Impact of the Cervicovaginal Microbiome on HIV Susceptibility: an Investigation of Mechanisms and Potential Interventions

Impact of the Cervicovaginal Microbiome on HIV Susceptibility: An Investigation of Mechanisms and Potential Interventions

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Citation Rice, Justin K. 2020. Impact of the Cervicovaginal Microbiome on HIV Susceptibility: An Investigation of Mechanisms and Potential Interventions. Doctoral dissertation, Harvard Medical School.

Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37364793

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Impact of the Cervicovaginal Microbiome on HIV Susceptibility: An Investigation of Mechanisms and Potential Interventions

Justin Rice

Harvard-M.I.T. Division of Health Sciences and Technology

Submitted in Partial Fulfillment of the Requirements for the M.D. Degree

February, 2020

Area of Concentration: Infectious Disease

Project Advisor: Douglas S Kwon, MD PhD

Prior Degrees: PhD (Linear Algebra/System Dynamics)

I have reviewed this thesis. It represents work done by the author under my guidance/supervision.

Table of Contents

Abstract 3

Introduction 4-7

Methods 8-11

Results 12-26

Discussion 27-29

Conclusions 30

Acknowledgements 30

References 31-40

Abstract

Sub-Saharan Africa has among the highest HIV infection rates in the world, with an estimated 980,000 new HIV infections in 2017 (Sidebé 2018). Since the majority of HIV transmission occurs through heterosexual sex (UNAIDS, 2014), understanding how HIV infection is established within the female genital tract (FGT) is critical for the development of HIV preventative interventions, and deserves further study. It has been known for decades that bacterial vaginosis (BV), a disease state characterized by increased FGT inflammation, increased pH, and colonization with non-Lactobacillus species, is associated with an increased risk of acquiring HIV (Sewankambo 1997), along with other sexually transmitted infections (STIs), and with health problems during pregnancy such as preterm birth (McGregor 2000). More recently it has also been shown that BV is associated with increased risk of female-to- male HIV transmission (Cohen 2012).

In this study, we conducted in vitro experiments on cervical tissue culture models of epithelial cells and fibroblasts. Our results confirmed prior observations regarding inflammation of BV associated bacteria such as Prevotella bivia, Lactobacilus iners, and Gardnerella vaginalis. At set time periods after the introduction of these pro-inflammatory species (with Lactobacillus crispatus for comparison) the supernatants were tested using ELISA assays for various pro- inflammatory cytokines (e.g. IL-6, IL-8), and cell death (via LDH), in order to determine which bacterial species induced pro-inflammatory cytokines. The same experiments were then repeated with the addition of various innate immune inhibitors targeting PAMP (Pathogen- Associated Molecular Pattern) sensing receptors such as TLR-4 (LPS), TLR-2 (peptidoglycan), and TLR-9 (unmethylated CpG) and down-stream inflammatory pathway facilitators (NFκB). The results of these experiments suggested specifically which signaling pathways were responsible for the innate-immune system activation and release of cytokines, and suggested potential interventions to block the process.

Strains of bacterial species that have been previously associated with an increased HIV risk were then isolated from the South African FRESH (Females Rising through Education Support and Health) cohort for further testing. Several strains of L. crispatus, G. vaginalis, and P. bivia from FRESH samples were isolated, and their DNA sequenced, to investigate the phylogenetic relationship of these bacteria. Furthermore, three novel FGT species were isolated in culture for the first time, and are now available for further research.

Introduction

1. Background:

It has been known for decades that infection with various STIs is correlated with an increased risk of HIV infection, as demonstrated by observational studies in a diverse set of high risk groups, e.g women who use drugs (Miller 2008), MSM in Belgium (Sasse 2009), and clients of sex workers in Mexico (Patterson 2009), with the highest contributing STI being found for concurrent HSV-2 infection (Braunstein 2009), and the highest hazard ratio seen with gonorrhea infection (7 fold increase in HIV incidence) (van de Wijgert 2009). See (Ward 2010) for a comprehensive review. The obvious confounders of increased risk behavior and exposure exist, but other studies show there to be an increased risk of HIV-infection in individuals after infection with another STI, after controlling for these confounders, e.g. (Menza 2009, Jin 2010), suggesting a causal relationship. Suggested mechanisms include HSV ulcers facilitating blood- blood transmission or active STIs in the HIV infected partner inducing increased viral genital tract shedding (Fox 2010), and the recruitment of neutrophils and active inflammation to the GU tract of the partner-at-risk, increasing CD4+ target cells (Johnson, 2008). Supporting this theory are genetic phylogeny experiments documenting that most heterosexual-sex-transmitted HIV infections can be traced back to a single virion (Haase 2010), whereas in individuals with concurrent STIs, post-hoc genetic analysis has shown that multiple virions are often responsible (Haaland 2019), suggesting a more permeable immunological barrier.

Bacterial vaginosis (BV), a disease state characterized by increased FGT inflammation, increased pH, and colonization with non-Lactobacillus species (such as G. vaginalis, Ureaplasma urealyticum, Mycoplasba hominis, Prevotella sps. Mobiluncus sps., etc (Zozaya- Hinchliffe 2010) and many fastidious bacteria specific to the FGT (Mitchell 2009), and associated with symptoms of gray vaginal discharge, unpleasant odor, and pruritis (Bilardi 2013) has also been associated with an increased risk of acquiring HIV (Sewankambo 1997), as well as other adverse sequelae such as preterm birth (McGregor 2000), chorioamnionitis, and post- gynecological surgery infections (Koumans 2001). BV and associated bacteria have also been linked with obesity in observational studies on humans (Brookheart 2019), and in mouse studies linking the two conditions via obesity-induced inflammation and systemic LPS (Si 2017). Regarding HIV, recent studies have confirmed the association between Lactobacillus dominated FGT microbiomes and decreased HIV prevalence (Borgdorff 2014), and shown that BV is associated with increased risk of female-to-male HIV transmission (Cohen 2012), likely due to increased FGT inflammation (Mitchell 2011). Furthermore, in (Gosmann 2017) a group of women at high risk for HIV infection with known FGT microbiomes was prospectively followed, and it was shown that women with certain non-lactobacillus dominated microbiomes had up to a 4X increased risk of contracting HIV, compared to women with L. crispatus dominated microbiomes, strongly suggesting a causal relationship between BV and HIV risk, and providing further impetus for research into BV and related mucosal factors. Recent research has also shown that the occurrence of BV-associated bacteria may be a significant risk factor for HIV infection in men as well, wherein the bacterial syndrome is called “Penile anaerobic dysbiosis” (Liu 2017), and is associated with significantly increased penile inflammation.

BV does not technically meet Koch’s postulates for a communicable disease – the original bacterial link with BV was G. vaginalis (Gardner 1954), which, when cultured alone, generally does not cause the disease state of BV. However, the condition has long been thought to be sexually transmitted, and Gardner himself noted that men would frequently re-infect their wives if untreated with tetracycline antibiotics themselves. More recent research has shown specifically that women are likely to re-experience BV after antibiotic treatment if they do not use condoms, and/or have sex with the same pre-treatment partner (Bradshaw 2013), that women transfer these BV-associated bacteria to their male partners (Liu 2015), and that circumcision- status of male partners in some situations has been associated with a decreased incidence of BV (Gray 2009), presumably by decreasing the incidence of these bacteria (and hence penile anaerobic dysbiosis) on the penis (Liu 2013).

In spite of the considerable sequelae and disease burden of BV, the understanding of the underlying pathophysiology and development the disease is still ongoing, in part due to the lack of an animal model in which it can be easily studied (Turovskiy 2010). However, it is generally believed that the “normal” commensal bacteria of lactobacilli contribute to the healthy conditions of the vagina by producing lactic acid and maintaining a low pH, thereby restricting pathogenic bacteria such as those mentioned above from taking hold. When such pathogenic bacteria do take hold, they are known to have complicated temporal dynamics (as shown via daily FGT swab collection and sequencing in (Muzny 2018)), with increased relative abundance of BV- associated bacteria around the start of menses (Ravel 2013). It has also been shown that there are numerous symbiotic relationships between the different species of bacteria, e.g. it is thought that G. vaginalis produces amino acids to aid colonization with other anaerobes, and that Prevotella bivia produces ammonia for Gardnerella (Pybus 1997). Other studies have suggested that the culture data, which many of these studies have relied on, may not well represent the FGT microbiome, since many of the anaerobic bacteria found in the FGT are fastidious and do not grow well on plates (Hiller 1986). It has also been found that one of the factors contributing to the difficulty of eradicating BV (to be discussed below) is the production of biofilm by G. vaginalis (Patterson 2007).

Nevertheless, given its severe sequelae and considerable symptoms, considerable work has been dedicated to treating BV, either via antibiotics or by some intervention to alter the FGT microbiome in other ways. The standard treatments of metronidazole or clindamycin have similar success rates in the 70-80% range (Ferris 1995), although relapse is experienced in approximately 30% of cases (Hay 2000). Part of the recurrence difficulty seems to be the aforementioned G vaginalis biofilm (Swidsinksi 2008) – the disease is not truly being eradicated, although significant work has been done on treating the biofilms directly (Gottschick 2016, Thellin 2016). Other research has shown that BV incidence involves a complex interaction between multiple species, and perhaps the transition from lactobacillus to other species is triggered by menses (Lambert 2013). Hence various studies have been done on the idea of supporting the ‘’good bacteria’’, e.g. giving probiotics (usually Lactobacillus species) to directly inhibit the pathogenic bacteria from growing, e.g. Lactobacillus rhamnosus and Lactobacillus fermentum, which have been shown to decrease the incidence of BV (Reid 2001, Reid 2003), and strangely, to increase the incidence of L crispatus in the FGT (Macklaim 2015). A variety of mechanisms may be at work, e.g. bacteriocins produced by Lactobacillus may kill a wide

5 spectrum of Gardnerella strains (Simoes 2001). Other work has shown that giving lactic acid by itself, or artificially lowering the pH of the FGT can alter the microbiome (O’Hanlon 2011 and Wilson 2005, and Decena 2006 ), cause an anti-inflammatory response (Hearps 2014) and hence perhaps help treat BV, although results have been mixed (Boeke 1993). Lactobacillus suppositories (Hemmerling 2010 and Bradshaw 2012) and even sucrose gel (Hu 2010) have also been investigated as possible interventions, with difficult-to-interpret results. Trials aimed at treating the male sexual partners have been so far unsuccessful (Mehta 2012). Other suggested interventions such as bacteriophage therapy (Oliveira 2015) aimed directly at altering the microbiome are also being developed, as well as several clinical trials investigating vaginal microbiota transplant (Kwon 2019, Johns Hopkins 2018). Such studies have already shown measured success in animal models, e.g. rhesus macaques and cynomolgus macaques (Daggert 2017).

The impetus for this diverse and ongoing research is obvious- besides the problematic symptoms and harmful sequelae, BV is extremely common, although with differential prevalence among ethnicities, with up to 20% of Americans with Asian ancestry colonized with non-Lactobacillus dominant vaginal communities, and up to 40% of African Americans, 20% of European ancestry, and 38% of Hispanics (Fettweis 2012, Ravel 2011). BV is even more common in parts of sub-Saharan Africa, with estimates of up to 63% in South Africa (Gosmann 2017), (where HIV acquisition rates are as high as 10% per year (Marrazzo 2015) among young women, and hence the importance of BV as a risk factor for HIV transmission is much higher).

2. State of the Field

Given the influence of BV on HIV transmission, and the prevalence and treatability of BV, recent research has been devoted to investigating if BV truly causally influences HIV susceptibility, and if so, the mechanism by which it does, with the aim of developing interventions for treating it. Recent in vitro work (Pyles 2014) presented promising first results showing that differentially culturing vaginal microbiomes in epithelial plate cultures could alter HIV infection and ARV efficacy, demonstrating a proof of concept, and prompting further investigation.

As for the mechanism, (Gosmann 2017), as mentioned previously, showed that individuals with a diverse population of anaerobic bacteria such as P. bivia, P. melaninogenica, and G. vaginalis in the FGT have up to a 4x increased risk for contracting HIV over individuals with microbiomes dominated by L. crispatus. Such individuals have upregulation of genes involved in NF-kB, Toll- like receptors (e.g. TLR-4), NOD-like receptors, and TNF-α signaling pathways and measurably higher levels of IL-1α, IL-1β, TNF-α, IFN-γ (Anahtar 2015). Ingenuity Pathway Analysis specifically predicted that TLR-4 (activated by LPS) was the most likely upstream signaling source, which was consistent with bacterial shotgun sequencing of the microbiomes, showing a prevalence of bacteria engaging in LPS biosynthesis. Women with this type of FGT bacterial microbiomes also have higher levels of CD4+ HIV-target cells in the FGT (Anahtar 2015, although this has been contested by Lennard 2018), accompanied by higher levels of cytokines

6 associated with the subclass of Th-17 cells (Gosmann 2017), which have been shown to be the first cells infected by HIV (Stieh 2016) in this environment. These demonstrations of a plausible mechanism – that some BV-associated bacteria trigger TLR-4 mediated activation in antigen presenting cells via NFκB, leading to increased levels of immune activation and the release of inflammatory cytokines in the FGT of individuals harboring these microbiota, hence leading to increased Th-17 CD4+ target cells in the FGT, and hence increased HIV susceptibility, was further supported by an experiment in (Gosmann 2017) that demonstrated that inoculation of germ-free mice with P. bivia, one of the risk-associated species, caused an increase in activated CD4+ cells (HIV target cells) in the FGT over mice inoculated with L. crispatus. While these results are very promising, and the most advanced in the field, further work elucidating the hypothesized mechanism of BV influence on HIV susceptibility is necessary.

Other research has suggested competing hypotheses for the mechanism, such as the provocative idea that BV associated bacteria, such as G vaginalis, may actually metabolize HIV pre-exposure prophylaxis (Klatt 2017) or at least that general FGT inflammation may decrease tenofivir efficacy by some unspecified mechanism (McKinnon 2018), although results have been mixed (Heffron 2017). Other ex-vivo work has suggested that lactobacillus itself may have a protective effect against HIV-1 infection (Palomino 2017). Female sex hormones and hormonal contraceptives have been implicated in modulating HIV susceptibility (Saba 2013) by increasing CD4+ target cells (Byrne 2016), as well in modulating the vaginal microbiome (Achilles 2013), and the two effects have been suggested to be synergistic, or at least linked (Wessels 2018).

3. Purpose of the Inquiry

In this project, we performed in-vitro FGT cell culture experiments seeking to test the causality of each part of the above hypothesized series of events, elucidating the mechanism behind the association between diverse FGT microbiomes and HIV susceptibility. Such evidence would suggest that an intervention in some part of this mechanism may lead to decreased HIV transmission risk, and suggest candidates for such an intervention, such as innate immune signaling blockades or FGT microbiome transplants.

In addition, to better investigate the variability among FGT bacteria species, a library of bacterial isolates was gathered from FRESH samples, and their phylogenetic relationship was determined, in order to investigate the relationship between standard laboratory models and BV- associated bacteria in the field. Such results can help clarify the variability in the inflammatory capacity of BV associated bacteria, and the utility of current bacterial models of BV and BV- induced HIV susceptibility. Such studies will also be useful in further categorizing different BV associated bacteria with the aim of characterizing the differences in metabolism, following up on the recent hypothesis of tenofovir metabolism by G. vaginalis (Klatt 2017).

In sum, such research will contribute scientifically to the field of HIV research by helping to clarify the role of the FGT microbiome in inflammation and HIV susceptibility, an important

7 measure that may lead to a better understanding and controlling of the HIV epidemic in sub- Saharan Africa.

Methods

1. Growing Ectocervical Cells

ECT1(E6E7, ATCC) cells were cultivated at 37C in a medium of Keratinocyte-Serum Free medium ( (GIBCO-BRL 17005-042) with 0.1 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, and additional calcium chloride 44.1 mg/L (final concentration 0.4 mM)) in 75cm^2 flasks and split between 60- and 90% confluence, and sub-cultivated 1:3. To remove cells for sub-cultivation, trypsin 0.03% was used

Cells were frozen in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM:F-12, ATCC Catalog No. 30-2006), 85%; fetal bovine serum, 10%; DMSO, 5% in liquid nitrogen.

2. Growing bacteria

FGT bacteria were cultured on the following media in anaerobic chamber (AS-580 Anaerobe Systems):

Lactobacillus crispatus: MRS plates and MRS broth

Prevotella Bivia: BHK plates and BHK broth

Prevotella Amnii: BHK plates and BHK broth

Gardnerella Vaginalis: V agar

Initially, CVL swabs were plated onto a variety of media, including BHK, LKV, CBA, V agar, chocolate agar, Wilkens Chalgren, and tryptic soy agar. After isolating pure colonies, samples were grown up on whole plates, then harvested into cryovials containing 1mL of 20% glycerol and thioglycolate.

3. Log growth data to find logarithmic growth time

MRS, Wilkins-Chalgrin, and Trypic Soy media were prepared in foil the night before, and introduced into the anaerobic chamber to reduce overnight. P. Bivia was inoculated into Trypic Soy and Wilkins Chalgrin, and L. crispatus was inoculated into MRS broth, and at each time point, OD was measured and 10^-1 through 10^-8 serial dilutions were plated. For plates found to have bacterial growth in the serial dilutions, individual colonies were counted, and CFUs were back calculated. Two experiments were performed in parallel. In experiment 1, time points were: 6, 9, 14, 22, 25, and 30 hours. In experiment 2, time points were: 11, 13, 16, 19, and 22 hours.

4. Correspondance between OD and CFU vs OD and bacterial #

Using an inoculation loop, bacteria were harvested from the appropriate culture media, added to 2mL PBS, agitated, and then serially diluted 1:5 until visually transparent (3-5 dilutions for each bacteria). OD was recorded for each bacterial dilution. 20uL specimens were then put in a Petroff-Hausser apparatus for bacterial counting, with 9 fields used for each sample, and the average count used to back-calculate bacterial concentration in the original samples.

Additionally, 1:10 serial dilutions were performed in PBS in a 96 well plate in duplicate, using multichannel pipets, up to 10^6 dilution, and 50uL were plated on MRS or BHK agar as appropriate. Colony forming units were then counted after incubation for 2-3 days, and original bacterial concentration was back-calculated.

5. Dose response for live vs dead P bivia

50,000 ECT1 cells were seeded into each well of a 96 well plate, and incubated in KSFM for 24 hours.

Sufficient P. Bivia was grown in liquid Tryptic Soy media, and L Crispatus was grown in liquid MRS media. The Petroff-Hausser chamber was used on serial concentrations of each media to dilute samples to 10^9 bacteria per mL, which was serially diluted down to 8*10^6 bacteria/ mL via a 1:3 dilution scheme (Schaefer, 2004). One set of bacterial serially diluted aliquots was then incubated at 60C for 30 minutes to kill all the bacteria, following (Eade et al, 2012). Bacterial samples were then added to ECT1 cells in KSFM at 37C for 24 hours. Supernatant was harvested and used for IL-8 assay via ELISA, as specified below.

6. Co-culture FGT bacteria w/ MatTek proprietary FGT tissue explant model

The MatTek EpiVaginal Tissue Model (MatTek, Ashland, MA) trans-well system was used. The disc-inserts were each washed with 1mL PBS and transferred to a 24 well plate with 500uL fresh VEC proprietary media each, and rested for 24hours. Bacteria were prepared by collection from broth culture as described above, centrifuged at 3200g for 10min, re-suspended in PBS, and diluted until the appropriate OD was reached via spectrophotometer, to produce 10^8CFU/mL solution.

When ready, MatTek inserts were transferred to special inset plate using forceps, and 500uL fresh VEC media was added.

Bacteria (50uL of appropriately diluted solution, or UP-PBS) was then pipetted gently to the top of the tissue, without contact, such that the total supernatant was 200-250uL. Tissue was then scarified:

Scarification: each insert was punched 12 times with an 18-guage needle, and care was taken not to perforate the insert all the way through. Inserts were then incubated for 1 hour at 37C to rest.

Cultures were then incubated for 24 hours at 37C. After this time, the supernatant was collected from each well, the remaining tissue was washed with UP-PBS which was also collected, and samples were centrifuged at 3200g at 10 minutes, then re-suspended in 100uL UP-PBS for future ELISAs.

For experiments in which inhibitors were used as well, they were added directly after the bacteria, before scarification. The following quantities were added:

TLR-2 inhibitor (Mab-mTLR from Invivogen) : 1ug added to each 200uL well (per dosing in (Roussel 2016))

TLR-4 inhibitor (LPS-RS) 5uL of 1mg/mL stock added to each 200uL well (our 25ug/mL is similar to 30ug/mL used in (Herzmann 2017))

TLR-9 inhibitor (ODN TTAGGG (A151) from Invivogen): 2uL of 500uM stock added to each 200uL well (dosing of 5uM per (Miranda 2012))

Bay (NFκB) inhibitor (Bay11-7082 from Invivogen): 10uL of 50mM was added to each well

7. Co-culture FGT bacteria w/ MatTek Langerhans Cells

After being received by the supplier, LCs were centrifuged to a pellet, then re-suspended in 2.5mL VEC media. 120,000 Langerhans cells were added per well on a 24 well plate, and an analogous experiment to that above was performed with the cells in solution of 100uL VEC media, incubated at 24 hours with analogous concentrations of bacteria and/or inhibitors. After incubation, supernatants were pipetted off, and saved in 96 well plates for future ELISAs.

8. ELISA Protocol

Plate Coating: 96 well plates were coated with capture antibody diluted from 250X by adding 48uL of stock solution to 12mL of coating buffer (itself diluted from 10X stock), then with the entire solution diluted to make 12mL, which was allocated out 100uL per well, onto flat-bottom ELISA plate by multipipette. Plate was then incubated overnight at 4C

Plates were then washed 3X with wash buffer, and plates were reblocked with 200uL/well ELISA/ELISPOT diluent, and incubated at RT for 1 hour, then after another washing, samples were added, and standard was added at appropriate dilutions and incubated for 2 hours. After

10 incubation and further round of washing, detection antibody was added, then washed 3 times, and strepavadin was added. After 30 minutes substrate was added, and 450nm fluorescence was measured, and standard curve was calculated for comparison / calibration.

9. LDH (Lactate Dehydrogenase) Protocol

Before starting, dye and stop-solution were thawed at room temperature, and catalyst was suspended in 1mL of miliQ water. The reactant solution was prepared by adding 133.3uL catalyst to 6mL of dye. Then, 100uL of this solution was added to each 100uL of sample in each well of the plate using a multi-pipetter, and incubated for 12 min at 37C, then 50uL of stop solution is added to each well, again by multi-pipetter, and the set-up is allowed to sit for an additional 12 minutes. Absorbance was then measured at 490nm.

10. Growing and Isolating Bacteria From FRESH Samples

The Females Rising through Education Support and Health (FRESH) study is a prospective cohort study being conducted in Kwazulu-Natal, South Africa, in which young women at risk for HIV are tested biweekly for new HIV infection. The study was conducted under Massachusetts General Hospital IRB Protocol # 2012P001812, and approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal. Informed consent was obtained.

Frozen cytobrush samples from 16 individuals believed from previous sequencing results to harbor complicate microbiomes were rapidly thawed in anaerobic chamber, diluted with 200uL UP-PBS, and removed. Cytobrush was then repeatedly rinsed with 4 cycles of 200uL UP-PBS via pipette, and resulting sample diluted in 1:10 fashion down to 1:10. 50uL samples were then pipetted and spread onto various agar media, which were incubated at 37C in anaerobic conditions until bacterial growth was observed. At this point, single colonies were plucked from the agar, and replated onto serial agar plates (of several varieties) to obtain pure colonies. Once fully grown up on plate, colonies were harvested into 1mL of 20% glycerol in thioglycolate solution for freezing and later DNA extraction and sequencing. Growth media used were: MRS, chocolate, CBA, LKV, TSA, V-agar, BHK.

Results

The following results are an account of original observations, experiments, and analysis performed by the author, often with the assistance and supervision of other lab members, who are specified at the end of each section.

1. Experiments Investigating CFU vs Bacterial Count (Dead or Alive).

In order to properly plan for FGT epithelial / bacterial co-culture experiments, in which precisely quantified samples of bacteria would be added to FGT cell cultures in an anaerobic chamber, it was necessary to find a precise and rapid method by which bacteria could be counted. Petroff- Hausser Counters, the gold standard, would require bacteria to be removed from the anaerobic chamber and laboriously counted. Optical density, which can be easily and rapidly measured within an anaerobic chamber, was the first option for such a rapid and reliable method (Sutton, 2011). To investigate the relationship between Optical Density and bacterial count, several bacterial species of interest; L. iners, P. amnii, and G. vaginalis, were anaerobically cultured in broth until non-transparent, then centrifuged and washed with UP-PBS, serially diluted, and the optical density was measured. Various dilutions were then put under the Petroff-Hausser counter for comparison, as shown in Figures 1, 2, and 3. As was noted early on in the experiment, the Petroff-Hausser counter was found to be difficult to use by the experimenters, due to clumping together of the bacteria and the fine nature of the counting. The initial counts were also noted to not have a very clear linear relationship with optical density, so the experiments were replicated over several days. The results showed that this counting method was not reproducible using the available technology and techniques (and personnel) – the reported variability of less than 5 percent between individuals counting was not achievable in our experiments, perhaps due to lack of the requisite experience (Elberg 1957).

Figure 1: Millions of L. Iners per milliliter, as quantified by Petroff Hauser Chamber, from 3 different days of experiments (red, blue, and green), as a function of Optical Density. Note the poor linear fit and lack of reproducibility.

Figure 2: Millions of G. Vaginalis per milliliter, as quantified by Petroff Hauser Chamber, from 2 different days of experiments (blue,and green), as a function of Optical Density. Note the poor linear fit and lack of reproducibility.

Given the need for precise control of the bacterial loads for the planned co-cultures, and the difficulty of using the Petroff Hausser chambers, other methods for bacterial quantification were sought out. Quantification of Colony Forming Units (CFUs) were identified as a possible alternative.

The aforementioned experiments were performed by myself and Mara Farcasanu.

Figure 3: Millions of P. Amnii per milliliter, as quantified by Petroff Hauser Chamber, from 3 different days of experiments (yellow, green, and gray), as a function of Optical Density. Note the poor linear fit and lack of replicability.

2. Bacterial Growth Curves as a Function of Time

In order to properly understand and plan for FGT epithelial / bacterial co-culture experiments, and to obtain mostly live bacteria in order to quantify CFUs (Colony Forming Units), it was necessary to understand the time dynamics of the growth of our bacteria in pure culture and hence be able to pick bacteria from the log-growth phase. Samples of ATCC P. bivia and ATCC L. crispatus were anaerobically cultured in Tryptic Soy and MRS broth, respectively, and 5 days after their last passage, were inoculated into Erlenmeyer flasks of the appropriate broth and incubated at 37C for 30 hours. During that time, serial measurements of CFUs, and OD (Optical Density) were taken. CFUs were measured by serially diluting and plating samples, then counting colonies after growth (see Figure 4) sufficient to distinguish individual groups.

Figure 4: L. crispatus (on yellow MRS agar), and P. bivia (on red TS agar) in serial dilutions, demonstrating method for calculating CFUs.

The combined CFU and OD data generated the curves seen in Figures 5 and 6. As can be seen, within the first 15-16 hours, the lag and exponential growth phases of both species had been passed, and the stationary phase was entered. This informed further experiments, in which bacteria used for inoculating later samples could be taken from the exponential growth phase.

Figure 5: P. bivia incubated anaerobically in pre-reduced Tryptic Soy broth, with serial measurements of optical density and Colony Forming Units (measured by taking serial dilutions, and plating onto Tryptic Soy agar.)

Figure 6: L. crispatus incubated anaerobically in pre-reduced MRS broth, with serial measurements of optical density and Colony Forming Units (measured by taking serial dilutions, and plating onto MRS agar.)

3. CFUs as a Function of Optical Density

Since future co-culture experiments would also require the rapid quantification and serial dilution of bacterial cultures, and since the total bacterial count (via Petroff-Hausser Chamber) was found to be difficult to use, the CFU vs OD during the log growth phase from the previous experiments was also plotted out, which showed a linear relationship (see Figures 7 and 8), consistent with published results for other bacteria, e.g. Pseudomonas aeruginosa in (Kim 2012).

Figure 7: P. Bivia samples from logarithmic growth curve anaerobically cultured in Tryptic Soy broth at 37C, showing roughly linear relationship between CFUs and OD.

The results shown in figures 7 and 8, and accompanying linear least-squares fits, provided a convenient means by which the appropriate numbers of CFUs could be quantified for addition to the later FGT co-cultures.

Figure 8: L. crispatus samples from logarithmic growth curve anaerobically cultured in MRS broth at 37C, showing roughly linear relationship between CFUs and OD.

4. Dose Response for Live vs Dead P. bivia

Given the promisingly linear and predictable relationship of OD and CFU, this made it appear to be a good candidate for quantifying bacteria for adding appropriate doses for the bacterial co- culture experiments. However, it was clear that CFUs were measuring something very different than bacterial count. Bacterial count included both live and dead bacteria. CFU, on the other hand, could undercount live bacteria – a single clump of multiple live bacteria would only create one colony, and would not count dead bacteria at all, even though the hypothesized mechanism – bacterial pathogen associated molecular patterns (PAMPs, e.g. LPS or flagellin) activating local toll like receptors and attracting CD4+ target cells to the area – would be activated by living as well as dead bacteria. Since the literature is full of both examples and counter-examples to this hypothesis, e.g. (Stathopoulou 2010), it was necessary to assay the bacteria of interest in order to confirm the relationship in this specific situation. Experiments were performed on cultured ectocervical cells (ECT1), with IL-8 produced (measured by ELISA) used as a proxy for inflammatory response, to find the dose-response relationship of live vs heat-killed P. bivia, with results shown in Figure 9. Bacteria were quantified via OD, and the above-determined linear relationship.

Figure 9: IL-8 produced by increasing doses of P. bivia, either live (gathered from Tryptic soy broth culture in log-growth phase), or heat killed, as specified in the Methods section. Values were averaged over 4 replications.

Experiments were performed on ECT1 cells, given the considerably greater ease and lower cost of cultivating ectocervical cells compared to the more sophisticated MatTek multi-layer preparation. Such a simplification was justified by the existing literature suggesting the presence of TLR-2, 3,4, 5, 6 expression in the FGT (Wira 2010, Herbst-Kralovetz 2008, Pioli 2004, Hirata 2005).

Given the close relationship between the IL-8 measured for the live vs heat-killed P. bivia, it was thought that CFU would be a sufficiently accurate proxy for the much more difficult to measure total bacteria (as counted by Petroff Hausser Chamber), and that the OD would be a practical method for quantifying it. These results also established that the PAMPs present on the suspected pathogenic bacteria would indeed trigger an immune response and release inflammatory cytokines in a dose-dependent manner when applied to FGT cells.

The author performed these experiments himself.

5. Co-cultured FGT Bacteria on MatTek multilayer FGT tissue model

Given the established inflammatory cytokine production of the ECT1 cells in response to P. bivia, and the dose dependent response, it was hypothesized that an experimental model more representative of in vivo would be the MatTek Epivaginal model, which is a stratified tissue culture insert comprised of human ectocervical epithelial and fibroblast cells, resembling the normal human vaginal epithelium. Some of the bacteria of interest as pathogenic species associated with increased HIV susceptibility in (Gosmann 2017) and with high prevalence in the FGT, such as P. bivia and amnii, along with L. crispatus, (species most identified with low HIV susceptibility), G. vaginalis (classically associated with BV), were grown in pure anaerobic culture on the MatTek platform. Pure LPS was included as a positive control, and untreated FGT tissue, LPS-RS (an inhibitor of TLR-4) (Baker 1990), along with LPS+LPSRS were included as negative controls. The IL-6 secreted in each of the above experimental conditions, as measured by ELISA, is shown in Figure 10.

As expected, the negative controls of untreated (no bacteria), LPS-RS, and LPSRS+LPS produced near zero levels of the inflammatory cytokine IL-6. Unexpectedly, LPS, which previous work (Gosmann 2017) suggested to be a prime upstream source of inflammation, did not result in very much IL-6, compared to P. amnii and P. bivia, which caused high expression of the inflammatory cytokine IL-6. In retrospect, this can be well understood by the need for soluble CD-14 for TLR-4 signaling (Zanoni 2011), which was not supplied in the experiment, and is not typically present in the vagina without other antigen stimulation (Duluc 2013).

Also unexpected were the low levels of IL-6 produced in response to G. vaginalis, a known pathogenic bacteria associated with BV and HIV susceptibility (Gosmann 2017). However, this can be understood in the context of more the sophisticated relationship of G. vaginalis to the other FGT bacteria. As is well known, G. itself produces very little LPS (Aroutcheva 2008), and is understood more as a symbiont with other more immunogenic bacteria, such as producing ammonia for P. bivia (Pybus 1997).

On the other hand, the high levels of IL-6 produced by P amnii and P bivia were just as expected, as were the low levels of IL-6 produced by L. crispatus, a ‘’protective’’ bacteria and L. iners, which, while thought to be less protective than L. crispatus, is also not associated with increased HIV susceptibility in previous experiments (Gosmann 2017).

The aforementioned experiments were performed by myself and Mara Farcasanu.

IL-6 (measured in pg/mL) Produced by Bacteria Co-cultured with MatTek EpiVaginal System

4500

4000

3500

3000

2500

2000

1500

1000

500

0 UT L crisp. P amnii P bivia G. vag L iners LPS LPSRS LPSRS+LPS

Figure 10: IL-6 (as measured by ELISA), produced on co-culture of various FGT bacteria and controls in the MatTek epivaginal trans-well model (including ectocervical epithelial cells and fibroblasts). (Diamonds in blue, green, and violet represent three experimental replicates)

6. IL-8 produced by MatTek Co-cultures is Decreased by Specific TLR Inhibitors

To follow-up on the aforementioned results, further experiments using the MatTek platform were performed, to try to identify the pathways responsible for the excitation of the immune system and production of the inflammatory cytokines. Given the reliably high IL-6 produced in response to P. bivia co-culture, and P. bivia’s association with HIV susceptibility, it was chosen as the best species for investigation with the inhibitors. Again, an untreated row of samples was used as a negative control, and uninhibited P. bivia was included as a positive control. The assay measured IL-8 due to the availability of that ELISA assay at a time when IL-6 ELISAs were not available. In addition, a separate but parallel experiment was performed on pure Langerhans Cells in suspension instead of the multilayer epithelial/fibroblast system of MatTek. The results of each experiment are shown in Figures 11 and 12.

Prevotella Bivia (PB) Cultured in MatTek Epivaginal System, With and Without Inhibitors

Figure 11: IL-8 produced by MatTek EpiVaginal transwell system in response to anaerobic co- culture with P bivia with and without the addition of inhibitors for TLR-2, TLR-4, TLR-9, and NFκB. Y-axis is in picograms/mL.

As expected, the untreated wells produced very little IL-8 as a negative control, and the positive control of untreated P. bivia produced high levels of IL-8, both for the assay of the MatTek epiVaginal model, and from the Langerhans cells in suspension (which led to much higher levels of IL-8 in every experimental condition except for the untreated negative control).

Interestingly, the amount of IL-8 produced was decreased when the TLR-4 inhibitors and NFκB inhibitors were added, as predicted by (Gosmann 2017), but not the TLR-9 inhibitors or TLR-2 inhibitors. This was also anticipated- TLR-2 is responsive to binding peptidoglycan, which is only present in small amounts in the largely gram-negative coliform bacteria (such as P. bivia) thought to be pathogenic in the FGT. TLR-9 is responsive to bacterial DNA, which would largely be unavailable for binding contained as it is within the live bacteria used in these experiments.

The aforementioned experiments were performed by myself and Mara Farcasanu.

5 xPrevotella 10 Bivia (PB) Co-culturedPB with with Langerhans and without inhibitorsCells, With and Without Inhibitors 3

2.5

1.5 pg/mL

0.5

0 UT PB PB+TLR2i PB+TLR4i PB+TLR9i PB+NFkBi

Figure 12: IL-8 produced by Langerhans Cells (supplied by MatTek) in response to anaerobic co-culture with P bivia with and without the addition of inhibitors for TLR-2, TLR-4, TLR-9, and NFκB. Note that the Langerhans Cells assays produced much higher levels of IL-8 than the Epivaginal model, as would be expected due to the very high levels of TLR’s expressed on dendritic cells. Of note, an error in the TLR-9i experiment was noted – both doses of inhibitor were put into the same trans-well, and none in the either.

7. Growing and Isolating Bacteria from FRESH FGT Samples

223 bacterial isolates were gathered from frozen cervico-vaginal lavage (CVL) or swabs from 16 individuals from the FRESH cohort in South Africa, the bacterial DNA was extracted and sequenced, and their genomes were compared with publicly available databases. 36 known individual bacterial species were identified (see Table 1), including Gardnerella piotii, a new species of Gardnerella that had not previously been isolated at the time this research was done, but was recently published (Vaneechoutte 2019). In addition, preliminary data suggests that three previously uncultured bacteria were isolated:

Order: Coriobacteriales, Family: Atopobiaceae, Genus: Fannyhessea, Species: unnamed Order: Mycobacteriales, Family: Mycobacteriaceae, Genus: Corynebacterium, Species: unnamed Order: Actinomycetales, Family: Bifidobacteriaceae, Genus: Bifidobacterium, Species: unnamed which can now be characterized and studied further in future research.

Anaerococcus_tetradius Atopobium_vaginae Campylobacter_ureolyticus Corynebacterium_amycolatum Corynebacterium_pseudogenitalium Cutibacterium_acnes Dialister_micraerophilus Fusobacterium_animalis Gardnerella_piotii Gardnerella_vaginalis Lactobacillus_crispatus Lactobacillus_iners Lactobacillus_jensenii Lactobacillus_salivarius Megasphaera_sp_UPII_135_E Mobiluncus_mulieris Peptoniphilus_lacrimalis Peptoniphilus_sp Peptostreptococcus_anaerobius Porphyromonas_uenonis Prevotella_amnii Prevotella_bivia Prevotella_buccalis Prevotella_colorans Prevotella_ihumii Prevotella_intermedia Prevotella_melaninogenica Prevotella_sp Prevotella_timonensis Staphylococcus_epidermidis Staphylococcus_haemolyticus Staphylococcus_hominis Stomatobaculum_sp Streptococcus_agalactiae Streptococcus_anginosus

Table 1: Identified bacteria from 16 cytobrush samples from FRESH samples (DNA isolation performed by Ian Herriott. Identification and phylogenic analysis performed by Matthew Hayward).

Furthermore, multiple new strains from many common FGT species of interest were isolated, adding significantly to the publically available reference genomes for study (see Figure 13), and in further characterizing the diversity and phylogeny of FGT bacteria associated with BV.

Quantification of New Genomes of BV- Associated FGT Bacteria Produced for Future Research

Figure 13: In peach color are the quantity of previously available reference genomes for the listed FGT bacterial species on the left. In purple are the genomes added by the isolates described in this thesis. (DNA isolation performed by Ian Herriott. Identification, phylogenetic analysis, and graph performed by Matthew Hayward).

This work was performed in conjunction with Ian Herriott (DNA isolation and sequencing), Seth Bloom (advice regarding bacterial culturing and isolation), and Matthew Hayward (phylogenetic analysis).

Discussion

1. Synthesis and Implications of Results After studying the growth dynamics of the bacteria of interest to determine the logarithmic growth stages of each, the CFUs and raw bacterial counts were compared with OD, in order to find an appropriately rapid and precise method of quantifying bacteria, such that the dose of bacteria could be manipulated as an independent experimental variable. The CFU’s were found to be linearly and precisely related to the OD. In order to confirm that CFU’s would not significantly underestimate the relevant bacteria, e.g. by discounting dead bacteria, assays of dead and alive P. bivia in different doses were co-cultured with pure cultured ectocervical cells, and the resulting IL-8 produced demonstrated that this was not the case, and that, especially in the lower doses, there was good correspondence in the inflammatory response to dead and alive bacteria.

With these preliminary results supporting the ability to use precise doses of live bacteria as a stimulus in co-culture with FGT cells to measure differential inflammatory responses, the stage was set for the first experiments with the sophisticated MatTek EpiVaginal trans-well system of fibroblasts and ectocervical cells. The first experiment on the MatTek platform demonstrated that co-culture with P bivia resulted in much more production of inflammatory IL-6 than G. vaginalis or L. iners, and confirmed that co-culture with L. crispatus resulted in very little inflammatory response. Since P. bivia was known to be associated with increased HIV susceptibility (as opposed to P. amnii, the other highly inflammatory bacteria), it was co-cultured in a further MatTek experiment, which showed that the inflammatory response induced in the FGT cells by P. bivia could be abrogated with selected inhibitors of the inflammatory response pathway, with the most effective inhibitors being the TLR-4 inhibitor and the NFκB inhibitor.

These preliminary results support the mechanisms proposed in (Anahtar 2015) and (Gosmann 2017), in demonstrating the first causal step in which certain bacteria associated with bacterial vaginosis could bring about increased local inflammation– via the LPS / TLR-4 and NFκB pathways – and release of pro-inflammatory cytokines, e.g. IL-6 and IL-8.

2. Limitations, and Alternative Hypotheses.

However, the research described in this report has significant limitations, and there may be alternative explanations for the results. While P. bivia was used for many of the experiments in this research due to its ease of culture, and association with inflammation and HIV susceptibility (Gosmann 2017). However, this severely limits the conclusions which can be drawn, and many other bacteria with similar associations, such as Megasphaera, Clostridium, Atopobium and Sneathia are also of likely importance in this relationship. As has long been suspected (Schwebke 2014), it is likely that these bacteria participate in complicated symbiotic relationships among themselves, and it may be that limiting the investigation to single bacterial species may fail to elicit some of the most important behavior of the system. In addition, while the bacteria used in these experiments were from commercially available samples (ATCC),

27 there could be significant variation between strains of the same species – e.g. between individuals or between strains prevalent in geographic locations.

As for the experimental design, due to costs and available funding, the number of samples in each experiment was severely limited, with some of the MatTek experiments having only 3 samples per condition. This obviously limits the ability to draw conclusions, as any recognized differences between the results for different experimental conditions could just be spurious results, given the inability to perform statistical analysis.

For similar reasons- limitations in funding and time, a limited number of assays were used to study the inflammatory response of the ECT cells and MatTek transwell systems – our study was limited to IL-6, IL-8, and LDH, instead of a more complete assay for inflammatory markers such as a commercially available Luminex Inflammation Multiplex Assay, which might provide more subtle hints as to the specific pathways involved. Likewise, and leading to the same limitations in the ability to draw conclusions, only a few inflammatory pathway inhibitors were used in the assays: inhibitors of TLR-2, TLR-4, TLR-9, and NFκB, even though ectocervical cells are well known to express many other PAMP receptors, e.g. TLR-1, TLR-3, TLR-6 (Herbst- Kralovetz 2008), and inhibitors to other down-stream inflammatory modulators are commercially available. Such experiments might have elicited other involved pathways, had they been conducted.

These limitations in the performed experiments are especially important in view of the limited abrogation of the inflammatory responses by the inhibitors that were used. As seen in Figures 11 and 12, while the TLR-4 inhibitor and NFκB inhibitor did decrease the measured IL-8 in co- culture with P bivia, the decrease was extremely moderate. This could be due to some experimental error, or perhaps also due to parallelism or synergism in the physiological mechanism by which the inflammatory response is mediated – more than one TLR or other PAMP receptor is likely involved in producing the inflammatory response, and several inhibitors used in combination would likely be needed to cause a more significant decrease in the production of inflammatory cytokines.

Other factors leading to a decreased ability to draw conclusions, the fact that the different MatTek experiments were assayed with different inflammatory cytokine ELISAs (IL-6 and IL-8), leads to a decreased certainty in the results. It is less clear that each experiment was conducted correctly, since the positive and negative controls cannot be compared between experiments to check for consistency. In a similar way, the data in Figure 9 was used to justify the equivalence of live vs heat-killed bacteria in the MatTek experiments, however, the bacterial concentrations in the experiments shown in Figure 9 were much higher than the concentrations used in the MatTek experiments, where the multiplicity of infections (MOIs) were much closer to physiologic levels.

Of course, in terms of extrapolating from these results to their implications in the human FGT, there are a number of limitations. The proprietary MatTek platform, while it has been extensively tested and is now used broadly in laboratories (Ayehunie 2011, Ayehunie 2015, Hearps 2017,

Garcia 2019) may not be precisely representative of the human FGT in terms of inflammatory responses to pathogenic bacteria. Additionally, while IL-8 is known to be elevated in women with high risk FGT microbiomes (Gosmann 2017, see Figure 3D), and IL-6 and IL-8 are known to be inflammatory cytokines, they may not be causative agents in attracting the CD4+ target cells for HIV that are expected.

3. Directions for Future Research

In the future, besides extending and replicating the experiments with more experimental conditions and dependent variables, as discussed above, there are multiple avenues of research that could be pursued in order to shore up and extend this research. For example, to investigate the physiological relevance of these results, P. bivia from multiple patients and geographical locations could be assayed for their pro-inflammatory effects in co-culture, to understand the inter-strain variation. Likewise, in addition to using multiple TLR inhibitors, by themselves and in combination, it has been suggested that L. Crispatus itself acts as an inhibitor of inflammation, either via lactic acid or by other means (Hearps 2017, Rizzo 2015), and this could be assayed by co-culturing L crispatus in combination with pathogenic FGT bacteria and FGT cells, e.g. the MatTek model, although care should be taken to tease out the difference between Lactobacillus-mediated-protection and the well documented lactic acid- mediated protection (Tachedjian 2017), as well as potential Lactobacillus-mediated (Palomino 2017) and lactic-acid mediated (Tyssen 2018) HIV inhibition.

In order to extend this research towards drawing conclusions about the human FGT, this approach can be extended to murine models. As suggested in (Gosmann 2016), germ-free mice with humanized immune systems can be inoculated with known FGT bacteria thought to be pathogenic and associated with increased HIV susceptibility (e.g. P. bivia, G. vaginalis, etc), and compared with controls for increased inflammatory cytokines (e.g. IL-6 and IL-8), and increased CD4+ and/or CD17+ target cells in the FGT. If these experiments were to produce results as anticipated, similar murine models (vs controls) could then undergo HIV challenges, with an anticipated increase in HIV infection rate in the mice inoculated with BV-associated FGT bacteria.

Regarding the isolation of FGT bacteria associated with BV, future research should be dedicated to further studying and characterizing the three new species that were isolated. Another interesting line of research would be to compare different sub-species strains of the various FGT bacteria to compare their inflammatory capacity – e.g. to compare P. bivia samples in Boston with P. bivia samples from South Africa in ability to induce IL-6 or IL-8 production from ectocervical cells.

Conclusions

In conclusion, the experimental results supported the hypothesis in (Gosmann 2017), with some limitations. Optical Density (OD) was found to be a reliable and precise proxy for CFU in the FGT bacteria of interest, and a dose dependent response was found between P. bivia CFUs and the release of inflammatory cytokines in vitro. Living and heat-killed P. bivia were found to evoke similar responses in ecto-cervical cells – demonstrating that heat-killed bacteria can safely be used in future experiments, for expediency and convenience.

Furthermore, the MatTek Epivaginal trans-well system was found to reliably produce an inflammatory response when co-cultured with P. bivia, one of the hypothesized pathogenic bacteria associated with BV and increased HIV susceptibility in (Gosmann 2017), much more so than L. crispatus, G. vaginalis, or L. iners. Additionally, TLR-4 and NFκB inhibitors were found to decrease this response, confirming a role of this pathway in the inflammatory response, as hypothesized in (Gosmann 2017). The data and experimental methods were limited in scope and in statistical power, but did support the hypothesis, and stand as a proof-of-concept for further work in this direction, as specified in the Discussion section.

Additionally, it was found that the relatively simple procedure of broadly culturing CVL samples from women suspected of having BV was able to provide interesting information on the phylogenetic relationships between various FGT bacteria, as well as yield previously un-cultured bacteria for future study.

Acknowledgements

The completion of this work would not have been possible without the patience, teaching efforts, and mentorship of Doug Kwon, and the day to day supervision, advice, and shoulder-to-cry-on of Mara Farcasanu. I am also sincerely grateful to Christina Gosmann, Angela Gasca-Lozano, Melis Anahtar, Zoe Rogers, Bjorn Corleis, Matthew Hayward, and Seth Bloom for their support and guidance, and for saving me from my own mistakes on a daily basis.

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