Hormonal contraceptive methods are highly effective at preventing unintended pregnancies and reducing maternal mortality and pregnancy-related morbidity, particularly in resource-limited settings. In sub-Saharan Africa where the HIV prevalence is highest, depot-medroxyprogesterone acetate (DMPA) represents the most commonly used method of contraception.1 However, some observational studies have suggested that women using DMPA may be at higher risk for HIV infection and may be more likely to infect their HIV-negative male partner (reviewed in Refs. 2 and 3). Although observational studies may be confounded by many factors including differences in sexual behavior, other studies point to biological mechanisms for increased HIV risk due to DMPA including changes in vaginal structure and immune modulatory effects. In some studies, DMPA was found to thin the vaginal epithelium to luteal phase levels and increased the number of HIV target cells in vaginal tissues although other studies found no effect of DMPA on thickness or immune cell populations.4–7 In pigtail macaques, DMPA reduces vaginal epithelial thickness and increases the percentage of CD3+ cells in a dose-dependent manner.8
Pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF), alone or in combination with emtricitabine (FTC) is a novel HIV prevention strategy that can reduce the risk of sexually acquired HIV infection in both men and women (reviewed in Ref. 9). PrEP with daily FTC/TDF has been approved in the US for HIV prevention in high-risk populations. Because women using DMPA could combine DMPA with PrEP to prevent HIV infection, it is important to understand if biological changes associated with DMPA may potentially reduce the prophylactic efficacy of FTC/TDF. We recently developed a pigtail macaque model of DMPA that recapitulates many of the biological effects of DMPA seen in women. This model uses a low DMPA dose that is sufficient to suppress ovulation while achieving reductions in vaginal epithelial thickness that are moderate in magnitude.8 The DMPA dose was defined through careful DMPA titration and analysis of several biologic and pharmacokinetic parameters including plasma MPA concentrations and changes in the vaginal epithelium. Here, we integrated this new DMPA model with a repeat low-dose vaginal simian HIV (SHIV) transmission model that demonstrated efficacy of FTC/TDF in normally cycling pigtail macaques.10
MATERIALS AND METHODS
Drugs and Viruses
TDF and FTC were kindly provided by Gilead Sciences and were given orally by gavage based on body weight as a single solution containing 20 mg/kg FTC and 22 mg/kg TDF.10 Macaques were anesthetized using standard doses of ketamine hydrochloride. The SHIV162p3 virus challenge stock was obtained from the National Institutes of Health AIDS Research repository and expanded in pigtail macaque peripheral blood mononuclear cells before this study.10
The efficacy of FTC/TDF in preventing vaginal transmission was evaluated using an established pigtail macaque model consisting of repeated vaginal exposures to a low dose (50 TCID50) of an R5-tropic SHIV162p3 isolate.10 The specific study design is shown in Figure 1A. SHIV exposures were performed once a week for up to 20 weeks by nontraumatic inoculation of 1 mL of virus into the vaginal vault through a sterile gastric feeding tube of adjusted length; exposures were stopped when a macaque became SHIV RNA positive. Anesthetized macaques remained recumbent for at least 15 minutes after each intravaginal inoculation. Six macaques received FTC/TDF and 6 received phosphate-buffered saline (PBS). Drug or PBS was given 24 hours before each virus exposure followed by a second dose 2 hours after exposure. The average age (13.1 and 13.7 years), weight (8.3 and 8.8 kg), and peak progesterone levels (4420 and 3763 ng/mL) before DMPA administration was similar between the treated and placebo groups (P > 0.5 for each comparison, Mann–Whitney two-tailed t test).
All 12 macaques received 6 cycles of 3 mg DMPA starting the month before the first virus challenge. Each DMPA cycle was 4 weeks in length except the third cycle, which was 5 weeks in length because of logistics that made it not possible to administer DMPA at the appropriate time. Blood samples were collected throughout the course of the study to monitor for progesterone, medroxyprogesterone acetate (MPA), and infection status. Progesterone levels were measured by liquid chromatography/mass spectrometry. MPA was quantified by a commercial radio immune assay (Immunometrics, London United Kingdom).8
SHIV RNA was quantified by a reverse transcriptase–polymerase chain reaction assay with a sensitivity of 50 RNA copies per milliliter. Proviral DNA was quantified using a double-stranded primer assay with a detection limit of 3 DNA copies per million peripheral blood mononuclear cells.10 Virus-specific serologic responses were measured using a synthetic-peptide enzyme immunoassay.10 Animals were considered protected if they remained seronegative and negative for SHIV plasma RNA as well as proviral DNA during the 20 weeks of PrEP and 16 weeks of follow-up. All animal procedures were approved by the CDC Institutional Animal Care and Use Committee.
Effect of DMPA on TFV and FTC Pharmacokinetics
At study completion, the 6 PrEP-protected macaques were maintained on DMPA and used to evaluate the effect of DMPA on FTC and TFV pharmacokinetics. FTC and TDF were administered by gavage as a single oral dose 2 weeks after DMPA injection when plasma MPA levels are highest.8 Drug concentrations were measured at 2, 5, 24, and 48 hours in plasma and vaginal secretions by high-performance liquid chromatography–tandem mass spectrometry.8 The 6 macaques underwent the same pharmacokinetic study in the absence of DMPA after a 6-month resting period. Peak drug concentrations and area under the curve (AUC) values in DMPA-treated and DMPA-untreated macaques were compared using a two-tailed Wilcoxon rank-sum test.
Plasma Progesterone and MPA in Macaques Receiving Monthly DMPA Cycles
A total of 12 pigtail macaques received 6 cycles of 3 mg DMPA starting the month before the first virus challenge. Figure 1A shows the experimental design. We monitored progesterone and MPA concentrations in plasma to ensure suppression of progesterone production and confirm that MPA levels with the 3 mg DMPA dose were within the range seen in women receiving 150 mg. Figure 1B shows that peak MPA levels in plasma (median = 2.4 ng/mL; minimum to maximum = 1.4–4.8 ng/mL) were similar to those seen in women (2.5 ng/mL)11 and sufficient to fully suppress progesterone production. The concentrations of MPA and progesterone in plasma in each of the 12 macaques before and during administration of DMPA are shown in Figure S1 (see Supplemental Digital Content, http://links.lww.com/QAI/A563). In 5 macaques (4 placebos, 1 FTC/TDF), progesterone transiently spiked coinciding with the missed DMPA dose at week 4 of the third cycle (week 9 of study). However, subsequent DMPA administration in these animals at week 10 rapidly suppressed progesterone (Figure S1).
Prophylactic Efficacy of FTC/TDF in Macaques Treated With DMPA
All 12 macaques treated with DMPA were exposed vaginally to SHIV162p3 once a week for up to 20 weeks. The first dose of DMPA was administered at week-4. The first SHIV RNA challenge was performed at week 1. Figure 1C shows that all 6 placebo controls were infected 2 each at exposures 3, 4, and 6. In contrast, all 6 macaques receiving FTC/TDF were uninfected despite receiving 14–17 more SHIV challenges than controls, reflecting a robust protection by FTC/TDF (P = 0.0007, log rank test).
Effect of DMPA on Systemic and Mucosal FTC and TFV Concentrations
The pharmacokinetic profile of FTC and TFV was evaluated in 6 macaques during their regular menstrual cycle or after administration of DMPA. Figure 2 shows the AUC and Cmax values for FTC and TFV in plasma and vaginal secretions. Plasma AUC0–24h and Cmax and values for FTC in the DMPA-treated animals (median = 12,245 ng·h/mL and 1488 ng/mL, respectively) were similar to those seen in DMPA-untreated macaques (median = 17,804 ng·h/mL and 2524 ng/mL, respectively) (P = 0.48 and P = 0.31 for AUC0–24h and Cmax comparisons). Plasma AUC0–24h and Cmax values for TFV were also similar among DMPA-treated (median = 4122 ng·h/mL and 267 ng/mL, respectively) and DMPA-untreated animals (median = 5230 ng·h/mL and 318 ng/mL, respectively) (P = 0.59 for both comparisons) (Fig. 2A).
In vaginal secretions, there was a possible trend toward higher AUC0–48h and Cmax values for FTC in DMPA-treated (median = 668,824 ng·h/mL and 57,889 ng/mL, respectively) than in DMPA-untreated (median = 122,646 ng·h/mL and 7230 ng/mL, respectively) animals (P = 0.09 and P = 0.06 for AUC0–48h and Cmax comparisons). Likewise, AUC0–48h and Cmax values for TFV in DMPA-treated (median = 179,095 ng·h/mL and 8784 ng/mL, respectively) were 2–3 times as high as those seen in DMPA-untreated (median = 51,235 ng·h/mL and 2577 ng/mL, respectively), although the difference was not statistically significant (P = 0.39 and P = 0.31) (Fig. 2B). Figure S2 (see Supplemental Digital Content, http://links.lww.com/QAI/A563) shows the pharmacokinetic curves for FTC and TFV in plasma and vaginal secretions.
Prevention of HIV infection and unintended pregnancies are both public health priorities. The wide use of DMPA for contraception in women and the new availability of PrEP with FTC/TDF for HIV prevention underscore the importance of understanding the interactions between DMPA, susceptibility to HIV-1, and PrEP efficacy.1 Randomized controlled clinical trials can assess the impact of DMPA on HIV acquisition and PrEP efficacy, but these trials are expensive and difficult to conduct. We used a macaque model that predicted FTC/TDF efficacy in women to assess to impact of a physiologic DMPA dose on PrEP. We demonstrate that FTC/TDF maintained high protection under continuous DMPA for 5 months. The efficacy seen in our study was identical to that seen in macaques with regular menstrual cycles10 and suggests little or no impact of DMPA on PrEP with FTC/TDF.
Our study design incorporates biologically relevant models of HIV transmission, risk, and prevention. We used pigtail macaques since they have normal lunar menstrual cycles and fluctuations in sex hormone levels that are similar to women.12 We modified this model by treatment with a physiologic 3 mg DMPA dose that effectively suppresses ovulation while resulting in plasma MPA exposure (AUC and Cmax) within the range seen in women receiving 150 mg of DMPA every 3 months.8 The 3-mg dose also results in moderate changes in vaginal epithelial thickness as opposed to the 30-mg dose historically used in macaques, which significantly reduces epithelial thickness.8 The vaginal challenge component used physiologic virus doses and repeated virus exposures to model high-risk human exposure. FTC and TDF were given orally at doses that mimic systemic drug concentrations in humans.10,13 Thus, the high protection observed in this model strongly suggests that the impact of DMPA on PrEP with FTC/TDF in women may be minimal. Because our results were seen with only 2 weekly doses of FTC/TDF, they suggest that daily dosing resulting in intracellular drug accumulation will also be effective.
We further conducted pharmacokinetic studies to examine the effect of DMPA on drug distribution. DMPA has been associated with increases in HIV target cells and cellular activation in vaginal mucosa in women.4,5 A high density of immune cells in the vaginal mucosa might conceivably require more drug coverage of susceptible target cells and alter drug protection thresholds by PrEP. Likewise, cellular activation enhances infectivity and is known to increase intracellular dNTP concentrations, which may reduce the antiviral activity of FTC-TP and TFV-DP by increasing competition with the natural dCTP and dATP substrates.14,15 We found that DMPA does not diminish vaginal drug exposures and, in fact, observed a possible trend toward higher drug concentrations in vaginal secretions but not in plasma. Although increases in vaginal drug levels due to DMPA require confirmation, it is tempting to surmise that prolonged vaginal thinning and increased epithelial permeability induced by MPA may have resulted in higher vaginal drug levels and thus, contributed to the high protection despite possible infiltration of activated target cells. This mechanism was also proposed as a potential explanation for the high and sustained vaginal absorption of FTC and TFV from vaginal gels seen in macaques treated with 30 mg of DMPA.16
We also note potential limitations in our study. First, virus challenges were nontraumatic and done in the absence of other cofactors that may increase HIV transmission risk such as sexually transmitted infections. Second, the shorter half-life of MPA in macaques compared with humans implied the need for monthly injections to efficiently model plasma MPA levels seen in women receiving 150 mg every 3 months. It is not known if shorter DMPA cycles resulting in more frequent MPA peaks and valleys might have affected our results.
A recent subanalysis of participants from the Partners PrEP Study that evaluated the efficacy of TDF or FTC/TDF for HIV-1 prevention found no evidence for a reduced efficacy of PrEP among women who used DMPA and men whose female partners used DMPA.17 Although these findings are not from a randomized clinical trial of DMPA and PrEP, they help support our observations in the macaque model, and also suggest that PrEP use in high HIV risk areas might overcome potential detrimental effects of DMPA on HIV-1 susceptibility.17 Animal model studies such as the one described here are well suited to evaluate multipurpose prevention technologies that incorporate PrEP and contraceptives.
The authors thank Dr. Brianna Skinner and Dr. Gregory Langham for serving as the attending veterinarians for this animal study protocol, Dr. David Garber for maintaining our cohort of animals and coordinating animal studies, and Dr. Ivana Massud, Mian-er Cong, and Susan Ruone for their help in processing some blood specimens.
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