Elicit: TAF Activation and Tenofovir Distribution in HIV-1 Cells

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TAF Activation and Tenofovir Distribution in HIV-1 Cells

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May 5, 2026

Tenofovir alafenamide (TAF) intracellular activation and tenofovir distribution to HIV-1 infected cells

TAF is activated intracellularly by cathepsin A to produce tenofovir diphosphate at 4- to 25-fold higher concentrations than TDF in peripheral blood mononuclear cells, with preferential distribution to lymphatic tissues and HIV-susceptible cells including CD4+ T cells, epithelial cells, and fibroblasts.

Abstract

Tenofovir alafenamide undergoes intracellular activation primarily through the lysosomal protease cathepsin A, which cleaves TAF to form intermediates that ultimately convert to the active metabolite tenofovir diphosphate (TFV-DP). Liver carboxylesterase 1 provides an alternative hepatic activation pathway. This intracellular activation mechanism enables TAF to achieve 4- to 25-fold higher TFV-DP concentrations in peripheral blood mononuclear cells compared to tenofovir disoproxil fumarate (TDF), while reducing systemic tenofovir exposure by 86-97%. TAF demonstrates preferential distribution to lymphatic tissues and accumulates in epithelial cells and fibroblasts of the female reproductive tract at 10- to 100-fold higher concentrations than in CD4+ T cells, with these cells releasing antiretroviral activity over multiple days to protect CD4+ T cells.

The enhanced intracellular TFV-DP loading translates to superior antiviral activity, with TAF demonstrating >600-fold greater potency than parent tenofovir in CD4+ T cells and a 3.5-fold higher resistance barrier than TDF. In viral breakthrough assays at physiologically relevant concentrations, TAF inhibited 40 of 42 K65R-containing isolates while TDF inhibited only 32 of 42, and TAF prevented breakthrough in 65 of 68 isolates with thymidine analog-associated mutations compared to 53 of 68 for TDF. The covalent hepatitis C protease inhibitors telaprevir and boceprevir potently inhibit cathepsin A-mediated TAF activation (IC50 0.16-0.27 μM), reducing anti-HIV activity 3- to 21-fold, while medroxyprogesterone acetate differentially suppresses TAF activity in genital versus blood CD4+ T cells. These findings support TAF’s clinical efficacy at lower doses than TDF with improved safety profiles and activity against resistant HIV-1 strains.

Methods

We analyzed 10 sources from an initial pool of 200, using 9 screening criteria. Each paper was reviewed for 7 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: TAF as Primary Intervention, Intracellular Mechanisms Focus, HIV-1 Infected Cell Models, In Vitro Study Design, Intracellular Concentration Measurements, Mechanistic Data Inclusion, HIV-1 Context Requirement, Intracellular Measurement Focus, Peer-Reviewed Research

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “Tenofovir alafenamide (TAF) intracellular activation and tenofovir distribution to HIV-1 infected cells”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract experimental design and methods used to study TAF intracellular activation or tenofovir distribution, including:

Extract all details about how TAF is activated intracellularly, including:

Extract quantitative data on tenofovir and metabolite levels in cells, including:

Extract comparative data between TAF and TDF (tenofovir disoproxil fumarate) regarding:

Extract information about factors that enhance or inhibit TAF activation or tenofovir distribution, including:

Extract data specific to TAF activity against HIV-1 resistant strains and how resistance affects drug distribution, including:

Extract findings about clinical implications of TAF activation and distribution, including:

Results

Characteristics of Included Studies

The review included 10 studies published between 2013 and 2023 examining TAF intracellular activation and tenofovir distribution to HIV-1 infected cells. Eight studies had full text available, while two were assessed using abstract-only information.

Study

Full text retrieved?

Study type

Primary focus

Cell types studied

Rujuta A. Bam et al., 2014

No

Primary study

CatA levels and intracellular metabolism across diverse donors

Primary CD4+ T-lymphocytes and monocyte-derived macrophages

C. Callebaut et al., 2015

Yes

Primary study

In vitro virology profile of TAF

MT-2 and MT-4 cell lines, primary human PBMCs, CD4+ T lymphocytes

G. Birkuš et al., 2015

Yes

Primary study

Intracellular activation mechanisms and enzyme involvement

HEK293T cells, HeLa cells, primary human CD4+ T lymphocytes

N. Margot et al., 2020

Yes

Primary study

Antiviral activity against TAM-containing HIV-1

MT-2 cells

N. Margot et al., 2015

Yes

Primary study

In vitro resistance characterization

MT-2 cells, PBMCs

N. Margot et al., 2016

No

Primary study

Resistance barrier driven by higher TFV-DP loading

HIV-1 infected cells

S. Cox et al., 2023

Yes

Primary study

Antiviral activity against K65R-containing isolates

Patient-derived HIV-1 isolates, MT-2 cells, HEK293T cells

Zheng Shen et al., 2017

Yes

Primary study

Hormonal contraceptive effects on TAF activity

Primary CD4+ T cells from blood and female reproductive tract tissues

Zheng Shen et al., 2019

Yes

Primary study

TAF accumulation and release from reproductive tract cells

Primary epithelial cells and fibroblasts from female reproductive tract

P. Ruane et al., 2013

Yes

Phase 1b clinical trial

Antiviral activity, safety, and pharmacokinetics of TAF monotherapy

HIV-1-positive adults

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Studies employed diverse experimental approaches including enzyme assays, drug uptake studies, resistance selection experiments, viral breakthrough assays, and clinical pharmacokinetic measurements. Experimental durations ranged from 24-hour incubations to multi-week resistance selection experiments lasting up to 74 days and a 10-day clinical monotherapy trial. Technical methods included HPLC, liquid chromatography with tandem mass spectrometry, luciferase-based viability readouts, and RT-PCR for gene expression analysis.

TAF Activation Mechanisms

Enzymatic Pathways

TAF undergoes intracellular activation primarily through the lysosomal protease cathepsin A (CatA). The activation occurs in the lysosomal compartment, where CatA cleaves the carboxyester bond in TAF to form a metastable metabolite. This metabolite then converts to TFV-Ala, which spontaneously converts to TFV due to the acidic pH in lysosomes. Subsequently, TFV undergoes two sequential phosphorylation reactions to form the active metabolite TFV-DP, involving adenylate kinase for the first phosphorylation step and a kinase with nucleotide diphosphate kinase activity for the second step.

Evidence for CatA’s central role comes from multiple experimental approaches. Overexpression of CatA in HEK293T cells increased intracellular TAF hydrolysis 2-fold, while knockdown of CatA expression using RNA interference in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs.

Alternative Activation Pathways

Liver carboxylesterase 1 (Ces1) represents an alternative pathway for TAF activation. Overexpression of Ces1 in HEK293T cells increased intracellular TAF hydrolysis 5-fold, and Ces1 is specifically important for the hepatic metabolism of TAF. TAF is converted to TFV by either CatA or Ces1 depending on the cell type, followed by intracellular phosphorylation to TFV-DP. This unique activation mechanism, distinct from TDF, contributes to TAF’s enhanced antiviral activity.

Intracellular Drug Concentrations and Distribution

Comparative TFV-DP Levels

TAF achieves substantially higher intracellular concentrations of the active metabolite TFV-DP compared to its predecessor TDF. The fold increases in TFV-DP concentrations varied across studies and doses:

Study

Cell type

Fold increase vs TDF

TAF dose

Time point

C. Callebaut et al., 2015

PBMCs

5-fold

Not specified

Not specified

N. Margot et al., 2020

PBMCs

4-fold

Not specified

Not specified

N. Margot et al., 2015

PBMCs

5- to 7-fold

Phase 1 study

Not specified

S. Cox et al., 2023

PBMCs

4-fold

4× TDF equivalent

Not specified

P. Ruane et al., 2013

PBMCs

7-fold (25 mg)
25-fold (40 mg)

25 mg and 40 mg

Day 21

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In blood and female reproductive tract (FRT) CD4+ T cells, intracellular TFV-DP concentrations were approximately 1-2 × 10^4 fmol/million cells after 24 hours of incubation with TFV or TAF.

Tissue-Specific Distribution

TAF demonstrates preferential distribution to lymphatic tissues and HIV-susceptible cells. In the female reproductive tract, epithelial cells and fibroblasts accumulate TFV-DP at substantially higher levels than CD4+ T cells, with epithelial cells showing 100-fold higher concentrations and fibroblasts showing 10-fold higher concentrations. Endometrial, endocervical, and ectocervical polarized epithelial cells pre-loaded with TFV or TAF released antiretroviral activity into their basolateral secretions 1, 2, and 3 days post-loading, providing sustained protection to CD4+ T cells. When TAF was added apically to epithelial cells, it was released basolaterally through Multidrug Resistant Protein transporters, taken up by fibroblasts, and released into secretions to partially protect CD4+ T cells.

Donor Variability

Levels of CatA and intracellular TAF metabolites differed minimally in CD4+ T cells and monocyte-derived macrophages among 13 demographically diverse donors tested. TAF’s relative range of antiviral activity across all tested donors was comparable to that of other HIV-1 reverse transcriptase inhibitors, with mean ±SD (range) EC50 values of 11.0 ±3.4 (6.6–19.9) nM in CD4+ T cells and 9.7 ±4.6 (2.5–15.7) nM in monocyte-derived macrophages.

Comparison with Tenofovir Disoproxil Fumarate

Potency and Activation Efficiency

TAF demonstrated markedly superior potency compared to parent TFV and the alternative prodrug TDF across multiple dimensions:

Parameter

TAF advantage

Source

Potency vs TFV in CD4+ T cells

600-fold more potent

Rujuta A. Bam et al., 2014

Potency vs TFV in macrophages

80-fold more potent

Rujuta A. Bam et al., 2014

Effective concentration

~300-fold lower than TFV for similar protection

Zheng Shen et al., 2017; 2019

Enhanced in vitro antiviral activity

600-fold vs TFV

G. Birkuš et al., 2015

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This enhanced potency stems from TAF’s more efficient intracellular conversion to TFV-DP, allowing for lower dosing requirements. In the phase 1b monotherapy study, TAF at 25 mg demonstrated greater antiviral effect at less than 1/10th the dose of TDF.

Mechanistic Differences

TAF and TDF employ fundamentally different activation pathways. TAF is converted intracellularly by CatA or Ces1, whereas TDF is quickly metabolized to TFV by gut and serum esterases. This distinction confers TAF with superior plasma stability. While TAF maintained activity in the presence of human serum, TDF activity was significantly reduced, demonstrating TAF’s improved plasma stability over TDF.

Pharmacokinetic Profiles

The intracellular versus systemic exposure profiles strongly favor TAF for safety and efficacy:

Study

TAF dose

Plasma TFV reduction

Intracellular TFV-DP increase

G. Birkuš et al., 2015

Not specified

Lower systemic levels

Higher concentrations in PBMCs

N. Margot et al., 2015

25 mg

86% reduction

5- to 7-fold increase

P. Ruane et al., 2013

8 mg

97% lower

Not specified

P. Ruane et al., 2013

25 mg

86% lower

7-fold increase

P. Ruane et al., 2013

40 mg

79% lower

25-fold increase

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This pharmacokinetic profile translates to clinical benefits, with TAF showing improved bone and kidney safety compared to TDF while achieving higher intracellular TFV-DP concentrations in PBMCs. Phase III data demonstrated that TAF was noninferior to TDF in virological suppression with more favorable effects on renal and bone parameters.

Activity Against Resistant HIV-1 Strains

Resistance Mutation Profiles

TAF activity was evaluated against multiple resistance mutations, with K65R and thymidine analog-associated mutations (TAMs: M41L, D67N, K70R, L210W, T215Y, K219Q) receiving particular attention.

Mutation type

TAF susceptibility change

Study

K65R alone

6.5-fold reduced

N. Margot et al., 2015

K65R alone

2.7- to 3.0-fold reduced

S. Cox et al., 2023

K65R + other RT mutations

1.2- to 27.6-fold reduced

S. Cox et al., 2023

Multiple TAMs

Variable reduction

N. Margot et al., 2020

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The presence of the M184V mutation increased TAF sensitivity compared to viruses without M184V. In TAM-containing HIV-1 site-directed mutants, the presence of M184V significantly increased TAF sensitivity compared to mutants without M184V. This hypersusceptibility associated with M184V contributes to TAF’s higher resistance threshold.

Resistance Barrier Comparison

TAF demonstrated a substantially higher resistance barrier than TDF across multiple experimental systems. In viral breakthrough assays mimicking physiologically relevant concentrations:

Study

TAF breakthrough

TDF breakthrough

Isolates tested

N. Margot et al., 2020

3/68 mutants

15/68 mutants

Site-directed and patient-derived mutants with TAMs

S. Cox et al., 2023

2/42 isolates (5%)

10/42 isolates (24%)

K65R-containing clinical isolates

Overall (S. Cox et al., 2023)

40/42 inhibited

32/42 inhibited

K65R-containing clinical isolates

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The resistance threshold for TAF was estimated to be 3.5-fold higher than for TDF, with TAF capable of inhibiting viruses with up to four TAMs, and up to six TAMs when M184V is present. This enhanced resistance barrier stems directly from the higher intracellular TFV-DP levels achieved by TAF, supporting higher resistance cutoffs for TAF compared to TDF in genotypic and phenotypic resistance algorithms.

Factors Affecting TAF Activation and Distribution

Drug Interactions

The covalent hepatitis C virus protease inhibitors telaprevir and boceprevir potently inhibited CatA-mediated TAF activation, with IC50 values of 0.27 μM and 0.16 μM respectively. At pharmacologically relevant concentrations, these drugs reduced anti-HIV activity of TAF in primary CD4+ T lymphocytes by 21-fold and 3-fold respectively. Clinical recommendations indicate avoiding coadministration of telaprevir or boceprevir with TAF in HIV/HCV-coinfected individuals. In contrast, there was no inhibition of CatA or significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV protease inhibitors, or various prescribed inhibitors of host serine proteases.

TAF demonstrated additive to synergistic interactions when combined with antiretroviral drugs from different classes including NRTIs, NNRTIs, integrase strand transfer inhibitors, and protease inhibitors.

Hormonal Contraceptive Effects

Medroxyprogesterone acetate (MPA) exhibited differential effects on TAF and TFV activity depending on cell type. In blood CD4+ T cells, MPA suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP concentrations. Conversely, in genital CD4+ T cells, MPA suppressed TAF inhibition of HIV infection and lowered TFV-DP concentrations without affecting TFV protection. These findings suggest MPA may decrease antiretroviral protection in individuals using these drugs intermittently for prevention. Other progestins including levonorgestrel, norethisterone, and progesterone did not affect TFV or TAF activity.

Cellular and Environmental Factors

Alterations in the vaginal microbiome, particularly the presence of anaerobic bacteria, can decrease antiretroviral efficacy. Inflammation from vaginal gels or sexually-transmitted infections undermines TFV effectiveness by recruiting target cells to the mucosal surface. These environmental factors may modulate TAF distribution and efficacy in the female reproductive tract.

Host factors including cathepsin A expression levels play a critical role in TAF activation. Ces1 expression is particularly important for hepatic TAF metabolism. The consistent intracellular metabolism and antiretroviral potency of TAF across multiple donors of variable gender, age, and ethnicity supports its clinical applicability.

Clinical Implications

Dosing and Efficacy

The phase 1b monotherapy study established a dose-response relationship for viral load decrease up to 25 mg TAF. At steady state, 8, 25, and 40 mg TAF yielded mean TFV plasma exposures 97%, 86%, and 79% lower respectively compared to 300 mg TDF. For the 25 and 40 mg doses, mean intracellular PBMC TFV-DP area under the curve was approximately 7-fold and 25-fold higher relative to 300 mg TDF. Significant reductions in plasma HIV-1 RNA from baseline to day 11 were observed for all TAF dose groups compared with placebo (P < 0.01), with median decreases of 1.08–1.73 log10 copies per milliliter.

TAF demonstrates potent antiviral activity at lower doses than TDF, which may translate into greater antiviral efficacy, a higher barrier to resistance, and an improved safety profile. The higher intracellular TFV-DP exposures may offer clinical benefits including improved resistance profiles and treatment durability.

Safety Considerations

The reduced systemic TFV exposure with TAF compared to TDF has important safety implications. Lower plasma TFV concentrations may reduce known TFV-associated nephrotoxicity by decreasing concentrations in renal proximal tubule cells, which express the organic anion transporters OAT1 and OAT3. Clinical dosing with TAF showed improved bone and kidney safety compared to TDF.

Monitoring and Resistance Testing

TAF concentrations in cervical vaginal lavages may not accurately reflect tissue concentrations or protection, suggesting that cervical vaginal lavages should not be used to monitor adherence or efficacy. The mucosal environment, including factors like local inflammation, contraceptive use, microbiome composition, and sexually-transmitted infections, should be considered when evaluating TAF efficacy.

Higher resistance cutoffs should be applied for TAF compared to TDF in genotypic and phenotypic resistance algorithms. TAF may be more effective in treating HIV-1 infections with resistance mutations, particularly those with TAMs and M184V. Clinical studies demonstrated TAF’s efficacy in maintaining viral suppression in patients with NRTI resistance.

Knowledge Gaps and Future Directions

Clinical validation of TAF’s higher resistance threshold remains limited due to ethical concerns regarding resistance induction studies. Further research is needed on TAF distribution and activation in genital tissues, particularly under conditions of inflammation and sexually-transmitted infections. Long-term durability and safety profiles require additional study in larger and longer-term clinical trials. Understanding the mechanisms of antiretroviral distribution and efficacy in different clinical scenarios, especially in the context of inflammatory conditions, represents an important area for future investigation.

References

Rujuta A. Bam, G. Birkuš, D. Babusis, T. Cihlář, S. Yant\ (2014).Metabolism and Antiretroviral Activity of Tenofovir Alafenamide in CD4+ T-Cells and Macrophages from Demographically Diverse Donors. Antiviral Therapy

C. Callebaut, G. Stepan, Ya-Wen Tian, Michael D. Miller\ (2015).In Vitro Virology Profile of Tenofovir Alafenamide, a Novel Oral Prodrug of Tenofovir with Improved Antiviral Activity Compared to That of Tenofovir Disoproxil Fumarate. Antimicrobial Agents and Chemotherapy

G. Birkuš, Rujuta A. Bam, Madeleine Willkom, C. Frey, Luong K. Tsai, and 3 more\ (2015).Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors. Antimicrobial Agents and Chemotherapy

P. Ruane, E. Dejesus, D. Berger, M. Markowitz, Lijie Zhong, and 2 more\ (2013).Antiviral Activity, Safety, and Pharmacokinetics/Pharmacodynamics of Tenofovir Alafenamide as 10-Day Monotherapy in HIV-1–Positive Adults. Journal of Acquired Immune Deficiency Syndromes

N. Margot, Renee R. Ram, M. Abram, R. Haubrich, C. Callebaut\ (2020).Antiviral Activity of Tenofovir Alafenamide against HIV-1 with Thymidine Analog-Associated Mutations and M184V. Antimicrobial Agents and Chemotherapy

N. Margot, Audun Johnson, Michael D. Miller, C. Callebaut\ (2015).Characterization of HIV-1 Resistance to Tenofovir Alafenamide In Vitro. Antimicrobial Agents and Chemotherapy

N. Margot, Yang Liu, Michael D. Miller, C. Callebaut\ (2016).High resistance barrier to tenofovir alafenamide is driven by higher loading of tenofovir diphosphate into target cells compared to tenofovir disoproxil fumarate. Antiviral Research

S. Cox, N. Margot, C. Callebaut\ (2023).Antiviral activity of tenofovir alafenamide (TAF) against HIV‐1 clinical isolates harboring K65R. Journal of Medical Virology

Zheng Shen, M. Rodríguez-García, Mickey V Patel, J. Bodwell, C. Wira\ (2019).Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells. Scientific Reports

Zheng Shen, M. Rodriguez-Garcia, Mickey V Patel, J. Bodwell, A. Kashuba, and 1 more\ (2017).Hormonal Contraceptives Differentially Suppress TFV and TAF Inhibition of HIV Infection and TFV-DP in Blood and Genital Tract CD4+ T cells. Scientific Reports

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Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells

Zheng Shen, M. Rodríguez-García, Mickey V Patel, J. Bodwell, C. Wira

Scientific Reports·

2019·

10 citations

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Study Methods

- Cell types used: Primary epithelial cells and fibroblasts from the human female reproductive tract. - Experimental approach: Pre-loading cells with TFV or TAF, testing secretions for HIV inhibition, and using LC-MS/MS for drug concentration measurement. - Duration of experiments: 24-hour incubations with TFV or TAF. - Technical methods: LC-MS/MS for intracellular TFV-DP measurement, RT-PCR for gene expression analysis. - Special conditions: Use of polarized epithelial cells, application of MRP-specific inhibitor MK571.

TAF Activation Mechanisms

- Specific enzymes involved: Cathepsin A - Cellular location of activation: Intracellular (specific location not detailed) - Metabolic pathway steps: TAF → TFV (via Cathepsin A) → TFV-DP (via two sequential phosphorylation reactions) - Evidence for enzyme involvement: Not mentioned - Kinetic parameters: Not mentioned - Alternative or secondary activation pathways: Not mentioned

Intracellular Drug Concentrations

- Concentrations of TFV-DP: ~340,000 fmol/million cells for 50% inhibition - Cell types: Epithelial cells, fibroblasts, CD4+ T cells - Time points of measurement: 24 hours - Units of measurement: fmol/million cells - Fold differences: Epithelial cells (100-fold), fibroblasts (10-fold) higher than CD4+ T cells - Differences between HIV-1 infected vs. uninfected cells: Not mentioned

TAF vs TDF Comparison

- Relative intracellular TFV-DP concentrations achieved: TAF achieves similar protection at ~300-fold lower concentrations than TFV. - Differences in activation mechanisms or pathways: TAF is converted to TFV via Cathepsin A, while TFV requires two phosphorylation steps. - Relative potency or antiviral activity: TAF is more potent, requiring lower concentrations for similar antiviral activity. - Systemic vs. intracellular drug exposure profiles: Not explicitly detailed, but TAF is administered at lower doses due to efficient intracellular conversion. - Advantages or disadvantages of TAF over TDF for drug distribution: TAF's higher potency and lower dosing requirements are advantages. - Fold differences in key parameters between the two prodrugs: TAF is ~300-fold more efficient in achieving necessary intracellular concentrations.

Factors Affecting Activation

- Host factors: Cathepsin A for TAF conversion to TFV - Drug interactions: Medroxyprogesterone acetate (MPA) inhibits conversion of TFV and TAF to TFV-DP - Cellular conditions: Alterations in vaginal microbiome (e.g., anaerobic bacteria) decrease ARV efficacy; inflammation from vaginal gels or STIs undermines TFV effectiveness - No specific quantitative effects (IC50, fold change in activity) or detailed clinical relevance provided

HIV-1 Resistance Context

Not mentioned (the paper does not discuss specific resistance mutations, TAF susceptibility in resistant vs. wild-type HIV-1, overcoming resistance with higher TFV-DP levels, breakthrough concentrations, or resistance barrier comparisons between TAF and TDF)

Clinical Relevance

- Physiologically relevant drug concentrations used: TAF achieves similar protection at concentrations ~300 fold lower than TFV. - Extrapolation to clinical dosing scenarios: TAF concentrations in CVL may not accurately reflect tissue concentrations or protection. - Implications for drug safety or efficacy: Factors like local inflammation, contraceptives, microbiome composition, and STIs modulate ARV efficacy. - Recommendations for clinical use based on mechanistic findings: CVL should not be used to monitor adherence or efficacy; consider mucosal environment factors. - Identified knowledge gaps or future research needs: Understanding ARV distribution and efficacy in inflammation and STIs. - Clinical trial data or real-world evidence referenced: CAPRISA 004 trial mentioned; implications for clinical trials discussed.

Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

a key early step in the HIV lifecycle. TFV and TAF, differ in their ability to enter cells. TFV with its inherent negative charge is poorly taken up by cells and is dependent on limited diffusion as well as energy dependent transporters [18] [19] [20] [21] . TAF, due to its neutral charge, readily diffuses into the cell, although transporters may also be involved in cell entry 22 . Thus TAF achieves similar protection against HIV infection at concentrations ~300 fold lower than TFV 7 . Intracellular TAF is readily converted to TFV via the actions of Cathepsin A. Once in the cell, TFV is converted into TFV-diphosphate (TFV-DP) through two sequential phosphorylation reactions 23 . It is TFV-DP, the active metabolite of TFV and TAF, which interferes with viral replication.

Previous studies by us evaluated the intracellular concentrations of TFV-DP (the active form of TFV) in purified immune and non-immune cells from the upper and lower human FRT 24 . We found that concentrations of TFV-DP were 100-fold higher in epithelial cells and 10-fold higher in fibroblasts when compared to CD4+ T cells and macrophages. In other studies, the distribution of TFV-DP was analyzed using combined confocal Raman spectroscopy (CRS) and optical coherence tomography (OCT) to measure the distribution of TFV in intact porcine vaginal tissues 25, 26 . Measured with sub-100-micron spatial resolution, the concentration of TFV following topical application was greatest in the epithelium and rapidly diminished deeper in the stroma. Taken together, these findings indicate a cell-specific distribution of TFV-DP in the reproductive tract and demonstrate that tissue biopsy concentrations may not reflect the physiologically-relevant concentrations of an ARV needed to prevent the sexual transmission of HIV. The recognition that ARVs are not uniformly distributed between cells in the reproductive tract emphasizes the need to understand the role of the tissue environment in modulating protection and susceptibility to HIV infection.

When TFV is applied topically into the vagina, it first enters epithelial cells at the apical (luminal) surface after which it is released basolaterally into the subepithelial (stromal) compartment, possibly through several mechanisms, including passive diffusion and/ or transporters, such as breast cancer resistant protein (BCRP) and P-glycoprotein (P-gp) or Multidrug Resistant Proteins (MRP) [27] [28] [29] . In contrast, following oral administration, TFV reaches the subepithelial stroma of the reproductive tract through the blood stream 30 . Important differences have been described in TFV-DP vaginal levels after topical and oral administration of TFV 31, 32 . Whether epithelial cells and fibroblasts act as a sink for TFV-DP to compromise protection, or release TFV slowly after initial uptake to maintain protection in the genital mucosa is unknown.

Using polarized primary epithelial cells and fibroblasts from the endometrium (EM), endocervix (CX) and ectocervix (ECX), we examined whether TFV-and/or TAF-derived intracellular accumulation of TFV-DP enhances or inhibits TFV-mediated protection against HIV by interfering with availability to CD4+ T cells. When epithelial cells were pre-loaded with TFV or TAF, we found that both ARVs were released basolaterally for at least 3 days at concentrations that provide partial protection of CD4+ T cells from HIV infection, and that ARV release was partially due to Multidrug Resistant Protein (MRP) transporters. These findings demonstrate that epithelial cells and fibroblasts contribute to mucosal protection against HIV throughout the FRT by acting as an ARV reservoir that extends partial protection following ARV administration.

Materials and Methods

ethics statement. All human subject work was carried out with the approval of the Dartmouth College Institutional Review Board. Approval to use tissues was previously obtained from the Committee for the Protection of Human Subjects (CPHS), and with written informed consent obtained from the patient before surgery. All samples were anonymized, and all investigations were conducted according to the principles expressed in the Declaration of Helsinki. source of tissue and blood. Human FRT tissues were obtained immediately following surgery from women who had undergone hysterectomies at Dartmouth-Hitchcock Medical Center (Lebanon, NH). Tissues from the endometrium (EM), endocervix (CX) and ectocervix (ECX) were collected from patients with benign conditions such as fibroids and prolapse (age from 26 to 61 years old). Tissue samples were distal from the sites of pathology and were without pathological lesions as determined by a pathologist. Blood donors were anonymous, no information regarding age or hormonal status was available and only female donors were used in this study.

Preparation of blood CD4+ t cells. Blood from women was obtained from our IRB-approved Blood Donor Program at Dartmouth-Hitchcock Medical Center. CD4+ T cells were purified with the CD4+ T cell isolation kit (Miltenyi Biotech) from frozen peripheral blood mononuclear cells (PBMC) as described before 5, 8 . Purified blood CD4+ T cells were activated in vitro using X-vivo 15 media (Lonza, Walkersville, MD) with Phenol Red plus Phytohemagglutinin (PHA, 2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann-La Roche Inc.) for 24 hr as described previously 5, 8 . Activated CD4+ T cells were plated at a density of 1 × 10 5 cells per well in round-bottom 96-well culture plates (Corning, Corning, NY) in 0.1 ml of Immune cell media consisting of X-vivo 15 Media supplemented with Phenol Red and 10% human AB serum (Valley Biomedical, Winchester, VA) prior to treatment. tissue processing. Tissues were rinsed with HBSS (Hanks balanced salt solution) supplemented with Phenol Red, 100 U/ml penicillin, 100 µg/ml streptomycin (all Thermo Scientific Hyclone, Logan, UT), and 0.35 mg/ml NaCO 3 (Thermo Fisher Scientific, Pittsburgh, PA) and processed as previously described 7, 8, 33, 34 . Tissues were then minced under sterile conditions into 1-2 mm fragments and digested at 37 °C for 1 hr using a mixture containing (final concentrations): 0.05% collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.01% DNase (Worthington Biochemical, Lakewood, NJ) in HBSS (Invitrogen Life Technologies, Grand Island, NY). Type IV collagenase was selected based on preliminary studies to ensure non-cleavage of surface markers 4, 6 . After digestion, cells were dispersed through a 250-µm nylon mesh screen (Small Parts, Miami Lakes, FL), washed, and resuspended in complete media consisting of DMEM/F12 medium without Phenol Red, supplemented with 10 mM HEPES (both GIBCO, Life Technologies, Grand Island, NY), 100 µg/ml primocin (InvivoGen, San Diego, CA), 2 mM L-glutamine, 2.5% heat-inactivated defined fetal Bovine Serum (FBS) (both from Thermo Scientific Hyclone) and 2.5% NuSerum (BD Biosciences, Bedford, MA). Epithelial cell sheets were separated from stromal cells by filtration through a 20-µm mesh filter (Small Parts). Epithelial cell sheets were retained on the filter, while stromal cells passed through.

Isolation and culture of FRT epithelial cells and stromal fibroblasts. Epithelial cell sheets were recovered by rinsing and backwashing the filter with complete medium, centrifuged at 500 g for 5 min and analyzed for cell number and viability as previously described 7, 24, 34 . To establish a cell culture system of polarized human FRT epithelial cells with both apical and basolateral compartments, FRT epithelial cells were cultured in Matrigel matrix (BD Biosciences) coated Falcon cell culture inserts in 24-well companion culture plates (Fisher Scientific). Apical and basolateral compartments contained 300 and 500 µl of complete medium, respectively, which was changed every 2 days. Tight junction formation of epithelial cell monolayers from EM and CX was assessed by periodically measuring transepithelial resistance (TER) using an EVOM electrode and Voltammeter (World Precision Instruments, Sarasota, FL), as described previously 35 . To keep the culture conditions similar, the same procedure was followed for culturing squamous ECX epithelial cells, which do not polarize.

Stromal fibroblasts were isolated as previously described 7, 34, 36 . Briefly, following removal of epithelial sheets, the flow-through containing stromal fibroblasts and immune cells was collected, centrifuged at 500 × g for 10 min, resuspended, cell number and viability determined. Freshly isolated stromal fibroblasts and immune cells were incubated in complete medium in a T75 cell culture flask (Fisher Scientific, Pittsburgh, PA). Media was replaced every 48 hr to remove non-adherent cells. Once cells reached confluence, they were trypsinized and plated at a concentration of 1 × 10 5 cells/well in 24-well culture plates (Fisher Scientific) for at least 48 hr prior to treatment.

tFV and tAF preparation. TFV in powder form was obtained from AIDS Research and Reference Reagent Program (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Tenofovir, catalog number 10199). A stock concentration of TFV 5 mg/ml was prepared by adding 1 ml of PBS to 5 mg of TFV powder, before being diluted in stripped media to the appropriate working concentration 7, 8, 24, 37 . TAF was kindly supplied by Gilead Sciences Inc. (Foster City, CA) and was dissolved in PBS at 10 mM, sterilely filtered (0.2 um) and the concentration checked by absorbance using a molar extinction coefficient of 11690 at 260 nm 7, 8 . Subsequent dilutions of TFV and TAF were made in complete media to the appropriate working concentrations.

Intracellular tFV-Dp measurement. TFV or TAF were added to polarized epithelial cells both apically (lumen) and basolaterally (tissue) compartments for 24 hr. For some experiments, TFV and TAF were added only apically as indicated in the Results. After treatment, cells were washed, harvested and lysed in 300 µl of 70% methanol, and stored immediately at -80 °C prior to TFV-DP evaluation as previously described 8, 24 . Intracellular TFV-DP concentrations were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and normalized values to fmol/million cells based on the number of cells per sample 32 .

Collection of secretions from epithelial cells and fibroblasts.

TFV or TAF were added to polarized epithelial cells both apically (lumen) and basolaterally (tissue) compartments for 24 hr, or only apically, as indicated. The time of measurement of TFV-DP used in this study and in our previous research 7, 8, 24 , was based on the initial clinical trial (CAPRISA 004) which used topical TFV applied in the vagina and reported significant protection against HIV acquisition in women when taken between 24 hr prior to sexual intercourse to 24 hr after sex 15 . Following incubation, cells were washed in media 3 times to remove extracellular TFV or TAF, after which epithelial cell inserts were transferred into new wells and fresh media was added to both the apical and basolateral compartments. Conditioned media (CM) were recovered from apical and basolateral compartments of cell inserts after 24 hr. Similarly, TFV or TAF was added to stromal fibroblasts grown to confluence in 24-well plates for 24 hr, after which they were washed repeatedly prior to the addition of fresh media to collect CM at 24 hr. For epithelial cell time course experiments, media in each compartment was replaced at 24 hr intervals (1, 2, and 3 days), CM was collected at each time point. To evaluate the interaction between epithelial cells and fibroblasts, polarized epithelial cell inserts were transferred to 24-well plates containing confluent subject-paired fibroblast cultures. TFV or TAF were added to polarized epithelial cells apically for 24 hr, after which basolateral CM was collected. At the end of this incubation, cell inserts and incubation chambers +/fibroblasts were washed thoroughly both apically and basolaterally so that both epithelial cells and fibroblasts were free of any extracellular TFV or TAF. Cells were then divided in to 2 separate groups consisting of EC inserts transferred to new wells (no fibroblasts), and fibroblasts in the lower chamber alone (no EC inserts). In each of these combinations, fresh media lacking ARVs was added and cells incubated for an additional 24 hr after which conditioned media (containing released intracellular ARVs) was recovered and analyzed for anti-HIV activity.

Inhibition of Multidrug Resistance-associated protein (MRps).

To evaluate the role of MRPs in ARV movement, epithelial cells were incubated with TFV or TAF for 20 hr, followed by the addition of human MRP-specific inhibitor MK571 (Sigma-Aldrich, St Louis, MO) for 4 hr. MK571 was added in 250 µl of complete media to both the apical and basolateral compartments and was present in the fresh media following cell rinsing and incubation. CM was collected from apical and basolateral compartments of cell inserts after 24 hr. CM was centrifuged at 10,000 g and stored at -80 °C until HIV infection assay. Untreated control cells were donor-matched to treated cells and processed in parallel at the same time. Cell viability was tested after treatment using the CellTiter 96 AQ ueous One Solution cell proliferation assay (Promega, Madison, WI, USA) and trypan blue staining (HyClone Laboratories, Inc., Logan, UT) as described before 7, 8 . All samples were assayed blind without any information provided as to cell origin and treatment.

HIV-infection. Activated blood CD4+ T cells were infected as previously described with minor modifications 5, 8 . Stock for HIV-BaL (R5) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo 38 . Briefly, activated blood CD4+ T cells were incubated with apical and basolateral epithelial cell or fibroblast CM for 24 hr and then washed with PBS prior to HIV infection. After washing, cells were incubated with HIV-BaL for 2 hr at an MOI of 0.1 and then washed to remove residual virus. Fresh 0.2 ml IL-2 supplemented immune cell media was added to each well and cells were incubated for 5 days, with half of the media from each well collected and replaced with fresh media on day 3. Released p24 in the culture media on day 5 was measured by p24 enzyme-linked immunosorbent assay (Advanced Bioscience laboratories, Rockville, MD) following the manufacturer's recommendations.

RNA isolation and quantitative Rt-pCR analysis. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was done with a two-step protocol as described previously 34, 36 . Total RNA was isolated from epithelial cells using RNeasy reagent (Qiagen, Valencia, CA) and QIAshredder columns according to the manufacturer's recommendations (Qiagen), and purified by elution through RNeasy columns (Qiagen) with on-column DNase digestion using the RNase-Free DNase set (Qiagen). For each specimen, 400 ng of total RNA was reverse-transcribed using the iScript complementary DNA (cDNA) synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's recommendations. Relative mRNA expression levels of genes of interest were measured using the 5′ fluorogenic nuclease assay in real-time quantitative PCR using TaqMan chemistry on the ABI 7300 Prism real-time PCR instrument (Applied Biosystems, Foster City, CA). The 5 members from human multidrug resistance-associated protein family (MRP1, 3, 4, 5 and 6) (ID nos. Hs01561483_m1, Hs00978452_m1, Hs00988721_m1, Hs00981089_m1, Hs01077866_m1, respectively) and β-actin (4333762 F) primer/MGB probe sets were obtained from Applied Biosystems assays-on-demand. The 4 epithelial cell tight junction genes analyzed included TJP1 (Zona Occluden 1) (ID nos. Hs01551861_m1), OCLN (Occludin) (ID nos. Hs00170162_m1), CLDN2 (ID nos. Hs00252666_s1) and 4 (ID nos. Hs00533616_s1) (Claudin 2 and 4). PCR was conducted using the following cycle parameters: 12 min at 95 °C for one cycle, followed by 40 cycles of 20 seconds at 95 °C and 1 min at 60 °C. Analysis was conducted using the sequence detection software supplied with the ABI 7300. The software calculates the threshold cycle (C t ) for each reaction and this was used to quantify the amount of starting template in the reaction. The C t values for each set of duplicate reactions were averaged for all subsequent calculations. A difference in C t values (ΔC t ) was calculated for each gene by taking the mean C t of each gene of interest and subtracting the mean C t for the housekeeping gene β-actin for each cDNA sample. The relative expression level of each gene was calculated using the formula 2 -ΔCt . statistics. Data analysis was performed using the GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).

A two-sided P value < 0.05 was considered statistically significant. Comparison of three or more groups was performed applying Kruskal-Wallis test for non-matched samples or Friedman test for matched samples, followed by Dunns-post test for multiple comparison correction. Comparison studies of HIV infections in the absence vs presence of inhibitor MK571 or accumulation of TFV-DP from apical and basolateral treatment and time course studies were analyzed using two-way ANOVA with Bonferroni post-test for multiple comparison correction.

Conversion of tFV and tAF into intracellular tFV-Dp by endometrial epithelial Cells.

In order to compare the protective effects of TFV and TAF, studies were undertaken to ensure that comparable concentrations of TFV-DP were present in polarized Endometrial (EM) epithelial cells following incubation with each ARV. These studies were carried out based on the recognition that TAF is administered clinically at lower doses than TFV and that intracellular conversion of TAF into TFV-DP is much more efficient than TFV 22 . EM epithelial cells were treated with different doses of TFV or TAF for 24 hr and intracellular levels TFV-DP were determined 7 . Using this data as a standard curve, we then calculated TFV and TAF doses that would result in equal amounts of intracellular TFV-DP to compare both drugs side by side. As seen in Fig. 1a and presented elsewhere 7 , we predetermined the dose for each ARV that would give the same intracellular concentration of TFV-DP. Incubation with TFV at 3 concentrations (33, 328, and 3277 μM) and TAF (0.1, 1, and 10 μM) resulted in equivalent amounts of intracellular TFV-DP. Exposure for 24 hr was chosen based on our previous publications 7, 24 and the initial clinical trial (CAPRISA 004) that reported significant protection against HIV acquisition in women that applied topical TFV in the vagina 24 hr prior to sexual intercourse 15, 39 . The findings presented here provide the foundation for comparisons in the following sections that measure the effectiveness of ARVs released into secretions for their ability to protect target cells from HIV infection.

Basolateral secretions from polarized endometrial epithelial cells incubated with tFV or tAF protect CD4+ t cells from HIV infection.

Based on our findings in Fig. 1a , the concentrations of TFV (3277 μM) and TAF (10 μM) were selected to evaluate whether preloaded polarized endometrial epithelial cells release their products in a way that confers protection against HIV infection. After incubation with TFV or TAF for 24 hr followed by extensive washing to remove excess ARVs, epithelial cells were incubated for an additional 24 hr in fresh media prior to recovery of apical and basolateral conditioned media (CM). This CM should contain any TFV or TAF released by epithelial cells. To test if released TFV from epithelial cells would confer protection, activated blood CD4+ T cells were incubated with apical or basolateral CM for 24 hr prior to in vitro HIV infection as detailed in methods. As shown in Fig. 1b , in a representative experiment, basolateral CM from TFV and TAF treated polarized epithelial cells, but not apical CM, significantly inhibited HIV infection of CD4+ T cells. As seen in Fig. 1c , when 10 patients were evaluated, we found that following epithelial preloading with TFV or TAF, basolaterally secreted CM protected against HIV infection (mean inhibition = 60% and 80% respectively). In all of these experiments, apical secretions showed no evidence of protection.

To determine whether protection by basolateral secretions persisted beyond 24 hr, CM was collected and replaced with fresh media daily for 3 days. As seen in Fig. 2a , protection by TFV and TAF in basolateral secretions persisted for 3 days in that each conferred partial protection against HIV infection of CD4+ T cells. By day 3, protection relative to media controls was lower than that measured on day 1. Despite this decline, significant inhibition of infection by HIV of activated CD4+ T cells was observed in 4/4 experiments. This progressive decline in HIV-protection correlated with a slow decline in intracellular TFV-DP levels in the epithelial cells (Fig. 2b, c ). Interestingly, combining the data for the percent inhibition of CD4+ T cells to TFV-DP levels for both TFV-and TAF-treated epithelial cells yielded a smooth curve showing a 50 percent inhibition corresponding to a TFV-DP concentration of ~340,000 fmol/million cells (Suppl. Fig. 1a ). This correlation between epithelial cell TFV-DP levels and protection to CD4+ T cells from HIV supports our hypothesis that protection is due to basolateral release of TFV and TAF into the basolateral secretions of epithelial cells. At no time was protection by ARVs evident in apical CM over the course of these experiments, demonstrating that EC-release of TFV is selective towards the subepithelial (tissue) compartment.

Recognizing that cell integrity is essential, we measured transepithelial resistance (TER) both prior to the addition of TFV and following its addition and wash out both apically and basolaterally for 3 days in culture. As seen in Fig. 2d , when TER was measured daily TFV treatment had no significant effect on TER, indicating that cell and barrier integrity was maintained throughout the course of our experiments. In parallel, we measured the impact of ARVs on tight junction gene expression and found that TFV had no effect on TJP1, OCLD, CLDN2 and CLDN4 mRNA expression in polarized epithelial cells (Suppl. Fig. 1b ). As a further indicator of cell viability, in separate studies, polarized EM epithelial cells were pretreated with TFV (3277 μM) and TAF (10 μM) for 24 hr, prior to measuring viability at 24 hr intervals for 3 days. As shown in Fig. 2e , no changes in viability were found for either ARV. These data demonstrate that the protective ARV levels found in the basolateral compartment in our studies are not due to ARV leakage through epithelial damage.

Recognizing that ARVs following preloading are preferentially released basolaterally, we investigated whether cell uptake of TFV and TAF also exhibited polarity. As seen in Fig. 2f , we found that directional application (apical vs basolateral) of TFV and/or TAF influenced TFV-DP concentrations within epithelial cells. When measured after 24 hr incubation, epithelial cells incubated basolaterally with TFV had 2-3 fold more TFV-DP than did matched cells incubated apically with TFV. In contrast, TAF applied to either the apical or basolateral surface of polarized epithelial cells resulted in comparable intracellular concentrations of TFV-DP.

Overall, these findings indicate that epithelial cells preloaded with either TFV or TAF gradually release ARVs basolaterally in a way that confers partial tissue protection of CD4+ T cells that persists for days. endocervical and ectocervical basolateral secretions from ARV-treated polarized epithelial cells protect against HIV infection. The possibility that the release pattern of ARVs was unique to endometrial epithelial cells prompted us to ask if epithelial cells from other sites in the FRT might share a similar pattern of ARV release. Purified endocervical (CX) and ectocervical (ECX) epithelial cells were grown to confluence, incubated with TFV or TAF for 24 hr and washed to remove excess ARVs after which CM was collected for 24 hr. When cells were preloaded with TFV or TAF, basolateral CM from CX (Fig. 3a ) and ECX (Fig. 3b ) epithelial cells protected against HIV infection of CD4+ T cells. Importantly, similar to EM epithelial cells, apical CM from CX and ECX cells had no protective effect against HIV infection. In other studies, we found that basolateral secretions collected at 24 hr intervals (days 1, 2 and 3) also protected against HIV infection, similar to that seen with EM epithelial cells (Fig. 3c ). Therefore, EM, CX and ECX epithelial cells shared a pattern of TFV and TAF-derived release that was gradual and primarily basolateral towards the underlying stroma.

Stromal fibroblasts from the FRT are a reservoir for gradual ARV release. Our previous studies indicated that in addition to epithelial cells, stromal fibroblasts from the FRT also concentrate intracellular TFV-DP approximately 10 fold higher than FRT CD4+ T cells 24 . To determine whether fibroblast secretions from the EM, CX and ECX were capable of protecting CD4+ T cells from HIV infection, fibroblasts grown to confluence in 24 well plates were incubated with either TFV (3277 μM) or TAF (10 μM) for 24 hr. Following repeated washes, cells were incubated for an additional 24 hr in fresh media prior to collection of CM for analysis. As shown in Fig. 4a, b , CM from EM as well as CX and ECX fibroblasts preloaded with TFV and TAF were capable of partially protecting activated CD4+ T cells from infection by HIV (30-50% protection).

protection by basolateral secretions from polarized eM epithelial cells incubated apically with ARVs. In topical Pre-exposure prophylaxis studies, TFV is deposited in the vagina at the epithelial cell surface after which it moves upstream into the ECX, CX and EM and into the underlying stroma containing CD4+ T cells 28, 32 . To mimic the movement of ARVs from the luminal surface, polarized epithelial cells were treated apically with TFV or TAF followed by basolateral CM collection after 24 hr incubation. Following multiple washes of epithelial cells, cells were incubated with fresh media for an additional 24 hr after which CM was collected. As seen in Fig. 4c , when CM from the initial incubation with TFV or TAF was evaluated, complete protection of CD4+ T cells from HIV infection was observed (Fig. 4c ; ARV-Incubation). Additionally, CM collected 24 hr after washout conferred partial protection (Fig. 4c ; ARV-Post-Incubation), demonstrating continued basolateral release of ARVs by epithelial cells following cell washing of initial ARV input. These findings indicate that under conditions of apical exposure, ARVs continue to be released basolaterally after the apical source of ARV is removed.

To further mimic the interactions of ARVs with the cells present below the epithelium, we apically treated polarized epithelial cells with TFV or TAF for 24 hr during which stromal fibroblasts were present beneath but not in contact with epithelial cells (Fig. 4d ). We found that basolateral CM collected during the first 24 hr from epithelial cells and stromal fibroblasts is able to fully protect CD4+ T cells from infection. Following repeated washings, epithelial cells and stromal fibroblasts were incubated separately (Post-incubation) for an additional 24 hr in fresh media. Under these conditions, both epithelial cells and fibroblasts released ARVs in sufficient concentrations to partially protect CD4+ T cells from HIV infection. This study demonstrates that under conditions of apical epithelial cell exposure, ARVs move to the basolateral compartment to be taken up by fibroblasts for subsequent release and protection of CD4+ T cells. These studies suggest that in situ, both epithelial cells and fibroblasts release ARVs that contribute to the protection of CD4+ T cells in stromal tissues of the FRT.

Multidrug resistance-associated proteins (MRp) are partially responsible for selective transport of ARVs into the subepithelial compartment. Our finding of preferential basolateral release by epithelial cells of ARVs at concentrations capable of protecting underlying cells from infection, suggested that ARV

release may in part be mediated through active transport. MRP1 and MRP3 have been reported to be selectively expressed in the basolateral surface of intestinal epithelial cells and mediate TFV transport outside the cell 40 . To determine whether primary FRT epithelial cells contain MRPs, polarized epithelial cells from the EM, CX and ECX were analyzed for the presence of MRP1, MRP3, MRP4, MRP5 and MRP6. As seen in Fig. 5a , epithelial cells from all 3 sites expressed MRPs, with MRP1 and MRP3 expressed at significantly higher levels than MRP4, MRP5 and MRP6.

Based on these findings, we hypothesized that MRP transporters might in part be responsible for the preferential basolateral release of TFV and TAF from polarized epithelial cells. To test this, EM epithelial cells were incubated with TFV or TAF for 20 hr after which the human MRP-specific inhibitor MK571 was added to apical and basolateral compartments for 4 hr 41 . MK571 was present in wash media and during the following 24 hr when cells were placed in fresh media. If MRPs are responsible for basolateral transport of TFV, we would expect that after MRP blockade less TFV would be released into the CM resulting in increased infection of CD4+ T cells by HIV. As see in Fig. 5b , MK571 partially reversed the protective effect of basolateral CM from ARV-treated epithelial cells, for both TFV and TAF by increasing HIV infection, but had no effect on viral infection in the absence of ARVs. To test if this effect could be maximized, MK571 was added at the start of incubation along with TFV or TAF for 24 hr prior to the collection of CM after washing cells and further incubation. We found that extended Cells were then incubated with fresh media that was replaced each day for 3 days, basolateral CM was collected daily. Activated CD4+ T cells were treated for 24 hr with CM. Following washout of CM, CD4+ T cells were infected after which secreted p24 levels measured by p24 ELISA as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100% (dashed line).

(n = 7 individual patients). Columns and horizontal lines represent the mean and SEM respectively. *p < 0.05. **p < 0.01. exposure did not further inhibit ARV release beyond that seen when MK571 was added for the last 4 hrs of culture (not shown).

To confirm that MK571 was biologically active, we measured intracellular TFV-DP levels in epithelial cells incubated with TFV and TAF for 24 hr in the presence of MK571. As seen in Fig. 5c , MK571 treatment resulted in a modest but significant increase in intracellular TFV-DP in epithelial cells. This increase in TFV-DP correlated with the loss of HIV protection when CD4+ T cells were incubated with basolateral CM from ARV-treated epithelial cells, suggesting a decreased efflux of TFV from epithelial cells. Overall, these findings indicate that MRPs contribute partially to the basolateral release of TFV in the direction of stromal tissues. Polarized EM epithelial cells were apically treated with TFV or TAF for 24 hr, after which basolateral CM was immediately collected (ARV-Incubation). Following rinsing, cells were incubated with fresh media (no TFV or TAF) for an additional 24 hr prior to CM collection (Post-Incubation 24 hr). Activated CD4+ T cells from blood were incubated with CM for 24 hr and washed and infected with HIV for 2 hr. Secreted viral p24 levels were measured after 5 days of infection (Methods). Data are normalized to the infection of CD4+ T cells in the absence of CM (media control) which is set to 100%. Bars represent EM tissues from 3 patients. (d) Effect of epithelial cells and fibroblasts on prevention of HIV infection. Polarized EM epithelial cells grown in the upper chamber of cell inserts were treated apically with TFV (3277 µM) or TAF (10 µM) for 24 hr in the presence of confluent fibroblasts (SF) from the same donor grown in the lower chamber (no cell contact). Following incubation, epithelial cells and stromal fibroblasts (EC + SF), basolateral CM was collected for analysis (ARV-Incubation). Following extensive washing to remove extracellular ARVs (Post-Incubation), epithelial cells alone (EC) and stromal fibroblasts alone (SF) were incubated in fresh media for 24 hr, after which CM were collected. CD4+ T cell protection against HIV infection for each CM was measured as described in Methods. Data are normalized to the infection of CD4+ T cells in the absence of CM (media control). Bars represent 4 patients. Blood CD4+ T cells were isolated from 2 donors. Column and horizontal lines represents the mean and SEM respectively. *p < 0.05. **p < 0.01.

Discussion

Overall, our studies demonstrate that following incubation with ARVs, EM, CX and ECX epithelial cells and fibroblasts retain and subsequently release TFV or TAF that provide downstream protection of CD4+ T cells from HIV acquisition. ARV release by polarized epithelial cells is selective towards the subepithelial (basolateral) compartment, where it can be taken up by underlying fibroblasts. Together, both epithelial cells and fibroblasts gradually release ARVs to protect CD4+ T cells. Following apical TFV or TAF administration, there is complete protection from HIV infection for the first 24 hr. However, after drug removal, epithelial cells and fibroblasts act as tissue reservoirs that slowly release TFV or TAF, conferring partial protection that persists for at least three days. These findings highlight a previously un-recognized role of the mucosal environment in protection against HIV acquisition by modulating availability of ARVs to HIV-target cells.

Previous studies using purified cells from the FRT to measure intracellular TFV-DP following incubation with TFV demonstrated that epithelial cells and fibroblast TFV-DP concentrations were 100-and 10-fold higher respectively than those found in CD4+ T cells 24 . These findings prompted us to ask whether epithelial cells and fibroblast accumulation of ARVs extends protection by acting as a reservoir, or compromises it by reducing drug availability to HIV target cells in the FRT. A major finding in the present study is that by gradually releasing TFV and TAF at concentrations sufficient to provide protection of CD4+ T cells from HIV infection, epithelial cells and fibroblasts extend ARV-mediated protection by providing a reservoir that lengthens the effectiveness of TFV and TAF beyond their initial application. Recognizing that ARVs can be administered orally or locally in the FRT, other studies have reported that the route of administration (oral vs. vaginal) influences the time needed for TFV to reach protective levels within HIV-target cells 31 . Following a single oral dose of TFV, the concentration of TFV and TFV-DP in the gastrointestinal (GI) mucosa is 100-fold greater than the FRT, and requires 7-14 days to reach a protective concentration in the FRT 32 . A possible reason for this delay could be that orally administered ARVs must GI cross the mucosa prior to entering circulation. Our studies indicating that TFV accumulates in FRT epithelial cells and fibroblasts, suggest that if a comparable concentration occurs in the GI tract cells, it may delay ARV levels reaching effective concentrations in the FRT. This is particularly relevant for TAF which was undetectable in most FRT tissues following a single oral dose, while still being detectable in circulation 42 . However, by acting as a local ARV reservoir, GI epithelial cells would also extend ARV protection for GI CD4+ T cells, which are susceptible to HIV infection. Whether FRT epithelial cells, and those from other mucosal sites, can act as a reservoir for other ARVs being considered for PrEP, such as dapivirine, remains to be determined, but could be an important factor in modulating their efficacy.

An important finding is that ARVs released by epithelial cells can be taken up and re-released by underlying fibroblasts. This suggests that in vivo, epithelial cells and fibroblasts work in tandem, with each contributing to a sustained tissue concentration that provides extended protection of HIV target cells. Although we did not measure the concentrations of TFV or TAF being released, based on our previous in vitro studies 8 , we know that the concentrations of TFV and TAF that result in partial protection of activated blood CD4+ T cells, as seen in this study, are very low (1-5 μM and 5-10 nM for TFV and TAF respectively) and several logs below the detectable limits for measuring TFV and TAF 7, 24 . Moreover, apical concentrations were below these concentrations (1-5 μM and 5-10 nM for TFV and TAF respectively), since we repeatedly found no evidence of protection. However, in vivo, tissue protection may be greater than predicted in our in vitro studies, because of the limited fluid volume within the interstitial space of the FRT, where concentrations of ARVs would be expected to be several folds higher than in our system. This conclusion is based on recent findings demonstrating that, whereas the stroma was thought to be dense connective tissue, the interstitium at mucosal sites is now recognized as a series of fluid-filled compartments 43 . Given the partial protection we observe with CM, we speculate that protection in vivo would be more complete, given the reduced interstitial volume and consequently higher concentration of secreted ARVs. Further studies are needed to obtain a more accurate profile of in vivo intracellular and interstitial concentrations of ARVs in FRT tissues, recognizing that even within the same tissue, drug distribution, and therefore efficacy, is likely to vary with cell type.

An unexpected finding in our study was that whereas basolateral secretions from TFV and TAF treated epithelial cells reduced HIV infection of CD4+ T cells, apical secretions from the same cells had no effect. The reasons for this unique phenotype are unclear but previous studies have shown that FRT epithelial cells can selectively secrete cytokines and chemokines via their apical or basolateral membranes 35, 44, 45 . To explore the underlying mechanisms responsible for basolateral release, we examined the expression of MRPs, which eliminate drugs from the cells, and found that FRT epithelial cells express MRP 1, 3, 4, 5 and 6. Chemical inhibition of MRP function resulted in a moderate but significant increase in intracellular TFV-DP levels in epithelial cells, and a decrease in CD4+ T cell protection by basolateral secretions from both TAF and TFV pre-loaded epithelial cells. These findings are consistent with increased TFV and TAF accumulation inside of epithelial cells when transporters are inhibited and suggest that MRPs are selectively expressed on the basolateral membrane of epithelial cells 40 . However, since protection was only partially reduced, this raises questions about the extent to which other transporters may also be involved in TFV and TAF efflux, since different families of transporters have been described in FRT cells, including BCRP and P-gp 27 . Another potential explanation for the partial effect seen in these studies is that initial levels of TFV and TAF in epithelial cells following preloading diffuse out and mask transporter function as ARVs exit the cell (Fig. 6 ). Alternatively, since MRPs are a family of anionic transporters 46 , some of these transporters may be insensitive to the inhibitor used in our studies. Additional mechanisms that mediate the selective transport of TFV from FRT epithelial cells remain to be elucidated.

An interesting finding in our study was that TFV and TAF were differentially taken up by epithelial cells when loaded apically (Fig. 2f ). Previous studies have demonstrated that the Organic Anion Transporter (OAT) proteins OAT1 and 3 are capable of transporting tenofovir into the cells lining the kidney proximal tubule 20, 47 . Other studies also demonstrated that endothelial cells express OAT1 and OAT3 receptors, however, we and others have previously measured these receptors and failed to detect their presence in primary epithelial cells and fibroblasts from throughout the FRT 18, 36 . Therefore, additional transporters other than OAT1 and OAT3 may be involved in the differential apical uptake between TFV and TAF. Others have demonstrated that TFV uptake was partially mediated through endocytosis in vaginal epithelial cell lines and T cells, while TFV disoproxil fumarate entered these cells by passive diffusion 18 . Whether this same mechanism may explain the difference in apical uptake between TFV and TAF by primary epithelial cells throughout the FRT remains to be determined. Further studies are needed to identify potential additional transporters and establish their role in the sustained HIV protection we report in this paper.

Our findings of preferential basolateral release of ARVs have important clinical implications. First, as shown by others 48 , when administered intravaginally to women, TFV in cervical vaginal lavages (CVL) decreases by 10-100 fold within 24 hr. When women have sex during this time, TFV concentrations further decrease in the vaginal lumen. These findings further support our conclusion that epithelial cells and fibroblasts serve as important sources of ARVs that are gradually released into underlying tissues to provide protection over a period of days. Second, it implies that TFV concentrations in CVL following topical or oral administration of TFV and TAF are not a good indicator of tissue concentrations and protection, and should not be used to monitor adherence or anti-HIV activity in clinical trials. Third, our findings offer another potential explanation for why TFV did not protect women with genital inflammation derived from STIs 49 , which probably increases target cell presence in the mucosa, and induces epithelial injury in the form of crypt abscesses, mucosal erosions and ulceration. Under these conditions immune cells would not be protected once the initial drug dose is washed out. Given our results, different factors that alter TFV-DP concentrations in epithelial cells and fibroblasts, such as inflammation 49 , sex hormones such as estradiol which is known to increase uterine epithelial cell intracellular TFV-DP concentrations 24 , or menopause which lowers concentrations 29, 50 , may be more relevant for protection than previously anticipated.

Beyond dosage, the efficacy of an ARV is modulated by several factors including local inflammation, use of contraceptives, composition of the microbiome, and presence of sexually-transmitted infections 8,51-53 . Our previous studies have demonstrated that medroxyprogesterone acetate (MPA), a commonly used contraceptive in Sub-Saharan Africa associated with increased risk of HIV acquisition, inhibits the conversion of TFV to TFV-DP in activated blood CD4+ T cells at concentrations similar to that used in this study, thus leading to decreased protection against HIV infection 8 . Similarly, MPA inhibits conversion of TAF to TFV-DP in FRT CD4+ T cells 8 . Alterations in the vaginal microbiome are also linked to decreased ARV efficacy. Anaerobic bacteria such as Gardnerella vaginalis commonly seen in bacterial vaginosis, decreased the pool of vaginal TFV available for HIV target cells faster than Lactobacillus species 51 . Lastly inflammation, either due to the composition of vaginal gels 54 or pre-existing sexually-transmitted infections 53 , can drive the recruitment of target cells to the mucosal surface bringing them into proximity of incoming HIV, which in turn can undermine the effectiveness of TFV 49 . Our studies add an additional component to the complexity of interactions that dictate ARV effectiveness. Our results of preferential basolateral release of ARVs suggest that TFV or TAF treatment would not protect against HIV infection events happening on the mucosal surface, induce under conditions of inflammation or STIs that attract target cells to the surface.

While there is evidence that TFV is released basolaterally by intestinal (Caco-2) and endometrial (HEC-1-A) epithelial cell lines, and that TFV is present in ex vivo treated tissue explants 55 , our study is novel in several aspects. First, ours is the first study to demonstrate that primary epithelial cells throughout the FRT accumulate TFV-DP inside the cells and, after the initial drug inoculum is cleared, gradually release TFV, and release it in sufficient amounts and in a sustained manner to protect CD4+ T cells from HIV infection. Second, we demonstrate for the first time that the same is true for TAF, and that the same effect can be achieved with much lower doses than TFV. Third, while previous studies have demonstrated the presence of TFV in the basolateral compartment, to the best of our knowledge, no formal comparison of basolateral and apical release has been performed until now. Lastly, our findings are unique in that they demonstrate the release of ARVs (TFV and TAF) by both columnar (EM, CX) and squamous (ECX) EC and underlying stromal fibroblasts from each of these sites. In doing so, both epithelial cells and stromal fibroblasts function as reservoirs that extend the time interval of protection beyond the initial uptake of TFV/TAF by mucosal CD4+ T cells, the primary targets of HIV infection.

As seen in our summary (Fig. 6 ), on entering cells TFV is converted to TFV-DP, its biologically active form, which is restricted to the intracellular environment on immune and non-immune cells 7, 8, 24 . Similarly, TAF enters cells and is sequentially converted to TFV, and then to TFV-DP 20 . As a part of this metabolic cycle, TFV-DP from TFV and TAF is converted back into TFV, which either leaves the cell or is metabolized to adenosine 20 . Our studies suggest that following washout, two distinct steps (diffusion and transport) regulate the efflux of TFV and possibly TAF and are involved in the protection of CD4+ T cells from HIV infection. We speculate that basolateral protection following ARV washout, is due to the gradual release of TFV and TAF that is retained inside the epithelial cells and fibroblasts as well as TFV-DP and TFV-MP until converted back to TFV. Upon efflux and uptake by CD4+ T cells, TFV is converted back to TFV-DP which exerts a protective effect by inhibiting viral replication. The gradual conversion of TFV-DP and release of TFV from epithelial cells and stromal fibroblasts steadily decreases over days following washout, and thus the protective effect against HIV infection also decreases. Also shown is the transfer of TFV from epithelial cells to stromal fibroblasts and the fibroblasts ability to gradually release TFV to protect CD4+ T cells from HIV infection. Further studies are needed to more fully define the mechanisms whereby epithelial cells and fibroblasts throughout the female reproductive tract function as reservoirs to extend protection of CD4+ T cells beyond the initial ARV tissue exposure.

In conclusion, our results demonstrate that epithelial cells and underlying stromal fibroblasts from throughout the FRT readily take up TFV and TAF and produce TFV-DP. After initial drug exposure, degradation of TFV-DP back to TFV as well as any residual TFV and TAF can be gradually released to confer protection of CD4+ T cells from HIV infection for several days. Our findings demonstrate a previously unrecognized role of the mucosal environment in controlling ARV-mediated protection against HIV acquisition and suggest that factors that modify retention of ARVs, and intracellular concentrations of their active metabolites, such as TFV-DP, in epithelial cells and fibroblasts may be more relevant for protection than previously anticipated.

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-38205-y.

Competing Interests:

The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acknowledgements

AcknowledgementsStudy supported by NIH grants AI102838 and AI117739 (CRW).We thank Richard Rossoll for technical assistance.We thank all study participants, Pathologists, Obstetrics and Gynecology surgeons, operating room nurses and support personnel at Dartmouth-Hitchcock Medical Center.

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