Efficacy of topical tenofovir against transmission of a tenofovir-resistant SHIV in macaques

Gel formulation

Tenofovir [(R)-9-(2-phosphonylmethoxypropyl)adenine] (TFV) was kindly provided by
Gilead Sciences; 1% TFV (wt/wt) was formulated in 2% hydroxyethyl cellulose (HEC)
gel as previously described 8], 9]. Gels were formulated at pH 6.5 to mimic the average vaginal pH of pigtailed macaques
21]. A matching placebo 2% HEC gel was used as a control.

Virus stocks

The wild-type SHIV162P3 and SHIV162P3
_K65R
virus stocks used were generated as described elsewhere 14]. The K65R substitution in SHIV162P3 confers ~5-fold reduction in susceptibility to
TFV and was introduced by site-directed mutagenesis with two nucleotide changes (

AA

A ? CGA) to minimize reversion of K65R in vivo 13]. Virus titer of challenge stocks was calculated in macaque PBMCs and diluted to 500
tissue culture infective dose (500 TCID
₅₀
) and stored separately in 1-ml aliquots in liquid nitrogen. Individual vials were
thawed on ice prior to each challenge.

Measurement of drug concentrations in plasma, vaginal lymphocytes, and peripheral
blood mononuclear cells (PBMCs)

TFV concentrations in plasma were measured in macaques 30 min after vaginal administration
of TFV gel, resulting in the analysis of 20 samples from each of the 6 macaques (120
total). Briefly, TFV was extracted from 100 µl of plasma by protein precipitation
with 500 µl of methanol containing 200 ng of 13C-labeled TFV as internal standard.
Supernatant containing the drug from precipitation was evaporated to near dryness
under vacuum and then re-suspended in HPLC buffer containing 9.9 mM of acetic acid,
5.9 mM of ammonium hydroxide, and 9.4 mM of formic acid (pH ~3). Drug levels were
analyzed by using liquid chromatography–mass spectrometry (LC–MS) 8], 24]. The assay had a lower limit of quantification (LLOQ) of 3 ng/ml and standard curve
R
²
values greater than 0.99.

We previously documented high intracellular TFV-DP concentrations in lymphocytes collected
from vaginal tissues in macaques (n = 3) sacrificed 4 h following a single dose of
vaginal 1% TFV gel 8]. To expand these data, we additionally measured TFV-DP levels in vaginal lymphocytes
in SHIV-infected macaques (n = 4) sacrificed 4 h after receiving a single vaginal
dose of 1% TFV gel. All tissue collection and processing procedures were conducted
by the same veterinarian pathologists, laboratory technicians, and using the same
tissue digestion and mononuclear cell enrichment protocols as described in the previous
study 8]. Briefly, vaginal tissue collected at time of necropsy was dissociated using enzyme
cocktails and lymphocyte purification procedures. Total cell populations were gated
and counted for mononuclear cells using a Muse Cell counter with CytoSoft Data Acquisition
and Analysis Software (Millipore, Billerica, MA). Intracellular TFV-DP concentrations
in vaginal lymphocytes were measured with an automated online weak anion exchange
solid-phase extraction method coupled with ion-pair chromatography–MS/MS 24]. TFV-DP levels were expressed as femtomoles (fmol) per million cells with a lower
limit of quantitation (LLOQ) of 2.5 fmol/sample.

To compare TFV-DP concentrations observed in vivo with those achieved in vitro, macaque
PBMCs were incubated with varying concentrations of TFV and intracellular TFV-DP concentrations
were measured at each dose. Briefly, PBMCs (5.0 × 10
⁶
) were incubated 2–4 h in RPMI media containing TFV concentrations within the range
of the 10–99% inhibitory concentrations (IC
_10–99
) [300, 60, 12, 2.4, 0.48, 0.096 µM] for wild-type and SHIV162P3
_K65R
virus 8], 13]. Following incubation, cells were washed extensively with saline buffer solution,
pelleted, and lysed in 1 ml of ice cold 80% MeOH. Intracellular TFV-DP levels were
measured as described above.

Efficacy of 1% TFV gel in preventing vaginal transmission of SHIV162P3
_K65R

The efficacy of TFV gel against vaginal transmission of SHIV162P3
_K65R
was evaluated in female pig-tailed macaques under conditions similar to those described
for wild-type SHIV162P3 8], 9]. Macaques received 3 ml of intravaginal placebo (n = 6) or 1% TFV (n = 6) gel 30 min
before each vaginal exposure to SHIV162P3
_K65R
. Challenges were performed twice per week (every 3–4 days) for 10 weeks or up to
20 exposures. Vaginal challenges were administered by atraumatic inoculation of 1 ml
of SHIV162P3
_K65R
(500 TCID) into the vaginal vault. The challenge dose was increased ~10 times higher
than wild type SHIV162P3 (from 50 to 500 TCID
₅₀
) to adjust for the lower transmissibility SHIV162P3
_K65R8], 9], 12], 14]. Blood was collected 30 min after each gel application to monitor for SHIV infection
and plasma drug levels. SHIV infection was determined by monitoring SHIV RNA in plasma
by RT-PCR 8]. The estimated time of infection was defined as 7 days (two challenges) prior to
SHIV positive to account for the eclipse period between virus inoculation and detection
of SHIV RNA in plasma 25]. Animals were considered protected if they tested negative for SHIV plasma RNA and
SHIV DNA in PBMCs and remained seronegative during the course of the study and the
following 10 weeks of washout in the absence of challenge and gel application. All
experiments were done under highly controlled conditions by the same personnel, using
the same virus stock, and procedures as described in previous studies 8], 9], 20]. These studies adhered to the Guide for the Care and Use of Laboratory Animals (Institute
for Laboratory Animal Research, 1996); all procedures were approved by the Institutional
Animal Care and Use Committees (IACUC) of both the Centers for Disease Control and
Prevention (CDC) and the Yerkes National Primate Research Center (Emory University).

Statistical analysis

The cumulative probability of macaques remaining uninfected after repeated low-dose
viral exposures was computed and graphically displayed using the product limit (Kaplan–Meier)
estimator. The log-rank test statistic was used to non-parametrically compare survival
curves between the control and treatment groups. Uninfected macaques were right censored
at the maximal exposure number (20 exposures). Intervention efficacy was calculated
as 1 ? (p
₁
/p
₀
), where p
₁
and p
₀
denote the proportion of infections for intervention and control animals, respectively.
Acute RNA viremias were compared using the Wilcoxon rank-sum test.