Natural Substrate Concentrations Can Modulate the Prophylactic Efficacy of Nucleotide HIV Reverse Transcriptase Inhibitors

Abstract

Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/10⁶ cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/10⁶ mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors.

INTRODUCTION

The human immunodeficiency virus (HIV)/AIDS pandemic remains one of our greatest public health challenges. Globally, an estimated 33.2 million people were living with HIV infection or AIDS in 2007. In that year, the annual incidence of new infections was an estimated 2.7 million, and there were an estimated 2.0 million HIV-related deaths (20). The ongoing high incidence of HIV infection and the incomplete coverage of basic HIV prevention tools underscore the need for new, highly effective biomedical HIV interventions to complement existing prevention strategies.

Oral administration of antiretroviral drugs prior to and during HIV exposure (preexposure prophylaxis [PrEP]) is a novel intervention to protect high-risk HIV-1-negative people from becoming infected (3, 12, 15). Drug candidates for oral PrEP have been selected from drugs currently approved for treatment of HIV-1-infected individuals. Among the drugs available, the well-established potency and tolerability of tenofovir disoproxil fumarate (TDF), the approved oral prodrug of the nucleotide analog tenofovir (TFV), makes it an attractive candidate for PrEP. A recently concluded human trial with a daily combination of TDF and emtricitabine (FTC) (Truvada) for HIV-seronegative men or transgender women who have sex with men has shown a 44% reduction in the incidence of HIV-1, giving the first indication that oral PrEP may potentially provide an additive effect with current proven HIV prevention measures (14).

TDF is the salt of a lipophilic and cell-permeant prodrug of TFV optimized for effective oral delivery. While oral administration of TDF improves the efficiency of lymphocyte drug loading over that with subcutaneous administration of TFV (5), the effect of the prodrug is to some extent limited by instability. GS7340 is an oral prodrug of TFV whose increased stability allows for further enhancement of the delivery of TFV into cells (6, 26). As with TDF, the active intracellular metabolite of GS7340 is TFV diphosphate (TFV-DP), which competes with the natural dATP substrate for incorporation by HIV reverse transcriptase (RT) and acts as a chain terminator. GS7340 has potent anti-HIV activity in culture, with a 50% effective concentration (EC₅₀) for HIV that is 1,000-fold greater than that of TFV (26). For HIV-infected patients, oral administration of 50 or 150 mg GS7340 for 14 days resulted in concentrations of TFV-DP in peripheral blood mononuclear cells (PBMCs) that were as much as 50-fold higher than those with 300 mg of TDF, and a better antiviral response (26a). The effective delivery of TFV into lymphoid cells and tissues by GS7340 suggests a great potential for this prodrug in PrEP. Since the intracellular half-life of TFV-DP exceeds 4 days (18, 31), use of the GS7340 prodrug might also reduce the frequency of drug dosing and allow for intermittent, coitally disassociated PrEP regimens.

Because of the favorable pharmacokinetic (PK) profile and potent antiviral activity of GS7340, we hypothesized that a single weekly drug dose might be sufficient to prevent infection. We tested this hypothesis by giving prophylactic GS7340 treatment to macaques 3 days prior to rectal exposure to simian-human immunodeficiency virus (SHIV). Paradoxically, we found that GS7340 was not protective despite resulting in high rectal and systemic TFV-DP levels. We explain this paradox by showing that rectal mononuclear cells have a high content of the natural dATP substrate, which renders TFV-DP less effective. These findings demonstrate that high systemic drug levels and potent antiviral activity are not sufficient to prevent rectal SHIV transmission in macaques, and they point to the importance of natural substrate concentrations at the virus point of entry for PrEP effectiveness.

MATERIALS AND METHODS

Drug preparation and administration.

GS7340 was prepared in 50 mM citric acid and was given orally at 13.7 mg/kg of body weight. TDF was prepared as described previously and was used orally at 22 mg/kg (11). On the bases of TFV equivalents, the TDF dose corresponds to 9.55 mg/kg of TFV and the GS7340 dose corresponds to 6.37 mg/kg of TFV, or about three-fourths of the oral TDF dose. All drugs were given by gavage to anesthetized macaques via a gastric feeding tube (11). TDF and GS7340 were provided by Gilead Sciences.

Efficacy of GS7340 against rectal SHIV transmission.

The efficacy of GS7340 in preventing rectal SHIV transmission was evaluated using a repeat-exposure macaque model described previously (11, 30, 33). Male Indian rhesus macaques were exposed rectally once weekly to a SHIV_SF162P3 chimeric virus that contains the tat, rev, and env coding regions of HIV-1_SF162 in a SIVmac239 background (National Institutes of Health AIDS Research and Reference Reagent Program [17]). The SHIV_SF162P3 challenge dose was 10 50% tissue culture infective doses (TCID₅₀) or 7.6 × 10⁵ RNA copies. Virus exposures (as many as 14) were carried out by nontraumatic inoculation of 1 ml of SHIV_SF162P3 into the rectal vault via a sterile gastric feeding tube of adjusted length (11, 30, 33). Macaques were anesthetized with standard doses of ketamine hydrochloride. Anesthetized macaques remained recumbent for at least 15 min after each intrarectal inoculation. Virus exposures were stopped when a macaque became SHIV RNA positive. Treated macaques that became infected continued receiving one weekly GS7340 dose for 12 to 14 weeks in order to monitor for the emergence of drug resistance. The Institutional Animal Care and Use Committee of the Centers for Disease Control and Prevention approved this study.

Infection monitoring by molecular and serologic testing.

SHIV RNA in plasma was quantified using a real-time RT-PCR assay described previously (33). This assay format has a sensitivity of 50 RNA copies/ml. SHIV_SF16P3 carrying the K65R mutation was detected at a low frequency in plasma by a sensitive allele-specific real-time PCR method described previously (19). Virus-specific serologic responses (IgG and IgM) were measured with a synthetic-peptide enzyme immunoassay (EIA) (Genetic Systems HIV-1/HIV-2 EIA; Bio-Rad, Redmond, WA). Animals were considered uninfected if they remained seronegative, negative for SHIV RNA in plasma, and negative for SHIV DNA in PBMCs during PrEP and during the following 70 days of washout in the absence of any drug treatment (11).

Separation of total mononuclear cells and cell subpopulations from blood and rectal tissues.

PBMCs were prepared using standard procedures. Red blood cells were lysed to minimize interference with TFV-DP determination (4). Lymphoid tissues were homogenized using a cell strainer, followed by Ficoll separation (Lymphocyte Separation Medium [LSM]; MP Biomedicals, Aurora, OH). Rectal tissues were dissociated using an enzyme cocktail containing collagenase type II, elastase, hyaluronidase, and DNase I by following procedures kindly provided by Francois Villinger. After digestion, cell suspensions were added to LSM in order to enrich the preparations with mononuclear cells. All cells were counted using a Guava cell counter (PBMCs) with CytoSoft data acquisition and analysis software (version 6.0.2; Millipore, Billerica, MA).

Total CD4⁺ cells, monocytes, and NK cells from blood and tissues were separated by magnetic cell sorting (MACS) using MACS technology according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). CD4⁺ cells were purified from blood and rectal tissues by negative selection using a CD4⁺ T cell isolation kit. Monocytes from blood were positively selected using a monoclonal anti-CD14 antibody. The CD14⁻ fraction was then enriched in NK cells by use of anti-CD16 antibodies. NK cells were included in the analysis because they are known virus reservoirs that can be persistently infected by HIV (36). After magnetic cell sorting, one fraction was used to measure intracellular TFV-DP as described below. Cell purity was evaluated by fluorescence-activated cell sorting (FACS) analysis. Median purity was 92.8% (minimum [min], 64%; maximum [max], 97%) for blood CD4⁺ cells, 94.3% (min, 88%; max, 98%) for blood CD14⁺ cells, and 79.5% (min, 52%; max, 98%) for blood CD16⁺ cells.

Measurement of intracellular TFV-DP and dATP in mononuclear cells from blood and tissues.

Intracellular TFV-DP and dATP concentrations were measured using an automated on-line weak anion-exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography-tandem mass spectrometry (MS-MS) using methods published recently (23). TFV-DP and dATP were monitored through 448→270 and 492→136 m/z fragments, respectively, with [¹³C₅]adenine-labeled TFV-DP as the internal standard. TFV-DP and dATP concentrations were measured in cells from blood (total mononuclear cells, CD4⁺ cells, monocytes, and NK cells), rectal tissues (total mononuclear cells and CD4⁺ cells), and lymphoid tissues (axillary, mesenteric, and inguinal lymph nodes) collected from 4 infected macaques at necropsy. Animals received a single oral dose of GS7340 orally 3 days prior to necropsy. Four additional macaques were used to evaluate the rate of decay in intracellular TFV-DP levels in PBMCs after a single GS7340 dose.

Measurement of TFV levels in plasma and rectal secretions.

The kinetics of distribution of TFV in plasma and rectal secretions were evaluated by administering a single dose of GS7340 to 4 macaques, followed by collection of blood and rectal secretions at 2 h, 5 h, and 24 h. Concentrations of TFV in plasma or rectal secretions were measured by high-performance liquid chromatography (HPLC)–MS-MS as described recently (24). Tenofovir isotopically labeled with [¹³C₅]adenine ([¹³C₅]-TFV) was used as an internal standard (Moravek Biochemicals, Brea, CA) (24).

Rectal secretions were collected in wicks (Weck-Cel surgical spear; Medtronic Ophthalmics, Jacksonville, FL) using a previously established protocol (8). Briefly, dry wicks (2 consecutive wicks/animal) were inserted 5 cm into the rectums of recumbent macaques and were maintained during 5 min. TFV was eluted from the wicks within 2 h using a phosphate buffer solution containing 0.25% bovine serum albumin and 10% Igepal (Sigma-Aldrich, St. Louis, MO) (8). After 15 min on ice to allow for the buffer to diffuse, each wick was transferred to a PCR purification column (QIAquick PCR purification kit; Qiagen) and was centrifuged at 14,000 rpm for 5 min at 4°C to extract TFV (8). The results are expressed as nanograms per milliliter of secretion.

Susceptibility of SHIV_SF162P3 to TFV-DP and impact of dATP concentrations on reverse transcription at high TFV-DP concentrations.

The susceptibility of wild-type (WT) SHIV_SF162P3 RT to TFV-DP was determined using the Amp-RT assay. The Amp-RT assay detects RT activity by using a known nonretroviral heteropolymeric RNA template derived from the encephalomyocarditis virus (EMCV) genome and a complementary EMCV-specific DNA primer (9). Testing conditions included triplicate RT reactions carried out with the RT activity contained in approximately 10⁵ particles of WT SHIV_SF162P3 (as measured by genomic RNA levels). An isogenic TFV-resistant SHIV_SF162P3 mutant containing the K65R mutation was used as a control. RT reactions were carried out in the absence or in the presence of TFV-DP (0.005 to 50 μM) and with a fixed concentration of 5 μM dATP. The other three deoxynucleoside triphosphates (dNTPs) were used at 20 μM each (10). The RT-generated EMCV cDNA was detected by real-time PCR amplification. To evaluate the impact of dATP on the ability of TFV-DP to block reverse transcription, the same RT input was tested using 5 μM TFV-DP and variable concentrations of dATP (0.5 to 500 μM).

Statistical analysis.

The Cox proportional hazards model was used to estimate instantaneous risk for infection, as a hazard ratio (HR), for controls relative to treated animals, assuming constant risk at all inoculations. Graphical methods of model assessment supported the use of Cox proportional hazards regression. The Wilcoxon rank sum statistic was implemented to test for group differences in magnitude of peak virus load. TFV-DP and dATP levels were compared using a mixed-effects model, with a random intercept and unstructured covariance to account for within-subject correlated measurements. WinNonlin software (version 5.2; Pharsight Corporation) was used to calculate the area under the concentration-time curve at 24 h (AUC₂₄) and drug half-lives. Statistical analyses were performed with SAS software (version 9.1; SAS Institute).

RESULTS

Pharmacokinetic profile of TFV in blood and rectal secretions.

The pharmacokinetic profile of GS7340 in blood and rectal secretions was evaluated at the first dose for 4 macaques and was compared with that previously seen at the first dose of TDF (8). GS7340 was given at 13.7 mg/kg and TDF at 22 mg/kg. On the bases of TFV equivalents, the GS7340 dose corresponds to 6.37 mg/kg of TFV and the TDF dose corresponds to 9.55 mg/kg of TFV. Figure 1 shows the levels of TFV achieved over 24 h in both plasma and rectal secretions. Plasma TFV concentrations peaked at 2 h with both GS7340 and TDF (Fig. 1A). The mean area under the concentration-time curve at 24 h (AUC₂₄) for TFV in plasma was 3,889 ng × h/ml (min, 3,117 ng × h/ml; max, 5,594 ng × h/ml) with GS7340, compared to 1,346 ng × h/ml (min, 1,130 ng × h/ml; max, 1,773 ng × h/ml) with TDF. The mean half-life of TFV in plasma was 6.3 h (min, 5.8 h; max, 6.7 h) after GS7340 dosing and 13 h (min, 8 h; max, 19 h) after TDF dosing. In rectal secretions, TFV levels peaked at 24 h with both prodrugs, with low or undetectable concentrations seen within 2 to 5 h (Fig. 1B). Thus, the overall kinetics of distribution of TFV in plasma and rectal secretions after GS7340 dosing were similar to those seen with oral TDF, although TFV levels appear to be higher with GS7340 than with TDF.

Fig. 1.

Pharmacokinetic profiles of GS7340 and TDF at the first dose in plasma (A), rectal secretions (B), and peripheral blood mononuclear cells (PBMCs) (C). Graphs show the median TFV or TFV-DP levels and the extreme values (min, max) observed in 4 animals. Levels of TFV-DP after TDF dosing were reported recently (8) and are added for comparison.

We also measured intracellular TFV-DP concentrations in PBMCs after a single GS7340 dose. Median TFV-DP concentrations, in femtomoles per 10⁶ cells, were 2,581 at 2 h, 4,845 at 5 h, 3,628 at 1 day, 1,597 at 3 days, and 966 at 7 days. Overall, these values are 50 to 100 times those seen at first dose in macaques receiving a human-equivalent dose of TDF (20 fmol/10⁶ cells at 2 h, 41 fmol/10⁶ cells at 1 day, and 26 fmol/10⁶ cells at 5 days) (Fig. 1C) (8).

Lack of prophylactic efficacy of GS7340 despite high TFV-DP levels in PBMCs.

We next investigated whether the high TFV-DP levels seen after oral GS7340 dosing could be sufficient to protect macaques against SHIV infection. We used a repeat low-dose rectal SHIV transmission model with weekly virus challenges. Six male Indian rhesus macaques received one weekly GS7340 dose followed by exposure to SHIV_SF162P3 3 days later. Infection rates were compared to those seen in 32 untreated controls, of which 3 were real-time controls and 29 were historical controls exposed to the same virus stock and dose under identical conditions. Figure 2a shows the cumulative percentage of uninfected animals relative to the number of virus exposures. Despite the high intracellular TFV-DP levels achieved with GS7340 at 3 days (∼1,200 fmol/10⁶ cells) (see below), 4/6 treated macaques became infected at challenges 2 (2 animals), 3 (1 animal), and 4 (1 animal), as did all 3 real-time untreated controls, which were infected at challenges 1, 3, and 5. The challenge series for the remaining 2 treated animals were stopped at exposure 5 after an interim analysis showed an absence of efficacy compared to the combined 32 real-time or historical controls (HR, 1.9; P, 0.23). Although they received only one weekly dose of GS7340, the peak viremia level in macaques infected during GS7340 prophylaxis (median, 5.5 log₁₀ RNA copies/ml; min, 3.1 log₁₀ RNA copies/ml; max, 5.9 log₁₀ RNA copies/ml) was significantly lower (P, 0.01) than that in untreated controls (median, 7.2 log₁₀ RNA copies/ml; min, 5.3 log₁₀ RNA copies/ml; max, 8.9 log₁₀ RNA copies/ml), a finding that is consistent with the potent antiviral activity of GS7340 and the long intracellular persistence of TFV-DP (Fig. 2B) (26a). We also monitored for drug resistance emergence during continuous exposure to one weekly dose of GS7340 for 12 to 14 weeks. None of the 4 infected macaques developed the K65R mutation associated with TFV resistance.

Fig. 2.

Prophylactic efficacy of one weekly GS7340 dose given 3 days prior to virus exposure. (A) Lack of protection against repeated rectal SHIV exposures. Each survival curve represents the cumulative percentage of infected macaques as a function of weekly virus exposure. (B) Acute viremia in macaques infected during GS7340 treatment. Red lines, animals with GS7340 failure (n = 4); blue lines, real-time untreated controls (n = 3); gray lines, historical untreated controls (n = 22). Median peak viremia levels in breakthrough animals were significantly lower than those in untreated macaques (P, 0.01 by the Wilcoxon rank sum test).

To confirm that breakthrough infections were not due to low TFV levels, we measured TFV-DP levels in PBMCs at each weekly virus exposure in all treated animals. Figure 3 shows that TFV-DP concentrations at 3 days were high and similar in infected (971 fmol/10⁶ cells; 95% confidence interval [95% CI], 731 to 1,211 fmol/10⁶ cells) and uninfected (1,365 fmol/10⁶ cells; 95% CI, 1,025 to 1,705 fmol/10⁶ cells) macaques (P = 0.06). Overall, TFV-DP levels seen 3 days after GS7340 dosing were 29-fold higher than those seen at peak (24 h) with oral TDF (41 fmol/10⁶ cells) (8).

Fig. 3.

Intracellular TFV-DP levels in PBMCs in infected and uninfected macaques. TFV-DP was measured over 7 weeks at the times of virus exposure for the 4 infected (red lines) and 2 uninfected (blue lines) animals. Mean TFV-DP levels in the two groups were comparable (P = 0.06).

High intracellular TFV-DP concentrations in cellular subpopulations from blood and in rectal and lymphoid tissues.

The failure to protect macaques with GS7340 was unexpected given the high intracellular TFV-DP concentrations seen in PBMCs 3 days after drug dosing (Fig. 1 and 3). To better understand this lack of prophylactic efficacy, we investigated whether GS7340 efficiently delivers TFV to different cell subpopulations and to tissues. We first measured TFV-DP in cell subpopulations from blood by administering a single dose of GS7340 to 4 infected male Indian rhesus macaques available from a different study, followed by blood collection 3 days afterwards and magnetic-bead separation of CD4⁺ cells, monocytes, and NK cells. Figure 4A shows representative FACS results for enriched CD4⁺, CD14⁺ (monocytes), and CD14⁻ CD16⁺ (NK cells) fractions. Figure 4B shows that median TFV-DP levels were above 500 fmol/10⁶ cells in all the cell fractions evaluated. Levels were 1,172 fmol/10⁶ cells in total PBMCs, 729 fmol/10⁶ cells in CD4⁺ cells, 544 fmol/10⁶ cells in monocytes, and 2,793 fmol/10⁶ cells in NK cells. These findings demonstrate that GS7340 effectively delivers TFV to all these cellular compartments, with TFV-DP levels that remain high 3 days after drug dosing. Overall TFV-DP concentrations were also higher than those seen at 24 h in two macaques receiving oral TDF (50 and 83 fmol/10⁶ cells in PBMCs, 35 and 34 fmol/10⁶ cells in CD4⁺ cells, 48 and 38 fmol/10⁶ cells in CD14⁺ cells, and 12 and 25 fmol/10⁶ cells in CD14⁻ CD16⁺ cells).

Fig. 4.

Intracellular TFV-DP levels in cell subpopulations from blood and tissues after a single GS7340 dose given 3 days prior to necropsy. (A) Representative FACS results for enriched CD4⁺, CD14⁺ (monocytes), and CD14⁻ CD16⁺ (NK cells) fractions purified from blood and used for drug level measurements. (B) Distribution of GS7340 in cell subpopulations from blood. Horizontal lines indicate median values. (C) TFV-DP levels in total mononuclear cells (MNCs) from inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), axillary lymph nodes (ALN), and rectal tissues (both total mononuclear cells and the enriched CD4⁺ cell fraction). Horizontal lines indicate median values.

We also measured TFV-DP concentrations in lymphoid tissue (mesenteric, axillary, and inguinal lymph nodes) and rectal tissue collected at necropsy from the same 4 animals (Fig. 4C). Median TFV-DP concentrations for lymphoid tissue were 590 fmol/10⁶ cells in inguinal lymph nodes (min, 345 fmol/10⁶ cells; max, 835 fmol/10⁶ cells), 438 fmol/10⁶ cells in mesenteric lymph nodes (min, 316 fmol/10⁶ cells; max, 828 fmol/10⁶ cells), and 542 fmol/10⁶ cells in axillary lymph nodes (min, 327 fmol/10⁶ cells; max, 872 fmol/10⁶ cells). Overall, these values were about 5- to 25-fold higher than those previously seen at 2 to 3 days after oral TDF dosing (20 to 100 fmol/10⁶ cells) (8). In rectal tissues, concentrations of TFV-DP in CD4⁺ cells purified from 2 macaques (1,302 and 488 fmol/10⁶ cells) and in total mononuclear cells from four animals (median, 377 fmol/10⁶ cells; min, 87 fmol/10⁶ cells; max, 1,618 fmol/10⁶ cells) were high and similar to those previously seen in 2 animals 2 to 3 days after oral TDF dosing (1,131 and 882 fmol/10⁶ cells) (8). Thus, GS7340 efficiently distributes TFV into circulating lymphocytes, lymphoid tissue, and rectal mononuclear cells and results in high intracellular TFV-DP concentrations at 3 days.

A high intracellular dATP content is associated with a lack of prophylactic efficacy.

TFV-DP competes with the natural dATP substrate for incorporation by reverse transcriptase (RT). Therefore, a possible explanation for the lack of prophylactic efficacy of GS7340 is that TFV-DP is not sufficiently active in rectal lymphocytes due to a high intracellular dATP content. To address this question, we measured intracellular dATP and TFV-DP concentrations, and we calculated dATP/TFV-DP ratios in mononuclear cells obtained from rectal tissues from the same 4 infected macaques described above. Figure 5 shows that dATP levels and dATP/TFV-DP ratios were about 100-fold higher in rectal mononuclear cells than in lymphoid tissues or PBMCs. These findings suggest that TFV-DP may be less active in blocking infection in rectal lymphocytes. We also measured dATP levels in one uninfected, untreated macaque that was euthanized for reasons unrelated to the study. dATP levels in rectal mononuclear cells (22,900 fmol/10⁶ cells) from this animal were also substantially higher than those seen in PBMCs (417 fmol/10⁶ cells) or lymphoid tissue (292 fmol/10⁶ cells in axillary lymph nodes, 165 fmol/10⁶ cells in inguinal lymph nodes, and 421 fmol/10⁶ cells in mesenteric lymph nodes [not shown]).

Fig. 5.

Intracellular dATP levels and dATP/TFV-DP ratios in cell subpopulations from blood and tissues after a single GS7340 dose given 3 days prior to necropsy. (A) dATP levels and dATP/TFV-DP ratios in cell subpopulations from blood. (B) dATP levels and dATP/TFV-DP ratios in inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), axillary lymph nodes (ALN), and rectal tissues (both total mononuclear cells [MNC] and the enriched CD4⁺ cell fraction). Horizontal lines indicate median values.

We also compared dATP levels and dATP/TFV-DP ratios between the 4 breakthrough animals receiving PrEP and the 2 uninfected animals (Fig. 6). dATP concentrations were measured longitudinally in PBMCs over 7 weeks during the challenge series. Mean dATP levels in the 4 infected animals (63.8 fmol/10⁶ cells; 95% CI, 52.4 to 75.1 fmol/10⁶ cells) were significantly higher than those seen in the 2 uninfected macaques (43.0 fmol/10⁶ cells; 95% CI, 26.9 to 59.1 fmol/10⁶ cells; P = 0.04). dATP/TFV-DP ratios in the infected animals (0.074 [95% CI, 0.055 to 0.093]) were also significantly higher than those seen in uninfected macaques (0.037 [95% CI, 0.010 to 0.064; P = 0.03]). Figure 6 also shows that dATP levels and dATP/TFV-DP ratios in infected animals were consistently higher during the complete challenge series.

Fig. 6.

Intracellular dATP levels (A) and dATP/TFV-DP ratios (B) in PBMCs from macaques exposed to SHIV_SF162P3 during GS7340 prophylaxis. dATP and TFV-DP levels were measured over 7 weeks at the time of virus exposure for the 4 infected (red lines) and 2 uninfected (blue lines) animals. Mean dATP levels and dATP/TFV-DP ratios were higher in the infected animals (P, 0.04 and 0.03, respectively).

Phenotypic reversion of SHIV_SF162P3 RT susceptibility to TFV-DP at high dATP concentrations.

We next explored biochemically the impact of variable dATP levels on the ability of TFV-DP to inhibit reverse transcription of a heteropolymeric RNA template in an in vitro assay (9, 10). A 5 μM concentration of TFV-DP inhibited WT SHIV_SF162P3 RT when the concentration of dATP was also 5 μM (a 1:1 ratio). However, 5 μM TFV-DP was not sufficient to block reverse transcription when the concentration of dATP was increased above 5 μM (Fig. 7B). These findings demonstrate that the susceptibility of SHIV_SF162P3 to TFV-DP can be diminished at high dATP/TFV-DP ratios.

Fig. 7.

Phenotypic reversion of SHIV_SF162P3 susceptibility to TFV-DP occurs at high dATP/TFV-DP ratios. (A) Inhibition of WT SHIV_SF162P3 RT by TFV-DP. Concentrations of 5 μM TFV-DP and 5 μM dATP (1:1 ratio) (arrow) can efficiently block WT RT but not a TFV-resistant RT containing the K65R mutation. Data are means from four separate experiments carried out in triplicate. (B) Phenotypic reversion and efficient completion of reverse transcription with TFV-DP can be achieved at high dATP concentrations. RT was incubated with 5 μM TFV-DP and increasing concentrations of dATP. Data are means from two separate experiments carried out in triplicate.

DISCUSSION

We demonstrate for the first time that high mucosal and systemic antiretroviral drug concentrations in cells that are primary targets for HIV infection do not necessarily translate into high prophylactic efficacy. As expected from the high cellular permeation of GS7340, intracellular TFV-DP levels in PBMCs and lymphoid tissues were much higher with GS7340 than with TDF despite the lower GS7340 dose used (8, 26). TFV-DP concentrations in PBMCs remained high for as long as 7 days, with levels that exceeded by 100-fold those seen at peak with TDF. In rectal lymphocytes, TFV-DP levels at 3 days were similarly high. Despite the favorable drug PK profile and high tissue TFV-DP levels, GS7340 did not protect macaques from rectal SHIV exposure. We relate these findings to intracellular drug pharmacodynamics in the rectal mucosa by showing that rectal lymphocytes have a high dATP content that likely rendered TFV-DP less effective in blocking early infection events (1). We also found that infection outcome was associated with dATP/TFV-DP ratios in PBMCs. Our results identify dATP/TFV-DP ratios as a negative correlate of protection with tenofovir and suggest that for nucleoside RT inhibitors, drug activity and prophylactic efficacy will depend on natural substrate concentrations at mucosal sites. Since high systemic TFV-DP levels that were sufficient to blunt acute viremias did not result in protection, our findings also suggest that active drug concentrations at the mucosa are more critical for PrEP effectiveness.

The finding of high dATP levels in rectal tissues was not completely surprising, since immune effector sites are in a state of physiological inflammation that results in cell activation and increased dNTP pools (1, 25, 27). In PBMCs, phytohemagglutinin (PHA) stimulation increases intracellular dNTP concentrations 4- to 13-fold (7). Since most of the CD4⁺ cells in the gut are CCR5⁺ activated memory CD4⁺ cells and are thus preferred cellular targets for HIV and SIV infection, high dATP pools might have reduced the ability of TFV-DP to block reverse transcription and prevent rectal infection (2, 28, 29). In contrast, a lower dATP content in PBMCs and less availability of activated target cells might increase the effectiveness of TFV against parenteral exposures. These two possible scenarios were noted in macaques receiving prophylactic treatment with oral TDF or subcutaneous TFV. While daily oral TDF did not prevent rectal infection during repeated exposures to SHIV_SF162P3, subcutaneous TFV successfully protected macaques parenterally exposed to a high dose of SIV_mne (33–35). It will also be important to determine how high dATP levels are in vaginal lymphocytes and if they can be affected by inflammation associated with sexually transmitted diseases (16). Additional studies comparing TFV-DP levels achieved in vaginal and rectal tissues after oral dosing, comparing dATP contents in rectal and vaginal lymphocytes, and assessing how all these relate to efficacy in preventing infection will provide valuable information on potential pharmacodynamic and chemoprophylactic differences between these two compartments.

In contrast to levels in circulating lymphocytes and lymphoid tissues, levels of TFV-DP in rectal tissues at 3 days were similar with GS7340 and TDF dosing. In both instances, high intracellular TFV-DP concentrations were associated with high extracellular TFV levels in rectal secretions. Such high TFV levels may originate partially from degradation of GS7340 or TDF to TFV by esterases present in the intestines and/or from trapping of TFV in mucus (22, 32, 37). The lack of protection seen despite such high tissue TFV-DP concentrations is worrisome and suggests that the threshold for protection against rectal transmission by tenofovir alone may be very high. This observation may also explain why the combination of TDF and FTC was more protective than TDF alone in our animal model, and it suggests that the additional antiviral activity provided by FTC is essential for the efficacy of PrEP against rectal transmission (11, 33).

Several important observations can be made from the PrEP breakthrough infections. First, acute viremias were blunted in the 4 PrEP animals who had breakthrough infections despite receiving only one weekly dose. The low viremias seen in these macaques demonstrate clearly the potent systemic antiviral activity of GS7340 and can be explained by the persistently high intracellular TFV-DP concentrations. These virologic responses are consistent with the 1.8 log₁₀ reduction in plasma virus loads seen in humans during GS7340 treatment (26a). The rapid decline in virus loads to undetectable levels within 4 to 8 weeks might also explain the lack of selection of the K65R mutation associated with TFV resistance. Similar blunted viremias have been noted in PrEP breakthrough infections with FTC or Truvada and have also been associated with reduced risks of resistance emergence (8, 11). Interestingly, all infections were initiated with WT viruses, suggesting that initial virus replication originates from cells that are not protected by PrEP either because of suboptimal TFV-DP concentrations or, possibly, because of high intracellular dATP levels in gut lymphocytes.

Several important limitations to our study should be considered. First, we did not measure TFV-DP concentrations in cellular subpopulations from rectal tissues that are also primary targets during early infection and might be inadequately protected by GS7340, such as macrophages or dendritic cells (25). Different molecular transport pathways for GS7340 and TFV might result in variable cellular drug exposures after GS7340 or TDF dosing. Second, our analysis of dATP contents in rectal lymphocytes was done 25 to 32 weeks after SHIV infection. It is not known whether the infection had contributed to the high dATP levels seen in rectal lymphocytes from these animals, although high dATP concentrations are consistent with the activation status of rectal lymphocytes (1, 25, 27). Also, SHIV_SF162P3 infections are not highly pathogenic; most animals appear to control infection (13, 21). Our analysis of dATP levels in one uninfected, untreated macaque suggests that neither infection status nor drug treatment might be responsible for the high dATP levels seen in our animals, although this observation needs to be further confirmed with more uninfected macaques. It will also be important to find out whether the high dATP contents seen in rectal lymphocytes from rhesus macaques are also observed in the gastrointestinal tracts of humans.

In summary, we show that GS7340 efficiently delivers TFV into peripheral lymphocytes, lymphoid tissues, and rectal tissue. However, we demonstrate that high rectal and systemic TFV exposure was not sufficient to prevent rectal SHIV transmission in macaques. We explain this paradox by showing high dATP concentrations in rectal mononuclear cells, which may reduce the ability of TFV-DP to block early infection events. Our results identify dATP/TFV-DP ratios as a negative correlate of protection by TFV and suggest that natural substrate concentrations in mucosal target cells may potentially modulate the prophylactic efficacy of tenofovir and may have broader implications for the entire nucleotide RT inhibitor drug class. Pharmacodynamic differences between rectal, vaginal, and systemic tissues may possibly impact the prophylactic efficacy of antiretroviral drugs against distinct routes of HIV transmission.