The human vaginal microbiota: from clinical medicine to models to mechanisms.

The composition of the vaginal microbiota is linked to numerous reproductive health problems, including increased susceptibility to infection, pregnancy complications, and impaired vaginal tissue repair; however, the mechanisms contributing to these adverse outcomes are not yet fully defined. In this review, we highlight recent clinical advancements associating vaginal microbiome composition and function with health outcomes. Subsequently, we provide a summary of emerging models employed to identify microbe-microbe interactions contributing to vaginal health, including metagenomic sequencing, multi-omics approaches, and advances in vaginal microbiota cultivation. Last, we review new in vitro, ex vivo, and in vivo models, such as organoids and humanized microbiota murine models, used to define and mechanistically explore host-microbe interactions at the vaginal mucosa.

Distinct maternofetal immune signatures delineate preterm birth onset following urinary tract infection.

Preterm birth is the leading cause of infant mortality resulting in over one million neonatal deaths annually. Maternal urinary tract infection (UTI) during pregnancy increases risk for preterm birth; however, biological processes mediating UTI-associated preterm birth are not well-described. We established a murine maternal UTI model in which challenge with uropathogenic E. coli resulted in preterm birth in about half of dams. Dams experiencing preterm birth displayed excessive bladder inflammation and altered uteroplacental T cell polarization compared to non-laboring infected dams, with no differences in bacterial burdens. Additional factors associated with preterm birth included higher proportions of male fetuses and lower maternal serum IL-10. Furthermore, exogenous maternal IL-10 treatment absolved UTI-associated preterm birth but contributed to fetal growth restriction in this model. Using urine samples from a cohort of human pregnancies with or without UTI, we correlated urinary cytokines with birth outcomes and urine culture status. These analyses yielded a non-invasive, highly predictive three-model system for evaluating preterm birth risk implicating cytokines IL-10, IL-15, IL-1ß, and IL-1RA. Our unique bimodal murine model coupled with patient samples provides a platform to investigate immunological and microbial factors governing UTI-associated preterm birth, revealing novel therapeutic opportunities to predict or prevent preterm birth.

Gestational diabetes augments group B Streptococcus infection by disrupting maternal immunity and the vaginal microbiota.

Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. Using a murine GDM model of GBS colonization and perinatal transmission, we find that GDM mice display greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing reveals differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase (yfhO), and altered host responses. GDM immune disruptions include reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered maternofetal cytokines. Lastly, we observe distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Here, we show a model of GBS dissemination in GDM hosts that recapitulates several clinical aspects and identifies multiple host and bacterial drivers of GBS perinatal disease.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbiota composition is associated with differential risk of urogenital infection. Although vaginal Lactobacillus spp. are thought to confer protection through acidification, bacteriocin production, and immunomodulation, lack of an in vivo model system that closely resembles the human vaginal microbiota remains a prominent barrier to mechanistic discovery. We performed 16S rRNA amplicon sequencing of wildtype C57BL/6J mice, commonly used to study pathogen colonization, and found that the vaginal microbiome composition varies highly both within and between colonies from three distinct vivaria. Because of the strong influence of environmental exposure on vaginal microbiome composition, we assessed whether a humanized microbiota mouse ( HMb mice) would model a more human-like vaginal microbiota. Similar to humans and conventional mice, HMb mice vaginal microbiota clustered into five community state types ( h mCST). Uniquely, HMb mice vaginal communities were frequently dominated by Lactobacilli or Enterobacteriaceae . Compared to genetically-matched conventional mice, HMb mice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia , but no differences were observed with uropathogenic E. coli . Specifically, vaginal Enterobacteriaceae and Lactobacillus were associated with the absence of uterine GBS. Anti-GBS activity of HMb mice vaginal E. coli and L. murinus isolates, representing Enterobacteriaceae and Lactobacillus respectively, were characterized in vitro and in vivo . Although L. murinus reduced GBS growth in vitro , vaginal pre-inoculation with HMb mouse-derived E. coli , but not L. murinus , conferred protection against vaginal GBS burden. Overall, the HMb mice are an improved model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens. IMPORTANCE: An altered vaginal microbiota, typically with little to no levels of Lactobacillus , is associated with increased susceptibility to urogenital infections, although mechanisms driving this vulnerability are not fully understood. Despite known inhibitory properties of Lactobacillus against urogenital pathogens, clinical studies with Lactobacillus probiotics have shown mixed success. In this study, we characterize the impact of the vaginal microbiota on urogenital pathogen colonization using a humanized microbiota mouse model that more closely mimics the human vaginal microbiota. We found several vaginal bacterial taxa that correlated with reduced pathogen levels but showed discordant effects in pathogen inhibition between in vitro and in vivo assays. We propose that this humanized microbiota mouse platform is an improved model to describe the role of the vaginal microbiota in protection against urogenital pathogens. Furthermore, this model will be useful in testing efficacy of new probiotic strategies in the complex vaginal environment.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Human Milk Oligosaccharides Reduce Murine Group B Streptococcus Vaginal Colonization with Minimal Impact on the Vaginal Microbiota.

Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage of healthy women and is a leading cause of neonatal bacterial infections. Currently, pregnant women are screened in the last month of pregnancy, and GBS-positive women are given antibiotics during parturition to prevent bacterial transmission to the neonate. Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial cells and a murine vaginal colonization model, we tested the impact of HMO treatment on GBS burdens and the composition of the endogenous microbiota by 16S rRNA amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. In addition, disruption of a putative GBS glycosyltransferase (Δsan_0913) rendered the bacterium largely resistant to HMO inhibition in vitro and in vivo but did not compromise its adherence, colonization, or biofilm formation in the absence of HMOs. We conclude that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with minimal impacts on the vaginal microenvironment. IMPORTANCE During pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. In addition, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum. Current recommended prophylaxis consists of administering broad-spectrum antibiotics to GBS-positive mothers during labor. Although antibiotics have significantly reduced GBS neonatal disease, there are several unintended consequences, including altered neonatal gut bacteria and increased risk for other types of infection. Innovative preventions displaying more targeted antimicrobial activity, while leaving the maternal microbiota intact, are thus appealing. Using a mouse model, we found that human milk oligosaccharides (HMOs) reduce GBS burdens without perturbing the vaginal microbiota. We conclude that HMOs are a promising alternative to antibiotics to reduce GBS neonatal disease.

Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment.

Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity.

CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.