Chlamydia trachomatis epidemiology and interaction with the cervicovaginal microbiome

Host-Microbiota Interactions and Chlamydia Trachomatis Infection Outcomes

The risk of sexually transmitted infection (STI) in humans depends on multiple biological factors, among which the occurrence of a cervicovaginal microbiota that is `permissive' to STI stands out. The microbial composition of a STI-permissive microbiota is similar to that observed in association with the syndrome of bacterial vaginosis, a condition that is generally defined by a high pH (>4.5), the absence of Lactobacillus spp. and an array of strict and facultative anaerobes such as Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp., and Prevotella spp. In contrast, a typical ‘non-permissive' microbiota is dominated by one of several species of Lactobacillus, a unique feature of the human cervicovaginal microbiota. The mechanism(s) by which a non-permissive cervicovaginal microbiota provides protection against STIs remains poorly understood, as no animal or cell culture model system developed to date satisfactorily reproduces the cervicovaginal mucosa in its natural environment as a target for experimental infection. As a consequence, our knowledge of the pathogenesis of STIs is incomplete, particularly as it pertains to the critical role of the human cervicovaginal microbiota. We have established extensive preliminary data that support the scientific premise of this project and states that a non- permissive indigenous microbiota interacts with the cervicovaginal epithelium to establish a homeostatic state that blocks STI and/or reduces disease severity. Conversely, a permissive microbiota disrupts host cell homeostasis, thereby allowing STI to progress. Advanced 3D organotypic models of the cervicovaginal mucosa that eliminate the inherent ethical and biological limitations of existing models will be used to test this hypothesis, using the most prevalent agent of STIs worldwide, Chlamydia trachomatis. We aim to develop a better understanding of the host-microbiota interactions and how they modulate the fate of C. trachomatis infection. In this project, 3D organotypic models colonized with different types of reconstructed microbiota will be exploited to ask specific questions about how different types of microbiota (permissive and non-permissive) modulate epithelial cell functioning (Aim 1) in relationship to their susceptibility/resistance to chlamydial infection (Aim 2). We will also assess the preclinical potential of the model for testing preventive (Aim 2) and therapeutic (Aim 3) interventions against STIs. For these studies, we will leverage the full force of state-of-the-art microscopic and RNA-sequencing methodologies to identify and characterize microbiota specific alterations of structural and host epithelial homeostasis and expand our understanding of the triangular relationship between the host, the microbiota and C. trachomatis.

There are approximately 20 million new sexually transmitted infections (STIs) in the U.S. each year that add $16 billion in medical costs to the healthcare system. One approach to prevention of STIs in women that has not been fully explored is harnessing the protective features of the vaginal microbiome. The vaginal microbiota play an important role in preventing colonization by pathogenic organisms. Vaginal Lactobacillus spp. provide broad-spectrum antimicrobial activity in part through their production of lactic acid, which creates an acidic and hostile environment to pathogens. To date, only studies based on bacterial cultivation techniques or microscopic evaluation are available to describe the temporal association between the vaginal bacteria and risk of STI. However, a majority of microbial species (>90%) resist cultivation and microscopy only provides morphological information. Complete characterization of the vaginal microbiota requires molecular approaches as we propose. We have shown using metagenomic analysis that specific genotypes of Lactobacillus spp. are associated with both chlamydial infection and vaginal microbiota instability. We are putting forth a novel hypothesis that, in addition to a low-Lactobacillus state, specific Lactobacillus genotypes are associated with increased risk for STIs, and that the mixture of D- and L- lactate isomers produced by Lactobacillus spp. differentially affects STI risk. Inflammation may cause destruction of the vaginal epithelium, allowing pathogens to access deeper tissues, and therefore, we also hypothesize that local immune responses associated with specific vaginal microbiota may facilitate STIs. We seek to utilize archived cervicovaginal lavage (CVL) samples collected from the 1999 NIH Longitudinal Study of Vaginal Flora in which 3,620 women were followed for one year with quarterly assessments. A total of 681 women were observed to acquire an incident Chlamydia trachomatis (CT), Neisseria gonorrhea (GC), or Trichomonas vaginalis (TV) genital infection. The repository samples provide a unique opportunity to characterize the vaginal microbiome prior to acquisition of STIs in a well-powered study. We will conduct a nested case-control study with incidence density sampling. Cases will be defined as women who had an incident STI and we will analyze the sample at the visit prior to the first STI detection. Controls will be matched on age, ethnicity and visit number to cases and will be women who did not develop a STI by the time of the case visit. Specific aims are to (1) Evaluate the association between vaginal microbiota and incidence of CT, GC, and TV genital infection using 16S rRNA gene analysis; (2) Compare levels of D- and L-lactic acid isomers in cases and controls; (3) Investigate if specific strains of Lactobacillus (or other bacterium) are associated with STI risk using metagenomic analysis; (4) Determine if cervicovaginal immune responses independently predict incident STIs. All four aims will be modeled in the context of comprehensive behavioral data. This study will provide a functional understanding of the vaginal microbiome's role in STI protection and may reveal novel targets to prevent STIs and improve women's health.

Chlamydia trachomatis (CT) is the most commonly reported bacterial sexually transmitted infection in the U.S. and untreated infections are a major cause of adverse sequelae, including pelvic inflammatory disease, infertility, and ectopic pregnancy. Screening programs have failed to curb rising infection rates. As most infections in women are asymptomatic and screening is only recommended annually, the incidence of CT is likely higher than reported. While antibiotic therapy is curative, successful biomedical prevention strategies are lacking. Most CT natural history studies evaluate CT in the short 1-2 week interval between screening and follow-up for treatment. Few studies have had longer follow-up. Overall, these studies suggest spontaneous clearance of CT (in the absence of antibiotic treatment) occurs in 11-44% of cases; however, the mechanisms are poorly understood. The vaginal microbiome (VMB) is a major factor in preventing CT acquisition, and the VMB may also aid in CT clearance by reducing CT proliferation and promoting effective immune responses. Thus, identifying modifiable vaginal microenvironmental features that play a role in spontaneous clearance of CT may lead to novel interventions. This proposal is submitted in response to PA-19-096 “Control of Sexually Transmitted Infections (STIs) Through a Comprehensive Understanding of the Natural History of Infection”. We propose to investigate the relations between spontaneous CT clearance and VMB (structure, function, metabolome), mucosal immunity, and CT serovar-specific features. This proposal will utilize archived cervicovaginal lavage samples collected from the Longitudinal Study of Vaginal Flora in which 3,620 cisgender women were followed quarterly for one year. Samples were retrospectively screened for CT after the study concluded and detected CT spontaneous clearance (n=311) and persistence (n=321) events. The specific aims utilize a repository with a long follow-up and will assess four domains that may drive the natural history of CT: 1) demographic, clinical, and behavioral factors, 2) vaginal microbiome and metabolome, 3) mucosal soluble markers of inflammation, 4) CT serovar composition. Our experienced, multi- disciplinary team will adapt, refine, and apply modern methods in longitudinal epidemiology with machine- learning and dimension-reduction techniques to assess high-dimensional, multi-omic data. We seek to identify immunologic, metabolomic, and bacterial candidates that are associated with spontaneous CT clearance. This epidemiologic study of over 600 archived samples presents the best available resource for identifying likely natural clearance and persistence mechanisms. Findings from the analyses would provide the cost-benefit justification for future confirmatory trials and experimental mechanistic studies. The results may lead to new CT vaccine approaches by pinpointing correlates of protection against clinically-relevant serovars and informing choice of adjuvants for optimal immune response. The data may also aid the design of antibiotic-sparing approaches, such as live biotherapeutic formulations, which could enhance CT control programs.

References

Klasner, C. et al. A Narrative Review on Spontaneous Clearance of Urogenital Chlamydia trachomatis: Host, Microbiome, and Pathogen-Related Factors. Sex. Transm. Dis. 51, 112–117 (2024).

Holm, J. B., Carter, K. A., Ravel, J. & Brotman, R. M. Lactobacillus iners and Genital Health: Molecular Clues to an Enigmatic Vaginal Species. Curr Infect Dis Rep 1–9 (2023) doi:10.1007/s11908-023-00798-5.