Pharmacological evaluation of an ex vivo cervicovaginal HIV prevention model

TL;DR

This study evaluates how well a lab model of cervical tissue can predict the effectiveness of HIV prevention drugs in different parts of the female genital tract.

Contribution

The study introduces a model to assess regional variability in drug efficacy for HIV prevention in the female genital tract.

Findings

Differences in drug-metabolizing enzyme expression were found between ecto- and endocervical tissues.

Tenofovir diphosphate levels were linked to reduced viral replication in ectocervical tissues.

Ectocervical explants showed consistent viral infectivity and dose-dependent drug inhibition.

Abstract

The female genital tract (FGT) is a unique compartment with physiologically distinct properties complicating the extrapolation of drug efficacy; critical gaps remain in understanding regional variability within the FGT itself. We performed an in-depth investigation across endo- and ectocervical tissues on the utility of the cervical explant model to evaluate pre-exposure prophylaxis (PrEP) efficacy.

Using normal cervical tissues, we evaluated gene expression of relevant drug metabolizing enzymes and transporters (DMETs) via qRT-PCR and compared ecto- and endocervix. To determine differences in drug phosphorylation and to assess antiretroviral (ARV) efficacy, we incubated explants in tenofovir and emtricitabine then measured intracellular metabolites. Viral infectivity and dose–response with ARVs was measured using viral RNA and p24 following HIV-1JR-CSF challenge.

ABCC4 expression was 3-fold lower in ectocervical tissues compared with endocervical, whereas CYP3A5 was 2-fold higher. IL-6 was correlated with ABCB1 (r = 0.52, P = 0.01) and ABCG2 (r = 0.56, P =0.005). Dose-normalized phosphorylation did not differ between endo- and ectocervix (P > 0.5). Infectivity of explants was low (53%) but did not differ by compartment. Intracellular tenofovir diphosphate concentrations were associated with a decrease in ectocervical viral replication (r =0.39, P < 0.05). There was a strong relationship between the proportion of explants infected and emtricitabine dose (P =0.02) but no relationship between intracellular emtricitabine triphosphate and protection.

We identified differences in DMET expression and ARV metabolism between ecto- and endocervical tissues, as well as correlations between DMET and IL-6. Ectocervical explants demonstrated consistent viral infectivity and dose-dependent inhibition. The model is useful in determining tenofovir diphosphate targets.

Figures

DMET expression by cervical compartment. The mRNA expression of 12 DMET genes in addition to IL-6 is shown relative to the housekeeping gene GAPDH (2−ΔCt) in ectocervix (left) and endocervix (right). Presented as median, IQR, and minimum/maximum, * P < 0.05.

Metabolism is comparable across cervical compartments. The percent metabolized on a molar level of the dose-normalized value of tenofovir (TFV) and emtricitabine (FTC) stratified by ectocervix (left) and endocervix (right). Both comparisons P > 0.05. Presented as median, IQR and minimum/maximum.

Dose-dependent HIV inhibition is observed in ectocervical explants. vRNA AUC0–4days percent inhibition in tenofovir (a), emtricitabine (b) and lamivudine (c), and dose–response measured by p24 percent inhibition at Day 4 in tenofovir (d), emtricitabine (e) and lamivudine (f). Means (diamonds) and medians (dashes) are shown.

Intracellular concentrations are higher in tissues protected from viral infection. Tenofovir diphosphate (TFVdp) (a) and emtricitabine triphosphate (FTCtp) (b) concentrations stratified by productive infection in RNA inhibition. Presented as median, IQR.

Tables

Table 1.. Intracellular TFVdp and FTCtp concentrations in ectocervical and endocervical explants

Dose	Intracellular concentrations, fmol/mg tissueMedian (IQR), N		# BLQ, %
	Ectocervix	Endocervix	Ectocervix	Endocervix
TFV 30 µg/mL	2.33 (1.06–38.0), 8	N/Aa	4 (50)	N/A
TFV 100 µg/mL	8.15 (1.12–59.5), 27	4.07 (1.90–162), 10	8 (30)	2 (20)
TFV 300 µg/mL	74.3 (3.48–711), 16	233 (176–1713), 6	3 (19)	1 (17)
FTC 30 µg/mL	13.4 (0.77–96.3), 10	N/Aa	4 (40)	N/A
FTC 100 µg/mL	2.35 (0.75–23.1), 29	0.69 (0.41–14.5), 8	12 (41)	5 (63)
FTC 300 µg/mL	3.60 (0.72 –15.7), 16	9.41 (4.90–22.9), 6	6 (38)	0 (0)

Full text

Introduction

An estimated 40.8 million people are living with HIV, with 1.3 million people newly infected with HIV in 2024;^1^ heterosexual women made up approximately 50% of those new HIV infections. Women need a variety of pre-exposure prophylaxis (PrEP) options that will fit their needs and provide consistent protection. The female genital tract (FGT) is a complex and heterogeneous compartment complicating predictions of PrEP efficacy;^2^ moreover, direct inter-compartment comparisons within the FGT (ectocervical, endocervical) are lacking. Development of reliable PrEP modalities will require determining effective concentrations in the FGT as well as understanding how the tissue microenvironment (e.g. drug transporters and metabolizing enzymes, inflammation, intercompartment variability) may influence efficacy. The cervicovaginal explant model has been a critical tool to advance understanding of HIV transmission. Cervicovaginal explant models have been used to test HIV preventive compounds, including tenofovir, dapivirine, and maraviroc.^3–7^  Ex vivo models allow for inclusion of the heterogeneous tissue microenvironment in evaluations of PrEP candidates.^8^

All current oral PrEP options include nucleos(t)ide reverse transcriptase inhibitors (e.g. tenofovir, emtricitabine, and lamivudine), which require intracellular phosphorylation to become the active metabolites. Intracellular phosphorylated metabolite concentrations may be influenced by expression of cellular membrane transporters and inflammation,^9^ whereas other PrEP candidates may be influenced by local expression of metabolizing enzymes.^10^ The role of drug metabolizing enzymes and transporters (DMETs) and their influence on the explant model is underexplored.

Here we performed an in-depth investigation of DMET expression, inflammation and viral infectivity across ecto- and endocervical compartments. To explore nucleotide phosphorylation in an ex vivo setting, refine viral quantification methods, and identify protective PrEP concentrations, we introduced common PrEP drugs (tenofovir, emtricitabine and lamivudine) to the model. These data will enhance our understanding of the FGT compartment and inform the utility of ex vivo cervicovaginal models for future PrEP development.

Materials and methods

Gene expression

As a prescreen to identify potential genes associated with cervical inflammation, RNAseq was performed using 24 archived cervical specimens collected from women undergoing gynaecological surgery at UNC Hospitals in Chapel Hill, NC. The panel included 31 candidate genes encompassing transporters and enzymes associated with disposition and metabolism of antiretrovirals (ARVs) (Table S1, available as Supplementary data at JAC Online). Cervicitis was defined by the pathologist on initial case reports.

Expression of genes identified from the RNAseq analysis (IL-6, ABCB1, ABCC1, ABCC2, ABCC4, ABCG2, SLC22A3, SLC29A3, CPY3A5, NME1, NME2, AK2 and PGK1) was quantified using real-time PCR in subsequent samples, using GAPDH to normalize expression. RNA was extracted from untreated and uninfected cervical tissue using the RNeasy blood and tissue kit (Qiagen, Hilden, Germany) and transcribed to cDNA using Superscript IV Vilo (Thermo Fisher Scientific, Waltham, MA, USA). We measured gene expression via off-the-shelf TaqMan assays (Table S2). The 2^−ΔCt^ method was used to determine expression relative to GAPDH.

Tissue procurement and experimental design

Cervical explant procedures were adapted from those previously described.^3,4,6,11,12^ Fresh cervical tissue was procured via University of Minnesota Bionet (Minneapolis, MN, USA), which obtained written informed consent from HIV-negative persons undergoing gynaecological surgery (IRB exempt STUDY00019053). Tissue was transported on ice in Iscove’s Modified Dulbecco’s Medium (Gibco) containing FBS, nystatin, penicillin/streptomycin and MEM, then processed immediately to remove underlying mucosa and create 3 mm explants with a biopsy punch. Demographic data including age, cervicitis history and comedications were recorded. Depending on experimental assignment, explants were stored in RNAlater (Qiagen), snap-frozen or cultured.

To compare the time course of viral RNA (vRNA) versus p24 (Figure S1a), explants were incubated with >10^5^ TCID_50_ HIV-1_JR-CSF_ for 3 h, washed extensively, then transferred to a prepared gelfoam raft and incubated for up to 14 days. Media were changed and supernatants collected for p24 on Days 4, 7, 10 and 14; explants were harvested at multiple timepoints for vRNA quantification. p24 was measured by ELISA (BioChain, Newark, CA, USA); vRNA was measured from extracted RNA of tissue homogenate using Precellys mixed bead tubes (Cayman Chemical, Ann Arbor, MI, USA) and the RNeasy mini kit (Qiagen), converted to cDNA with Superscript IV Vilo (Invitrogen, Carlsbad, CA, USA), and measured via an RT-qPCR assay designed to detect mRNA copies of the viral protein nef from the D4-A7 splice junction in the HIV-1_JR-CSF_ genome, which we previously demonstrated as able to rapidly detect viral replication in explants.^6^

To examine ARV intracellular metabolites, explants were incubated with tenofovir or emtricitabine for 24 h, then collected, weighed, snap-frozen and analysed by LC-MS/MS. Metabolism of phosphorylated ARV (tenofovir diphosphate, emtricitabine triphosphate) was expressed as the molar percent of metabolite relative to parent drug incubation concentration.

For determination of PrEP efficacy (Figure S1b), explants were incubated with or without ARV (tenofovir, emtricitabine, lamivudine) at 0, 30, 100 or 300 μg/mL for 24 h, exposed to >10^5^ TCID_50_ HIV-1_JR-CSF_ for 3 h, washed extensively, then transferred to a prepared gelfoam raft and incubated for up to 4 days with drug concentrations in media maintained throughout. As above, vRNA was measured by the spliced RT-qPCR assay. One explant was collected at the end of 3 h viral incubation to account for residual viral inoculum and this was used as the baseline sample. Percent inhibition was calculated by the percent difference in vRNA copies/mg tissue between the control and drug-treated tissues. Viral peak was defined as the maximum percent change from baseline, AUC_0–4d_ was calculated as fold change from baseline, and productive infection was defined as 1.5-fold change (≥50%) above baseline. This definition of infection was defined a priori based on prior experiments as a way to normalize variability across inocula with variable infectivity. Expanded methods can be found in the Supplemental methods.

Statistical analysis

Drug concentrations and gene expression data were log-transformed for statistical analyses. Incubation dose was normalized by expressing tenofovir diphosphate and emtricitabine triphosphate as a molar percentage of tenofovir or emtricitabine incubation concentrations. Comparisons between groups were done using Student’s t-test and paired t-tests, while associations between continuous variables were performed using Pearson’s test. Statistics were performed in SAS 9.4 (Cary, NC, USA). Data are expressed as median (IQR) unless otherwise noted.

Results

Expression of DMET genes in cervical tissues

Of 31 genes initially screened in archived RNA, 5 met genome-wide significance with a false discovery rate of <5% (Figure S2). Four other genes were identified using t-test without adjusting for multiple comparisons. Three genes [ABCB1, ABCG2 (efflux transporters) and SLC29A3 (uptake transporter)] were up-regulated in tissues with cervicitis whereas six [ABCC1, ABCC5 (efflux transporters), CYP3A5 (metabolizing enzyme), NME1, NME2 and PGK1 (nucleotide kinases)] were down-regulated. These genes were further explored in subsequent explant studies to validate altered expression in inflammatory states and associations with intracellular NRTI metabolism. Additional genes were examined due to their known association with tenofovir, emtricitabine or lamivudine metabolism including ABCC4, an efflux transporter, and AK2, a phosphorylating enzyme of tenofovir.^9^

Expression of candidate genes in ecto- and endocervical tissue is summarized in Figure 1. ABCC4 was 3-fold lower in ectocervical tissues (0.0033; IQR 0.0014–0.00089; n = 17) compared with endocervical (0.0104; IQR 0.0032–0.0196; P =0.01; n = 16); conversely, CYP3A5 was 2-fold higher in ectocervical tissues (0.0011; IQR 0.0007–0.0032; n =8; endocervical: 0.0005; IQR 0.0003–0.0008; P = 0.02; n =12). In a subset of samples where both ectocervix and endocervix were available from the same donors, only ABCC4 remained significantly lower in ectocervix in a paired analysis (P = 0.049, n = 10). Two DMET genes were associated with IL-6 gene expression: ABCB1 (r = 0.52, P =0.01, n = 23) and ABCG2 (r =0.56, P =0.005, n = 23). Whereas no relationship was observed within endocervix, ectocervix IL-6 was correlated with ABCB1 (r = 0.8, P =0.003, n = 11), ABCG2 (r = 0.85, P = 0.0008, n = 11), SLC22A3 (r = 0.78, P =0.005, n =11) and NME1 (r =0.65, P =0.02, n =12).

DMET expression by cervical compartment. The mRNA expression of 12 DMET genes in addition to IL-6 is shown relative to the housekeeping gene GAPDH (2−ΔCt) in ectocervix (left) and endocervix (right). Presented as median, IQR, and minimum/maximum, * P < 0.05.

Intracellular metabolism of nucleos(t)ide analogues in cervical explants

Generally, there was an increase in metabolite concentrations with increasing incubation dose, with the exception of median emtricitabine triphosphate concentrations at 30 µg/mL being higher than the median at 100 or 300 µg/mL (Table 1). Intracellular phosphorylation was not statistically significant between the two compartments (Figure 2). When limited to 100 µg/mL and 300 µg/mL doses to remove bias from samples below the limit of quantitation (BLQ), the percent metabolized remained similar between ectocervix and endocervix when dosed with tenofovir (P = 0.53, n =43 ectocervix, 16 endocervix) or emtricitabine (P = 0.95, n = 45 ectocervix, 14 endocervix). In cases where endocervix and ectocervix were available from the same donor, there was no difference between metabolism in paired analyses (tenofovir: P =0.5, n = 15; emtricitabine: P = 0.2, n= 13). Results were consistent when comparing untransformed concentrations at individual doses (Table 1).

Metabolism is comparable across cervical compartments. The percent metabolized on a molar level of the dose-normalized value of tenofovir (TFV) and emtricitabine (FTC) stratified by ectocervix (left) and endocervix (right). Both comparisons P > 0.05. Presented as median, IQR and minimum/maximum.

Spliced RNA assay detects early infection in explant model

To compare p24 in culture supernatant and spliced vRNA in tissue homogenate as methods to quantify infection in the explant model, time course experiments were performed in 12 tissue cases with sampling occurring over 14 days post infection (Figure S3). In 10 cases, ectocervix vRNA fold change was greater than 50% above baseline, suggesting productive infection. Seven (of 10 infected) showed continued increasing vRNA over time, further supporting productive infection. This productive infection could be seen as early as Day 2 post infection. In contrast, only three cases (of 10 infected) showed increasing p24, suggesting sustained replication but not until Days 9–11. Data in endocervix (n = 10) showed four cases with productive infection. None of these showed increasing vRNA over time whereas only 2 of 10 showed increasing p24 over time.

Determinants of infectivity in cervical explants

Using the 4 day model, the percent of ectocervical and endocervical tissues productively infected was similar: 28/52 (54%) of ectocervical tissues and 3/6 (50%) of endocervical (P > 0.9). Viral replication also did not differ between ectocervical (n = 52) and endocervical (n = 6) explants over 4 days (viral AUC_0–4days_: 4.9; IQR: 3.0–7.3; versus 4.3; IQR: 3.4–7.0; P = 0.9). In paired comparison, where ectocervix and endocervix are from the same donor (n = 6), viral replication was on average 4-fold higher in ectocervix, but this did not meet significance (viral AUC_0–4days_, P = 0.5). Gene expression of IL-6 was not predictive of infectivity in cervical explants. IL-6 was not correlated with viral AUC_0–4days_, or viral peak (P = 0.9). IL-6 expression was similar in tissues with and without productive infection (P = 0.2).

Dose–response relationships in cervical explants

We examined the dose–response of tenofovir, emtricitabine and lamivudine vRNA (viral AUC_0–4days_) inhibition and total p24 reduction (Figure 3). A general dose–response was observed with tenofovir in vRNA and p24 measurements and a median of 80% viral inhibition was observed at 300 µg/mL tenofovir (Figure 3a). Although not statistically significant, the proportion of explants infected generally decreased with increasing dose (P = 0.2). The p24 dose–response was less pronounced over the dose range (Figure 3d).

Dose-dependent HIV inhibition is observed in ectocervical explants. vRNA AUC0–4days percent inhibition in tenofovir (a), emtricitabine (b) and lamivudine (c), and dose–response measured by p24 percent inhibition at Day 4 in tenofovir (d), emtricitabine (e) and lamivudine (f). Means (diamonds) and medians (dashes) are shown.

There was a strong relationship between the proportion of explants infected and the emtricitabine dose (P = 0.02). There was a strong dose–response observed, with 100% vRNA inhibition observed at the highest dose tested (300 µg/mL) (Figure 3b). Like tenofovir, there was a general, yet less pronounced, dose–response relationship between p24 reduction and dose (Figure 3e).

There was no relationship between lamivudine dose and proportion of explants infected (P = 0.99). There was no dose–response relationship between lamivudine dose and vRNA inhibition (Figure 3c). There was a subtle dose–response relationship between lamivudine dose and p24 inhibition, with no effect seen at 30 or 100 µg/mL and 25% inhibition at 300 µg/mL (Figure 3f).

Relationship between intracellular concentrations and viral replication in cervical explants

Intracellular tenofovir diphosphate was significantly correlated with reduction in vRNA (r = 0.39, P < 0.05, n = 38) but not in reduction of p24 (r = 0.26, P = 0.1, n = 41). The mean concentration of tenofovir diphosphate in protected explants was 30.4 fmol/mg (n = 6) compared with 4.3 fmol/mg in explants that were productively infected (n = 39) (Figure 4). Although no statistically significant relationship between emtricitabine triphosphate and vRNA inhibition was observed (r = −0.13, P = 0.4, n = 40), concentrations of emtricitabine triphosphate were >20-fold lower in tissues that were productively infected (0.85 fmol/mg, n = 34) compared with those that were not (18.6 fmol/mg, n = 12) (Figure 4). In addition, there was a surprising negative correlation between emtricitabine triphosphate and p24 inhibition (r = −0.34, P = 0.03). Lamivudine triphosphate concentrations were not available.

Intracellular concentrations are higher in tissues protected from viral infection. Tenofovir diphosphate (TFVdp) (a) and emtricitabine triphosphate (FTCtp) (b) concentrations stratified by productive infection in RNA inhibition. Presented as median, IQR.

Discussion

To improve the utility of the explant model to evaluate PrEP compounds, we sought to characterize factors that influence drug distribution within the FGT. In addition to drug distribution factors, such as DMET and inflammation, influence on infectivity and concentration–response relationships were examined using an ex vivo cervical explant challenge model. We examined inter-compartmental differences between ecto- and endocervical regions, compared viral quantification methods, and evaluated metabolism of antiretrovirals. These parameters provide a critical foundation for future applications of the explant model to evaluate PrEP efficacy and identify pharmacological targets in the FGT.

First, we examined expression of DMET genes in ecto- and endocervical tissues to determine the importance of localization within the lower FGT. Although most genes examined were not significantly different between ecto- and endocervix, CYP3A5 expression was higher in ectocervical tissues. Nucleos(t)ides are not substrates of CYP3A5; however, this differential expression between tissue sites could have consequences for the metabolism of other ARVs used in treatment or prevention of HIV.^10^ The influence of inflammation on the expression of DMET genes and intracellular drug concentrations was also examined. IL-6 was associated with the expression of SLC22A3, ABCB1 and ABCG2, suggesting that local inflammation may influence the disposition of substrates for these transporters.

Intracellular tenofovir diphosphate was not correlated with inflammation or expression of any DMET genes. Given that ABCC4 encodes MDR protein 4 (MRP4), an efflux transporter of tenofovir, we anticipated a correlation between increased ABCC4 and decreased tenofovir diphosphate due to efflux of tenofovir.^13^ Although we saw a trending decrease of tenofovir diphosphate as ABCC4 expression increased, it did not reach statistical significance. Despite higher expression of ABCC4 in endocervix, this did not confer any difference in tenofovir diphosphate. One limitation of our analysis is that we did not examine protein expression, nor did we genotype these samples although functional variants of ABCC4 are known to exist.^14^ We saw similar inverse relationships between ABCC4 expression and tenofovir diphosphate in both ecto- and endocervix. Like the 2014 findings of Zhou et al.,^15^ that these tissues have similar mRNA MRP4 expression, our study demonstrated similar mRNA expression levels of MRP4 efflux transporters in ectocervix and endocervix, although it is important to note that mRNA expression does not always predict functional protein. We previously performed immunohistochemistry to quantify MRP4 protein, finding that expression in endocervical glandular mucosa tissues was significantly higher when compared to ectocervical squamous epithelium.^16^

Culture supernatant p24 has been the most common method to quantify viral growth; however, a minimum 9–15 days is required to achieve reliable viral growth in cervicovaginal explants.^17^ Our group previously described a spliced RNA assay able to detect viral growth within 3 days.^6^ Here, we directly compared viral reduction by vRNA and p24 as measures of viral infection. Spliced RNA could consistently detect infection at timepoints earlier than p24, as early as Day 2 post infection, leading to shorter culture durations and greater efficiency. This agrees with Rollenhagen and Asin,^18^ who reported that differences could not be detected by p24 until Day 7, whereas there was detectable infection by PCR at Day 5.

We observed variability in the infectivity of our explants, with roughly 50% of exposed tissues not becoming infected. However, we did not observe a significant difference in infectivity between endocervix and ectocervix. Other researchers have observed variability in viral replication from varying FGT compartments. Trifonova et al.^19^ used polarized ectocervical and endocervical explants to demonstrate that although endocervix had fewer susceptible cells, greater viral replication (by p24) was observed. Meanwhile, Dezzutti et al.^20^ illustrated that infected ectocervix explants showed a drastically greater replication rate of HIV-1 compared with other vaginal regions. Our data further refine our understanding of variability in infectivity across the FGT. Although there are some data that suggest the endocervix is a critical site of HIV transmission in humans,^21,22^ our findings suggest that ectocervical tissue (which has greater surface area and therefore opportunity for greater number of replicates) may be reasonable to use.

Using our explant model, we were able to evaluate the efficacy of three nucleos(t)ide analogues in ectocervical tissue. Given that these drugs require intracellular phosphorylation to their active metabolites, it is important to quantify the extent to which our model’s tissues can phosphorylate parent tenofovir and emtricitabine to tenofovir diphosphate and emtricitabine triphosphate, respectively. Modest differences in the concentrations of phosphorylated metabolites between endocervical and ectocervical were observed. Regional variations in tenofovir diphosphate concentrations have been previously reported although it is unclear whether these differences are due to differential distribution of tenofovir to tissue sites or differential metabolism due to cell populations of distinct sites.^23–25^ Although we did not observe significant correlations between IL-6 mRNA and tenofovir diphosphate or emtricitabine triphosphate, inflammation likely affects cellular activation within tissues, which could contribute to variable metabolism.^26,27^

The ex vivo tissue model has been used to estimate efficacy and assess antiviral inhibition prior to Phase III clinical trials.^3–5,28–30^ Previous cervicovaginal explant experiments with tenofovir found similar HIV inhibition efficacy.^4–6^ Rohan et al.^3^ observed 100% inhibition with a higher dose of 1 mg/mL, which was meant to simulate the 1 mg/mL tenofovir gel in development at the time. Although these incubation doses are higher than those achieved in tissues with clinically relevant dosing, when looking at intracellular concentrations of the active metabolites, these concentrations are similar to those observed in clinical trials. It is plausible that ex vivo conversion of parent to metabolite is less extensive than in vivo. This highlights the importance of measuring the intracellular metabolite directly in the ex vivo model to accurately translate clinical relevance.

To our knowledge, no prior viral cervical explant challenges with emtricitabine or lamivudine have been performed. Along with tenofovir, emtricitabine is part of currently approved oral PrEP regimens, but its role in the effectiveness of these regimens has been understudied. Given the low penetration of tenofovir to the FGT, emtricitabine may be a critical driver of efficacy, especially as the two drugs may have a synergistic effect on viral inhibition.^31^ Our findings suggest more consistent inhibition from emtricitabine than with tenofovir although we could not model an EC_50_.

Although our experiments sought to improve the pharmacological aspects of the explant model, there were several limitations. In our determination of the percent of parent drug that is phosphorylated, we did not measure parent drug directly and therefore our calculation assumes that all the parent drug is available for phosphorylation. Another limitation is that our observed dose–response was not linear across the dose range. Although the highest dose tested consistently had the highest inhibition across all three drugs tested, there was little discrimination between the lower doses tested. Despite this, this range of drugs and doses tested exceeds those commonly tested in other models, which typically focus on one dose.^3–5,12^

Despite the variability in the explant model, these ex vivo evaluations provide unique insight into the dose–response relationship in female genital tissues. We found minor differences in infectivity or drug phosphorylation between endocervix and ectocervix. This supports the utility of the model in that extrapolation can be made across populations despite variability in donor-specific factors. Models that can fully recapitulate the microenvironment of the FGT are sorely needed to fully optimize current and future PrEP regimens. Future models that can incorporate the presence of sexually transmitted infections, vaginal dysbiosis, or hormone differences found in pre- and postmenopausal women, could accelerate future PrEP candidates leading to more prevention options for women.

Supplementary Material

dkag103_Supplementary_Data