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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Hormonal Contraceptives Differentially Suppress TFV and TAF Inhibition of HIV Infection and TFV-DP in Blood and Genital Tract CD4+ T cells

Zheng Shen, M. Rodriguez-Garcia, Mickey V Patel, J. Bodwell, A. Kashuba, C. Wira

Scientific Reports·

2017·

11 citations

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Study Methods

- Cell types used: Primary CD4+ T cells from blood and female reproductive tract (FRT) tissues. - Experimental approach: Incubation with TFV and TAF to assess anti-HIV effects; measurement of intracellular TFV-DP concentrations. - Duration of experiments: 24-hour incubation with TFV and TAF; HIV infection measured on day 5. - Technical methods: Liquid chromatography with tandem mass spectrometry (LC-MS/MS) for TFV-DP; p24 enzyme-linked immunosorbent assay for HIV infection. - Special conditions: Use of Medroxyprogesterone acetate (MPA) to assess its impact on TFV and TAF efficacy.

TAF Activation Mechanisms

- Specific enzymes involved: Cathepsin A for initial hydrolysis of TAF to TFV; adenylate kinase (AK) for first phosphorylation of TFV to TFV-MP; a kinase with nucleotide diphosphate kinase activity for second phosphorylation; pyruvate kinase (PK) for conversion of TFV-MP to TFV-DP. - Cellular location of activation: Lysosomal. - Metabolic pathway steps: TAF → Cathepsin A → TFV → AK → TFV-MP → Nucleotide diphosphate kinase → TFV-DP. - Evidence for enzyme involvement: Not specified in the paper. - Kinetic parameters: Not mentioned in the paper. - Alternative or secondary activation pathways: Not identified in the paper.

Intracellular Drug Concentrations

- Concentrations of TFV-DP: Approximately 1-2 × 10^4 fmol/million cells - Cell types: Blood CD4+ T cells, FRT CD4+ T cells - Time points: After 24 hours of incubation with TFV or TAF - Units of measurement: fmol/million cells - Fold differences: MPA reduced TFV-DP in blood CD4+ T cells; MPA lowered TAF-derived TFV-DP in FRT 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 equal intracellular TFV-DP concentrations with approximately 1500-fold less TAF than TFV. - Differences in activation mechanisms or pathways: TAF is activated by Cathepsin A, whereas TFV's activation pathway is not specified in the paper. - Relative potency or antiviral activity: TAF has increased anti-HIV efficacy compared to TFV. - Systemic vs. intracellular drug exposure profiles: Not directly compared in the paper. - Any advantages or disadvantages of TAF over TDF for drug distribution: TAF has advantages in terms of reduced toxicity and preferential accumulation in lymphoid tissues. - Fold differences in key parameters between the two prodrugs: TAF is effective at up to 300-fold lower levels than TFV.

Factors Affecting Activation

- Drug interactions: MPA suppresses TFV's anti-HIV effect in blood CD4+ T cells and TAF's effect in genital CD4+ T cells. - Host factors: Unknown mechanisms may involve cell-specific actions related to drug uptake and conversion. - Cellular conditions: MPA affects intracellular TFV-DP concentrations differently in blood and genital CD4+ T cells. - Chemical inhibitors or enhancers: MPA is a chemical inhibitor of TFV and TAF in specific contexts. - Quantitative effects: Not explicitly mentioned in terms of IC50 or fold change. - Clinical relevance: MPA may decrease ARV protection in individuals using these drugs intermittently for prevention.

HIV-1 Resistance Context

Not mentioned (the paper does not discuss specific resistance mutations, 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: The study uses concentrations of TFV and TAF that result in approximately 50% inhibition of HIV infection. - Extrapolation to clinical dosing scenarios: The findings suggest that MPA could compromise ARV efficacy, particularly with low TFV concentrations or poor adherence. - Implications for drug safety or efficacy: MPA may increase the risk of HIV infection by reducing TFV and TAF efficacy. - Recommendations for clinical use based on mechanistic findings: Future MPT studies should consider ARV/contraceptive interactions to maximize anti-HIV protection. - Identified knowledge gaps or future research needs: Further research is needed on TAF distribution and activation in genital tissues. - Clinical trial data or real-world evidence referenced: The study references clinical trials where women taking MPA may have experienced reduced ARV efficacy.

HIV prevention research is focused on combining antiretrovirals (ARV) and progestin contraceptives to prevent HIV infection and pregnancy. The possibility that progestins compromise ARV anti-HIV activity prompted us to evaluate the effects of progestins on tenofovir (TFV) and TFV-alafenamide (TAF) on HIV infection and intracellular TFV-diphosphate (TFV-DP) concentrations in blood and genital CD4+ T cells. Following incubation of blood CD4+ T cells with TFV or TAF, Medroxyprogesterone acetate (MPA), but not Levonorgestrel, Norethisterone or progesterone, suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP. In contrast, with genital CD4+ T cells, MPA suppressed TAF inhibition of HIV infection and lowered of TFV-DP concentrations without affecting TFV protection. These findings demonstrate that MPA selectively compromises TFV and TAF protection in blood and genital CD4+ T cells and suggests that MPA may decrease ARV protection in individuals who use ARV intermittently for prevention.

oral dosing, a minimum adherence to 6 of 7 doses/week (85%) was required to protect lower female genital tract tissues from HIV, while adherence to 2 of 7 doses/week (28%) was required to protect colorectal tissue 14 .

We have previously reported that female sex hormones have the potential for modifying TFV conversion to TFV-DP 15 . Also of relevance is the recent study that bacterial vaginosis-associated bacteria can affect TFV levels and HIV transmission risk 16 . While topical ARV administration offers advantages of higher vaginal drug concentrations and lower systemic toxicity, it is unclear whether protective concentrations of ARVs can be reached in the entire FRT after local ARV administration [17] [18] [19] . Protection of the entire FRT along with regional lymph nodes is necessary, given the evidence that HIV-target cells are present throughout the FRT 15, [20] [21] [22] [23] and studies in non-human primate models showing that infection can occur in all the major anatomical regions of the FRT 21, 22 .

A new pro-drug of TFV, Tenofovir alafenamide (TAF; formerly known as GS-7340), has been approved for HIV treatment 24 . TAF presents advantages compared to TFV, including increased anti-HIV efficacy, reduced toxicity, and preferential accumulation in lymphoid tissues and HIV susceptible cells [24] [25] [26] . Under clinical conditions, TAF is administered at lower doses than tenofovir (TFV), owing to the more efficient intracellular conversion of TAF into TFV-DP than TFV [24] [25] [26] . Moreover, TAF is a prodrug formulation of TFV that exerts antiviral activity at up to 300-fold lower levels. While TFV tissue concentrations after vaginal administration of TFV have been investigated, tissue concentrations of TAF are still under investigation and no studies using topical administration of TAF are available. TAF conversion to TFV inside the cells is initiated by the esterase activity of Cathepsin A, followed by subsequent reactions probably occurring within lysosomes to form TFV 27 , after which the same kinases are utilized to form TFV-DP. Despite the advantages that TAF offers for HIV treatment, it is not currently approved for Pre-Exposure Prophylaxis (PrEP) 28 . However, its ability to protect against mucosal HIV infection and determination of genital tissue concentrations are being investigated 20, 29, 30 .

In addition to HIV, a major health issue for women of reproductive age is unintended pregnancy. To meet the needs of reproductive age women, current HIV prevention research is leading towards multipurpose prevention technologies (MPTs). MPTs deliver hormonal contraceptives and ARVs to simultaneously prevent unintended pregnancy and HIV infection 31, 32 . However, several studies have reported contradictory findings about the interactions between ARVs and hormonal contraceptives 33, 34 . These findings emphasize the need to determine whether hormonal contraceptives interact with ARVs to decrease the effectiveness of ARVs in protecting against HIV.

In this study we evaluated possible interactions between progestin hormonal contraceptives (MPA, norethindrone (NET) and levonorgestrel (LNG)) and ARVs (TFV and TAF) that could result in decreased anti-HIV activity of ARVs. We compared interactions using CD4+ T cells from blood and from FRT tissues. Our experimental approach was to use lower concentrations of TFV and TAF that resulted in approximately 50% inhibition of HIV infection, and concentrations approximately 1500-fold higher to measure intracellular TFD-DP. We found that MPA, but not LNG or NET, was able to reverse the protective effects of ARVs. Unexpectedly, we found that MPA decreased TFV anti-HIV activity in blood CD4+ T cells and TAF anti-HIV activity in tissue CD4+ T cells. Increased infection after MPA treatment was associated with decreased intracellular TFV-DP, the active drug form with anti-HIV activity. These findings highlight the need to test potential hormonal contraceptive and ARV (topical and oral) interactions in blood and tissue cells when developing MPTs, since anti-HIV effectiveness may be differentially compromised by contraceptives based upon anatomical location.

Results

Intracellular TFV-DP concentrations in CD4+ T cells from Blood and FRT tissues. The amount of TFV applied vaginally (40 mg total at a concentration of 10 mg/ml) in HIV prevention trials 9 and the levels of TAF found in blood after oral administration [24] [25] [26] are very different. Therefore, to accurately compare the effects of TFV and TAF, we predetermined the dose for each ARV which would give the same intracellular concentration of TFV-DP and therefore the same levels of protection against HIV infection. As seen in Fig. 1 , blood CD4+ T cell incubation for 24hr with TFV (1 mg/ml: 3277 μM) or TAF (1 µg/ml: 2 μM) resulted in equivalent amounts of intracellular TFV-DP. Under identical culture conditions, incubation of CD4+ T cells from the FRT also resulted in intracellular concentrations of TFV-DP that were not significantly different from blood CD4+ T cells. Moreover, when CD4+ T cells from the endometrium (EM, black circle), endocervix (CX, open circle) and ectocervix (ECX, triangle) were analyzed, intracellular concentrations of TFV-DP between sites in the FRT were the same and not significantly different from the concentrations measured in blood CD4+ T cells. Each symbol represents blood CD4 + T cells from different female donors or FRT CD4+ T cells from an individual patient. These data indicate that irrespective of the source of CD4+ T cells analyzed, the doses of ARV selected result in intracellular concentrations of TFV-DP for blood and FRT CD4+ T cells that are approximately 1-2 × 10 4 fmol/ million cells. These data provide the foundation for studies in the following sections, which determine whether progestational contraceptives compromise both TFV and TAF mediated anti-HIV immune protection as well as intracellular concentrations of TFV-DP.

Comparison of TFV and TAF Inhibition of HIV Infection of CD4+ T Cells from Blood and

Endometrium. The doses of TFV and TAF used in Fig. 1 are very efficient at suppressing HIV infection of CD4+ T cells in vitro, and therefore could mask any potential modifications of ARV effectiveness by contraceptives. For this reason, dose response studies were undertaken to identify the concentration of TFV and TAF that resulted in approximately 50% inhibition of HIV infection, in order that changes in effectiveness in either direction (increase or decrease) could be detected. Following activation for 24hr, purified blood CD4+ T cells were treated with TFV (0.33-3277 μM) or TAF (0.01-10,000 nM) for another 24hr prior to extensive washout and infection with BaL (MOI 0.1) as described in Methods. In dose response studies, we found that inhibition of HIV infection by TFV (Fig. 2a ) was lost between 1-10 μM, while TAF (Fig. 2b ) inhibition of HIV infection was lost at doses between 1-10 nM.

To determine whether CD4+ T cells from the endometrium and blood are equally protected against HIV infection by TFV and TAF, CD4+ T cells were incubated with increasing doses of TFV and TAF for 24hr prior to infection with Bal (MOI 0.1). As seen in Fig. 2c , whereas blood CD4+ T cell infection was partially decreased by TFV starting at 1 μM, the same 1 μM dose of TFV had no anti-HIV effect on endometrial CD4+ T cells. The anti-HIV effect of TFV was also significantly reduced in endometrial CD4+ T cells at 5 and 10 μM when compared to blood CD4+ T cells. In contrast to TFV, as shown in Fig. 2d , TAF inhibited HIV infection to the same extent in blood and endometrial CD4+ T cells with no differences seen at the 3 concentrations tested. Overall, these findings indicate that endometrial CD4+ T cells are different from blood CD4+ T cells in terms of their sensitivity to TFV but not TAF.

Effect of MPA on TFV inhibition of HIV Infection and intracellular TFV-DP in CD4+ T cells from blood.

The possibility that some hormonal contraceptives interfere with TFV and/or TAF effectiveness resulting in a reduced protection against HIV acquisition prompted us to determine whether MPA might compromise the effectiveness of ARVs in preventing HIV infection of CD4+ T cells. Figure 3a shows the analyses of CD4+ T cells from 5 different blood donors, demonstrating that MPA suppresses the efficacy of TFV at 1 and 5 μM leading to increased HIV infection of CD4+ T cells. Further, it indicates that MPA alone has no effect on cell viability, since in the absence of TFV (Fig. 3a ) and TAF (Fig. 3c ) infection by HIV was not affected. An increase in secreted p24 levels indicates a significant loss of TFV protection against HIV infection in the presence of MPA compared to control TFV alone.

Given the suppression of TFV-mediated protection against HIV infection of HIV target cells by MPA, we investigated whether the effect of MPA might be due to altered intracellular concentrations of tenofovir diphosphate (TFV-DP). To determine the relationship between TFV and TFV-DP concentrations, blood CD4+ T cells were pre-treated with MPA (1 × 10 -7 M) for 24hr followed by incubation with TFV (3277 μM) for an additional 24hr prior to measuring intracellular TFV-DP. We found that the lower TFV concentrations used in the HIV studies resulted in intracellular TFV-DP concentrations that were below the limit of detection. As shown in Fig. 3b , intracellular TFV-DP in 4 separate experiments were significantly lower when cells were treated with MPA. Overall, these findings indicate that MPA reduces both protection against HIV infection by TFV as well as intracellular TFV-DP concentrations in blood CD4+ T cells.

Lack of effect of MPA on TAF inhibition of HIV Infection of blood CD4+ T cells.

Using the experimental approach described above for TFV, we asked whether MPA inhibits TAF-mediated protection of CD4+ T cells. Using concentrations of TAF identified in HIV infection dose-response experiments (Fig. 2 ), we selected the optimal range of TAF (1-10 nM) for preventing HIV infection. As seen in Fig. 3c in 4 separate experiments, MPA had no effect on TAF-mediated inhibition of HIV infection by HIV of blood CD4+ T cells. As a part of these studies, we asked whether MPA altered the conversion of TAF into TFV-DP. Figure 3d demonstrates that MPA had no effect on the concentration of intracellular TFV-DP when compared to control cells. These findings demonstrate, that even when evaluated at concentrations resulting in similar intracellular TFV-DP levels, MPA exerts differential effects on TFV and TAF inhibition of HIV infection of blood CD4+ T cells. Whereas MPA suppressed TFV-mediated HIV infection and lowered intracellular concentrations of TFV-DP, it had no effect on either parameter in the presence of TAF.

Effect of MPA on TFV and TAF Inhibition of HIV Infection and Intracellular TFV-DP Concentrations in endometrial CD4+ T cells.

Recognizing that CD4+ T cells in the human FRT are unique and distinct from those in the blood 15, 35 , we asked whether MPA alters TFV-and/or TAF-mediated HIV protection and intracellular TFV-DP concentrations in FRT CD4+ T cells. As seen in Fig. 4a , MPA had no effect on TFV (1, 5, and 10 μM) inhibition of HIV infection of endometrial CD4+ T cells. In contrast, MPA treatment decreased the protective effect of TAF at 5 nM against HIV infection of endometrial CD4+ T cells from 4 patients. Analysis of intracellular TFV-DP concentrations in endometrial CD4+ T cells indicated that, whereas MPA had no effect on TFV-DP concentrations derived from TFV (3277 mM), MPA significantly lowered intracellular TFV-DP concentrations derived from TAF treatment of endometrial CD4+ T cells obtained from 7 different patients (Fig. 4b ). These findings indicate that MPA selectively impacts endometrial CD4+ T cells in a way that is opposite to that seen with blood CD4+ T cells both in terms of TFV and TAF protection against HIV infection and intracellular TFV-DP concentrations.

Lack of effect of progestins and progesterone on TFV and TAF inhibition of HIV Infection and intracellular TFV-DP Concentrations in Blood CD4+ T cells.

To determine whether the effect of MPA on TFV inhibition of blood CD4+ T cell HIV infection is a characteristic of other progestin contraceptives and naturally occurring progesterone, we examined the effects of LNG, NET and progesterone on reversing HIV infection of blood CD4+ T cells. Analysis of each progestin was undertaken under conditions identical to that used to analyze the effect of MPA on suppressing CD4+ T cell protection. The concentration of steroids used in these studies (1 × 10 -7 M), which corresponds to or exceeds that reached in blood, was selected in order to observe any potential inhibitory effects 36 . As shown in Fig. 5a and c , we found that LNG, NET, and progesterone had no effect on TFV-or TAF-mediated protection against HIV infection of blood CD4+ T cells. Moreover, when intracellular TFV-DP concentrations derived from TFV or TAF treatment were measured (Fig. 5b and d ), LNG, NET and progesterone had no effect on TFV-DP concentrations from either ARV. These findings suggest that the effects of MPA are not characteristic of all progestin compounds or of naturally occurring progesterone.

Effect of MPA, LNG, NET and progesterone on intracellular TFV-DP from TAF treatment of endometrial CD4+ T cells.

Since we observed a suppressive effect of MPA effect on TAF conversion to TFV-DP in endometrial CD4+ T cells (Fig. 4b ), we investigated whether this occurred with other progestins. Endometrial CD4+ T cells were pre-incubated with NET, LNG, or progesterone for 24hr prior to incubation with TAF and hormonal contraceptives for an additional 24hr. As seen in Fig. 6 , LNG, NET and progesterone had no effect on intracellular TFV-DP concentrations in endometrial CD4+ T cells in 4 separate experiments, in contrast to the decreased concentrations observed after MPA treatment.

Discussion

The present study evaluated the effect of progestin contraceptives on TFV and TAF inhibition of blood and endometrial CD4+ T cell infection by HIV, and their conversion into intracellular TFV-DP. We found under conditions in which intracellular concentrations of TFV-DP are the same in blood and endometrial CD4+ T cells, that MPA but not LNG, NET or progesterone, both suppresses the protective effect of TFV on HIV infection and lowers intracellular concentrations of TDV-DP in blood CD4+ T cells. In contrast, MPA suppresses TAF inhibition of HIV infection and lowers intracellular TFV-DP concentrations in endometrial CD4+ T cells. Our in vitro findings demonstrate that protection against HIV infection varies with each ARV, each hormonal contraceptive, and the source of CD4+ T cells. To the best of our knowledge, these findings are the first to demonstrate that TFV and TAF anti-HIV effects can be reversed by some progestational contraceptives. Based on these findings, future HIV prevention and MPT studies should consider potential ARV/contraceptive interactions that may compromise ARV efficacy.

An important finding in our study is that TFV and TAF effectively protected blood and tissue CD4+ T cells from HIV infection in vitro. We also demonstrated that CD4+ T cells from the FRT (EM, CX and ECX) efficiently convert TAF into TFV-DP, with equal intracellular concentrations attained when cells were incubated with approximately 1500-fold less TAF than TFV. Interestingly, recent studies investigating tissue TAF concentrations after single dose oral administration, could not consistently detect TAF or TFV-DP in genital tissues (vagina) 30 . We have previously reported that primary epithelial cells from EM, CX and ECX convert TAF into TFV-DP 20 , with no pro-inflammatory effects, in contrast to TFV 20, 37 . As TAF is being considered for PrEP after success in non-human primate studies 29 , our results suggest that once present in the FRT, TAF would be very effective in preventing HIV infection, arguing towards exploring local administration of TAF in the FRT.

A major finding in our study is that MPA treatment, but not LNG or NET, was able to decrease intracellular TFV-DP concentrations, offering a potential explanation for the loss of anti-HIV activity. The mechanisms through which MPA exerts its differential effects on CD4+ T cells from the reproductive tract are unknown but could involve cell specific actions including ARV uptake/transport and/or enzymes that convert TAF and TFV to their active forms or increase their degradation. Whereas previous studies postulated that entry of TFV and TAF into immune and non-immune cells is by diffusion or taken up by organic anionic transporters 38, 39 , a recent study provides evidence that TFV is taken up by CD4+ T cells through a relatively inefficient, energy-dependent, non-receptor-mediated endocytic-like pathway 40 . In contrast, recent studies with TAF suggest that uptake into cells is by passive diffusion 41 (Herold, B. personal communication). Beyond the level of uptake, achieving an effective concentration is the result of dosage and a composite of factors involving metabolic activation and/ or degradation. The first phosphorylation of TFV to TFV-MP is known to utilize adenylate kinase (AK). The second requires a kinase with nucleotide diphosphate kinase activity. More recently, it has been shown that pyruvate kinase (PK) has a major role in the conversion of TFV-MP to TFV-DP 42 . In contrast, TAF is converted to TFV by a series of reactions initiated by Cathepsin A which results in conversion to TFV after which it utilizes the pathways described above. Our findings that MPA decreases the concentrations of TFV-derived TFV-DP in CD4+ T cells from blood and TAF-derived TFV-DP in FRT CD4+ T cells, both of which correlate with reduced anti-HIV activity, raises the likelihood that MPA exerts its effects on blood and reproductive tract CD4+ T cells through different mechanisms. Given that blood samples used in this study were different from the FRT tissues, future studies are needed to confirm these findings using matched samples, to determine the exact mechanisms involved. The unique effect of MPA on immune cell function, in contrast to NET and LNG, has been reported by Huijbregts et al. 43 . One reason for the specific effects of MPA could be due to the dynamics of steroid hormone-receptor interactions. Despite sharing structural similarities, the four progestins each have different affinities for multiple steroid hormone receptors 44 . In particular, MPA has a stronger affinity for both the glucocorticoid receptor (GR) and androgen receptor (AR) than the other three progestins 45, 46 . Recognizing that GR signaling is often immune-suppressive, and CD4+ T cells do not normally express nuclear progesterone receptor (PR) in non-pregnant women 47 , it is likely that MPA mediates its effects on CD4+ T cells via the GR 48 . However, T cells also express membrane PR 49 and nuclear AR 50 . Thus, we cannot definitively rule out the contribution of these receptors to the differential effects seen in this study. Overall, these studies suggest that differential receptor expression and activity between blood and endometrial CD4+ T cells might be a mechanism by which MPA exerts cell-specific effects on TFV and TAF efficacy. Further studies are needed to define the precise hormone receptors through which progestins exert their effects in CD4+ T cells.

We found that MPA in the presence of TFV and TAF selectively increases the infection of blood and FRT CD4+ T cells by HIV respectively, whereas MPA in the absence of ARVs had no effect. Other studies, however, provide contradictory information. Sampah et al. showed that MPA decreased HIV infection of blood CD4+ T cells, and that this was not associated with decreased levels of activation markers (CD25, CD69, CD38) or the (co)-receptors CD4, CCR5, or CXCR4 51 . In contrast, Huijbregts et al. demonstrated that MPA increased the infection of blood CD4+ T cells, and prevented the downregulation of CXCR4 and CCR5 on activated CD4+ T cells 43 . The reasons for this are unclear, but are likely due to differences in experimental protocols. We introduced MPA after exposure of the CD4+ T cells to HIV, to ensure that differential activation of CD4+ T cells was not a confounding factor, whereas Sampah and Huijbregts treated CD4+ T cells with MPA prior to infection with HIV. We and Huijbregts used replication-competent HIV-BaL while Sampah employed a single-cycle pseudovirus. Further, our maximal concentration of MPA was 100 nM, while the other studies ranged between 7.5 nM (Sampah) and 1000 nM (Huijbregts). Thus, the direct effect of MPA on infection of CD4+ T cells remains unclear, but is likely affected by multiple factors including dose, time of exposure, cell activation state, and viral strain amongst others.

The mechanism involved in the selective MPA inhibition of TFV in blood CD4+ T cells but not in FRT CD4+ T cells and the inhibition of TAF in FRT CD4+ T cells but not in blood CD4+ T cells is currently under investigation. This appears to be yet another example 35 that blood CD4+ T cells are functionally different from FRT CD4+ T cells. This appears to be yet another example 35 that blood CD4+ T Cells are functionally different from FRT CD4+ T Cells. These results suggest that MPA differently affects either the transporters or enzymes involved in TFV/TAF metabolism in the two different cell types. Although specific effects of MPA in the two cell types on the common enzymes utilized by TFV and TFV generated from TAF to form TFV-DP cannot be ruled out, it is quite possible that the effect of MPA might be at specific steps not shared by the two drugs. Specific steps involved might include the energy requiring transport of TFV into the cell and/or the conversion of TAF via Cathepsin A (and subsequent steps) to TFV 27, 40 . Studies are under way to identify the mechanisms through which MPA exerts its differential effects on blood and tissue CD4+ T cells.

Our findings may represent a potential contributing factor to the failure of HIV prevention trials with TFV, in which women were taking hormonal contraceptives, mainly MPA 7, 8 . Our results suggest that under clinical conditions, when TFV concentrations are high, MPA is not sufficient to reverse anti-HIV activity. However, when TFV concentrations are low, as could be envisioned by poor adherence 11 , MPA could potentially increase the risk of HIV infection by decreasing the anti-HIV activity of TFV. No formal comparison between chemical contraceptives and HIV acquisition was performed in the majority of PrEP trials, as most of the participants were taking MPA 7, 9 . However, in a secondary analysis of Partners PrEP, HIV-1 prevention among women using DMPA was efficacious and not significantly different from women using no hormonal contraception 52 . Recognizing that ARV protection was effective in this trial, these findings are consistent with our results demonstrating that high doses of ARV are effective in preventing infection in the presence of MPA. In addition, when considering topical administration of ARVs, it is unclear whether TFV reaches the upper FRT at concentrations to confer protection 17, 18 , and whether this would also be true for TAF. As HIV target cells exist throughout the FRT [21] [22] [23] 35, 53 , MPA could increase the risk of HIV infection in the upper relative to the lower FRT, where ARV concentrations would be expected to be lower after topical (genital) administration. Less lear would be the effect of MPA when ARVs are given orally, recognizing that drug concentrations are sometimes higher in the upper FRT than the lower tract 54 .

It is worth noticing that the TFV and TAF concentrations that protect CD4+ T cells from HIV infection in our in vitro study are significantly lower than those measured in tissues from clinical trials 55 , which suggest that the limit for protection is much higher in vivo than that measured in vitro. Based on our previous studies 15 , it is clear that TFV and its active metabolite TFV-DP are not equally distributed between different cell types in FRT tissues. Rather, because of the concentration of TFV-DP in epithelial cells and fibroblasts, which can be 100-1000 fold higher than that measured in CD4+ T cells 15 , whether under in vivo conditions, relatively small changes in TFV-DP due to MPA result in increased risk of HIV infection remains to be determined.

In conclusion, our results demonstrate that MPA, but not LNG or NET, is able to reverse TFV and TAF anti-HIV protection in blood and tissue CD4+ T cells differentially. As ARVs become increasingly widespread (topical and oral), either due to incorporation in MPTs or treatment for HIV+ individuals, it is essential to understand how they interact with common contraceptives used by millions of women worldwide. Our study demonstrates another mechanism by which MPA may increase the likelihood of HIV acquisition in women -by compromising ARV efficacy. Based on these findings, future MPT studies need to consider the likelihood of ARV/ contraceptive interactions that may compromise ARV efficacy, and increase the risk of HIV infection. Further, our findings highlight the need for testing ARV and hormonal contraceptive interactions in genital tissues to select the appropriate pairs that maximize anti-HIV protection in the FRT and prevent of unintended pregnancy.

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 27 to 51). 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 Leuko Paks from women were obtained from our IRB-approved collection facility at Dartmouth-Hitchcock Medical Center. CD4+ T cells were purified with the CD4+ T cell isolation kit (Miltenyi Biotech) following isolation of peripheral blood mononuclear cells (PBMC) by standard Ficoll density gradient centrifugation 35, 56 . Freshly isolated blood CD4+ T cells were activated in vitro using Xvivo 15 media 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 24hr as described previously 56 . Activated CD4+ T cells were plated at a density of 1 × 10 5 cells per well in round bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media consisting of X-VIVO 15 Media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped 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). Tissues were minced into 1-2 mm fragments and digested at 37 °C for 1hr 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 35 . 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 removed from stromal cells by filtration through a 20-µm mesh filter (Small Parts). Stromal cells were then washed and counted and dead cells removed using a Dead cell removal kit (Miltenyi Biotec, Auburn, CA).

Isolation of tissue CD4+ T cells.

Following removal of dead cells, CD4+ T cells were isolated by negative magnetic bead selection with the CD4+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications, as previously described 35 . Anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4+ T cell population was higher than 90%. Isolated CD4+ T cells were activated for 24 hr with PHA and IL-2 as described for blood CD4+ T cells. Activated CD4+ T cells were plated at a density of 1 × 10 5 cells per well in round-bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media.

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 15, 20, 37 . TAF was kindly supplied by Gilead Sciences Inc. (Foster City, CA) and was dissolved in PBS at 10 mM, sterilely filtered (0.2um) and the concentration checked by absorbance using a molar extinction coefficient of 11690 at 260 nm. Subsequent dilutions of TFV and TAF were made in immune cell media to the appropriate working concentrations. TFV or TAF were added to CD4+ T cells for 24 hr prior to HIV infection or TFV-DP determination. Untreated control cells were donor-matched to treated cells. 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 20, 37 and no changes in viability were found.

Hormone preparation. Medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich, St. Louis, MO), Levonorgestrel (LNG) and Norethisterone (NET) and progesterone (Calbiochem, Gibbstown, NJ) were dissolved in 100% ethanol for an initial concentration of 1 × 10 -3 M, evaporated to dryness and suspended in immune cell complete media to a concentration of 1 × 10 -5 M. Further dilutions were made to achieve a final working concentration of 1 × 10 -7 M. As a control, an equivalent amount of ethanol without dissolved hormone was initially evaporated.

Intracellular TFV-DP measurement. Fresh isolated CD4+ T cells from tissues and activated CD4+ T cells from blood were pre-treated with hormone or hormonal contraceptives for 24hr prior to and during incubation with TFV (3277 μM) or TAF (2 μM) for an additional 24hr. 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 13 . 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 13 . All samples were assayed blind without any information provided as to cell origin and treatment.

HIV-infection. Activated blood or endometrial CD4+ T cells were infected as previously described with minor modifications 56 . 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 57 . Briefly, activated blood or endometrial CD4+ T cells were treated with either TFV (1, 5 and 10 μM) or TAF (1, 5 and 10 nM) for 24hr and then washed to remove extracellular TFV or TAF. After washing, cells were incubated with HIV-BaL for 2hr at an MOI of 0.1 and then washed to remove residual virus. Fresh 0.2 ml IL-2 supplemented immune cell media with or without MPA, NET, LNG or progesterone (1 × 10 7 M) was added to each well and cells then incubated for 5 days, with half of the media from each well collected and replaced with fresh media with or without hormone on day 3. MPA was used at a concentration of 1 × 10 -7 M in all studies based on clinical investigations demonstrating that peak serum MPA levels reach this concentration after intramuscular administration [58] [59] [60] . This experimental sequence was followed in all HIV infection studies when it was found in initial studies that 48hr preincubation of CD4+ T cells with MPA suppressed cell activation and interfered with HIV infection. Released p24 in culture media on day 5 was measured by p24 enzyme-linked immunosorbent assay (Advanced Bioscience laboratories, Rockville, MD) following the manufacturer's recommendations.

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 two groups was performed applying Mann-Whitney U test. Comparison of three or more groups was performed applying Kruskal-Wallis, followed by Dunns-post test for multiple comparison correction. Comparison of HIV infections between CD4+ T cells from blood and tissues in the absence vs presence of hormone studies were analyzed using two-way ANOVA with Bonferroni post-test for multiple comparison correction.

Author Contributions

Conceived and designed the experiments: Z.S., M.R.G., M.V.P., J.E.B., C.R.W. Performed the experiments: Z.S., M.R.G. Analyzed the data: Z.S., J.V.F., J.E.B., M.R.G., A.D.M.K., C.R.W. Contributed reagents, materials, analysis tools: C.R.W., A.D.M.K. Wrote the paper: C.R.W., Z.S., M.V.P., M.R.G., A.D.M.K., J.E.B. All the authors read and approved the manuscript.

Additional Information

Competing Interests: The authors declare that they have 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.Flow cytometric analysis was carried out in DartLab, the Shared Resource at Dartmouth supported by (P30CA023108-37) and (P30GM103415-15).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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