lacticaseibacillus casei (previously known as Lactobacillus casei) and Lacticaseibacillus rhamnosus (previously known as Lactobacillus rhamnosus).As used herein, Limosilactobacillus fermentum, Lacticaseibacillus casei and Lacticaseibacillus rhamnosus are referred to as Lactobacillus and are included in the meaning of Lactobacillus.

[0099] The substantially complete vaginal microbiota preparation of the present invention comprises less than 5% of the genera Gardnerella, Atopobium and Prevotella. In some embodiments, the substantially complete vaginal microbiota preparation of the present invention comprises less than 10%, less than 7%, less than 5%, less than 2%, less than 1%, less than 0.5% or less than 0.1% of the genera Gardnerella, Atopobium and Prevotella. The preparation of the present invention can further comprise less than 5% of the genera Gardnerella vaginalis, Atopobium and Prevotella and Fannyhessa vaginae. Gardnerella vaginalis has been historically misclassified and has recently been reclassified as belonging to the genus Bifidobacteria. Thus, the terms Gardnerella vaginalis and Bifidobacterium vaginalis are used interchangeably. Atopobium vaginae and Fannyhessea vaginae are also used interchangeably throughout this application.

[0100] A substantially complete vaginal microbiota preparation isolated from an animal or human body (an isolated substantially complete vaginal microbiota preparation) is provided herein. Thus, a substantially complete vaginal microbiota preparation is used interchangeably with an isolated substantially complete vaginal microbiota preparation.

[0101] Lactobacillus crispatus dominant preparation

[0102] Provided herein is a substantially complete vaginal microbiota preparation dominated by Lactobacillus crispatus, in which Lactobacillus crispatus is present in greater amounts than each of Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. Preferred aspects and embodiments of the substantially complete vaginal microbiota preparation dominated by Lactobacillus crispatus are provided below.

[0103] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus in a relative amount of about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus crispatus in a relative amount of greater than 90%, 95%, 99%, or 99.9% of all detectable bacterial species in the preparation. In some embodiments, the species Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri are not present in detectable amounts in the preparation.

[0104] In other aspects, the substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus and Lactobacillus iners, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus gasseri and / or Lactobacillus jensenii. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus iners, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners constituting up to 5%, 10%, 20%, or 30% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus iners, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes about 5-40% of all detectable bacterial species in the preparation, such as 5-10%, 10-20%, 20-30% or 30-40%. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus iners, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes at least 5%, 10%, 20%, or 30% of all detectable bacterial species in the preparation.

[0105] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus gasseri and / or Lactobacillus iners. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes at least 0.01% to about 45% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes about 0.01-5%, 5-10%, 10-20%, or 30-40% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes about 0.01%-5% of all detectable bacterial species in the preparation.

[0106] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus gasseri. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners and Lactobacillus jensenii each constituting at least 0.01% to about 20% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus jensenii each constitute at least 0.01 to about 15% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus jensenii each constitute about 2% to 15% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus jensenii each constitute about 2%-10% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes about 0.01-5%, 5-10%, or 10-20% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes about 0.01-5%, 5-10%, or 10-20% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus jensenii each constitute up to 15% of all detectable bacterial species.

[0107] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus constitutes at least 50%, 60%, 70%, or 80% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus gasseri constitute 0.01%-20%, 0.2-15%, or 0.2-10% of all detectable bacterial species in the preparation, respectively. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus gasseri each constitute between 0.01% and about 5% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, Lactobacillus crispatus constitutes at least 60% of all detectable bacterial species in the preparation, and further, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus gasseri each constitute from 0.01 to about 10% of all detectable bacterial species in the preparation.

[0108] Lactobacillus iners dominant preparation

[0109] The present invention further provides a substantially complete vaginal microbiota preparation dominated by Lactobacillus iners, in which Lactobacillus iners is present in greater amounts than each of Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri. Preferred aspects and embodiments of the substantially complete vaginal microbiota preparation dominated by Lactobacillus iners are provided below.

[0110] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus iners in a relative amount of about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus iners in a relative amount of greater than 90%, 95%, 99%, or 99.9% of all detectable bacterial species in the preparation. In some embodiments, the species Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri are not present in detectable amounts in the preparation.

[0111] In other aspects, the substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus gasseri. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus constitutes less than 20%, less than 10%, or less than 5% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus constitutes 0.01-10%, 0.01-5%, 0.01-2% or 0.01-1% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and wherein Lactobacillus iners constitutes at least 70% and Lactobacillus crispatus constitutes less than 20% of all detectable bacterial species in the preparation.

[0112] In other aspects, the substantially complete vaginal microbiota preparations of the invention comprise Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparations do not contain detectable amounts of Lactobacillus gasseri and / or Lactobacillus crispatus. In some embodiments, the preparations comprise Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes less than 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes 0.01%-0.05% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes at least 70% and Lactobacillus jensenii constitutes less than 1% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners constituting at least 80% and Lactobacillus jensenii constituting less than 0.1% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners constituting at least 90% of all detectable bacterial species in the preparation and Lactobacillus jensenii constituting less than 0.1%.

[0113] In other aspects, the substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus jensenii and / or Lactobacillus crispatus. In some embodiments, Lactobacillus jensenii and / or Lactobacillus crispatus constitute less than 2%, 1%, 0.5%, 0.1% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus gasseri constitutes less than 5%, 4%, 3%, 2%, or 1% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus gasseri constitutes 0.01-5%, 1-4%, or 1-3% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes at least 70% and Lactobacillus gasseri constitutes less than 5% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners constituting at least 80% of all detectable bacterial species in the preparation and Lactobacillus gasseri constituting less than 5%. In some embodiments, the preparation comprises Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus iners constituting at least 90% of all detectable bacterial species in the preparation and Lactobacillus gasseri constituting less than 5%.

[0114] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus gasseri. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus crispatus and Lactobacillus jensenii each constituting at least 0.01% to about 10% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus and Lactobacillus jensenii each constitute at least 0.01% to about 5% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus and Lactobacillus jensenii each constitute about 0.01% to 1% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii constitutes less than 2%, 1%, 0.5%, 0.2% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus constitutes less than 2%, 1%, 0.5%, 0.2% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus jensenii constitute less than 2%, 1%, 0.5%, and 0.2%, respectively, of all detectable bacterial species in the preparation.

[0115] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus jensenii. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, with Lactobacillus crispatus and Lactobacillus gasseri each constituting at least 0.01% to about 10% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus and Lactobacillus gasseri each constitute at least 0.01% to about 5% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus and Lactobacillus gasseri each constitute at least 0.01% to about 1% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus gasseri constitutes less than 2%, 1%, 0.5%, 0.2% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus crispatus constitutes less than 2%, 1%, 0.5%, 0.2% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus iners, Lactobacillus crispatus, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners and Lactobacillus gasseri constitute less than 2%, 1%, 0.5%, and 0.2%, respectively, of all detectable bacterial species in the preparation.

[0116] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus iners constitutes at least 50%, 60%, 70%, or 80% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus gasseri respectively constitute 0.01%-25%, 0.1-20%, or 0.2-15% of all detectable bacterial species in the preparation. In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus gasseri each constitute between 0.01% and about 5% of all detectable bacterial species in the preparation.In some embodiments, the preparation comprises Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, Lactobacillus iners constitutes at least 50% of all detectable bacterial species in the preparation, and further, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus gasseri each constitute from 0.01 to about 25% of all detectable bacterial species in the preparation.

[0117] Lactobacillus jensenii dominant preparation

[0118] The present invention further provides a substantially complete vaginal microbiome preparation dominated by Lactobacillus jensenii, in which Lactobacillus jensenii is present in greater amounts than each of Lactobacillus crispatus, Lactobacillus iners and Lactobacillus gasseri. Preferred aspects and embodiments of the substantially complete vaginal microbiome preparation dominated by Lactobacillus jensenii are provided below.

[0119] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus gasseri, which together constitute between about 80 and 99.9% of the relative amount of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus jensenii in a relative amount of greater than 60%, 65%, 70%, or 75% of the total detectable bacterial species in the preparation. In some embodiments, each of Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus gasseri constitutes between 0.01 and about 20% of the total detectable bacterial species in the preparation. In some embodiments, each of Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus gasseri constitutes 0.05 to about 15% of all detectable bacterial species in the preparation. In some embodiments, Lactobacillus jensenii constitutes at least 65% of all detectable bacterial species in the preparation, and further, each of Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus gasseri constitutes 0.01 to about 15% of all detectable bacterial species in the preparation.

[0120] Lactobacillus gasseri dominant preparation

[0121] The present invention further provides a substantially complete vaginal microbiome preparation dominated by Lactobacillus gasseri, in which Lactobacillus gasseri is present in greater amounts than each of Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri. Preferred aspects and embodiments of the substantially complete vaginal microbiome preparation dominated by Lactobacillus gasseri are provided below.

[0122] In one aspect, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus gasseri in a relative amount of about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation comprises Lactobacillus gasseri in a relative amount of greater than 90%, 95%, 99%, or 99.9% of all detectable bacterial species in the preparation. In some embodiments, the species Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri are not present in detectable amounts in the preparation.

[0123] In other aspects, a substantially complete vaginal microbiota preparation of the invention comprises Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus iners, and / or Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation; and less than 5% of the genera Gardnerella, Atopobium, and Prevotella. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus iners and / or Lactobacillus jensenii. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus crispatus and / or Lactobacillus jensenii. In some embodiments, the preparation does not contain detectable amounts of Lactobacillus iners and / or Lactobacillus crispatus. In some embodiments, the preparation comprises Lactobacillus gasseri in a relative amount of at least 40%, 45%, 50%, 55%, 60%, 65% or more of all detectable bacterial species in the preparation.

[0124] The substantially complete vaginal microbiota preparation and pharmaceutical compositions comprising same are suitable for administration to a subject, preferably vaginal administration. The subject may be a human.

[0125] In one aspect, the substantially complete vaginal microbiota preparation provided herein is suitable for vaginal administration.In a further aspect, the substantially complete vaginal microbiota preparation provided herein is for use in treating inflammation or inflammation-related disorders.In a further aspect, the substantially complete vaginal microbiota preparation provided herein comprises an effective amount of vaginal lactobacilli to colonize the recipient's vaginal niche and treat inflammation.

[0126] Lactobacilli in the vaginal niche

[0127] Lactobacilli are the major microorganisms in the vaginal microbial community and play a major role in maintaining a healthy urogenital tract. They can prevent the attachment and growth of pathogenic microorganisms and / or the overgrowth of pathogenic commensals through a mechanism believed to involve the secretion of anti-adhesins, hydrogen peroxide, bacteriocins and the fermentation of glycogen to lactic acid, thereby creating an acidic environment hostile to pathogens and pathogenic commensals. The genus Lactobacillus comprises a phenotypically heterogeneous group of Gram-positive, aerotolerant anaerobic, lactic acid-producing bacteria. Other typical characteristics include catalase-negative and rod-shaped, and generally have DNA with a low content of guanine (G) and cytosine (C), less than about 50%. They are members of the phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae. Those skilled in the art will be able to identify Lactobacillus species using standard techniques.

[0128] Species of lactobacilli can be identified phenotypically and genetically, for example, based on the 16S rRNA (ribosomal RNA) sequence (or the DNA encoding 16S rRNA, commonly referred to as 16S rDNA). Genetic analysis can be performed using standard techniques, such as whole genome sequencing analysis, as well as widely used typing approaches based on nucleotide variations in hundreds of DNA sequences and a few gene fragments: for example, Multi-locus Sequence Typing (MLST), Multi-locus Variable number of tandem repeat Analysis (MLVA), rMLST and cgMLST, as discussed in Marcos Perez-Losada, M. et al., "Microbial sequence typing in the genomic era", Infection, Genetics and Evolution, Vol. 63, Sep. 2018, p. 346-359.

[0129] Other identification techniques include vaginal pH, Nugent score, Whiff test, gas-liquid chromatography analysis of glucose fermentation products, total anaerobic organism concentration, total aerobic organism concentration, enzyme activities (e.g., lipase, phospholipase A2 and phospholipase C, hydrogen peroxide production).

[0130] In some embodiments, the substantially complete vaginal microbiota preparation described herein does not include (is not derived from) isolated and / or culture-grown bacterial strain(s). The substantially complete vaginal microbiota preparation can be prepared, for example, from ectocervical secretions (vaginal fluid) collected from the vaginal canal of a female donor with healthy vaginal flora. The substantially complete vaginal microbiota preparation can be collected using standard techniques using commercially available collection devices such as menstrual fluid collection devices (soft cups or soft disks), syringes, tubes, spatulas or beakers, or absorbent matrices. In some embodiments, the menstrual fluid collection device is a vaginal self-sampling device. The vaginal self-sampling device can be used, for example, by a donor to collect vaginal fluid or ectocervical secretions without the assistance of another person.

[0131] Optionally, the collected material is centrifuged. The centrifugation step may be performed to facilitate collection without physically separating the vaginal fluid components. In some embodiments, the substantially complete vaginal microbiota preparation comprises one or more of mucus (e.g., secreted by the cervix), excreted epithelial cells, vaginal leakage fluid, and bacteria other than lactobacilli found in secretions from female donors. It is believed that the mucus and other components of secretions (including other bacteria) are beneficial for the growth and survival of Lactobacillus upon administration to the urogenital tract, thereby supporting engraftment in the female recipient. This provides an advantage over compositions comprising isolated strain(s). Thus, in some embodiments, the substantially complete vaginal microbiota preparation is not cultured (e.g., for the purpose of strain isolation) or grown in vitro, but rather is preferably stored in a refrigerator at 4° C. or frozen immediately after collection (e.g., frozen at 0° C., −20° C., −80° C., −190° C.), or optionally spray-dried or freeze-dried.

[0132] If desired, the ectocervical secretion can be further processed, e.g., prior to refrigeration or freezing, e.g., by filtration for sterility and / or to remove residual particles, aggregates and cells, and by adding diluents to reach a desired volume, concentration (e.g., CFU / mL) and / or viscosity, e.g., as discussed herein. Optionally, the substantially complete vaginal microbiota preparation is maintained refrigerated or frozen until formulated into a dosage form and / or dispensed into an applicator or dispenser.

[0133] Other advantages of the substantially complete vaginal microbiota preparations described herein include that in some embodiments, substantial engraftment does not require the use of mucoadhesive excipients (e.g., hydrocolloids, such as xanthan gum, locust bean gum alginate, etc.) to increase the ability of lactobacilli to adhere to the mucosa and colonize, which are sometimes used to increase the engraftment of isolated and expanded strains. This minimizes the risk of inducing adverse side effects, such as allergic reactions, from mucoadhesive excipients and increases tolerance. Another advantage of the substantially complete vaginal microbiota preparations described herein is the efficient and long-lasting engraftment / colonization of lactobacilli in the vaginal mucosa. In some embodiments, the colonization effect persists for at least 1, 2, 3, 4, 5 or more menstrual cycles after administration of the substantially complete vaginal microbiota preparation.

[0134] One of the further advantages of the method and preparation of the present invention is that pretreatment with antibiotics is not necessary to successfully treat inflammation in dysbiosis subjects, which was not possible before.This is likely due to the use of smaller sample concentrations or single strains, but the preparation provided herein contains not only the entire ecosystem of vaginal microflora, but also a higher concentration and / or amount of vaginal microflora in the preparation.Finally, the underuse or overuse of antibiotics is directly related to the problem of increasing antibiotic resistance.The present preparation does not rely on the use of antibiotics before or during treatment, and therefore this is a further advancement and advantageous effect of the present invention.

[0135] Pioneer species

[0136] Another advantage of the substantially complete vaginal microbiota preparation is the presence of the substantially complete ecosystem of donor vaginal microbiota.In certain embodiments, even lactobacilli present at very low levels, such as 0.01% or even 0.001%, can be successfully transferred to recipient by administering the preparation.Without being bound by theory, it is believed that the substantially complete ecosystem provides optimal growth conditions for pioneer species, such as Lactobacillus jensenii, Lactobacillus gasseri, and / or Lactobacillus iners, which preferentially colonize and grow in the suboptimal recipient dysbiosis vaginal niche, for example at higher pH.

[0137] In some embodiments, the pioneer species of the invention are characterized by being the first to colonize and grow in the dysbiotic vaginal niche, thereby being able, for example, to liberate metabolic products (including amino acids) and lower the pH, which is expected to promote the growth of other lactobacilli present in the preparation and in the vaginal cavity.

[0138] By providing a substantially complete ecosystem, the vaginal Lactobacillus species included in the substantially complete vaginal microbiota preparation can act in concert to reverse dysbiosis and, in certain embodiments, result in "twinning" of the vaginal microbiome, e.g., recreating a near copy of the donor's healthy vaginal microbiota in the recipient's vaginal niche.

[0139] Lactobacillus species appear to have species-specific optimum pH ranges for proliferation and growth. L. crispatus typically grows well in a pH range of about 3.7-4.0, more optimally at pH 3.7-3.9. L. iners typically grows best in a pH range of 3.9-4.2. L. gasseri and L. jensenii grow best at pH values ​​of about 4.0-5.2. L. jensenii, in particular, can grow well at high pH.

[0140] In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which constitute about 80-99.9% of all detectable bacterial species in the preparation, and L. crispatus is present in a relative concentration of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or about 99.9%, and further, the survival and / or growth rate of L. jensenii after administration is higher than the survival and / or growth rate of L. crispatus.

[0141] In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which constitute about 80-99.9% of all detectable bacterial species in the preparation, with L. crispatus and L. iners both being present at about 30-45%, and further, the survival and / or proliferation rate of L. jensenii is higher compared to the survival and / or proliferation rate of L. crispatus following administration.

[0142] In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which constitute about 80-99.9% of all detectable bacterial species in the preparation, with L. crispatus and L. iners both being present at about 30-45%, and further, the survival and / or proliferation rate of L. gasseri following administration is higher compared to the survival and / or proliferation rate of L. crispatus.

[0143] In some embodiments, the substantially complete vaginal microbiota preparation comprises one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, which constitute about 80-99.9% of all detectable bacterial species in the preparation, with L. crispatus and L. iners both present at about 30-45%, and further, the survival and / or proliferation rate of L. iners following administration is higher compared to the survival and / or proliferation rate of L. crispatus.

[0144] In some embodiments, the substantially complete vaginal microbiota preparation comprises L. iners, L. gasseri and / or L. jensenii species capable of growing at a pH of about 3.8-5.0, 4.0-5.0, 4.0-4.8, 4.0-4.6, 4.0-4.5, 4.2-5.0, 4.2-4.8, 4.2-4.6, 4.4-4.8, or pH 4.4-5.0. In some embodiments, growth of L. iners, L. gasseri and / or L. jensenii reduces the pH of the recipient's vaginal niche to a pH suitable for growth of L. crispatus. In some embodiments, growth of L. iners, L. gasseri and / or L. jensenii reduces the pH of the recipient's vaginal niche to a pH suitable for growth of L. crispatus. In some embodiments, after administration of the preparation, at least one of L. iners, L. gasseri and / or L. jensenii has the highest engraftment and / or proliferation rate that is higher than the rate of L. crispatus.

[0145] In embodiments, successful administration and treatment of dysbiosis with a substantially complete vaginal microbiota preparation transfers the donor's vaginal ecosystem to the recipient, including minor species that may be present in low relative abundance.

[0146] After administration of a substantially complete vaginal microbiota preparation to a dysbiotic recipient, pioneer species, such as L. iners, L. gasseri and / or L. jensenii, may have a growth advantage due to their specific tolerance of a higher pH range that is compatible with the resident pH in the dysbiotic vaginal niche. Thus, although the Lactobacillus species contained in the ecosystem may not colonize and grow at the same rate after administration, depending on the pH of the dysbiotic vaginal niche, a subset of lactobacilli, such as pioneer species of the ecosystem (e.g., L. iners, L. gasseri and / or L. jensenii), may be favored to grow first. This initial growth of pioneer species makes the dysbiotic vaginal niche habitable for "follower species," such as L. crispatus, which prefer a lower pH to grow. Growth of pioneer species such as L. iners, L. gasseri and / or L. jensenii can help lower the pH level, for example from pH 4.8 to 4.0, thereby creating more suitable conditions for the growth of L. crispatus.

[0147] Interactions between different Lactobacillus species present in a substantially complete vaginal microbiota preparation are predicted to influence the success of the treatment of dysbiosis: this beneficial effect would not be observable with isolated strains or species.

[0148] In some embodiments, the lactobacilli contained in the substantially complete vaginal microbiota preparation described herein can colonize and establish (engraft) the human vagina. In some embodiments, colonization and engraftment occurs even during menstrual discharge. The lactobacilli contained in the substantially complete vaginal microbiota preparation can remain present in the vagina after administration for several menstrual cycles. In some embodiments, the lactobacilli colonize the vaginal microbial niche upon vaginal administration for one or more (e.g., 2, 3, 4, 5 or 6) menstrual cycles. The residence time can be determined, for example, using nucleic acid sequencing of specific lactobacilli, such as, for example, by comparing the results of sequencing before and after administration of a substantially complete vaginal microbiota preparation described herein, for example, determining the identity of the newly added members (e.g., one or more lactobacilli) to the recipient's microbial community, and then determining (e.g., via nucleic acid sequencing) at a given time interval (e.g., before or after a menstrual cycle, optionally over several menstrual cycles) that all or a specific subset of the newly added members are still substantially present at the specified time interval. In some embodiments, the residence time of the recipient's microbiome can be compared to the donor's microbiome. Residence time can vary depending on various factors, including hormone levels (e.g., estrogen), diet, sexual activity, vaginal acidity, and the presence or absence of reproductive tract infections and other microbial perturbations, such as treatment with antibiotics.

[0149] Successful colonization and engraftment of lactobacilli contained in a substantially complete vaginal microbiota preparation may be indicated by one or more of the following in the vaginal cavity and vaginal microbial niche (e.g., when compared to the baseline of the same subject (e.g., prior to administration and engraftment) or of an untreated control subject): decreased pH, increased lactic acid content, decreased abundance of antibiotic resistance genes, decreased amount of fungal DNA, decreased toxin content, decreased virulence factors, decreased inflammatory cytokines and chemokines, decreased immune cell infiltration, decreased amount of total bacterial DNA, decreased amount of total pathogenic DNA, increased viscoelasticity, increased sialoglycan content, decreased relative or absolute abundance of pathogenic commensals or pathogens, or any combination thereof.

[0150] stability

[0151] The lactobacilli contained in the substantially complete vaginal microbiota preparation or in the pharmaceutical composition formulated with the substantially complete vaginal microbiota preparation described herein are generally viable for up to one week when stored in a refrigerator at 4° C., and for up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months or more) when stored frozen at about −18° C., or preferably at about −80° C. The viability decreases over time. The substantially complete vaginal microbiota preparation or pharmaceutical composition described herein is suitable for administration if it retains at least 30%, 40%, 50%, 60%, 70%, or at least 80% viable bacteria before use. Viable cells are generally capable of settling and engrafting. Viability refers to the percentage of viable bacteria in a population. In some embodiments, the substantially complete vaginal microbiota preparation described herein is further processed, for example by adding a diluent such as saline, for example by formulating a pharmaceutical composition. In some embodiments, the substantially complete vaginal microbiota preparations or compositions thereof described herein can be further processed, e.g., freeze-dried (lyophilized) for easy storage, packaging, e.g., formulation and transportation into compositions, and rehydrated prior to administration.

[0152] Some compositional features

[0153] Compositions, such as pharmaceutical compositions, comprising a substantially complete vaginal microbiota preparation are provided herein. In some embodiments, the pharmaceutical composition comprises i) a substantially complete vaginal microbiota preparation, e.g., one or more Lactobacillus species, and optionally one or more residual components of ectocervical secretions (e.g., vaginal mucus, vaginal discharge, other vaginal fluids, and vaginal epithelial cells), ii) a pharma- ceutically acceptable buffer or diluent or other excipient, e.g., saline (e.g., to adjust the viscosity and / or isotonicity / osmolality of the composition), iii) one or more acidifying agents, e.g., lactic acid or boric acid (e.g., to adjust the pH of the composition, e.g., below pH 5, or between pH 3.0 and 4.5), iv) one or more other active agents, e.g., a prebiotic (e.g., to generate a synbiotic mixture with the substantially complete vaginal microbiota preparation), a spermicide (e.g., to kill or inactivate residual sperm in the preparation), a hormone (e.g., estrogen), an anti-inflammatory agent, and the like. In a preferred embodiment, the pharmaceutical composition comprises (i) and (ii). In one embodiment, the pharmaceutical composition consists of (i) and (ii). In one embodiment, the pharmaceutical composition comprises (i) and (ii) but not (iii) and (iv). In one embodiment, the pharmaceutical composition comprises (i), (ii) and (iii) but not (iv). In one embodiment, the pharmaceutical composition comprises (i), (ii), (iii) and a spermicide. In some embodiments, the pharmaceutical composition does not comprise a prebiotic, a hormone or an anti-inflammatory agent.

[0154] Acidifier

[0155] The compositions comprising the substantially complete vaginal microbiota preparation described herein may optionally include one or more acidifying agents, such as organic acids or salts thereof. In some embodiments, the acidifying agent can be used to lower the pH of the composition, for example to pH 5.5 or below 5.0, for example to pH 3.5-4.5, or pH 3.0-4.5. In lowering the pH, such acidifying agents may act as antibacterial agents (e.g., to inhibit Candida or pathogenic bacteria). Acidifying agents include, for example, lactic acid, acetic acid, ascorbic acid, citric acid, folic acid, sorbic acid, or boric acid. In other embodiments, the acidifying agent is administered separately from the composition comprising the substantially complete vaginal microbiota preparation; for example, before, simultaneously with, or after administration of the composition (e.g., in a different dosage form, such as a suppository, cream, gel, powder, douche, or the like), for example, for 1-7 days, or for 1-10 days. In these embodiments, the acidifying agent can be used to promote the survival and engraftment of lactobacilli contained in the substantially complete vaginal microbiota preparation. The acidifying agent can also be useful as a spermicidal agent, for example to kill or inactivate residual sperm that may be present in the preparation. In one embodiment, spermicidal activity is provided by adding lactic acid. In one embodiment, lactic acid can be provided as a racemic mixture of D- and L-isomers, or in different suitable ratios, for example, including only D-lactic acid or only L-lactic acid.

[0156] In some embodiments, the composition comprising a substantially complete vaginal microbiota preparation is acidified by adding 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 5% of an acidifying agent (e.g., lactic acid). In preferred embodiments, the acidifying agent is added at 0.5%-1.5%, or 0.2%-2% (w / w), or a similar concentration compatible with a healthy vagina.

[0157] viscosity

[0158] The composition comprising the substantially complete vaginal microbiota preparation described herein may comprise one or more pharma- ceutically acceptable buffers or diluents or other excipients. In a preferred embodiment, the diluent is saline (e.g., 0.9% NaCl), e.g., sterile saline. In one embodiment, the diluent is used to adjust the viscosity of the composition, e.g., to reduce the viscosity of the substantially complete vaginal microbiota preparation derived from cervico-vaginal secretions, thereby increasing the ability to administer the composition to a female recipient, e.g., using an applicator or dispenser or similar vaginal delivery system. In other embodiments, one or more excipients are used to formulate the composition into different dosage forms, e.g., suppositories, creams, or dissolving films or tablets.

[0159] In some embodiments, the dosage form is liquid, solid or semi-solid. Solid dosage forms preferably include tablets, capsules or films. Semi-solid dosage forms preferably include suppositories, ointments, gels, creams or rigid foams. In a preferred embodiment, the dosage form is a gel.

[0160] When adjusting the viscosity for administration to the vaginal cavity of a female recipient, for example, using an applicator or dispenser, care should be taken to adjust the viscosity so that it can be easily dispensed into the vaginal cavity (e.g., without the need to apply significant force to expel the liquid composition), but avoid making the composition a fluid / liquid that does not remain in the vaginal cavity for a significant period of time after administration. Therefore, a suitable viscosity should be selected to prevent rapid expulsion from the vaginal cavity. The composition comprising the substantially complete vaginal microbiota preparation described herein is preferably of a viscosity that allows the majority of it to remain in the vagina for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes when the female recipient is in an upright position, but administration is preferably performed, for example, in a lithotomy position with the female recipient lying down. Viscosity can be measured, for example, using a viscometer.

[0161] Spermicide

[0162] The composition comprising the substantially complete vaginal microbiota preparation described herein may contain one or more spermicides. Caution should be exercised when using nonoxynol-9 (N-9) due to its membrane-disrupting properties (increasing the permeability of vaginal tissue by causing damage to the cervix epithelium) and has been shown to be harmful to Lactobacillus species. Other spermicides include octoxynol-9, sodium cholate, and benzalkonium chloride. In one embodiment, lactic acid is used. In other embodiments, lactic acid may be used in combination with, for example, citric acid.

[0163] Prebiotics

[0164] As used herein, a "prebiotic" is a growth substrate that increases the growth of bacteria (such as lactobacilli) contained in a substantially complete vaginal microbiota preparation, as can be measured, for example, in vitro. If desired, but not generally necessary, prebiotics can be added to the compositions described herein, for example, to create a synbiotic mixture, for example, to increase the growth of bacteria contained in a substantially complete vaginal microbiota preparation upon administration to the urogenital tract of a female recipient. This can increase colonization and engraftment success under certain circumstances. In other embodiments, prebiotics can be used to maintain the engrafted preparation and / or overall vaginal health.

[0165] If prebiotics are desired, they should be carefully selected so that they cannot be significantly metabolized by (e.g., to avoid promoting the growth of) any yeasts (e.g., Candida species), pathogenic commensals or pathogens (e.g., E. coli and other gram-negative bacteria) that may be present in the female recipient's urogenital tract. Prebiotics include, for example, lactitol, lactulose, and optionally other oligosaccharides and soluble fibers, such as fructooligosaccharides (FOS), glucooligosaccharides (GOS), and inulin.

[0166] Other activators

[0167] If desired, other active agents may be added to the compositions described herein, e.g., antibacterial, antifungal, antibacterial, antiviral, antibiotic, antiparasitic (e.g., with activity against Trichomonas vaginalis), anti-inflammatory agents, etc., for combating bacterial or fungal infections and / or sexually transmitted diseases in the recipient female. When formulating these agents into the pharmaceutical composition, care must be taken so as not to substantially interfere with the activity and effectiveness of the substantially intact vaginal microbiota preparation (and lactobacilli) contained in the composition.

[0168] In some embodiments, the pharmaceutical composition comprises a form of estrogen. Adequate levels of estrogen play a role in the trophism of the vaginal mucosa, as estrogen increases the cellular content of glycogen.

[0169] In some embodiments, the pharmaceutical composition includes thiosulfate, for example to enhance the anti-pathogenic effect of lactobacilli.

[0170] If desired, the pharmaceutical composition can further contain an antibiotic, such as metronidazole, or one or more antibiotics from the following classes: macrolides (e.g., azithromycin, clarithromycin, and erythromycin), tetracyclines (e.g., doxycycline, tigecycline), fluoroquinolones (e.g., gemifloxacin, levofloxacin, ciprofloxacin, and mosifloxacin), cephalosporins (e.g., ceftriaxone, defotaxime, ceftazidime, cefepime), penicillins (e.g., amoxicillin, amoxicillin and clavulanic acid, ampicillin, piperacillin, and ticarcillin), and optionally beta-lactams. amase inhibitors (e.g., sulbactam, tazobactam, and clavulanic acid), such as ampicillin-sulbactam, piperacillin-tazobactam, and ticarcillin and clavulanate, aminoglycosides (e.g., amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), penems or carbapenems (e.g., doripenem, ertapenem, imipenem, and meropenem), monobactams (e.g., aztreonam), oxazolidinones (e.g., linezolid), vancomycin, glycopeptide antibiotics (e.g., telavancin), and the like.

[0171] In some embodiments, the pharmaceutical composition can further contain an antibacterial agent (antibiotic or antifungal agent) selected from metronidazole, tinidazole, secnidazole, clindamycin, nystatin, azithromycin, erythromycin, ofloxacin, doxycycline, levofloxacin, amoxicillin, and fluconazole.

[0172] If desired, the pharmaceutical composition can contain an agent for treating mycobacterial infections. Agents suitable for treating mycobacterial infections include aminoglycosides (e.g., capreomycin, kanamycin, streptomycin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin), isodianides and isodianide analogs (e.g., ethionamide), aminosalicylic acid, cycloserine, diarylquinolines, ethambutol, pyrazinamide, prothionamide, rifampin, and the like.

[0173] If desired, the pharmaceutical compositions can contain a suitable antiviral agent, such as remdesivir, oseltamivir, zanamavir, amantidine or rimantadine, ribavirin, ganciclovir, valganciclovir, foscavir, Cytogam® (cytomegalovirus immune globulin), pleconaril, rupintrivir, palivizumab, motavizumab, cytarabine, docosanol, denotivir, cidofovir, and acyclovir.

[0174] If desired, the pharmaceutical compositions can contain suitable antifungal agents, such as polyenes (eg, nystatin and natamycin) and imidazole antifungals (eg, flucanozole and clotrimazole).

[0175] If desired, the pharmaceutical composition can contain one or more suitable steroids. For example, the composition can include androgenic / anabolic steroids, estrogens, progestogens, corticosteroids, neurosteroids, estradiol, estropipate, premarin, drospirenone, norethisterone, levonorgestrel, testosterone, fluoxymesterone, methylesterosterone, oxandrolone, and oxymetholone.

[0176] Combination therapy

[0177] Also provided herein are methods of treating a subject in need of treatment, e.g., a human female, e.g., a subject exhibiting a dysbiosis (e.g., a dysbiosis in the urogenital tract), by administering an effective amount of a composition, e.g., a pharmaceutical composition comprising a substantially complete vaginal microbiota preparation as described herein.

[0178] It will be understood by those skilled in the art that the administration regimen for treatment may include one or more other therapies in combination with the administration of the substantially complete vaginal microbiota preparations and compositions described herein. In some embodiments, the method for treating vaginal dysbiosis described herein may further include administering a standard of care treatment. For example, the method for treating vaginal dysbiosis described herein may further include administering an antibacterial agent, an antifungal agent, an antibacterial agent, an antiviral agent, an antibiotic, an antiparasitic agent (e.g., with activity against Trichomonas vaginalis), an anti-inflammatory agent, and the like. Current standard of care antibacterial agents (antibiotics, antifungals) include metronidazole, tinidazole, secnidazole, clindamycin, nystatin, azithromycin, erythromycin, ofloxacin, doxycycline, levofloxacin, amoxicillin, and fluconazole. In another example, the methods for treating vaginal dysbiosis described herein may further comprise administering thiosulfate, for example, to aid in recolonization of the vaginal microflora and prevent regrowth of pathogenic agents and thus recurrence.

[0179] Any of the therapeutic agents described herein can be considered a) for formulation as a pharmaceutical composition with a substantially complete vaginal microbiota preparation, or b) as a combination therapy. These combination therapies can be administered together or separately, and simultaneously (substantially simultaneously) or sequentially (e.g., before or after) with the administration of the substantially complete vaginal microbiota preparation and composition described herein. They can be administered using different routes of administration and dosage forms, such as orally (e.g., as a pill) or topically (e.g., as a gel). The use of combination therapies should be evaluated to determine that the treatment does not substantially interfere with the activity and effectiveness of the substantially complete vaginal microbiota preparation (and lactobacilli) contained in the composition, and if implemented, the regimen (e.g., timing, dosing, sequencing, etc.) should be adjusted to minimize interference.

[0180] Vaginal Delivery System

[0181] Vaginal delivery systems (eg, Figures 13A, 13B, 13C) are also provided herein.

[0182] A vaginal delivery system can be used to administer a composition comprising a substantially complete vaginal microbiota preparation as described herein, for example, to a woman (e.g., vaginal cavity). The vaginal delivery system provides a means for administering a composition to the vaginal cavity. It can be composed of a device or dosage form suitable for vaginal delivery of a composition comprising a substantially complete vaginal microbiota preparation as described herein. For example, a suitable device includes an open end (e.g., a tip) for insertion into the vaginal cavity and a dispensing end (e.g., a plunger or piston) for expelling the composition through the open end. The composition comprising a substantially complete vaginal microbiota preparation can be filled through either end, e.g., packaged, and can be pre-filled or separate from the composition. If desired, the packaging is sterile. The device can then be used directly for administration (if pre-filled) or filled with the composition immediately (or shortly) before administration by the user. Alternatively, the device is filled with the composition and stored under refrigeration for several days to about a week (or frozen for longer storage) before administration. In some cases, for example, when the device is an applicator or dispenser, after the composition comprising the substantially complete vaginal microbiota preparation is released into the vaginal cavity, the device is removed from the vaginal cavity. Suitable devices include applicators and dispensers, such as those commonly used in medicine and science. Applicators and dispensers may be manually operated or may include electromechanical assistance.

[0183] If desired, the vaginal delivery system is calibrated, e.g., with a volume indication or a maximum volume indication or control, e.g., to aid in administration. In one embodiment, the vaginal delivery system is a single-use device that is discarded after each use. In another embodiment, the vaginal delivery system is a multiple-use device that must be cleaned and optionally sterilized after each use. Both single-use and multiple-use applicators may be of any suitable material, e.g., an inert polymeric material such as polystyrene or polypropylene.

[0184] Exemplary, non-limiting embodiments of vaginal delivery systems are shown in Figures 13A, 13B, and 13C with optional features.

[0185] In other embodiments, the vaginal delivery system comprises a vaginal suppository that remains in the vaginal cavity until it dissolves.When formulating a vaginal suppository or tablet, care must be taken not to damage the bacteria (lactobacilli) contained in the substantially complete vaginal microbiota preparation, for example, when pressure or heat is applied during the formulation process to inactivate the formulation.Measures should be taken to reduce the effect of heat and pressure on the microorganisms.In further embodiments, the vaginal delivery system comprises an absorbent product that comprises a composition that comprises a substantially complete vaginal microbiota preparation, such as, for example, a feminine hygiene diaper, a panty liner, a sanitary napkin, a tampon, a panty guard or an incontinence guard.

[0186] The vaginal delivery system described herein may optionally comprise a composition comprising a substantially complete vaginal microbiota preparation in a dry form, e.g., freeze-dried or spray-dried, and then optionally formulated into a dosage form as described herein, or reconstituted before use, e.g., with sterile water, a weak acid solution, a gel, or a buffer. If the composition is not reconstituted, rehydration may be achieved in the vaginal cavity, for example, with the aid of resident vaginal fluid, depending on the exact formulation of the dosage form. The method of preserving viable bacteria by freeze-drying can facilitate long-term storage of microorganisms. Those skilled in the art will be able to freeze-dry bacteria using standard techniques.

[0187] In all of these embodiments, dyes, fragrances, pH buffers, drying or resuspending agents, or other standards for formulation may be incorporated into the compositions and devices.

[0188] In all of these embodiments, the devices and compositions described herein may be packaged in suitable packaging, such as bottles, vials, blister packs, cartridges, applicators or dispensers. In one embodiment, the vaginal delivery system comprises a dosage form comprising the pharmaceutical composition or substantially complete vaginal microbiota preparation of the present invention suitable for administering the composition or preparation to the vaginal cavity, the dosage form being an applicator, dispenser or suppository. In a further embodiment, the pharmaceutical composition or substantially complete vaginal microbiota preparation is in liquid or semi-solid form. Solid dosage forms include capsules, tablets and films. Semi-solid dosage forms include suppositories, ointments, gels, creams or hard foams.

[0189] Dosage forms and preparations

[0190] Multiple dosage forms comprising a substantially complete vaginal microbiota preparation suitable for a vaginal delivery system are contemplated herein, including suspensions, sprays, gels, creams, ointments, powders, (gelatin or vegetable cellulose) capsules, douches or douches, foams, films, eggs, vaginal inserts (e.g., tampons), tablets, disks, wafers (e.g., dried on a film by evaporation), or microencapsulated products using excipients and formulation techniques known to those skilled in the art. Particularly preferred dosage forms include formed gels, lyophilized gels, tablets, frozen formulations, and films.

[0191] A number of suitable excipients, such as bulking agents, polymers, carbon sources, mucoadhesives, or pH adjusting and / or buffering agents, can be used to formulate a substantially complete vaginal microbiota preparation.

[0192] The carbon source excipient may act as a carbon source for the microbiota contained in the substantially complete vaginal microbiota preparation. Such carbon courses include mannitol, maltodextran, and guar gum. Some excipients may further function as mucoadhesives or viscosity agents. The bulking agent may include one or more of mannitol, microcrystalline cellulose, maltodextran, guar gum, inulin, or alginic acid (e.g., sodium alginate). The polymer may include a structural polymer. In some embodiments, the polymer includes one or more of mucin, hyaluronic acid, polyvinyl alcohol, sodium CMC, polyvinylpyrrolidone, hydroxypropyl methylcellulose, Carbopol (e.g., Carbopol 934), and poloxamer (e.g., Poloxamer 407). Mucoadhesives include, but are not limited to, alginic acid (sodium alginate) and sodium CMC. Viscosity agents include, but are not limited to, guar gum and Carbopol 934.

[0193] Some excipients function as pH adjusters and / or buffers, such as lactate and acetate buffers.

[0194] A suitable formulation will exhibit little or no flow on suitable vertical surfaces and will maintain high bacterial viability (eg CFU counts) both upon formulation and during (long-term) storage.

[0195] Desired formulation selection parameters include, for example, mucoadhesion (e.g., of the reconstituted product in the vaginal tract); viscosity (of the reconstituted product) (e.g., the final viscosity of a gel-based product should be pourable at ambient temperature and should preferably solidify at 37° C. (at body temperature, e.g., in the vaginal tract); total sugar content (of the reconstituted product) (e.g., ideally below physiological concentrations (approximately 0.5-1.0 mg / mL)); volume of the reconstituted product (e.g., up to 3 mL); hydration / disintegration rate (of the gel / matrix) (e.g., , sufficient physical integrity to provide the desired release rate); pH (e.g., about pH 3.4-3.9 (e.g., to promote inhibition of competitive vaginal bacteria); water activity / water content (e.g., of the dry formulation) (e.g., 0.5-3% water (e.g., for long-term dry formulation stability)); microbial diversity (e.g., relative abundance of Lactobacillus species, e.g., L. crispatus, L. gasseri, L. jensenii, and L. iners); total dose / potency (e.g., preferably at least about 1×10 per dose). 5 CFU / VCC, at least about 10 6 CFU / VCC, at least about 10 7 CFU / VCC or at least about 10 8 CFU / VCC (per dose), shelf life (unreconstituted) at various temperatures, and microbial limits (e.g., absence of microorganisms such as Pseudomonas aeruginosa, Candida albicans, Staphylococcus aureus, Ph Eur standards 5.1.4, 2.6.12 and 2.6.13).

[0196] Suitable testing methods include plate counts (e.g., MRS agar) for e.g. viable count, dose determination, shelf life; rheometers for e.g., mucoadhesion and viscosity, pH meters, Karl Fisher / water activity meters, standard assays such as the Ph Eur test for microbial load.

[0197] The substantially complete vaginal microbiota preparation can be freeze-dried,

[0198] For example, it is formulated into gels and tablets, as well as other dosage forms that can be filled with lyophilized products, such as capsules. The substantially complete vaginal microbiota preparation can also be formulated into freezable gels and freezable liquid media (e.g., including glycerol). The substantially complete vaginal microbiota preparation can also be formulated into (air-dried) films, which can be, for example, in the form of a disk. The loss of viability ranges from about 0.5 log to 1 log during the formulation stage, depending on the excipients and dosage form.

[0199] In a preferred embodiment, the substantially complete vaginal microbiota preparation of the present invention is provided in a dosage form selected from a tablet, a preformed gel, a lyophilized gel, a liquid formulation, a frozen formulation, a film-forming formulation or a film.

[0200] Preformed and frozen gels

[0201] The substantially complete vaginal microbiota preparation of the present invention may be included in a preformed gel. In some embodiments, the preformed gel is provided in a vial, for example for drawing up into a syringe. In some embodiments, the preformed gel is provided with a suitable applicator, for example as shown in FIG. 13. The preformed gel may be stored frozen or refrigerated to maintain the stability of the substantially complete vaginal microbiota preparation. In some embodiments, the preformed gel comprises hyaluronic acid. In some embodiments, the hyaluronic acid is included at a concentration of about 0.3-3%. In some embodiments, the hyaluronic acid is included at a concentration of about 0.5-2%. In some embodiments, the preformed gel comprising hyaluronic acid may be frozen at -80°C. In some embodiments, the frozen preformed gel comprising hyaluronic acid is substantially stable for a period of at least 3 months, 6 months, 9 months, 12 months, 18 months or more.

[0202] Freeze-dried gel

[0203] The substantially complete vaginal microbiota preparation of the present invention may be included in a lyophilized gel. The lyophilized gel may be stable and may be stored for long periods at 2-8°C. The lyophilized gel comprises a lyophilized substantially complete vaginal microbiota preparation and a gel-forming excipient. The lyophilized gel may be provided in a vial. The lyophilized gel may be reconstituted prior to administration (e.g., in a clinical or home environment) with a reconstitution agent. In some embodiments, the reconstitution agent comprises a gel, a gel-forming agent, or a liquid. The liquid may comprise water, saline, or another liquid suitable for reconstitution and subsequent administration to a subject.

[0204] In certain embodiments, the lyophilized gel comprising the substantially complete vaginal microbiota preparation of the present invention further comprises sodium carboxymethylcellulose (Na-CMC). In some embodiments, the Na-CMC is present at a concentration of about 1-3%. In some embodiments, the Na-CMC is present at a concentration of about 2%. In certain embodiments, the lyophilized gel comprises the substantially complete vaginal microbiota preparation of the present invention and hyaluronic acid. In some embodiments, the hyaluronic acid is present at a concentration of about 0.3-3%. In some embodiments, the hyaluronic acid is present at a concentration of about 0.5-2%. In certain embodiments, the lyophilized gel comprises the substantially complete vaginal microbiota preparation of the present invention, as well as hyaluronic acid and sodium carboxymethylcellulose (Na-CMC) at the above concentrations. The lyophilized gel comprising NA-CMP, hyaluronic acid, or a combination of both, is stable at 2-8° C. or −20° C. for at least 3 months, 6 months, 9 months, 12 months, 18 months, or more. In some embodiments, the lyophilized gel comprising hyaluronic acid can be frozen at -80° C. In some embodiments, the lyophilized gel comprising hyaluronic acid is stable for at least 3 months, 6 months, 9 months, 12 months, 18 months or more. In some embodiments, the lyophilized gel and the reconstituting agent are provided as a kit.

[0205] Film-forming formulations

[0206] The substantially complete vaginal microbiota preparation of the present invention may be included in a film-forming formulation. The substantially complete vaginal microbiota preparation of the present invention may be formulated with a polymeric excipient, which has bioadhesive properties and film-forming capabilities. Exemplary polymeric excipients for film-forming formulations include polyvinyl alcohol (PVA), sodium lactate, and lactic acid. In certain embodiments, the film-forming formulation includes polyvinyl alcohol (PVA). In some embodiments, PVA is included at a concentration of about 10-25%, 10-20%, or about 12-15%. In certain embodiments, the film-forming formulation includes PVA at a concentration of about 12%. In some embodiments, the film-forming formulation includes PVA at a concentration of, for example, about 10-25%, 10-20%, 12-15%, or 12%, and is air-dried. In some embodiments, the film-forming formulation is provided as a disk or wafer. In some embodiments, the film-forming formulation is provided as a mucoadhesive pessary or patch. The film-forming formulation is substantially stable for at least 3 months, 6 months, 9 months, 12 months, 18 months or more at 2-8° C. In some embodiments, the film-forming formulation disperses or dissolves rapidly upon contact with fluid, such as endocervical fluids, to form a viscous bioadhesive gel within the vagina. In some embodiments, the formation of a bioadhesive dispersion is retained within the vagina for an extended period of time.

[0207] tablet

[0208] The substantially complete vaginal microbiota preparation of the present invention may be included in a tablet. The tablet may include an agent having gel-forming properties, mucoadhesive properties, or a combination of both. The tablet may further include an excipient, such as a swelling agent, a bulking agent, lactic acid, Carbopol, HPMC, alginate, or sodium carboxymethylcellulose (Na-CMC). In some embodiments, the bulking agent includes microcrystalline cellulose, HPMC / PVP, maltodextran, or Poloxamer 407. In certain embodiments, the tablet includes the substantially complete vaginal microbiota preparation of the present invention and Na-CMC. In certain embodiments, the tablet includes the substantially complete vaginal microbiota preparation of the present invention and polyvinylpyrrolidone (PVP). The tablet may further be substantially stable, e.g., substantially retain viability of Lactobacilli. The tablet may be substantially stable at 2-8°C or room temperature (about 25°C) for a period of at least 3 months, 6 months, 9 months, 12 months, 18 months, or more. In certain embodiments, a tablet comprises a substantially complete vaginal microbiota preparation of the present invention and sodium carboxymethylcellulose (Na-CMC) and is stable for at least 3 months, 6 months, 9 months, 12 months, 18 months or more at 2-8° C. or at room temperature (about 25° C.). In certain embodiments, a tablet comprises a substantially complete vaginal microbiota preparation of the present invention and polyvinylpyrrolidone (PVP) and is stable for at least 3 months, 6 months, 9 months, 12 months, 18 months or more at 2-8° C. or at room temperature (about 25° C.).

[0209] Liquid formulations

[0210] The substantially complete vaginal microbiota preparation of the present invention may be included in a liquid formulation. In some embodiments, the liquid formulation may be provided as a frozen formulation. In some embodiments, the liquid formulation is provided in a kit together with a gelling agent. The liquid formulation may include a cryoprotectant such as glycerol, where the glycerol concentration is optionally less than 25%, less than 20%, less than 15%, or less than 10%. In some embodiments, the liquid formulation may be frozen, for example at -20°C or -80°C, and substantially preserves Lactobacillus viability. In some embodiments, the liquid formulation further includes lactic acid. In a preferred embodiment, the liquid formulation includes a cryoprotectant such as lactic acid and glycerol. In some embodiments, the frozen liquid formulation including a cryoprotectant and optionally lactic acid is substantially stable for a period of at least 3 months, 6 months, 9 months, 12 months, 18 months, or more. In some embodiments, the liquid formulation including the substantially complete vaginal microbiota preparation is provided in a kit together with a gelling agent. In some embodiments, the frozen liquid formulation comprising a substantially complete vaginal microbiota preparation is provided in a kit together with a gelling agent. In some embodiments, the frozen liquid formulation not comprising a substantially complete vaginal microbiota preparation is provided in a kit together with a lyophilized gel, the lyophilized gel comprising a substantially complete vaginal microbiota preparation.

[0211] kit

[0212] Also provided herein is a kit comprising a vaginal delivery system comprising one or more dosage forms or devices described herein and other agents, optionally including instructions for a user. In some embodiments, the dosage form or device and the composition are in separate sterile packages. In some embodiments, the dosage form and / or composition comprises multiple doses. In some embodiments, the dosage form or device is ready to use. In another embodiment, a substantially complete vaginal microbiota preparation or composition thereof needs to be reconstituted. In such an embodiment, the kit further comprises a medium for reconstitution. The kit may comprise a single dosage form or multiple dosage forms.

[0213] Methods of manufacture of preparations and compositions and donor considerations

[0214] Aspects of the invention include methods of producing the substantially complete vaginal microbiota preparations described herein. These methods include selecting a suitable female donor to provide a sample of vaginal fluid, preferably ectovaginal secretions, and processing the sample to provide a composition (such as a pharmaceutical composition) comprising the substantially complete vaginal microbiota preparation described herein. The donor female is generally healthy, does not exhibit vaginal microbiota dysbiosis, and is optionally subjected to one or more additional health tests. The substantially complete vaginal microbiota preparations provided herein include (i) one, two, three, or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species in the preparation; and (ii) less than 5% of the genera Gardnerella, Atopobium, and Prevotella. For example, a method for producing a substantially complete vaginal microbiota preparation includes providing a microbiota sample (e.g., ectocervical secretions) from a healthy female donor and releasing the processed sample from quarantine (e.g., based on meeting one or more predetermined (quality) parameters, e.g., parameters obtained by performing one or more of steps 5(a) to 5(e) below), e.g., as a (pharmaceutical) composition for administration to a female recipient in need of a substantially complete vaginal microbiota preparation.

[0215] Generally, providing and processing a microbiota sample (e.g., ectocervical secretions) from a healthy female donor can include one, two, three, four, five or more steps, and any combination, e.g., any two, three, four, five or more steps, including: (1) adding a diluent (e.g., saline), buffer, or other excipient to the microbiota sample to create a diluted sample; (2) removing a portion of the diluted microbiota sample for testing (e.g., nucleic acid sequencing); (3) freezing or pre-cooling the remaining microbiota sample for freezing; (4) storing the refrigerated or frozen microbiota sample in isolation, and / or (5) refrigerating or freezing. The collected microbiota sample may be held in quarantine until completion of any combination of (a), (b), (c), (d) and / or (e): (a) testing the donor to exclude the presence of infectious pathogens (e.g., blood, vaginal swab, and / or urine sample testing), (b) confirming the composition and viability of the donor sample's microbiota (e.g., lactobacilli), (c) further confirming the health of the female donor with multiple post-screening tests performed within a period of 30-90 days (alternatively, 10-120 days, or 30-60 days) after donation, (d) testing for the presence of sperm cells, and / or (e) testing the pH of the sample (e.g., FIG. 14). All steps (1)-(5) and 5(a)-5(e) are optional and may be performed sequentially or in parallel in any particular order desired.

[0216] In one embodiment, a method for producing a substantially complete vaginal microbiota preparation comprises: A. Providing a microbiota sample from a donor female urogenital tract; step A includes one, two, or three of steps (1), (2), (3), or any combination thereof, and both steps (4) and (5): (1) Adding a diluent to the microbiota sample to prepare a diluted sample; (2) Removing a portion of the diluted microbiota sample for testing (e.g., nucleic acid sequencing); (3) precooling the remaining microbiota samples for refrigeration or freezing; (4) Storage of refrigerated or frozen microbiome samples under quarantine; (5) storing the refrigerated or frozen microbiota sample in isolation until any combination of: (a) testing the donor to exclude the substantial presence of one or more communicable pathogens (e.g., testing blood and / or ectocervical secretions, and / or urine samples), (b) confirming the composition and viability of the microbiota, or (c) further confirming the health status of the female donor by multiple post-screening tests performed within 30 to 90 days after donation; and B. Releasing a refrigerated or frozen microbiota sample from isolation to define a substantially complete vaginal microbiota preparation.

[0217] Step (1) may include, for example, adding an acidifying agent (e.g., to adjust the pH of the sample); adjusting the viscosity of the sample (e.g., to aid in administration as described herein); adjusting tonicity / osmolality; and / or adding one or more other active agents such as those described herein, including spermicides, antibacterial agents, hormones, anti-inflammatory agents, and optionally prebiotics. In one embodiment, the acidifying agent is lactic acid.

[0218] For step (2), the microbiota sample (e.g., vaginal fluid / secretion sample) is preferably at least 75 mg or 100 mg, more preferably at least 150 mg, and a portion is removed for nucleic acid sequencing. The microbiota sample can be 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg. In further embodiments, the microbiota sample (e.g., vaginal fluid / secretion sample) is at least 200 mg, 300 mg, 400 mg, 500 mg, 500 mg, 700 mg, or more. In one embodiment, the microbiota sample (e.g., vaginal fluid / secretion sample) is at least 500 mg.

[0219] To evaluate the microbial community of donor microbiota sample and select suitable donor female, sequencing is carried out.Preferably, the presence of one, two, three, four or five different bacterial species from the genus Lactobacillus is detected in donor microbiota sample by nucleic acid sequencing.Most preferably, the presence of one (dominant) bacterial species from the genus Lactobacillus is detected in donor microbiota sample by nucleic acid sequencing. In some embodiments, nucleic acid sequencing determines that the donor microbiota sample contains 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.9%, 80-99.9%, 75%-95%, 85%-95%, 85%-99.9%, or 90%-99.9% lactobacilli of one species out of the total of all detectable species in the preparation. In some embodiments, nucleic acid sequencing determines that the donor microbiota sample contains 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99.9%, about 99.5%, about 99.9%, 80-99%, 75%-95%, 85%-95%, 85%-99.9%, or 90%-99.9% lactobacilli of more than one species (e.g., two, three, four or five species) out of the total of all detectable species in the preparation. Preferably, nucleic acid sequencing determines that the donor microbiota sample contains Lactobacillus species selected from a) Lactobacillus crispatus; (b) Lactobacillus iners; (c) Lactobacillus jensenii; (d) Lactobacillus gasseri, or any combination thereof (including combinations of two, three, or all four species).

[0220] Optionally, nucleic acid sequencing is performed to identify any pathogens or pathogenic symbionts in the donor sample, e.g., to determine that the donor sample is substantially free of pathogens and pathogenic symbionts. For example, nucleic acid sequencing is performed to detect the presence of one or more of the genera Gardnerella, Atopobium, and / or Prevotella. In some embodiments, nucleic acid sequencing is performed to detect the presence of one or more of the genera Gardnerella, Atopobium, Prevotella, and / or Fannyhessa vaginae. In some embodiments, nucleic acid sequencing is performed to detect the presence of one or more of the genera Gardnerella vaginalis, Bacteroides, Mobiluncus, Sneathia, and Mycoplasma hominis. In some embodiments, nucleic acid sequencing is performed to detect the presence of one or more of Escherichia, Enterococcus, Pseudomonas, Proteus, Klebsiella, Streptococcus, Staphylococcus, Gardnerella, Ureaplasma, Bacteroides, Peptococcus, Neisseria, Serratia, Corynebacterium, Clostridium, and Candida. The presence of any one or more of these species indicates replacement of healthy vaginal lactic acid-producing bacteria with undesirable species and signals the presence of vaginal dysbiosis. Donor samples containing more than about 1%, 3%, 5%, 8%, 10% or more than about 15% of species belonging to one or more undesirable species are not used for further processing to generate a substantially complete vaginal microbiota preparation as described herein. In one embodiment, donor samples containing greater than about 5% species belonging to one or more undesirable species (e.g., Gardnerella, Atopobium, and / or Prevotella) are not used for further processing to generate a substantially complete vaginal microbiota preparation as described herein.

[0221] Optionally, nucleic acid sequencing is performed to identify the presence of any antimicrobial resistance (AMR) genes in the donor sample, for example, to determine that the donor sample is substantially free of antimicrobial resistance (AMR) genes. Antimicrobial resistance (AMR) genes include genes that confer resistance to one or more antibiotics (e.g., as described and cataloged in the NCBI National Database of Antibiotic Resistant Organisms (NDARO)), including, for example, aminoglycosides, beta-lactams, tetracyclines, and sulfonamides. It will be understood that due to the widespread use of antibiotics, the cutoff is not zero. Some reasonable allowance is made for the presence of AMR genes. The exact cutoff can be determined by one of skill in the art, for example, based on the nature of the AMR genes (e.g., the level of health concern) and public health recommendations.

[0222] Alternatively, or additionally, tests are performed to demonstrate that the donor sample is substantially free of one or more of gram-negative toxins (e.g., endotoxins or lipopolysaccharides (LPS) and other secreted exotoxins and enterotoxins), virulence factors / bacterial virulence factors and / or colonization factors (e.g., motility, adherence, invasiveness, etc.).

[0223] Optionally, a test is performed (e.g., by microscopy) to identify the presence of any human sperm (spermatozoa) in the donor sample, for example to determine that the donor sample is substantially free of human sperm (spermatozoa).

[0224] Step (3) may include pre-cooling for subsequent refrigeration (e.g., 4° C.) or freezing (e.g., −18° C. or −80° C.) depending on the desired period for storage, isolation and use for administration. This step may also include, for example, freeze-drying (lyophilizing) for easy storage, packaging, formulation and transportation. Furthermore, this step may optionally include viability testing, e.g., upon refrigeration, freezing or lyophilization, e.g., viability of lactobacilli contained in the substantially complete vaginal microbiota preparation.

[0225] Steps (4) and (5) include storing the refrigerated, frozen or lyophilized microbiota sample in quarantine and holding the stored microbiota sample in quarantine until certain tests performed on the donor microbiota sample and / or the sample donor are completed. Quarantine is released and the sample is released for use as a substantially complete vaginal microbiota preparation or composition (e.g., a pharmaceutical composition), e.g., for administration to a recipient female, when the sample and / or donor pass one or more predetermined tests (sample quality and / or female donor health tests). These include one or more of the following: (a) testing the donor to rule out the presence of infectious pathogens (e.g., blood, vaginal swabs, and / or urine sample testing); (b) confirming the microbiota composition and viability of the donor sample (e.g., lactobacilli), and / or (c) further confirming the health of the female donor with multiple post-screening tests performed within a period of 30 to 90 days (alternatively, 10 to 120 days, or 30 to 60 days) after donation.

[0226] Testing in step 5 to exclude the presence of transmissible (and potentially infectious) pathogens (e.g., blood, vaginal swab, and / or urine sample testing) may include determining that the female donor is substantially free of any one or more (two or more, three or more, or four or more) of: (i) bacteria involved in bacterial vaginosis (e.g., Gardnerella and Mobiluncus); (ii) yeast (e.g., Candida, Cryptococcus, and Saccharomyces species); (iii) sexually transmitted pathogens (including Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis); (iv) bacteria involved in urinary tract infections (e.g., E. coli, Staphylococcus, Chlamydia, and Mycoplasma); and (v) viruses (e.g., HIV, human papilloma virus (HPV), Hepatitis B virus, Hepatitis C virus, HSV-2).

[0227] Step 5 testing may include determining that the female donor is substantially free of any one or more sexually transmitted infections or diseases (STIs, STDs), including chlamydia infection, chancroid, hair lice (pubic lice), genital herpes, genital warts, Hepatitis B, human immunodeficiency virus / acquired immune deficiency syndrome, human papilloma virus, trichomoniasis, molluscum contagiosum, pelvic inflammatory disease, syphilis, gonorrhea, and yeast infection.

[0228] The testing in step 5 may include determining that the female donor does not exhibit dysbiosis in the vaginal canal, for example, by one or more established tests for bacterial vaginosis (BV) and bacterial infection. Such tests include the Amsel criteria, the Nugent Gram stain scoring system, and the Hay-Ison criteria. Alternatively, other methods may be used to determine the absence of dysbiosis, for example, using the BV Blue test or the Affirm Microbial Identification Test.

[0229] The Amsel criteria include the presence of three of the following four symptoms: (a) a thin, homogeneous, foul-smelling discharge; (b) a vaginal pH of >4.5; (c) an amine odor from the vaginal fluid upon addition of 10% KOH; and (d) the presence of "clue" cells (vaginal epithelial cells with adherent bacteria that obscure the cell margins) (Amsel et al., Am. J. Med. 74:14-22 (1983)).

[0230] The Nugent Gram stain scoring system involves evaluating normally prepared Gram stains for the relative abundance of three morphotypes of bacteria and then calculating a so-called Nugent score based on the amount of large Gram-positive rods (lactobacilli morphotypes; lactobacilli reduction scored as 0-4), small Gram-negative and variable rods (Bacteroides and Gardnerella morphotypes; scored as 0-4), and curved Gram-variable rods (Mobiluncus genus morphotypes; scored as 0-2). Nugent scores can range from 0-10, with scores of 0-3 considered normal (non-BV), 4-6 intermediate, and 7-10 positive for BV.

[0231] The Hay-Ison criteria (or Ison-Hay scoring system) describes five grades of flora: a) grade 0, epithelial cells without bacteria; b) grade I, normal vaginal flora (only lactobacillus morphotypes); c) grade II, reduced numbers of lactobacillus morphotypes with mixed bacterial flora; d) grade III, only mixed bacterial flora, with few or no lactobacillus morphotypes; e) grade IV, only gram-positive cocci.

[0232] Grades 0, I, and IV are seen in women without BV. Grade II is intermediate and is not seen in women with BV as defined by the Amsel criteria. Grade III is consistent with BV diagnosed by the Amsel criteria. Grade III flora is indicative of BV (CAIson and PE Hay, Sex Transm Infect. 2002 Dec;78(6):413-5).

[0233] The BV BLUE test (Gryphus diagnostics) detects sialidase activity, an enzyme produced by BV-associated bacteria such as Gardnerella vaginalis, Bacteroides spp., Prevotella spp., and Mobiluncus spp. A vaginal fluid sample is placed in a test vessel containing a chromogenic substrate for sialidase. After incubation, a developer is added and if the sample contains high levels of sialidase, a blue or green color is seen. Samples without sialidase or with low levels of this enzyme produce a yellow color in the reaction.

[0234] The AFFIRM Microbial Identification Test (Beckton Dickinson) is a DNA probe-based diagnostic test for the differential detection and identification of three vaginitis-associated organisms: Candida spp., G. vaginalis, and T. vaginalis.

[0235] In some embodiments, the donor female is selected from generally healthy premenopausal women aged 18 years or older with regular and predictable menstrual cycles. In some embodiments, the donor female is selected from generally healthy postmenopausal women. In some embodiments, the donor female is selected from both premenopausal and postmenopausal women. The donor may require oral contraceptives, hormonal contraceptives, hormonal intrauterine devices, or may not require contraceptives. The donor is substantially free of vaginal symptoms such as odor, discharge, or itching. Optionally, donors will refrain from using or engaging in one or more of the following during the sample donation period, for example from initial donor screening to final donation: a) using inserted vaginal female products, e.g., tampons, menstrual cups, sex toys, although sanitary napkins are permitted; b) using other vaginal products, e.g., cleansing products, spermicides, lubricants, sanitary powders and sprays; c) having vaginal and anal intercourse; d) bathing, swimming, sitting in a hot tub, and / or e) wearing thong underwear (or all of a-e).

[0236] Testing above a set of predefined thresholds for pathogen and / or pathogenic commensal loads (e.g., viral, fungal, and bacterial), where individual maximum thresholds can be set, e.g., above zero, e.g., substantially free of): (a) a health history of one or more of the following: bacterial vaginosis, recurrent yeast infection, trichomoniasis, syphilis, human papillomavirus (HPV) including genital warts, high-grade Pap smear, herpes, pelvic inflammatory disease, recurrent urinary tract infection, and mycoplasma, or any combination thereof; (b) a history of one or more of the following: HIV, Hepatitis A / B / C, syphilis, human T-lymphotropic virus (HTLV)-I / II, WNV, Epstein-Barr virus (EBV), rubella, toxoplasma gondii, herpes simplex virus (HSV)-1 / 2, chlamydia infection, gonorrhea, mycoplasma genitalium, Trichomonas Donors may be excluded if they exhibit one or more of the following: (a) positive tests for Lactobacillus vaginalis, HPV, and other yeasts or bacteria considered pathogenic / abnormal and / or exhibiting antibiotic resistance, or any combination thereof; (c) vaginal fluid / ectovaginal secretions that are not dominated by one of the common vaginal Lactobacillus species, as determined, for example, by qPCR; and / or (d) a history of gonorrhea or chlamydial infection (e.g., within 12 months prior to screening), or any combination of (a), (b), (c), and (d).

[0237] Additionally, donors may be excluded if they exhibit one or more of the following: hysterectomy, insertion or removal of an intrauterine device, cervical cryotherapy, or cervical laser therapy (e.g., within 2 months prior to screening), any condition requiring regular use of systemic antibiotics, use of long-acting hormone therapy, social, medical, or psychiatric conditions, such as a history of drug or alcohol abuse, menopause (e.g., defined as amenorrhea lasting more than 12 months with no known alternative cause), irregular menstrual cycles, use of other medications, or any combination thereof. Preferably, donors are not currently pregnant or breastfeeding.

[0238] If desired, donors exhibiting one or more of cytomegalovirus (CMV), rubella and varicella zoster virus (VZV) IgG, or any combination thereof, can be matched only with CMV positive and / or rubella and / or VZV positive recipients or excluded.

[0239] Method of administration

[0240] Aspects of the present invention include methods for vaginal administration of a composition comprising a substantially complete vaginal microbiota preparation as described herein to a human female subject. The methods may include using a device for administration, for example, these methods are typically performed by a health care provider (e.g., in a clinic). For example, a composition comprising a substantially complete vaginal microbiota preparation is provided frozen, for example, in a cryovial, then thawed, preheated to 37° C., and then dispensed by a health care provider into a syringe / applicator, which is then administered to the recipient. Alternatively, the methods include using alternative dosage forms as described herein, such as, for example, suppositories, tablets, capsules, films, creams, etc. The methods can be performed by the recipient themselves (e.g., at home), if desired, for example, by self-administration. The methods can also include other health care-related activities, such as diagnosing health problems and providing standard care, in addition to providing a substantially complete vaginal microbiota preparation or composition as described herein. The activities can include one or more combination therapies as provided herein. For example, the methods of administration described herein may further include administering an antibacterial agent, an antifungal agent, an antibacterial agent, an antiviral agent, an antibiotic, an antiparasitic agent (e.g., with activity against Trichomonas vaginalis), an anti-inflammatory agent, and the like.

[0241] In embodiments where administration is performed using a device, the device typically includes an open end (e.g., a tip) for insertion into the vaginal cavity and a dispensing end (e.g., a plunger or piston) for expelling the composition through the open end. The administration step includes a) introducing the open end into the vaginal cavity, b) expelling the composition into the vaginal cavity, and c) removing the device from the vaginal cavity (after administering the desired dose). Administration is preferably performed with the recipient in a lithotomy position, e.g., with the female recipient in a lithotomy position. In some embodiments, the recipient will remain in the lithotomy position for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes before returning to an upright position to allow sufficient residence time of the substantially complete vaginal microbiota preparation or composition in the vaginal cavity, e.g., sufficient contact time with the vaginal mucosal or endometrial surfaces. In some embodiments, administration is targeted to high areas in the vaginal cavity, e.g., near the vaginal fornix.

[0242] If necessary, the recipient female's menstrual cycle is taken into consideration when determining the timing of administration. For example, treatment can be avoided during menstruation. In some embodiments, times other than during menstruation are preferred for carrying out administration, including, for example, during a time window including preovulation and premenstruation.

[0243] In some embodiments, the exact steps, timing and length of the procedure will vary between recipients (e.g., as determined by a health care provider) to provide optimal conditions for the bacteria (e.g., lactobacilli) contained in the substantially complete vaginal microbiota preparation to colonize and establish (engraft) the recipient woman's vagina.

[0244] Treatment and Recipient Considerations

[0245] Suitable female recipients include women exhibiting dysbiosis of the vaginal microbiota and women in need of treatment for dysbiosis, e.g. dysbiosis associated with infection (e.g. pathogens) and / or (chronic) inflammation.

[0246] An aspect of the present invention relates to a method for restoring a healthy human vaginal microbiota balance in the female urogenital tract. The method comprises administering to a female subject in need of restoring a balance of the vaginal microbiota an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein, for the purpose of providing a community of microbial species that can colonize or inhabit the human vagina or vaginal epithelium, impede the growth of pathogenic microorganisms, and provide a microbial niche that is not pro-inflammatory (e.g., anti-inflammatory). The method comprises administering to a female subject in need of a balance of the vaginal microbiota an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein, for the purpose of maintaining a healthy vaginal microbiota or a balance of a healthy vaginal microbiota.

[0247] In one aspect, the invention provides a pharmaceutical composition for use in treating inflammation of the female urogenital tract in a human subject, wherein the female subject exhibits a dysbiotic microbiota of the urogenital tract, the method comprising administering to the subject an effective amount of the pharmaceutical composition, the pharmaceutical composition comprising a substantially complete vaginal microbiota preparation,

[0248] (i) The preparation comprises (i) containing one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, constituting >80% to >99.9% of all detectable bacterial species in the preparation; and (ii) containing <5% of the genera Gardnerella, Atopobium, and Prevotella; the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or diluent.

[0249] Inflammation and inflammation-related disorders, conditions, or diseases are used interchangeably and include acute and chronic inflammation. Dysbiotic vaginal microbiota can be associated with inflammation.

[0250] The subject may be an asymptomatic subject, characterized as not having the Amsel criteria, Nugent score, or other measures of symptoms associated with bacterial vaginosis (BV). In one embodiment, the recipient is an asymptomatic dysbiotic woman.

[0251] Vaginal douching

[0252] To reduce the amount of pathogens in the vaginal cavity of a dysbiosis recipient, in some embodiments, the vaginal cavity is rinsed, e.g., a vaginal wash is performed, prior to administration of the pharmaceutical composition.

[0253] Vaginal doubling may be performed using an absorbent material pre-soaked in a doubling solution. In one embodiment, the absorbent material is gauze. In some embodiments, a tampon-shaped gauze can be used. Doubling is typically performed by a medical professional, such as a gynecologist, with the recipient in the lithotomy position and the pre-soaked absorbent material used with pliers to cleanse the surface of the vaginal epithelium. After doubling, excess doubling solution can be absorbed by a dry absorbent material, for example, a dry tampon-shaped gauze.

[0254] Vaginal douching can be performed with any solution suitable for reducing the amount of pathogens in the vaginal cavity, including, but not limited to, saline, antiseptic, or lactic acid. In some embodiments, vaginal douching includes saline douching, lactic acid douching, or antiseptic douching. Suitable antiseptic solutions include chlorhexidine and povidone-iodine. In certain embodiments, vaginal douching is performed with saline. In another particular embodiment, vaginal douching is performed with lactic acid, optionally with a pH of about 3.5 to 4.5. In certain embodiments, vaginal douching is performed with lactic acid, optionally with a pH of about 3.8 to 4.3 or 3.8 to about 4.0. In further particular embodiments, vaginal douching is performed with an antiseptic solution, optionally with chlorhexidine or povidone-iodine. In certain embodiments, vaginal douching is performed with povidone-iodine, and the solution comprises about 10% povidone-iodine. In certain embodiments, the vaginal cleansing is performed with chlorhexidine, the solution comprising about 0.5% chlorhexidine.

[0255] Homeostasis of the vaginal microbiota

[0256] The balance of healthy human vaginal microbiota includes the establishment (e.g., including engraftment) of various selected microbial species (including one or more Lactobacillus species) in which the relative number of each species or the total number of vaginal Lactobacilli (e.g., Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri) is in homeostasis when measured after administration of a composition comprising a substantially complete vaginal microbiota preparation as described herein.Homeostasis is generally a state in which the relative abundance of each species in a population does not change substantially over a given period of time, for example, at least 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 30 days, 60 days, 90 days, 6 months, or at least 12 months, or for example, over several (1, 2, 3, 4, 5, 6) menstrual cycles in women of reproductive age. Homeostasis may be measured, for example, by metagenomic sequencing, 16S sequencing, and / or qPCR, such as MLST and MLVA. If desired, CFU counts may be performed, for example, to measure the persistence of healthy bacterial colony forming units.

[0257] The balance of a healthy human vaginal microbiota in a homeostatic state (preferably rich in lactobacilli, e.g. at least 50%, 60%, 70% or at least 80% lactobacilli, e.g. selected from one or more of L. crispatus, L. iners, L. gasseri and L. jensenii) is stable for a certain period of time, e.g. conferring resistance to disturbances caused by vaginal pathogens (non-pathogenic) and / or providing an anti-inflammatory environment. However, a healthy vaginal microbiota does not require the complete absence of all pathogens. Many healthy women have low levels of yeasts and / or pathogenic commensals present in their microbiota.

[0258] In some embodiments, the method for restoring a balance of healthy human vaginal microbiota in a female urogenital tract comprises providing a non-pro-inflammatory or anti-inflammatory environment in the urogenital tract. In some embodiments, a composition comprising a substantially complete vaginal microbiota preparation described herein, when administered (e.g., for treatment), provides anti-inflammatory activity to the urogenital tract. In some embodiments, a composition comprising a substantially complete vaginal microbiota preparation described herein, when administered (e.g., for treatment), does not cause local or systemic inflammation or immune response in a subject or recipient. In some embodiments, a composition comprising a substantially complete vaginal microbiota preparation described herein, when administered (e.g., for treatment), promotes local or systemic anti-inflammatory or immune response in a subject or recipient. In some embodiments, a composition comprising a substantially complete vaginal microbiota preparation described herein, when administered (e.g., for treatment), provides for the maintenance of a balance of anti-inflammatory and pro-inflammatory mediators in vaginal epithelial cells of the urogenital tract.

[0259] Inflammatory markers

[0260] Pro-inflammatory cytokines that may be modulated by the methods described herein include, for example, IL-1β, IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17, IL-23, and TNF-α. In one embodiment, administration of a substantially complete vaginal microbiota preparation results in a reduction or stabilization of levels of inflammatory markers, including one or more of IL-1a, IFN-γ, IL-18, IL-12, and MMP-10. In some embodiments, anti-inflammatory markers are increased following administration of a preparation of the invention.

[0261] Anti-inflammatory and pro-inflammatory mediators upon administration and engraftment of lactobacilli contained in the substantially complete vaginal microbiota preparations described herein can be measured using appropriate commercially available biomarker panels by collecting vaginal samples (e.g., vaginal fluid / secretion) or systemic samples (e.g., blood).

[0262] Provided herein is a method for treating (chronic) inflammation of the female urogenital tract. The method comprises administering to a female subject in need of treatment an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein. Subjects treated by the methods described herein include women who exhibit inflammation (possibly chronic inflammation) that may result in a number of adverse health outcomes, such as urinary tract infections (UTIs).

[0263] Dysbiosis

[0264] Provided herein is a method for treating dysbiosis of the female urogenital tract. The method comprises administering to a female subject in need of treatment an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein. Subjects treated by the methods described herein include women who exhibit an imbalance in the vaginal microbiota that can overgrow pathogenic microorganisms and pathogenic commensals, resulting in dysbiosis, inflammation and / or infection. The method includes treating vaginal infections (vaginitis), such as bacterial vaginosis, vaginal candidiasis and trichomoniasis, and combinations thereof, with a composition comprising a substantially complete vaginal microbiota preparation as described herein.

[0265] Vaginal dysbiosis is a common condition that affects approximately 30-40% of female adult subjects. In some embodiments, the dysbiotic female subject does not exhibit any symptoms (asymptomatic). In other embodiments, the dysbiotic female subject suffers from vaginal symptoms characterized by the Amsel criteria, the Nugent score criteria, or the Hay-Ison criteria.

[0266] Bacterial vaginosis (BV) is the most common vaginal infection and is associated with complications during pregnancy and an increased risk of sexually transmitted diseases. It is caused by an imbalance of the naturally occurring bacterial flora, resulting in overgrowth of lactobacilli by a mixed flora of anaerobic bacteria. A pH above 4.5, among other diagnostic criteria, is considered to be indicative of bacterial vaginosis. BV can be diagnosed using the Amsel criteria, Nugent score, Hay-Ison criteria, or another appropriate diagnostic method. Current treatment options rely on oral or vaginal administration of classical antibiotics such as metronidazole and clindamycin, but there is a high recurrence rate of over 50% within 3 months after the initial exposure. Alternative treatment options include vaginal acidification with naturally occurring acids such as lactic acid or acetic acid.

[0267] In one embodiment, a female subject exhibiting one or more diagnostic symptoms of BV is treated with an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein. In one embodiment, the treatment comprising administration of an effective amount of a composition comprising a substantially complete vaginal microbiota preparation further comprises a combination therapy (as described herein) comprising providing a standard of care treatment for BV, such as administration of antibiotics, e.g., metronidazole and clindamycin, and / or vaginal acidification with an acidifying agent, e.g., lactic acid or acetic acid, either prior to, concurrently with, or after administration of the composition comprising a substantially complete vaginal microbiota preparation to the female subject.

[0268] Vaginal candidiasis is a fungal infection of any of the Candida species (yeasts), of which Candida albicans is the most common. Most Candida infections are treatable with minimal complications such as redness or itching. However, complications can also be severe or even fatal if left untreated in certain populations, such as immunocompromised patients. External use of detergents or internal disturbances (hormonal or physiological) can disrupt the normal vaginal flora, resulting in an overgrowth of Candida cells that cause infection. Pregnancy and use of oral contraceptives have also been reported as risk factors. In clinical practice, candidiasis is commonly treated with antifungal agents such as clotrimazole, nystatin, fluconazole, and ketoconazole. However, certain yeasts, such as C. albicans, can develop resistance to said antifungal agents.

[0269] In one embodiment, a female subject exhibiting one or more diagnostic symptoms of candidiasis is treated with an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein. In one embodiment, the treatment comprising administration of an effective amount of a composition comprising a substantially complete vaginal microbiota preparation further comprises a combination therapy (as described herein), e.g., providing a standard of care for candidiasis, such as administering an antifungal agent, e.g., clotrimazole, nystatin, fluconazole and / or ketoconazole, either prior to, simultaneously with, or after administration of the composition comprising a substantially complete vaginal microbiota preparation to the female subject.

[0270] Trichomoniasis is a sexually transmitted infection of the urogenital tract, caused by the parasite Trichomonas vaginalis. Symptoms include inflammation of the cervix, urethra, and vagina, resulting in itching and burning. Treatment options include the use of antibiotics / protozoal drugs (e.g., metronidazole) or antiparasitic drugs (e.g., tinidazole). However, as with most antibacterial drugs, resistance can develop.

[0271] In one embodiment, a female subject exhibiting one or more diagnostic symptoms of trichomoniasis is treated with an effective amount of a composition comprising a substantially complete vaginal microbiota preparation as described herein. In one embodiment, the treatment comprising administration of an effective amount of a composition comprising a substantially complete vaginal microbiota preparation further comprises a combination therapy (as described herein) comprising providing a standard of care for trichomoniasis, e.g., administering an antifungal agent, such as an antibiotic / protozoal agent (e.g., metronidazole) or an antiparasitic agent (e.g., tinidazole), either prior to, concurrently with, or following administration of a composition comprising a substantially complete vaginal microbiota preparation to the female subject.

[0272] definition

[0273] The following definitions apply to all embodiments disclosed herein: For example, the term "substantially complete vaginal microbiota preparation" shall have the same meaning when used in the context of the first embodiment relating to the vaginal delivery system, or when used in the context of the second embodiment relating to the pharmaceutical composition, or when used in the context of any other embodiment disclosed herein.

[0274] General items

[0275] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In addition, it is understood that methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features of the present disclosure will be apparent from the following detailed description and claims.

[0276] The singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprises" means "includes." The abbreviation "eg" comes from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "eg" is synonymous with the term "for example." The term "optional" or "optionally" means that the subsequently described event, circumstance, or substitution may or may not occur, and that the description includes instances when the event or circumstance occurs as well as instances when it does not occur.

[0277] Unless otherwise indicated, all numbers expressing amounts of ingredients, times, and the like used in the specification or claims should be understood to be modified by the term "about." Accordingly, unless otherwise implicitly or explicitly indicated, or unless otherwise understood by one of ordinary skill in the art that the context has a more determinative configuration, the numerical parameters recited are approximations that may depend upon the desired properties and / or limits of detection sought under standard testing conditions / methods known to those of ordinary skill in the art.

[0278] In a preferred embodiment, the term "about" is intended to permit deviations from the indicated numerical value of ±10%, and even more preferably ±5%.

[0279] The term "comprising" should be understood to simultaneously disclose the term "consisting" as a preferred option. For example, if a composition is said to comprise three components, this also discloses a composition consisting of these three components as a preferred embodiment.

[0280] When the term "comprising" is used to refer to (a) pharma- ceutical active compound(s), bacterium(s), etc., it should be understood that this also simultaneously discloses that the pharma- ceutical active compound(s), bacterium(s), etc. is preferably the only pharma- ceutical active compound(s), bacterium(s), etc. For example, when a donor sample is said to contain Lactobacillus crispatus and Lactobacillus iners, this simultaneously preferably discloses that the donor sample contains Lactobacillus crispatus and Lactobacillus iners as the only bacteria. When a donor sample is said to contain Lactobacillus crispatus, Lactobacillus iners, and an excipient, this simultaneously preferably discloses that the donor sample contains Lactobacillus crispatus and Lactobacillus iners as the only bacteria, plus an excipient. It is further disclosed that the donor sample consists of Lactobacillus crispatus, Lactobacillus iners, and excipients.

[0281] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that may be used in connection with the present invention.

[0282] As used herein, the "urogenital tract" or "urogenital tract" includes the uterus, fallopian tubes, ovaries, vagina, cervix, vulva, and urinary tract. In some instances, used herein are teachings and examples that specifically call out the "vaginal canal," "vaginal cavity," or "vagina." Those skilled in the art will understand that these examples and teachings are exemplary only and are non-limiting, and thus apply, where appropriate, to other anatomical portions of the urogenital tract or urogenital tract in addition to the vagina.

[0283] "Dysbiosis" as used herein refers to a microbial imbalance in which the amount of normally dominant species is reduced and less dominant species become more abundant and / or predominant. Vaginal dysbiosis is a microbial imbalance in the vagina that is an abnormality of a healthy state. Dysbiosis is generally associated with one or more of (a) a qualitative and quantitative change in the content or amount of the microbiota itself, (b) a change in its metabolic activity, and / or (c) a change in its local distribution. A dysbiotic human vaginal microbiota balance refers to a population of vaginal microorganisms that contribute to or establish an environment that promotes inflammation of the tissues of the vagina and / or allows or promotes the colonization or proliferation of one or more pathogenic microorganisms. Dysbiosis also refers to a disturbance of vaginal homeostasis. In some embodiments, a dysbiotic vaginal microbiota generally results in an elevated pH, e.g., a pH greater than 4.5, e.g., a pH of 5.0, 5.5, 6.0, 6.5, 7.0 or higher, compared to a healthy microbiota. In some embodiments, vaginal dysbiosis is characterized by a reduction in Lactobacillus species and an increase in vaginal anaerobic bacterial diversity. Vaginal dysbiosis is associated with an increased risk of upper genital tract infections or pelvic inflammatory disease (PDI) and sexually transmitted diseases. Dysbiosis can be characterized by the relative abundance of selected pathogens, e.g., >20% of selected pathogens, and low relative abundance of vaginal lactobacilli, e.g., <10% of vaginal lactobacilli (L. crispatus, L. iners, L. jensenii, L. gasseri).

[0284] As used herein, dysbiotic subjects include subjects who have vaginal symptoms (symptomatic subjects) and subjects who do not have any vaginal symptoms (asymptomatic subjects), where symptoms are characterized by the Amsel criteria, Nugent score and / or Hay / Ison score.

[0285] In contrast, the terms "normal" or "healthy" "vaginal flora" or "vaginal niche" "microbiota" or "condition" or similar terms refer to a condition in which a woman has no vaginal complaints and does not exhibit vaginal pathology (e.g., no signs or symptoms corresponding to or resulting from a vaginal lesion), the condition of the vagina is such that it is relatively less susceptible to sexually transmitted diseases and pathogens, generally has a low pH, e.g., pH 4.5 or less, e.g., 3.2-4.5; and is generally dominated by lactic acid-producing bacteria (e.g., Lactobacillus species). Normal vaginal microbiota or normal flora is a community of microorganisms that are localized in the vagina in a normal, healthy state, i.e., in a non-pathological, non-pathogenic and / or non-dysbiotic state.

[0286] "Cervical vaginal secretions" ("CVS") or "vaginal fluid" refers to the mixture of mucus secreted by a woman's cervix, excreted epithelial cells, vaginal discharge, and bacteria found in the vagina.

[0287] "Isolated bacterial strain" refers to a strain that has been separated from other strains (e.g., from the vaginal bacterial community, e.g., from a sample of ectocervical secretions or vaginal fluid) and grown in vitro in a culture containing said strain. An isolated bacterial strain is substantially free of contaminants or components that accompany the material from which it is derived in its natural state (e.g., vaginal mucus and epithelial cells, etc.).

[0288] By "culture-independent method" is meant a method that does not involve the isolation and / or in vitro growth of, for example, a bacterial strain in culture.

[0289] The term "microbe" is used synonymously with the term "microorganism" and includes bacteria (archaea, eubacteria), yeast, fungi, and viruses. The term "species" is used herein to refer to a taxonomically and / or genetically distinct group of microorganisms. A species may include one or more distinguishable (e.g., by sequencing) strains.

[0290] The term "microbiota" refers to a community of microorganisms localized in a distinct shared environment ("microbial niche"). For example, the "vaginal microbiota" is a community of one or more species of microorganisms localized or found in the vagina. The terms "microflora" or "flora" are used interchangeably with the term "microbiota". A healthy or normal microbiota refers to a community of commensal microorganisms that colonize (inhabit) a specific microbial niche of the host, such as the vagina. Bacteria are the most numerous microbial component of the normal flora.

[0291] As used herein, "mucosa" refers to the mucosa membrane. Mucus is a secretion produced by and covering the mucosa. Mucus is viscous and is typically produced by mucosal cells (e.g., goblet cells) found in the mucosa and submucosal glands, and is rich in antiseptic enzymes (such as lysozyme), immunoglobulins, inorganic salts, proteins such as lactoferrin, and glycoproteins (mucins). Mucosal surfaces include the epithelial lining of the reproductive tract (vagina), for example, lactobacilli can colonize the vaginal mucosal surface.

[0292] As used herein, the term "effective amount" refers to an amount (e.g., of a composition or preparation) to be administered to a typical subject (e.g., a female recipient) that is sufficient to produce a desired beneficial or therapeutic effect in the subject. The desired beneficial or therapeutic effect includes, for example, prevention and / or treatment of dysbiosis, inflammation or infection or the urogenital tract, and also includes restoration and / or rebalancing of the vaginal microflora (e.g., to achieve homeostasis), e.g., an anti-inflammatory and / or anti-pathogenic state of the urogenital tract and vaginal microflora. An effective amount is, for example, an amount sufficient to alleviate at least one or more symptoms, delay the onset of a symptom, alter the course of a symptom (e.g., slow the progression of a symptom), or reverse a symptom (e.g., at the dosage and duration required). An effective amount generally causes a statistically significant measurable change.

[0293] The effective amount in the delivery system varies depending on the specific drug, the intended use, the expected release rate and the expected time for the system to provide treatment. A variety of devices with different sizes can be formulated to administer the dosage. Those skilled in the art can easily determine the effective amount of active agent required for each specific application and delivery system. The effective amount can be determined, for example, in clinical trials and animal tests. The term "effective amount" is used interchangeably with the term "therapeutically effective amount".

[0294] A "lyophilized" or "freeze-dried" composition refers to a composition from which water has been removed, e.g., for easy storage and transportation. Such compositions can be rehydrated prior to use (e.g., administration).

[0295] As used herein, the term "viable" refers to cells (e.g., bacterial cells) that can survive in a given condition (e.g., storage for a period of time under specific storage conditions, including temperature, humidity), and generally can colonize and reproduce (e.g., in the urogenital tract) after exposure to the condition. Percent viability refers to the proportion of viable cells in a population. For example, percent viability can refer to the proportion of lactobacilli in a pharmaceutical composition that survive (e.g., refrigerated, frozen, and / or stored) and colonize upon application to the vaginal mucosal surface.

[0296] The abbreviation "cfu" means "colony forming unit."

[0297] The abbreviation "VCC" means viable cell count (determined by viability staining).

[0298] As used herein, the phrases "excipient", "pharmaceutical acceptable carrier", "diluent" or "buffer" refer to an inactive pharmaceutical acceptable material, ingredient, composition or vehicle that is added to form part of the final formulation and / or maintains a drug or other agent in a form for delivery to a subject. Each carrier is preferably compatible with the other components of the formulation, e.g., the carrier does not reduce the effect of the active ingredient or agent on the treatment, e.g., the carrier is pharmaceutical inactive. In some embodiments, the pharmaceutical acceptable carrier can be a carrier other than water (e.g., including a cream, emulsion, gel, liposome, nanoparticle, film, ointment and / or vaginal device). In some embodiments, pharmaceutical compositions are provided that include a substantially complete vaginal microbiota preparation together with a pharmaceutical acceptable carrier and / or diluent (e.g., saline). These compositions allow for easy administration of the preparation by means known to those skilled in the art. In some embodiments, a buffering agent, e.g., a weak acid or base, is added that maintains acidity at a selected level (e.g., between pH 3.5 and 4.5) and prevents rapid changes in acidity.

[0299] The terms "contacting", "administering" or "submitting", more specifically "vaginal application" or "vaginal administration", are used interchangeably herein and refer to a device, dosage form or pharmaceutical composition provided or given to a subject (e.g., a female recipient) or to a particular organ or other physiological site (e.g., the urogenital tract or vaginal cavity or a portion thereof, e.g., the vaginal wall (mucosa or endometrial surface) or vaginal vault) and to a subject or site for the purpose of colonizing and engrafting a desired bacterial community (e.g., including lactobacilli), e.g., as included in the substantially complete vaginal microbiota preparation described herein. In one embodiment, administration is performed topically. In one embodiment, administration is performed vaginally, e.g., in the context of treating a disease or disorder, e.g., to improve vaginal health.

[0300] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a pharma- ceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry, or an active agent in combination with an additional active agent (e.g., a substantially complete vaginal microbiota preparation as described herein). A pharmaceutical composition is physiologically and pharmacologically acceptable. In general, a compound, material (device), composition, and / or dosage form is "pharmaceutical," "therapeutic," or "pharmaceutical acceptable" if it is suitable for use in contact with human tissues without excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of sound medical judgment (e.g., by a physician or regulatory agency), commensurate with a reasonable benefit / risk ratio (e.g., the desired benefit(s) versus side effects (adverse events)). A pharmaceutical composition generally includes agents for buffering and preservation in storage, and may include buffers and carriers for appropriate delivery, depending on the route of administration. A pharmaceutical composition can be conventionally administered in unit doses. The term "unit dose" refers to physically discrete units suitable as unitary administration to subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in combination with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0301] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. Dosage varies depending on several factors, including frequency of administration (e.g., daily, more than once a week, more than once a month, or more than once every three months); size and tolerance of the individual; severity of the condition; intended outcome (e.g., treatment, prevention, modulation, or restoration of the microbial community), and route of administration. Baseline doses can be administered and modified based on the subject's initial response. For example, a single dose of a composition comprising a substantially complete vaginal microbiota preparation as described herein may be administered in the amount of 10 4 ~10 10 In other embodiments, a single dose of the composition may range from 10 3 ~10 12 , 10 4 ~10 9 , 105 ~10 9 , 10 5 ~10 8 , 10 6 ~10 9 , 10 7 ~10 9 , or 10 7 ~10 10 The range may be in colony forming units (per dose).

[0302] "Dosage form" refers to the particular physical form of a pharmaceutical composition, depending, for example, on the dosage required to deliver a desired amount of active agent and the route of administration. For example, the dosage form may be a suppository, tablet, capsule, film, cream, etc., or a device, such as an applicator or dispenser for vaginal administration. The dosage form may be a single or multi-use dosage form.

[0303] As used herein, the terms "treat", "treatment", and "treating" refer to prophylactic and therapeutic treatments, the purpose being to prevent, reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition or symptom associated with a disease or disorder. Treatment includes improving symptoms or markers (of a disease or condition), as well as stopping or at least slowing the progression or worsening of symptoms that would be expected in the absence of treatment. Therapeutic efficacy can be measured by monitoring one or more symptoms or clinical markers, e.g., compared to the subject's condition and symptoms before administration, or to a control subject not receiving treatment. In one example, treating a vaginal infection refers to reducing the amount of infectious agents (e.g., the number of cells or viral particles), reducing the severity of symptoms, and / or reducing the frequency of symptoms. Treatments that reduce the level of pathogens to those suppressed by the immune system or to a state established by a healthy vaginal microflora (e.g., the infection is no longer detectable, e.g., by symptoms or common diagnostic techniques) are considered to be effective as the term is used herein.

[0304] As used herein, the term "colonization" or "engraftment" refers to the colonization of an environment (e.g., a microbial niche), e.g., the vagina or vaginal epithelium, by a microorganism, e.g., a bacterium (e.g., lactobacilli), such that a viable population of the microorganism remains present in the niche for a period of time. Depending on the period of time that the microorganism remains present in the niche, the engraftment can be transient or stable. Colonization and engraftment (and residence time) can be quantified, for example, by counting the number of colony forming units (CFU) / gram and / or by nucleic acid sequencing of the microorganisms contained in one or more vaginal samples taken over a period of time.

[0305] The term "subject" refers to a human (e.g., a human female) subjected to treatment, observation or testing. In one embodiment, the subject is an adolescent subject. In one embodiment, the subject is pregnant. In one embodiment, the subject is of childbearing age. In one embodiment, the subject is a premenopausal subject. In one embodiment, the subject is a postmenopausal subject. In one embodiment, the subject is a recipient, e.g., a female recipient, of a composition comprising a substantially complete vaginal microbiota preparation as described herein. In one embodiment, the subject is a donor female who provides a microbial sample. In some embodiments, the subject is a human female suffering from or exhibiting a clinical condition associated with a microbial imbalance (e.g., a dysbiosis, an infectious disease, or an inflammatory condition). In some embodiments, the subject is a human female who prophylactically uses the compositions and preparations described herein.

[0306] The terms "increased," "increasing," "enhancing," or "activating" can mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a reference level, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase between 2-fold and 10-fold or more.

[0307] The terms "lower," "reduced," "reduction" or "decrease," "downregulation" or "inhibit" refer to a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of 100% or less (i.e., levels that are absent (or levels below the limits of detection) compared to a reference sample), or any decrease between 10-100% compared to a reference level.

[0308] The term "substantially" means to a large or significant degree. For example, in the case of a sample that is "substantially free" of a pathogen, the term means that the sample is largely or essentially, but possibly not completely, free of the pathogen. A sample that is substantially free of a selected pathogen may refer to a sample that contains about 5%, <5%, <4%, <3%, <2%, or <1% of the selected pathogen. In certain embodiments, the term "substantially free" includes <5%.

[0309] The terms "inflammation," "inflammatory disorder," "disease associated with inflammation," and "condition associated with inflammation" are used interchangeably and refer to any pathology, disorder, disease, or condition that includes or is characterized by inflammation. Inflammation can be acute inflammation or chronic inflammation. EXAMPLES

[0310] Working Example

[0311] Example 1: Screening of women to identify donors and recipients

[0312] This example describes the process of identifying suitable donors based on analysis of vaginal microbiome composition and the absence of pathogens. A schematic of the screening is shown in FIG.

[0313] Participant Enrollment and Vaginal Samples

[0314] Healthy women with no history of vaginal health problems were recruited. Participants were informed and consented to provide a vaginal swab for microbiome analysis and a separate swab for HPV analysis. A total of 96 women consented and provided samples. For the last 60 women screened, instead of vaginal swabs, cervicovaginal secretion (CVS) samples were obtained using a vaginal self-sampling device (e.g., a flexible menstrual cup inserted into the vaginal cavity, such as the Soft Disc Menstrual Cup by The Flex Company, also sold under the name "Flex disc"), allowing both microbiome and biomarker analysis, as well as a dedicated swab for HPV screening. Both sample types were obtained by self-sampling by the donors after thorough instructions to the donors on the correct sampling procedure.

[0315] Microbiome Analysis

[0316] Microbiome composition from vaginal swabs or CVS samples was determined by shotgun DNA sequencing analysis. Swabs for vaginal microbiome analysis were kept at 4°C for up to 48 hours before DNA extraction. CVS samples were diluted with saline to reduce viscosity, aliquoted (see Example 3 for procedure), and stored at -80°C before DNA extraction. DNA was extracted from both sample types using the Molysis Complete5 kit (MolZym), which uses differential lysis method to extract microbial DNA and remove human DNA. Shotgun sequencing and bioinformatics analysis of the microbiome were performed by Seqbiome (Cork, IE). All samples were prepared for shotgun metagenomic sequencing following Illumina Nextera XT Library Preparation Kit guidelines using unique double indexing for multiplexing with the Nextera XT Index Kit (Illumina). Samples were sequenced at the Teagasc DNA sequencing facility on an Illumina NextSeq 500 sequencing platform with v2.5 kit (300 cycle kit / 150bp PE sequencing) using standard Illumina sequencing protocols. The quality of the raw sequencing data was assessed using FastQC. Samples that did not meet the minimum quality check cutoff or a sequencing depth of at least 4M reads were resequenced. The shotgun metagenomic sequencing data was then processed by an analysis workflow utilizing the Kneaddata wrapper tool. Quality filtering and host genome decontamination (human) were performed utilizing Trimmomatic and Bowtie2. Taxonomic classification of the quality filtered reads was further performed using the Kraken2 species classifier using a customized version of the Genome Taxonomy Database (GTDB), which also contains reference sequences belonging to archaeal, fungal and viral genomes. The Kraken2 classification was then passed to the Bracken tool to estimate species-level abundance. The vaginal microbiome composition was determined for each putative donor and recipient by obtaining information on the presence and relative abundance of bacterial species.

[0317] In this study, to be eligible as a donor, the vaginal microbiome composition had to meet two criteria: containing at least 80% vaginal lactobacilli (L. crispatus, L. iners, L. jensenii, L. gasseri) and containing less than 5% selected pathogens (Atopobium spp., Prevotella spp., B. vaginale, and F. vaginae). The vaginal microbiome composition of 61 of the 96 women (64%) met these two criteria and was considered a healthy vaginal microbiome. The relative abundance of lactobacilli present in the vaginal microbiota is shown in Figure 3, where each bar represents the microbiome composition of a single healthy donor. The relative abundance of species is shown on the y-axis. The microbiomes of the screened donors differed in their relative composition. In some instances, the microbiome was dominated by a single species, e.g., L. crispatus, while other microbiomes showed a heterogeneous population of lactobacilli, e.g., including a combination of L. crispatus, L. iners, and L. jensenii. Notably, even small relative abundances, e.g., less than 0.05%, could be detected (see Table 2).

[0318] Of the 96 women screened, 28% (n=27) exhibited a dysbiotic microbiome (defined as >20% selected pathogens and <10% vaginal lactobacilli) (see Figures 2 and 4). Women who exhibited a dysbiotic vaginal microbiome had no symptoms of vaginal disease and were in good overall health. The remaining 8% (n=8) fell between the healthy and dysbiotic microbiome classifications, did not exhibit vaginal disease symptoms, or contained species not classified / defined by the screening criteria (Figure 4). These women were not eligible as putative donors or recipients.

[0319] The microbiome composition of the healthy cohort (n = 61) had a median vaginal lactobacilli abundance of 99.26% and selected pathogens of 0.03%. In contrast, the dysbiotic cohort (n = 27) was characterized by a median vaginal lactobacilli abundance of 0.34% and selected pathogens of 87.21%. The undefined cohort (n = 8) was characterized by a median vaginal lactobacilli abundance of 35.76% and selected pathogens of 61.83%.

[0320] This suggests that the selection criteria (see FIG. 2) can reliably distinguish between healthy and dysbiotic vaginal microbiomes with high certainty. Only healthy vaginal microbiomes were considered as putative donors and subjected to further stringent screening procedures.

[0321] HPV screening

[0322] Human papillomavirus (HPV) is a very common viral infection, some strains of which are associated with cervical cancer. The prevalence of HPV-positive women is high. For vaginal swabs taken for HPV screening during the donor screening visit, DNA extraction was performed using a Qiagen QIAmp DNA mini kit. DNA extracted from each donor sample was used in a SeeGene Anyplex II HPV 28 kit and run semiquantitatively on a BioRad CFX96 Dx qPCR instrument, which was set up and calibrated for this assay (TrioLab, DK). The semiquantitative method provides three quantity levels for 28 different genotypes of HPV, including all known high-risk genotypes. Only HPV-negative samples were considered suitable putative donors.

[0323] Confirmation and testing of pathogens by medical personnel

[0324] To minimize adverse effects for the recipients, putative donors (women with a healthy microbiome and who passed HPV screening) were further screened by gynecologists for the presence of pathogens, pathogenic commensals and sexually transmitted diseases, and underwent medical and gynecological examinations to evaluate for the presence of cancer or other diseases such as endometriosis. This procedure included further HPV testing.

[0325] Standard diagnostic testing was performed for HIV, Hepatitis A, B, and C, Cytomegalovirus, Treponema, urinary tract infection, HPV, Chlamydia infection, Gonorrhea, Trichomonas, genital herpes, Candida, Mycoplasma, and Streptococcus types A, B, C, and G. During the medical examination, the putative donors underwent a general health check, including medical history and medication use, demographics, heart rate, and blood pressure measurements.

[0326] For approved donors, the same testing was repeated at a follow-up visit after the last donation over the specified period (see FIG. 1). Samples remained in quarantine and were not released for administration to recipients until the approved donor passed the testing at the follow-up visit and all samples passed all quality checks (see Example 4).

[0327] Donor Selection

[0328] Donors were selected through the above screening procedures, including microbiome analysis, pathogen screening, and medical and gynecological examinations. Only subjects with healthy vaginal microbiomes, no positive pathogen test, and no abnormal findings in medical and gynecological examinations were considered suitable donors and enrolled in the program (see also Example 2).

[0329] Following the screening protocol, 12 (13%) of the 96 screened women were enrolled in the donor program and initiated their donor visit. Of these, three were unable to provide sufficient donor support or failed during follow-up screening. Overall, of all 96 women enrolled for screening, 9% fully passed all screening criteria, resulting in nine approved donors (n=9) (Table 1).

[0330] Table 1 provides an overview of the pathogens and pathogenic commensals screened for in donor subjects who passed HPV screening (n=38). [Table 1]

[0331] The composition of substantially complete vaginal microbiota preparations of all screened subjects (n=96), including fully approved donors (n=9), is summarized in Table 2 .

[0332] Table 2. Vaginal microbiota composition of the four cohorts: (i) healthy, (ii) fully accepted donors, (iii) dysbiosis, and (iv) undefined. Median and interquartile range (IQR) (%) quantified bacterial relative abundance to the total amount of detectable species in sample preparations are shown. [Table 2]

[0333] The "healthy", "dysbiotic" and "undefined" cohorts were classified based on the relative abundance of vaginal lactobacilli and pathogens as described above (see also Figure 2). "Fully approved donors" refer to subjects who, in addition to passing the first screen, also successfully passed additional screening to exclude STIs and other infectious and medical conditions, e.g., as described above. Subjects (n=9) who passed all screening criteria and were qualified as suitable donors had a median vaginal lactobacilli concentration of 99.62% and selected pathogens of 0.01%. Approved donors had the highest abundance of selected vaginal lactobacilli and the lowest abundance of vaginal pathogens of the group. Fully approved donors were not limited to a vaginal microbiota composition dominated by L. crispatus, but included a mixture of lactobacilli species or were dominated by L. iners.

[0334] Recipient Selection

[0335] Recipients were recruited and enrolled in clinical trials. In one study, healthy volunteer women aged 18-45 years were recruited for screening and participation. Only women without symptoms of vaginal disease and generally in good health were enrolled for screening. In another study, a single patient with vaginal symptoms was enrolled. All recipients were classified as having vaginal dysbiosis (having a dysbiotic vaginal microbiome) according to the inclusion criteria defined above (see Figure 2): selected pathogens Atopobium spp., Prevotella spp., B. vaginale, and F. vaginae >20% and vaginal lactobacilli <10%.

[0336] Example 2: Obtaining Donor CVS Samples

[0337] This example describes a process for obtaining a vaginal microbiota sample from a donor, the sample comprising substantially the complete vaginal microbiota.

[0338] Donor visit setting

[0339] Donors who successfully passed the screening process described in Example 1 were asked to provide a cervix-vaginal secretion sample (CVS) within 40 days between two gynecologist / pathogen testing visits (see FIG. 1). Each donor made 10-15 donations at any time during the 40 days, except for days during menstruation and one day thereafter. Donor donations were spaced at least 16 hours apart.

[0340] During the donation period, donors were required to adhere to the following restrictions: no vaginal or anal intercourse; no swimming in lakes, jacuzzis, or swimming pools; and no use of intravaginal products (e.g., tampons, soap). At each visit, donors filled out a questionnaire to verify adherence to these restrictions along with general health questions.

[0341] After the donation period, the donors underwent a follow-up examination at a gynecologist. Only if this follow-up examination was passed were the samples considered for release and use for administration (see FIG. 1, Example 1). Until that point and until all samples had passed all quality checks (see Example 4), all samples remained in quarantine.

[0342] CVS donor self-collection

[0343] Cervical vaginal secretions (CVS) samples were obtained by self-collection using a vaginal self-sampling device after thorough instructions. Donors obtained their CVS samples in a dedicated, hygienically designed donor room. The donor room was washed with 70% ethanol after each donor and subjected to biweekly environmental monitoring to check for Enterobacteriaceae, total aerobic microbial counts, and yeasts and molds. This setup maximizes cleanliness and minimizes processing time, compared to, for example, home sampling. Only a single contamination was detected in one of the donor samples consisting of skin bacteria, caused by the donor not following the sampling procedure correctly.

[0344] The vaginal self-sampling device was a single-use menstrual cup with a flexible / pliable ring and a plastic foil cup (described in Example 1). For donor donation of CVS, it was not worn like a menstrual cup over the cervix, but instead used as a large swab by partially folding it and inserting it into the vagina, leaving it in a longitudinal position for approximately 10 seconds, and twisting it along its longitudinal axis while removing it. Donors placed the vaginal self-sampling device in a provided labeled and pre-weighed sterile 50 mL tube. After 15-20 minutes, the process was repeated a second time with a new vaginal self-sampling device placed in another sterile 50 mL tube.

[0345] The soft and flexible material of the Vaginal Self-Sampling Device, in conjunction with its partial fold insertion into the vaginal canal, allows for effective collection of CVS from the surfaces of the vaginal cavity without damaging or irritating the vagina. The Vaginal Self-Sampling Device is intended and safe for vaginal use, however, to ensure maximum donor safety and control, each batch of devices was screened for Enterobacteriaceae contamination and found to be free of any contamination.

[0346] The procedure described herein is believed to have several advantages over using vaginal self-sampling devices for long periods as collection devices on the cervix.In the above-mentioned method, the bacteria contained in the CVS sample spend less time in contact with the vaginal self-sampling device, and the amount of vaginal mucus and vaginal bacteria collected increases.The vaginal epithelium contains the substrate of the vaginal microbiome.Therefore, the sample obtained in this way has a higher concentration of viable vaginal lactobacilli and mucus, and a lower amount of fluid from the endometrium.

[0347] Example 3: Production of a substantially complete vaginal microbiota preparation

[0348] This example demonstrates the production of a substantially complete vaginal microbiota preparation from ectocervical secretions.

[0349] Process for producing a substantially complete vaginal microbiota preparation

[0350] A schematic diagram of the sample processing is shown in Figures 5A and 5B. At each visit, donors were provided with two pre-labeled and pre-weighed 50 mL sterile tubes and two vaginal self-sampling devices along with a questionnaire (see Examples 1 and 2). The CVS was collected by centrifugation at 190 x g and ambient temperature for 5 minutes. The low speed allows the CVS to be collected from the device without phase separation into layers. All further steps were performed under sterile conditions at ambient temperature. The self-sampling device was discarded from the tube and the CVS sample weight was determined. Samples with a total (combined) weight of less than 200 mg were discarded. Samples with visible blood were discarded.

[0351] The sample in each tube was mixed with 1 mL of saline to reduce viscosity, after which the two samples were combined and aliquoted for storage and further testing. The two combined samples were then divided into several aliquots for quality control (see Example 4) and storage, as shown in FIG. 5B.

[0352] Cryovials containing samples for storage were placed in a CoolCell (Corning) and then placed at -80°C. The CoolCell has a controlled cooling rate of 1°C / min, which ensures that maximum viability of the samples is maintained. After a minimum of 2 hours, samples were transferred to a regular -80°C storage box labeled "Quarantine". Samples were released only after all release criteria were met (see Figure 5B and above).

[0353] Example 4: Quality Control

[0354] This example describes the quality control of a substantially complete vaginal microbiota preparation produced from ectocervical secretions, so that it can be safely used for administration.

[0355] Overall Procedure

[0356] A schematic diagram of the sample processing procedure with details regarding quality control is shown in Figure 5A and Figure 5B. The procedure is highly optimized so that the volumes used for analysis and quality control are as low as possible to minimize the loss of substantially intact vaginal microbiota preparations. Samples used for pH measurements are discarded after the measurement. Holding vials are maintained for at least one year after administration for safety reasons (e.g., to check for STIs in case the recipient develops one). All first and last samples from each donor were subjected to qPCR analysis to confirm the absence of multidrug resistance genes (MDR genes).

[0357] Quality control analysis

[0358] pH was measured using a micro pH probe. Technical triplicate measurements were performed and repeated if the difference between measurements was >0.2. Samples used for pH measurements were discarded after the measurements. Microscopic examination was performed. Absence of sperm cells was determined by microscopy and optically using acid phosphate paper, a sensitive and selective method for identifying sperm cells. Viability was tested by colony forming unit (CFU) counts on de Man, Rogosa and Sharpe (MRS) agar plates or by viable cell count (VCC) for donors in which L. crispatus, L. jensenii, or L. gasseri predominated at screening. For donors in which L. iners predominated at screening, this method could not be used. Instead, viability of these samples was tested using BacLight Live / Dead stain (ThermoFisher Scientific) and counting live cells / mL in a Thoma cell counting chamber under a microscope. Within one week of using the samples for administration, the stability vials were used to test cell viability and dose calculations upon vaginal administration.

[0359] DNA extraction was performed using the Molysis Complete5 kit (Molzym) according to the manufacturer's instructions to obtain sufficient bacterial sequence reads to perform an in silico engraftment check after dosing based on metagenomic data (see Example 1 for the method used). Multidrug resistance (MDR) marker qPCR was performed using the same DNA used for shotgun sequencing using the SeeGene Allplex Entero-DR qPCR assay kit on a BioRad CFX96 Dx qPCR instrument calibrated for the assay. This kit allows single or multiple detection of carbapenemase genes (NDM, KPC, OXA-48, VIM, IMP), extended-spectrum beta-lactamase (ESBL) genes (CTX-M), and vancomycin resistance genes (VanA, VanB).

[0360] Example 5: Characterization of the donor microbiome

[0361] This example describes a substantially complete vaginal microbiota preparation suitable for vaginal administration.

[0362] Microbiome composition during the donor period

[0363] It is known that a woman's vaginal microbiome can fluctuate across the menstrual cycle or in response to sexual behavior (Gajer et al., 2012, Science Translational Medicine). For donors with mixed species composition who did not use contraception (Table 3), variation between species was observed across the menstrual cycle (Figure 6A,B), but the overall abundance of L. crispatus, L. iners, L. jensenii, and L. gasseri in the sample remained stable at >80%. The microbiomes of donors with a highly L. crispatus-dominated microbiome remained highly stable over time (e.g., Donor 7, Figure 6G).

[0364] The microbiomes of L. iners-dominant donors were mostly stable, but in one case, a shift between L. iners and L. crispatus was observed toward the end of the donor period (Figure 6I). This donor did not have a menstrual cycle due to contraception. The stability of the microbiome observed in the donor is likely assisted by the restrictions that the donor adheres to, e.g., not having sexual intercourse.

[0365] Some donors returned for a second donation visit, indicated by vertical lines in Figure 6. For donors 2, 5, and 7, there was a one-month gap between the two visits, and for donor 1, there were 1 and 5 months. For donor 6, there was a two-month gap between the two visits, and she switched from using birth control pills at the first visit to not using hormonal contraception at the second visit.

[0366] In some donor samples, certain species (mostly L. jensenii) were present in small but very consistent amounts (0.3-3.5%). In one of these donors, even smaller amounts of L. iners were consistently present (0.07-0.33%). Even smaller amounts of both L. iners (0.02-0.10%) and L. jensenii (0.01-0.03%) were consistently observed in yet another L. crispatus-dominant donor. In two donors with mixed species composition, the relative abundance sometimes dropped to as low as 0.27%, but none of the species present in the mixture disappeared completely (below the level of detection) over the donor period. Overall, this means that the vaginal microbiome is a stable ecosystem, pointing to a symbiotic relationship between different Lactobacillus species in one person. This is further demonstrated by the observations of vaginal administration of the preparation, as described in Example 7.

[0367] In addition to the four major vaginal lactobacilli (L. crispatus, L. iners, L. jensenii, L. gasseri), other lactobacilli: L. acetotolerans, L. acidophilus, L. amylovorus, L. gallinarum, L. gigeriorum, L. helveticus, L. johnsonii, L. kefiranofaciens, L. kitastonis, L. paragasseri, L. psittaci, Lactobacillus sp002911475, L. taiwanensis, L. ultunensis, L. coleohominis, L. reuteri, and L. vaginalis were regularly observed to be consistently present in low amounts in donor samples (mostly <0.05%). The stable presence of these lactobacilli further points to a complex ecosystem that may be collectively required to promote and maintain a healthy vaginal niche.

[0368] Some other lactobacilli appeared sporadically in a subset of donor samples. L. helveticus was not observed in the two L. iners-dominated donors, and only appeared in the last visit of one donor who shifted from L. iners-dominated to L. crispatus-dominated, which may suggest that certain species prefer to live together with each other, while other species may not be compatible in the ecosystem. In contrast to the reports in the literature that L. iners-dominated donors were not considered suitable for administration, the data suggest that the L. iners-dominated microbiome of the fully approved donor has a very stable and healthy ecosystem (see also the inflammation marker data in Example 8), making it a suitable donor for practically complete vaginal microbiota preparation.

[0369] Although L. gasseri was rarely observed as the dominant species (Figure 3), it was typically found in low abundance in some donors (Figure 6, Table 1). L. gasseri was detected at relative concentrations of 0.01–2.23% in L. crispatus-dominated donor 4 (Figure 6D); at relative concentrations of 0.01–0.07% in mixed-microbiome donor 1 (Figure 6A); and at relative concentrations of 0.3–7.14% in L. iners-dominated donor 8 (Figure 6H). The consistent maintenance of low abundance Lactobacillus species suggests that low abundance species may play a previously unknown role in the maintenance or stability of the vaginal ecosystem.

[0370] Physical properties of the obtained samples

[0371] Sample weights varied between donations from the same donor and between donors, but averaged >200 mg. CFU and pH remained stable and within defined thresholds of <4.5 (Table 3). Overall, this stability for both microbiome composition and sample characteristics indicates the health and stability of the donor microbiome.

[0372] Table 3 summarizes the characteristics of the CVS and substantially complete vaginal microbiome preparations produced. Each row contains data for one donor during 10-15 donations over a 40-day period. The "Time Window" column lists the days on which the donor provided the number of samples listed in the "Number of Donors" column. Donor IDs are the same as in the microbiome graph in Figure 6. Weight is the combined weight of the two samples provided by the donor before the addition of saline. Dose is the final amount in mL in the substantially complete microbiome preparation vial used to administer to the recipient after the entire volume for analysis has been removed (see Figure 5B). The maximum dose is 1.8 mL to fit in a cryovial. Viable cells / dose was determined by CFU plate counts on MRS medium grown anaerobically for 2-3 days for all species except for the two L. iners dominant donors (8 and 9). For these, viable cells / dose was determined by live / dead staining and counting of viable cells in a counting chamber under a microscope. Abbreviations: hIUD: hormonal intrauterine device; pill: oral contraceptive. [Table 3]

[0373] We further evaluated whether repeated CVS donations would affect the composition of the samples. Donors donated two samples twice a day at 6-hour intervals and three times a day at 4-hour intervals. Both repeated CVS donation procedures resulted in a significant increase in pH (an increase of about 0.4), as well as a decrease in sample weight (to about 200 mg or less) and CFU (1 log), making repeated sampling within a single day inappropriate. This suggests that the microbiome requires more than 6 hours to fully regenerate after sampling. As a result, donors could give samples up to once a day with a donation interval of about 16 hours.

[0374] Stability of substantially complete vaginal microbiota preparations

[0375] The stability of substantially complete vaginal microbiota preparations containing different vaginal microbiome compositions was tested after different storage periods ranging from 2 weeks to 11 months (Figure 7). Stability was tested in 50 μL aliquots, which were found to be representative of the full volume vials (data not shown).

[0376] In general, viability remained high over time for all species, with no significant loss observed (mean loss in viability less than 1 log). No significant differences in viability between species were observed. Other researchers have reported a 2 log loss in viability after 9 months for vaginal microbiota compositions not dominated by L. crispatus. The improved stability seen here may be at least partially attributable to the collection method of obtaining ectocervical secretions provided herein, which does not rely on the collection cup being worn inside the donor subject for an extended period of time, but instead exposes vaginal mucus and bacteria to the collection cup for only a short period of time. Without wishing to be bound by theory, it is believed that the longer lactobacilli reside in the menstrual cup without contacting or being able to attach to the vaginal epithelium, the worse the viability, which may be particularly relevant for sensitive species such as L. iners.

[0377] The process of obtaining and processing samples described herein provides substantially complete vaginal microbiota preparations that are stable over extended periods of time for all test species compositions.

[0378] Furthermore, we evaluated whether the mode of thawing the samples affected the viability of the lactobacilli contained in the preparation. When administering the preparation intravaginally in the clinic, clinicians typically pre-warm all necessary materials to 30-37°C. Samples were thawed and warmed to approximately 30°C in two alternative ways and then tested for viability: (i) thawed from -80°C to 4°C, then to room temperature (RT) and 30°C, or (ii) following the same procedure but without the 4°C step.

[0379] Figure 8A shows the results, where "CFU fresh" is the CFU obtained when seeding fresh samples immediately after sampling and before storage. "CFU thawed at 4°C and RT" is the CFU after removing the sample from -80°C storage and gradually warming it by placing it at 4°C for 30 minutes, then at room temperature (RT) for 30 minutes, then at 30°C. "CFU thawed by RT" follows the same procedure but omits the 4°C step.

[0380] CFU and cell viability remained stable in all samples tested, with the former method yielding slightly better viability in two samples, whereas the latter method was slightly better in the third sample (Figure 8A). However, the differences were small and generally acceptable viability was maintained. This suggests that the 4°C step could be omitted and method (ii) was used for clinical implementation during vaginal administration of the preparation.

[0381] Repeated freeze-thaw cycles

[0382] Another aspect of stability is the ability to withstand repeated freeze-thaw cycles. This is of practical relevance when recipients are unable to attend appointments to receive substantially complete vaginal microbiota preparations, and samples must be re-frozen for administration at a later time point. Alternatively, shipment may be delayed, necessitating re-freezing after thawing in the middle. Therefore, the effect of freeze-thaw cycles on samples was evaluated, as well as long-term incubation at room temperature (RT).

[0383] Figure 8B shows preliminary data, where "CFU fresh" is the CFU obtained when seeding fresh samples immediately after sampling and before storage. "1 freeze / thaw cycle" is the CFU after the sample was removed from -80 °C storage, left at room temperature for approximately 40 min, and pre-warmed to approximately 30-37 °C for approximately 30 min. "2 freeze / thaw cycles" is the CFU after the sample was stored at -80 °C for 2-3 weeks between thaw cycles, with this freeze / thaw cycle repeated one more time but without heating to 30-37 °C. "4 h thaw" is the CFU after the sample was removed from -80 °C storage and left at room temperature (RT) for 4 h instead of 30 min before seeding. The results show that undergoing two freeze / thaw cycles reduces viability more than only undergoing one cycle. The L. crispatus-L. jensenii mixture was the most robust and was able to withstand repeated freeze / thaw cycles better than the other tested compositions (Figure 8B).

[0384] Prolonged incubation at RT (4 hours) reduced viability by more than or close to 1 log and should be avoided. These preliminary data demonstrate that all samples are able to maintain cell viability for at least one freeze-thaw cycle if not left at RT for extended periods. This is useful in a clinical setting where samples can be re-frozen and do not need to be discarded.

[0385] Example 6: Matched donors and recipients for vaginal administration of preparations

[0386] To evaluate whether donor samples could be selected in vitro prior to administration, the following assay was performed. CVS material from recipient 2, which was B. vaginale dominant (previously called Gardnerella vaginalis) (Figure 8B), was processed according to the biobanking protocol (see Example 8), inoculated into Gardnerella vaginalis medium (ThermoFisher Scientific, PB5067A) and grown overnight at 37°C in an anaerobic jar equipped with a gas pack. After adding 1 mL of sterile saline to the plate, colonies were scraped, resuspended, and diluted to a density of approximately 0.5 McFarland standard. 100 μL of diluted recipient sample was inoculated into four new Gardnerella vaginalis plates and air-dried for 5 minutes. Four sterile holes, each approximately 3 mm in diameter, were then punched into each plate. A substantially complete vaginal microbiota preparation from the donor was deposited into each hole (two replicates of undiluted sample and two replicates of 5-fold diluted sample). The plates were incubated anaerobically at 37°C in an anaerobic jar equipped with a gas pack. The presence and size of the halo formed around the holes was measured after 5 and 7 days. The halo is a zone around the well characterized by a clear change in appearance that can be determined visually. If the hole contained an agent that negatively affected the growth of pathogenic cells (bacterial exclusion zone), the absence or inhibition of the growth of pathogenic cells from the recipient could be observed around the hole. The larger the halo around the well, the stronger the inhibition / exclusion. Since a substantially complete entire vaginal microbiota preparation was administered, the effect observed here is the result of the complete composition. Thus, the halo assay provides a readout to evaluate which donor is suitable and has the best chance of inhibiting the cellular growth of pathogens, e.g., pathogens present in the vaginal cavity of the recipient. The ability of lactobacilli-dominated microbial preparations to inhibit the growth of resident pathogens in a niche (e.g., the vaginal cavity of the recipient) is expected to aid in engraftment and stable colonization upon administration to the niche.The Gardnerella vaginalis medium does not contain antibiotics to which the lactobacilli are sensitive and therefore represents an antibiotic-free administration.

[0387] The results are summarized in Table 4. [Table 4]

[0388] Undiluted samples from donors 1 and 5 showed equally large halos (3 mm) indicating a strong inhibitory effect on pathogen growth. The same samples were diluted 5-fold, resulting in a decrease in efficacy, which can be seen by the reduction in the halo for donor 1. In the case of donor 5, dilution abolished the inhibitory effect. Samples from donors 4 and 9 were not effective in inhibiting pathogen growth at any concentration.

[0389] These data suggest that donor 1 may potentially be a better match for administration of a substantially complete vaginal microbiota preparation to this recipient than donors 5, 4, and 9, increasing the likelihood of successful treatment of the dysbiotic microbiome present in this recipient. Therefore, this sample from donor 1 was used to administer to recipient 2 in a clinical setting (described in Example 7 below).

[0390] Example 7: Treatment of a dysbiotic microbiome by vaginal administration of a preparation

[0391] This example demonstrates how a substantially complete vaginal microbiota preparation can be used to revert a recipient's dysbiotic microbiome to a donor's healthy microbiome by vaginal administration, thereby treating the dysbiotic microbiome.

[0392] Administration and Results of Substantially Complete Vaginal Microbiota Preparations

[0393] Substantially intact vaginal microbiota preparation samples were first thawed by transferring from -80°C to room temperature for 40 minutes. The samples were then placed in a 37°C incubator for 30 minutes. The tube containing the sample was then mixed by gently inverting it five times. The sample was drawn up into a 5mL syringe and applied to the recipient's vagina in the lithotomy position using an insemination catheter attached to the 5mL syringe. After the sample was inserted into the vagina, the recipient was instructed to lie in a horizontal position for 30 minutes.

[0394] Two representative changes in dysbiotic vaginal microbiota after administration of a substantially complete vaginal microbiota preparation are shown in FIG. 9. Recipient 1 received three doses (one dose / day for three consecutive days) (FIG. 9A), while recipient 2 received only a single dose (FIG. 9B). To increase the chances of success for recipient 2, an in vitro donor-recipient matching assay was performed as described in Example 6. The best performing sample in that assay from donor 1 (see Table 4) was successfully used for administration in recipient 2. The recipient was not pretreated with antibiotics before or during administration of the preparation. After administration of the preparation, the changes in the recipient's microbiome indicate that the substantially complete vaginal microbiota preparation can change the dysbiotic microbiome to one more similar to that of the donor by administering the substantially complete vaginal microbiota preparation alone (i.e., without antibiotic treatment).

[0395] All recipients were enrolled based on their microbiome at screening, which generally occurred one month prior to the visit. At the visit for administration of the substantially complete vaginal microbiota preparation, their microbiome was checked again as a baseline measurement. Recipient 1 was asymptomatic, while recipient 2 had vaginal disease symptoms at the time of administration. Recipient 2 reported a significant reduction in symptoms one week after administration of the preparation, and complete resolution of all vaginal disease symptoms two weeks later.

[0396] The recipient's post-treatment microbiome appeared similar to that of the donor (which may be termed "microbiome twinning") and remained fairly stable. Overall, the post-treatment recipients clustered together with the donor samples in an unsupervised principal component analysis (PCA) plot, whereas the pre-treatment recipients formed a separate cluster (Figure 10). This suggests that the recipient's vaginal microbiome after treatment with the preparation was not only altered from a dysbiotic state, but also resembled the vaginal microbiome composition of healthy, non-dysbiotic donors.

[0397] The recipient microbiome composition before and after administration of the substantially complete vaginal microbiota preparation(s), as well as the composition of the respective donor, is summarized in Table 5. The table shows the relative abundance of species in the donor and recipient samples (determined by shotgun sequencing). After administration of the preparation, "microbiome twinning" was observed in each case, i.e. donor species cultured in the recipient vaginal niche included species present in low abundance in the donor microbiome and absent in the recipient before administration of the preparation. The relevant species for which this phenomenon is observed are highlighted in grey columns.

[0398] The recipient's post-administration microbiome contained species present in low abundance in the donor microbiome (Table 5; where L. is Lactobacillus and B. is Bifidobacterium), suggesting that an entire stable ecosystem was transferred from the donor to the recipient. These species were also observed to be stably present at all donation visits for most donors (see Example 5). Transfer of the entire ecosystem to the recipient by administration of a substantially complete vaginal microbiota preparation may allow successful engraftment and colonization of the Lactobacillus-dominated donor microbiome even in the absence of antibiotic pretreatment, which is used to eradicate existing pathogens and pathogenic commensals and create a substantially empty microbial niche. This may explain why previously tested single-species or single-strain preparations generally do not perform well. Alternatively or additionally, specific substances may be required in the substantially complete vaginal microbiota preparation (e.g., mucin, lactic acid, etc.) to support initial engraftment.

[0399] Moreover, from 2 weeks after administration onwards, the microbiome of recipient 2, although of mixed species composition, was nearly identical to that of the donor, and 1 week after administration, L. jensenii was the first predominant Lactobacillus species. This may indicate the presence of a "pioneer species" that paves the way for the colonization of other species. This phenomenon may be explained by different substrate utilization or pH optimum of different species. However, in some of the other recipients, such as recipient 1, after 1 week, the microbiome was already nearly identical to that of the donor. The variation of species within the microbiome observed in the donor was also observed in the recipient, suggesting an ecosystem shift. The material used for recipient 1 was from the same donor as that used for recipient 2, but from a different visit. Across those samples, the species were the same, but their relative abundances differed, indicating that species abundance is not necessarily the determining factor. Moreover, the virtually complete vaginal microbiome preparation was effective both with single and multiple doses.

[0400] Engraftment is considered successful if the entire ecosystem is present in the donor material, thus requiring a substantially complete preparation.

[0401] Other researchers have previously suggested a detrimental role for L.iners. However, L.iners is present in the donor microbiomes tested. In women who meet the stringent screening criteria described herein, L.iners may also play a beneficial role in indicating a healthy and stable microbiome. This confirms the selection process that uses all four major lactobacillus species in the administration of the preparation.

[0402] As a blinded approach, we compared the post-procedure recipient microbiome with a library of all donor microbiomes (metagenomic sequencing data) to confirm engraftment of a specific donor microbiome in one of the recipient's vaginal niches. It was possible to identify the correct donor sample from the donor library based on its closeness to the recipient microbiome, suggesting successful engraftment of the donor microbiome in the recipient.

[0403] Thus, this example demonstrates that administering a substantially complete vaginal microbiota preparation to a dysbiotic recipient successfully treated a dysbiotic vaginal microbiome, and that following administration, the recipient's microbiota composition closely resembled that of the donor. Although pre-administration antibiotic treatment was thought to be necessary, as suggested by others, these data demonstrate that antibiotic pretreatment is not necessary when a substantially complete vaginal microbiota preparation containing a substantially complete ecosystem is provided. [Table 5-1] [Table 5-2]

[0404] Example 8: Elevated markers of vaginal inflammation in dysbiotic women

[0405] This example demonstrates that the dysbiotic vaginal microbiome represents a different local inflammatory state than healthy women.

[0406] Biobanking of screening cohort CVS for biomarker analysis

[0407] CVS samples were collected as described for substantially complete vaginal microbiota preparations (see Example 2), with the following differences: samples were placed on ice immediately upon receipt and kept chilled throughout the entire processing procedure; cold sterile saline was used instead of room temperature, and samples were frozen at -80°C without the use of CoolCell; instead of adding 1 ml of sterile saline per sample, 2.5 μL was added per mg of sample, so that all samples were weight normalized before analysis; quality checks other than sequencing were omitted (CFU, pH, sperm cell testing).

[0408] Analysis of inflammatory proteins

[0409] Bacterial vaginosis (BV)-associated and dysbiotic vaginal microbiota are associated with increased concentrations of several pro-inflammatory cytokines, such as IL-1a, IL-1b, IL-8, IL-12, IL-18, and FMS-related tyrosine kinase 3 ligand (FLT-3L), as well as matrix metalloproteinases (MMPs). To investigate the inflammatory status of asymptomatic and dysbiotic women and healthy women in a screening cohort, an O-link analysis (BioXpedia, Aarhus, DK) was performed. O-link is a highly specific targeted proteomic method in which qPCR is performed on proteins by using antibody-conjugated oligonucleotides. Different panels of multiwell plates targeting different subsets are available, such as the inflammation panel used here to quantify 92 inflammatory markers.

[0410] A clear separation based on inflammatory protein markers was observed between the dysbiosis and healthy groups in the screening cohort (Figure 11A). Only one healthy microbiome sample clustered with the dysbiosis sample, which could be due to an undetermined infection not detected by bacterial metagenomic sequencing, e.g., Candida (this data was generated before gynecological examination and pathogen screening, and all women were asymptomatic). Several pro-inflammatory cytokines known to be upregulated in BV, e.g., IFNγ, IL-1α, IL-12, IL-18 and MMP-10 (see references and Figure 11B) (belonging to Th1 cytokines (IFNγ, IL-12), innate / inflammatory related cytokines (IL-1, IL-18) and metalloproteinases (e.g., MMP-10)), were also upregulated in dysbiosis participants. The horizontal line represents the median value.

[0411] Example 9: Administration of a substantially complete vaginal microbiota preparation reduces the inflammatory state of the vaginal niche

[0412] This example describes the alteration of the dysbiotic vaginal microbiome induced by administration of a substantially complete vaginal microbiota preparation by reducing local inflammatory conditions.

[0413] Biobanking of donor and recipient CVS for biomarker analysis

[0414] At several time points, CVS samples taken from recipient 2 were used for Olink inflammatory biomarker analysis. Samples were processed in the same manner as the biobanked samples from the screening cohort in Example 8. For each donor, including those used for recipient 2, no dose was administered at visit 5, but instead was processed according to the biobanking method. This resulted in a number of aliquots for biobanking purposes only.

[0415] Analysis of inflammatory proteins

[0416] To allow samples from recipient 2 to be compared to dysbiosis and healthy vaginal microbiome samples without batch-to-batch variability between Olink plates, a subset of samples from the screening (based on sample availability) were run again on the same Olink inflammation plates as the recipient samples. Biobanking samples of the donors used for recipient 2 were run on the same plates.

[0417] A subset of the remigration screening cohort samples again showed a clear separation based on inflammatory protein markers between the dysbiotic and healthy groups (Figure 12A). The donor biobanking samples clustered together with the healthy / lactobacilli dominant screening samples. Before administration, i.e. before administration of the substantially complete vaginal microbiota preparation to the recipients, the samples of recipient 2 clustered with the dysbiotic samples and separated from the donor and healthy cohorts (Figure 12A). One month after administration of the preparation to the recipients, the recipient microbiome clustered with the healthy cohort, which can be seen by the "1m later" data point in the PCA plot of Figure 12A, which is very close to the solid circle representing the healthy subject. Thus, administration of the substantially complete vaginal microbiota preparation shifted the recipient vaginal microbiome from the dysbiotic microbiome to that of the healthy donor (Figure 12A). The post-administration and donor dots do not overlap exactly, which may be due to slight differences in the donor samples (same donor, but different donation and preparation dates) that were assayed for Olink and used to administer to the recipient. Additionally, healthy donor microbiomes also vary in the relative abundance of lactobacilli (see Figure 6A-C), so slight variation between donors and recipients is expected.

[0418] Although limited by the small number of available data points, selected BV-associated pro-inflammatory markers that were differentially expressed in the dysbiotic and healthy cohorts (FIG. 11B) were unchanged or showed a trend towards being downregulated following treatment (FIG. 12B), suggesting that treatment of dysbiosis by administration of a substantially complete vaginal microbiota preparation may treat or ameliorate local inflammation in the vaginal cavity.

[0419] Example 10: Clinical Trials

[0420] This example describes two clinical trials aimed at evaluating the efficacy of using a substantially complete vaginal microbiota preparation as provided herein as a treatment for vaginal dysbiosis.

[0421] Two clinical trials will be conducted to investigate and map shifts in the vaginal microbiome as a result of treatment with a substantially complete vaginal microbiota preparation.

[0422] The primary objective of the first study is to evaluate the effect of three consecutive administrations of substantially complete vaginal microbiota preparations from healthy donors to healthy, asymptomatic volunteer women screened as having vaginal dysbiosis based on criteria defined from metagenomic sequencing of vaginal swab samples. The substantially complete vaginal microbiota preparations will be administered three times on three different and three consecutive visits, and subjects and their vaginal microbiomes will be followed for approximately six months after the first administration. The study is a single-center, randomized, double-blind, placebo-controlled, two-arm, parallel-group study in n=40 healthy volunteer women aged 18-45 years. It includes a total of 11 study visits (Figure 14).

[0423] The primary objective of the second study is to evaluate the effect of up to three separate administrations, separated by follow-up visits, from healthy donors to healthy asymptomatic or symptomatic female volunteers who have been screened as having vaginal dysbiosis based on criteria defined from metagenomic sequencing of vaginal swab samples. Single-dose treatments will be administered up to three times, separated by follow-up visits, and subjects and their vaginal microbiomes will be followed until approximately six months after the first administration. Each administration will be assessed individually for success in shifting the recipient's dysbiotic microbiome. A second administration will be administered only if a follow-up visit after the first administration determines that the first administration did not successfully shift the vaginal microbiome to a healthy Lactobacillus-dominant taxonomic profile. Similarly, a third administration will be administered only if a follow-up visit after the second administration indicates that a dysbiotic vaginal microbiome remains. This study is a single-center, randomized, double-blind, placebo-controlled, two-arm, parallel-group study in n=48 healthy female volunteers aged 18-40 years. It includes a total of 10 study visits (Figure 16).

[0424] The investigational product is a cryovial containing approximately 1.4-1.8 mL of a substantially complete vaginal microbiota preparation. The donors used to obtain the substantially complete vaginal microbiota preparation underwent the screening and follow-up procedures described herein. The reference product (placebo) is provided in a cryovial similar to the investigational product, but containing only approximately 1.3 mL of sterile saline.

[0425] The study includes a screening visit where all criteria for participation are checked and a vaginal swab is obtained to check if predefined dysbiosis criteria based on metagenomic sequencing are met (see below). If all criteria are confirmed, a baseline visit, Visit 2, is scheduled to be performed around day 8 (Study 1) or day 10 (Study 2) of each individual subject's next menstrual cycle. In addition to the baseline assessment, subjects are randomized to the "active" substantially complete vaginal microbiota preparation or "placebo" group in a 3 (active):1 (placebo) ratio and receive their first dose. For Study 1, Visits 2, 3 and 4 (performed on days 8, 9 and 10 of the same menstrual cycle as Visit 2) consist of receiving one dose each for a total of three doses. For Study 2, a second dose is administered on day 10 of the participant's cycle if Dose 1 is unsuccessful. Dose 3 is also administered at Visit 6 on day 10 of the participant's cycle if Dose 2 is unsuccessful.

[0426] In total, in Study 1, seven follow-up visits (Visits 5-11) will be scheduled to occur up to six months after the last dose. In Study 2, a total of three follow-up visits (Visits 7-9) will be scheduled to occur up to six months after the first dose. All follow-up visits will be scheduled to occur around day 8 (Study 1) or day 10 (Study 2) of the menstrual cycle for subjects with one menstrual cycle between visits. At all follow-up visits, metagenomic sequencing assessment of the microbiota in vaginal samples will be performed.

[0427] Participants must meet all of the following criteria to be eligible to participate:

[0428] Be able and willing to give written informed consent.

[0429] ≧18~≦45 (Test 1) or ≧18~≦40 (Test 2).

[0430] Be judged by the investigator to be in general health.

[0431] Absence of vaginal symptoms (Study 1 only).

[0432] Being premenopausal.

[0433] Fulfilling the following predefined criteria for vaginal dysbiosis: a combined Lactobacilli relative abundance of less than 10% and a combined Gardnerella + Atopobium + Prevotella relative abundance of more than 20% based on metagenomic sequencing of vaginal samples.

[0434] Have regular, predictable menstrual cycles of known length or have had at least 3 months of amenorrhea due to the use of long-acting progestins or hormonal contraceptives.

[0435] Be willing to answer questions about your personal medical, sexual and behavioral history.

[0436] Willingness to undergo 3 or up to 3 procedures with SCVMP.

[0437] Willingness to self-collect cervicovaginal secretions and vaginal swab samples at the clinic.

[0438] Be willing to abstain from vaginal intercourse, unless using a condom, without the use of supplemental spermicide or lubricant, for at least one menstrual cycle (28 days) after treatment.

[0439] Be willing to avoid bathing, swimming, sitting in a hot tub, or wearing thongs for at least one menstrual cycle (28 days) after treatment.

[0440] Be willing to refrain from using insertable vaginal female products (e.g., tampons, menstrual cups, sexual toys), vaginal cleansing products, spermicides, lubricants, or other vaginal products that have not been approved for use by the investigator for at least one menstrual cycle (28 days) after treatment.

[0441] Not becoming pregnant during study participation and willing to use condoms during sexual intercourse during study participation (Study 2 only).

[0442] Participants will be excluded from the study if any of the following criteria are present:

[0443] Participants were postmenopausal, defined as having amenorrhea for more than 12 consecutive months with no other known cause other than the use of long-acting progestins or hormonal contraceptives.

[0444] Participants who are pregnant, breastfeeding and / or have been pregnant within the past 2 months, or for Study 2, are planning / hoping to become pregnant within the next 6 months.

[0445] For Study 1, for participants of current childbearing potential but not using an effective contraceptive method: Complete abstention from sexual intercourse for 2 weeks prior to treatment, throughout the clinical trial, until completion of follow-up procedures, or for 2 weeks after discontinuation of study participation if the participant discontinues the study early. (Participants utilizing this method must agree to use an alternative method of contraception when they should become sexually active and will be queried as to whether they have abstained for the previous 2 weeks when they present to the clinic for their final visit.) b. Having a male partner who has been surgically sterilized prior to the screening visit and who is the participant's only male partner. c. Use an acceptable method of contraception, such as spermicide, mechanical barrier (e.g., male condom, female diaphragm), or birth control pill. Participants must continue to use this method for at least 1 week after the end of the study. Use of any non-hormonal intrauterine device (IUD) or contraceptive implant with published data showing highest expected failure rate <1% / year. Participants must have the device inserted for at least 3 months prior to the first screening visit, throughout the study, and for 2 weeks after completion of the study.

[0446] Women in same-sex relationships or likely to have sexual relations with other women throughout the study (Study 1 only).

[0447] Participants with HIV / AIDS or other immune deficiencies.

[0448] Participants who tested positive for HIV, chlamydia, gonorrhea, Mycoplasma genitalium and / or trichomonas vaginalis; (for Study 2) and / or urine hCG screening.

[0449] Participants with current vaginal Candida infection requiring procedures that in the opinion of the investigator contraindicate participation in this study (Study 1 only).

[0450] Participants may not be receiving any treatment, including experimental drugs. If participants were taking part in recent experimental trials, these must have been completed at least 30 days prior to the study.

[0451] Participants who had undergone any type of procedure involving trauma to the cervix within the last 2 months prior to screening (i.e., IUD removal, cervical cryotherapy, or cervical laser treatment).

[0452] Participants with any known condition requiring regular use of antibiotics that suggests the participant is likely to require antibiotic treatment during the study.

[0453] Use of systemic and / or vaginally applied antibiotics within the last month before screening (Study 1 only).

[0454] Participant with any social, medical, or psychiatric condition that, in the opinion of the Investigator, would make the participant less likely to be suitable for the study or would complicate the interpretation of the data from the participant.

[0455] Participants with a history of drug or alcohol abuse that, in the opinion of the Investigator, makes the participant less likely to be suitable for the study or complicates the interpretation of data from the participant (Study 1 only).

[0456] Participants with a history of gynecologic cancer, gynecologic conditions, or surgical gynecologic history that, in the investigator's opinion, would preclude participation (Study 1 only).

[0457] Participants with abnormal findings on screening tests that, in the opinion of the investigator, would preclude participation.

[0458] A modified clinical trial identical to study 1, but in which antibiotics were further administered in addition to a substantially complete vaginal microbiota preparation, is shown in FIG.

[0459] Preliminary results

[0460] Across the two trials, the following success rates were observed in shifting the vaginal microbiome from dysbiosis to a Lactobacillus -dominant healthy vaginal microbiome with a taxonomic profile similar or nearly similar to the donor sample used for administration of the substantially complete vaginal microbiota preparation: Data were stratified by contraceptive method in the recipient and are presented only for recipients randomized to the active intervention (not placebo recipients) (Table 6). [Table 6]

[0461] Three of eight dysbiotic subjects (38%) treated using non-IUD hormonal contraception successfully shifted from a dysbiotic vaginal microbiome to a healthy vaginal microbiome. Similarly, three of nine dysbiotic subjects (33%) treated without hormonal contraception successfully shifted from a dysbiotic vaginal microbiome to a healthy vaginal microbiome. Thus, a total of six of 17 subjects (35%) in these two groups successfully shifted from a dysbiotic vaginal microbiome to a healthy vaginal microbiome. All seven dysbiotic subjects wearing intrauterine devices (IUDs) were unsuccessful in reverting to a dysbiotic vaginal microbiome. Of the seven participants who used IUDs, four had the hormonal IUD brand Mirena, one had the hormonal IUD brand Jaydess, one had an unknown hormonal IUD brand, and one participant had a copper IUD with an unknown brand. The data suggest that, at least for recipients not using an IUD during treatment, approximately one-third of subjects were successfully treated with a substantially complete vaginal microbiome preparation. This success rate was achieved even in the absence of (pre)treatment with antibiotics. This suggests that successful treatment of dysbiosis by administering a substantially complete vaginal microbiome preparation as described herein is feasible in the absence of antibiotics and the adverse effects associated with their use.

[0462] Example 11: Multiparameter evaluation of different formulations using simulated vaginal fluid

[0463] Various suitable dosage forms were evaluated for formulating the substantially complete vaginal microbiota preparation described herein, including formed gels, freeze-dried gels, tablets, and films.Several excipients were evaluated to obtain suitable dosage forms, including mannitol, microcrystalline cellulose, mucin (porcine, Sigma), hyaluronic acid (Sigma), maltodextrin, guar gum (Sigma), inulin (Sigma), alginic acid (sodium alginate, DuPont), polyvinyl alcohol (PVA Parteck SRP 80, Merck), sodium CMC (Ac-Di-sol, sodium carboxymethylcellulose, DuPont), polyvinylpyrrolidone (Kollidon (PVP), BASF), hydroxypropyl methylcellulose (Methocel K 4 M (HPMC), Colorcon), poloxamer (poloxamer 407 (Kolliphor), BASF), Carbopol (Carbopol 934, Serva), lactic acid, and acetate buffer.

[0464] Basic preparations

[0465] For example, gels formed were prepared using excipients including hyaluronic acid, sodium alginate, HPMC / PVP, and poloxamer 407. The gels were pH adjusted to maintain a pH of approximately pH 3.4 to pH 3.9. Combinations of lactate and acetate buffers were evaluated.

[0466] Tablets were prepared using excipients, e.g., microcrystalline cellulose, HPMC / PVP, maltodextran, and bulking agents including poloxamer 407, as well as compression. Lyophilized excipients were also evaluated to determine if they improved mucoadhesion and / or gelation. A combination of lactate and acetate buffer salts was evaluated for inclusion. Tablet tensile strength was targeted at approximately 1.0 MPa to ensure lack of excessive fracture and good processability. The pH target for the reconstituted tablet (e.g., inside the vaginal cavity) was approximately pH 3.4 to pH 3.9. Disintegration profiles in low liquid volume environments (e.g., inside the vaginal cavity) were also evaluated.

[0467] For lyophilized gels, several excipients were lyophilized including, for example, hyaluronic acid, sodium alginate, HPMC / PVP, and poloxamer 407. All gels were pH adjusted to maintain a pH of approximately pH 3.4 to pH 3.9. Lactate and acetate buffer combinations were evaluated. The target lyophilized appearance was a clean, uniform cake. Target water content was generally less than 3% w / w water (e.g., to increase drug substance viability). Target reconstitution time in the vial was less than 2 minutes with hand swirling.

[0468] For films (air dried), various concentrations of PVA were evaluated. For example, PVA was supplemented with sodium CMC and other excipients to modify drying time, final flexibility of the film, mucoadhesion, etc. Lactate and acetate buffer combinations were evaluated, and pH effect on immersion in simulated vaginal fluid was evaluated. Target film properties included sufficient flexibility for application (e.g., to the vaginal canal), non-stickiness, acceptable drying time for ease of processing, adequate film thickness, and disintegration profile in a low liquid volume environment (e.g., inside the vaginal cavity), as well as effective release and implantation of the drug substance. The pH target for the reconstituted film (e.g., inside the vaginal cavity) was approximately pH 3.4 to pH 3.9.

[0469] All components were evaluated for optimization of viscosity (e.g., at 37°C, e.g., inside the vaginal cavity), enhancement of mucoadhesion (e.g., to epithelial, mucosal surfaces, e.g., inside the vaginal cavity) and / or positive impact on drug substance stability (e.g., bacterial viability during formulation and storage). An ideal formulation would show little or no flow on suitable vertical surfaces and maintain high bacterial viability (e.g., CFU counts) both during formulation and during (long-term) storage.

[0470] Formulation selection parameters evaluated include: mucoadhesion (e.g., of the reconstituted product in the vaginal tract); viscosity (of the reconstituted product) (e.g., the final viscosity of a gel-based product should be pourable at ambient temperature and should preferably solidify at 37°C (at body temperature, e.g., in the vaginal tract); total sugar content (of the reconstituted product), e.g., ideally below physiological concentrations (approximately 0.5-1.0 mg / mL); volume of the reconstituted product, e.g., up to 3 mL; hydration / disintegration rate (e.g., gel / matrix), e.g. for example, sufficient physical integrity to provide a desired release rate; pH, for example, about pH 3.4-3.9 (for example, to promote inhibition of competitive vaginal bacteria); water activity / water content (for example, in dry formulations), for example, 0.5-3% water (for example, for long-term dry formulation stability); microbial diversity, for example, the relative abundance of Lactobacillus species, e.g., L. crispatus, L. gasseri, L. jensenii, and L. iners; total dose / potency, for example, preferably 1×10 per dose. 5 CFU / VCC of 1×10 6 CFU / VCC of 1×10 7 CFU / VCC or 1×10 8 These include CFU / VCC (per dose), shelf life (unreconstituted) at various temperatures, and microbial limits, e.g., absence of microorganisms such as Pseudomonas aeruginosa, Candida albicans, Staphylococcus aureus (Ph Eur standards 5.1.4, 2.6.12 and 2.6.13).

[0471] Testing was performed using standard assays including, e.g., plate count (e.g., MRS agar) or viable cell count (VCC, e.g., using the Quantom Tx Automated Counting System (AM620) with fluorescent stains) for e.g., viable count, dose determination, shelf life; rheometers, pH meters, Karl Fisher / water activity meters for e.g., mucoadhesion and viscosity; Ph Eur tests, for example, for microbial load.

[0472] Gel formulation

[0473] A gel formulation was prepared using the following excipients:

[0474] 0.5% and 2% hyaluronic acid

[0475] HPMC+Carbopol+PVP

[0476] Poloxamer 20% + Sodium Alginate 2%

[0477] PVA 3% + NaCMC 2%

[0478] Poloxamer 20% + Guar Gum 2%

[0479] 0.5% and 2% guar gum

[0480] Mucin 5%

[0481] Inulin 8%

[0482] The flow rate under gravity (viscosity, mPa·sec / shear rate 1 / sec) at ambient temperature was determined for hyaluronic acid 0.5%, HPMC Kollidon 25+Carbopol, guar gum 0.5%, simulated mucus and inulin using 0.5 mL on a 56 mm glass slide. The values ​​obtained ranged from 300 to 1×10 at shear rates of 0 to 3, depending on the formulation. 7 The viscosity ranged from 0.5 to 7,000 mPa·sec at a shear rate of 100 1 / sec. Guar gum (0.5%) and hyaluronic acid (0.5%) did not appear to form gels and resulted in low viscosities. Viscosity versus temperature from 15°C to 45°C (with a fixed shear rate of 10 s-1) was also determined. Poloxamer showed thermoreversible gelation behavior with an increase in viscosity at about 25°C (about 10,000 to about 12,000 mPa·sec), whereas the other formulations mostly showed small changes in viscosity at various levels ranging from about 500 to 12,000 mPa·sec at 15°C and about 200 to 11,000 at 45°C, depending on the formulation.

[0483] The next step involved evaluating which gel bases could be lyophilized and reconstituted to form acceptable gels. The appearance of the "cake" was also evaluated to indicate uniformity.

[0484] Simulated vaginal fluid (SVF) was generated using the following composition: 35 mg NaCl, 14 mg KOH, 22 mg calcium hydroxide, 20 mg lactic acid, 10 mg acetic acid, 1.6 mg glycerol, 50 mg glucose dissolved in 10 mL water and adjusted to pH 4.2 with HCL.

[0485] A single vial was used to simulate a single utero-vaginal secretion donation with SVF, and the volumes used were believed to represent the range of approximately 0.5 mL to 1.5 mL expected from a typical donation. After determining the appropriate excipients, these excipients were used to form gels by adding 1.5 mL of SVF. The addition of SVF allows for accurate pH balancing and simulates the addition of a substantially complete vaginal microbial preparation to the excipient base. The samples were manually shaken in the vial to form a uniform gel and then lyophilized. Several excipients produced acceptable appearances after lyophilization. PVA, guar gum, and HA, formed acceptable (clear) gels; Kolliphor P 407 (Poloxamer 407) and Methocel K4M formed a white crystalline cake; maltodextrin and PVP exhibited discoloration (e.g., yellow), while sodium alginate, mucin, and NaCMC produced heterogeneous cakes with discoloration (e.g., brown). Surprisingly, mannitol, which typically forms a consistently acceptable cake, is crystalline, likely due to the salt content in the SVF.

[0486] The resulting gels were reconstituted with 1.5 mL of deionized water after lyophilization. HA, guar gum, and PVA were easily reconstituted, with reconstitution times of 2:30, 2:30, and 1:30 min, respectively. HA formed a homogeneous gel at pH 4.6. Guar gum formed a heterogeneous gel at pH 4.5. PVA formed a homogeneous liquid at pH 4.7 with some foaming. PVP, NaCMC, poloxamer, sodium alginate, and HPMC formed semi-homogeneous gels. All required more than 3 min for reconstitution. PVP dissolved slowly at a pH of 4.1. NaCMC formed a non-homogeneous viscous liquid at pH 4.9. Poloxamer showed some foaming at pH 5.4. HPMC formed a gel at pH 4.4 with some foaming. Sodium alginate was non-homogeneous at pH 5.5. Mannitol formed a homogeneous liquid and some crystals remained at pH 4.4.

[0487] A mucoadhesion test was performed to test the effect of the formulation under gravity during application. For this, a mucosal surface (such as that present in the vaginal cavity) was simulated. 500 mg of mucin was compressed into a disk a using a flat 2 cm tooling to a pressure of about 3 tons. These were then glued to a substrate, for example the bottom of a plastic box. Each disk was then wetted with about 200 μL of deionized water and rubbed with a gloved finger until the mucin surface was tacky to the touch. About 0.5 mL of sample (a full dosage unit in the case of tablets and PVA films) was then applied in a horizontal position and allowed to stand for 2 minutes. The box was then raised so that the samples were in a vertical position, and a visual evaluation was made of the adhesive properties (mucoadhesion test) of the guar gum, poloxamer, mannitol, PVP, hyaluronic acid, sodium alginate, CMC, HPMC, and PVA gel samples. Hyaluronic acid, NaCMC, sodium alginate, and HPMC showed the best mucoadhesion in this test.

[0488] Since poloxamers showed thermoreversible behavior in previous studies, the mucoadhesion test was repeated with pre-heated plastic boxes and mucin disks at 37° C. The gel formed was more viscous than at ambient temperature and was found to be retained on the surface of the moistened mucin disk, indicating that poloxamers may have the appropriate viscosity at body temperature.

[0489] After adjusting the concentrations, 3% (45 mg) carbopol, 4% (60 mg) sodium alginate, 3% (45 mg) HPMC and 1.9% (30 mg) sodium CMC, as well as 24% (360 mg) poloxamer were also evaluated for syringeability. These were prepared with SVF:lactate buffer mixtures. The inclusion of lactate buffer in the pH range of about 3.4-4 can promote engraftment of lactobacilli in vivo, for example, by minimizing competition of undesirable bacterial taxa potentially present in existing microbial niches (e.g., in the vaginal tract). The excipients were mixed with 1.5 mL of SVF+lactate buffer. Both carbopol and poloxamer formed homogenous gels with some air bubbles dispersed as they settled after shaking by hand. Sodium alginate and HPMC+Na-CMC did not form homogenous gels.

[0490] A lyophilized gel format as a dosage form for a substantially complete vaginal microbial preparation can be produced, for example, by blending with excipients followed by lyophilization and packaging, for example, in a vial. The vial can then be reconstituted, for example, in a clinic environment with water to form a gel within the vial prior to application of the reconstituted drug product, and the composition comprising the substantially complete vaginal microbial preparation can be administered to the vaginal tract of a subject, for example, by using an applicator, such as a syringe.

[0491] The frozen gel represents a dosage form for a substantially complete vaginal microbial preparation that is blended with gelling excipients (optionally with an appropriate lactate buffer), and the liquid gel form is then frozen (at -80°C) and stored in either vials or pre-filled syringes (see, e.g., Figure 13).

[0492] Tablet Evaluation

[0493] In addition to the gel, a tablet-pessary was produced as an additional dosage form. The following aspects were considered: selection of excipients suitable for compression and lyophilization to form tablets, as well as selection of excipients to provide an acceptable level of mucoadhesion, optionally including the optional inclusion of a lactate buffer with a pH target of about pH 3.5-4, with the goal of obtaining a single tablet prepared from an individual cervicovaginal fluid donation. All of the excipients evaluated that provided both acceptable possible gel formation and acceptable lyophilization (e.g., to form a reasonably free-flowing powder for further processing), including Carbopol, HPMC, gelatin, sodium alginate, poloxamer, NaCMC, and pectin, were evaluated (along with additional excipients) for tabletting quality in a manual tablet press. Tablets were compressed at about 1 ton a with 5.5 mm tooling to form tablets. As previously described, tablets were gently added to a wet mucin disk and mucoadhesion was evaluated.

[0494] All tablets remained attached for 5 minutes. Tablets were rinsed every minute to assess the effect of adhesion. NaCMC began to swell into a gel. Carbopol, HPMC, alginate, NaCMC tablets showed adequate tablet integrity. Poloxamer formed tablets but with relatively low strength. Pectin and gelatin tablets showed low hardness and some brittleness.

[0495] Polymer Film

[0496] Film formation / casting is a potential pathway to maintain the viability of living biomaterials. To investigate this, the following excipient test samples were generated:

[0497] 3% PVA

[0498] 20% PVA

[0499] PVA 3%, NaCMC 2%

[0500] PVA 20%, NaCMC 5%

[0501] All samples were mixed as needed to create gels / films, transferred to plastic blister packs (size 0), and stored at ambient conditions for approximately 48 hours to solidify.

[0502] Both samples containing 3% PVA did not form a suitable film. The film containing NaCMC was brittle and difficult to remove from the blister mold. The 20% PVA sample formed a film and could be easily removed from the blister mold. The film was flexible and resistant to tearing.

[0503] To assess whether it was possible to form a film containing a substantially intact vaginal microbial preparation, a "sandwich" approach to film preparation was attempted. This was performed with an upturned Karl Fischer lid (with PTFE lining) to produce a mold with a flat disk design. A 12% PVA solution (from the previous 20%) was used to reduce the initial viscosity and allow for more consistent sample preparation by pipette. Preparation was performed as follows:

[0504] 0.25 mL of 12% PVA was pipetted into each mold, followed by 0.5 mL of SVF (to simulate an essentially complete vaginal microbial preparation). An additional 0.25 mL of 12% PVA was pipetted on top to complete the "sandwich." The molds were allowed to solidify.

[0505] After about 48 hours, a disk was formed that was flexible and left no obvious residue on the PTFE lining, suggesting that PTFE may be a suitable material for molding and that a film was easily recovered from this surface.

[0506] The films were gently added to the mucin discs and mucoadhesion was assessed as described previously. The PVA discs showed no migration from the mucin surface, indicating good mucoadhesion. A wash with approximately 2 mL of SVF was performed after 5 min, but this did not displace the PVA discs from the mucin surface. [Table 7]

[0507] Example 12: Formulation of a dosage form containing a substantially complete vaginal microbiota preparation

[0508] Based on excipient data obtained from formulation studies using simulated vaginal fluid (Example 11) and viability studies performed (data not shown), a number of formulations containing substantially complete vaginal microbiota preparations (SCVMPs) were generated.

[0509] Lyophilized gel 1:

[0510] This formulation contained NaCMC and a substantially complete vaginal microbiota preparation and was subjected to lyophilization.

[0511] 30 mg of NaCMC was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, then lyophilized.

[0512] Lyophilized gel 2:

[0513] The formulation contained poloxamer and a substantially complete vaginal microbiota preparation and was subjected to lyophilization.

[0514] 240 mg of Poloxamer 407 was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, then lyophilized.

[0515] Cryogel 1:

[0516] The formulation contained hyaluronic acid and a substantially complete vaginal microbiota preparation and was subjected to freezing at -80°C.

[0517] 45 mg of hyaluronic acid was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, which was then frozen at -80°C.

[0518] Cryogel 2:

[0519] The formulation contained poloxamer and a substantially complete vaginal microbiota preparation and was subjected to freezing at -80°C.

[0520] 240 mg of Poloxamer 407 was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, which was then frozen at -80°C.

[0521] Lyophilized tablet 1:

[0522] This formulation contained NaCMC and a substantially complete vaginal microbiota preparation and was subjected to lyophilization.

[0523] 30 mg of NaCMC was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, then lyophilized. Once lyophilized, 300 mg of NaCMC was added to the material and compressed to 1 ton.

[0524] Lyophilized tablet 2:

[0525] The formulation contained poloxamer and a substantially complete vaginal microbiota preparation and was subjected to lyophilization.

[0526] 30 mg of Poloxamer 407 was weighed and transferred to a lyophilization vial. 1 SCVMP was transferred to a lyophilization vial. 1 mL of lactate buffer was used to rinse any remaining SCVMP out of the vial, which was then transferred to a lyophilization vial. The vial was capped and swirled by hand to form a uniform gel, then lyophilized. Once lyophilized, 300 mg of PVP (Kollidon) was added to the material and compressed to 0.5 tonnes.

[0527] PVA Film Disc

[0528] The formulation contained 12% PVA and a substantially intact vaginal microbiota preparation sandwiched between two PVA layers (approximately 0.25 mL of PVA per layer).

[0529] Following the formulation of substantially complete vaginal microbiota preparations, viable cell counts (VCC) were determined at t=0. Viability was also measured after 1 and 2 months, assessing both VCC and CFU. [Table 8]

[0530] The substantially complete vaginal microbiota preparation can be freeze-dried and formulated into, for example, gels and tablets, as well as other dosage forms that can be filled with freeze-dried products, such as capsules. The substantially complete vaginal microbiota preparation can also be formulated into freezable gels, and into freezable liquid media (e.g., including glycerol). The substantially complete vaginal microbiota preparation can also be formulated into (air-dried) films, which can be, for example, in the form of a disk. The loss of viability during the formulation stage ranges from about 0.5 log to 1 log, depending on the excipients and dosage form.

[0531] References Anahtar MN,Byrne EH,Doherty KE,Bowman BA,Yamamoto HS,Soumillon M,Padavattan N,Ismail N,Moodley A,Sabatini ME,Ghebremichael MS,Nusbaum C,Huttenhower C,Virgin HW,Ndung'u T,Dong KL,Walker BD,Fichorova RN,Kwon DS.2015.Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract.Immunity 42:965-976.https: / / doi.org / 10.1016 / j.immuni.2015.04.019 Lennard K,Dabee S,Barnabas SL,Havyarimana E,Blakney A,Jaumdally SZ,Botha G,Mkhize NN,Bekker L-G,Lewis DA,Gray G,Mulder N,Passmore J-AS,Jaspan HB.2018.Microbial composition predicts genital tract inflammation and persistent bacterial vaginosis in South African adolescent females.Infect Immun 86:e00410-17.https: / / doi.org / 10.1128 / IAI.00410-17. Masson L,Mlisana K,Little F,Werner L,Mkhize NN,Ronacher K,Gamieldien H,Williamson C,Mckinnon LR,Walzl G,Abdool Karim Q,Abdool Karim SS,Passmore J-AS.2014.Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection:a cross-sectional study.Sex Transm Infect 90:580-588.https: / / doi.org / 10.1136 / sextrans-2014-051601 Fichorova RN,Morrison CS,Chen P-L,Yamamoto HS,Govender Y,Junaid D,Ryan S,Kwok C,Chipato T,Salata RA,Doncel GF.2020.Aberrant cervical innate immunity predicts onset of dysbiosis and sexually transmitted infections in women of reproductive age.PLoS One 15:e0224359.https: / / doi.org / 10.1371 / journal.pone.0224359. Garrett N.,Mtshali,A.,Osman,F.et al.Impact of point-of-care testing and treatment of sexually transmitted infections and bacterial vaginosis on genital tract inflammatory cytokines in a cohort of young South African women.Sex Transm Infect.2021 Dec;97(8):555-565. Camargo Campos AC,Candido Murta EF,Michelin MA,and Reis C.Evaluation of Cytokines in Endocervical Secretion and Vaginal pH from Women with Bacterial Vaginosis or Human Papillomavirus.Obstetrics and Gynecology Volume 2012,Article ID 342075. Cherne MD, Cole AL, Newberry L, Schmidt-Owens M, Deichen M, Cole AM.Matrix Metalloproteinases Expressed in Response to Bacterial Vaginosis Disrupt the Endocervical Epithelium,Increasing Transmigration of HIV.Infect Immun.2020 Mar 23;88(4):e00041-20.

[0532] Enumerated Items 1. A vaginal delivery system comprising: a) a dosage form suitable for administering the composition to the vaginal cavity; and b) the vaginal delivery system, comprising a composition comprising a substantially complete vaginal microbiota preparation, wherein (i) the preparation comprises one to five bacterial species from the genus Lactobacillus, and (ii) the one to five bacterial species constitute about 80-99% of the preparation, and the composition further comprises a pharma- ceutically acceptable carrier or diluent.

[0533] 2. The vaginal delivery system of item 1, wherein the dosage form is an applicator, dispenser or suppository.

[0534] 3. The vaginal delivery system according to item 1 or 2, wherein the preparation contains about 80-99% Lactobacillus crispatus.

[0535] 4. The vaginal delivery system according to item 1 or 2, wherein the preparation contains about 80-99% Lactobacillus iners.

[0536] 5. The vaginal delivery system according to item 1 or 2, wherein the preparation contains about 80-99% Lactobacillus jensenii.

[0537] 6. The vaginal delivery system according to item 1 or 2, wherein the preparation comprises about 80-99% Lactobacillus gasseri.

[0538] 7. The vaginal delivery system according to item 1 or 2, wherein the preparation comprises about 80-99% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0539] 8. The vaginal delivery system of any one of the preceding items, wherein said preparation is substantially free of pathogens and pathogenic commensals.

[0540] 9. The vaginal delivery system of item 8, wherein the pathogen or pathogenic commensal is one or more of the genera Gardnerella, Atopobium, and Prevotella.

[0541] 10. The vaginal delivery system of item 8, wherein the preparation contains less than 5% of the genera Gardnerella, Atopobium, and Prevotella.

[0542] 11. The vaginal delivery system of any one of the preceding items, wherein the preparation is substantially free of antimicrobial resistance (AMR) genes.

[0543] 12. The vaginal delivery system of any one of the preceding items, wherein the preparation is substantially free of human sperm (spermatozoa).

[0544] 13. The vaginal delivery system of any one of the preceding items, wherein the composition further comprises a spermicide.

[0545] 14. The vaginal delivery system of any one of the preceding items, wherein the composition further comprises an acidifying agent.

[0546] 15. The vaginal delivery system of any one of the preceding items, wherein the composition further comprises lactic acid.

[0547] 16. The vaginal delivery system of any one of the preceding items, wherein the preparation comprises a glycan.

[0548] 17. The vaginal delivery system of item 16, wherein the glycan is mucus.

[0549] 18. The vaginal delivery system of any one of the preceding items, wherein the preparation comprises fewer than 20 species other than the Lactobacillus genus.

[0550] 19. The vaginal delivery system of any one of the preceding items, wherein the composition has a pH of 4.5 or less.

[0551] 20. The dosage form is 10 2 ~10 10 8. The vaginal delivery system of any one of the preceding claims, comprising CFU of lactobacilli.

[0552] 21. A vaginal delivery system according to any one of the preceding items, wherein the composition is dispensed or formulated into a dosage form.

[0553] 22. The vaginal delivery system of any one of the preceding items, wherein the dosage form is sterile packaged.

[0554] 23. The vaginal delivery system of item 2, wherein the applicator or dispenser comprises an open end (e.g., a tip) for insertion into the vaginal cavity and a dispensing end (e.g., a plunger or piston) for expelling the composition.

[0555] 24. A vaginal delivery system according to any one of the preceding items, wherein the preparation is obtained by a culture-independent method.

[0556] 25. The vaginal delivery system of any one of the preceding items, wherein the dosage form is calibrated (e.g., has a volume indication or maximum volume indication or control).

[0557] 26. The vaginal delivery system of any one of the preceding items, wherein the dosage form is a single-use or multi-use device.

[0558] 27. The vaginal delivery system of any one of the preceding items, wherein the dosage form is designed from an inert polymeric material, such as polystyrene or polypropylene.

[0559] 28. A pharmaceutical composition formulated for vaginal administration comprising a substantially complete vaginal microbiota preparation, wherein (i) the preparation comprises one to five bacterial species from the genus Lactobacillus, and (ii) the one to five bacterial species constitute about 80-99% of the preparation, and the composition further comprises a pharma- ceutical acceptable carrier or diluent.

[0560] 29. The pharmaceutical composition according to item 28, wherein the preparation contains 80-99% Lactobacillus crispatus.

[0561] 30. The pharmaceutical composition according to item 28, wherein the preparation contains 80-99% Lactobacillus iners.

[0562] 31. The pharmaceutical composition according to item 28, wherein the preparation contains about 80-99% Lactobacillus jensenii.

[0563] 32. The pharmaceutical composition according to item 28, wherein the preparation comprises about 80-99% Lactobacillus gasseri.

[0564] 33. The pharmaceutical composition according to item 28, wherein the preparation comprises about 80-99% of one, two, three or four of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.

[0565] 34. The pharmaceutical composition of any one of the preceding items, wherein the preparation is substantially free of pathogens and pathogenic commensals.

[0566] 35. The pharmaceutical composition according to item 34, wherein the pathogen or pathogenic symbiont is one or more of the genera Gardnerella, Atopobium and Prevotella.

[0567] 36. The pharmaceutical composition according to item 34, wherein the preparation contains less than 5% of the genera Gardnerella, Atopobium and Prevotella.

[0568] 37. The pharmaceutical composition of any one of the preceding items, wherein the preparation is substantially free of antimicrobial resistance (AMR) genes.

[0569] 38. The pharmaceutical composition of any one of the preceding items, wherein the preparation is substantially free of human sperm (spermatozoa).

[0570] 39. The pharmaceutical composition of any one of the preceding items, wherein the composition further comprises a spermicide.

[0571] 40. The pharmaceutical composition according to any one of the preceding items, wherein the composition further comprises an acidifying agent.

[0572] 41. The pharmaceutical composition of any one of the preceding items, wherein the composition further comprises lactic acid.

[0573] 42. The pharmaceutical composition of any one of the preceding items, wherein the preparation comprises a glycan.

[0574] 43. The pharmaceutical composition according to item 42, wherein the glycan is mucus.

[0575] 44. The pharmaceutical composition of any one of the preceding items, wherein the preparation includes fewer than 20 species other than the Lactobacillus genus.

[0576] 45. The pharmaceutical composition of any one of the preceding items, wherein the composition has a pH of 4.5 or less.

[0577] 46. ​​The pharmaceutical composition according to any one of the preceding items, wherein the preparation is obtained by a culture-independent method.

[0578] 47. A method for vaginally administering a composition comprising a substantially complete vaginal microbiota preparation to a human female subject in need thereof using a device, the device comprising an open end (e.g., a tip) for insertion into the vaginal cavity and a dispensing end (e.g., a plunger or piston) for expelling the composition through the open end, the method comprising: a) introducing said open end into the vaginal cavity; b) discharging said composition into said vaginal cavity, whereby the composition is administered to the vaginal cavity.

[0579] 48. The method according to item 47, wherein the device is an applicator or dispenser described in any one of items 2 to 27.

[0580] 49. The method according to item 47, wherein the composition is a pharmaceutical composition according to any one of items 28 to 46.

[0581] 50. The method of any of the preceding items, wherein expelling the composition comprises contacting the vaginal mucosa or endometrial surface.

[0582] 51. The method according to any one of items 47 to 50, wherein the subject is in need of improving vaginal health.

[0583] 52. The method according to any one of items 47 to 51, wherein administration to the subject is performed while the subject is in lithotomy position.

[0584] 53. The method of any one of items 47 to 52, wherein the composition is of suitable viscosity to allow a majority of the composition to remain in the vaginal cavity for at least 5, 10, 20, or 30 minutes when the subject is in an upright position.

[0585] 54. The method according to any one of items 47 to 53, wherein the device is calibrated (e.g., has a volume indication or a maximum volume indication or control).

[0586] 55. The device comprises about 10 2 ~10 10 55. The method according to any one of items 47 to 54, wherein CFU of lactobacilli are administered.

[0587] 56. The method according to any one of items 47 to 55, wherein the administration is performed once, twice or three times within 3 to 21 days.

[0588] 57. The method according to any one of items 47 to 55, wherein administration is performed once every 1 to 4 weeks over a period of 1...

Claims

1. 1. A pharmaceutical composition for use in a method of treating inflammation of the female urogenital tract in a human female subject, said subject exhibiting a dysbiotic microbiota of said urogenital tract, said method comprising administering to said subject an effective amount of said pharmaceutical composition, said pharmaceutical composition comprising a substantially complete vaginal microbiota preparation; The preparation comprises: (i) comprising one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species of the preparation; and (ii) containing less than 5% of the genera Gardnerella, Atopobium, and Prevotella; and (iii) comprising mucus, excretory epithelial cells, and / or vaginal discharge fluid, optionally wherein said mucus is mucus secreted by the cervix; The pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent.

2. 2. The pharmaceutical composition for use according to claim 1, characterized in that the subject has not been treated with antibiotics prior to or during treatment of the inflammation; and / or the subject has not been diagnosed with bacterial vaginosis; and / or the subject does not wear an intrauterine device (IUD), optionally wherein the IUD is a hormonal IUD or a non-hormonal IUD.

3. 3. A pharmaceutical composition for use according to claim 1 or 2, characterized in that it is administered to the vaginal cavity of the subject in need thereof, optionally characterized in that vaginal cleansing is performed before administering the pharmaceutical composition, optionally characterized in that the vaginal cleansing is performed with saline, lactic acid or a disinfectant, and further optionally the disinfectant is povidone iodine or chlorhexidine. (A) the composition does not contain an added cryoprotectant; and / or (B) the preparation further comprises: (a) lactic acid at a concentration of about 0.5-2%, optionally at a concentration of about 1-1.5%; and / or Approximately 80-99.9% of all detectable bacterial species in the preparation (a) Lactobacillus crispatus; (b) Lactobacillus iners; (c) Lactobacillus crispatus and Lactobacillus iners; (d) Lactobacillus crispatus and Lactobacillus jensenii; (e) Lactobacillus crispatus, Lactobacillus iners and Lactobacillus jensenii; (f) Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii; (g) Lactobacillus iners and Lactobacillus jensenii; (h) Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri; (i) Lactobacillus iners and Lactobacillus gasseri; or (j) A pharmaceutical composition for use according to any one of claims 1 to 3, comprising Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri.

5. 5. The pharmaceutical composition for use according to claim 4, wherein for (c) to (j), Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and / or Lactobacillus gasseri are present in a greater relative amount than one or more of the other vaginal lactobacilli present in the preparation.

6. wherein treating said inflammation treats or reduces the risk of said subject developing a disease, disorder or unwanted condition associated with inflammation of the female genitourinary tract; and / or 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein less than 10% of all detectable bacterial species of the dysbiotic vaginal microbiota in the urogenital tract of the female subject belong to the genus Lactobacillus and at least 20%, 30%, 40%, 50%, 60%, 70% or more of all detectable bacterial species of the dysbiotic vaginal microbiota are pathogens or pathogenic commensals selected from the group consisting of the genera Gardnerella, Atopobium and Prevotella.

7. A substantially complete vaginal microbiota preparation or a pharmaceutical composition comprising the same, said preparation comprising (i) comprising one, two, three or four bacterial species of the genus Lactobacillus selected from Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, constituting about 80-99.9% of all detectable bacterial species of the preparation; and (ii) containing less than 5% of the genera Gardnerella, Atopobium, and Prevotella; and (iii) comprising mucus, excretory epithelial cells, and / or vaginal discharge fluid, optionally wherein said mucus is mucus secreted by the cervix; The preparation and / or pharmaceutical composition comprises a pharma- ceutically acceptable carrier or diluent.

8. (A) the preparation or pharmaceutical composition is in the form of a suspension, spray, gel, cream, ointment, semisolid foam, film, powder, capsule, douche or douche solution, egg, vaginal insert, tampon, tablet or microencapsulated product; and / or (B) the preparation or pharmaceutical composition comprises at least about 10 6 viable bacterial cells, optionally at least about 10 7 viable bacterial cells; and / or (C) the pharma- ceutically acceptable carrier or diluent is saline; and / or (D) the preparation or pharmaceutical composition comprises additional lactobacilli other than Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, or Lactobacillus gasseri, wherein the additional lactobacilli are present at a concentration of about 0.01-1% of all detectable bacterial species in the preparation; and / or (E) The preparation or pharmaceutical composition according to claim 7, wherein the preparation is obtained by a culture-independent method.

9. A dosage form comprising the pharmaceutical composition or the substantially complete vaginal microbiota preparation according to claim 7 or 8, formulated for vaginal administration, said dosage form being a solid, semi-solid, liquid or film-forming formulation; In some cases: (i) the solid dosage form is selected from a capsule, a tablet, optionally comprising polyvinylpyrrolidone (PVP) or sodium carboxymethylcellulose (Na-CMC), a lyophilized gel, optionally comprising sodium carboxymethylcellulose (Na-CMC) or hyaluronic acid, and a film, optionally comprising polyvinyl alcohol (PVA); or (ii) the semi-solid dosage form is selected from a suppository, an ointment, a preformed gel, a cryogel, a cream, or a foam; or (iii) The dosage form, wherein the preformed gel or cryogel comprises hyaluronic acid.

10. A vaginal delivery system for administration to the vaginal cavity comprising a pharmaceutical composition or a substantially complete vaginal microbiota preparation according to any one of claims 7 to 8 in a dosage form suitable for an applicator, dispenser or suppository. (i) the pharmaceutical composition or the substantially complete vaginal microbiota preparation is in liquid or semi-solid form, optionally the pharmaceutical composition or the substantially complete vaginal microbiota preparation is in gel or film form; or 11. The vaginal delivery system of claim 10, wherein (ii) the pharmaceutical composition or the substantially complete vaginal microbiota preparation is lyophilized, optionally with a gel-forming excipient, optionally formulated in a storage container separate from the applicator or dispenser, and further optionally, the lyophilized pharmaceutical composition or the lyophilized substantially complete vaginal microbiota preparation is reconstituted and then combined with the applicator or dispenser.

12. 10. A method for intravaginal administration of a pharmaceutical composition or a substantially complete vaginal microbiota preparation according to claim 7 or 8 to a human female subject, wherein the composition or preparation is administered using a device, the device comprising an open end for insertion into the vaginal cavity and a dispensing end for expelling the composition or preparation through the open end, the method comprising: a) introducing the open end into the vaginal cavity; b) discharging said composition or preparation into said vaginal cavity; thereby administering the composition or preparation to the vaginal cavity.

13. 1. A method for producing a substantially complete vaginal microbiota preparation, comprising: A. Providing a microbiota sample from a donor female urogenital tract; step A includes one, two, or three of the following steps (1), (2), (3), or any combination thereof, and both steps (4) and (5): (1) adding a diluent to the microbiota sample to create a diluted sample; (2) removing a portion of the diluted microbiota sample for testing (e.g., nucleic acid sequencing); (3) pre-cooling the remaining microbiota sample for refrigeration or freezing; (4) storing the refrigerated or frozen microbiota sample in isolation; (5) storing the refrigerated or frozen microbiota sample in isolation until any combination of: testing the donor (e.g., testing blood, and / or ectocervical secretions, and / or urine samples) to exclude the substantial presence of one or more infectious pathogens; (b) confirming the composition and viability of the microbiota; or (c) further confirming the health status of the female donor by multiple post-screening tests performed within 30 to 90 days after donation; and B. The method further comprising releasing the refrigerated or frozen microbiota sample from isolation to define the substantially complete vaginal microbiota preparation.

14. A substantially complete vaginal microbiota preparation obtainable by the method according to claim 13.

15. 1. An isolated and substantially complete vaginal microbiota preparation suitable for vaginal administration, comprising: (i) the preparation contains Lactobacillus iners and Lactobacillus gasseri, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, the preparation contains less than 5% of the genera Gardnerella, Atopobium, and Prevotella, and the preparation contains mucus, excreted epithelial cells, and / or vaginal leakage fluid, optionally wherein the mucus is mucus secreted by the cervix; or (ii) the preparation contains Lactobacillus iners and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, the preparation contains less than 5% of the genera Gardnerella, Atopobium, and Prevotella, and the preparation contains mucus, excretory epithelial cells, and / or vaginal leakage fluid, optionally wherein the mucus is mucus secreted by the cervix; or (iii) the preparation comprises Lactobacillus iners and Lactobacillus crispatus, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, the preparation comprises less than 5% of the genera Gardnerella, Atopobium, and Prevotella, and the preparation comprises mucus, excreted epithelial cells, and / or vaginal leakage fluid, optionally wherein the mucus is mucus secreted by the cervix; or (iv) the preparation contains Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, the preparation contains less than 5% of the genera Gardnerella, Atopobium, and Prevotella, and the preparation contains mucus, excreted epithelial cells, and / or vaginal leakage fluid, optionally wherein the mucus is mucus secreted by the cervix; or (v) the preparation comprises Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii, which together constitute about 80-99.9% of all detectable bacterial species in the preparation, and further comprises less than 5% of the genera Gardnerella, Atopobium, and Prevotella, and the preparation comprises mucus, excretory epithelial cells, and / or vaginal leakage fluid, optionally wherein the mucus is mucus secreted by the cervix.

Images