Composition comprising lactobacillus species for vaginal therapies

Abstract

Compositions and methods for treating patients are disclosed. An example composition may include a vaginal microbiota composition. The vaginal microbiota composition may include a mixture of bacteria. The mixture of bacteria may include Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii / Lactobacillus mulieris and Lactobacillus rhamnosus.

Description

COMPOSITION COMPRISING LACTOBACILLUS SPECIES FOR VAGINAL THERAPIESTechnical Field

[0001] The present disclosure pertains to compositions and methods for treating patients.Background

[0002] A wide variety of compositions and methods have been developed for treating diseases and / or conditions, for example diseases and / or conditions of the reproductive system, digestive track, liver, immune system, and the like. Of the known compositions and methods, each has certain advantages and disadvantages. There is an ongoing need to provide alternative compositions cand methods for treating diseases and / or conditions.Brief Summary

[0003] In a first aspect there is provided a composition for use in therapy, comprising (or consists of): at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species, the / said Lactobacillus bacterial species comprising Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii / Lactobacillus mulieris, and / or Lactobacillus rhamnosus, the composition optionally comprising one or more pharmaceutically acceptable adjuvants and / or excipients.

[0004] Further aspects and teachings are provided herein below and in accordance with the claims appended hereto.

[0005] The composition may comprise 1 , 2, 3 or all 4 of the aforementioned Lactobacillus species. In one embodiment, the composition comprises at least Lactobacillus rhamnosus, which may be present in an amount (%age of total Lactobacillus species present in the composition) of between 10 - 100%, typically, 25 - 95%, such as 40 - 90%. In one embodiment, the composition comprises a cocktail of all 4 of the aforementioned Lactobacillus species, with each species being present in an independent amount of between 10 - 50%, such as 15 - 30%. Conveniently, each species may beprovided in an approximately equal percentage to each other. Typically, this may be between approximately 20 - 25%.

[0006] This disclosure provides design, material, manufacturing method, and use alternatives for compositions and methods for treating patients. In one teaching a vaginal microbiota composition is disclosed. As used herein, the term “vaginal” or “vagina” is understood to relate not just to the vaginal region, but to include any of the urogenital area and so should be broadly interpreted, unless the context dictates otherwise. In addition to the aforementioned Lactobacillus species, the vaginal microbiota composition may comprise in addition, one or more of bacteria including, Lactobacillus iners, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, and Lactobacillus delbrueckii. When any of these additional Lactobacillus species are present in a composition, they may be present in any suitable amount. Typically, they may independently be present in an amount of 0.5%-10%, such as 1 %- 5%. The compositions of the present disclosure may, in some instances, not include bacterial species other than from the genus Lactobacillus. Thus, in some instances, the compositions may contain bacteria consisting of, or consisting essentially of Lactobacillus species.

[0007] In accordance with the disclosure, the compositions described herein, may be of use in a method for restoring beneficial vaginal microbial communities and / or increasing resistance to vaginal disease, including bacterial vaginosis (BV) and / or sexually transmitted disease. Usefulness of the composition may be enhanced, through administration of an acidifying agent, before, during and / or after administration, such as lactic acid. Conveniently, a mixture of D- and L- forms of lactic acid may be added. Typically, the ratio of D- to L- lactic acid may be between !:8 to 1 :2, such as 1 :6 - 1 :3 (w / w).

[0008] The composition can also be filtered for sterility and to remove particles, aggregates and cells, for administration as a filtrate, and optionally mixed with isolated and cultured Lactobacillus bacteria, or spray dried or lyophilized (optionally with one or more cryoprotectants) and, optionally, packaged into single dosage unit applicators.

[0009] Alternatively or additionally to any of the embodiments above, the composition comprises or includes one or more lyophilized strains of bacteria.

[0010] Alternatively or additionally to any of the embodiments above, the composition is disposed in a capsule.

[0011] Alternatively or additionally to any of the embodiments above, the composition is disposed in a suppository.

[0012] Alternatively or additionally to any of the embodiments above, the composition is disposed in a soluble shell.

[0013] A composition for use in a method for treating an infection is disclosed. The method comprises: administering the composition, such as a vaginal composition of any one of the herein described embodiments to a patient with an infection. The composition may serve to modulate, such as attenuate an immune response of epithelial cells (such as vaginal epithelial cells) against pathological microbial species (such as in a biofilm) being present in the vagina. The immune response may be associated with the expression of one or more inflammatory markers and the compositions described herein may serve to reduce expression and / or function of said markers. Accordingly, in a further embodiment there is provided a composition for use in a method of treating inflammation, and in particular inflammation arising from an infection, particularly arising from a vaginal disease, including bacterial vaginosis (BV) and / or sexually transmitted disease. In an embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6 and CXCL8.

[0014] In a further aspect there is provided a method of treating an infection, wherein the method comprises administering the composition, such as a vaginal composition of any one of the herein described embodiments to a patient with an infection. The composition may serve to modulate, such as attenuate an immune response of epithelial cells (such as vaginal epithelial cells) against pathological microbial species (such as in a biofilm) being present in the vagina. The immune response may be associated with the expression of one or more inflammatory markers and the compositions described herein may serve to reduce expression and / or function of said markers. Accordingly, in a further embodiment there is provided a method of treating inflammation, and in particular inflammation arising from an infection, particularly arising from a vaginal disease, includingbacterial vaginosis (BV) and / or sexually transmitted disease. In an embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6 and CXCL8.

[0015] In a further aspect there is provided the use of a composition, such as a vaginal composition of any one of the herein described embodiments, in the manufacture of a medicament for the treatment of an infection. The composition may serve to modulate, such as attenuate an immune response of epithelial cells (such as vaginal epithelial cells) against pathological microbial species (such as in a biofilm) being present in the vagina. The immune response may be associated with the expression of one or more inflammatory markers and the compositions described herein may serve to reduce expression and / or function of said markers. Accordingly, in a further embodiment there is provided the use of a composition in the manufacture of a medicament for the treatment of inflammation, and in particular inflammation arising from an infection, particularly arising from a vaginal disease, including bacterial vaginosis (BV) and / or sexually transmitted disease. In an embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6 and CXCL8.

[0016] Alternatively or additionally to any of the embodiments above, wherein the infection includes one or more of bacterial vaginosis, a candidiasis infection, a human papilloma virus infection, a urinary tract infection, a sexually transmitted infection, and a gynecological cancer.

[0017] A method for treating a patient is disclosed. The method comprises: administering the composition, such as a vaginal composition of any one of the herein described embodiments to a patient.

[0018] Alternatively or additionally to any of the herein described embodiments, wherein administering the vaginal microbiota composition to the patient includes one or more of preventing preterm birth, preventing miscarriages, treating infertility, treating interstitial cystitis, and treating polycystic ovary syndrome.

[0019] The above summary of some embodiments is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The Detailed Description, which follows, more particularly exemplify these embodiments.Detailed Description

[0020] For the following defined terms, these definitions shall be applied, unless a different definition is given in the claims or elsewhere in this specification.

[0021] All numeric values are herein assumed to be modified by the term “about”, whether or not explicitly indicated. The term “about” generally refers to a range of numbers that one of skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In many instances, the terms “about” may include numbers that are rounded to the nearest significant figure.

[0022] The recitation of numerical ranges by endpoints includes all numbers within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0023] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0024] It is noted that references in the specification to “an embodiment”, “some embodiments”, “other embodiments”, etc., indicate that the embodiment described may include one or more particular features, structures, and / or characteristics. However, such recitations do not necessarily mean that all embodiments include the particular features, structures, and / or characteristics. Additionally, when particular features, structures, and / or characteristics are described in connection with one embodiment, it should be understood that such features, structures, and / or characteristics may also be used in connection with other embodiments whether or not explicitly described unless clearly stated to the contrary.

[0025] The term ‘supernatant’ as sued herein refers to the liquid phase of a culture which has been subjected to some form of separation procedure. For example, a bacterial culture (such as a culture comprising any of the bacterial species described herein) subject to a gravity-based separation technique (e.g. centrifugation) results in a pellet comprising the cultured bacteria and a liquid phased which is known as the supernatant. The supernatant will comprise chemicals, metabolites and other compounds produced by the bacteria during the period of culture. As such, it is possible to derive the therapeutic benefits attributed to the bacteria themselves, through the use of supernatant(s) derivedfrom cultures of the various bacteria described herein, which supernatant(s) comprise therapeutic compounds produced by the cultured bacteria.

[0026] The human microbiome (or human microbiota) is the aggregate of microorganisms that reside on the surface and in deep layers of skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal, urogenitary, and / or vaginal tracts of humans inclusive of microorganisms related to reproductive health such as the placental microbiome. The human microbiome is comprised of bacteria but may also include fungi, phages, viruses, archaea, and the like. Some of these organisms perform tasks that are useful for the human host, but the function of the majority of the organisms that make up the human microbiome is still being studied. Under normal circumstances, these microorganisms do not cause disease to the human host, but instead participate in maintaining health.

[0027] The vaginal microbiota may play a role in a number of different health conditions. Some of these conditions may include infections such as bacterial vaginosis, candidiasis infection, human papilloma virus infections, urinary tract infections, and / or sexually transmitted infections. Other conditions may include gynecological cancers (e.g., cervical cancer), preterm birth, miscarriages, infertility, interstitial cystitis, polycystic ovary syndrome, and the like. Restoring, normalizing, and / or otherwise shifting the makeup of the vaginal microbiota may help to alleviate these and other conditions. Disclosed herein are compositions and methods for treating patients. At least some of the compositions include vaginal microbiota compositions. At least some of the compositions for use in methods of treatment, include methods for treating bacterial vaginosis, treating candidiasis infections, treating human papilloma virus infections, treating urinary tract infections, treating sexually transmitted infections, treating gynecological cancers (e.g., cervical cancer), preventing preterm birth, preventing miscarriages, treating infertility, treating interstitial cystitis, treating polycystic ovary syndrome, and the like.

[0028] As used herein, the term “urogenital” region refers to the region of the anus and the genitalia. In certain embodiments, the female urogenital region comprises the cervix, vagina, vulva, clitoris, urethral meatus, urethral meatus, vulval vestibule, perineum, and / or anus. In one teaching, the term “urogenital” region refers to the region of the distal urinary tract and the genitalia. In certain embodiments, the female urogenital regioncomprises the cervix, vagina, vulva, clitoris, introitus, urethral meatus, urethral fold, vulval vestibule, and / or perineum.

[0029] As used herein, the term “vaginal microbiota” refers to the collective microorganisms that normally colonize the vulva, clitoris, vestibule, and vagina As used herein, the term “vaginal microbiota” refers to the collective microorganisms that normally colonize the gential region and are non-pathogenic. The vaginal microbiota may refer to that of the vaginal microbiota (e.g., vaginal mucosal microbiota) of a female subject, the cervical microbiota of a female subject, vulval microbiota of a female subject, or any combination thereof.

[0030] In healthy, reproductive-age women, bacteria of the genus Lactobacillus tend to dominate the vaginal microbiome. There are four species consistently identified across women; Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, andLactobacillus jensenii. These species are listed in order of their frequency of dominance among women of white, black, Asian, and Hispanic ethnicities, Generally, lactobacilli dominance of the vaginal microbiome is associated with health, This dominance of a single genus or species creates a low diversity microbiome. Increases in vaginal microbiome diversity are associated with poor health and a higher risk of infection. For example, bacteria from the genera Sneathia, Megasphaera, Atopobium (aka Fannyhessea), Peptoniphilus, Dialister, Prevotella, Mobiluncus and / or Gardnerella may tend to correlate with an unhealthy vaginal microbiota, as they have been identified in women with other indicators of poor vaginal health (high Nugent score and vaginal pH).These generalizations, however, can differ in different populations.

[0031] The vaginal microbiome may vary between pre-puberty, pre-menopausal, and post-menopausal women. For example, pre-puberty women may have a relatively low relative abundance of Lactobacillus and a relatively high diversity in microbes (as well as have a relatively thin vaginal epithelium, a relatively thin layer of mucus, and a relatively low amount of glycogen). Pre-menopausal women may have a relatively high relative abundance of Lactobacillus and a relatively low diversity in microbes (as well as have a relatively thick vaginal epithelium, a relatively thick layer of mucus, relatively high estrogen levels, and a relatively low amount of glycogen). Post-menopausal women may have a moderate relative abundance of Lactobacillus and a relatively low or lower diversity in microbes (as well as have a relatively thin vaginal epithelium, a relatively thin layer ofmucus, relatively moderate estrogen levels, and a relatively moderate amount of glycogen). Post-menopausal women symptomatic of a vaginal infection (e.g., bacterial vaginosis) may have a lower relative abundance of Lactobacillus and a relatively high diversity in microbes. Collectively, compositions that tend to include or otherwise increase the relative abundance of Lactobacillus, as well as help to lower the microbial diversity, may be beneficial.

[0032] Bacterial vaginosis (“BV”), as used herein, refers to the overgrowth of one of several non-Lactobacillus types of bacteria normally present in the vagina, upsetting the natural balance of vaginal bacteria.

[0033] In at least some instances, the compositions disclosed herein (e.g., vaginal microbiota compositions) utilize vaginal microbiota collected from vaginal microbiota donors. However, other sources of vaginal microbiota are contemplated including bacterial cultures, etc. Potential vaginal microbiota donors may be identified and may undergo a number of screening processes. In some instances, potential donors may be screened using a health history questionnaire. An example questionnaire may be the same as or similar to that used by the Red Cross for screening of potential blood donors. In some of these and in other instances, potential donors may be screened for common infectious diseases and other conditions. Such screening may include blood tests, stool tests, urine tests, vaginal swab tests and the like. Such tests may include testing for the presence of HIV, hepatitis (hepatitis A, B, and / or C), syphilis, C. difficile, bacterial pathogens, ova and parasites, and / or the like. These are just examples. The health of donors may be monitoring by administering blood tests, analyzing stool samples, analyzing urine samples, analyzing vaginal secretions, periodically updating health history, etc. Vaginal swabs may be collected to assess vaginal health via Gram staining and Nugent scoring, white blood cell counts, and detection of the presence of yeast. Urine and / or vaginal swabs may be collected to test for sexually transmitted infections.

[0034] After a potential donor has been determined to be healthy enough to donate, the donor is provided with a sample collection kit (e.g., a sample self-collection kit). The sample collection kit may include donor instructions, a vaginal DNA / RNA collection kit for sequencing analysis (e.g., OMNIgene vaginal collection kit ORM-130, commercially available from DNAgeneotek™), and a specimen collection and transportation kit (e.g., an eSwab 480C Copan Liquid Amies Elution Swab Collection and Transport System,commercially available from Copan Diagnostics). Donors 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 hand washing, a swab from the vaginal DNA / RNA collection kit can be used to collect the vaginal specimen by inserting the swab a few inches into the vagina and swabbing the vaginal wall for about 20 seconds. The swab can then be inserted into a tube containing a stabilizing buffer and the tube / specimen can be further analyzed. The second sample may be used for culturing vaginal microbes. For example, a swab from the specimen collection and transportation kit can used to collect the vaginal specimen. The swab can then be inserted into a tube containing liquid Amies solution (e.g., which may preserve aerobic, anaerobic, and fastidious bacteria for up to 48 hours). Upon collection, the donor places the sample tubes and materials inside a biohazard bag, and then drops the specimen off at the donation facility.

[0035] To process the sample for sequencing (e.g., shotgun sequencing), first the protease (e.g., QIAGEN Protease) may be rehydrated using sterile PCR-certified water to generate an 80 mg / mL solution (this may include inverting the sample 10 times or more). The sample (e.g., the vaginal DNA / RNA collection kit sample) may be removed from the refrigerator and vortexed for 30 seconds. The collection tube may be shook three times to bring the solution containing the sample to the bottom of the tube. Five microliters of the rehydrated protease may be added to the collection tube containing the vaginal swab sample. The sample may be inverted 10 or more times. The sample may then be incubated for 2 hours in a 50°C incubator. The sample may then be vortexed for 30 seconds. The collection tube may then be shook three times to bring the solution containing the sample to the bottom of the tube. The swab may be removed from the tube (which may include pressing the swab on the side of the tube to recover sample absorbed into the swab). 500 microliters of the sample may be aliquoted into each of two 2-mL cryovials for storage and the samples can be stored at -80°C until ready for sequencing analysis (which may be conducted at a suitable processing / analysis facility such as Divergent™ in St. Paul, MN).

[0036] To process the sample for culturing, the sample (e.g., the specimen collection and transportation kit sample) may be placed into an airlock of an anaerobic chamber and introduced into the chamber. With the swab still in place, the tube can be vigorously vortexed for 5 second to release the sample from the swab tip. This is the undiluted or10° dilution. The swab can then be removed. The sample may undergo a 10-fold serial dilution by transferring 100 microliters into a tube containing 900 microliters of sterile normal saline (0.9%). The sample may be vortex vigorously for 5 seconds and / or mixed by inversion. This is the 10-1dilution. 100 microliters of the 10-1dilution may be transferred to a new tube containing 900 microliters of sterile normal saline (0.9%). The sample may be vortex vigorously for 5 seconds and / or mixed by inversion. This is the 10-2dilution. The process may be repeated until the specimen has been diluted out to a 10-6dilution.

[0037] In triplicate, spread plates can be prepared on CDC Anaerobe 5% sheep blood agar, De Man, Rogosa and Sharpe (MRS) agar, and 50% MRS agar plates from the following dilution tubes: 10-3, 104, 10-5. Briefly, from the appropriate tube, 100 pL may be transferred directly onto the surface of an agar plate and spread the inoculum across the plate surface using a sterile L-shaped spreader and a plate rotator. The plates may be incubated at 35°C under anaerobic conditions for 24-120 hrs. The plates may be checked daily for emergence of new colonies. If plates begin to dry out, wrap them in parafilm. Colonies may be observed and colony counts and morphology may be recorded (which may include taking a capture photo of plates to assist with documentation of colony morphology). Individual colonies for further characterization may be selected and streaked on new plates. If desired, selective media may be utilized such as Bifidobacterium selective agar (BSA), Enterococcosel agar (ECA), Cetrimide Agar (CA), Mannitol Salt Agar (MSA), or the like.

[0038] In some instances, Gram staining may be utilized. When doing so, a specimen may be applied to a glass slide. If staining a liquid culture, a sterile inoculating loop may be used to directly apply culture to the slide as a smear. If staining a colony from a plate, a drop of sterile saline may be applied to the center of a slide using an inoculating loop, and then a small amount of a single colony may be collected and added to the drop. If possible, the drop and sample can be gently mixed to yield a thin, uniform smear. The drop may be allowed to dry. The slides can be methanol fixed by flooding with absolute methanol for 1 -2 minutes, and then rinsing with tap water. The fixed smear may be flooded with the primary stain (e.g., crystal violet) for 1 minute. The primary stain may be removed by gently rinsing with tap water. The fixed smear may be flooded with a secondary stain / mordant (e.g., Gram iodine) for 1 minute, followed by gently rinsing with tap water. The slide can be decolorized until the solvent running from the slide is colorless (e.g., about 3-60 seconds) and the slide can be washed gently with tap water. The slide canthen be flooded with counterstain (e.g., safranin; basic fuchsin can be substituted if initial counterstain results are not satisfactory) for 1 minute. The slides can be washed with tap water and allowed to dry. The smear can be exampled under an oil immersion lens and the results / observation may be recorded.

[0039] Other sample testing can be performed including catalase testing, which may include the addition of hydrogen peroxide to differentiate between Staphylococci and Streptococci. Another sample test that may be performed may include a coagulase test, which may include the addition of plasma to differentiate between Staphylococci and Streptococci.

[0040] Isolates that are determined to be Lactobacillus may be further characterized to determine their antibiotic susceptibility profiles. This may include suspending Lactobacillus colonies from an overnight plate in broth comprised of 90% Iso-sensitest broth and 10% MRS broth until the suspension is equivalent to a McFarland standard of 1. Next, a sterile swab may be dipped in the inoculum and squeezed against the wall of the tube to eliminate excess liquid. The swab may be streaked over the entire agar surface (rotating two or more times to ensure even distribution). After allowing excess moisture to be absorbed, a minimum inhibitory concentration (MIC) test strip can be applied (e.g., which may include applying the strip with the scale facing upwards and the code of the strip to the outside of the place). The strip may be pressed onto the surface of the agar while ensuring that the whole length of the antibiotic gradient is in complete contact with the agar surface, repositioning if necessary). The plate can be inverted and incubated at 35°C for 24-48 hours (or longer). The results can be interpreted.

[0041] Isolated strains can be cryopreserved and stored. This may include inoculating an overnight culture with a well-isolated colony selected from a streak plate. If many colonies are being selected, a 96-well plate format may be used. A suitable broth may be used (e.g., Lactobacillus may likely use MRS). The culture may be allowed to incubate at 35°C overnight in an anaerobic chamber incubator. If the culture is not turbid by the following morning, additional incubation time may be utilized. Once culture appears visually turbid, 200-300 microliters may be transferred to a new 96-well plate containing a cryoprotectant (e.g., 50-60 microliters of 50% glycerol). The final concentration of glycerol may be 10%. The 96-well plates may be sealed and stored in a -80°C freezer.

[0042] A cryoprotectant is any agent that prevents the formation of ice crystals, which can rupture cell membranes and hence a cryoprotectant refers to a substance that is used to protect biological cells or tissues from the effects of freezing. Cryoprotectants include extracelluar cryoprotectants that do not penetrate bacterial cell walls, and intracellular cryoprotectants that penetrate bacterial cell walls. Examples of cryoprotectants include, dextrose, betaine, glycine, sucrose, polyvinyl alcohol, polyethylene glycol (PEG), Pluronic F-127, mannitol, tween 80, ethylene glycol, 1 ,3-propanediol, hydroxypropyl cellulose, glycerol, PEG / glycerol mix, milk (e.g., skim milk), and propylene glycol. In some embodiments, PEG may be preferred.

[0043] As suggested herein, individual microbe strains (e.g., individual strains or species of bacteria) may be isolated from the swabs. This may include one or more processes such as plating onto agar media selective for the desired strains and subsequently “purifying” strains as single-colony isolates. The individual microbe strains may be grown / cultured and be species-identified / characterized by sequencing, biochemical tests, colony morphology, cellular morphology, microscopic mortality, and susceptibility to different representative antibiotics. The microbe strains may vary. In some instances, the microbe strains may include one or more of Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii / L mulieris, and optionally one or more of Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus 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 genera, fungi, combinations thereof, and the like.

[0044] It should be noted that herein reference is made to Lactobacillus jensenii and L. mulieris. Lactobacillus mulieris was identified as a new bacterial species in 2020 and analysis suggested that a number of L. jensenii species should be reclassified as L.mulieris. As such, and for the purposes of this disclosure, Lactobacillus jensenii and L. mulieris are use conjointly and are to be considered as synonymous.

[0045] In some instances, each of the components may be lyophilized. The total number of microorganisms in the example composition may be on the order of about 1 x 105to 1x1015CFU / ml, or about 1 x 106to 1x1012CFU / ml, or about 1 x 107to 1x1010CFU / ml, or 1 x 108to 1x109CFU / ml. Thus, the number of microorganisms may be understood to relate to the number of microorganisms prior to lyophilisation. The composition may be disposed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or the like. In some instances, the composition may be administered orally. In other instances, the composition may be administered locally (e.g., by inserting the capsule, suppository, soluble shell, etc. into the vagina).

[0046] The example compositions may include strains of Lactobacillus crispatus, Lactobacillus gasseri / L. mulieris, Lactobacillus jensenii, and Lactobacillus rhamnosus with desirable characteristics. For example, strains of each of the components may be selected to have a pH reducing activity, produce lactic acid, have an inhibitory activity on other microorganisms, having other unique activities, combinations thereof, and / or the like.

[0047] An example composition may include a mixture of lyophilized Lactobacillus crispatus, lyophilized Lactobacillus gasseri, lyophilized Lactobacillus jensenii / L. mulieris and lyophilized Lactobacillus rhamnosus. Each of the components may be isolated from a donor. For example, the composition may include about 20-85% Lactobacillus rhamnosus, or about 25-75% Lactobacillus rhamnosus, or about 30-60% Lactobacillus rhamnosus, or about 40% Lactobacillus rhamnosus. 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 crispatus, or about 0.5-4% Lactobacillus crispatus, or about 1 % Lactobacillus crispatus. One or more of the components may be derived / isolated from a donor specimen and / or bacterial culture. The total number of microorganisms in the example composition may be on the order of about 1 x 105to 1x1015CFU / ml, or about 1 x 106to 1x1012CFU / ml, or about 1 x 107to 1x1010CFU / ml, or1 x 108to 1x109CFU / ml, prior to lyophilisation. The composition may be disposed in a suitable delivery vehicle such as a capsule, suppository, soluble shell, and / or the like. In some instances, the composition may be administered orally. In other instances, the composition may be administered locally (e.g., by inserting the capsule, suppository, soluble shell, etc. into the vagina).

[0048] The example compositions may include strains of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jenseniil L. mulieris and Lactobacillus rhamnosus with desirable characteristics. For example, strains of each of the components may be selected to have a pH reducing activity, produce lactic acid, typically a combination of D- and L- forms, have an inhibitory activity on other microorganisms, having other unique activities, combinations thereof, and / or the like.

[0049] As indicated above, the total number of microorganisms in the example composition may be on the order of about 1 x 105to 1x1015CFU / ml, or about 1 x 106to 1x1012CFU / ml, or about 1 x 107to 1x1010CFU / ml, or 1 x 108to 1x109CFU / ml, prior to lyophilisation. This may correspond to the total number of microorganisms in a single capsule, suppository, soluble shell, and / or the like. Alternatively, this may correspond to the total number of microorganisms in a suitable dosage of the composition (e.g., which may include one or more capsules, suppositories, soluble shells, and / or the like).

[0050] A single capsule, suppository, soluble shell, and / or the like may be described as an encapsulated drug product or dose, which may be administered to a patient. This may include administering the dose to a patient using a suitable dosing regimen. This may include administering one or more doses to the patient. For example, the 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 the patient. In some of these and in other instances, the encapsulated drug products may be administered to the patient on one or more days. For example, the doses may be administered over 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or more days. In some of these and in other instances, the doses may be administered to the patient one or more times per day to the patient. For example, the doses may be administered 1 , 2, 3, 4, 5, 6, or more times per day to the patient.

[0051] One example dosing regimen may include administering two doses to the patient two times per day for two days. Another example dosing regimen may include administering four doses to the patient two times per day for two days. Another example dosing regimen may include administering four doses to the patient two times per day for four days. Another example dosing regimen may include two doses per day to the patient for four days. Another example dosing regimen may include one dose per day to the patient for eight days. These are just examples. In at least some of these examples, eachdose may include a lyophilized material comprising a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus rhamnosus.

[0052] In at least some instances, the vaginal microbiota composition, following administration of a composition as described herein, may be substantially (e.g. <1x102- 1x103CFU / ml) free of bacteria from the genus Sneathia, Megasphaera, Atopobium, Peptoniphilus, Dialister, Prevotella, Mobiluncus and / or Gardnerella. For example, the vaginal microbiota composition may be free of bacteria from the genera Atopobium, Gardnerella, and Prevotella.

[0053] The vaginal microbiota compositions disclosed herein may be used to treat a number of different health conditions. For example, the vaginal microbiota compositions may be used for treating bacterial vaginosis, treating candidiasis infections, treating human papilloma virus infections, treating urinary tract infections, treating sexually transmitted infections, treating gynecological cancers (e.g., cervical cancer), reducing and / or preventing preterm birth, reducing and / or preventing miscarriages, treating infertility, treating interstitial cystitis, treating polycystic ovary syndrome, and the like.

[0054] Vaginal microbiota compositions are also contemplated that include the combination of a full spectrum vaginal microbiota combined or mixed with one or more additional microbes (e.g., such as those collected and isolated from donors). For the purposes of this disclosure, the full spectrum vaginal microbiota may be understood to be a collection of microorganisms present in the vagina of a typical female. The full spectrum vaginal microbiota is not purposefully manipulated to change the presence or absence of any particular microorganism in the sample and, instead, is intended to represent the full population of organisms in the sample. It can be appreciated that the makeup of the vaginal microbiota may differ form person to person. Because of this, the full spectrum vaginal microbiota may have differences. In some instances, vaginal microbiota compositions are contemplated that include full spectrum vaginal microbiota that is combined, mixed, or doped with one or more of Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii / L. mulieris and optionally, one or more of Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii. Inat least some instances, both the full spectrum vaginal microbiota and the additional microbes are sourced from vaginal microbiota donors.

[0055] The bacteria / microbes may be disposed within a suitable vessel such as a capsule, suppository, soluble shell, and / or the like for oral or local delivery. In some instances, a simulated vaginal fluid may be incorporated into the vaginal microbiota composition. In some of these and in other instances, the vaginal microbiota composition may include glycogen. The total volume contained within the vessel / suppository may be on the order of about 100 microliters to about 10 milliliters.

[0056] In at least some instances, the bacteria / microbes in the vaginal microbiota composition may include lyophilized bacteria / microbes. This may include a lyophilization process in which bacteria / microbes are freeze-dried. The bacteria / microbes and / or the lyophilized bacteria / microbes may be disposed within a suitable vessel such as a capsule, suppository, soluble shell, and / or the like for oral or local delivery.

[0057] Treatment of patients may include the administration of a vaginal microbiota composition to the patient. One aim of the treatment may be to normalize the vaginal microbiota of the patient by effectively replacing the vaginal microbiota of the patient with the vaginal microbiota composition. This may include administering the vaginal microbiota composition directly to the vagina of the patient by implantation, suppository, or another suitable route of administration.

[0058] In at least some instances, a plurality of vaginal microbiota compositions may be stored or banked in a suitable storage device / facility. The storage device may include temperature control device such as a refrigeration device (e.g., a 4°C refrigeration device), freezer (e.g. a -20°C freezer), deep freezer (e.g., a -80°C freezer), or the like. In some instances, an indexing system may also be coupled to or otherwise associated with the vaginal microbiota composition bank. The indexing system may include data such as donor data that can be used to match a vaginal microbiota composition from a given donor with a suitable patient. The donor data may include age at collection, reproductive status (non-menopausal, menopausal) at collection, race, menstrual stage at collection, history of bacterial vaginosis, history of candidiasis infection, history of human papilloma virus, other medical history (e.g., history of C. difficile infection, history of cancer diagnosis / treatment, etc.), combinations thereof, and / or the like. A clinician may utilizedonor data to tailor a treatment regime to a particular patient. For example, a patient with bacterial vaginosis may be treated with a vaginal microbiota composition derived from a donor with a history of successful amelioration of bacterial vaginosis.

[0059] In some instances, donor data may include pregnancy history. For example, some donors may have a history of a high probability of becoming pregnant when seeking pregnancy or otherwise have a real or perceived elevated fertility. Vaginal microbiota compositions including microbes from such donors may be administered to patients experiencing fertility challenges in order to attempt to increase or boost the fertility of the patient. In such cases, the vaginal microbiota composition may be administered as an oral capsule. Alternatively, the vaginal microbiota composition may be administered as a transplant and / or otherwise inserted directly into the vagina of the patient.

[0060] U.S. Patent No. 9,675,648 is herein incorporated by reference.

[0061] U.S. Patent No. 9,629,881 is herein incorporated by reference.

[0062] U.S. Patent No. 10,226,431 is herein incorporated by reference.

[0063] U.S. Patent Application Pub. No. US 2018 / 0289750 is herein incorporated by reference.

[0064] Examples

[0065] The disclosure may be further clarified by reference to the following Examples and with reference to the figures , which show:

[0066] Figure 1 : Biofilm forming ability of lactobacilli isolates. (A) Crystal violet assay of monospecies biofilms at 24 hours. Macroscopic images were taken prior to leaching the dye (top), with the graph displaying absorbance readings after ethanol leaching (bottom). Graph displays mean and standard deviation from n=3. Statistics are one-way ANOVA with Tukey’s post-hoc for multiple comparisons. p<0.001 refers to comparisons between BPL5 and all other isolates. (B) Live / dead fluorescent staining of monospecies biofilms at 24 hours using SYTO9 (live) and propidium iodide (PI; dead) dyes. Image taken at x40 obj on AMG EVOS fl microscope.

[0067] Figure 2: Lactobacilli treatment of bacterial vaginosis biofilms. Experimental overview (A), followed by viable levels of G. vaginalis (B), F. vaginae (C), M. curtisii (D) and P. bivia (E). Each individual species was summed (F) and the composition of viable cells within biofilms calculated (G).

[0068] Figure 3: Supernatant treatments. Viable counts from BVAB biofilms (left) after application of lactobacilli supernatant. pH values from lactobacilli soups with and without pH adjustment are shown in the table.

[0069] Figure 4: Lactic acid against G. vaginalis. Showing levels of D- and L-lactic acid produced by probiotic isolates at 24 (A) and 48 (B) hours. pMIC of D-Lactic acid (C) and L-lactic acid (D) are shown against G. vaginalis. Synergy analysis of D- and L-lactic acid (E), and biofilm treatment using lactic acid (F).

[0070] Figure 5: Cocktail treatment. Treatment of BVAB biofilms using lactobacilli cocktails. Data shows biofilm composition after 4-hrs (A) and 24-hrs (B) treatments. Actual quantities are also shown for 24-hr biofilms (C).

[0071] Figure 6: Lactobacilli treatment attenuates the inflammatory potential of BV biofilms against VK2 cells. (A-C): BV bacteria (G. vaginalis, F. vaginae, P. bivia and M. curtsii) or lactobacilli (L crispatus, L. gasseri, L. mulieris, L. rhamnosus) were applied7 as planktonic cocktails at 1x10 CFU / mL on top of a VK2 monolayer for 24-hours. (D-F):7BV biofilms alone or BV biofilms treated with a lactobacilli cocktail at 1x10 CFU / mL were sonicated into 1 mL KSFM media and applied to a VK2 cell monolayer for 24-hours. (G): Live / dead imaging of VK2 cells following addition of sonicated biofilms. Stains are SYTO9 (green) and propidium iodide (red). Images taken at x20 obj on an EVOS fluorescent microscope.

[0072] Materials and Methods

[0073] Crystal violet biomass assay

[0074] Monospecies biomass was assessed using the crystal violet assay in 24 well microtiter plates. Following 24 hour incubation, biofilms were gently washed three times in sterile PBS to remove dead and unadhered cells. After washing, biofilms were dried by15 minute incubation at 55°C before 500 pL of 0.05% crystal violet solution (w / v) was applied to each well. Biofilms were incubated at room temperature for 15 minutes and then washed by dipping into beakers containing tap water. To leach dye, 500 pL of 100% ethanol was applied and gently mixed by pipetting 8 times. From each biofilm, 5 x 75 pL aliquots were transferred to a new flat-bottom 96 well microtiter plate. Well absorbance was read at 570nm using a FLUOstar Omega microplate reader. The average of five aliquots per well was used to give final biomass readings for each biofilm. All absorbance readings were corrected against media controls.

[0075] Live / dead fluorescent microscopy

[0076] Monospecies lactobacilli biofilms were imaged using the LIVE / DEAD™ BacLight™ bacterial viability kit (Invitrogen, Paisley, UK). This kit contains SYTO9 (3.34 pM) and propidium iodide (20 pM) dyes which fluoresce green and red respectively. Cells with an intact membrane will stain green, and cells with a compromised membrane (dead or dying) staining red. For this assay, 24 hour monospecies biofilms were gently washed once with sterile distilled water. A dye master solution was prepared by adding SYTO9 and propidium iodide at a 1 :1 volume ratio in sterile distilled water. The dyes were then applied to biofilms at 250 pL to cover the bottom of microtiter plates, and incubated in the dark for 15 minutes. After staining, dyes were removed and biofilms washed a further three times with sterile distilled water. Fluorescent imaging was performed using an EVOS (AMG EVOS fl) at x40 obj magnification.

[0077] Multi-species biofilm formation

[0078] Multi-species BVAB biofilms were grown using a ‘G. vaginalis primed system’, whereby this organism is allowed to colonise for 24 hours, followed by addition of remaining bacteria. This is similar to previously developed dual-species biofilm models in which G. vaginalis was initially cultured in isolation, followed by additional organisms (Machado et al., 2013, Castro et al., 2019, Castro et al., 2022). For this model, G. vaginalis was standardised to =1x108CFU / mL (OD550 0.2), and diluted 1 :10 in NYC III broth to 1x107CFU / mL. From here, 500 pL was applied to wells of a 24 well microtiter plate containing sterile 13 mm NuncTM ThermanoxTM coverslips (Fisher Scientific). Microtiter plates were incubated for 24 hours under anaerobic conditions at 37°C. After incubationmedia was removed and F. vaginae, P. bivia and M. curtisiiwere standardised separately to =1x108CFU / mL (OD550 0.15, 0.2 and 0.2 respectively). Each species was then combined to create a cocktail containing 1x107CFU / mL final concentration of each bacteria. Plates were incubated for a further 24 hours under anaerobic conditions at 37°C. On day 3, treatments (lactobacilli) were applied.

[0079] Probiotic treatment of multi-species biofilms

[0080] For probiotic treatment of biofilms, lactobacilli isolates were cultured in NYC III medium for 24 hours under anaerobic conditions at 37°C. Once turbid in growth, each isolate was standardised to =1x108 CFU / mL (OD600 0.5) in NYC III. For monospecies biofilm treatments, three concentrations of probiotic were applied to biofilms (=1x107, 1x108and 1x109CFU / mL). Cells were either directly applied (1x108 CFU / mL), diluted 1 :10 in NYC III (1x107CFU / mL) or concentrated to 1x109CFU / mL. For concentrating, falcon tubes containing 1x108CFU / mL were centrifuged at 3500 RPM for 10 minutes and resuspended in 10X less volume. For example, if 25 mL of broth was centrifuged then it was resuspended in 2.5 mL fresh sterile NYC III media. Once final concentrations were obtained, accessory pathogens were gently removed from biofilms and 500 pL of each probiotic was applied to BVAB biofilms for 4 or 24 hours.

[0081] For cocktail treatment, the same concentrations of total cells were applied (=1x107, 1x108and 1x109CFU / mL). For these experiments, each species was concentrated to 1x109CFU / mL and added in equal volumes to a final 4 species cocktail. This meant the concentration of each species was =2.5x108CFU / mL, creating a final cocktail concentration of =1x109CFU / mL which was confirmed by Miles-Misra colony counting. From here, this was diluted 1 :10 and 1 :100 to create cocktails containing =1x108and 1x107CFU / mL respectively. Once prepared, cocktail treatment was applied by adding 500 pL of each concentration to BVAB biofilms for 4 or 24 hours.

[0082] PMAxx™ treatment

[0083] Following multi-species biofilm treatment on day 4, coverslips were gently washed three times by dipping into petri dishes containing sterile PBS and placed into bijoux’s containing 1 mL PBS. To remove bacteria from coverslips, bijouxs were sonicatedat 35 kHz for 10 minutes, after which the sample was split into 500 pL aliquots. One of the samples was treated with PMAxx™ dye (Biotium, California, USA), using a method adapted from a previously published protocol (Latka et al., 2022). In brief, PMAxx™ was applied at 25 pM and tubes were gently flicked to mix. Tubes were then incubated in the dark at 4°C for 15 minutes. This process was repeated three times in total, taking the final PMAxx™ concentration to 75 pM. After PMAxx™ treatment, samples were then exposed to the PMA-lite device (Biotium) for a further 15 minutes.

[0084] DNA extraction

[0085] DNA was extracted from all samples using the MasterPure Complete DNA and RNA Purification Kit following their ‘Cell samples’ protocol (Biosearch Technologies, Hoddesdon, UK). For this protocol, PMAxx™ treated bacterial cells were pelleted by centrifugation at 10000 RPM for 5 minutes. The supernatant was discarded and pellets resuspended in 300 pL tissue and cell lysis solution supplemented with 1 pL of proteinase K (50 pg / pL). Samples were incubated on a hot plate at 65°C for 15 minutes with pulse vortexing every 5 minutes. Each sample was then placed in a cold-room at 4°C for 10 minutes, after which 150 pL MPC protein precipitation reagent was added. This solution was pulse vortexed and centrifuged at 4°C for 10 minutes at 15000 x g. The supernatant was then removed and applied to fresh DNAse free Eppendorf tubes containing 500 pL of 100% isopropanol, followed by 30 - 40 tube inversions. All samples were centrifuged at 4°C for 10 minutes at 15000 x g, supernatants were removed and pellets washed twice with 500 pL 70% ethanol. The remaining pellet was then resuspended in 35 pL TE buffer. DNA was stored at -20°C until further use.

[0086] Quantitative PCR (qPCR)

[0087] All qPCR reactions were performed on a ViiA 7 real-time PCR system (Applied Biosystems) using 20 pL reactions. Each reaction mixture consisted of 10 pL 2x PowerUp SYBR green mastermix (Fisher Scientific), 7 pL Hyclone molecular grade water, 1 uL forward primer (10 pM), 1 pL reverse primer (10 pM) and 1 pL of DNA. This resulted in final primer concentrations of 0.5 pM per reaction. After preparation, plates were incubated with the following cycling conditions on the ViiA 7 analyser; 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 3 s followed by 60°C for 30 s (with melt curve). Primer sequencesused throughout this study targeting each species (or genus) are highlighted below (table 1). For each organism, a standard curve was prepared using DNA extracted from known concentrations of the target bacteria (1x103- 1x108CFU / mL). This standard curve served to quantify the level of bacteria in each sample. No template controls (NTCs) containing 1 pL Hyclone molecular grade water instead of DNA were included alongside each reaction.

[0088] Table 1 : Primer sequences used during this study for qPCR to determine the composition of biofilm samples. Original references are highlighted.

[0089] Conditioned media

[0090] Conditioned media was prepared by initially standardising each isolate (LC01 , LG01 , LJ01 , BPL5 or combined as a cocktail) to 1x107CFU / mL in NYC III broth media. For each culture, 15 mL was added to 75 cm3cell culture flasks (CORNING Costar) and incubated for 24 hours under anaerobic conditions at 37°C to facilitate biofilm formation. The following day, media was removed and biofilms washed three times in sterile PBS,before 15 mL fresh media was applied for a further 24 hours. After incubation, biofilm supernatants were harvested as follows; supernatants were transferred to sterile 50 mL tubes and centrifuged at 3500 RPM. The residual supernatants were then transferred to a fresh 50 mL tube and sterile filtered using a syringe attached to a 0.22 pM filter.

[0091] Supernatants were used fresh for treatments. This involved preparation of a 5x NYC III broth solution containing 1.25 g of HEPES (Sigma-Aldrich, Gillingham, UK), 7.5 g of Protease-peptone (Sigma-Aldrich), 1.875 g of Yeast extract (Fisher chemical, Loughborough, UK), 2.5 g of Sodium chloride (NaCI; Fisher chemical) to 40 mL of distilled water. This solution was autoclaved at 121 °C and 10 mL of glucose (v / v at 25 g / 100 mL in H2O, equivalent to 2.5 g / 100 mL) was added, alongside 50 mL of heat-inactivated horse serum (GibcoTM, Fisher Scientific, Renfrew, UK). The final solution was 100 mL and contained 5x of each NYC III component.

[0092] Treatments were applied at 80% conditioned media, which consisted of 80% lactobacilli supernatant or water as control, plus 20% 5x NYC III broth. As such, all variables contained equal nutrient proportions. For follow-up experiments, this conditioned media was pH adjusted by addition of 3000 mg / L sodium bicarbonate and 50 mM HEPES or appropriate water vehicle control. This pH adjustment has been previously used to neutralise lactobacilli broths for use in vaginal epithelial cell co-culture experiment (Anton et al., 2022).

[0093] Media supplementation with citric and lactic acid

[0094] Media was pH adjusted using citric or lactic acid. For citric acid experiments, 20 mM sodium citrate (Sigma-Aldrich) was added to a 50 mL aliquot of NYC III (w / v = 0.258 g in 50 mL). From here, citric acid (w / v, Sigma-Aldrich) was gently added with continual pH measurements. At specific pH values (untreated, pH 5, pH 4.5, pH 4), 10 mL was aliquoted into a fresh 50 mL falcon tube and filter sterilised (0.22 pM). This pH adjusted media was applied to preformed BVAB biofilms as a treatment. For lactic acid, media was supplemented using D-lactic acid (Cambridge Bioscience, catalogue number 4003254.0250) or L-lactic acid (Sigma-Aldrich, catalogue number L1750-10G) to defined concentrations. This was then applied to biofilms to assess planktonic minimum inhibitoryconcentrations against G. vaginalis (defined as the concentration at which >90% growth is inhibited), synergy assessment and biofilm efficacy.

[0095] Planktonic and biofilm inhibition

[0096] Planktonic minimum inhibitory concentrations (pMIC90) for D- and L-lactic acid were performed using the CLSI M11-A8 broth microdilution method in NYC III, as reported previously for BVAB and lactobacilli (Landlinger et al., 2021 , Arroyo-Moreno et al., 2022). In brief, G. vaginaliswas initially standardised to 1x108CFU / mL in NYC III, and then further diluted to 1x106 CFU / mL in sterile media (1 :100). D- and L-lactic acid were purchased as powders and prepared to stock concentrations of 100 g / L (D-lactic acid) and 1000 g / L (L- lactic acid) in sterile distilled water. Working concentrations were prepared by dilution in NYC III medium and serially diluted 1 :2 in sterile media. D- and L-lactic acid were initially added at 100uL to 96-well round bottom microtiter plates at double the required concentration, followed by 100uL of 1x106CFU / mL G. vaginalis. The final bacterial concentration in wells was therefore 5x105 CFU / mL as is recommended for pMIC assays (Wiegand et al., 2008). Plates were incubated for 24 hours under anaerobic conditions at 37°C. The pMIC was visually determined by growth inhibition and confirmed by absorbance at 550nm on a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

[0097] Colony counting

[0098] Colony forming units (CFU / mL) analysis was performed on BVAB biofilms exposed to conditioned media and media supplemented with citric and lactic acid in 24- well microtiter plates. For this, treatments were removed and biofilms were gently washed twice with sterile PBS. After washing, 1 mL of PBS was applied to wells and biofilms removed by scraping. Colony counting was performed using the Miles and Misra method (Miles & Misra, 1938), in which serially diluted biofilms were applied in triplicate (20 pL) to Columbia blood agar plates. Plates were incubated for 48 h anaerobically, and CFU / mL estimation was performed by using the average of triplicate values.

[0099] Lactic acid measurements

[0100] Lactic acid measurements were conducted by staff at Rebiotix Inc. In brief, probiotic lactobacilli were incubated for 24 or 48 hours in MRS broth. From here, lactic acid measurements were performed on clarified supernatants. All measurements were conducted using the D- / L-Lactic Acid (D- / L-Lactate)(Rapid) assay kit by Neogen Megazyme.

[0101] Synergy assessment

[0102] A synergy assessment was performed between D-lactic acid and L-lactic acid using the checkerboard assay. This assay was performed on planktonic G. vaginalis at 5x105CFU / mL (same as pMIC), and the plate layout is as follows. Experiments were performed using a protocol developed by Emery Pharma © available at; (https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy- study-checkerboard-testing / ). In brief, plates were prepared containing a standard curve consisting of D- or L-lactic acid alone, and various concentrations of a combination of both D- and L-lactic acid. The inhibitory dose of each acid in isolation and in combination were used to determine a fractional inhibitory concentration index (FICi) which is calculated as below;Where; A and B are the MIC90of each acid in combination (in a single well), and MIC(a) and MIC (b) are the MIC90of each drug individually. Values <0.5 are synergistic, whilst values >4 indicate antagonism.

[0103] Culturing VK2 cells

[0104] The VK2 / E6E7 cell line (referred to as VK2 herein) was established in 1996 from the normal vaginal mucosal tissue taken from a premenopausal woman undergoing anterior-posterior vaginal repair surgery. These cells were used in all experiments described within this report. The original cell line was immortalised at passage 3 (p3), and all experiments were conducted with cells at p<10. For routine culture, VK2 cells were grown in KSFM media supplemented with 0.1 ng / ml human recombinant EGF, 0.05mg / mL bovine pituitary extract and additional calcium chloride at 44.1 mg / L (final concentration 0.4 mM). Cells were grown in 75cm3flasks (T75) until 80-90% confluent.

[0105] Cell splitting was performed by removing media from the T75 flask, gently rinsing with 2 mL 0.25% Trypsin I 0.03% EDTA solution. This 2 mL was removed and replaced with a fresh 3 mL Trypsin-EDTA and flasks were incubated for 5 minutes at 37°C under 5% CO2. After incubation, trypsin was neutralised by addition of 7 mL DMEM:F12 containing 10% foetal bovine serum. Cells were then placed in a 15 mL tube and centrifuged at 1000 RPM for 10 minutes. The supernatant was discarded and cells were resuspended in KSFM with (for subculture) or without (for experiments) the addition of 100 U / mL penicillin and 100 pg / mL streptomycin. For experimentation, cells were seeded at 1x105cells / well in 24-well plates using antibiotic free KSFM for 24 hours prior to addition of bacteria. Comparable cell concentrations have been previously used in host-bacterial co-culture work in the vagina (Anton et al., 2022) and the oral cavity (Brown et al., 2019).

[0106] Supernatant lactate dehydrogenase

[0107] Following exposure of VK2 cells to bacteria, supernatants were harvested and clarified by centrifugation at 10,000 RPM for 10-minutes. After clarification, lactate dehydrogenase (LDH) measurements were performed using the CyQUANT LDH cytotoxicity kit (Thermo Fisher, catalogue number C20301). This assay measures LDH present in cell culture supernatants as a marker of cytotoxicity. LDH is a cytoplasmic enzyme which will be released by dead or dying cells as membrane integrity is compromised. For this assay, 50 pL of clarified supernatant was mixed with 50 pL assay buffer and incubated for 30-minutes at room temperature. After incubation, the reaction was stopped by addition of 50 pL stop solution and absorbance was read at 490nmon a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) with correction at 680nm.

[0108] Supernatant ELISAs

[0109] Measurement of IL-6 and CXCL8 were also performed on clarified supernatants using sandwich enzyme-linked immunosorbent assays (ELISAs). These assays were purchased as premade kits from PEPROTECH; Human IL-6 TMB EDK (900-T16) andHuman IL-8 TMB EDK (900-T18, also referred to as CXCL8). Experiments were performed exactly as described by manufacturers. Cell culture supernatants were diluted 1 :2 or 1 :4 with PEPROTECH assay buffer to ensure each sample fell within range of the standard curve. Concentrations were determined by interpolating absorbance values from samples against a 5PL curve fitted against standards.

[0110] Propidium iodide imaging

[0111] Dead VK2 cell imaging was performed using propidium iodide (PI), which is a fluorescent membrane impermeable dye. As such, PI is unable to pass the intact cell membrane of viable cells and thus selectively fluoresces dead or dying cells with compromised membrane integrity. For VK2 cell imaging, cells were washed twice with sterile PBS before PI was added at 1 pg / mL. Cells were incubated for 15 minutes in the dark at room temperature before the dye was removed and cells were washed a further twice in sterile PBS. Imaging was then performed using an EVOS (AMG EVOS fl) at x10 obj magnification. For certain experiments, PI was combined with SYTO9 at the same concentration which fluorescently labels viable cells green.

[0112] Bacterial addition to VK2 cells

[0113] For planktonic work, bacterial isolates were standardised to 1x107 CFU / mL in KSFM media and directly applied to VK2 monolayers for 24 hours. For biofilm sonicate co-culture experiments, BVAB biofilms were grown and treated using a lactobacilli cocktail on 13 mm NuncTM ThermanoxTM coverslips (Fisher Scientific) under anaerobic conditions at 37°C. Following treatment, coverslips were gently washed three times by dipping into petri dishes containing sterile PBS and placed into bijoux’s containing 500 pL KSFM. To remove bacteria from coverslips, bijoux’s were sonicated at 35 kHz for 10 minutes, after which the 500 pL sample was applied directly on top of pre-adhered VK2 cells, and was incubated at 37°C with 5% CO2 for 24-hours. The following day, supernatants were harvested and LDH, CXCL8 and IL-6 were assayed as described above. Live / dead fluorescent imaging was also performed on VK2 cells.

[0114] Examples Section

[0115] Bacterial vaginosis (BV) is characterized by an imbalance in the vaginal microbiome, associated with a reduction in commensal lactobacilli and the formation of polymicrobial anaerobic biofilms. Currently, BV is managed using antibiotics (metronidazole and clindamycin), which exhibit unacceptably high rates of disease recurrence. Re-establishing a healthy microbiome using probiotics is an attractive treatment approach particularly following antibiotic failure. As such, this study sought to investigate the efficacy of four vaginal derived lactobacilli against an in vitro polymicrobial biofilm model representative of BV.

[0116] Example 1 Lactobacillus monospecies biofilm formation

[0117] Four Lactobacillus strains (L crispatus - LC01, L. gasseri - LG01, L. jensenii - LJ01 and L. rhamnosus - BPL5) were studied for their ability to form biofilms. As can be seen from Figure 1 , it is demonstrated that probiotic lactobacilli have different biofilm forming capabilities, but L. rhamnosus is particularly efficient at forming biofilms.

[0118] Example 2 Model bacterial vaginosis (BV) biofilm treatment using probioticsA BV model was developed and is based on the biofilm foundation of keystone pathogen Gardnerella vaginalis, and the introduction of three accessory BV pathogens, Prevotella bivia, Mobiluncus curtisii and Atopobium vaginae, which is representative of the dysbiotic clinical scenario. Four lactobacilli were isolated from healthy volunteers (L crispatus, L. gasseri, L. mulieris, L. rhamnosus). Anti-biofilm activity was assessed using live / dead qPCR. Additionally, the host response to treated biofilms was investigated using VK2 vaginal epithelial cells (Figure 2A).

[0119] The model allows for the study of direct therapeutic interventions which can be incorporated following biofilm development. Live / dead qPCR allows for a viability based compositional analysis to demonstrate treatment efficacy of probiotic based Lactobacillus interventions - monospecies and cocktail treatment (Figs 2B-G). As can be seen, this demonstrates that the four probiotic lactobacilli reduce the viable number of bacterial- vaginosis associated bacteria within preformed BV model species biofilms at a range of doses.

[0120] Example 3 pH dependence and lactic acid form of antibacterial effect

[0121] The 4 Lactobacillus strains as used in Example 1 were used in order to consider if any antibacterial effect was pH dependent. As can be seen from Figure 3, the antibacterial effect is pH (acidic) dependent, as the use of a buffer in order to maintain pH of the model BV biofilm, resulted in a cessation of any antibacterial effect. The pH of untreated biofilms was approximately pH 6 at 4 hours, and pH 4.3- 4.6 after 24 hour incubation with Lactobacillus. Additionally, this demonstrates that supernatants from lactobacilli elicit contact-independent killing of BVAB biofilms and that this effect is pH dependent.

[0122] Further studies looked at the effect of Lactic acid against G. vaginalis. Figures 4A and B show the relative amounts of D- and L-lactic acid produced by the four Lactobacillus strains after 24 (Figure 4A) and 48 hours (Figure 4B) respectively. As can be seen from Figures 4C and 4D, different inhibitory concentration levels against G. vaginalis, are observed between D- and L-lactic acid. Figures 4E and 4F show that there is a synergistic inhibitory effect against G. vaginalis, when using D- and L-lactic acid in combination. Figure 4F shows that a combination of D- and L-lactic acid is best in providing an antibacterial effect. Given the relative levels of D- and L-lactic acid produced by the 4 strains (see Figures 4A and 4B) and biofilm generation ability of Lactobacillus species (see Figure 1), a combination of L. rhamnosus and at least one other tested Lactobacillus strain may be most efficacious in terms of providing a suitable combination of D- and L- lactic acid and antibacterial effect.

[0123] Example 4 BV model biofilm composition before and after Lactobacillus treatment

[0124] As Demonstrated in Figure 5 lactobacilli applied as a cocktail can effectively kill preformed BV biofilms by 24hr - Figure 5A at 4hours and Figure 5B at 24 hours. Although lactobacilli species colonised to varying degrees, L. rhamnosus (BPL5) was the dominant Lactobacillus species at 4hr and 24hrs (see Figures 5A and 5B). Figures 50 and 5D show the same data as presented in part B, however this is the absolute number of viable cells present as opposed to them presented in a percentage format in fig5B

[0125] Example 5 The effect of Lactobacilli treatment in attenuating an inflammatory potential of model BV biofilms against vaginal mucosal cells.

[0126] As can be seen from Figure 5, planktonic and biofilm forming BV model bacteria result in the production of an inflammatory marker (lactase dehydrogenase) and inflammatory cytokines (IL-6 and CXCL8). However, addition of a Lactobacilli cocktail (L crispatus, L. gasseri, L. mulieris, L. rhamnosus), resulted in a significant reduction of all markers when added to planktonic bacteria and a significant reduction of LDH and CXCL8 when added to a BV cocktail.

[0127] Conclusions

[0128] All lactobacilli elicited an anti-bacterial effect against pre-formed BV biofilms, and perform favourably when compared with antibiotic treatment of the same model (3); Each isolate was able to colonise BV biofilms when applied as a cocktail, with L. rhamnosus the most abundant species after 24-hours; Conditioned media from each species significantly reduced the bioburden of BV biofilms, which was reversed upon pH neutralisation with HEPES and sodium bicarbonate; and Treatment with lactobacilli significantly attenuated the inflammatory potential of BV biofilms against VK2 cells.References:Machado, A., Jefferson, K. K. & Cerca, N. 2013. Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation. Int J Mol Sci, 14, 12004-12.Castro, J., Machado, D. & Cerca, N. 2019. Unveiling the role of Gardnerella vaginalis in polymicrobial Bacterial Vaginosis biofilms: the impact of other vaginal pathogens living as neighbors. ISME J, 13, 1306-1317.Castro, J., Sousa, L. G. V., Franga, A., Podpera Tisakova, L., Corsini, L. & Cerca, N. 2022. Exploiting the Anti-Biofilm Effect of the Engineered Phage Endolysin PM-477 to Disrupt In Vitro Single- and Dual-Species Biofilms of Vaginal Pathogens Associated with Bacterial Vaginosis. Antibiotics (Basel), 11 .Latka, A., Van Simaey, L., Reynders, M., Cools, P., Rogier, T., Lebbe, B., Corsini, L., Landlinger, C. & Vaneechoutte, M. 2022. Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis. Antibiotics (Basel), 11 .Zozaya-Hinchliffe, M., Lillis, R., Martin, D. H. & Ferris, M. J. 2010. Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis. J Clin Microbiol, 48, 1812-9.Pacha-Herrera, D., Vasco, G., Cruz-Betancourt, C., Galarza, J. M., Barragan, V. & Machado, A. 2020. Vaginal Microbiota Evaluation and Lactobacilli Quantification by qPCR in Pregnant and Non-pregnant Women: A Pilot Study. Front Cell Infect Microbiol, 10, 303. Anton L, Ferguson B, Friedman ES, Gerson KD, Brown AG, Elovitz MA. Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses. Microbiome. 2022; 10(1 ):119. Published 2022 Aug 4. doi: 10.1186 / s40168-022- 01317-9Landlinger, C., Tisakova, L., Oberbauer, V., Schwebs, T., Muhammad, A., Latka, A., Van Simaey, L., Vaneechoutte, M., Guschin, A., Resch, G., Swidsinski, S., Swidsinski, A. & Corsini, L. 2021. Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo. Pathogens, 10.Arroyo-Moreno, S., Cummings, M., Corcoran, D. B., Coffey, A. & McCarthy, R. R. 2022. Identification and characterization of novel endolysins targeting Gardnerella vaginalis biofilms to treat bacterial vaginosis. NPJ Biofilms Microbiomes, 8, 29.Wiegand, I., Hilpert, K. & Hancock, R. E. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc, 3, 163-75.Brown, J. L., Johnston, W., Delaney, C., Rajendran, R., Butcher, J., Khan, S., Bradshaw, D., Ramage, G., & Culshaw, S. (2019). Biofilm-stimulated epithelium modulates the inflammatory responses in co-cultured immune cells. Scientific reports, 9(1), 15779. https: / / doi.Org / 10.1038 / S41598-019-52115-7

Claims

Claims1 . A composition for use in therapy, comprising: at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species, the / said Lactobacillus bacterial species comprising Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii / Lactobacillus mulieris, and / or Lactobacillus rhamnosus, the composition optionally comprising one or more pharmaceutically acceptable adjuvants and / or excipients.

2. The composition for use according to claim 1 , comprising a mixture of Lactobacillus species, wherein said mixture comprises L. rhamnosus and / or a supernatant derived from a culture of L. rhamnosus and at least one of said other identified Lactobacillus species and / or a supernatant(s) derived from cultures of the / said other Lactobacillus bacterial species.

3. The composition for use according to claims 1 or 2 comprising a mixture of all 4 identified species and wherein L. rhamnosus is present in a greater amount than each of the other identified species.

4. The composition for use according to any preceding claim, wherein the composition comprises about 20-85% Lactobacillus rhamnosus, preferably about 25-75% Lactobacillus rhamnosus, more preferably about 30-60% Lactobacillus rhamnosus, and more preferably about 40% Lactobacillus rhamnosus.

5. The composition for use in therapy according to claim 1 , wherein the composition is for reducing microbial species, such as bacterial, yeast, fungi and / or viral species, associated with a vaginal infection / condition.

6. The composition for use in therapy according to claim 5, wherein the bacterial, yeast, fungi and / or viral species is present within the vagina as a biofilm.

7. The composition for use in therapy according to either of claims 5 or 6, wherein the bacterial species associated with a vaginal infection / condition comprise oneor more of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and / or Mobiluncus curtisii.

8. The composition for use in therapy according to any of claims 5 - 7, wherein the reduction in microbial species corresponds with a recolonization by one or more of said administered Lactobacillus bacterial species.

9. The composition for use in therapy according to any preceding claim, wherein the composition is for use in treating a biofilm and / or planktonic microbial species.

10. The composition for use according to any preceding claim wherein the composition reduces the pH at the site of administration.11 . The composition for use according to claim 10, wherein the reduction in pH is due, at least in part, by the / said Lactobacillus bacterial species producing D- and / or L- lactic acid.

12. The composition for use according to claim 11 , wherein the reduction in pH is due, at least in part, by the / said Lactobacillus bacterial species producing a combination of both D- and L- lactic acid.

13. The composition for use according to claim 12 wherein the ratio (w / w) of D- lactic acid to L-lactic acid is 1 :8 to 1 :2, such as 1 :6 - 1 :

314. The composition for use according to any preceding claim wherein said at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species modulates (e.g. attenuates) an immune response of epithelial vaginal cells against pathological microbial species being present in the vagina.

15. The composition for use according to claim 14, wherein modulation of the immune response comprises a reduction of one or more inflammatory markers and / or cytokines.

16. The composition for use according to claim 15, wherein modulation of the immune response comprises a reduction in one, two or all three of lactate dehydrogenase, interleukin 6 (IL-6) and / or interleukin 8 (CXCL8).

17. The composition for use in therapy according to any preceding claim, wherein the composition is for use in treating a vaginal infection or condition, wherein the infection / condition includes one or more of bacterial vaginosis (BV), dysplasia, a candidiasis infection, a human papilloma virus infection, a urinary tract infection, a sexually transmitted infection, or a gynecological cancer.

18. The composition for use in therapy according to any preceding claim, wherein said at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species is disposed in a capsule.

19. The composition for use in therapy according to any preceding claim, wherein said at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species is disposed in a suppository.

20. The composition for use in therapy according to any preceding claim, wherein said at least one or more Lactobacillus bacterial species, and / or supernatant(s) derived from cultures of the / said Lactobacillus bacterial species is disposed in a soluble shell.

21. The composition for use in therapy according to any preceding claim, wherein said composition comprises 1 x 105to 1x1015CFU / ml, 1 x 106to 1x1012CFU / ml, 1 x 107to 1x1010CFU / ml, or 1 x 108to 1x109CFU / ml of at least said one or more Lactobacillus bacterial species.

22. The composition according to any preceding claim, wherein the composition comprises a mixture of lyophilized bacteria.

23. A method for treating an infection, the method comprising:administering the composition of any preceding claim to a patient with an infection, wherein the infection includes one or more of bacterial vaginosis, a candidiasis infection, a human papilloma virus infection, a urinary tract infection, a sexually transmitted infection, and a gynecological cancer.

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