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.

Figure 1. Refined vaginal epithelial multilayer co-cultures that support HIV-1 infection.

Optimized conditions for establishment of co-cultures that contain HIV-1 susceptible cells are illustrated in panels A–J. Panels A–G represent outcomes with VEC/TZM-bl co-cultures as indicated by H&E staining of 6 um sections (A) or immunolabeling of a section showing the presence of CD4+ TZM-bl cells (B, arrows indicate positive cells) in the context of VEC labeled with cytokeratin and DAPI (C). TZM-bl cells provided a b-gal reporter system to detect HIV-1 infection via staining with X-gal (blue precipitate) as shown in panels D–G. Panel D shows X-gal localization of HIV-1 infection (blue box) of an uncolonized culture while E–G illustrate cultures apically colonized with clinical VBM (CSTI, CSTIII and CSTIVB, respectively). Co-cultures with primary human MDM were established as illustrated by H&E staining (H), CD4 immunolabeling ((I); green cells indicated by arrows are MDM) in the context of cytokeratin and DAPI labeled VEC (J). HIV-1_SX infection was evaluated over time in triplicate multilayer VEC/TZM-bl (K) or VEC/MDM co-cultures (L). The transwell culture system provided sampling of each chamber (apical in blue, cell fraction in black and basal chamber in red) representing different aspects of the viral infection process.

Figure 2. VMB colonization of VEC/TZM-bl multilayer co-cultures recreated the complex bacterial communities present in clinical samples.

To confirm that the environment created by VEC multilayer cultures could support colonization by in tact VMB, established cultures in 96 well format (N≥6/time point) were inoculated apically with ∼1,000 bacterial genomes and followed kinetically by qPCR (A and B). A representative VMB of CST III (Nugent score, NS 2) and a CST IVB (NS8) types are depicted to illustrate that overall bacterial load (blue line) increased over the 64 h evaluation with a coincident increase in host genomes (black line) indicating culture health. In both graphs, green lines represent bacteria associated with healthy vaginal status and red lines are considered pathogenic. Quantified data for the bacteria targeted by the qPCR from these and other representative VMB are presented in Table 1. The lower panels depict SEM evaluations of a set of parallel cultures (2/time point) colonized by a CST IVB VMB with community diversity. Colonized cultures were fixed at the indicated times and visualized to illustrate the increasing number and diversity of bacterial morphotypes as well as the production of extracellular polymeric substances coating and connecting individual bacteria (lower right panel).

Figure 3. HIV-1 burden was significantly altered by VMB colonization of VEC multilayer co-cultures.

To directly study the impact of representative VMB upon HIV-1 infection and replication VEC/TZM-bl (A) or VEC/MDM (B) co-culture multilayers were established in 96 well format. Matured cultures were colonized and virally challenged as described in the methods. Representative healthy (dark green bars) or pathogenic (blue bars) VMB, single species bacteria or the probiotic Lactobacilli mixture (light green bars) are indicated on the x-axes. Mean genomic HIV-1 titer is shown on the y axes. Statistical comparisons were completed by one-way ANOVA with Bonferroni's or Dunnett's multiple comparison correction. A “*” indicates a significant difference (P<0.05) relative to the uncolonized viral burden. A “#” indicates a significant difference (P<0.05) with the suppressive CST III VMB. Triplicate cultures were created and the study was replicated twice. Poly-I:C (0.1 mg/ml) was utilized as an inducer of HIV-1 infection and replication and was applied at the time of colonization.

Figure 4. TFV efficacy was altered by representative VMB colonization and impacted the composition of VMB.

Panel A depicts the anti-HIV-1 efficacy of TFV in VEC/TZM-bl cultures (N = 3) colonized as indicated with representative VMB. The white bars indicate HIV-1 titers in untreated parallel cultures and confirm the previous results (Fig. 3). The presence of TFV (blue bars) consistently reduced the numbers of viral genomes but, as shown in the red bars, a non-significant trend indicated that the level of reduction was VMB-dependent. Panels B and C, respectively, depict the most suppressive and most enhancing VMB community profiles in the absence (white bars) or presence (blue bars) of TFV (N = 2 cultures/condition). The fold change in detected bacterial genomes associated with TFV application is shown for each of the selected organisms as a loss (red bar on left of the vertical axis) or gain (right of axis) in number relative to the untreated cultures.