Patent Grant
12005089
The present invention is generally in the field of treatment of infections of the vagina and sexually transmitted diseases.
Approximately one-third of all women currently have bacterial vaginosis (BV), a condition where the vaginal microbiota is not dominated by lactobacilli. Another one-third of women have mixed vaginal microbiota (“intermediate BV”), and only one-third of women have healthy, lactobacilli-dominated microbiota. Women with BV are known to have >2-6-fold increased susceptibility to numerous sexually transmitted infections (STI), including HIV, herpes (HSV), gonorrhea, chlamydia, and other viral, bacterial, and protozoan pathogens. STI transmission rates from women to men are higher if the woman has BV. Pregnant women with BV are much more susceptible to miscarriage, premature delivery, and post-partum endometriosis. Strong links have also been established between BV and increased incidence of pelvic inflammatory disease and urinary tract infections.
Perhaps most alarming, few people have heard of BV, and even fewer know how to identify if they have it. The standard of care is vaginal or oral antibiotics. However, the effectiveness of antibiotics is limited by mutation leading to antibiotic-resistance, and estimates of BV relapse 4 weeks after antibiotic treatment are as high as 70%. Although BV is an incredibly important global women's health issue, there is currently no known long-term cure. Several attempts have been made to colonize the vagina with large doses of specific probiotic strains of lactobacilli, but the results have been disappointingly modest. In contrast to probiotic strategies in which a single strain of dormant lactobacilli is placed into an environment that is detrimental to its survival, isolating one important player in a complex mix of factors appears to be too simplistic of an approach to be fully effective.
Studies have been conducted involving the introduction of probiotic lactobacillus strains in isolation, which have demonstrated modest results. One strain in particular, Lactobacillus crispatus CTV-05, has been demonstrated to achieve colonization in the vaginas of women without BV and was demonstrated as safe and tolerable in a Phase 2 trial in women with BV. Fecal transplants have been demonstrated to be safe, and have had as high as 94% effectiveness at eradicating C. difficile infection in clinical studies. Probiotic products have also been used, as reported by Antonio M A, et al. J Infectious Dis 199(10); 1506-1513 (2009), who studied microbial composition over time, and demonstrates that pregnant women that have healthy, term pregnancies are more likely to have Lactobacillus crispatus-dominated microbiota, and their microbial communities are more stable over time. Romero et al. Microbiome, 2:4 http://www.microbiomejournal.com/content/2/1/4 (2014) was the first report of the temporal dynamics of vaginal microbiota in healthy, reproductive age women. This paper discusses that Lactobacillus crispatus dominated communities are more stable, and therefore, less often associated with transitions to a state of bacterial vaginosis (BV). Gajer et al. Science Translational Medicine, 4(132) 132ra52 (2012) demonstrated in pregnant women that L. crispatus colonization is more stable, and that L. iners is more conducive to the development of BV. Verstraelen et al. BMC Microbiology 9(116) (2009) describes the first, and now unethical, studies of BV-associated bacteria, with pregnant women. Gardnerella vaginalis alone was insufficient in initiating BV in 12 out of 13 women with lactobacillus-dominated vaginal microbiota. However, 11 of 15 women inoculated with cervicovaginal fluid from women infected with G. vaginalis developed symptoms, indicating that other environmental factors were needed for the bacteria to thrive. Criswell et al. Obstet and Gynecol. 33(2) 195-199 (1968) describes the prevalence of BV amongst women who have sex with women. They found that of 58 monogamous couples, 95% were concordant for the presence or absence of BV, which was statistically very distinct from the normal distribution expected in the female population. This would indicate that vaginal microbiota transfer must occur as a result of transfer of vaginal fluids. Marrazzo et al. JID. 185:1307-13 (2002) described BV relapse rates of up to 70% one month after antibiotic treatment. See also Larsson and Forsum, APMIS. 113: 305-16 (2005).
The introduction of beneficial bacterial communities within the environmental milieu that supports their survival appears to be more effective than introducing isolated bacterial strains. No such communities have been identified or tested for treatment of BV, however.
It is therefore an object of the present invention to provide a method and materials for treating BV.
It is another object of the present invention to identify “donor” participants with the characteristics necessary for providing donor samples for treatment and prevention of BV.
It is another object of the present invention to provide methods and materials for CVS transplants to increase the effectiveness of standard antibiotic treatments for treating bacterial vaginosis.
Method and materials for treating bacterial vaginosis (BV) are disclosed herein. Methods and materials for transplanting cervicovaginal secretions (“CVS”) to increase the effectiveness of standard antibiotic treatments for BV are also described. Methods for identifying “donor” participants with the characteristics necessary for providing donor samples for treatment and prevention of BV have also been developed.
Instead of isolating and purifying a particular strain, CVS from one or more women with vaginal microbiota dominated (>50%) by species typically found in the human vagina, e.g. Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, is transplanted to women with BV as a method for restoring beneficial vaginal microbial communities and/or increasing resistance to sexually transmitted disease and BV recurrence. Efficacy can be enhanced, or the properties of the endogenous CVS improved, through administration of an acidifying agent, before and/or after transplantation, such as lactic acid. The CVS 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 and, optionally, packaged into single dosage unit applicators.
The method is based on the following:
Only certain lactobacillus communities and strains are truly healthy and protective against vaginal infection and should be transplanted to women after treatment for bacterial vaginosis.
The whole bacterial communities must be introduced in the vaginal environment to establish colonization, rather than isolated bacterial strains.
Components of cervicovaginal mucus itself are beneficial to bacterial growth and survival, indicating that the mucus itself is the ideal vehicle for introduction of microbiota into the vagina.
Vaginal microbiota transplantation can be supported by repeated vaginal delivery of lactic acid or other similar compounds.
Examples demonstrate the role of healthy CVS in disease resistance, and the effect of pH on CVS properties. The examples also describe the collection and transplantation of healthy beneficial CVS into women at risk for, or after treatment for, BV.
Probiotic, as used herein, utilizes the World Health Organization's 2001 definition of “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host”. Probiotics must be alive when administered, have viability and reproducibility based on in vivo testing, and during use and storage.
Microbial flora refers to the microorganisms that normally live in the gastrointestinal tract, skin, nose, etc. In a healthy human, the internal tissues, e.g. blood, brain, muscle, etc., are normally free of microorganisms. However, the surface tissues, i.e., skin and mucous membranes, are constantly in contact with environmental organisms and become readily colonized by various microbial species. The mixture of organisms regularly found at any anatomical site is referred to as the normal flora, except by researchers in the field who prefer the term “indigenous microbiota”. Bacteria are the most numerous microbial components of the normal flora.
Microbiota, a term created by Jeffrey Gordon, refers to the collection of microbial species that form a microbial community. This includes the normal flora and “harmful” ones. “Microbiome,” refers to the collection of genes present in the genomes of microbial species present in a community.
Bacterial vaginosis (“By”), 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.
Cervicovaginal secretions refers to the mixture of mucus secreted by the cervix, shed epithelial cells, vaginal transudate, and bacteria found in the vagina of a woman.
Sexually transmitted diseases (“STD”) are any of various diseases or infections (such as syphilis, gonorrhea, chlamydia, and genital herpes) that are usually transmitted by direct sexual contact and include some (as hepatitis B and AIDS) that may be contracted by other than sexual means.
As used herein, a “dissolution agent” is an acid, or salt thereof, that is added to the vaginal area, vaginal secretions, cervicovaginal secretions, or formulations containing cervicovaginal secretions. The secretions may be in dry (lyophilized) or wet form.
Cryoprotectant is any agent that prevents the formation of ice crystals, which can rupture cell membranes.
Diluent is any solution, optionally containing a cryoprotectant and/or a dissolution agent. Diluent may optionally be balanced for a desired osmolarity.
A. CVS Transplants
The materials for transplant are secretions collected using standard techniques from women with vaginal microbiota dominated (>50%) by species of lactobacillus typically found in the human vagina, e.g. Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, who are healthy, free of sexually transmitted disease and bacterial vaginosis, and have a low pH in the secretions. As described in Example 1, these can be collected using commercially available materials such as Instead Soft Cup menstrual fluid device, beaker, syringe, or absorbent matrix.
The secretions are preferably stored in the refrigerator at 4° C. for up to 1 week, or in certain cases, immediately frozen after collection and stored for up to several months, before being implanted into the recipient. Samples must maintain at least 20% viable bacteria prior to use.
The identity and relative abundance of bacteria in the CVS are determined by 16S rRNA pyrosequencing. The sequencing data is then used to identify a community state, and only samples classified within the community states of lactobacillus that are typically found in the human vagina, including Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii, will be considered for transplant. This community state often includes other species of Lactobacillus in smaller fractions. Genetic sequencing techniques and assignment of community states have been defined by the laboratory of Jacques Ravel at the University of Maryland.
B. Additives: Dissolution Agents, Cryoprotectants and Diluents
The secretions may be in dry (lyophilized) or wet form. Suitable dissolution agents include nitrogen-free organic acid having at least one carboxylic acid group and a total of from 2 to about 20 carbon atoms, a phosphoric acid containing compound, a sulfonated polyphosphoric acid compound, a polyphosphonate having three or more phosphonate groups, an enzyme; or salts thereof; or combinations thereof. Examples include lactic acid, citric acid, tartaric acid, gluconic acid, glycolic acid, hydroxysuccinic acid, galactaric acid, hydroxypropionic acid, lactic acid, glyceric acid, hydroxybutyric acid, hydroxyisobutyric acid, hydroxy methylbutyric acid, bis(hydroxymethyl) propionic acid, gibberellic acid, hydroxyoctadecanoic acid, di-tert-butyl hydroxybenzoic acid, benzilic acid, hydroxyl fluorenecarboxylic acid, hydroxydecanoic acid, hydroxynaphthalenecarboxylic acid, hydroxybenzenedicarboxylic acid, hydroxymethylbenzoic acid, hydroxyphenylacetic acid, mandelic acid, hydroxymethoxybenzoic acid, methoxysalicylic acid, hydroxyoctanoic acid, hydroxy cinnamic acid, dihydroxycinnamic acid, dihydroxy-hydrocinnamic acid, hydroxyphenylpropionic acid, dihydroxytartaric acid, hydroxymethoxycinnamic acid, chlorohydroxybenzoic acid, chloromandelic acid, chloro phthalic acid, salicylic acid, chlorosalicylic acid, citrazinic acid, dibromo hydroxybenzoic acid, dichlorohydroxy-benzoic acid, dichlorosalicylic acid, galactouronic acid, glucuronic acid, hydroxypropanedioic acid, hydroxyphenyl propionic acid, lactic acid, methoxysalicylic acid, trihydroxybenzoic acid, or their partial salts and combinations thereof. Another group of organic acids are various hydroxyl free and nitrogen free saturated or unsaturated dicarboxylic acids having from 2 to about 20 carbon atoms and can contain nitrogen atoms. Examples include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, fumaric acid, decanedoic acid, camphoric acid, benzenedicarboxylic acid, phthalic acid, cyclohexanedicarboxylic acid, cyclohexanediacetic acid, octanedioc acid, homophthalic acid, phenylmalonic acid, cyclopentanediacetic acid, nonanedioic acid, benzylmalonic acid, phenylenediacetic acid, phenylsuccinic acid, bromosuccinic acid, carboxyphenacetic acid, cyclobutanedicarboxylic acid, cyclohexanedicarboxylic acid, decanedicarboxylic acid, dibromosuccinic acid, dichlorophthalic acid, diethylmalonic acid, diglycolic acid, dimethylmalonic acid, dimethyl pentanedioic acid, dimethylsuccinic acid, ethylmalonic acid, glutamic acid, hexenedioic acid, imino diacetic acid, methylmalonic acid, methylsuccinic acid, naphthalene dicarboxylic acid, oxalacetic acid, oxopentanedioic acid, undecane dicarboxylic acid, dipicolinic acid, or their partial salts, and combinations thereof.
Cryoprotectant is any agent that prevents the formation of ice crystals, which can rupture cell membranes. Cryoprotectants include extracelluar cryoprotectants that do not penetrate bacterial cell walls, and intracellular cryoprotectants that penetrate bacterial cell walls. Examples of extracellular cryoprotectants include sucrose, dextrose and polyvinylpyrrolidone (PVP). Examples of intracellular cryoprotectants include dimethyl sulfoxide (DMSO) glycerol (glycerine).
Diluent is any solution, optionally containing a cryoprotectant and/or a dissolution agent. Diluent may optionally be balanced for a desired osmolarity. Exemplary diluent may be a solution of sodium chloride and lactic acid.
In a preferred embodiment, the recipient may also receive daily vaginal treatment with a food acid such as a lactic acid gel, spray or powder before and/or after transplantation to encourage Lactobacillus growth. Daily treatment may occur for up to 1 week before and/or after transplantation. The preferred concentration range of lactic acid to promote Lactobacillus survival is 1-1.5% lactic acid. Lactic acid is preferred to other types of food acid such as vinegar, lemon juice, and acetic acid, although these may also be utilized.
A. Treatment of BV
Certain types of lactobacillus and compositions of vaginal microbial communities confer stability and resistance to bacterial vaginosis (BV). These are preferably obtained from women with vaginal microbiota dominated (>50%) by species of lactobacillus typically found in the human vagina, e.g. Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii, who are healthy, free of sexually transmitted disease and bacterial vaginosis, and have a low pH in the secretions. These are administered to women with BV, as identified clinically with Amsel's criteria, and confirmed in the laboratory by Nugent scoring (Nugent et al., Journal of Clinical Microbiology, 29(2):297-301 (1991)). In a preferred embodiment, women with recurrent BV (requiring >3 treatment courses in 1 year) will first be treated with standard antibiotic treatment to reduce the bacterial load in the vagina. Twenty-four hours after the final antibiotic dose, the recipient will then receive a CVS transplant. The recipient will remain supine for at least 1 hour. Vaginal swabs will be collected from the recipient before transplant, after transplant, and at standard intervals (1 month, 2 months) after transplant. Characterization of vaginal microbiota will be done by 1) Amsel's criteria, 2) Nugent score, and 3) 16S rRNA sequencing. “Success” will be defined as a lack of BV relapse at 1 and 2 months after transplant, as assessed by Amsel's criteria and Nugent score. 16S rRNA sequencing will reveal the relative abundance of vaginal bacteria, and the degree of similarity to the composition of the transplanted sample.
B. Increased Resistance to STDs
CVS transplants also have barrier properties to sexually transmitted pathogens such as HIV are compromised in BV. However, these mucus barrier properties do not appear to be restored, even after successful antibiotic treatment, which is likely because the lactobacillus species most commonly associated with BV (strains of L. iners) also have a negative impact on the vaginal mucus barrier. Transplantation of more beneficial lactobacillus types, including the vaginal microbial community and the mucus environment in which they live, is a promising method for re-establishing healthy bacterial communities that do not compromise the structural and adhesive properties of CVS in the vaginas of women with recurrent BV.
Several samples have been obtained with high Nugent scores and pH>4.5 (together indicating BV) which look normal to the naked eye or are very thick in consistency, which may be a further reflection of the diversity of BV-associated microbiota and their effects on CVS. Changes in the local microstructure of BV samples, as probed by 1000 nm mucus penetrating particles (MPP) (
Although the CVS samples were acidic after treatment, it was found that the L/D lactic acid ratios were similar to when the participants had BV. This further supports the belief that certain strains of L. iners, the type of lactobacilli most commonly present in the vagina after BV treatment, may not be associated with increased protective benefits in the vagina.
C. Dosage Unit Formulations
The CVS or a sterile filtered and pH adjusted CVS can be packaged into single dosage units for ease of administration. Typically these would be in a dispenser or applicator, sterile packaged, which has a tip for insertion into the vagina, and a plunger to expel the packaged formulation.
In other embodiments, the CVS can be lyophilized or spray dried and stored frozen or in a sterile container, for resuspension immediately prior to use. The CVS can be resuspended with sterile water, a weak acidic solution, gel, or buffer.
In yet another embodiment, the spray dried formulation can be formulated as a disk or wafer, which is inserted into the vagina where it hydrates and repopulates the vaginal mucosa.
In all of these embodiments, dyes, perfumes, pH buffering agents, drying or resuspending agents, or other materials standard in the probiotic field can be incorporated into the formulations.
Typically, the dosage unit formulations contain between 103 and 1015 colony forming units (CFU) of any one of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii. In some aspects, the dosage units provide between 103 and 1015 CFU of any combination of Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii.
The present invention will be further understood by reference to the following examples.
For CVS transplants to be successful, bacteria in the “donor” sample must be in an environment that is beneficial to their survival. Lactobacilli thrive in an acidic environment with high lactic acid concentration, which is inhospitable to many other types of bacteria, including those commonly associated with BV. Thus, it is important that, upon mixing with the vaginal secretions of a woman with BV, the environment must become acidified.
Materials and Methods
Ensemble-averaged geometric mean square displacements (<MSD>) as a function of time scale for 1000 nm MPP, which diffuse more rapidly in CVS from women with BV than from women with healthy microbiota, and for HIV, were measured in BV-CVS as well as in CVS from women with healthy microbiota (not on hormonal contraceptives). W=theoretical MPP/HIV diffusion rate in water.
The Percentage of HIV with Log10 (MSD) values at a time scale of 1 s in CVS from women collected before and after antibiotic treatment for BV were calculated.
Results
Materials and Methods
Mixing experiments were performed to demonstrate killing of BV-associated bacteria in freshly obtained BV-CVS. In these experiments, the “supernatant” (the fluid containing bacteria, soluble factors, etc. after centrifuging a CVS sample to separate the large molecular weight mucin components and cells) of healthy CVS samples was mixed with BV-CVS. The mixture was then plated to determine the effectiveness of the BV-associated bacteria “killing”.
Mixing experiments were performed in which CVS with pathogen trapping ability was mixed 1:1 with CVS that did not trap pathogens (one participant on hormonal contraceptives (HC), and one participant with BV).
For CVS transplants to be successful, bacteria in the “donor” sample must be in an environment that is beneficial to their survival. Lactobacilli thrive in an acidic environment with high lactic acid concentration, which is inhospitable to many other types of bacteria, including those commonly associated with BV. Thus, it is important that, upon mixing with the vaginal secretions of a woman with BV, the environment must become acidified.
Results
As shown in
As shown in
This experiment was conducted with 3 other sample combinations, with similar results: the pH of the CVS mixture immediately after mixing was in the range ideal for lactobacillus growth, the CVS mixtures completely trapped virus after incubation overnight, and the L/D ratio decreased to a level similar to the healthy CVS sample.
Materials and Methods
Production of Cell-Free Sterile-Filtered CVS (CF-L-CVS):
Whole CVS from individuals with lactobacilli was centrifuged at 20,000×g for 2 minutes. The collected supernatant was then spun again over a 0.2 micron spin filter.
Microdialysis:
CF-L-CVS was further pipetted into dialysis chambers and sealed with a square of 1K MWCO dialysis membrane. Chambers were floated in a medium with one of the following dialysates for 2 hours, the dialysates being saline (normal saline, 0.9% sodium chloride, pH 7), saline (containing 25 mM glycylglycine buffer and adjusted with hydrochloric acid and sodium hydroxide as needed) with pH to match the CF-L-CVS (sample pH ranges ˜3-4), or saline (containing 25 mM glycylglycine buffer and adjusted with hydrochloric acid and sodium hydroxide as needed) with matching pH and matching lactic concentration as the CF-L-CVS (sample ranges 0.5-1.5% lactic acid). Both saline buffers contained adjusted sodium chloride such that each buffer had the same osmolality. The dialysis was sufficient to reduce the lactic acid concentration of CVS by ˜95%, unless the dialysate was supplemented with matching concentration of lactic acid.
BV-Associated Bacteria Inactivation Assay:
BV-CVS was diluted 1:100 into one of the following media, (i) saline, (ii) CF-L-CVS, (iii) CF-L-CVS that was dialyzed against saline, (iv) CF-L-CVS that was dialyzed against saline with matching pH as the CF-L-CVS, or (v) CF-L-CVS that was dialyzed against saline with matching pH and matching lactic concentration as the CF-L-CVS. Diluted BV-CVS in a medium was incubated under anaerobic conditions at 37° C. before being plated onto Brucella broth 5% sheep blood plates and incubated anaerobically for 2-3 days, before colony forming units (CFUs) were counted.
Results
As shown in
Approximately one-third of all women currently have BV, a condition where the vaginal microbiota is not dominated by lactobacilli. Another one-third of women have mixed vaginal microbiota (“intermediate BV”), and only one-third of women have healthy, lactobacilli-dominated microbiota. Women with BV have >2-6-fold increased susceptibility to numerous sexually transmitted infections (STI), including HIV, herpes (HSV), gonorrhea, chlamydia, and other viral, bacterial, and protozoan pathogens. STI transmission rates from women to men are also higher if the woman has BV. Pregnant women with BV are much more susceptible to miscarriage, premature delivery, and post-partum endometriosis. Strong links have also been established between BV and increased incidence of pelvic inflammatory disease and urinary tract infections. The current clinical treatment for BV includes vaginal or oral antibiotic treatment. Current experimental methods for treating BV include lactic acid gels/washes and vaginal probiotics, and have had minimal success.
A method for transplantation of CVS from a woman with healthy vaginal microbiota to a woman with BV as a means for restoring healthy vaginal microbiota has been developed. CVS transplants should be effective to cure or reduce symptoms or severity of BV, thereby having a major impact on women's sexual and reproductive health.
Materials
Donor participants will be recruited from the Johns Hopkins hospital area. They will be asked to provide CVS samples and vaginal swabs on the initial visit for screening. If they are selected as a donor, the donor candidate will be asked to return for a more thorough screening visit that involves a routine gynecological exam, blood samples to screen for HIV and other infectious diseases, and vaginal swabs for sexually transmitted infection screening. Donor participants will agree to abstain from sexual contact between the screening visit and returning to provide a CVS sample for donation to a recipient candidate. Donor participants, due to the nature of their CVS, are at a markedly reduced risk of acquiring sexually transmitted infections.
CVS samples are obtained by insertion of an INSTEAD SOFT CUP® menstrual fluid device (as described in active protocols HIRB00000526, NA_00038105, and NA_00090758).
Methods
“Recipient” participants will be recruited from the Johns Hopkins Outpatient Center (JHOC). The recipients will be prescribed antibiotic treatment by a medical professional at the clinic. On the seventh (final) day of antibiotic treatment, the recipient will undergo a routine gynecological exam, provide vaginal swab and CVS samples, and then receive the CVS transplant.
Collected donor CVS samples are loaded into a syringe and inserted into the vagina; similar procedures are used to administer and collect fluid (cervicovaginal lavage collection) as described in active protocol NA_00036496.
Control subjects receive a standard vaginal applicator loaded with vaginal placebo gel (hydroxyethylcellulose, HEC). The recipient will be asked to remain supine for 1 h after the procedure. After the procedure, the recipients will be counseled about the benefits of condom use and the negative impacts of vaginal products on the success of the transplant. The recipients will be given journals for logging information about sexual activity and vaginal product use, as well as swabs for taking twice weekly self-samples. The recipient will be scheduled to return for 1 month and 2 month follow-up visits and instructed to bring their swab samples and journals to these visits. The follow up visits will involve a routine gynecological exam and the same sample collections as the initial visit and will take about 1 h.
Results
Definition of Treatment Failure or Participant Removal Criteria.
Donor participants will be removed from the study at any visit that they do not fulfill the criteria (bacterial flora type, lack of sexually transmitted infections, no gynecological abnormalities, no high-risk sexual behavior) for providing CVS donor samples. Recipients will not be removed from the study if they do not follow the counseled suggestions for condom use, as the impact of sexual behavior is of interest for the relative success of the CVS transplants. Even if the recipients become pregnant, the visits at month 1 and 2 are simply observational rather than interventional, and therefore do not pose a risk to the recipient participants. In the event that the recipients have BV relapse or acquire a sexually transmitted infection, they will be referred back to the clinic where they were originally treated, and will be followed through the 2 month window. There is only 1 “dose” involved in the CVS transplant procedure. If the participants have a BV relapse or acquire a sexually transmitted infection, they will be referred back to their medical provider at JHOC for treatment. CVS transplants are not intended as an ongoing therapy at this time.
Primary Outcome Variable.
The primary outcome will be whether the recipient participations are classified as having BV as assessed by standard Amsel's criteria in the clinic and by Nugent scoring at the 1 month and 2 month follow up visits.
Secondary Outcome Variables.
The secondary outcome will be the composition of the recipient participant's vaginal microbiota at the 1 month and 2 month follow-up visits, which will be determined by RNA analysis of the vaginal swabs collected.
The recipient participants can benefit by improved rates and longer duration of BV resolution, as standard antibiotic treatment often leads to relapse within a month. As such, the women that receive only standard treatment will likely have recurrent BV symptoms in addition to the numerous sexual and reproductive risks associated with BV. In contrast, a successfully CVS transplant could provide prolonged resolution of symptoms and resistance to relapse.
Materials
Cryoprotectant solution contains between 1% and 15% glycerol in saline.
Lactic acid-based diluent containing between about 0.5 and 1.5% lactic acid with sodium chloride and balanced to osmolality 200-300 mOsm, and a pH of between 3.0 and 4.0.
Methods
“Donor” Identification
Donors will be screened to ensure:
Freezing and Thawing
The CVS from various donors were collected and frozen at various conditions described below.
The samples of frozen CVS were thawed by immersing sample tubes in 37° C. water bath for 10 minutes. The samples were then transferred to incubator set at 37° C. for 20 minutes, then diluted and plated.
Colony forming units of samples H260 and H257 (ID numbers denote individual patient samples) before freezing (Fresh), and after thawing, at a given dilution, were counted and presented in Tables 3-6 below.
Results
Data for freezing conditions suggest that each sample should be evaluated for optimal freezing conditions and use of a cryoprotectant. In some cases, CVS itself serves as the best cryoprotectant.
H260 (optimal conditions include 3.75% glycerol/saline with slow freeze).
H257 (Optimal Conditions Appear to be Slow Freeze without Glycerol)
This application is a continuation-in-part of International Application no. PCT/US2015/065002, filed on Dec. 10, 2015, which claims priority to and benefit of U.S. Provisional Application No. 62/091,970 “CVS Transplantation for Treatment of Bacterial Vaginosis” filed on Dec. 15, 2014, the disclosures of which are hereby incorporated herein by reference in their entirety.
This invention was made with Government Support under NIH grants R01HD062844, R21/R33A1094519, and R21/R33A1079740 by the National Institutes of Health. The Government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20160279182 A1 | Sep 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62091970 | Dec 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/065002 | Dec 2015 | US |
| Child | 15174540 | US |
