Vaginal microbiome composition

Abstract

Compositions and methods for treating patients are disclosed. Exemplary compositions can include a vaginal microbiota composition. The vaginal microbiota composition can include a mixture of bacteria. The mixture of bacteria can include Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus.

Description

[Technical Field]

[0001] This disclosure relates to compositions and methods for treating patients. [Background technology]

[0002] A wide variety of compositions and methods have been developed to treat diseases and / or conditions, such as those of the reproductive system, gastrointestinal tract, liver, and immune system. Among the known compositions and methods, each has its own specific advantages and disadvantages. There is an ongoing need to provide alternative compositions and methods for treating diseases and / or conditions. [Overview of the project] [Means for solving the problem]

[0003] This disclosure provides alternative designs, materials, manufacturing methods, and uses for compositions and methods for treating patients. Vaginal microbiome compositions are disclosed. The vaginal microbiome composition includes Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus helveticus, and Lactobacillus plantarum. It contains a mixture of bacteria including Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, and Lactobacillus delbrueckii.

[0004] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 25-75% Lactobacillus crispatus.

[0005] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 30-60% Lactobacillus crispatus.

[0006] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 40% Lactobacillus crispatus.

[0007] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 10 - 40% Lactobacillus iners.

[0008] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 15 - 30% Lactobacillus iners.

[0009] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 20% Lactobacillus iners.

[0010] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 1 - 10% Lactobacillus gasseri.

[0011] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 2 - 8% Lactobacillus gasseri.

[0012] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 5% Lactobacillus gasseri.

[0013] Instead of any of the above embodiments, or in addition thereto, the bacterial mixture contains 1 - 10% Lactobacillus jensenii.

[0014] In place of or in addition to any of the above embodiments, the bacterial mixture may contain 2-8% Lactobacillus jensenii.

[0015] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 5% Lactobacillus jensenii.

[0016] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus reuteri.

[0017] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5-4% Lactobacillus reuteri.

[0018] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus reuteri.

[0019] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus acidophilus.

[0020] In place of or in addition to any of the above embodiments, the bacterial mixture contains approximately 0.5–4% Lactobacillus acidophilus.

[0021] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus acidophilus.

[0022] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus vaginalis.

[0023] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5–4% Lactobacillus vaginalis.

[0024] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus vaginalis.

[0025] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus rhamnosus.

[0026] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5–4% Lactobacillus rhamnosus.

[0027] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus rhamnosus.

[0028] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1 to 5% Lactobacillus johnsonii.

[0029] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5-4% Lactobacillus johnsonii.

[0030] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus johnsonii.

[0031] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus helveticus.

[0032] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5-4% Lactobacillus helveticus.

[0033] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus helveticus.

[0034] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1-5% Lactobacillus plantarum.

[0035] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5–4% Lactobacillus plantarum.

[0036] In place of or in addition to any of the above embodiments, the bacterial mixture contains about 1% Lactobacillus plantarum.

[0037] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 0.1–5% Lactobacillus fermentum.

[0038] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.5–4% Lactobacillus fermentum.

[0039] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus fermentum.

[0040] In place of or in addition to any of the above embodiments, the bacterial mixture contains 0.1–5% Lactobacillus salivarius.

[0041] In place of or in addition to any of the above embodiments, the bacterial mixture may contain 0.5–4% Lactobacillus salivarius.

[0042] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus salivarius.

[0043] In place of or in addition to any of the above embodiments, the bacterial mixture may contain 0.1-5% Lactobacillus delbrueckii.

[0044] In place of or in addition to any of the above embodiments, the bacterial mixture may contain 0.5–4% Lactobacillus delbrueckii.

[0045] In place of or in addition to any of the above embodiments, the bacterial mixture comprises 1% Lactobacillus delbrueckii.

[0046] In place of or in addition to any of the above embodiments, the bacterial mixture comprises one or more freeze-dried bacterial strains.

[0047] In addition to or instead of any of the embodiments described above, the bacterial mixture is placed inside a capsule.

[0048] In place of, or in addition to, any of the above embodiments, the bacterial mixture is placed in the suppository.

[0049] In place of, or in addition to, any of the above embodiments, the bacterial mixture is placed in a soluble shell.

[0050] A method for treating an infectious disease is disclosed. The method comprises administering a vaginal microbiome composition of any one of the above embodiments to a patient having an infectious disease.

[0051] In lieu of or in addition to any of the embodiments described above, the infection includes one or more of the following: bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, and gynecological cancer.

[0052] A method for treating a patient is disclosed. This method involves administering to the patient one of the vaginal microbiome compositions of the above embodiments.

[0053] In place of or in addition to any of the above embodiments, administering a vaginal microbiome composition to a patient includes one or more of the following: preventing premature birth, preventing miscarriage, treating infertility, treating interstitial cystitis, and treating polycystic ovary syndrome.

[0054] A vaginal microbiome composition is disclosed. The vaginal microbiome composition comprises a suppository and a mixture of freeze-dried bacteria placed inside the suppository, the mixture of freeze-dried bacteria being Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus helveticus This includes Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, and Lactobacillus delbrueckii.

[0055] A vaginal microbiome composition bank is disclosed. The vaginal microbiome composition bank includes a first container containing a first population of one or more microorganisms arranged therein, the first population of which is collected from a first donor; a second container containing a second population of one or more microorganisms arranged therein, the second population of which is collected from a second donor; and an index system including donor data from the first and second donors.

[0056] A vaginal microbiome composition bank is disclosed. The vaginal microbiome composition bank includes a first container containing a first population of one or more microorganisms, the first population of which is collected from a first donor, and a second container containing a second population of one or more microorganisms, the second population of which is collected from a second donor.

[0057] In place of or in addition to any of the above embodiments, the vaginal microbiome composition bank includes an index system containing donor data from a first donor and a second donor.

[0058] A vaginal microbiome composition bank is disclosed. The vaginal microbiome composition bank includes a first container containing a first population of one or more freeze-dried microorganisms, the first population of which is collected from a first donor; a second container containing a second population of one or more freeze-dried microorganisms, the second population of which is collected from a second donor; and an index system including donor data from the first and second donors.

[0059] A vaginal microbiome composition bank is disclosed. The vaginal microbiome composition bank includes a first container containing a first population of one or more freeze-dried microorganisms, the first population of which is collected from a first donor, and a second container containing a second population of one or more freeze-dried microorganisms, the second population of which is collected from a second donor.

[0060] In place of or in addition to any of the above embodiments, the vaginal microbiome composition bank includes an index system containing donor data from a first donor and a second donor.

[0061] The above-described outlines of some embodiments are not intended to describe each disclosed embodiment or all implementations of this disclosure. The detailed descriptions that follow illustrate these embodiments more specifically. [Modes for carrying out the invention]

[0062] The terms defined below shall apply unless otherwise provided in the claims or elsewhere in this specification.

[0063] All numerical values ​​are assumed to be qualified by the term “about,” whether expressly indicated herein or otherwise. The term “about” generally refers to a range of numerical values ​​that a person skilled in the art would consider equivalent to the stated value (e.g., having the same function or result). In many cases, the term “about” may include digits rounded to the maximum number of significant figures.

[0064] Numerical ranges specified by endpoints include all numbers within that range (for example, 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0065] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise. As used herein and in the appended claims, the term "or" is used to mean "and / or" unless the context explicitly indicates otherwise.

[0066] It should be noted that references in the specification such as “a certain embodiment,” “several embodiments,” or “other embodiments” indicate that the embodiments described may include one or more specific features, structures, and / or characteristics. However, such descriptions do not necessarily mean that all embodiments include a particular feature, structure, and / or characteristic. Furthermore, if a particular feature, structure, and / or characteristic is described in relation to one embodiment, it should be understood that such feature, structure, and / or characteristic may also be used in relation to other embodiments, whether explicitly stated or not, unless the opposite is explicitly stated.

[0067] The human microbiome (or human microflora) is an aggregate of microorganisms that reside on the surface and deep layers of human skin, in saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tract, genitourinary tract, and / or vaginal canal, including microorganisms associated with reproductive health, such as the placental microbiome. The human microflora consists of bacteria, but may also include fungal phages, viruses, and archaea. While some of these organisms perform useful tasks for the human host, the functions of the majority of organisms that make up the human microflora are still under investigation. Under normal circumstances, these microorganisms do not cause disease in the human host; instead, they are involved in maintaining health.

[0068] The vaginal microbiota can play a role in many different health conditions. Some of these conditions include infections such as bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infections, and / or sexually transmitted infections. Other conditions include gynecological cancers (e.g., cervical cancer), premature birth, miscarriage, infertility, interstitial cystitis, and polycystic ovary syndrome. Restoring, normalizing, and / or otherwise shifting the composition of the vaginal microbiota may help alleviate these and other conditions. This specification discloses compositions and methods for treating patients. At least some of the compositions include vaginal microbiota compositions. At least some of the methods include methods for treating bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infections, sexually transmitted infections, gynecological cancers (e.g., cervical cancer), preventing premature birth, preventing miscarriage, treating infertility, treating interstitial cystitis, and treating polycystic ovary syndrome.

[0069] In healthy women of reproductive age, bacteria of the Lactobacillus genus tend to dominate the vaginal microbiome. Four species have been consistently identified across women: Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii. These species are listed in order of their dominance frequency among Caucasian, Black, Asian, and Hispanic women. In general, Lactobacillus dominance in the vaginal microbiome is associated with health status. Dominance by a single genus or species creates a less diverse microbiome. Increased diversity in the vaginal microbiome is associated with a higher risk of ill health and infection. For example, bacteria from the genera Sneathia, Megasphaera, Atopobium, Peptoniphilus, Dialister, Prevotella, and / or Gardnerella may tend to correlate with an unhealthy vaginal microbiome because they are identified in women using other indicators of vaginal ill health (high Nugent score and vaginal pH). However, these generalizations may differ in different populations.

[0070] The vaginal microbiome can vary among pre-pubescent, pre-menopausal, and post-menopausal women. For example, pre-pubescent women may have a relatively low relative abundance of Lactobacillus and relatively high microbial diversity (as well as relatively thin vaginal epithelium, relatively thin mucosa, and relatively low levels of glycogen). Pre-menopausal women may have a relatively high abundance of Lactobacillus and relatively low microbial diversity (as well as relatively thick vaginal epithelium, relatively thick mucosa, relatively high estrogen levels, and relatively low levels of glycogen). Post-menopausal women may have a moderate relative abundance of Lactobacillus and relatively low or lower microbial diversity (as well as relatively thin vaginal epithelium, relatively thin mucosa, relatively moderate estrogen levels, and relatively moderate levels of glycogen). Post-menopausal women exhibiting symptoms of vaginal infections (e.g., bacterial vaginosis) may have a lower abundance of Lactobacillus and relatively high microbial diversity. In summary, compositions containing or otherwise tending to increase the relative abundance of Lactobacillus, as well as compositions that help reduce microbial diversity, may be useful.

[0071] In at least some examples, the compositions disclosed herein (e.g., vaginal microbiome compositions) utilize vaginal microbiomes collected from vaginal microbiome donors. However, other sources of vaginal microbiomes, including bacterial cultures, are possible. Potential donors can be identified and may undergo numerous screening processes. In some cases, potential donors may be screened using health history questionnaires. Exemplary questionnaires may be the same as or similar to those used by the Red Cross for screening potential blood donors. In some and other examples, potential donors may be screened for common infections and other conditions. Such screenings may include blood tests, stool tests, urine tests, vaginal swab tests, etc. Such tests may include testing for the presence of HIV, hepatitis (types A, B, and / or C), syphilis, Clostridium difficile (C. difficile), bacterial pathogens, eggs and parasites, and / or other similar substances. These are just examples. The donor's health can be monitored by performing blood tests, analyzing stool samples, analyzing urine samples, analyzing vaginal discharge, and regularly updating their health history. Vaginal swabs may be collected to assess vaginal health through Gram staining and Nugent scoring, white blood cell count, and detection of yeast presence. Urine and / or vaginal swabs may be collected to test for sexually transmitted infections.

[0072] After a potential donor is determined to be sufficiently healthy to provide samples, the donor is provided with a sample collection kit (e.g., a self-collection kit). The sample collection kit may include a donor information sheet, a vaginal DNA / RNA collection kit for sequencing analysis (e.g., the OMNIgene vaginal collection kit ORM-130, commercially available from DNAgeneotek®), and a sample collection and transport kit (e.g., the eSwab 480C Copan Liquid Amies Elution Swab Collection and Transport System, commercially available from Copan Diagnostics). The donor may be asked to sign an informed consent form. Each donor may be asked to collect two samples. The first sample may be used for sequencing analysis. For example, after handwashing, a vaginal sample can be collected using a swab from the vaginal DNA / RNA collection kit by inserting the swab several inches into the vagina and swab the vaginal wall for approximately 20 seconds. The swab can then be inserted into a tube containing stabilizing buffer, and the tube / sample can be further analyzed. The second sample may be used to culture vaginal microorganisms. For example, a vaginal sample can be collected using a swab from a sample collection and transport kit. The swab can then be inserted into a tube containing liquid amies solution (for example, this can preserve aerobic, anaerobic, and hard-to-culture bacteria for up to 48 hours). At the time of collection, the donor places the sample tube and material inside a biohazard bag and then leaves the sample at the providing facility.

[0073] To prepare the sample for sequencing (e.g., shotgun sequencing), a first protease (e.g., QIAGEN protease) can be rehydrated using sterile PCR-certified water to produce an 80 mg / mL solution (this may involve inverting the sample more than 10 times). The sample (e.g., vaginal DNA / RNA collection kit sample) can be removed from the refrigerator and vortexed for 30 seconds. The collection tube can be shaken three times to bring the solution containing the sample to the bottom of the tube. 5 microliters of rehydrated protease can be added to the collection tube containing the vaginal swab sample. The sample can be inverted more than 10 times. The sample can then be incubated in a 50°C incubator for 2 hours. The sample can then be vortexed for 30 seconds. The collection tube can then be shaken three times to bring the solution containing the sample to the bottom of the tube. The swab can be removed from the tube (this may involve pressing the swab against one side of the tube to collect the sample absorbed by the swab). A 500-microliter sample can be divided into two 2 mL cryogenic storage vials, which can then be stored at -80°C until ready for sequence analysis (which can be performed at a suitable processing / analysis facility such as Divergent™ in St. Paul, MN).

[0074] To prepare a sample for culture, the sample (e.g., sample collection and transport kit sample) can be placed in the airlock of an anaerobic chamber and introduced into the chamber. With the swab still inside, the tube can be vigorously vortexed for 5 seconds to release the sample from the tip of the swab. This can be undiluted or 10 0 This is a dilution. The swab can then be removed. The sample can undergo a 10-fold serial dilution by transferring 100 microliters to a tube containing 900 microliters of sterile physiological saline (0.9%). The sample may be vigorously vortexed for 5 seconds and / or mixed by inversion. This is a 10 -1 It is a dilution. 100 microliters -1The dilution can be transferred to a new tube containing 900 microliters of sterile physiological saline (0.9%). The sample may be mixed by vigorously vortexing for 5 seconds and / or by inversion. This is 10 -2 It is a dilution. The sample is 10 -6 This process can be repeated until the substance is diluted to its original concentration.

[0075] In three ways, the smear culture plate is placed on a CDC anaerobic bacteria 5% sheep blood agar plate, a De Man, Rogosa and Sharpe (MRS) agar plate, and a 50% MRS agar plate, with the following dilution tubes: 10 -3 , 10 -4 , 10 -5 It can be prepared from the following: In short, 100 μL can be directly transferred from a suitable tube onto the surface of an agar plate and spread across the entire plate surface using a sterile L-shaped spreader and plate rotator. The plate can be incubated at 35°C under anaerobic conditions for 24 to 120 hours. The plate can be checked daily for the appearance of new colonies. Once the plates begin to dry, wrap them with Parafilm. If colonies can be observed, the number and morphology of the colonies can be recorded (this may include taking capture photographs of the plates to aid in documented verification of colony morphology). Individual colonies can be selected for further characterization and streaked onto new plates. If necessary, selective media such as Bifidobacterium selective agar (BSA), Enterococcus cell agar (ECA), Cetrimide agar (CA), and Mannitol salt agar (MSA) may be used.

[0076] In some cases, Gram staining may be used. If this is done, the specimen can be applied to a glass slide. When staining liquid cultures, the culture can be applied directly to the slide as a smear using a sterile inoculation loop. When staining colonies from plates, a droplet of sterile saline can be applied to the center of the slide using an inoculation loop, and then a small amount of a single colony can be collected and added to the droplet. If possible, the droplet and the specimen can be gently mixed to obtain a thin, uniform smear. The droplet can be dried. The slide can be methanol-fixed by pouring anhydrous methanol for 1-2 minutes and then rinsing with tap water. A primary stain (e.g., crystal violet) can be poured onto the fixed smear for 1 minute. The primary stain can be removed by gently rinsing with tap water. A secondary stain / modant (e.g., Gram iodine) can be poured onto the fixed smear for 1 minute and then gently rinsing with tap water. The slide can be destained until the solvent moving away from the slide becomes colorless (e.g., about 3-60 seconds), and the slide can be gently washed with tap water. Then, a counterstain can be poured onto the slide for 1 minute (e.g., safranin; if the result of the initial counterstain is unsatisfactory, it can be replaced with basic fuchsin). The slide can be washed with tap water and dried. The smear can be examined under an oil immersion lens, and the results / observations can be recorded.

[0077] Other sample tests can be performed, including a catalase test, which may involve adding hydrogen peroxide to differentiate between staphylococci and streptococci. Another sample test that can be performed is a plasma coagulation test, which may involve adding plasma to differentiate between staphylococci and streptococci.

[0078] Isolates determined to be Lactobacillus may be further characterized to determine their antibiotic susceptibility profiles. This may involve suspending Lactobacillus colonies from a plate cultured overnight in a broth consisting of 90% iso-sensitest broth and 10% MRS broth until the suspension is equal to 1 part McFarland turbidity standard solution. A sterile swab may then be immersed in the inoculum and pressed against the wall of a tube to remove excess liquid. The swab may be streaked across the entire agar surface (rotated two or more times to ensure uniform distribution). After absorbing excess moisture, a minimum inhibitory concentration (MIC) test strip may be applied (for example, this may involve applying the test strip with the scale facing upwards and the code on the test strip facing outwards). The test strip may be pressed against the agar surface with the entire length of the antibiotic gradient in complete contact with the agar surface and repositioned as needed. The plate may be inverted and incubated at 35°C for 20–48 hours (or longer). These results can be interpreted.

[0079] Isolated strains can be cryopreserved and stored. This may involve inoculating a culture overnight with well-isolated colonies selected from a streaked plate. If many colonies are selected, a 96-well plate format may be used. A suitable broth may be used (e.g., Lactobacillus tends to use MRS). The culture can be incubated overnight at 35°C in an anaerobic chamber incubator. If the culture is not cloudy by the following morning, an additional incubation time may be taken. Once the culture appears visually cloudy, 200-300 microliters can be transferred to a new 96-well plate containing a cryoprotectant (e.g., 50-60 microliters of 50% glycerol). The final glycerol concentration may be 10%. The 96-well plate can be sealed and stored in a freezer at -80°C.

[0080] As suggested herein, individual microbial strains (e.g., individual strains or bacterial species) can be isolated from a swab. This may involve one or more processes, for example, plating the strain on a selective agar medium for the desired strain, and then "purifying" the strain as a single colony isolate. Individual microbial strains can be grown / cultured and the species can be identified / characterized by sequencing, biochemical tests, colony morphology, cell morphology, microscopic mortality, and susceptibility to different representative antibiotics. Microbial strains can vary.In some cases, the microbial strains include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus helveticus, and Lactobacillus plantarum. This may include one or more of the following: plantarum, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii, Gardnerella vaginalis, non-Lactobacillus bacteria, Atopobium vaginae, Prevotella bivia, other species of these genera, Megasphaera, Sneathia, Dialister, Peptoniphilus, fungi, and combinations thereof.

[0081] Vaginal microbiome compositions are thought to consist of a single isolated strain or a mixture of two or more isolated strains. Exemplary compositions may contain about 20–85% Lactobacillus crispatus, or about 25–75% Lactobacillus crispatus, or about 30–60% Lactobacillus crispatus, or about 40% Lactobacillus crispatus. In some of these, and in other examples (for example, in addition to or instead of any one or more of the microorganisms listed above), the exemplary composition may contain about 10–50% Lactobacillus iners, or about 10–40% Lactobacillus iners, or about 15–30% Lactobacillus iners, or about 20% Lactobacillus iners. In some of these, and in other examples (for example, in addition to or in place of one or more of the microorganisms listed above), the exemplary composition may contain about 1-10% Lactobacillus gasseri, or about 2-8% Lactobacillus gasseri, or about 5% Lactobacillus gasseri. In some of these, and in other examples (for example, in addition to or in place of one or more of the microorganisms listed above), the exemplary composition may contain about 1-10% Lactobacillus jensenii, or about 2-8% Lactobacillus jensenii, or about 5% Lactobacillus jensenii.In some of these, and in other examples (for example, in addition to or in place of one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus reuteri, or about 0.5–4% Lactobacillus reuteri, or about 1% Lactobacillus reuteri. In some of these, and in other examples (for example, in addition to or in place of one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus acidophilus, or about 0.5–4% Lactobacillus acidophilus, or about 1% Lactobacillus acidophilus. In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus vaginalis, or about 0.5–4% Lactobacillus vaginalis, or about 1% Lactobacillus vaginalis. In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus rhamnosus, or about 0.5–4% Lactobacillus rhamnosus, or about 1% Lactobacillus rhamnosus.In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus johnsonii, or about 0.5–4% Lactobacillus johnsonii, or about 1% Lactobacillus johnsonii. In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus helveticus, or about 0.5–4% Lactobacillus helveticus, or about 1% Lactobacillus helveticus. In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus plantarum, or about 0.5–4% Lactobacillus plantarum, or about 1% Lactobacillus plantarum. In some of these, and in other examples (for example, in addition to or in place of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus fermentum, or about 0.5–4% Lactobacillus fermentum, or about 1% Lactobacillus fermentum.In some of these, and in other examples (for example, in addition to or instead of any one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus salivarius, or about 0.5–4% Lactobacillus salivarius, or about 1% Lactobacillus salivarius. In some of these, and in other examples (for example, in addition to or in place of one or more of the microorganisms listed above), the exemplary composition may contain about 0.1–5% Lactobacillus delbrueckii, or about 0.5–4% Lactobacillus delbrueckii, or about 1% Lactobacillus delbrueckii.

[0082] An example composition is approximately 40% Lactobacillus crispatus, approximately 20% Lactobacillus iners, approximately 5% Lactobacillus gasseri, approximately 5% Lactobacillus jensenii, approximately 1% Lactobacillus reuteri, approximately 1% Lactobacillus acidophilus, approximately 1% Lactobacillus vaginalis, approximately 1% Lactobacillus rhamnosus, and approximately 1% Lactobacillus johnsonii. It may also contain approximately 1% Lactobacillus helveticus, approximately 1% Lactobacillus plantarum, approximately 1% Lactobacillus fermentum, approximately 1% Lactobacillus salivarius, and approximately 1% Lactobacillus delbrueckii.

[0083] Exemplary compositions may include Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus. For example, the composition may include about 20 - 85% Lactobacillus crispatus, or about 25 - 75% Lactobacillus crispatus, or about 30 - 60% Lactobacillus crispatus, or about 40% Lactobacillus crispatus. The composition may include about 1 - 10% Lactobacillus gasseri, or about 2 - 8% Lactobacillus gasseri, or about 5% Lactobacillus gasseri. The composition may include about 1 - 10% Lactobacillus jensenii, or about 2 - 8% Lactobacillus jensenii, or about 5% Lactobacillus jensenii. The composition may include about 0.1 - 5% Lactobacillus rhamnosus, or about 0.5 - 4% Lactobacillus rhamnosus, or about 1% Lactobacillus rhamnosus. In some cases, each of the components may be lyophilized. The total number of microorganisms in the exemplary composition is about 1×10 5 ~1×10 15 CFU, or about 1×10 6 ~1×10 12 CFU, or about 1×10 7 ~1×10 10The order may be CFU. The composition can be placed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or other similar. In some cases, the composition may be administered orally. In other cases, the composition may be administered topically (e.g., by inserting a capsule, suppository, soluble shell, etc., into the vagina).

[0084] Exemplary compositions may include strains of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus having the desired properties. For example, each strain of the component may be selected to have pH-lowering activity, produce lactic acid, and have inhibitory activity against other microorganisms having other unique activities, in combination thereof, and / or other similar properties.

[0085] Exemplary compositions may include a mixture of lyophilized Lactobacillus crispatus, lyophilized Lactobacillus gasseri, lyophilized Lactobacillus jensenii, and lyophilized Lactobacillus rhamnosus. Each component may be isolated from a donor. For example, a composition may contain about 20–85% Lactobacillus crispatus, or about 25–75% Lactobacillus crispatus, or about 30–60% Lactobacillus crispatus, or about 40% Lactobacillus crispatus. The composition may contain approximately 1-10% Lactobacillus gasseri, or approximately 2-8% Lactobacillus gasseri, or approximately 5% Lactobacillus gasseri. The composition may also contain approximately 1-10% Lactobacillus jensenii, or approximately 2-8% Lactobacillus jensenii, or approximately 5% Lactobacillus jensenii. The composition may contain approximately 0.1–5% Lactobacillus rhamnosus, or approximately 0.5–4% Lactobacillus rhamnosus, or approximately 1% Lactobacillus rhamnosus. Each component may be derived / isolated from a donor sample. The total number of microorganisms in the exemplary composition is approximately 1 × 10⁶. 5 ~1 × 10 15 CFU, or approximately 1 × 10⁻⁶ 6 ~1 × 10 12CFU, or approximately 1 × 10⁻⁶ 7 ~1 × 10 10 The order may be CFU. The composition can be placed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or other similar. In some cases, the composition may be administered orally. In other cases, the composition may be administered topically (e.g., by inserting a capsule, suppository, soluble shell, etc., into the vagina).

[0086] Exemplary compositions may include a mixture of lyophilized Lactobacillus crispatus, lyophilized Lactobacillus gasseri, lyophilized Lactobacillus jensenii, and lyophilized Lactobacillus rhamnosus. Each component may be isolated from a donor. For example, a composition may contain about 20–85% Lactobacillus crispatus, or about 25–75% Lactobacillus crispatus, or about 30–60% Lactobacillus crispatus, or about 40% Lactobacillus crispatus. The composition may contain approximately 1-10% Lactobacillus gasseri, or approximately 2-8% Lactobacillus gasseri, or approximately 5% Lactobacillus gasseri. The composition may also contain approximately 1-10% Lactobacillus jensenii, or approximately 2-8% Lactobacillus jensenii, or approximately 5% Lactobacillus jensenii. The composition may contain about 0.1–5% Lactobacillus rhamnosus, or about 0.5–4% Lactobacillus rhamnosus, or about 1% Lactobacillus rhamnosus. Each component may be derived from a bacterial culture. The total number of microorganisms in the exemplary composition is about 1 × 10⁶ 5 ~1 × 10 15 CFU, or approximately 1 × 10⁻⁶ 6 ~1 × 10 12CFU, or approximately 1 × 10⁻⁶ 7 ~1 × 10 10 The order may be CFU. The composition can be placed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or other similar. In some cases, the composition may be administered orally. In other cases, the composition may be administered topically (e.g., by inserting a capsule, suppository, soluble shell, etc., into the vagina).

[0087] Exemplary compositions may include a mixture of lyophilized Lactobacillus crispatus, lyophilized Lactobacillus gasseri, lyophilized Lactobacillus jensenii, and lyophilized Lactobacillus rhamnosus. Each component may be isolated from a donor. For example, a composition may contain about 20–85% Lactobacillus crispatus, or about 25–75% Lactobacillus crispatus, or about 30–60% Lactobacillus crispatus, or about 40% Lactobacillus crispatus. The composition may contain approximately 1-10% Lactobacillus gasseri, or approximately 2-8% Lactobacillus gasseri, or approximately 5% Lactobacillus gasseri. The composition may also contain approximately 1-10% Lactobacillus jensenii, or approximately 2-8% Lactobacillus jensenii, or approximately 5% Lactobacillus jensenii. The composition may contain approximately 0.1–5% Lactobacillus rhamnosus, or approximately 0.5–4% Lactobacillus rhamnosus, or approximately 1% Lactobacillus rhamnosus. One or more of the components may be derived / isolated from a donor sample, and one or more components may be derived from a bacterial culture. The total number of microorganisms in the exemplary composition is approximately 1 × 10⁶ 5 ~1 × 10 15 CFU, or approximately 1 × 10⁻⁶6 ~1 × 10 12 CFU, or approximately 1 × 10⁻⁶ 7 ~1 × 10 10 The order may be CFU. The composition can be placed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or other similar. In some cases, the composition may be administered orally. In other cases, the composition may be administered topically (e.g., by inserting a capsule, suppository, soluble shell, etc., into the vagina).

[0088] Exemplary compositions may include strains of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus having the desired properties. For example, each strain of the component may be selected to have pH-lowering activity, produce lactic acid, and have inhibitory activity against other microorganisms having other unique activities, in combination thereof, and / or other similar properties.

[0089] As shown above, the total number of microorganisms in the example composition is approximately 1 × 10⁻⁶. 5 ~1 × 10 15 CFU, or approximately 1 × 10⁻⁶ 6 ~1 × 10 12 CFU, or approximately 1 × 10⁻⁶ 7 ~1 × 10 10 The order may be in units of CFU. This may correspond to the total number of microorganisms in a single capsule, suppository, soluble shell, and / or other similar items. Alternatively, this may correspond to the total number of microorganisms in a preferred dosage form of the composition (for example, this may include one or more capsules, suppositories, soluble shells, and / or other similar items).

[0090] A single capsule, suppository, soluble shell, and / or other similar product may be described as an encapsulated formulation or dose that can be administered to a patient. This may involve administering a dose to a patient using a preferred dosing regimen. This may involve administering one or more doses to a patient. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may be administered to a patient. In some of these, and in other examples, the encapsulated formulation may be administered to a patient once or more times a day. For example, a dose may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times, or more times a day. In some of these, and in other examples, a dose may be administered to a patient once or more times a day. For example, a dose may be administered to a patient 1, 2, 3, 4, 5, 6, or more times a day.

[0091] One exemplary dosing regimen may involve administering two doses to a patient twice daily for two days. Another exemplary dosing regimen may involve administering four doses to a patient twice daily for two days. Another exemplary dosing regimen may involve administering four doses to a patient twice daily for four days. Another exemplary dosing regimen may involve administering the patient once daily for eight days. These are merely examples. In at least some of these examples, each dose may include a lyophilized material containing a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus.

[0092] In at least some examples, the vaginal microbiome composition may not contain bacteria from the genera Sneathia, Megasphaera, Atopobium, Peptoniphilus, Dialister, Prevotella, and / or Gardnerella. For example, the vaginal microbiome composition may not contain bacteria from the genera Atopobium, Gardnerella, and Prevotella.

[0093] The vaginal microbiome compositions disclosed herein can be used to treat a number of different health conditions. For example, the vaginal microbiome compositions can be used to treat bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infections, sexually transmitted infections, gynecological cancers (e.g., cervical cancer), reduce and / or prevent premature birth, reduce and / or prevent miscarriage, treat infertility, treat interstitial cystitis, and treat polycystic ovary syndrome.

[0094] Vaginal microbiome compositions are also considered to include broad vaginal microbiome combinations that are combined with or mixed with one or more additional microorganisms (e.g., those collected and isolated from a donor). For the purposes of this disclosure, broad vaginal microbiome can be understood as a collection of microorganisms present in the vagina of a typical woman. Broad vaginal microbiome is not intentionally manipulated to alter the presence or absence of any particular microorganism in the sample, but rather intended to represent the entire population of organisms in the sample. The composition of vaginal microbiome can vary from person to person. For this reason, broad vaginal microbiomes can differ. In some cases, the vaginal microbiome composition may include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus helveticus, and Lactobacillus plantarum. It is thought to contain a broad vaginal microbiome combined with, mixed with, or supplemented with one or more of the following: Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, and Lactobacillus delbrueckii. In at least some cases, both the broad vaginal microbiome and the additional microorganisms are supplied from a vaginal microbiome donor.

[0095] Bacteria / microorganisms may be placed in suitable containers such as capsules, suppositories, soluble shells, and / or other similar forms for oral or topical delivery. In some cases, simulated vaginal fluid may be incorporated into the vaginal microbiome composition. In some of these, and in other examples, the vaginal microbiome composition may contain glycogen. The total volume contained within the container / suppository may be on the order of approximately 100 microliters to approximately 10 milliliters.

[0096] In at least some examples, the bacteria / microorganisms in the vaginal microbiome composition may include lyophilized bacteria / microorganisms. This may involve a lyophilization process in which the bacteria / microorganisms are freeze-dried. The bacteria / microorganisms, and / or lyophilized bacteria / microorganisms, may be placed in suitable containers such as capsules, suppositories, soluble shells, and / or other similar containers for oral or topical delivery.

[0097] Treatment of the patient may include administering a vaginal microbiome composition to the patient. One objective of the treatment may be to normalize the patient's vaginal microbiome by effectively replacing the patient's vaginal microbiome with a vaginal microbiome composition. This may include administering the vaginal microbiome composition directly into the patient's vagina by implantation, suppositories, or another preferred route of administration.

[0098] In at least some examples, multiple vaginal microbiome compositions may be stored or banked in a suitable storage device / facility. The storage device may include temperature control devices such as refrigerators (e.g., 4°C refrigerators), freezers (e.g., -20°C freezers), ultra-low temperature freezers (e.g., -80°C freezers), or other similar devices. In some examples, an index system may also be linked to or otherwise associated with the vaginal microbiome composition bank. The index system may include data such as donor data that can be used to match a vaginal microbiome composition from a given donor to a suitable patient. Donor data may include age at collection, reproductive status at collection (non-menopausal, menopausal), race, menstrual period at collection, history of bacterial vaginosis, history of candidiasis, history of human papillomavirus, other medical history (e.g., history of Clostridium difficile infection, history of cancer diagnosis / treatment, etc.), combinations thereof, and / or other similar information. Clinicians can use donor data to tailor treatment regimens for specific patients. For example, a patient with bacterial vaginosis may be treated with a vaginal microbiome composition derived from a donor who has successfully treated bacterial vaginosis in the past.

[0099] In some cases, donor data may include pregnancy history. For example, some donors may have experience of being more likely to become pregnant when seeking pregnancy or otherwise having an actual or recognized increase in fertility. Vaginal microbiome compositions containing microorganisms from such donors may be administered to patients experiencing infertility challenges in an attempt to increase or enhance the patient's fertility. In such cases, the vaginal microbiome composition may be administered as an oral capsule. Alternatively, the vaginal microbiome composition may be administered as a graft and / or inserted directly into the patient's vagina by other means.

[0100] U.S. Patent No. 9,675,648 is incorporated herein by reference.

[0101] U.S. Patent No. 9,629,881 is incorporated herein by reference.

[0102] U.S. Patent No. 10,226,431 is incorporated herein by reference.

[0103] The specification of U.S. Patent Application Publication No. 2018 / 0289750 is incorporated herein by reference. [Examples]

[0104] This disclosure can be further clarified by reference to the following embodiments, which are essentially predictive, serve to illustrate some embodiments, and do not limit this disclosure in any way.

[0105] Example 1 A vaginal microbiome composition can be prepared. The vaginal microbiome composition consists of approximately 40% Lactobacillus crispatus, approximately 20% Lactobacillus iners, approximately 5% Lactobacillus gasseri, approximately 5% Lactobacillus jensenii, approximately 1% Lactobacillus reuteri, approximately 1% Lactobacillus acidophilus, approximately 1% Lactobacillus vaginalis, approximately 1% Lactobacillus rhamnosus, and approximately 1% Lactobacillus johnsonii. It contains a bacterial mixture including Lactobacillus johnsonii, approximately 1% Lactobacillus helveticus, approximately 1% Lactobacillus plantarum, approximately 1% Lactobacillus fermentum, approximately 1% Lactobacillus salivarius, and approximately 1% Lactobacillus delbrueckii.

[0106] The bacterial mixture can be placed in a suppository. The total volume contained in the suppository (e.g., the volume of the material containing the bacterial mixture) may be on the order of approximately 100 microliters to approximately 10 milliliters.

[0107] This suppository can be administered to a patient. For example, the suppository can be administered to a patient to treat one or more of the following conditions: bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, and gynecological cancer.

[0108] This suppository can be administered to a patient. For example, the suppository can be administered to a patient to prevent premature birth, prevent miscarriage, treat infertility, treat interstitial bladder, treat polycystic ovary syndrome, and / or a combination thereof.

[0109] Example 2 - Exemplary Lactobacillus jensenii strain for use in vaginal microbiome compositions A strain of Lactobacillus jensenii, designated strain 8001-D01-M006, was isolated from donor specimens deposited during donation (e.g., donated as described herein). Donors were eligible by donating vaginal swabs and urine specimens, which were sent to Fairview Diagnostics Laboratory for Gram staining and Nugent scoring (GRAM) and tested for the following STIs: Trichomonas vaginalis (TVPCR), Neisseria gonorrhoeae (GCPCR), and Chlamydia trachomatis (CHPCR).

[0110] During the preparation of the working cell stock of 8001-D01-M006, setting the cultured cell density was not considered, but overnight culture was initiated by selecting single-well isolated colonies from previously prepared streak-smear plates. This overnight culture was diluted 1:5 in fresh broth the following morning and grown for at least 4 hours, after which the culture was combined with glycerol to prepare a 10% glycerol stock. Generally, the preparation of a glycerol stock using this method yields approximately 1 × 10⁶ units. 9 This yields a stock with a cell density of CFU / ml.

[0111] OmniGene and ESwab vaginal swab samples (8001-D01) were provided for metagenomic sequencing (OmniGene) and culture and qPCR identity testing (ESwab). ESwab sample 8001-D01 for culture and qPCR identity testing was introduced into an anaerobic chamber. The ESwab sample was briefly vortexed, and then 50 μL of the sample was taken out. The sample aliquots were pelletized by centrifugation. DNA was extracted from the pellet, and the sample was evaluated by qPCR to determine the Lactobacillus species identity.

[0112] An additional 100 μL of sample was taken from the original Eswab sample. This aliquot was serially diluted 10-fold, and then 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was plated onto MRS agar and then incubated at 35°C for 24–72 hours.

[0113] Lactobacillus species were identified on agar plates based on presumed morphology and Gram staining. Individual colonies with morphology consistent with the presumed Lactobacillus colonies were picked up using a sterile toothpick, placed on fresh MRS agar or LAMVAB agar (a medium selective for Lactobacillus), and then inoculated into MRS broth in the wells of a 96-depth plate. The 96-depth plates containing Lactobacillus species grown in MRS broth were incubated for 24 hours. Patch plates were incubated at 35°C for 24–72 hours.

[0114] Strain 8001-D01-M006 was picked from one of the original MRS agar dilution plates, spread onto grid spot #6 on an MRS agar patch plate and grid spot #6 on a LAMVAB plate, and inoculated into MRS broth in well F7. The broth culture of strain 8001-D01-M006, grown in well F7 of a 96-well deep plate, was transferred to well F7 of a 96-well PCR plate and to well F7 of two 96-well microtiter plates (each well containing 50% glycerol, resulting in a final glycerol concentration of 10% upon addition of the 8001-D01-M006 culture). The cultures were pelletized in 96-well PCR plates, DNA was extracted, and the samples were tested by qPCR to determine the Lactobacillus species identity (where possible). Strain 8001-D01-M006 was identified as Lactobacillus jensenii by qPCR. The culture and a 2×96 well microtiter plate (cryopreservation plate) containing 10% glycerol were sealed with adhesive foil seals and then cryopreserved at -70°C or below.

[0115] Strain 8001-D01-M006 was recovered from frozen 96-well microtiter plates for growth and characterization. One 96-well microtiter plate containing the culture and 10% glycerol was introduced into an anaerobic chamber and thawed at room temperature for 1 hour. The culture in well F7 was gently mixed by pipetting and then streaked onto MRS agar for isolation. The plates were incubated at 35°C for 24–72 hours.

[0116] Single-well isolated colonies were selected from streak plates and used to inoculate overnight cultures in pre-reduced MRS broth (18-24 hours at 35°C). The following day, the overnight cultures were diluted in fresh MRS broth, incubated for approximately 4 hours, then combined with 50% glycerol (final concentration of 10% glycerol), divided into cryopreservation vials (1 ml per vial), and stored frozen at -70°C or below.

[0117] Strain 8001-D01-M006 was isolated and propagated using pre-reduced MRS agar and broth. The lot of the culture medium used was recorded for sample 8001-D01 on the P249-01 sample culture worksheet. The completed culture medium preparation worksheets for each lot of MRS broth and MRS agar were stored in the P249 binder. In short, MRS agar and MRS broth growth media were prepared in-house using Hardy Diagnostic's Criterion® Lactobacillus MRS broth (grams / liter) containing the following ingredients: dextrose (20.0 g), pepsin digest of animal tissue (10.0 g), beef extract (10.0 g), yeast extract (5.0 g), sodium acetate (5.0 g), disodium phosphate (2.0 g), ammonium citrate (2.0 g), Tween 80 (1.0 g), magnesium sulfate (0.1 g), and manganese sulfate (0.05 g). The MRS agar plates use the same Criterion® Lactobacillus MRS broth as a base and contain 15 g / L of BD Difco agar. The MRS broth and agar also contain 0.5 g / L of L-cysteine.

[0118] This strain, 8001-D01-M006, was identified by qPCR and Strainview sequencing. qPCR detects Lactobacillus species and seven different Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) by comparing the dissociation curve profile of the amplicon generated from an unknown isolate with that of a known positive control strain. Strainview sequencing was performed using Diversigen. The strain was found to be free of antibiotic resistance genes and pathogenicity genes when analyzed using the Strainview pipeline.

[0119] Strain 8001-D01-M006 may be an example of a suitable Lactobacillus jensenii strain that can be used in vaginal microbiome compositions.

[0120] Example 3 - Exemplary Lactobacillus gasseri strain for use in vaginal microbiome compositions A Lactobacillus gasseri strain designated as strain 8018-IN-M001 was isolated from donor specimens deposited during donation (e.g., donated as described herein). Donors were eligible by donating vaginal swabs and urine specimens, which were sent to Fairview Diagnostics Laboratory for Gram staining and Nugent scoring (GRAM) and tested for the following STIs: Trichomonas vaginalis (TVPCR), Neisseria gonorrhoeae (GCPCR), and Chlamydia trachomatis (CHPCR).

[0121] During the preparation of the 8018-IN-M001 working cell stock, setting the culture cell density was not considered, but overnight culture was initiated by selecting single-well isolated colonies from previously prepared streak-smear plates. This overnight culture was diluted 1:5 in fresh broth the following morning and grown for at least 4 hours, after which the culture was combined with glycerol to prepare a 10% glycerol stock. Generally, the preparation of a glycerol stock using this method yields approximately 1 × 10⁶ units. 8 ~7×10 8 This yields a stock with a cell density of CFU / ml.

[0122] OmniGene and ESwab vaginal swab samples (8018-IN) were provided for metagenomic sequencing (OmniGene) and culture and qPCR identity testing (ESwab). ESwab samples 8018-IN for culture and qPCR identity testing were introduced into an anaerobic chamber. The ESwab samples were briefly vortexed, and then 50 μL of the sample was taken out. The sample aliquots were pelletized by centrifugation. DNA was extracted from the pellets, and the samples were evaluated by qPCR to determine the Lactobacillus species identity.

[0123] An additional 100 μL of sample was taken from the original Eswab sample. This aliquot was serially diluted 10-fold, and then 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was plated onto MRS agar and then incubated at 35°C for 24–72 hours.

[0124] Lactobacillus species were identified on agar plates based on presumed morphology and Gram staining. Individual colonies with morphology consistent with the presumed Lactobacillus colonies were picked up using a sterile toothpick, placed on fresh MRS agar or LAMVAB agar (a medium selective for Lactobacillus), and then inoculated into MRS broth in the wells of a 96-depth plate. The 96-depth plates containing Lactobacillus species grown in MRS broth were incubated for 24 hours. Patch plates were incubated at 35°C for 24–72 hours.

[0125] Strain 8018-IN-M001 was picked from one of the original MRS agar dilution plates, spread onto grid spot #1 on an MRS agar patch plate and grid spot #1 on a LAMVAB plate, and inoculated into MRS broth in well A7. The broth culture of strain 8018-IN-M001, grown in well A7 of a 96-well deep plate, was transferred to well A7 of a 96-well PCR plate and to well A7 of two 96-well microtiter plates (each well containing 50% glycerol, resulting in a final glycerol concentration of 10% upon addition of the 8018-IN-M001 culture). The cultures were pelleted in 96-well PCR plates, DNA was extracted, and the samples were tested by qPCR to determine the Lactobacillus species identity (where possible). Strain 8018-IN-M001 was identified as Lactobacillus gasseri by qPCR. The culture and a 2×96 well microtiter plate (freezing plate) containing 10% glycerol were sealed with adhesive foil seals and then frozen at -70°C or below.

[0126] Strain 8018-IN-M001 was recovered from frozen 96-well microtiter plates for growth and characterization. One 96-well microtiter plate containing the culture and 10% glycerol was introduced into an anaerobic chamber and thawed at room temperature for 1 hour. The culture in well A7 was gently mixed by pipetting and then streaked onto MRS agar for isolation. The plates were incubated at 35°C for 24–72 hours.

[0127] Single-well isolated colonies were selected from streak plates and used to inoculate overnight cultures in pre-reduced MRS broth (18-24 hours at 35°C). The following day, the overnight cultures were diluted in fresh MRS broth, incubated for approximately 4 hours, then combined with 50% glycerol (final concentration of 10% glycerol), divided into cryopreservation vials (1 ml per vial), and stored frozen at -70°C or below.

[0128] Strain 8018-IN-M001 was isolated and propagated using pre-reduced MRS agar and broth. The lot of the culture medium used was recorded for sample 8018-IN on the P249-01 sample culture worksheet. The completed culture medium preparation worksheets for each lot of MRS broth and MRS agar were stored in the P249 binder. In short, MRS agar and MRS broth growth media were prepared in-house using Hardy Diagnostic's Criterion® Lactobacillus MRS broth (grams / liter) containing the following ingredients: dextrose (20.0 g), pepsin digest of animal tissue (10.0 g), beef extract (10.0 g), yeast extract (5.0 g), sodium acetate (5.0 g), disodium phosphate (2.0 g), ammonium citrate (2.0 g), Tween 80 (1.0 g), magnesium sulfate (0.1 g), and manganese sulfate (0.05 g). The MRS agar plates use the same Criterion® Lactobacillus MRS broth as a base and contain 15 g / L of BD Difco agar. The MRS broth and agar also contain 0.5 g / L of L-cysteine.

[0129] This strain, 8018-IN-M001, was identified by qPCR and Strainview sequencing. qPCR detects Lactobacillus species and seven different Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) by comparing the dissociation curve profile of the amplicon generated from an unknown isolate with that of a known positive control strain. Strainview sequencing was performed using Diversigen. The strain was found to be free of antibiotic resistance genes and pathogenicity genes when analyzed using the Strainview pipeline.

[0130] Strain 8018-IN-M001 may be an example of a suitable Lactobacillus gasseri strain that can be used in vaginal microbiome compositions.

[0131] Example 4 - Exemplary Lactobacillus crispatus strain for use in vaginal microbiome compositions A strain of Lactobacillus crispatus, designated strain 8001-D01-M004, was isolated from donor specimens deposited during donation (e.g., donated as described herein). Donors were eligible by donating vaginal swabs and urine specimens, which were sent to Fairview Diagnostics Laboratory for Gram staining and Nugent scoring (GRAM) and tested for the following STIs: Trichomonas vaginalis (TVPCR), Neisseria gonorrhoeae (GCPCR), and Chlamydia trachomatis (CHPCR).

[0132] During the preparation of the working cell stock of 8001-D01-M004, setting the cultured cell density was not considered, but overnight culture was initiated by selecting single-well isolated colonies from previously prepared streak-smear plates. This overnight culture was diluted 1:5 in fresh broth the following morning and grown for at least 4 hours, after which the culture was combined with glycerol to prepare a 10% glycerol stock. Generally, the preparation of a glycerol stock using this method yields approximately 1 × 10⁶ units. 7 ~1 × 10 9 This yields a stock with a cell density of CFU / ml.

[0133] OmniGene and ESwab vaginal swab samples (8001-D01) were provided for metagenomic sequencing (OmniGene) and culture and qPCR identity testing (ESwab). ESwab sample 8001-D01 for culture and qPCR identity testing was introduced into an anaerobic chamber. The ESwab sample was briefly vortexed, and then 50 μL of the sample was taken out. The sample aliquots were pelletized by centrifugation. DNA was extracted from the pellet, and the sample was evaluated by qPCR to determine the Lactobacillus species identity.

[0134] An additional 100 μL of sample was taken from the original Eswab sample. This aliquot was serially diluted 10-fold, and then 10 -3 , 10 -4 , 10 -5 100 μL of the dilution was plated onto MRS agar and then incubated at 35°C for 24–72 hours.

[0135] Lactobacillus species were identified on agar plates based on presumed morphology and Gram staining. Individual colonies with morphology consistent with the presumed Lactobacillus colonies were picked up using a sterile toothpick, placed on fresh MRS agar or LAMVAB agar (a medium selective for Lactobacillus), and then inoculated into MRS broth in the wells of a 96-depth plate. The 96-depth plates containing Lactobacillus species grown in MRS broth were incubated for 24 hours. Patch plates were incubated at 35°C for 24–72 hours.

[0136] Strain 8001-D01-M004 was picked from one of the original MRS agar dilution plates, spread onto grid spot #4 on an MRS agar patch plate and grid spot #4 on a LAMVAB plate, and inoculated into MRS broth in well D7. The broth culture of strain 8001-D01-M004, grown in well D7 of a 96-well deep plate, was transferred to well D7 of a 96-well PCR plate and to well D7 of two 96-well microtiter plates (each well containing 50% glycerol, resulting in a final glycerol concentration of 10% upon addition of the 8001-D01-M004 culture). The cultures were pelleted into 96-well PCR plates, DNA was extracted, and the samples were tested by qPCR to determine the Lactobacillus species identity (where possible). Strain 8001-D01-M004 was identified as Lactobacillus crispatus by qPCR. The culture and a 2×96 well microtiter plate (freezing plate) containing 10% glycerol were sealed with adhesive foil seals and then frozen at -70°C or below.

[0137] Strain 8001-D01-M004 was recovered from frozen 96-well microtiter plates for growth and characterization. One 96-well microtiter plate containing the culture and 10% glycerol was introduced into an anaerobic chamber and thawed at room temperature for 1 hour. The culture in well D7 was gently mixed by pipetting and then streaked onto MRS agar for isolation. The plates were incubated at 35°C for 24–72 hours.

[0138] Single-well isolated colonies were selected from streak plates and used to inoculate overnight cultures in pre-reduced MRS broth (18-24 hours at 35°C). The following day, the overnight cultures were diluted in fresh MRS broth, incubated for approximately 4 hours, then combined with 50% glycerol (final concentration of 10% glycerol), divided into cryopreservation vials (1 ml per vial), and stored frozen at -70°C or below.

[0139] Strain 8001-D01-M004 was isolated and propagated using pre-reduced MRS agar and broth. The lot of culture medium used was recorded for sample 8001-D01 on the P249-01 sample culture worksheet. The completed culture medium preparation worksheets for each lot of MRS broth and MRS agar were stored in the P249 binder. In short, MRS agar and MRS broth growth media were prepared in-house using Hardy Diagnostic's Criterion® Lactobacillus MRS broth (grams / liter) containing the following ingredients: dextrose (20.0 g), pepsin digest of animal tissue (10.0 g), beef extract (10.0 g), yeast extract (5.0 g), sodium acetate (5.0 g), disodium phosphate (2.0 g), ammonium citrate (2.0 g), Tween 80 (1.0 g), magnesium sulfate (0.1 g), and manganese sulfate (0.05 g). The MRS agar plates use the same Criterion® Lactobacillus MRS broth as a base and contain 15 g / L of BD Difco agar. The MRS broth and agar also contain 0.5 g / L of L-cysteine.

[0140] This strain, 8001-D01-M004, was identified by qPCR and Strainview sequencing. qPCR detects Lactobacillus species and seven different Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) by comparing the dissociation curve profile of the amplicon generated from an unknown isolate with that of a known positive control strain. Strainview sequencing was performed using Diversigen. The strain was found to be free of antibiotic resistance genes and pathogenicity genes when analyzed using the Strainview pipeline.

[0141] Strain 8001-D01-M004 may be an example of a suitable Lactobacillus crispatus strain that can be used in vaginal microbiome compositions.

[0142] Example 5 - Exemplary Lactobacillus rhamnosus strain for use in vaginal microbiome compositions An exemplary strain of Lactobacillus rhamnosus, designated as strain BPL5, is disclosed in U.S. Patent No. 10,588,926, the entirety of which is incorporated herein by reference.

[0143] Strain BPL5 may be an example of a suitable Lactobacillus rhamnosus strain that can be used in vaginal microbiome compositions.

[0144] Example 6 - pH-lowering ability and lactic acid production of a strain when grown anaerobically in MRS broth for 48 hours. The in vitro ability of strains BPL5 (Lactobacillus rhamnosus; Example 6), 8001-D01-M006 (Lactobacillus jensenii; Example 2), 8001-D01-M004 (Lactobacillus crispatus; Example 4), and 8018-IN-M001 (Lactobacillus gasseri; Example 3) to lower the pH of MRS broth over time was evaluated. To evaluate the pH reduction, each strain was inoculated into MRS broth and grown overnight (under anaerobic conditions). The following day, the overnight culture was allowed to grow to approximately 1.0 OD. 600nm (This is approximately 1 × 10 8 (Equivalent to CFU / mL) was adjusted, and then further diluted in fresh MRS broth (approximately 1 × 10⁻⁶). 6 The target starting concentration is CFU / mL (for 8001-D01-M006, 8001-D01-M004, and 8018-IN-M001). Strain BPL5 will be tested separately at different starting concentrations (approximately 1 × 10⁻¹⁰). 7The cultures were tested in CFU / mL. All cultures were incubated anaerobically for 48 hours, and pH was measured by taking samples at 0 and 48 hours of growth. All strains were able to significantly lower the broth pH by 48 hours, with Lactobacillus rhamnosus and Lactobacillus crispatus strains showing the most significant pH reduction (Table 1). Both BPL5 (Lactobacillus rhamnosus) and 8001-D01-M004 (Lactobacillus crispatus) strains were able to lower the broth pH to below 4.0 by 48 hours (3.78 and 3.77, respectively). Strains 8001-D01-M006 (Lactobacillus jensenii) and 8018-IN-M001 (Lactobacillus gasseri) also reduced broth pH by 48 hours (4.02 and 4.06, respectively), but not to the same extent as Lactobacillus rhamnosus and Lactobacillus crispatus.

[0145] [Table 1]

[0146] Additional samples were collected at 48 hours, filtered, and removed for bacterial removal. The supernatant, free of these cells, was then analyzed to detect levels of D-lactic acid and L-lactic acid. D-lactic acid and L-lactic acid were quantified using the D- / L-Lactic Acid (D- / L-Lactate) (Rapid) Assay Kit (catalog number K-DLATE) manufactured by Megazyme. This kit is designed to quantify both D-lactic acid and L-lactic acid using an enzymatic reaction employing lactate dehydrogenase and glutamate dehydrogenase. The amount of NADH produced by the oxidation of lactate is stoichiometric with respect to the starting concentration of lactate. The kit was used according to the manufacturer's recommended procedure. All strains tested produced detectable levels of lactate (Table 2). The 8001-D01-M004 (Lactobacillus crispatus) strain produced the maximum levels of D-lactic acid and L-lactic acid (20.9 g / L) and the maximum amount of D-lactic acid (13.5 g / L), while the BPL5 (Lactobacillus rhamnosus) strain produced the maximum amount of L-lactic acid (17.3 g / L) and a very small amount of D-lactic acid (0.8 g / L). Both Lactobacillus crispatus and Lactobacillus gasseri strains produced large amounts of both lactic acid isomers, and both strains produced more D-lactic acid than L-lactic acid. Lactobacillus jensenii strain 8001-D01-M006 primarily produced D-lactic acid (12.4 g / L) and a very small amount of L-lactic acid (0.3 g / L).

[0147] [Table 2]

[0148] These results demonstrate that strains BPL5 (Lactobacillus rhamnosus; Example 6), 8001-D01-M006 (Lactobacillus jensenii; Example 2), 8001-D01-M004 (Lactobacillus crispatus; Example 4), and 8018-IN-M001 (Lactobacillus gasseri; Example 3) may be suitable materials for use in vaginal microbiome compositions.

[0149] Example 7 - Exemplary Vaginal Microbiota Composition Exemplary vaginal microbiome compositions can be formed by combining Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus. In at least some examples, Lactobacillus jensenii may be the strain described in Example 2. In at least some examples, Lactobacillus gasseri may be the strain described in Example 3. In at least some examples, Lactobacillus crispatus may be the strain described in Example 4. In at least some examples, Lactobacillus rhamnosus may be the strain described in Example 5.

[0150] It should be understood that this disclosure is, in many respects, illustrative. Modifications can be made in detail, particularly with respect to shape, size, and step arrangement, without exceeding the scope of this disclosure. This may include, to the extent appropriate, the use of any feature of one exemplary embodiment being used in other embodiments. The scope of the invention is, of course, defined in the language in which the appended claims are expressed.

Claims

1. A vaginal microbiome composition, It contains a mixture of bacteria including Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus. A vaginal microbiome composition comprising a mixture of bacteria containing 25-75% Lactobacillus crispatus, 1-10% Lactobacillus gasseri, 1-10% Lactobacillus jensenii, and 0.1-5% Lactobacillus rhamnosus.

2. The vaginal microbiome composition according to claim 1, wherein the bacterial mixture contains 30-60% Lactobacillus crispatus.

3. The vaginal microbiome composition according to claim 1 or 2, wherein the bacterial mixture comprises 40% Lactobacillus crispatus.

4. The vaginal microbiome composition according to any one of claims 1 to 3, wherein the bacterial mixture contains 2 to 8% Lactobacillus gasseri.

5. The vaginal microbiome composition according to any one of claims 1 to 4, wherein the bacterial mixture comprises 5% Lactobacillus gasseri.

6. The vaginal microbiome composition according to any one of claims 1 to 5, wherein the bacterial mixture contains 2 to 8% Lactobacillus jensenii.

7. The vaginal microbiome composition according to any one of claims 1 to 6, wherein the bacterial mixture contains 5% Lactobacillus jensenii.

8. The vaginal microbiome composition according to any one of claims 1 to 7, wherein the bacterial mixture contains 0.1 to 5% Lactobacillus acidophilus.

9. The vaginal microbiome composition according to any one of claims 1 to 8, wherein the bacterial mixture contains 0.5 to 4% Lactobacillus rhamnosus.

10. The vaginal microbiome composition according to any one of claims 1 to 9, wherein the bacterial mixture comprises 1% Lactobacillus rhamnosus.

11. The vaginal microbiome composition according to any one of claims 1 to 10, wherein the mixture of bacteria is arranged in a capsule.

12. The vaginal microbiome composition according to any one of claims 1 to 11, wherein the mixture of bacteria is placed in a suppository.

13. The vaginal microbiome composition according to any one of claims 1 to 12, wherein the mixture of bacteria is arranged in a soluble shell.

14. The aforementioned bacterial mixture is 1 × 10 ５ ~1 x 10 １５ A vaginal microbiome composition according to any one of claims 1 to 12, comprising CFU.

15. The aforementioned bacterial mixture is 1 × 10 ６ ~1 x 10 １２ A vaginal microbiome composition according to any one of claims 1 to 12, comprising CFU.

16. The aforementioned bacterial mixture is 1 × 10 ７ ~1 x 10 １０ A vaginal microbiome composition according to any one of claims 1 to 12, comprising CFU.

17. The vaginal microbiome composition according to any one of claims 1 to 16, wherein the bacterial mixture comprises a freeze-dried bacterial mixture.

18. For use in the treatment of infectious diseases, A pharmaceutical composition comprising a vaginal microbiome composition according to any one of claims 1 to 16, wherein the infection comprises one or more of the following: bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, and gynecological cancer.

Images