Efficient immune control of intestinal nematodes largely depends on the development of protective type 2 immune responses driven by CD4+ Th2 cells in concert with type 2 innate lymphoid cells. Both cell types produce the Th2 cytokines IL-4, −5, and −13 leading to concerted immune effector mechanisms. These mechanisms fortify intestinal barriers via enhanced mucus production, increased epithelial cell turnover, intestinal fluid influx and hypercontraction of smooth muscle cells (Sorobetea et al., 2018). Nematode infections additionally support the activation and expansion of regulatory T cells (Tregs) (Maizels et al., 2012), a phenomenon linked to the anti-inflammatory effect of nematode infections in several models of autoimmunity (McSorley and Maizels, 2012).

Whether the microbiota affects the development of immune effector mechanisms during nematode infections has only recently received attention. One study has reported that low dose antibiotic treatment during H. polygyrus infection increased the abundance of members of the Lactobacillaceae and Enterobacteriaceae bacterial families (Reynolds et al., 2014). Lactobacilli abundance correlated positively with worm burdens, and, importantly, with higher numbers of Foxp3+ Tregs in gut-associated lymphoid tissue, suggesting Lactobacilli-mediated induction of Treg responses (**Figure 3C**). However, increased Lactobacilli abundance was observed only in C57BL/6 mice permissive for long-lasting infections with high worm burdens, while more resistant BALB/c mice did not display this phenotype. Interestingly, administration of Lactobacilli to more resistant mice increased their susceptibility to infection (Reynolds et al., 2014). In addition, H. polygyrus-dependent changes in intestinal microbiota composition have been correlated to the suppression of allergic airway inflammation. High concentrations of shortchain fatty acids (SCFA) resulted from the increased abundance of Clostridiales bacteria in H. polygyrus infected mice (**Figure 3C**; Zaiss et al., 2015). The SCFA increase supports mucosal Treg responses associated with the reduced susceptibility of mice to allergic airway inflammation (Zaiss et al., 2015). Furthermore, Ohnmacht et al. (2015) found that the microbiota is needed for the induction of highly activated RORγT + Foxp3+ Tregs, which could be induced via the introduction of a cocktail of Clostridia species in germfree mice, most likely via the provision of the SCFA butyrate (**Figure 3C**). Importantly, the conditional removal of the RORγT + Treg subset rendered H. polygyrus-infected mice more resistant to infection due to the development of a more robust Th2 response. Together, these observations suggest that the microbiota can indirectly control intestinal Th2 responses via the induction and maintenance of suppressive Treg subsets in the context of intestinal nematode infections (**Figure 3C**; Ohnmacht et al., 2015).

T. muris infection, on the other hand, leads to a notable decrease in Bacteroides producing SCFA, and a concomitant decrease in Foxp3+ Tregs in the lamina propria during chronic infection (Houlden et al., 2015). Moreover, Tregs in T. muris infection appeared less prone to release the antiinflammatory cytokine IL-10. Therefore, chronically infected mice were more susceptible to intestinal inflammation and displayed poor worm expulsion due to the development of Th1 responses counteracting Th2 immunity (**Figure 3D**; Holm

nematode Heligmosomoides polygyrus, the increased abundance of Lactobacilli and Clostridiales producing short chain fatty acids (SCFA)have been shown to induce the expansion of Foxp3+ regulatory T cells (Treg) in the gut, while SFB additionally induce the development of a highly potent immunosuppressory subset of Foxp3+RORγT + Treg, thus indirectly suppressing Th2 cell responses during infection. (D) A decrease in SCFA-producing Bacteroides and elevated abundances of Lactobacilli during chronic T. muris infection correlate with decreased intestinal Treg numbers and importantly, these Tregs appeared less prone to secrete IL-10 during chronicity.

et al., 2015). Thus, in nematode infection, the commensal microbiota and, importantly, specific bacterial groups, appear essential for both the induction and suppression of host immune responses. Contextually, this can contribute to resistance or susceptibility to infection, depending on the bacterial community and the invading parasite species.

# Microbiota-Dependent Immune Responses in Mosquitoes

Multiple reports in the past have highlighted the ability of bacteria to inhibit the establishment of Plasmodium infection in the mosquito host (Gonzalez-Ceron et al., 2003; Dong et al., 2009; Cirimotich et al., 2011; Tchioffo et al., 2013; Bahia et al., 2014). Consistently, some antibiotic treatments promote parasite development (Beier et al., 1994; Dong et al., 2009; Gendrin et al., 2015, 2016). Plasmodicidal properties have been reported for diverse microbes and must rely on some general rather than species-specific mechanisms (Lowenberger et al., 1999; Gonzalez-Ceron et al., 2003; Dong et al., 2009; Cirimotich et al., 2011; Tchioffo et al., 2013).

Female mosquitoes alternate between sugar and blood feeding. Such diverse diets expose the gut to strong physiological and oxidative challenges (reviewed in Sterkel et al., 2017). Uptake of proteins and lipids from iron and heme-rich blood induces massive metabolic and transcriptional changes in the midgut (**Figure 4**). Nutrient abundance sets off bacterial proliferation which, in turn, induces expression of immune genes (Dong et al., 2009). Blood intake elevates hydrogen-peroxide levels in the mosquito hemolymph and activates reactive oxygen species (ROS) detoxification responses (Kumar et al., 2003; Molina-Cruz et al., 2008; de Almeida Oliveira et al., 2012). However, how bloodfeeding affects ROS levels in the midgut, and the underlying mechanisms remain unknown (**Figure 4B**). What is clear is that ROS inhibition after blood feeding causes lethal systemic bacterial infections, suggesting a crucial link between oxidative stress and immune activation (Molina-Cruz et al., 2008).

The peritrophic matrix (PM) provides the first line of defense against blood meal-induced oxidative stress and bacterial infections (**Figures 1**, **4B**). The cardia cells of mosquito larvae continuously secrete sleve-shaped PM (type II), whereas adult epithelial midgut cells synthesize type I PM only after blood feeding (Wigglesworth, 1930; Waterhouse, 1953). Feeding on complex organic matter continuously exposes larvae to bacteria. In contrast, blood feeding induces massive bacterial proliferation in the gut and renders adult females particularly vulnerable to microbial infections (Dong et al., 2009; Linenberg et al., 2016). Such differences in mosquito exposure to microbes at these developmental stages likely contribute to constant PM secretion in larvae vs. inducible PM secretion in adults. Interestingly, PM synthesis in adults requires midgut microbiota. Bacterial proliferation after blood feeding upregulates expression of hundreds of genes, including the genes encoding PM proteins such as glucosamine-fructose-6-phosphate aminotransferase (GFAT) and chitin synthase enzyme 1 (CHS1; Rodgers et al., 2017). Conversely, antibiotic treatment before blood feeding compromises the integrity of PM by inhibiting expression of GFAT and CHS1, but also the genes encoding peritrophic proteins 1 and 14, whereas it upregulates expression of the PM-degradation chitinase genes A and B (Rodgers et al., 2017; Song et al., 2018). The PM of mosquitoes, therefore, serves as an inducible protective mechanical barrier against bacteria.

The immune-deficiency (IMD) pathway, initially identified in Drosophila, is the major immune pathway that control bacterial infections by coordinated expression of antimicrobial peptide (AMPs) genes (Buchon et al., 2009; Broderick et al., 2014). In Anopheles, the pathway is activated by the recognition of DAP-type and lysine-type peptidoglycans by the peptidoglycan recognition protein LC (PGRP-LC; Meister et al., 2009). Although relatively understudied in the mosquito gut, several lines of evidence suggest that IMD is functional in this tissue. A. gambiae mosquitoes with intact microbiota display higher basal expression levels of immune genes compared to antibiotic-treated controls (**Figure 4A**; Dong et al., 2009). These genes encode a range of AMPs, signal-transducing serine proteases, IMD pathway components and immune genes such as fibrinogen-related and thioester-containing proteins among others (Dong et al., 2009). Importantly, silencing of a transcriptional activator of the NF-kB family, REL2 and of PGRP-LC receptor, promotes the proliferation of gut microbiota and increases mosquito susceptibility to Plasmodium infection (Dong et al., 2009; Meister et al., 2009). The proliferation of bacteria is believed to activate the IMD pathway via the PGRP-LC receptor. Since bacterial proliferation 24 h after blood feeding coincides with ookinete transversal of the midgut epithelium, it may also lead to the IMD-mediated killing of Plasmodium parasites (**Figure 4B**; Dong et al., 2009; Meister et al., 2009; Linenberg et al., 2016). Indeed, clearance of the mosquito microbiota by antibiotics before infection increases mosquito susceptibility to Plasmodium, whereas bacteria inoculation by feeding decreases it in a PGRP-LC-dependent manner (Meister et al., 2009).

Wolbachia, intracellular maternally transmitted bacteria, confer protection to their arthropod hosts against a range of pathogens (Hedges et al., 2008; Teixeira et al., 2008; Kambris et al., 2009; Moreira et al., 2009; Hughes et al., 2011). Wolbachia has been identified in natural Anopheles populations in Burkina Faso and Mali (Baldini et al., 2014; Gomes et al., 2017). Importantly, Wolbachia infections (experimental transovarian of the Asian malaria vector A. stephensi, natural infections of A. coluzzii and somatic infections in A. gambiae) significantly decrease the prevalence of P. falciparum-infected mosquitoes (Hughes et al., 2011; Bian et al., 2013; Shaw et al., 2016; Gomes et al., 2017). Although the mechanisms that cause mosquito resistance to Plasmodium remain to be elucidated, experimental Wolbachia infections induce expression of a series of immune effectors and ROS which could potentiate parasite killing (Kambris et al., 2009; Bian et al., 2013).

In addition to inducing the immunity-mediated Plasmodium killing, some bacteria show direct plasmodicidal activity. Enterobacter isolates from Zambian Anopheles kill P. falciparum in the midgut when administrated with the infectious blood meal (Cirimotich et al., 2011). This has been attributed to direct inhibition of ookinete development by the bacteria-produced ROS (Cirimotich et al., 2011). Soluble factors released by Serratia marcescens also inhibit P. falciparum ookinete development in the mosquito (Bahia et al., 2014). Inhibition of P. berghei ookinetes in vivo was linked to the length of the flagella and the motility of S. marcescens (Bando et al., 2013). Currently, it is unclear whether this inhibition is direct or immunitymediated. Furthermore, oral administration of Chromobacterium sp. isolated from A. aegypti in Panama, induces high mortality in mosquito larva and adults, whereas the surviving mosquitoes exhibit low Plasmodium infection loads (Ramirez et al., 2014). Biochemical analyses identified romidepsin, the histone deacetylase inhibitor, as the plasmodicidal factor, uncovering an essential role of histone modifications in the mosquito stages of

Plasmodium. Surprisingly, administration of romidepsin did not affect mosquito survival, suggesting that another bacterial factor mediates the Chromobacterium-induced mortality (Saraiva et al., 2018).

How plasmodicidal properties of mosquito microbiota correlate with dynamics of Plasmodium transmission in the field remains unknown. In laboratory conditions, Serratia sp. and Enterobacteriaceae curb Plasmodium infection (Bando et al., 2013; Tchioffo et al., 2013; Bahia et al., 2014). In contrast, two semi-field studies in Cameroon associated the same bacteria with higher P. falciparum loads (Boissière et al., 2012; Tchioffo et al., 2016). Most of the experimental studies examined the role of microbes in Plasmodium infections using microbiotafree mosquitoes, and the results of the few field studies stress the importance of studying the tripartite interactions between mosquitoes, microbes and parasites in natural conditions.

## BACTERIAL FEEDING VS. BACTERIAL CLEARANCE

# Manipulating Host Microbiota as a Treatment Strategy Against Protozoan Parasites

Treatment of giardiasis primarily relies on the administration of antibiotics like metronidazole. However, due to rising levels of resistance, frequent reports of side effects and clinical failures, there is a growing need for the development of novel alternative treatment strategies (**Table 1**; Ansell et al., 2015). In the past, it has been shown that the use of Lactobacilli as probiotics in the treatment of giardiasis holds promise. In G. duodenalis-infected gerbils, administration of L. johnsonii La1 leads to reduced infection rates, lack of pathological damage to the epithelial cell layer and absence of immune cell infiltration and inflammation in mice receiving the probiotic compared to the placebo group (Humen et al., 2005). One proposed mechanism of the observed anti-giardial activities of L. johnsonii La1 has been attributed to the metabolic generation of unconjugated bile salts (Pérez et al., 2001; Travers et al., 2016). Very recently, Allain et al. tested in vitro and in vivo the anti-giardial activity of three bile salt hydrolases (BSH), enzymes naturally produced by L. johnsonii La1 (Allain et al., 2018). In vitro treatment of G. duodenalis with increasing concentrations of recombinant BSH47 and BSH56 enzymes revealed a dose-dependent giardicidal activity of both enzymes in the presence of bile. Importantly, the group further demonstrated the anti-giardial activity of rBSH47 after administration to G. duodenalis-infected suckling mice, supporting the view of L. johnsonii La1 as a probiotic candidate for the treatment of Giardia infections.

Along the same line, Enterococcus faecalis SF68, a lactic acid bacterium indigenous to the mammalian commensal microbiota, has also been suggested as a potential probiotic treatment for Giardia. Oral administration of this strain prior to G. lamblia infection led to elevated levels of total IgA levels in small intestine during the acute stage of infection, as well as higher levels of specific anti-Giardia IgA and systemic IgG compared to control animals. Nevertheless, parasitological data were less conclusive and the use of E. faecalis SF68 as a probiotic supporting anti-Giardia immunity merits further investigations (Benyacoub et al., 2005). Overall, it appears that administration of probiotic Lactobacilli in Giardia-infected mice reduces epithelial damage, attenuates overt inflammation and fosters enzymatic giardicidal activity.

Treatment of toxoplasmosis, on the other hand, relies primarily on the administration of pyrimethamine and sulfonamide drugs (Alday and Doggett, 2017). Recently, the administration of Bifidobacterium animalis subsp. lactis to mice TABLE 1 | Administration modes, outcomes and proposed mechanisms of action of probiotic bacteria as potential treatment strategies against intestinal parasitic infections in murine hosts and mosquito vectors.

chronically infected with T. gondii demonstrated enhanced serum levels of anti-T. gondii IgG and higher numbers of CD19+ B-lymphocytes, a reduction in brain cysts, and attenuated intestinal villi inflammation in the probiotic-treated group (Ribeiro et al., 2016). Conversely, previous work in gnotobiotic mice colonized by probiotic E. coli strains revealed that these microbes contribute significantly to intestinal inflammation during T. gondii-infection (Bereswill et al., 2013). These results indicate that the use of probiotic bacterial strains in the treatment of intestinal parasite infections is highly contextual and reports on the potential benefits and risks for host health should be considered in the future design of novel probiotics.

### Probiotic Administration and Microbiota Manipulation During Nematode Infections

In intestinal helminth infections, few in vivo or in vitro studies have demonstrated mechanisms of action of probiotic bacteria favoring either infection resistance or susceptibility. Treatment of T. muris-infected mice with viable or dead probiotic Lactobacillus casei led to the maintainance of high worm burdens in the chronic phase of infection and concurrently suppressed cellular and Th1/Th2 cytokine responses in secondary lymphoid organs (Dea-Ayuela et al., 2008). On the other hand, administration of live Lactobacillus rhamnosus JB-1 to T. muris-infected mice resulted in accelerated worm expulsion and elevated gut tissue concentrations of IL-10, but not Th2 or Th1 cytokines. Administration of L. rhamnosus enhanced worm expulsion in mice deficient in mucin 2 (Muc2−/−), a key component of the intestinal mucus layer led to elevated goblet cell frequencies compared to medium-treated Muc2−/− mice, suggestive of probiotic-dependent induction of additional mucins involved in worm expulsion (McClemens et al., 2013).

The administration of probiotic bacteria against gastrointestinal protozoan parasite infections has gained attention as a novel and effective treatment strategy (**Table 1**). Numerous studies have highlighted a range of beneficial aspects of probiotic bacteria including support for intestinal antibody responses, reduction in immunopathology and elevated anti-inflammatory cytokine responses, as well as potential competition for nutrients with the invading intestinal parasite species. On the other hand, the introduction of certain probiotic bacteria during intestinal parasite infections could also have a number of detrimental effects, including increasing the numbers of intestinal Tregs and thus potentially facilitating nematode survival or providing support for damaging inflammatory reactions during infection.

# Microbiota Manipulations in Mosquitoes as a Vector Control Strategy

The environmental bias of the mosquito microbiome poses a significant challenge for its manipulation for vector control purposes. However, several approaches have been explored for introducing into natural mosquito populations bacteria with anti-Plasmodium properties (**Table 1**). Wolbachia has been successfully exploited in A. aegypti mosquitoes to interrupt the transmission of dengue virus (Hoffmann et al., 2011, 2014). Efficient Wolbachia application for malaria control requires rapid bacterial spread in the mosquito populations. This spread relies on Wolbachia ability to induce cytoplasmic incompatibility (CI) in the host, which is manifested by the sterility of individuals with different Wolbachia infection status (Bordenstein and Werren, 2007). CI has not been observed in Wolbachia naturally occurring in A. gambiae. Instead, the bacteria appear to induce a modest acceleration of egg-laying rates (Shaw et al., 2016). For experimentally-introduced Wolbachia, successful maternal transmission and CI has been reported in A. stephensi (Bian et al., 2013). Another study has shown inhibition of vertical Wolbachia transmission in A. gambiae and A. stephensi by resident microbiota (Hughes et al., 2014), while other attempts to stably introduce Wolbachia in A. gambiae have been unsuccessful (Kambris et al., 2009; Hughes et al., 2011).

Recently, mosquito colonization with genetically engineered Serratia has been proposed as an alternative transmissionblocking strategy (Wang et al., 2017). In addition to the plasmodicidal activity of S. marcescence described above, the engineered Serratia strains express one or multiple antimalarial effector proteins and successfully decrease P. falciparum infections in A. stephensi (Wang et al., 2017).  However, the persistence of genetically engineered Serratia under laboratory conditions was only demonstrated for three successive generations with a substantial drop in bacterial loads already in the second generation (Wang et al., 2017).

A surprising link between immune activation and changes in microbiota composition of the mosquito reproductive organs has been reported recently. Transgenic mosquitoes that expressed an active form of the REL2 transcriptional factor in the midgut after a blood meal were reported to inhibit bacterial proliferation and Plasmodium invasion (Dong et al., 2011). Unexpectedly, activation of the IMD pathway modified the mosquito mating behavior leading to the preferential mating of transgenic males with wild-type females (Pike et al., 2017). Although the exact mechanism is unclear, the authors proposed that transgene expression inhibits bacterial proliferation in the male reproductive organs and facilitates the spread of the transgene (Pike et al., 2017). Surprisingly, female mating preference was not affected by the transgene expression. Whether such behavioral manipulation was caused by a particular bacterial species or the overall bacterial loads and whether transgenesis-induced changes were male-specific remains to be investigated.

Another interesting approach to mosquito control exploits the entomopathogenic bacteria Chromobacterium sp. and the fungus Beauveria bassiana that exhibit plasmodicidal activities. As discussed above, Chromobacterium interferes with Plasmodium through histone modification processes and induces mortality by an as yet unknown mechanism (Saraiva et al., 2018). B. bassiana, on the other hand, downregulates IMD-mediated immunity and expression of dual oxidase DUOX, causing midgut dysbiosis and systemic infections by opportunistic bacteria that kill mosquitoes (Wei et al., 2017). Therefore, disrupting or manipulating the mosquito microbiome often has fatal consequences for the vector

that may directly or indirectly contribute to the inhibition of Plasmodium development.

### CONCLUSIONS AND FUTURE PERSPECTIVES

Along the entire length of the mammalian intestinal tract from the small intestine infected by Giardia, T. gondii, and H. polygyrus, to the large intestine where T. muris thrives, there is evidence of a critical and vastly complex relationship between the host immune system, the resident microbiota and the invading pathogens. Alterations of the host microbiota can both affect the integrity and efficiency of protective immune responses, as well as the development of the parasite and its infection efficacy. However, we still lack a more comprehensive understanding of the causality vs. correlation between specific compositional shifts of the commensal bacterial communities in the gut and the induction of host immune mechanisms during intestinal parasite infections. More specifically, the majority of current studies are performed in laboratory conditions, often with microbiota-free animals. Despite their limited sample size, field and semi-field studies provide valuable insights into the microbiome complexity of natural Anopheles populations and the effects of natural microbial communities on Anopheles vectorial capacity and anti-parasitic responses in mice deserve further investigation.

The intestinal microbiota of mice and mosquitoes naturally faces vastly contrasting environmental and biochemical challenges due to major differences in host development, physiology and diet among many other factors (**Table 2**). The mouse microbiota displays higher complexity and species diversity than the resident microbial community in the mosquito, but at the same time experiences less drastic variations and expansion compared to those seen in freshly blood-fed mosquito females. While the significant expansion of γ-Proteobacteria documented during acute T. gondii infection in mice contributes to immunopathology due to the activation of neutrophils and ROS-producing inflammatory monocytes, γ-Proteobacteria are important players in limiting vector competence of mosquitoes for Plasmodium. More importantly, the generation of ROS is a crucial immune response mechanism in insects, induced in response to a range of microorganisms. Considering the importance of murine resident γ-Proteobacteria in the induction of ROS-producing inflammatory monocytes against T. gondii, it merits further investigations whether a similar bacteria-mediated process is limiting vector capacity of mosquitos after uptake of Plasmodium with the blood meal.

Following each blood meal, the mosquito midgut synthesizes a semipermeable peritrophic matrix that shields the gut epithelium from direct exposure to potentially harmful external stimuli and invading microorganisms. Similarly, the murine gastrointestinal tract contains a viscous layer of mucus enriched in mucin glycoproteins protecting the intestinal epithelial layer (**Table 2**). Microbes enhance mucus secretion in the mammalian gut similarly to what has been suggested for PM deposition in the mosquito. Further studies are required to see if distinct microbes support the peritrophic membrane and thereby limit Plasmodium infection.

Changes in microbiota induced during intestinal parasite infections may provide valuable information on new intervention strategies against these pathogens. A better mechanistic understanding of how the microbial status and infectioninduced microbiome alterations of an individual affects immune responses to parasites and of how the microbiome could be targeted to reduce infection success of parasites and arthropod vectorial capacity necessitates further studies. So far, the diversity in study designs using a gnotobiotic, mono-colonized, antibiotic-treated or standard-pathogenfree mouse and vector models offers invaluable tools for the elucidation of effector mechanisms responsible for the induction and maintenance of immune responses and to what extent specific members of the commensal microbiota are involved. Nevertheless, variations in study design like

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the colonization status of the host/vector, host age, sex, commercial provider, parasite strain and infection dosage, microbiota culturing and sampling techniques, as well as differences in data analysis at the phylum, order, family, or genus level present considerable challenges to microbiota research. Caution must also be taken in the interpretation of studies on microbiota-dependent immunity and the translation of their findings outside the laboratory, as inbred lab-reared mice/vectors experienced a considerable change in bacterial diversity/composition compared to wild animals, which provide a more representable animal model. Future studies should consider parallel investigations of wild rodent models and vectors from natural populations for a more comprehensive understanding of the tripartite interactions between host, microbiota and invading intestinal parasites. The administration of cocktails of probiotic bacteria with varying stimulatory effector functions, rather than single bacterial groups would represent successful future probiotic treatment strategies. As such, further studies focusing on how commensal and probiotic bacterial communities communicate and regulate each other in the context of gastrointestinal infections would be required in humans, laboratory and free-living mice and mosquito species alike.

### AUTHOR CONTRIBUTIONS

All authors contributed to the work presented in this manuscript. EL and SH conceived the original framework of this paper. IY and SZ performed the literature search and wrote the manuscript. EL, SR, GC, and SH edited and critically revised the manuscript.

### FUNDING

The study was supported by the German Research Foundation by the Research Training Group 2046 (GRK 2046) within project A1 and B4 to EL and SH. SZ received a stipend from the GRK 2046 and IY received funding from the Graduate School Scholarship Program (GSSP) of the German Academic Exchange Service (DAAD).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yordanova, Zakovic, Rausch, Costa, Levashina and Hartmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Conceptual Design of a Universal Donor Screening Approach for Vaginal Microbiota Transplant

Kevin DeLong1,2†, Sabrine Bensouda1,3†, Fareeha Zulfiqar 1,2, Hannah C. Zierden1,4 , Thuy M. Hoang1,5, Alison G. Abraham2,6, Jenell S. Coleman7 , Richard A. Cone8 , Patti E. Gravitt 6,9, Craig W. Hendrix 3,5, Edward J. Fuchs 3 , Charlotte A. Gaydos 6,10 , Ethel D. Weld3,5,10 \* and Laura M. Ensign1,2,4,5,7,10 \*

### Edited by:

*Gilda Tachedjian, Burnet Institute, Australia*

### Reviewed by:

*Rita Verhelst, Hogeschool Gent, Belgium Tania Crucitti, Centre Pasteur du Cameroun, Cameroon*

### \*Correspondence:

*Ethel D. Weld<EMAIL_ADDRESS>Laura M. Ensign<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

### Specialty section:

*This article was submitted to Microbiome in Health and Disease, a section of the journal Frontiers in Cellular and Infection Microbiology*

> Received: *16 May 2019* Accepted: *09 August 2019* Published: *28 August 2019*

### Citation:

*DeLong K, Bensouda S, Zulfiqar F, Zierden HC, Hoang TM, Abraham AG, Coleman JS, Cone RA, Gravitt PE, Hendrix CW, Fuchs EJ, Gaydos CA, Weld ED and Ensign LM (2019) Conceptual Design of a Universal Donor Screening Approach for Vaginal Microbiota Transplant. Front. Cell. Infect. Microbiol. 9:306. doi: 10.3389/fcimb.2019.00306* *1 The Center for Nanomedicine, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 2 Department of Ophthalmology, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 3 Division of Clinical Pharmacology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 4 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, United States, 5 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 6 Department of Epidemiology, Johns Hopkins Medical Institutions, Baltimore, MD, United States, 7 Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States, 8 Department of Biophysics, Johns Hopkins University, Baltimore, MD, United States, 9 Department of Global Health, George Washington University, Washington, DC, United States, 10 Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States*

The success of fecal microbiota transplant (FMT) in treating recurrent *Clostridioides difficile* infection has led to growing excitement about the potential of using transplanted human material as a therapy for a wide range of diseases and conditions related to microbial dysbiosis. We anticipate that the next frontier of microbiota transplantation will be vaginal microbiota transplant (VMT). The composition of the vaginal microbiota has broad impact on sexual and reproductive health. The vaginal microbiota in the "optimal" state are one of the simplest communities, dominated by one of only a few species of Lactobacillus. Diversity in the microbiota and the concomitant depletion of lactobacilli, a condition referred to as bacterial vaginosis (BV), is associated with a wide range of deleterious effects, including increased risk of acquiring sexually transmitted infections and increased likelihood of having a preterm birth. However, we have very few treatment options available, and none of them curative or restorative, for "resetting" the vaginal microbiota to a more protective state. In order to test the hypothesis that VMT may be a more effective treatment option, we must first determine how to screen donors to find those with minimal risk of pathogen transmission and "optimal" vaginal microbiota for transplant.  We further characterized key physicochemical properties of donor cervicovaginal secretions (CVS) and the corresponding composition of the vaginal microbiota to delineate criteria for inclusion/exclusion. We anticipate that the framework described here will help accelerate clinical studies of VMT.

Keywords: microbiota, fecal microbiota transplant (FMT), bacterial vaginosis (BV), Lactobacillus, urinary tract infection (UTI), cervicovaginal secretions (CVS), sexually transmitted infections

# INTRODUCTION

Evidence continues to accumulate that demonstrates the critical role that bacteria play in human health and disease. It is often cited that the commensal bacteria colonizing our epithelial surfaces, glands, and fluids outnumber the cells that make up our bodies (Abbott, 2016; Sender et al., 2016). It has even been suggested that the gut microbiota should be considered an additional organ (Baquero and Nombela, 2012; Liu, 2016). The gut microbiota is perhaps the most extensively characterized, and has been shown to influence a wide range of diseases and disorders affecting the gut and beyond (Clemente et al., 2012; Valdes et al., 2018). Indeed, the premise of microbiota transplantation originated in the gut with the development of fecal microbiota transplantation (FMT) as a strategy for treating recurrent Clostridioides difficile (C. difficile) infection. The tremendous clinical success of FMT has led to explorations in using FMT for treating inflammatory bowel disease, obesity, liver disease, depression, food allergies, antibiotic resistance, malnutrition, multiple sclerosis, and more (Borody et al., 2013). Further, FMT has motivated the study of other forms of microbiota transfer, including skin microbiota transplant (Myles et al., 2018; Perin et al., 2018) and vaginal microbiota transfer from mother to babies born by Cesarean section (Dominguez-Bello et al., 2016).

Despite being one of the simpler commensal bacteria communities, cervicovaginal microbiota play a key role in sexual and reproductive tract health. The earliest attempts to characterize the cervicovaginal microbiota were conducted in the mid 1800s, a time when puerperal sepsis killed at least 15% of women giving birth in Europe and America (Hallett, 2005). In 1879, Louis Pasteur reported observation of streptococci in the blood of women with puerperal fever (Dunn, 2005). He believed this may have resulted from mechanical trauma that allowed bacteria in the vagina to enter the bloodstream. Later in 1892, Doderlein reported that women who had Grampositive, lactic acid-producing bacilli in their vagina at the time of childbirth were less likely to develop puerperal sepsis after delivery (Doderlein, 1892; Thomas, 1928). Although there has been increasing awareness of the broad spectrum of "normal" (Smith and Ravel, 2017; Anahtar et al., 2018), it is generally considered that the "optimal" vaginal microbiota communities are dominated by one of only a handful of species of Lactobacillus (Linhares et al., 2011; Petrova et al., 2015). Diversity in the vaginal microbiota and lack of dominance by Lactobacillus species can be described clinically as bacterial vaginosis (BV), a condition that has been linked to increased risk of sexually transmitted infection acquisition and transmission (Cherpes et al., 2003; Wiesenfeld et al., 2003; Allsworth et al., 2008; Atashili et al., 2008; Cohen et al., 2012), urinary tract infections (Sumati and Saritha, 2009; Stapleton, 2016), and fertility and pregnancy outcomes (Hyman et al., 2014; Petricevic et al., 2014; García-Velasco et al., 2017; Stout et al., 2017). Further, rates of BV relapse after standard antibiotic treatment can be as high as 70% within 3 months (Larsson and Forsum, 2005). Beyond BV, vaginal microbiota have also been implicated in recurrent yeast infections (Zhou et al., 2009; Liu et al., 2013), colonization with group B streptococcus (Rosen et al., 2017; van de Wijgert, 2017), and potentially reproductive tract cancers (Xu et al., 2014; Kyrgiou et al., 2017). Many have drawn attention to the limitations of currently available treatments for BV and the need for innovation in approaches for modifying the vaginal microbiota (Bradshaw and Sobel, 2016; Martin and Marrazzo, 2016).

Vaginal microbiota transplant (VMT) has the potential to revolutionize the way we view and treat conditions affecting the female reproductive tract. Unfortunately, study of vaginal microbiota in preclinical animal models is severely limited by the fact that dominance of the vaginal microbiota and acidification by Lactobacillus species is a uniquely human phenomenon (Miller et al., 2016; Witkin and Linhares, 2017). Even our primate cousins have low vaginal colonization by Lactobacillus species, and it has been suggested that the normal rhesus macaque vaginal microbiota is a good model for human BV (Spear et al., 2010; Mirmonsef et al., 2012; Yildirim et al., 2014). Unlike FMT, there is no previous history of anecdotal clinical implementation of VMT.  Thus, a logical next step is to determine whether cervicovaginal secretions (CVS) can be used to transplant vaginal microbiota from a donor to a recipient in a clinical setting. Undoubtedly, ensuring safety and tolerability are top priorities. Here, we describe the development of a universal donor screening protocol intended for maximal risk reduction to mitigate potential transmission of infectious pathogens, as well as collection, characterization, and testing procedures for donor CVS.

# MATERIALS AND METHODS

### Ethics Statement

The sample collection and testing procedures described here were approved by the Johns Hopkins University Institutional Review Boards as a part of study IRB00131437. Informed consent was obtained from all human subjects prior to participation.

## Screening Questionnaire

We have developed a questionnaire for pre-screening potential VMT donors. The questionnaire includes all questions listed in the FDA Guidance for Industry for Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (Section IV, Donor screening, §1271.75, section E). In addition, we included screening questions that are consistent with what has been observed to impact vaginal microbiota and stability of vaginal microbiota communities, including sexual history, sexual behavior, and vaginal product usage. We also included questions about medical history, and travel history (e.g., potential exposure to Zika or Ebola) that could have an impact on risk of incident sexually transmitted infections. For the pilot donor screening study described herein, we used an abbreviated questionnaire with the primary goal of correlating testing outcomes with self-reported sexual behavior, vaginal symptoms, vaginal product usage, and self-reported history of reproductive tract and sexually transmitted infections. Participant demographics and questionnaire data can be found in **Table 1**.

### Test List

The list of clinical tests and laboratory characterizations with their submeasures and readouts with normal ranges, if applicable, can be found in **Supplementary Tables 1** and **2**, respectively. We followed the FDA Guidance for Industry for Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (Section VI, Donor Testing, §1271.85, sections A and B) for testing of leukocyte-rich cells or tissues, which also required the use of a FDA-licensed, cleared, or approved donor screening test where such a test is available (Memorial Blood Centers, St. Paul, MN). Additional testing was conducted by Johns Hopkins Medical Institutions Medical Laboratories, including tests for herpes viruses, hepatitis A, Toxoplasma gondii, Epsteinbarr virus (EBV), rubella virus, general immunocompetence, pregnancy, and bacterial and fungal cultures. Several tests were subsequently sent to Quest Diagnostics, as indicated by test codes beginning in "Q" in **Supplementary Table 1**. It was noted after the first four participants that many did not recall their hepatitis A vaccination status while testing positive for hepatitis A IgG, so a test for hepatitis A IgM was added. Additional laboratory characterizations for sexually transmitted infections, included nucleic-acid amplification tests (NAAT) for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium, and Human papilloma viruses (HPV), as well as quantitative polymerase chain reaction (qPCR) and 16S rDNA sequencing to determine composition of vaginal microbiota communities. As 16S rDNA sequencing runs typically require batching of samples into a 96 well plate format, we sought to develop a qPCR approach that was predictive of the relative abundance of Lactobacillus species and Gardnerella vaginalis and could be readily performed on individual samples on the same day for rapid screening and eligibility determination.

### Sample Collection

Potential study participants were recruited from a list of participants from the investigator's prior studies that (i) had agreed to be contacted for potential participation in future studies, and (ii) had provided Lactobacillus-dominated CVS samples as part of these prior studies. To be included in the study, all participants identified as female and were pre-menopausal between the ages of 18–45 yrs. The potential participants were informed that at the time of sample collection, they must not be currently menstruating or within 3 days of their last menstrual period, they must be currently healthy and free of vaginal symptoms, and must not have used vaginal products or had vaginal intercourse in the prior 3 days. Participants were advised to drink extra water in the time leading up to their appointment to ensure proper hydration for blood collection. Participants were consented using an interactive question and answer approach that explained the purpose of the study, the associated risks, and the testing information that was to be obtained. The participants signed a consent form for HIV testing in the state of Maryland. The participants then filled out the abbreviated questionnaire and TABLE 1 | Participant demographics and questionnaire data\*.

\**The following symptoms "you currently have" were not selected by any participant: pain during intercourse; abdominal or pelvic pain; vaginal irritation; pain during urination. The following options for conditions that "you have ever been diagnosed with" were not selected by any participant: trichomoniasis, gonorrhea, syphilis, pelvic inflammatory disease, other please specify. The following options for product use within the past 6 months were not selected by any participant: feminine hygiene spray, feminine hygiene powder, norforms, vaginal acid gel.*

DeLong et al. Universal Donor Screening for VMT

asked questions for clarification as needed. The participants were then given a bag containing 5 unwrapped vaginal swabs [2 × BD Eswabs (Becton Dickinson), 1 × Mini-tip flocked swabs with viral transport media (Becton Dickinson), 1 × Digene HC2 DNA collection brushes (Qiagen), 1 × Aptima vaginal swab (Hologic)], a Softdisc menstrual fluid collection device (The Flex Company), a sterile wrapped urine specimen cup, and a 50 mL conical tube (Corning Falcon). The participants were shown diagrams to instruct them on how to use each swab and how to insert the Softdisc to collect CVS using a previously described method (Boskey et al., 2003). It was emphasized that the participants take their time collecting each specimen carefully, to ensure that none of the materials touched unintended surfaces, and to return for replacement swabs if any were dropped or contacted a surface other than the vaginal wall. The participant then went to a self-locked single stall restroom for sample self-collection. Upon their return to the clinical area, the participant was prepared for blood collection. The total volume of blood collected across various tubes to perform all tests was ∼50 mL. Specimens were then grouped for immediate overnight shipping to Memorial Blood Centers, transport to the JHMI Medical Laboratories, and transport on ice to the respective laboratory facilities for testing and characterization. Three participants had menstrual blood in their CVS, either trace amounts still present despite reporting 3 days post the end of their menstrual period, or because of an unanticipated early start to their period. They returned within 2–20 days to provide another CVS sample and vaginal Eswab for 16S rDNA sequencing, which were the samples used for the data reported herein.

### Laboratory Characterizations Panther System

Aptima (Hologic) swabs were transported on ice and stored at 4 ◦C for <30 days until testing by the Aptima Combo 2 for chlamydia and gonorrhea, by Aptima TV for trichomonas, and by Aptima MG for Mycoplasma genitalium.

### Human Papilloma Virus Assay

The Digene HC2 vaginal brushes were transported on ice and stored at 4◦C for <60 days until testing. The Roche Linear Array HPV Genotyping Test is a qualitative test that detects 37 human papillomavirus genotypes including 17 high risk types, 15 lowrisk types, and 5 unknown-risk/probable-high-risk (pHR) types (Muñoz et al., 2003; de Villiers et al., 2004). The tests were performed according to the manufacturer's protocol (Woo et al., 2007), with broad-spectrum amplification and reverse line blot hybridization for genotype discrimination (Gravitt et al., 1998; Low et al., 2015).

### CVS Characterization

Specimens were transported from the clinical location to the laboratory in a cooler on ice. Immediately upon return to the laboratory, the 50 mL conical tube containing the Softdisc was centrifuged at 1,000 RCF for 2 min to collect the CVS. The CVS was transferred to a 1.5 mL Eppendorf tube using a 50 µL Wiretrol (Drummond Scientific). The approximate sample volume was noted (average 0.25 ± 0.14 mL, range 0.1–0.5 mL). We were able to characterize 3 out of 4 Amsel's criteria for BV diagnosis, including: (i) pH >4.5, (ii) basic amine "fishy" odor upon mixing with 10% KOH (positive whiff test), and (iii) presence of clue cells in the wet mount. A sample had to meet all 3 criteria to be categorized as BV based on Amsel's criteria. CVS sample pH was measured using a Mettler Toledo EL20 pH meter with a micro-combination pH electrode MI-411 (Microelectrodes, Inc.). Slides were prepared for wet mount by rolling a swab coated in CVS on a standard microscope slide followed by the addition of 10 µL of normal saline and covered with a glass coverslip. The wet mount slide was observed for the presence of clue cells using differential interference contrast (DIC) microscopy. The whiff test was performed by dipping a cotton swab in the CVS, pipetting 100 µl of 10% KOH onto the swab, and using a gloved hand to waft air over to determine whether a fishy odor was produced. Another swab covered in CVS was rolled onto a second standard microscope slide and left to air dry for gram staining and Nugent scoring for diagnosis of BV (Nugent et al., 1991). For lactic acid measurements, ∼10 µL of CVS was transferred to a preweighed Eppendorf tube to obtain the sample mass (6–20 mg). The CVS was diluted with 490 µL of normal saline and frozen at −20◦C until performing the assay. The diluted samples were thawed and centrifuged at 1000 RCF for 5 min to pellet mucus solids. The supernatant was processed per manufacturer's instruction in a 96 well plate format using a D/L-lactic acid kit (R-Biopharm).

### Multiple Particle Tracking to Assess HIV-1 Virion Mobility

Fluorescently labeled HIV-1 pseudoviruses were prepared as previously described (Nunn et al., 2015; Hoang et al., submitted). HIV virions (0.3 µL) were pipetted into 20 µL of undiluted CVS in a custom-made glass slide with a circular sample well, gently mixed, and immediately sealed with a glass coverslip. Twenty second videos of viral motion were recorded at room temperature using a Zeiss Axio Observer inverted epifluorescence microscope equipped with a 100x/1.46 NA oil-immersion objective and an EM-CCD camera (Evolve 512; Photometrics). The image resolution was 25 nm/pixel and sequential images were captured at a frame rate of 15 Hz. A minimum of 5 videos were collected per CVS sample. Virion trajectories were analyzed using automated MATLAB-based particle tracking software with a minimum of 16 frames (∼1 s) of consecutive tracking as previously published (Suh et al., 2005; Lai et al., 2007; Schuster et al., 2015).

### 16S Sequencing and Analysis DNA Extraction

BD Eswab fluid (150 µL) or 10 mg of CVS were resuspended in 180 µL of lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.0, 2 mM EDTA) with 20 mg/mL of lysozyme and incubated for 1 h at 37◦C. Following lysozyme treatment, DNA was extracted using the DNeasy R Blood and Tissue kit (QIAGEN R , Hilden, Germany), including the addition of 20 µL of proteinase K and incubation for 40 min at 56◦C for pretreatment of gram-positive bacteria. Following DNA extraction, DNA concentrations were measured by NanoDrop.

### DNA Amplification/Library Preparation

Library preparation was performed by the JHMI Deep Sequencing and Microarray Core at the Johns Hopkins Medical Institute according to the Illumina 16S metagenomic library preparation protocol (Illumina, San Diego, CA) with primers that amplified the V4 region of the 16S gene. Paired-end sequencing of the pooled library was performed with the MiSeq system (Illumina, San Diego, CA), generating 2 × 250 reads. Adaptor and barcode trimming was also performed by the JHMI Deep Sequencing and Microarray Core at the Johns Hopkins Medical Institute.

### Sequence Processing

QIIME 1.9.1 was used to join paired reads, demultiplex samples, identify and filter out chimeras using USEARCH 6.1 (Edgar, 2010), perform open-reference OTU picking and make taxonomic assignments (Caporaso et al., 2010). Joining required a minimum of 15 base calls of overlap and <20% dissimilarity between matching sequences. Quality control during demultiplexing required 60% of base calls in a read to have a quality score above 17 with no more than 9 consecutive base calls below 17. The reference database used for OTU picking and taxonomic assignments is a custom reference database that combined an existing vaginal microbiome 16S database (Srinivasan et al., 2012) with the V4 regions of additional species represented in the VaHMP V1-V3 database (Fettweis et al., 2012).

### Sequencing Data Analysis

Reads were rarefied in R (R 2017, R version 3.4.3) and kmeans clustering was performed to assign samples to CSTs. To compare vaginal swab and CVS samples, Inverse Simpson Indices were calculated in R using the vegan (2.5–4) diversity command and a Bray-Curtis dissimilarity matrix, and NMDS analysis was performed using the vegan metaMDS command and a Bray-Curtis dissimilarity matrix. Stress of the NMDS analysis was 0.1725. PERMANOVA statistics were calculated using the vegan adonis command.

### Bacterial Culturing

Lactobacillus crispatus (BEI Resources strain EX533959VC06), Lactobacillus jensenii (ATCC strain 25258), Lactobacillus iners (BEI Resources strain UPII 60-B), Lactobacillus gasseri (ATCC strain 33323), and Gardnerella vaginalis (BEI Resources strain JCP7275) were all grown in either MRS (L. crispatus, L. jensenii, L. gasseri) or NYC III (L. iners, G. vaginalis) liquid broth. Bacteria grown in MRS were plated on MRS agar plates while bacteria grown in NYC III were plated on BBLTM Brucella Agar plates with 5% Sheep Blood with Hemin and Vitamin K1 (Becton Dickinson). Plates were incubated in anaerobic jars with GasPakTM EZ anaerobe container system (Becton Dickinson) at 37◦C for 2–3 days before CFUs were counted (O'Hanlon et al., 2011).

### Quantitative PCR and Standard Curve Generation Quantitative PCR

qPCR of vaginal samples and cultured bacteria was performed on Applied Biosystems, QuantStudio 3. Bacterial species-specific primers (Integrated DNA Technologies) for the 16S rRNA gene were used at a concentration of 100 nM (**Supplementary Table 3**) (Jespers et al., 2012). To standardize the qPCR, a fixed volume (2 µL) of DNA was used for cultured standards, CVS, and swab samples. The program started with an initial incubation at 95◦C for 20 s, followed by 40 cycles with denaturing at 95◦C for 1 s followed by anneal/extend at 60◦C for 20 s. Data is shown for qPCR of CVS; the correlation coefficients for Ct values between CVS and swab was 0.97 for L. crispatus, 0.96 for L. iners, 0.96 for L. jensenii, 0.96 for L. gasseri, and 0.85 for G. vaginalis.

### Species-Specific qPCR Standard Curve Generation

Bacteria were cultured overnight in liquid media (see above) then diluted 1:10 into fresh media and incubated for an additional 3– 12 h to minimize the amount of dead bacteria. The subcultures were serially diluted 10-fold 5 times in triplicate. Serial dilutions were then plated as described above and used for DNA extraction. Following qPCR, the cycle thresholds (Ct) of the dilutions were plotted against the log of the CFU concentration for each species. Linear fitting provided an equation that was used to predict the CFU concentration in CVS based on the Ct for each species of bacteria.

### Calculation of Lactobacilli Fraction

To calculate the fraction of a particular species of Lactobacillus using 16S sequencing data, the number of reads assigned to that species was divided by the total number of reads assigned to all Lactobacilli. To calculate the predicted fraction of a particular species of Lactobacillus using qPCR data, the predicted concentration (CFU/mL) for a particular species was divided by the total predicted CFU/mL for all Lactobacilli species.

### Statistical Analysis

PERMANOVA statistics were calculated on the Bray-Curtis dissimilarity command using the adonis command of the vegan package in R. Inverse Simpson indices were compared using the student's t-test. Correlation analysis and significance for qPCR standard curves and comparisons between specific Lactobacillus species representation from 16S rDNA sequencing and qPCR was performed in GraphPad Prism 8.1.0.

# RESULTS

### Participant Demographics

Participant demographics are shown in **Table 1**. The median age of the participants was 26.5 yrs with a range of ages from 23 to 35 yrs. Two of the 20 (10%) of participants were Hispanic or Latino. The majority of participants were White (12/10, 60%) (**Table 1**).

# Notable Findings From Self-Reported Questionnaires

The participants answered questions about their sexual history, history of sexually transmitted infections, vaginal symptoms, and vaginal product use. A summary of key questionnaire data is provided in **Table 1**. Birth control usage was high, with participants reporting None (4/20, 20%), Condoms (2/20, 10%), Oral contraceptives (5/20, 25%), and copper or progestin intrauterine device (IUD) (9/20, 45%). Two participants (10%) reported BV in the past, one of which appeared to have active BV by Amsel's criteria (also noted below) and one who had the appearance of mixed bacteria on wet mount and gram staining. Prior human papilloma virus (HPV) infection was reported by one participant (5%). Prior yeast infection was reported by 12/20 participants (60%). One participant reported prior UTI. Prior chlamydia infection or treatment for potential exposure was reported by 4/20 participants (25%). Prior herpes infection was reported by 1 participant who was also on viral suppressive therapy (also noted below). Self-reporting of vaginal symptoms was rare and did not correlate well with other test results that were criteria for exclusion. For example, vaginal discharge (2/20, 10%) was reported by two participants with Lactobacillusdominated microbiota, and not by the one participant that apparently had active BV. Vaginal product use was also rarely reported and did not correlate with other negative findings. Interestingly, the 1 participant that reported use of boric acid did not report prior incidence of BV, which is the situation where boric acid is most likely to be used. There was a wide range of reported total numbers of sexual partners (0–29). The number of new sexual partners during the past month was low (0–1), and the percentage of participants reporting that their current male partner was circumcised was high (15/20, 75%). All participants in the study reported no tobacco use and no prior pregnancies resulting in birth of a baby. Many of the participants also had some extent of medical records that were accessible to supplement the self-reported questionnaire answers. It was noted that one participant was on testosterone therapy, presumably as part of gender affirming therapy.

### Notable Findings From Testing Blood Tests

As expected, a significant proportion (50%, 10/20) of the participants were CMV positive. Thus, while stool donors are typically not tested for CMV (Woodworth et al., 2017), we intend to ensure that CVS from CMV positive donors is used only in CMV positive recipients. Additionally, we found that 5/20 (25%) were positive for HSV-1 IgG, and two of these participants noted having oral cold sores in the past on follow-up. Out of an abundance of caution, we suggest that this be an exclusion criterion for potential VMT donors. Two participants out of 20 (10%) were HSV-2 IgG positive, where one was unaware of any past symptoms of infection and tested negative on the swabbased herpes NAT, and the other was on viral suppressive therapy. As expected, all participants (20/20, 100%) had rubella IgG and varicella zoster virus (VZV) IgG present due to past vaccination or exposure. Further, all participants tested were negative for rubella and VZV IgM, indicating there was no active infection. Likewise, 16/20 (80%) of participants were reactive for hepatitis A (HAV) IgG, but all participants tested were negative for HAV IgM. All participants (20/20) tested negative for current hepatitis B (HBV) and C (HCV) infections. One participant's bloodwork was indicative of microcytic anemia, which was consistent with a family history of thalassemia reported to the study team doctor on follow-up. While thassalemia does not have known impact on immunity or vaginal microbiota, for early investigations of VMT, a participant with thassalemia may be excluded under investigator discretion.

### Swab Tests

We observed that there was not necessarily concordance between self-reporting of past yeast infection (12/20, 60%) and the presence of culturable yeast from a vaginal swab (6/20, 30%).

FIGURE 1 | Stacked bar graph of vaginal bacteria phylotypes as determined by 16S rDNA sequencing of CVS. Samples are organized according to community state types (CSTs) as indicated by the colored bar on top of the graph. From left to right, green = *L. crispatus* (LC); yellow-green = *L. iners*/*L. crispatus* mix (LI/LC); yellow = *L. iners* (LI); red = polymicrobial (P). Each column represents an individual sample (*n* = 20 total). The height of each color indicates the relative abundance of a specific bacterial phylotype, as indicated in the legend. The top 19 abundant phylotypes are included, with all additional phylotypes summed and labeled as "Other".

Three of the participants that had culturable yeast did not report prior yeast infection, and 9 of the participants that reported past yeast infection did not have yeast reported after fungal culture. Regardless, all six participants with cultivable yeast (typically Candida albicans) did not have any symptoms of a yeast infection on follow-up. Bacteria species not considered to part of the normal urogenital flora were detected in 4/20 (20%) of participants. Two of these participants had culturable Staphylococcus bacteria but reported no symptoms of active infection on follow-up. One of the participants had light growth of numerous bacteria species including Escherichia coli and Bacteroides species, but reported that she dropped the swab on the floor when the study team doctor contacted for follow-up. One of the participants had moderate growth of Corynebacterium species on culture, which are typically innocuous. However, this participant was also the only participant (1/20, 5%) to test positive for Mycoplasma genitalium, and was instructed to see her physician on follow-up. All participants (20/20) tested negative for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. One 1/20 participants (5%) tested negative for all strains of HPV, though another 6/20 (30%) had only "weak" or "very weak" positives that may warrant additional confirmatory testing. All participants (20/20, 100%) were negative for HPV16 and 18, the strains responsible for most HPV-related cancers. Given the intermittent nature of viral shedding, we suggest that vaginal swabs be collected with every donor CVS sample to test for HPV and herpes viruses.

### Urine Tests

All participants tested negative for pregnancy, Chlamydia trachomatis, and Neisseria gonorrhoeae.

### CVS

One participant did not report any history of BV, but the CVS sample characteristics were indicative of BV by fulfilling the 3 Amsel's criteria tested (positive whiff test, pH 4.62, visible clue cells/biofilm in wet mount). The presence of a polymicrobial bacteria community was confirmed by 16S rDNA sequencing (see below) and Nugent score (8–10). One participant had no notable exclusion criteria based on the questionnaire and screening tests, but the CVS itself appeared ovulatory (egg-white appearance, spinnbarkeit).

### Microbiota Communities Were Consistent Between Swabs and CVS

There have been differing reports as to whether the sampling location and sampling method affects the composition of the vaginal microbiota (Kim et al., 2009; Virtanen et al., 2017). The Softdisc method described here collects material from the entirety of the cervicovaginal canal, which is then pooled together during centrifugation. Thus, we used 16S rDNA sequencing to characterize the vaginal microbiota communities for paired CVS and vaginal swab samples to determine whether the results would be similar. As shown in **Figure 1**, we found that 19/20 (95%) of the participants had CVS dominated by Lactobacillus spp., which was consistent with testing negative for BV based on the three Amsel's criteria characterized. CVS samples clustered into four distinct community state types (CSTs), three of which were predominated by Lactobacillus spp. (L. crispatus, L. iners, or a L. crispatus/L. iners mix). When comparing sequencing of DNA extracted from CVS samples and vaginal swabs, paired CVS samples and vaginal swabs were not discernible from each other using NMDS analysis (**Figure 2A**) and applying PERMANOVA did not reveal a significant difference between the distribution of the CVS samples or vaginal swabs (p = 0.986). The paired samples from each participant clustered into concordant CSTs and did not show a significant difference in diversity, as measured by the inverse Simpson Index (p = 0.7138) (**Figure 2B**).

## Standardized qPCR Correlates With the Relative Abundance of Specific Bacterial Species

Because 16S rDNA gene sequencing is a high-throughput approach, samples are typically batched together in large numbers. For the purpose of screening CVS samples to rapidly determine individual donor and sample eligibility, we sought to determine whether qPCR could be used to provide rapid compositional information that was reflective of sequencing. Using species-specific primers for L. crispatus, L. jensenii, L. iners, L. gasseri, and G. vaginalis, we performed 5 qPCR reactions on DNA extracted from each CVS sample. By comparing the qPCR results with the relative abundance obtained from the 16S sequencing, we determined that in the case of G. vaginalis, a Ct cutoff of 20 readily distinguished three samples that had >10% relative abundance (red bars in the last three columns in **Figure 1**) from samples with low relative abundance of G. vaginalis (**Figure 3A**). Furthermore, Ct < 20 reliably predicted dominance by Lactobacillus species (**Figure 3A**, **Supplementary Figure 1A**), though not necessarily the relative abundance of each species in mixed samples. Therefore, to allow rapid predictive CST classification before 16S rDNA sequencing, we generated qPCR standard curves from laboratory strains of L. crispatus, L. jensenii, L. iners, and L. gasseri to relate Ct values with the concentration of colony forming units (CFU) per volume across four logs in concentration in serial dilutions. Strong linearity was observed in all four species over four logs in bacteria concentration (insets in **Figure 3B**, **Supplementary Figure 1B**). The comparison of the predicted fractional representation based on qPCR and the fractional representation based on the 16S rDNA sequencing is shown for the dominant species (L. crispatus, L. iners) in **Figure 3B** and for the minor species (L. jensenii, L. gasseri) in **Supplementary Figure 1B**. In all four cases, we observed strong correlations between the predicted relative abundance based on qPCR and the relative abundance based on 16S rDNA sequencing.

## CVS Physicochemical Properties Correlate With the Presence of Lactobacillus

The protective role of the lactic acid and low pH of the vagina appears to be important for maintaining vaginal health (Olmsted et al., 2005; Aldunate et al., 2013; Hearps et al., 2017; Tachedjian et al., 2017), suggesting that the physicochemical characteristics of the CVS may be more important than the particular species

using species specific primers for *G. vaginalis*, *L. crispatus*, and *L. iners*. Individual data points are color coded for each group based on sequencing. Dashed lines indicate Ct = 20, our suggested threshold. (B) Individual CVS samples according to the predicted fraction of *L. crispatus* or *L. iners* relative to the combined lactobacilli concentrations determined by qPCR compared to that obtained from the 16S rDNA sequencing. Insets show the standard curve used to estimate the concentration of the indicated species and calculate the predicted fraction by qPCR.

present. As shown in **Figure 4A**, dominance by L. crispatus is generally associated with lower pH. In comparison, samples dominated by L. iners or a mixture of L. crispatus and L. iners had a slightly higher average pH, though still below the clinical cutoff for BV (>4.5, gray dotted line). Indeed, only the one sample that was polymicrobial had a pH above 4.5 (**Figure 4A**). We also

BV according to Amsel's criteria. Individual data points are color coded for each group based on sequencing. Linear regression line shown, *r* 2 = 0.37, *p* = 0.004. (D) Nugent score as a function of pH. Individual data points are color coded for each group based on sequencing. The gray dotted lines correspond to the threshold for Nugent score considered negative for BV (≤3) and the clinical cutoff for BV according to Amsel's criteria (pH 4.5).

found the relative concentrations of the D- and L- isomers of lactic acid (D-LA, L-LA) were reflective of the bacteria present in the CVS sample. Namely, there were relatively higher levels of D-LA compared to L-LA in CVS samples dominated by L. crispatus, where the opposite was true in samples dominated by L. iners (**Figure 4B**). For the samples that were a mixture of L. iners and L. crispatus, the concentrations for D-LA and L-LA were more similar, with the exception of one sample that had higher D-LA concentrations (**Figure 4B**), which may reflect the metabolic contributions of both species. The total concentration of lactic acid (D + L) generally increased with decreasing pH, meaning that L. crispatus-dominated samples typically had higher total LA concentrations, though there also were L. iners-dominated samples and L. crispatus/L. iners mix samples within the cluster (**Figure 4C**). We further found that the ability of the CVS to adhesively trap fluorescently labeled HIV-1 virions correlated with the Lactobacillus spp. present. L. crispatus-dominated CVS consistently trapped virions (**Supplementary Figure 2A**), supporting the mucosal barrier function to pathogens. However, virion mobility was more variable in L. iners and L. iners/L. crispatus mix CVS samples, where some samples trapped virions and other samples allowed for permissive diffusion of virions (**Supplementary Figure 2A**). Generally, virion mobility increased with increasing CVS pH, with the transition being relatively sharp around pH 4.2 (**Supplementary Figure 2B**).

Thus, as pH is relatively easy to measure in real time and is an indicator of dominance by Lactobacillus spp. and the amount of lactic acid produced, we suggest a pH cutoff of ≤4.2 for use in VMT. Similarly, we found that a cutoff pH of ≤4.2 also correlated well with scoring negative for BV by Nugent (≤3) (**Figure 4D**). A few samples that were Lactobacillus-dominated by sequencing and pH < 4.5 had high Nugent scores in the intermediate (4–6) or BV (7–8) range. Two of these samples were dominated by L. iners but had significant amounts of Gardnerella vaginalis (Nugent scores 6 and 8). Based on these data, we suggest that Nugent score ≤2 is sufficient to identify Lactobacillus-dominated samples with high lactic acid content and low pH suitable for VMT.

# Potential Eligibility Findings

The full lists of clinical and laboratory tests performed can be found in **Supplementary Tables 1, 2**, respectively. FDA testing requirements indicate that a potential donor of leukocyte-rich cells or tissues that is not sexually intimate with the recipient must be tested for HIV (types 1 and 2), HBV, HCV, Treponema pallidum; HTLV (types 1 and 2), CMV, Chlamydia trachomatis, and Neisseria gonorrhea. We propose that for VMT, a potential donor candidate should also be tested for HAV, HSV-1/2, VZV, EBV, rubella virus, Toxoplasma gondii, HPV, Trichomonas vaginalis, and Mycoplasma genitalium, cultivable yeast/fungi, and cultivable bacteria. The test list employed here included redundant serological testing and vaginal swab-based testing of herpes viruses (HSV-1/2, VZV). All active infections should be excluded. Evidence of past exposure to CMV, VZV, and rubella (including vaccination) should be matched between donors and recipients. We suggest that the evidence of past exposure to HSV-1 or HSV-2 should be an exclusion criterion. The presence of cultivable yeast in the absence of symptoms of an active yeast infection and/or evidence of yeast growth in the wet mount preparation need not be an exclusion criterion unless the participant indicates having had a history of yeast infections (>1 in the past). We suggest similar guidelines that only UTI occurring on more than 1 occasion be grounds for exclusion. The HCT/P guidelines stipulate chlamydia infection is only an exclusion if it has occurred in the prior 12 months, though we suggest that infections occurring >12 months ago should be considered along with other history and could potentially be grounds for exclusion at investigator discretion upon review. The presence of cultivable bacteria that are not considered "normal" urogenital flora (terminology used by the pathology laboratory) would result in exclusion. We suggest that self-reported product use need not be an exclusion criteria alone, but that donor participants should be instructed to avoid insertion of any vaginal products (including tampons) during the entire CVS collection period. Our results suggest that there is not a clear rationale for setting a maximum limit for number of sexual partners for inclusion as a CVS donor. However, we suggest that donor participants should be required to avoid participating in vaginal, rectal, or oral intercourse, as well as use of sex toys, digital penetration, etc. during the entire CVS collection period. As the effects of exogenous hormones on the vaginal microbiota are not well-studied, we determined that this would be an exclusion criteria for participating as a VMT donor. Further, while the procedures for participation as a donor do not likely pose any risk to pregnant women, we propose that the potential impacts of hormones and other factors on the vaginal microbiome during pregnancy be grounds for exclusion. Ovulatory CVS or CVS collected at the time of menses would not be ideal for transplant due to low bacteria density and elevated pH, but would not exclude a donor from providing additional CVS samples in the future.

For the purposes of screening a potential donor's vaginal microbiota composition, our results suggest that qPCR can be used to perform rapid characterization of the relative abundances of the four common Lactobacillus species that is predictive of the relative abundance by 16S rDNA sequencing. We prefer the use of CVS isolated with the menstrual cup collection approach to a vaginal swab for this purpose, because with isolated CVS, sample mass can be standardized. In contrast, the amount of CVS collected with a vaginal swab is not known or easily standardized, which would affect the threshold Ct value between samples. Of course, use of this qPCR approach would require individual labs to generate standard curves using reference bacteria and standardization using individual instrumentation and reagents. Using our qPCR instrument and a standard mass of 10 mg CVS, we found that Ct < 20 (>10% relative abundance by 16S sequencing) for G. vaginalis should be an exclusion criterion. Similarly, dominance by Lactobacillus species was predicted by Ct < 20. Together with Nugent scoring, pH measurement, and lactic acid measurements, dominance by Lactobacillus species can be quickly confirmed in individual CVS samples. Further, all samples should still be characterized by confirmatory 16S rDNA sequencing, which is standard in the field. Also, we would suggest that a standard plating method and counting of CFUs should be used to determine the overall Lactobacillus "dose." While the HIV mobility assay is informative and correlative with other CVS physicochemical properties, the equipment and materials required are specialized and need not be considered a standard screening approach.

Based on the test results and exclusion criteria discussed above, we found that 7/20 (35%) of our participants may be eligible for CVS donation as part of a future VMT study. However, we anticipate that the actual success rate for participation as a CVS donor in a clinical trial will be much lower than 35% for several reasons. This small pilot study used an abbreviated screening questionnaire, as described in the Materials and Methods. The participants were selected from a pool of participants from the study team's prior clinical studies, increasing the likelihood that they would have Lactobacillusdominated vaginal microbiota and fulfill the donor criteria. Of note, the majority of the participants were White or Asian, which is consistent with the observation that White and Asian women in the U.S. are less likely to have BV (Allsworth and Peipert, 2007; Fettweis et al., 2014; Peebles et al., 2019). However, efforts should be made to recruit a racially/ethnically diverse donor pool, as the potential impact of race/ethnicity on the success of VMT is unknown. Additionally, the more stringent cut-off values suggested based on the participants in this study (Nugent ≤ 2, pH ≤ 4.2) may need to be adjusted for a larger and more racially/ethically and/or geographically diverse donor pool. Further, the "universal" donor approach would typically consist of an initial full blood, swab, and urine screening like that described here, followed by a 30–60 day period of sequential CVS sample collections (excluding menses and ovulation if not using hormonal contraceptives). Out of an abundance of caution, we propose that donors be willing to abstain from vaginal intercourse and vaginal product use during the full duration of CVS collection, which was not an eligibility criterion for this small pilot study. Although not performed here, we also suggest that every donor CVS sample be tested for the presence of semen with a test like the ABAcard P30 test for the forensic identification of semen. We further propose that due to the intermittent nature of viral shedding (Wald et al., 1995; Phipps et al., 2011; Gravitt and Winer, 2017), vaginal swabs be collected to test for herpes viruses and HPV at the time of every CVS sample. After collection of the last CVS sample, the donor should go through the full blood, swab, and urine testing procedure again after a quarantine period of ∼30 days. It is likely that this series of additional testing will result in additional screening failures.

### DISCUSSION

Although the first modern literature report of fecal microbiota transplant (FMT) was documented in 1958 (Eiseman et al., 1958), it was not until 2013 that the first randomized clinical trial for treating recurrent C. difficile infection with FMT was reported (van Nood et al., 2013). Although the study did not involve blinding, it was considered to unequivocally represent the potential of FMT as a safe and effective treatment (Sampath et al., 2013). The prior year, the FMT process was greatly streamlined by demonstrating that equivalent efficacy could be achieved with (i) stool from standard volunteer donors rather than donors identified by the patient, and (ii) stool frozen prior to use (Hamilton et al., 2012). These findings paved the way for various stool banks around the country that screen potential donors and collect, process, and freeze stool for distribution to clinical providers. OpenBiome, a non-profit that reportedly shipped over 43,000 treatments since starting its service in 2013, was an early innovator in developing thorough screening and quarantining procedures for frozen, ready-to-use stool preparations to increase patient access and safety1 . The development of the FMT field is an obvious source of inspiration for initiating study of other forms of microbiota transplantation, such as VMT. Thus, our goal here was to conceptualize a universal VMT donor screening process that is uniquely suited to what we understand about vaginal microbiota and sexually transmitted infections. For example, unique considerations for VMT donor participants include the number of sexual partners, practice of certain sexual behaviors and frequency, and use of vaginal products. Further, any history of sexually transmitted infections should be grounds for exclusion. The stringency of the universal donor screening process inherently limits the availability of eligible donor participants, which is why OpenBiome reports only 3% of their participants screened are deemed eligible for stool donation (Dubois et al., 2015) 1 . It is possible that if VMT were to be successful, a similar model for donor screening with CVS banking and distribution could be implemented. Further, the development of a rationally designed biotherapeutic product using clinical outcome data from VMT studies would be a logical next step, both from a safety perspective as well as the much larger potential patient population. The trajectory of the FMT field is similarly informative in this regard, and will aid in accelerating the development of other microbiota-based therapeutic strategies.

Much of what we understand about the dynamics of vaginal microbiota was learned in studies that employed vaginal swabs, self-collected or physician collected, as the means for isolating bacteria (Aagaard et al., 2012; Gajer et al., 2012; DiGiulio et al., 2015). Other studies have reported the physicochemical properties of undiluted CVS or cervicovaginal lavage fluids, which gives us added indirect measures of bacteria composition and some aspects of metabolic function (Lai et al., 2009a,b, 2010; O'Hanlon et al., 2011; Chappell et al., 2014; Nunn et al., 2015). However, we and others have begun characterizing both the CVS and the composition of the vaginal microbiota to begin to correlate community structure and function (Nunn et al., 2015; Hoang et al., submitted). Here, we further demonstrate that the community structure is strikingly similar whether using a vaginal swab or an aliquot of the CVS as the biological matrix for DNA extraction, which supports the use of vaginal swabs as a reliable and valid representation of the native mucosal environment. Similar to what was described for Lactobacillus bacteria in culture, we found that the relative concentrations of D- and L-lactic acid were reflective of the Lactobacillus spp. present in the CVS (Witkin et al., 2013). We also found that L. crispatus-dominated CVS tended to have higher total lactic acid content, lower pH, and more effective immobilization of fluorescently-labeled HIV virions, which is consistent with prior observations made by our group and others (Nunn et al., 2015; O'Hanlon et al., 2019). Our data shown here also suggested that using a CVS pH cut-off of ≤4.2 and a Nugent score cut-off of ≤2 would be most suitable for VMT, which is slightly more stringent than the criteria defined for categorizing samples as negative for BV (Amsel et al., 1983; Nugent et al., 1991).

The procedures and data shown herein are not intended to be wholly inclusive of every consideration for screening potential donors for VMT. Further, to operate under an investigational new drug application (IND), there are additional considerations for Chemistry, Manufacturing, and Controls (CMC). In the case of stool transplants, which were known to be efficacious for treating recurrent C. difficile prior to regulation by the FDA, we have yet to identify relevant measures of potency. In contrast to a small molecule drug or biologic, we do not know the key "active" component in stool, and thus cannot quantify the concentration or activity of that component. Further, the clinical success of using processed stool with 10% glycerol added prior to freezing (a standard approach for freezing bacteria stocks) was reported without characterization of the stool before and after freezing (Hamilton et al., 2012; Satokari et al., 2015). Many studies since have looked at the effect of processing and storage on the stability of particular bacteria or the overall bacteria community (Costello et al., 2015; Fouhy et al., 2015), though there is still not a general consensus on what is important for a successful clinical outcome. In the case of VMT, donor CVS is largely considered a monoculture of one Lactobacillus species, and thus, lends itself to performing characterization of the colony forming units (CFU) per unit volume before and after freezing. We can also determine the effect of cryoprotectants and other media on the Lactobacillus viability, which is an area of active study in our group. Of note, however, although we can directly characterize the "potency" (concentration in CFU/mg) of the Lactobacillus, it is also possible that the lactic acid, mucins, or other components of the host environment could play a role in the potential success of the transplantation process. While vaginal microbiota can be considered relatively monomicrobial, there is also evidence here and described by others that more than one species of Lactobacillus, such as L. crispatus and L. iners, can cohabitate (Ravel et al., 2011; Gajer et al., 2012). Further, although other species may be in the minority, it has yet to be determined whether the overall community structure with major and minor players is important. We anticipate that the trajectory of VMT will likely follow that of FMT, wherein there are many parallel efforts in academia and industry to determine what minimum cultivable components can be manufactured to produce uniform, standardized products with similar therapeutic efficacy as stool (Kelly et al., 2015; Hoffmann et al., 2017; Ott et al., 2017).

1OpenBiome (2019). Available online at: https://www.openbiome.org/impact (accessed March 09, 2019).

In addition to intensive and thorough screening of potential donor participants for VMT, it is also important to consider testing of potential recipient participants. An inherent risk of the VMT procedure is transmission of a sexually transmitted pathogen. For various reasons associated with participant safety and for determining the overall safety of the VMT procedure, the baseline infection status of the potential recipient should be known. Thus, potential recipients should be screened similarly to potential donor participants, but the inclusion and exclusion criteria would differ. In addition to the obvious consideration that BV would be an exclusion for donors but not for recipients, it is also feasible that a controlled herpes infection or HPV would not necessarily be an exclusion criterion for potential recipient participants. Because of the transient and intermittent nature of viral shedding, potential recipient participants would ideally be screened at multiple time points prior to undergoing the VMT procedure to maximize detection of asymptomatic viral shedding of HSV-1, HSV-2, and HPV. Further, samples for testing should be taken immediately prior to the VMT procedure, because the results will help interpretation of any notable findings that may occur during recipient follow-up, including whether they may be attributable to the donor CVS. As the field of VMT is in its infancy, ensuring safety and tolerability of the procedure is of paramount importance in the first clinical studies. Various standardized adverse event grading systems are available, such as the Division of AIDS (DAIDS) Female Genital Grading Table for Use in Microbicide Studies for local vaginal/reproductive tract adverse events. Further, follow-up testing of recipient participants can also determine whether a potential shift in vaginal microbiota composition impacts future risk of acquiring sexually transmitted infections. It is also conceivable that as VMT may be explored for other indications, additional follow-up considerations may be timing of childbirth and birth outcomes, or recurrence of conditions such as urinary tract infection or yeast infection.

Another important aspect to consider is the VMT procedure itself. As we describe, the donor CVS was collected using a non-absorbent polymer-based cup, allowing for collection of undiluted material by centrifugation. However, similar to FMT, dilution with fluid such as sterile saline would ease loading into a device for application and administration to the recipient. Thus, we anticipate that similar to FMT, dosing would be determined by the ratio of sample mass to diluent fluid volume. This is in contrast to the dosing approach for probiotic products, where the concentration of CFU/dose can be standardized during the manufacturing procedure—with CVS donation, the mucus itself may be as important as the bacterial colony count. However, in the case of CVS, we observe a wide range of CFU/mg between participants, which is also reflective of the functional differences of the dominant bacteria. As such, when working with CVS from individual donors, it is not straightforward to standardize the dose based on the concentration of CFU. This is perhaps particularly true for Lactobacillus species found in CVS, because when they are the predominant species, they are present in lower numbers of CFU than the total number of CFU when the community is polymicrobial (Hill, 1993). Indeed, the ability of lactobacilli to colonize and compete out other bacterial species in CVS does not appear to be directly dependent on their concentration.

In the discussion of how microbiota transplantation must be regulated differently than standard drugs and biologics, it is worth noting that VMT and vaginal microbiota transfer from mother to babies born by Cesarean section, or "vaginal seeding," uses the same "drug." However, the nature of the screening procedures and the handling of the CVS are quite different, and thus, VMT has many more parallels to FMT than vaginal seeding. For example, the notion of universal donors cannot apply to vaginal seeding, as the donor must be the mother. Thus, screening failure rules out the possibility of conducting the procedure at all. Further, because of the relevant time constraints, the vaginal seeding procedure also rules out the possibility of performing thorough testing of the CVS at the time of collection (1 h before Cesarean section) and quarantining in the frozen state until test results are known. The CVS collection procedure for vaginal seeding is also quite different and requires no subsequent processing; absorbent gauze or swabs are used to "soak up" the CVS for direct transfer to the baby's skin after birth. Thus, the definition of the "dose" is also different, in that a single dose would just be the amount of CVS that is collected in the gauze while it is left in the mother's vagina. Finally, the use of the term "transfer" rather than "transplant" reflects the fact that, by definition, transplanted material is placed in the same area of the body from which it was obtained. In the case of vaginal seeding, the CVS is swabbed onto the baby's mouth, nose, and skin in order to mimic the process of passing through the vaginal canal during birth. In contrast, VMT is a true transplant of CVS from the vagina of the donor to the vagina of the recipient.

## CONCLUSION

This project represents a multidisciplinary effort to establish a protocol for comprehensive screening and characterization of donor CVS for the purpose of VMT, including a parallel rigorous screening process for potential recipients of VMT. The limitations of this pilot study include the small sample size and selection of participants from past study participant pools known to the study team to have optimal microbiota, which would not be generalizable to larger populations. It is possible that the requirement for sexual abstinence throughout the CVS sample collection period, or the requirement for providing multiple sequential self-collected swabs and CVS samples intensively over a month-long period, may constrain enrollment in future studies. The stringency of our exclusion criteria for donors, motivated by the ethical and clinical imperative to avoid potential transmission of pathogens, is both necessary and challenging from the perspective of recruitment. However, once a donor has been identified, using this protocol, as a safe donor, she could ideally donate CVS on multiple appropriately screened occasions; the idea of a "super-donor" with no identified past or current infections and with favorable Lactobacillus-dominated microbiota is one that should be explored and is of potential high impact to the project and the field. It is certain that as the field of microbiota transplant expands, unique regulatory questions and dynamic scientific, ethical, and clinical considerations will continue to arise (Hoffmann et al., 2017). These should be met with equally dynamic innovative approaches that incorporate optimized human safety considerations while recognizing the potential value of microbiota transplantation in addressing BV and its varied adverse impacts on human health.

### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. As of submission, the raw sequencing data is not publicly available, but public links or the data itself will be supplied on request.

### AUTHOR CONTRIBUTIONS

LE, EW, CG, PG, EF, CH, RC, and JC contributed conception and design of the study. LE wrote the first draft of the manuscript and KD, SB, and EW wrote sections of the manuscript. KD, SB, FZ, HZ, TH, LE, CG, and PG conducted experiments, oversaw staff testing of clinical specimens, and/or collected clinical specimens. EW, EF, and CH oversaw the clinical study and coordinated clinical activities. KD, SB, FZ, HZ, TH, PG, EW, AA, and LE performed data analysis and/or data interpretation. All authors contributed to the manuscript revision, read, and approved the submitted version.

### FUNDING

We received regulatory guidance from the Institute for Clinical and Translational Research (ICTR) funded by a grant from the NIH through NCATS (UL1TR001079). We also

### REFERENCES

acknowledge funding from the Burroughs Wellcome Fund Preterm Birth Initiative (grant 1015020). KD was supported by a Hartwell Foundation Postdoctoral Fellowship, SB was supported by the Doctoral Diversity Program (DDP), HZ was supported by a National Science Foundation Graduate Research Fellowship, and TH was supported by a PhRMA Foundation Pre Doctoral Fellowship.

### ACKNOWLEDGMENTS

We would like to acknowledge several people that gave valuable perspectives on the potential value of VMT in the early stages of conception, including: Judy Lee, MD; Jenny Robinson, MD; Jim Segars, MD; Thomas Moench, MD; Anne Burke, MD, MPH; Karen Carroll, MD; Sue Tuddenham, MD; Khalil Ghanem, MD; Rich Ambinder, MD; Noel Mueller, PhD. We also would like to thank several people who provided valuable guidance and points to consider toward the regulatory aspects, including: Carol Kobrin, PhD; Angela Ondo; Vic Lemas; Janice Davis Sproul; Karie Brooks; Kunal Parikh, PhD. We also want to give a special thank you to the OpenBiome team who shared their experience with starting the stool bank and working with the FDA (Mark Smith, PhD and Carolyn Edelstein, PhD). Thank you to Mark Marzinke, PhD, for providing guidance on how to group tests to minimize the volume of blood that needed to be collected. We would also like to thank Diane Hoffmann, JD, MS, for organizing the Microbiota Transplant Symposium at the University of Maryland. We also thank Samuel Lai, PhD, for generously providing fluorescently labeled HIV virions.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2019.00306/full#supplementary-material

properties of cervicovaginal fluid. Am. J. Obstet. Gynecol. 211:226 e1–7. doi: 10.1016/j.ajog.2014.03.041

without universal Lactobacillus dominance. ISME J. 8, 2431–2444. doi: 10.1038/ismej.2014.90

Zhou, X., Westman, R., Hickey, R., Hansmann, M. A., Kennedy, C., Osborn, T. W., et al. (2009). Vaginal microbiota of women with frequent vulvovaginal candidiasis. Infect. Immun. 77, 4130–4135. doi: 10.1128/IAI.00436-09

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 DeLong, Bensouda, Zulfiqar, Zierden, Hoang, Abraham, Coleman, Cone, Gravitt, Hendrix, Fuchs, Gaydos, Weld and Ensign. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Distinct Immune Responses Elicited From Cervicovaginal Epithelial Cells by Lactic Acid and Short Chain Fatty Acids Associated With Optimal and Non-optimal Vaginal Microbiota

David J. Delgado-Diaz 1,2, David Tyssen1 , Joshua A. Hayward1,2, Raffi Gugasyan1,3 , Anna C. Hearps 1,4 and Gilda Tachedjian1,2,5 \*

*1 Disease Elimination Program and Life Sciences Discipline, Burnet Institute, Melbourne, VIC, Australia, 2 Department of Microbiology, Monash University, Clayton, VIC, Australia, 3 Department of Immunology, Monash University, Melbourne, VIC, Australia, 4 Department of Infectious Diseases, Monash University, Melbourne, VIC, Australia, 5 Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia*

### Edited by:

*Shai Bel, Bar-Ilan University, Israel*

### Reviewed by:

*Gil Sharon, California Institute of Technology, United States Andrew T. Gewirtz, Georgia State University, United States*

\*Correspondence: *Gilda Tachedjian<EMAIL_ADDRESS>
### Specialty section:

*This article was submitted to Microbiome in Health and Disease, a section of the journal Frontiers in Cellular and Infection Microbiology*

> Received: *30 July 2019* Accepted: *11 December 2019* Published: *10 January 2020*

### Citation:

*Delgado-Diaz DJ, Tyssen D, Hayward JA, Gugasyan R, Hearps AC and Tachedjian G (2020) Distinct Immune Responses Elicited From Cervicovaginal Epithelial Cells by Lactic Acid and Short Chain Fatty Acids Associated With Optimal and Non-optimal Vaginal Microbiota. Front. Cell. Infect. Microbiol. 9:446. doi: 10.3389/fcimb.2019.00446* Non-optimal vaginal microbiota, as observed in bacterial vaginosis (BV), is typically characterized by a depletion of beneficial lactobacilli and an abundance of numerous anaerobes. These non-optimal conditions are associated with subclinical cervicovaginal inflammation and an increased risk of HIV infection compared to women colonized with optimal vaginal microbiota dominated by lactobacilli. Lactic acid (LA) is a major organic acid metabolite produced by vaginal lactobacilli that elicits anti-inflammatory effects from cervicovaginal epithelial cells and is dramatically depleted during BV. However, it is unclear if LA retains its anti-inflammatory activity in the presence of vaginal microbiota metabolites comprising short chain fatty acids (SCFAs) and succinic acid, which are also produced by an optimal vaginal microbiota. Furthermore, the immunomodulatory effect of SCFAs and succinic acid on cervicovaginal epithelial cells at higher concentrations present during BV is unknown. Here we report that in the presence of physiologically relevant concentrations of SCFAs and succinic acid at pH 3.9 (as found in women with lactobacillus-dominated microbiota) LA induced an anti-inflammatory state in cervicovaginal epithelial cells and inhibited inflammation elicited by the toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid and Pam3CSK4. When cervicovaginal epithelial cells were treated with a vaginal microbiota metabolite mixture representative of BV, containing a lower concentration of LA but higher concentrations of SCFA/succinic acid at pH 7, no anti-inflammatory was observed. Rather, the vaginal microbiota metabolite mixture representative of BV dysregulated the immune response of cervicovaginal epithelial cells during prolonged and sustained treatments. This was evidenced by increased basal and TLR-induced production of pro-inflammatory cytokines including tumor necrosis factor-α, but decreased basal production of chemokines including RANTES and IP-10. Further characterization of individual components of the BV vaginal microbiota mixture suggested that acetic acid is an important vaginal microbiota metabolite capable of eliciting diverse immunomodulatory effects on a range of cervicovaginal epithelial cell targets. These findings indicate that elevated levels of SCFAs are a potential source of cervicovaginal inflammation in women experiencing BV, and support the unique anti-inflammatory properties of LA on cervicovaginal epithelial cells as well as a role for LA or LA-producing lactobacilli to reverse genital inflammation associated with increased HIV risk.

Keywords: vaginal microbiota metabolites, lactic acid, bacterial vaginosis, inflammation, short chain fatty acids, succinic acid, cervicovaginal epithelium

# INTRODUCTION

The lower female reproductive tract epithelium plays important roles as a physical and immunological barrier against infection (Anderson et al., 2014); however its function is influenced by many factors including the effects of commensal vaginal microbiota and their metabolic products (O'Hanlon et al., 2011, 2013; Ravel et al., 2011; Aldunate et al., 2013; Anderson et al., 2014; Hearps et al., 2017; Tachedjian et al., 2017). The lower female reproductive tract microbiota is dynamic (Gajer et al., 2012) and can vary considerably between women of different ethnicities and geographical locations (Ravel et al., 2011; Gajer et al., 2012; Gosmann et al., 2017), although a predominance of beneficial lactobacilli is considered an optimal microbiota that is associated with eubiosis and favorable reproductive and sexual health outcomes (Aldunate et al., 2015; Fettweis et al., 2019; McKinnon et al., 2019).

Lactic acid (LA) is an organic acid metabolite predominately produced by lactobacilli (Boskey et al., 2001) and is present in the lower female reproductive tract at a concentration of ∼110 mM (∼1.0% ± 0.2% w/v) and a pH < 4.5 in women with lactobacillus-dominated microbiota (Boskey et al., 2001; O'Hanlon et al., 2013). There are two isoforms of LA, L-LA, and D-LA, which are produced in different ratios within the lower female reproductive tract by various Lactobacillus spp. (Witkin et al., 2013). At a vaginal pH below its pKa (3.86), LA is predominantly present in its protonated form (Aldunate et al., 2013) which elicits an anti-inflammatory response from cervicovaginal epithelial cells in vitro (Hearps et al., 2017). LA has antimicrobial activities against human immunodeficiency virus (HIV) (Aldunate et al., 2013), herpes simplex virus (Conti et al., 2009; Isaacs and Xu, 2013), Neisseria gonorrhoeae (Graver and Wade, 2011), Chlamydia trachomatis (Gong et al., 2014), and bacteria associated with bacterial vaginosis (BV) (O'Hanlon et al., 2011), and inhibits the infection of epithelial cells with Chlamydia trachomatis in vitro (Edwards et al., 2019).

In contrast, the lower female reproductive tract of women colonized with a dysbiotic or non-optimal microbiota, such as in BV, is dominated by anaerobes, a dramatic depletion of beneficial lactobacilli and an elevated vaginal pH > 4.5. This altered microbiota is associated with distinct vaginal concentrations of organic acid metabolites including short chain fatty acids (SCFA) and succinic acid produced by commensal bacteria (reviewed in Aldunate et al., 2015). In addition to LA, which is the predominant metabolite of a lactobacillus-dominated microbiota, acetic and succinic acid, and to a lesser extent propionic and butyric acid, are major microbiota metabolites found within the lower female reproductive tract, particularly during BV where their concentration increases dramatically, up to ∼120 and 20 mM for acetic and succinic acid, respectively (Al-Mushrif et al., 2000; Chaudry et al., 2004; Mirmonsef et al., 2011, 2012b; Gajer et al., 2012).

BV is also associated with subclinical genital inflammation (Eschenbach et al., 1988) and an increased risk of transmitting and contracting HIV (Eschenbach et al., 1988; Atashili et al., 2008; Cohen et al., 2012). Women diagnosed with Nugent-BV, as determined by the Nugent score that enumerates bacterial morphotypes using a Gram stain (McKinnon et al., 2019), are at a 1.53-fold (95% CI = 1.24–1.89) higher risk of acquiring HIV than women without BV (Low et al., 2011), while young South African women colonized with a pro-inflammatory, diverse vaginal microbiota, as determined by next generation sequencing i.e., Molecular-BV (McKinnon et al., 2019), are at a 4.4-fold (95% CI = 1.17–16.61) higher risk of acquiring HIV than women colonized by L. crispatus-dominated microbiota (Gosmann et al., 2017). Another study in African women showed that bacterial diversity and specific BV-associated taxa (e.g., Eggerthella species type I, Parvimonas species types I and 2 and Megasphaera spp.) were associated with increased risk of HIV acquisition (McClelland et al., 2018). The pro-inflammatory female reproductive tract milieu associated with non-optimal vaginal microbiota is thought to increase HIV susceptibility by recruiting activated HIV target cells to the genital mucosa, decreasing epithelial integrity and changing the barrier properties of cervicovaginal mucus (Lewis et al., 2013; Borgdorff et al., 2016; Zevin et al., 2016; Gosmann et al., 2017).

Although the mechanisms contributing to the proinflammatory cervicovaginal milieu due to non-optimal vaginal microbiota (e.g., BV) have not been completely elucidated, overgrowth of vaginal commensal anaerobic bacteria such as Gardenerella vaginalis, Prevotella spp., Atopobium vaginae, and Molibuncus spp. may expose vaginal epithelial cells to pathogen-associated molecular patterns, stimulating pathogen recognition receptors such as toll-like receptors (TLRs) and eliciting an inflammatory response (Lavelle et al., 2010; Byrne et al., 2015; Gosmann et al., 2017). Organic acid metabolic products from BV-associated bacteria such as SCFAs and succinic acid at concentrations observed during BV (Al-Mushrif et al., 2000; Chaudry et al., 2004; Mirmonsef et al., 2011) elicit a pro-inflammatory response from peripheral blood cells and enhance TLRassociated inflammation (Mirmonsef et al., 2012a); however the impact of SCFAs on cervicovaginal epithelial cells has not been reported.

We have previously shown that protonated LA at low pH (3.9), present during vaginal eubiosis (O'Hanlon et al., 2013; Tyssen et al., 2018), elicits an anti-inflammatory response and dampens TLR-induced pro-inflammatory responses from cervicovaginal epithelial cells (Hearps et al., 2017). However, it is unknown whether other vaginal microbial metabolic products present in the lower female reproductive tract such as SCFAs and succinic acid modulate this effect, or if they have immunomodulatory effects of their own. Accordingly, we investigated whether L-LA in combination with other vaginal microbiota metabolites, and at a pH relevant to vaginal eubiosis, maintains its antiinflammatory effects. In addition, we investigated if individual or combinations of SCFAs and succinic acid, at conditions typically observed in BV, modulate the production of cytokines and chemokines relevant to increased HIV risk from cervicovaginal epithelial cells.

### MATERIALS AND METHODS

## Cervicovaginal Epithelial Cell Culture and Stimulations

Ectocervical Ect1/E6E7 (Ect) and vaginal VK2/E6E7 (VK2) epithelial cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in serum-free keratinocyte medium, supplemented with 0.1 ng /ml human recombinant epithelial growth factor, 0.05 mg/ml bovine pituitary extract (all from Life Technologies, Carlsbad, CA), 0.4 mM CaCl2, 50 U/ml penicillin and 50µg/ml streptomycin. Primary human cervicovaginal epithelial cells derived from discarded surgical tissue (Ayehunie et al., 2006) were obtained from MatTek (Ashland, MA) and were maintained in media as above but with 10 ng/ml human recombinant epithelial growth factor.

Cervicovaginal epithelial cells (100,000 per well) were seeded into either 6.5 mm diameter polycarbonate transwell inserts containing 0.4µm pores (Corning, Corning, NY) in 24-well tissue culture plates or into 96-well tissue culture plates and cultured for 7 days to obtain a confluent cell monolayer. Epithelial monolayers were treated with combinations of vaginal microbiota metabolites, comprising L-LA, butyric acid, succinic acid (all from Sigma Aldrich, St. Louis, MO), acetic acid (Merck, Kenilworth, NJ), and propionic acid (MP Biomedicals, Santa Ana, CA). To mimic a lactobacillus-dominant microbiota (defined as eubiosis), cells were treated with media containing the predominate metabolites found during eubiosis and at concentrations and pH representative of this state. We assessed L-LA at 33 mM given our previous work showing that this concentration had significant immunomodulatory effects on cervicovaginal cells but minimal impacts on cell viability (Hearps et al., 2017). Acetic, succinic, propionic, and butyric acids are typically present at low to undetectable levels in vaginal fluid from women with eubiosis (Al-Mushrif et al., 2000; Mirmonsef et al., 2011; O'Hanlon et al., 2013) but can reach levels up to ∼4.6 mM for acetic acid (O'Hanlon et al., 2013) and 0.6 mM for succinic acid (Al-Mushrif et al., 2000) in women with lactobacillus-dominated vaginal microbiota, thus concentrations of 4 mM for acetic and 1 mM for succinic, butyric and propionic acid were assessed as part of the eubiosis mixture. Eubiosis treatment media was adjusted to pH 3.9 to mimic the physiological pH of women with lactobacillusdominated microbiota (O'Hanlon et al., 2013).  To mimic these conditions, cells were treated with media containing L-LA 6 mM (reduced to ∼20% of that in the eubiosis mixture to reflect the approximate 5-fold reduction in LA concentration observed in women with BV), acetic acid 100 mM, succinic acid 20 mM, propionic acid 4 mM, and butyric acid 4 mM at pH 7. These concentrations of SCFAs and succinic acid were selected to reflect those reported in women experiencing BV, where levels of acetic acid can reach up to ∼120 mM (Chaudry et al., 2004; Mirmonsef et al., 2011), succinic acid up to ∼22 mM (Al-Mushrif et al., 2000) and propionic and butyric acids up to ∼4 mM (Al-Mushrif et al., 2000). This BV metabolite mixture was adjusted to pH 7 before adding to cells, to reflect the upper limit of vaginal pH commonly found in women with BV (Brabin et al., 2005; Sánchez-Hernández et al., 2012). To assess the effect of individual vaginal microbiota metabolites in the context of BV, cells were treated with each metabolite alone at the same concentration as in the BV mix. Vaginal microbiota metabolites were added in the absence or presence of the TLR1/2 agonist Pam3CSK4 (Pam, 1µg/ml) or the TLR3 agonist high-molecular weight polyinosinic:polycytidylic (PIC, 20µg/ml) (both from InvivoGen, San Diego, CA) to simulate pathogen challenge. The pH stated for each treatment media reflects that of the media added to the apical chamber for experiments conducted in transwells, and of the entire well for experiments conducted in culture plates.

Cells in transwell inserts were stimulated apically with vaginal microbiota metabolite-containing media for 1 h, washed with PBS, fresh media (containing no stimulant) was then added and cells were incubated for a further 18 h. Previous optimization identified the pH of treatment media in the apical chamber was maintained during this 1 h stimulation period (Hearps et al., 2017). For experiments involving prolonged stimulation with BV-relevant SCFA, transwells were not able to maintain the desired conditions for these extended periods due to diffusion between apical and basal compartments, thus cells were treated in tissue culture plates. For these experiments, cells cultured in 96-well plates were stimulated continuously with treatment media for 24 h. Supernatants were collected from the wells or the apical chamber of the transwell inserts and stored at −80◦C for subsequent cytokine analysis. Cell viability was assessed following stimulation and supernatant collection using the CellTiter 96 Aqueous One Solution Cell proliferation assay (Promega, Madison, WI) as published (Hearps et al., 2017). Cell viability for all treatments remained above 70% of untreated cells (data not shown and as previously reported for LA) (Hearps et al., 2017) indicating minimal effects on cell viability under the conditions tested.

### Cytokine and Chemokine Quantitation

The production of cytokines interleukin (IL)-1 receptor antagonist (IL-1RA), IL-6, tumor necrosis factor α (TNFα), IL-1β, and chemokines IL-8, C-C motif chemokine ligand 5 (RANTES), interferon gamma-induced protein 10 (IP-10), and macrophage inflammatory protein 3 alpha (MIP-3α) in culture supernatant was quantified using ProcartaPlex Multiplex Immunoassays (Affymetrix, Santa Clara, CA). Samples were analyzed on a Luminex 200 (Bio-Rad, Hercules, CA), or MAGPIX system (Merck Millipore, Burlington, MA), and analysis was performed using manufacturer-provided software. For some treatments, values for IP-10 were above the limit of quantification of the assay and were assigned the maximum value of the standard curve.

# RESULTS

## The Anti-inflammatory Effect of L-LA on Cervicovaginal Epithelial Cells Persists in the Presence of Other Organic Acid Metabolites Relevant to Vaginal Eubiosis

Our previous studies examined the immunomodulatory effect of LA alone on cervicovaginal epithelial cells (Hearps et al., 2017); however LA exists in combination with other organic acid metabolites from female reproductive tract-resident bacteria in the vaginal lumen (Aldunate et al., 2015). Our previous study demonstrated that L- and D-LA elicit similar responses from cervicovaginal epithelial cells with regard to cytokines and chemokines associated with increased risk of HIV transmission in women (Masson et al., 2015; Hearps et al., 2017). Accordingly, we performed experiments using one of the LA isomers (L-LA). To determine if L-LA maintains its anti-inflammatory effect when combined with other organic acid metabolites, cervicovaginal epithelial cells were treated in transwell inserts with a combination of vaginal microbiota metabolites relevant to eubiosis with and without TLR stimulation for 1 h, shown previously to be sufficient for L-LA to elicit its anti-inflammatory effects (Hearps et al., 2017), and cytokine and chemokine production were analyzed 18 h post-treatment. The eubiotic condition tested represents the maximum level of SCFAs and succinic acid measured in women carrying an optimal vaginal microbiota (Al-Mushrif et al., 2000; Mirmonsef et al., 2011, 2012b; O'Hanlon et al., 2013). Prior to addition to the apical compartment, the pH of treatment media was adjusted to 3.9 to mimic the physiological acidity in women with an optimal, lactobacillus-dominated vaginal microbiota (Aldunate et al., 2013).

Ectocervical (Ect) and primary ectocervical cells, treated with L-LA in combination with the eubiotic vaginal microbiota metabolite mixture elicited an 8.5-fold (p = 0.01) and 2.6-fold (p = 0.02) increase in the production of the anti-inflammatory cytokine IL-1RA as compared to untreated Ect and primary cells, respectively, which was similar to the effect elicited by L-LA alone (**Figure 1A** and **Supplementary Figure 1A**). A heightened production of IL-1RA was also observed from vaginal VK2 cells treated with eubiotic vaginal microbiota metabolite mixture containing L-LA compared to untreated cells, although this did not reach statistical significance (**Supplementary Figure 2A**). Furthermore, the production of IL-1RA by eubiotic and L-LA treatments was also observed in the presence of TLR1/2 (Pam) and TLR3 (PIC) agonists, which mimic bacterial and viral challenge, respectively (**Figure 1A** and **Supplementary Figures 1A**, **2A**).

Basal production of IL-6 and MIP-3α from Ect cells was significantly decreased by both the eubiotic treatment and L-LA (p = 0.01 for both, **Figures 1B,F**) consistent with our previous findings (Hearps et al., 2017), and a similar trend for IL-6 was also seen in VK2 cells (**Supplementary Figure 2B**). Similar to L-LA alone, the eubiotic treatment elicited a minimal 2 fold increase in the production of IL-1β from all cell types (**Supplementary Figures 1F, 2F, 3A**), which physiologically is likely to be mitigated by the substantially higher production of the antagonist IL-1RA elicited by L-LA (7420-, 5812-, and 10166 fold excess of IL-1RA to IL-1β in Ect, VK2 and primary cells, respectively (**Figure 1** and **Supplementary Figures 1, 2**). Taken together, these data indicate that the anti-inflammatory effect of L-LA on unstimulated cervicovaginal epithelial cells persists within a complex mixture of vaginal microbiota organic acids representing a eubiotic vaginal environment.

### A Eubiotic Vaginal Microbiota Metabolite Mixture Containing L-LA Inhibits Pro-inflammatory Responses From TLR Stimulated Cervicovaginal Epithelial Cells

Genital inflammation, driven by a highly diverse vaginal microbiota, and increased cervicovaginal levels of proinflammatory cytokines and chemokines are associated with higher levels of HIV transmission (Masson et al., 2015; Gosmann et al., 2017). Beneficial vaginal lactobacilli (e.g., L. crispatus) dampen inflammatory and inhibit pro-inflammatory responses mediated by TLR agonists (Rose et al., 2012; Doerflinger et al., 2014) and vaginal pathogens (Santos et al., 2018), and we have demonstrated that this effect is mediated, at least in part, by LA (Hearps et al., 2017). Here, we asked whether this effect is maintained in the presence of physiological concentrations of other vaginal organic acid metabolites.

Apical stimulation of Ect cells with PIC and Pam, elicited an increased production of pro-inflammatory immune mediators including IL-6, IL-8, IP-10, RANTES, and MIP-3α, with PIC eliciting a greater response than Pam stimulation except for IL-8 and MIP-3α (**Figures 1B–F**). Similar to the effect of L-LA alone, in the context of PIC stimulation the eubiotic treatment significantly dampened the production of IL-6, IL-8, IP-10, MIP-3α (p < 0.01 for all) and RANTES (p = 0.02) from Ect cells (**Figures 1B–F**). A similar inhibitory effect was observed for Pam stimulated Ect cells, which was statistically significant for IL-6, IP-10, and MIP-3α production (p = 0.03 for all). TNFα production from Ect cells was largely below the limit of quantification, except for PIC-stimulated cells, with this response being completely inhibited by L-LA (**Supplementary Figure 3B**).

We next confirmed this result in VK2 and primary cervicovaginal cells and observed a similar ability of the eubiotic

multiplex assay. Graphs show mean and standard error of the mean from *n* = 4 independent assays. Mann-Whitney tests were used to estimate statistical significance between treatment groups. + denotes a significant difference (*p* < 0.05) as compared to untreated (UT) cells. *P* ≤ 0.05 and ≤ 0.01 are denoted as \* and

\*\*, respectively. L-LA, L-isomer of lactic acid; TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

treatment to inhibit TLR-mediated inflammation, similar to that observed with L-LA alone (**Supplementary Figures 1, 2**). These data demonstrate that the ability of L-LA to mitigate the proinflammatory response of cervicovaginal epithelial cells to TLR agonists mimicking bacterial and viral PAMPs is retained in the presence of physiological concentrations of vaginal microbiota organic acid metabolites found in women with a lactobacillusdominated microbiota.

## Short Exposure of Cervicovaginal Epithelial Cells to Vaginal Microbiota Metabolites Associated With BV Do Not Elicit Immunomodulatory Effects

BV is associated with vaginal inflammation, but whether this is mediated by an effect of vaginal microbiota metabolites produced by BV-associated bacteria on the production of inflammatory mediators by epithelial cells is not known. To determine if SCFAs and succinic acid, which are found at higher concentrations in the vagina of women with BV (Al-Mushrif et al., 2000; Chaudry et al., 2004; Mirmonsef et al., 2011; Gajer et al., 2012), have immunomodulatory effects on cervicovaginal epithelial cells, a combination of vaginal microbiota metabolites representing BV conditions was established and added apically to cervicovaginal epithelial cells seeded in transwells for 1 h. This mixture contained vaginal microbial metabolites at concentrations which represented the upper limits of what is reported in vivo for women with BV (acetic acid 100 mM, succinic acid 20 mM, propionic acid 4 mM, and butyric acid 4 mM) and at pH 7, to reflect the higher pH found in this state ranging from pH 4.5 to 8 (Brabin et al., 2005). In contrast to the eubiotic vaginal microbiota metabolite combination, a mixture of vaginal microbiota metabolites representing BV conditions did not significantly change the production of the anti-inflammatory cytokine IL-1RA compared to untreated cells, nor did it significantly alter the production of any other immune mediators tested from either unstimulated or TLR-stimulated Ect cells (**Figure 2**). A similar lack of immunomodulatory effect was also observed in VK2 and primary cells (data not shown). These findings suggest that unlike L-LA under eubiotic conditions, which elicited a significant immunomodulatory effect, vaginal microbiota organic acid metabolites representative of those found in BV conditions do not alter the inflammatory response of cervicovaginal epithelial cells when added to cells for comparable periods of time.

## BV-Related Vaginal Microbiota Metabolites Have Immunomodulatory Effects on Cervicovaginal Epithelial Cells Only During Prolonged and Sustained Treatments

Given the above findings, and a previous report demonstrated a pro-inflammatory effect of SCFAs on peripheral blood cells, including neutrophils, after longer treatment periods

Mann-Whitney tests were used to estimate statistical significance between treatment groups. + denotes a significant difference (*p* < 0.05) as compared to untreated

(Mirmonsef et al., 2012b), we hypothesized that vaginal microbiota metabolites may need prolonged and sustained contact with lower female reproductive tract epithelial cells to elicit an observable effect. The transwell system allows the diffusion of molecules from the apical to basolateral compartments. Therefore, to sustain the concentration and pH of the established treatments for longer periods, cervicovaginal epithelial cells were seeded into 96-well plates. Cells were treated with the combination of vaginal microbiota metabolites representing BV conditions, with and without TLR stimulation at pH 7 for 24 h.

(UT) cells. TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

Prolonged treatments of Ect cells under these conditions with a mixture of vaginal microbiota metabolites representing BV caused an increase in the production of TNFα from unstimulated cells (p = 0.03, **Figure 3C**) and potentiated the TLR-elicited production of TNFα for Pam and BV-stimulated cells compared to cells treated with Pam alone (p = 0.03). A similar effect was also seen in VK2 cells (**Supplementary Figure 4B**). The BV vaginal microbiota metabolite mix also increased basal TNFα production from primary cervicovaginal cells, but intriguingly the opposite effect was observed in PIC-stimulated cells (**Supplementary Figure 4F**). The BV metabolite mixture also potentiated TLR-induced IL-8 production, with this effect being unique to Ect cells (**Figure 3B** and data not shown).

In contrast to this enhanced production of the proinflammatory mediators TNFα and IL-8, the BV vaginal microbiota metabolite mixture dampened the PIC and Pamstimulated production of RANTES from all three cell types tested (p < 0.05 for all) (**Figure 3E** and **Supplementary Figures 4D,H**). Similarly, PIC-stimulated production of IP-10 was significantly inhibited by the BV vaginal microbiota metabolite mixture in all cell types (**Figure 3D** and **Supplementary Figures 4C,G**), while Pam-stimulated production was inhibited in VK2 and primary cells (p = 0.03 for both). Furthermore, the BV mixture inhibited basal production of IP-10, but this effect was only observed in primary cells (**Supplementary Figure 4G**). There was also a trend for the BV mixture to inhibit PIC-induced IL-6 production, although this was only significant in primary cells (p = 0.03) (**Figure 3** and **Supplementary Figure 4**). Similar results were observed when cells were treated with a BV mixture lacking L-LA (data not shown), consistent with our previous findings that the immunomodulatory effects of L-LA are only observed at a low pH (i.e., 3.9) (Hearps et al., 2017).

Taken together, these data demonstrate that prolonged and sustained treatments of cervicovaginal epithelial cells with a mixture of SCFAs and succinic acid recreating BV conditions results in a dysregulation of inflammatory responses, with a heightened basal and TLR-simulated production of the potent inflammatory cytokine TNFα, but a dampening of TLR-elicited production of IL-6 and the chemokines RANTES and IP-10. Although most of these effects were consistent between the three cell types tested, some effects appear to be cell-type

dependant (e.g., TLR-induced TNFα production in primary cells and potentiation of IL-8 production in Ect cells), highlighting the subtle but potentially important differences in the immune response of different lower female reproductive tract epithelial cell types.

### Individual Vaginal Microbiota Metabolites Elicit Dysfunctional Immunomodulatory Effects on Lower Female Reproductive Tract Epithelial Cells After Prolonged and Sustained Treatments

Having identified immunomodulatory effects of sustained treatments with a mixture of BV vaginal microbiota metabolites, we sought to identify which individual metabolite might be responsible for these dysfunctional effects. Lower female reproductive tract epithelial cells were therefore treated with individual SCFAs or succinic acid in 96 well plates under the same concentrations and pH conditions as above.

The pro-inflammatory effect of the BV mixture on increased TNFα production was largely recapitulated by treatment of cells with acetic acid alone (**Figures 4A–C**) with a consistent effect observed for all three cell types. Treatment with acetic acid alone also reproduced the potentiation of TLR-induced TNFα production in Ect and VK2 cells, and the converse effect in primary epithelial cells (**Figures 4A–C**). Butyric acid alone had a similar but non-significant effect on enhancing TLRelicited TNFα production in Ect cells (**Supplementary Figure 5**), while no effects were observed for succinic acid or propionic acid alone in any cell type tested (data not shown). These data suggest that acetic acid, and to a lesser extent butyric acid, are likely the metabolites responsible for the proinflammatory effects of the BV mixture on TNFα production in these cells.