Fig 1.
Effects of vaginal bacteria on TFV degradation.
(A)Relative abundance of CVLs from 44 women with and without diagnosed BV ranked in order of increasing TFV degradation rates. The 19 most abundant phyla are shown. Shannon diversity plots showing alpha diversity in CVLs. Blue, BV negative by Nugent score at the time of collection; Red, BV positive by Nugent score at the time of collection. (B) % TFV remaining after 24 hours at 37C in Lactobacillus dominant vs. non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance; Black, cells + TFV control. (C) Rate of TFV degradation (% lost/hour) vs. %Lactobacillus in CVLs after 24-hour incubation. Rate of degradation calculated as % remaining/24 hours.
Fig 2.
Effects of vaginal bacteria on TAF degradation.
(A) Relative abundance of CVLs from 44 women with and without diagnosed BV ranked in order of increasing TAF degradation rates. The 19 most abundant phyla are shown. Shannon diversity plots showing alpha diversity in CVLs. Blue, BV negative by Nugent score at the time of collection; Red, BV positive by Nugent score at the time of collection. (B) % TAF remaining after 24 hours at 37C in Lactobacillus dominant vs. non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance; Black, cells + TFV control. (C) Rate of TAF degradation (% lost/hour) vs. %Lactobacillus in CVLs after 24-hour incubation. Rate of degradation calculated as % remaining/24 hours.
Fig 3.
Effects of vaginal bacteria on DPV degradation.
(A) Relative abundance of CVLs from 44 women with and without diagnosed BV ranked in order of increasing DPV degradation rates. The 19 most abundant phyla are shown. Shannon diversity plots showing alpha diversity in CVLs. Blue, BV negative by Nugent score at the time of collection; Red, BV positive by Nugent score at the time of collection. (B) % DPV remaining after 24 hours at 37C in Lactobacillus dominant vs. non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance; Black, cells + TFV control. (C) Rate of DPV degradation (% lost/hour) vs. %Lactobacillus in CVLs after 24-hour incubation. Rate of degradation calculated as % remaining/24 hours.
Fig 4.
Fraction of drug remaining based on Nugent score diagnosed BV+/- women.
(A) % TFV remaining after 24 hours at 37C in BV diagnosed vs non-BV diagnosed CVLs. Orange, BV negative; Purple, BV positive. Black dots indicate less than 50% Lactobacillus dominance despite BV negative diagnoses. Gray dots indicate greater than 50% Lactobacillus dominance despite BV positive diagnoses. (B) % TAF remaining after 24 hours at 37C in BV diagnosed vs non-BV diagnosed CVLs. Orange, BV negative; Purple, BV positive. Black dots indicate less than 50% Lactobacillus dominance despite BV negative diagnoses. Gray dots indicate greater than 50% Lactobacillus dominance despite BV positive diagnoses. (C) % DPV remaining after 24 hours at 37C in BV diagnosed vs non-BV diagnosed CVLs. Orange, BV negative; Purple, BV positive. Black dots indicate less than 50% Lactobacillus dominance despite BV negative diagnoses. Gray dots indicate greater than 50% Lactobacillus dominance despite BV positive diagnoses.
Fig 5.
Intracellular active metabolite formation after 24 hours grouped by Lactobacillus dominance or non-dominance.
(A) Amount of intracellular TFV-DP formed post-TFV administration after 24 hours at 37C in Lactobacillus dominant versus non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance. (B) Amount of intracellular TFV-DP formed post-TAF administration after 24 hours at 37C in Lactobacillus dominant versus non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance. (C) Amount of intracellular DPV formed post-DPV administration after 24 hours at 37C in Lactobacillus dominant versus non-Lactobacillus dominant CVLs. Orange, greater than 50% dominance; Purple, less than 50% dominance. (D) Rate of TFV-DP formation (amount/hour) vs. %Lactobacillus in CVLs after 24-hour incubation with TFV. Rate of degradation calculated as amount formed/24 hours. (E) Rate of TFV-DP formation (amount/hour) vs. %Lactobacillus in CVLs after 24-hour incubation with TAF. Rate of degradation calculated as amount formed/24 hours. (F) Rate of DPV formation (amount/hour) vs. %Lactobacillus in CVLs after 24-hour incubation with DPV. Rate of degradation calculated as amount formed/24 hours.
Fig 6.
Uptake efficiency of Jurkat cells in converting drug into active metabolite after 1 hour.
Uptake efficiency is defined as the amount of intracellular drug divided by the amount of drug added extracellularly. (A) Uptake efficiency of Jurkat cells in converting TFV into TFV-DP based on 1) TFV + cells alone (black) 2) TFV + cells + G. vaginalis (purple) 3) TFV + cells + L. iners (orange) 4) TFV + cells + L. crispatus (green) 5) TFV + cells + L. iners + G. vaginalis (red). Bacteria were added 15 minutes prior to incubation at 37C and added at a ratio of bacteria to cells of 2.5:1. Co-culture experiments (4) were added at a 1:1 ratio of L. iners: G. vaginalis and an overall ratio of total bacteria to cells at 2.5:1. N = 12. (B) Uptake efficiency of Jurkat cells in converting TAF into TFV-DP based on 1) TAF + cells alone (black) 2) TAF + cells + G. vaginalis (purple) 3) TAF + cells + L. iners (orange) 4) TFV + cells + L. crispatus (green) 5) TFV + cells + L. iners + G. vaginalis (red). Bacteria were added 15 minutes prior to incubation at 37C and added at a ratio of bacteria to cells of 2.5:1. Co-culture experiments (4) were added at a 1:1 ratio of L. iners: G. vaginalis and an overall ratio of total bacteria to cells at 2.5:1. N = 12. (C) Uptake efficiency of Jurkat cells in accumulating intracellular DPV based on 1) DPV + cells alone (black) 2) DPV + cells + G. vaginalis (purple) 3) DPV + cells + L. crispatus (orange) 4) TFV + cells + L. crispatus (green) 5) TFV + cells + L. iners + G. vaginalis (red). Bacteria were added 15 minutes prior to incubation at 37C and added at a ratio of bacteria to cells of 2.5:1. Co-culture experiments (4) were added at a 1:1 ratio of L. crispatus: G. vaginalis and an overall ratio of total bacteria to cells at 2.5:1. N = 12.
Fig 7.
HIV-1LAI infection of CEM-GFP cells after incubation at 37C.
(A). % Infection of CEM-GFP cells over time up to 72 hours post-incubation with 1) CEM-GFP cells + HIV-1LAI 2) CEM-GFP cells + HIV-1LAI + TFV 3) CEM-GFP cells + HIV-1LAI + TFV + L. crispatus 4) CEM-GFP cells + HIV-1LAI + TFV + G. vaginalis. CEM-GFP cells were incubated for 30 minutes with bacterial inoculum. TFV was added and allowed to incubate for 1.5 hours prior to HIV-1LAI addition. Purple G. vaginalis; Orange, L. crispatus; Black, virus alone; Gray, virus and TFV. N = 6. (B) % Infection of CEM-GFP cells over time up to 72 hours post-incubation with 1) CEM-GFP cells + HIV-1LAI 2) CEM-GFP cells + HIV-1LAI + TAF 3) CEM-GFP cells + HIV-1LAI + TAF + L. crispatus 4) CEM-GFP cells + HIV-1LAI + TAF + G. vaginalis. CEM-GFP cells were incubated for 30 minutes with bacterial inoculum. TAF was added and allowed to incubate for 1.5 hours prior to HIV-1LAI addition. Purple G. vaginalis; Orange, L. crispatus; Black, virus alone; Gray, virus and TAF. N = 6. (C) % Infection of CEM-GFP cells over time up to 72 hours post-incubation with 1) CEM-GFP cells + HIV-1LAI 2) CEM-GFP cells + HIV-1LAI + DPV 3) CEM-GFP cells + HIV-1LAI + DPV + L. crispatus 4) CEM-GFP cells + HIV-1LAI + DPV + G. vaginalis. CEM-GFP cells were incubated for 30 minutes with bacterial inoculum. DPV was added and allowed to incubate for 1.5 hours prior to HIV-1LAI addition. Purple G. vaginalis; Orange, L. crispatus; Black, virus alone; Gray, virus and DPV. N = 6. (D) Rate of TFV degradation versus % infection of CEM-GFP cells 48 hours after incubation with virus. Purple G. vaginalis; Orange, L. crispatus; Black, TFV alone. N = 6. (E) Rate of TAF degradation versus % infection of CEM-GFP cells 48 hours after incubation with virus. Purple G. vaginalis; Orange, L. crispatus; Black, TAF alone. N = 6. (F) Rate of DPV degradation versus % infection of CEM-GFP cells 48 hours after incubation with virus. Purple G. vaginalis; Orange, L. crispatus; Black, DPV alone. N = 6.
Fig 8.
Degradation kinetics over time.
(A) Rate of TFV degradation over time up to 72 hours. Purple G. vaginalis; Orange, L. crispatus; Black, TFV alone. N = 6. (B) Rate of TAF degradation over time up to 72 hours. Purple G. vaginalis; Orange, L. crispatus; Black, TAF alone. N = 6. (C) Rate of DPV degradation over time up to 72 hours. Purple G. vaginalis; Orange, L. crispatus; Black, TAF alone. N = 6.
Fig 9.
TFV remaining after Incubation with endocytosis inhibitor, DMA.
(A) TFV remaining after 24 hours based on 1) TFV alone (black) 2) TFV + DMA (red) 3) TFV + Jurkat (gray) 4) TFV + Jurkat + DMA (dark orange) 5) TFV + G. vaginalis (purple) 6) TFV + G. vaginalis + DMA (dark green) 7) TFV + Jurkat + G. vaginalis (dark purple) 8) TFV + Jurkat + G. vaginalis + DMA (dark blue). Jurkat cells were exposed to 100uM DMA for 30 min prior to treatment with TFV (1mg/mL) and G. vaginalis. Co-culture experiments were added at a 2.5:1 ratio of bacteria to cells. N = 5. (B) TFV remaining after 24 hours based on 1) TFV alone (black) 2) TFV + DMA (red) 3) TFV + Jurkat (gray) 4) TFV + Jurkat + DMA (dark orange) 5) TFV + L. iners (orange) 6) TFV + L. iners + DMA (dark green) 7) TFV + Jurkat + L. iners (yellow) 8) TFV + Jurkat + L. iners + DMA (teal). Jurkat cells were exposed to 100uM DMA for 30 min prior to treatment with TFV (1mg/mL) and L. iners. Co-culture experiments were added at a 2.5:1 ratio of bacteria to cells. N = 5.
Fig 10.
Validation of ODE model of ART drug kinetics.
Comparison of experimental data (blue) to model-predicted uptake efficiency (orange) of Jurkat cells in converting. (A) TFV to TFV-DP based; (B) TAF to TFV-DP; and (C) DPV based on 1) TFV + cells alone 2) TFV + cells + G. vaginalis 3) TFV + cells + L. iners 4) TFV + cells + L. iners + G. vaginalis (ns denotes p > 0.05). Uptake efficiency is defined as the amount of intracellular drug divided by the amount of drug added extracellularly.
Fig 11.
Time after MNZ treatment magnifies the effect of G. vaginalis on active drug formation.
Simulations of active drug formation after 1hr, 2hr and 4hr post-treatment with MNZ at varying relative amounts of G. vaginalis to L. iners (GV:LI ratio) from 0.01-fold less G. vaginalis to L. iners to 100-fold more G. vaginalis to L. iners for (A) TFV; (B) TAF; and (C) DPV. The percent change from 0.01-fold GV:LB to 100-fold GV:LB active drug concentrations is listed in the legend.