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Monday

Vaginal microbicides are a female-controlled HIV prevention option whose early development faced challenges that necessitated the search for safety biomarkers in clinical trials. We started out with a group of 30 healthy Caucasian women in Antwerp, Belgium and characterized cellular and soluble immunity and the vaginal microbiome in the lower female genital tract. We also conducted both a cross-sectional and longitudinal characterization of the female genital tract in 430 sexually active women from Sub-Saharan Africa (Kenya, Rwanda and South Africa).

As generally expected, bacteria that are generally linked with good vaginal health, L. crispatus and L. vaginalis, were associated with lower levels of pro-inflammatory cytokines and higher levels of protective antimicrobial proteins. In contrast, BV-associated A. vaginae, G. vaginalis and P. bivia were associated with increased pro-inflammatory cytokines. Vaginal dysbiosis (specifically BV) was characterized by a clear pro-inflammatory signature and reduced levels of IP-10, SLPI and total protein.

Based on our findings, we propose pro-inflammatory IL-1 (α and β), IL-6, IL-8, IL-12(p70); anti-inflammatory IL-1RA; the chemokine IP-10; and the antimicrobial protein SLPI as a smaller panel of soluble biomarkers for consideration for safety measurement of candidate vaginal microbicides. Additionally, the assessment of microbicide safety should include clinical examination for visible signs of vaginal epithelia irritation and the effect of candidate microbicides on cellular markers of inflammation as well as the vaginal microbiome. An ideal outcome would be point-of-care tools that can be easily used in clinical setting in resource-limited settings to identify women with increased susceptibility to HIV infection.


Wednesday

Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3

1Department of Pharmaceutics, University of Washington, Seattle, WA, USA

2Washington National Primate Research Center, University of Washington, Seattle, WA, USA

3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA

4Department of Immunology, University of Toronto, Toronto, Canada

5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada

Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria increase HIV transmission rates by inducing activation and accumulation of neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage.

Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.

Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p<0.0001) or with neutrophils and Lactobacillus spp. (p<0.0001).

Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.


Wednesday

Mariya I. Petrova

KU Leuven, Belgium

The vaginal microbial community is typically characterized by abundant lactobacilli. Lactobacillus iners, a fairly recently detected species, is frequently present in the vaginal niche. However, the role of this species in vaginal health is unclear, since it can be identified in normal conditions as well as during vaginal dysbiosis, such as bacterial vaginosis, a condition characterized by an abnormal increase in bacterial diversity and lack of typical lactobacilli. Compared to other Lactobacillus species, L. iners has more complex nutritional requirements and a Gram-variable morphology. L. iners has an unusually small genome, indicative of a symbiotic or parasitic lifestyle, in contrast to other lactobacilli that show niche flexibility and genomes of up to 3-4 Mbp. The presence of specific L. iners genes, such as those encoding iron-sulfur proteins and unique σ-factors, reflects a high degree of niche specification. The genome of L. iners strains also encodes inerolysin, a pore-forming toxin related to vaginolysin of Gardnerella vaginalis. Possibly, this organism may have clonal variants that in some cases promote a healthy vagina, and in other cases are associated with dysbiosis and disease. Future research should examine the exact role of this species and its relationship with the host.


Wednesday
Probiotic fermented food for Africa
04:16pm - 04:16pm Eastern - February 6, 2019

In the poorest parts of the world, how do we empower people to use their local resources in a manner than provides health and economic benefits to all? When a group of women in Mwanza, Tanzania set up a community kitchen producing probiotic yoghurt, in 2004, their entrepreneurial talent laid the foundation for a program now reaching over 260,000 people each week in east Africa, including people in refugee camps. The development of a sachet containing starter culture Streptococcus thermophilus C106 and either Lactobacillus rhamnosus GR-1 or GG (Yoba 2012), probiotic strains documented by over one thousand published studies, provided the critical technological advance to empower people across the region to make not only probiotic yoghurt but also cereal and fruit juices. Research has shown that the fermentation process not only inhibits pathogens, but also removes highly toxic aflatoxins present in many roadside-sold foods. The microenterprise business model reaches all elements of the value chain providing thousands of jobs for women, men and youth and netting profits for production units, especially when they sell 20-100 litres yoghurt per sachet. Life-changing cases have been documented with some owners employing 20 staff, buying many cows, sending their children to good schools, and moving from mud huts to modern homes. These microbiota-directed foods provide health-promoting, affordable nutrition with the potential to reach millions of people around the world.


Wednesday

Heleen Schuster MDa,b, Annelot Breedveld MScc, Andries Budding MD PhDa, Paul Savelkoul PhDa,d

aDepartment of Medical Microbiology and Infection Control, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; bDepartment of Obstetrics and Gynecology, Amsterdam UMC, Univ of Amsterdam, Amsterdam, Netherlands; cDepartment of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; dDepartment of Medical Microbiology, Maastricht University Medical Centre, Maastricht, Netherlands


As knowledge on the role and composition of the vaginal microbiome is accumulating, more interest is directed to microbe:immune crosstalk. Local immunoglobulins (Igs) play an important role as mediator of mucosal immunity and homeostasis. In the vaginal mucosa, IgG is the dominating antibody, while at other mucosal surfaces IgA is most prevalent. In the gut, alterations in IgA coating of commensal bacteria are associated with inflammatory bowel disease and Clostridium difficile infection. Less is known about the role of local Igs in the female genital tract. We aim to provide insight in the IgA and IgG coating of vaginal bacteria using a combination of flow cytometry based techniques and microbiota profiling.


Vaginal swabs were collected from healthy volunteers. Fluorescence-Activated Cell Sorting (FACS) was used to sort micro-organisms according to IgA and IgG coating. The bacterial composition was analyzed with bacterial profiling technique IS-pro, using fragment variability of the 16S-2S rDNA intergenic spacer region.


Lactobacillus crispatus and L. iners dominated the vaginal microbiota of healthy volunteers and show large numbers of IgA and IgA/IgG coated Lactobacilli. Bacteria with only IgG on their surface were not detected. The proportions of Ig coated bacteria varied among individuals with similar microbial compositions.


Vaginal bacteria show varying amounts of IgA and IgA/IgG coating between different healthy volunteers. The high amount of Ig coated vaginal commensal bacteria demonstrate the importance of local Igs in the mucosal environment. Disturbances of the coating might lead to increased local inflammation and can possibly identify women at risk for diseases of the reproductive tract and adverse pregnancy outcomes. These promising results indicate a putative role of Igs in the maintenance of a healthy microbiome.


Wednesday

Praveen Thumbikat1, Stephen F. Murphy1, Jonathan F. Anker1, Daniel J. Mazur1, Anthony J. Schaeffer1, Christel Hall1

1Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

Gram-positive bacteria are considered an ubiquitous but incidental finding in urine and prostatic secretion cultures from male patients with chronic pelvic pain syndrome (CPPS) However, we hypothesized that gram-positive traditional non-uropathogenic bacteria can play a direct role in CPPS pathogenesis. Staphylococcus epidermidis, Staphylococcus haemolyticus, and Enterococcus faecalis comprised 62% of prostate-localizing bacteria from CPPS patients. A subset of these induced high NFκB expression, contained pathogenic virulence factors, and were associated with increased clinical symptoms. Here we show that an immunogenic subset of the most common gram-positive prostate-localizing bacterial strains can directly cause pelvic tactile allodynia and voiding dysfunction. Intra-urethral infection of mice with immunogenic bacterial isolates from each strain, but not an S. epidermidis isolate from a healthy control, recapitulated symptoms seen in patients. Mechanistically, these pathogenic gram-positive bacteria induced unique prostate inflammation, involving T cells, NK cells, and cytokine production. We demonstrate for the first time that the prevalent, yet untreated traditional non-uropathogenic gram-positive bacteria can play a causative role in CPPS pathogenesis.