Cultivated Vaginal Microbiomes Alter HIV-1 Infection and Antiretroviral Efficacy in Colonized Epithelial Multilayer Cultures

Abstract

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents

“…Recent studies employing cultivation-independent molecular approaches showed that the vaginal microbiome (VMB) is much more complex than previously appreciated and that conventional culture techniques are limited, allowing the detection of only a small fraction of the microorganisms in the vaginal flora (29,30). Therefore, detailed studies on the effects of QAS on the VMB, employing genetically controlled ex vivo models in combination with cultivation-independent methods (31,32), should be performed in the future. This kind of approach will allow a more detailed analysis of the effects of QAS not only on healthy VMB but also on dysbiotic VMB collected from patients with bacterial vaginosis (32), the most common cause of vaginal infection in women of childbearing age, a main factor of increased susceptibility to infection by HIV and other STI (29).…”

“…Therefore, detailed studies on the effects of QAS on the VMB, employing genetically controlled ex vivo models in combination with cultivation-independent methods (31,32), should be performed in the future. This kind of approach will allow a more detailed analysis of the effects of QAS not only on healthy VMB but also on dysbiotic VMB collected from patients with bacterial vaginosis (32), the most common cause of vaginal infection in women of childbearing age, a main factor of increased susceptibility to infection by HIV and other STI (29). QAS, being neutral salts, will not alter vaginal pH directly, but indirect effects through alterations of the VMB cannot be excluded at this stage.…”

Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions

“…DNA or RNA was extracted on an automated MagNA Pure 96 (Roche). VMBs were characterized by customized qPCR array as described previously [17]. Any VMB samples molecularly positive for STI, yeast or Y chromosome were excluded from the study.…”

“…Immortalized human VEC (V19I and BVEC02I) were cultured as described [20] and used to produce stratified squamous vaginal multilayers [17, 18]. Briefly, VEC from one of 2 donors were plated in 24 (10 6 cells/transwell) or 96 (10 5 cells/transwell) well transwell format and cultured at 37°C with 5% CO 2 .…”

“…To test the hypothesis that increased CO 2 may be a predisposing factor for creating dysbiotic VMBs dominated by anaerobic bacteria, we utilized our ex vivo human vaginal mucosal culture system [17, 18]. The air-interfaced, apical surface of the VEC cultures support colonization with intact VMBs transplanted from samples collected from woman during routine gynecological exams.…”