Intermittent prophylaxis with oral truvada protects macaques from rectal SHIV infection.

Abstract

HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.

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DOI: 10.1126/scitranslmed.3000391
, 14ra4 (2010);
2 Sci Transl Med
, et al. J. Gerardo García-Lerma
from Rectal SHIV Infection
Intermittent Prophylaxis with Oral Truvada Protects Macaques

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AIDS AND HIV
Intermittent Prophylaxis with Oral Truvada Protects
Macaques from Rectal SHIV Infection
J. Gerardo García-Lerma,1* Mian-er Cong,1James Mitchell,1Ae S. Youngpairoj,1
Qi Zheng,1Silvina Masciotra,1Amy Martin,1Zsuzsanna Kuklenyik,2Angela Holder,1
Jonathan Lipscomb,1Chou-Pong Pau,1John R. Barr,2Debra L. Hanson,1Ron Otten,1
Lynn Paxton,1Thomas M. Folks,1,3Walid Heneine1
(Published 13 January 2010; Volume 2 Issue 14 14ra4)
HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care,
preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid trans-
mission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the
efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada
(TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-
acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication
and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada
to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a
week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed
by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the
long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus
exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We
saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of
blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an
antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They
strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.
INTRODUCTION
More than a quarter century after the description of the first cases of
AIDS, HIV has spread to virtually every country in the world, infecting
65 million people and killing 25 million (1). An effective HIV vaccine
remains elusive, although the recent finding of a modestly successful
two-vaccine prime-boost vaccination strategy gives new hope to vac-
cine development (1, 2). A multicomponent approach that includes be-
havioral, structural, and biomedical interventions will likely be essential
to curb the HIV epidemic (3, 4).
Daily preexposure prophylaxis (PrEP) with antiretroviral drugs is a
promising intervention to protect high-risk HIV-negative people from
becoming infected. Multiple lines of evidence favor this approach.
Antiretroviral drugs effectively prevent HIV transmission at birth, during
breastfeeding, and after occupational exposure (5–7). Daily PrEP with the
HIV reverse transcriptase inhibitors tenofovir (TFV) disoproxil fumarate
(TDF), emtricitabine (FTC), or Truvada (an FTC-TDF combination) can
prevent or delay simian immunodeficiency virus (SIV) or simian-human
immunodeficiency virus (SHIV) transmission in macaques in a dose-
dependent manner (8–10). These observations have provided the basis
for evaluating the efficacy of daily PrEP in preventing HIV transmission
in humans, and several clinical trials to evaluate HIV transmission with
TDF or Truvada are now ongoing in high-risk populations (3, 4). Al-
though these trials will provide the first proof-of-concept data on the ef-
ficacy of PrEP in humans, intermittent drug administration is a far more
feasible strategy than daily PrEP. Intermittent PrEP (iPrEP) is likely to be
more practical to implement and more cost-effective; it may reduce the
riskofdrugtoxicitiesandpotentiallyincreaseadherenceandminimizethe
emergence of drug resistance.
We hypothesized that intermittent dosing designed to deliver
long-acting drugs at the viral entry sites will be as effective as daily
dosing in blocking the earliest stages of virus replication and pre-
venting mucosal transmission. We based our rationale on the proved
efficacy of short-course prophylaxis with the long-acting drug nevir-
apine in preventing HIV transmission at birth (7), the relatively small
size of founder virus populations that initiate an HIV infection in the
mucosa (11–13), and the high FTC and TFV concentrations achieved
in the male and female genital tract after oral administration (14, 15).
Our recent findings in macaques showing complete protection from
rectal SHIV exposures by a two-dose subcutaneous regimen contain-
ing a high dose of TFV with FTC provided the first proof of concept
for iPrEP as a chemoprevention strategy against sexual transmission
(8). However, the high drug doses and subcutaneous drug delivery sys-
tem used may produce an overestimation of iPrEP efficacy. Therefore,
more studies with orally administered drugs that provide drug expo-
sures and distribution kinetics similar to those in humans are necessary
to better evaluate iPrEP efficacy and inform clinical trial designs.
Here, we repeatedly exposed macaque monkeys rectally to SHIV to
assess the efficacy of different iPrEP dosing regimens with oral Truvada
and to define windows of protection for preexposure or postexposure dos-
1Division of HIV/AIDS Prevention, National Center for HIV, Hepatitis, STD, and Prevention,
Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, USA.
2Division of Laboratory Sciences, National Center for Environmental Health, Centers for
Disease Control and Prevention, Atlanta, GA 30329, USA.3Southwest National Primate
Research Center, Southwest Foundation for Biomedical Research, San Antonio, TX 78245,
USA.
*To whom correspondence should be addressed. E-mail: GGarcia-Lerma@cdc.gov
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ing. The macaque model resembles human HIV transmission in many
ways (16). First, the SHIV challenge dose is lower and more physiologic
than what is conventionally used in the single high-dose challenge models.
This SHIV contains an R5-tropic HIV-1SF162envelope similar to naturally
transmitted viruses. Second, exposures to virus arerepeated up to 14 times
tomimichigh-riskhumanexposures.Thesingle-doseapproachonlymea-
sures protection against one transmission event per animal, whereas the
repeated-challenge model evaluates protection against multiple transmis-
sion events and may be a more robust model for testing biomedical inter-
ventions (8, 9, 17). We show that significant protection can be achieved
with a single dose of Truvada given 1 to 7 days before exposure followed
by a second dose 2 hours after exposure. We also show that drug admin-
istration around the time of exposure may be effective but that protection
is lost if the first dose is delayed 24 hours after exposure.
RESULTS
Effect of preexposure and postexposure doses on
iPrEP effectiveness
We recently showed that an intermittent regimen consisting of two sub-
cutaneous doses of FTC (20 mg/kg) and TFV (22 mg/kg) given 2 hours
before and 22 hours after virus exposure was fully protective in the
repeat-exposure macaque model of rectal transmission (8). Although
the dose of TFV used in these animals was higher by factors of 3 to 4
than that used in humans, these findings demonstrated that an iPrEP
strategy might be effective. To explore whether the preexposure dose
was sufficient for full protection and to assess the contribution of the post-
exposure dose, we gave a group of six macaques the same subcutaneous
FTC-TFV dose 2 hours before each rectal exposure without the +22-hour
postexposure dose. We also evaluated, in a second group of six macaques,
the effectiveness of this regimen when given at 24 and 48 hours after each
exposure [postexposure prophylaxis (PEP)] without the preexposure dos-
ing. Protection from 14 weekly rectal virus challenges with SHIV162p3
was evaluated against 32 untreated controls; 5 were real-time controls
and 27 were recent or historic controls from experiments done with
the same virus stock, inoculum size, and inoculation protocol (8).
A single subcutaneous FTC-TFV dose given 2 hours before exposure
(−2 hours) was not fully protective, and two of the six macaques were
infected during the 14 challenges (challenges 8 and 13) (Fig. 1). This
result indicates that protection can be achieved by the preexposure
dose but that the treatment after exposure is necessary for maximal
iPrEP effectiveness. In contrast, three of the six macaques receiving
PEP were infected during the first two challenges; the challenge series
was stopped at this point after an interim analysis showed absence of
efficacy relative to untreated controls (P > 0.5, log-rank test for differ-
ences in survival curves). These findings show the inability of this
short-course PEP regimen to control virus spread after mucosal infec-
tion occurs (Fig. 1). Overall, these results indicate that a preexposure
drug dose that can block the initial establishment of infection is critical,
highlight the importance of drug availability before virus exposure, and
suggest that both a preexposure and a postexposure drug dose are nec-
essary for maximum effectiveness.
Protection by preexposure dose followed by a second
postexposure dose
To define the window of protection of the preexposure dosing, we de-
signed several interventions in which we administered FTC and TDF
at different times relative to the virus challenge. To better model the
situation in humans, we gave FTC and TDF orally at doses that resulted
in exposure to Truvada similar to that in humans (FTC, 20 mg/kg;
TDF, 22 mg/kg) (Fig. 2A) (8). Macaques received the first dose of
Truvada 22 hours (group 1), 3 days (group 2), or 7 days (group 3)
before each virus exposure followed by a second dose 2 hours after
exposure. An additional group of nine control macaques was main-
tained without drug administration at the same time. All animals were
exposed weekly to SHIV162p3 rectally for up to 14 weeks.
Consistent with previous data, all nine controls became infected
within one to five challenges. In contrast, all three iPrEP regimens
showed protection (Fig. 2B). Five of six animals from group 1 (−22
hours/+2 hours), five of six animals from group 2 (−3 days/+2 hours),
and four of six animals from group 3 (−7 days/+2 hours) remained
uninfected after the 14 weekly challenges [P < 0.0001 for each group
relative to 32 untreated control macaques (9 real-time and 23 recent
and historic controls)]. Relative to all untreated controls, the risk of
infection by these three interventions was reduced by factors of 16.7
(group 1, P = 0.006), 15.4 (group 2, P = 0.008), and 9.3 (group 3, P =
0.003).
To better understand the relation between protection and drug
pharmacokinetics, we performed a separate experiment with different
animals to calculate the half-lives of active TFV-diphosphate (TFV-
DP) and FTC-triphosphate (FTC-TP) in peripheral blood mono-
nuclear cells (PBMCs). FTC-TP and TFV-DP were measured in four
macaques for 12 days after oral administration of a single dose of Tru-
vada. The estimated mean half-life for TFV-DP was 115 hours (range,
78 to 170 hours), similar to that seen in humans (150 hours; range, 60
Fig. 1. Protection by subcutaneous FTC-TFV administered before and/or
after virus exposure. Each survival curve represents the cumulative percent-
age of uninfected macaques as a function of weekly (−2-hour/+22-hour,
−2-hour, and control groups) or biweekly virus exposures (+24-hour/+48-
hour group). Uninfected animals remained negative after 14 exposures and
a mean washout observation period of 13 weeks. The challenge series in
the +24-hour/+48-hour group was stopped after an interim analysis done
at week 2 showed absence of efficacy (P > 0.5). The full protection
conferred by the −2-hour/+22-hour FTC-TFV regimen has been reported
(8). The group of control animals includes 5 real-time controls and 27 his-
toric and recent controls exposed to the same virus stock and dose with
the same inoculation method. The five real-time controls were infected at
challenges 1, 2 (two animals), and 3 (three animals).
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to >175 hours) (18, 19) (Fig. 3). FTC-TP concentrations declined at a
higher rate (half-life, 24 hours; range, 15 to 49 hours) and followed
kinetics that were also similar to those seen in humans (20). These
findings suggest that the high level of protection by the different pre-
exposure dosing strategies may be related to the long persistence of
TFV-DP and FTC-TP in PBMCs.
Protection by prophylaxis related to viral exposure
To investigate the effectiveness of drug administration around the
time of exposure, we administered orally to two groups of macaques
a two-dose Truvada regimen starting 2 hours before (group 4) or 2
hours after (group 5) exposure (Fig. 2A). Animals from both groups
received a second dose of Truvada 24 hours after the first dose. The
−2-hour/+22-hour regimen conferred significant protection [hazard
ratio (HR) reduction by a factor of 4.1, P = 0.02], with three of the
six macaques protected during the 14 challenges (P = 0.0004 relative
to controls) (Fig. 2B). Infection of these three macaques was not as-
sociated with lower plasma drug concentrations at exposure or with
delayed or reduced drug absorption, as indicated by the analysis of
area under the curve (AUC24 hours) and plasma drug half-life values
(figs. S1 and S2). Significant protection was also seen in group 5 ma-
caques that received the +2-hour/+26-hour PEP regimen (HR reduc-
tion by a factor of 4.0, P = 0.03), with three of the six animals protected
Fig. 2. Protection by exposure-independent and event-driven prophy-
laxis with Truvada. (A) Study design and interventions with oral Truvada.
All macaques received two weekly doses of Truvada by oral gavage and
were exposed once a week at the indicated time points, with the excep-
tion of macaques from group 5. Group 5 macaques were exposed and
treated every 2 weeks to minimize residual drug exposure due to long
intracellular FTC and TFV persistence. Truvada was administered at the
indicated times (short black bars) relative to viral exposure. (B) Reduc-
tion in the risk of infection (HRs) by intermittent prophylaxis with oral
Truvada. Each survival curve represents the cumulative percentage of
uninfected macaques as a function of virus exposure. For comparison,
the previously reported survival curve showing protection of daily PrEP
with oral Truvada (continuous red line) is shown (8). The group of con-
trol animals includes the 9 real-time controls and 23 historic and recent
controls exposed to the same virus stock and dose using the same in-
oculation method. The nine real-time controls were infected at chal-
lenges 1 (six animals), 3 (two animals), and 5 (one animal).
Fig. 3. Intracellular FTC-TP and TFV-DP concentrations in macaque PBMCs.
The mean (±SEM) FTC-TP and TFV-DP concentrations observed in four ma-
caques after administration of a single dose of FTC and TDF by oral gavage
are shown. Mean half-lives (t1/2) of FTC-TP and TFV-DP are also indicated.
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during the 14 challenges (P = 0.0004 relative to controls) (Fig. 2B). We
also explored whether a higher dose of Truvada could increase the
effectiveness of exposure-driven iPrEP by orally administering to a
group of six macaques (group 6) twice the human equivalent dose
of both FTC (40 mg/kg) and TDF (44 mg/kg) 2 hours before and
24 hours after exposure. Five of the six animals were protected and
only one was infected at challenge 4 (P < 0.0001 relative to
controls) (fig. S3). An examination of plasma drug concentrations
in the infected animal showed consistently low FTC and TFV con-
centrations during the first 7 to 9 weeks, which suggests that in this
particular macaque, infection might have been facilitated by sub-
optimal drug absorption (fig. S3).
Pharmacokinetics of FTC and TFV in rectal secretions
and tissues
Optimal drug concentration at the mucosa is essential for effective
iPrEP. We evaluated the pharmacokinetic profiles of FTC and TFV
in both plasma and rectal secretions by administering to four ma-
caques a single dose of Truvada orally followed by collection of blood
and secretions at 2, 5, and 24 hours (Fig. 4A). Concentrations of TFV
at 2 and 5 hours in secretions were low or undetectable but increased
substantially in all animals at 24 hours (median = 1186 ng/ml, range =
279 to 59,920). In contrast, FTC was detected in secretions in all ma-
caques at 2 hours (median = 100 ng/ml, range = 41 to 618) and in
three of the four animals at 5 hours (median = 114 ng/ml, range = 95
to 240), albeit at lower concentrations than in plasma (Fig. 4A). Simi-
lar to that observed with TFV, FTC concentrations in secretions also
increased considerably at 24 hours (median = 743 ng/ml, range = 243
to 28,504) (Fig. 4A). Thus, the pharmacokinetic profile of FTC and
TFV in rectal secretions was different from that seen in blood in which
drug concentrations peaked around 2 hours and substantially de-
creased at 24 hours. At 24 hours, median FTC and TFV concentra-
tions in secretions were higher by factors of 17 and 50 than those seen
in plasma, respectively (Fig. 4A).
The kinetics of TFV-DP and FTC-TP were further evaluated in rec-
tal tissue by orally administering to seven macaques a single dose of
Truvada followed by necropsies to collect tissues after 2 hours and 1,
2, 3, or 7 days. Intracellular drug concentrations were compared to
those seen in blood and axillary, mesenteric, and inguinal lymph node
specimens collected in parallel. TFV-DP concentrations at 2 hours were
similar in blood, rectal tissue, and lymphoid tissue (Fig. 4B). However,
TFV-DP concentrations in rectal tissues collected from four macaques
at 1 day (361 and 156 fmol per 106cells), 2 days (1131 fmol per 106
cells), or 3 days (882 fmol per 106cells) were substantially higher than
Fig. 4. Extracellular and intracellular FTC
and TFV concentrations in macaques af-
ter oral administration of a single dose
of Truvada. (A) Extracellular FTC and TFV
concentrations in plasma and rectal secre-
tions from four macaques. Secretions were
collected at three time points with rectal
wicks as detailed in the Supplementary Ma-
terial. Data represent the median (range)
FTC and TFV concentrations seen in four
animals. (B) TFV-DP concentrations in
mononuclear cells from blood, rectal tissue,
and axillary, mesenteric, and inguinal lymph
nodes collected from seven macaques that
received Truvada 2 hours and 1, 2, 3, or 7
days before scheduled necropsy. Individual
TFV-DP concentrations in each specimen
are shown. The number of animals evalu-
ated at each time point is also indicated.
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those seen in blood or lymph nodes, a finding that was consistent with
the high extracellular TFV concentrations in rectal secretions at 24
hours and long TFV-DP persistence in mononuclear cells (Figs. 3
and 4A). Degradation of FTC-TP during tissue processing was evident
by the detection of FTC diphosphate, FTC monophosphate, and FTC
and precluded an accurate analysis of FTC-TP concentrations. Never-
theless, we were able to measure FTC-TP in rectal tissues from two
animals at 2 hours (358 fmol per 106cells) and 24 hours (390 fmol per
106cells).
Plasma virus loads and drug resistance emergence
in iPrEP failures
The mean peak viremia was significantly lower in iPrEP failures (5.6 log10
RNA copies per milliliter) relative to untreated controls (7.1 log10RNA
copies per milliliter) (P = 0.009) (Fig. 5). Virus loads also declined sig-
nificantly faster in iPrEP failures (0.32 log10per week) during continued
treatment (median = 13.5 weeks; range, 9 to 16) than in control animals
(0.13 log10per week, P < 0.0001) (Fig. 5). None of the animals infected
during iPrEP developed the K65R or M184V mutations associated with
TFV or FTC resistance during a median follow-up period of 13 weeks
on PrEP. A detailed description of the infection kinetics for each iPrEP
failure is shown in fig. S4.
DISCUSSION
We used macaque monkeys repeatedly exposed rectally to SHIV to
investigate the effectiveness of intermittent treatment schedules with
the antiretroviral drug Truvada orally administered at human equiva-
lent doses. We tested dosing regimens initiated 1, 3, or 7 days before
exposure and showed that, as with daily PrEP, all were protective
against infection (8). We selected these drug-dosing intervals to eval-
uate whether one to two weekly doses of Truvada followed by a
booster dose after exposure to the virus were sufficient to prevent trans-
mission. We attribute the extended window of protection to the long
intracellular persistence of FTC and TFV in PBMCs; drug half-lives
were within the range of those observed in humans (18–20). We fur-
ther show efficacy for prevention when drug treatment is initiated
within 2 hours of virus exposure, thus providing evidence that event-
driven drug dosing might also be effective. We saw increased protection
in this scenario when the dose of Truvada was doubled. Together, these
findings indicate a range of possible iPrEP designs and highlight the
promise of this strategy as an alternative to daily PrEP in preventing
HIV transmission. If the ongoing daily PrEP clinical trials show ef-
ficacy in humans, intermittent dosing such as described here may
provide a next-generation chemoprevention strategy with potential
benefits, including cost-effectiveness, greater patient adherence,
and reduced risks of drug toxicities.
Our observations with a potent subcutaneous FTC and high-
dose TFV combination suggested that treatment is needed both
before and after exposure for maximum protection. We admin-
istered FTC and TFV subcutaneously to these animals to facilitate
a rapid tissue distribution. The finding that a single subcutaneous
dose of this regimen given 2 hours before exposure was protective
highlights the importance of efficiently blocking early replication in
the mucosa, an observation that was confirmed by the lack of pro-
tection seen when the first dose was delayed until 24 hours after
exposure. The inability to protect these macaques further demon-
strates that founder virus populations are rapidly established mu-
cosally, as previously noted in macaques exposed vaginally to
SIVmac251, and, importantly, the difficulty in controlling virus
spread from productively infected cells (13, 21, 22). Thus, waiting
24 hours after exposure to provide FTC and TFV may have allowed
the earliest mucosal infections to proceed because reverse tran-
scription can be completed in 4 to 6 hours in activated T cells (23).
With the exception of one animal that showed unusually low
drug concentrations, we could not establish an association be-
tween infection outcome and plasma drug concentrations. However,
our analysis was limited because the number of drug measure-
ments associated with infection was small and only a few animals
were infected in our studies. Our observation that the efficacy of
exposure-related iPrEP can be increased by doubling the Truvada
dose suggests that drug concentrations may correlate with protec-
tion and also strengthens the possibility of using iPrEP tied to viral
exposure if the risks of drug toxicity do not increase when the dose
of Truvada is doubled. It will be important to define the threshold
for plasma and intracellular drug concentrations that are asso-
ciated with protection.
It is noteworthy that we found that FTC and TFV concentrations
in rectal secretions were low during the first 5 hours and were highest
at 24 hours. Low tissue drug concentrations shortly after drug admin-
istration may increase risks of infection and partially explain the ap-
parent lower effectiveness seen when treatment was contingent on
exposure (6 of 12 animals from groups 4 and 5 infected), although a
lower efficacy of these regimens could not be statistically ascertained.
The high TFV and FTC concentrations in rectal secretions 24 hours
after oral drug administration may partially originate from degradation
of TDF to TFV by esterases in the intestines and from elimination of
FTC in feces (24). A combination of local and systemic drug absorption
may protect more susceptible target cells at the mucosa and contribute to
Fig. 5. Blunted acute viremia in macaques infected during iPrEP. Indi-
vidual virus load kinetics in breakthrough infections under continued
drug exposure (n = 6, red lines) and in untreated control macaques with
sufficient follow-up (n = 22, black lines) are shown. Time 0 indicates the
peak plasma virus load. Mean peak viremia was significantly lower in
iPrEP failures (P = 0.009). Virus loads also declined significantly faster
in iPrEP failures than in control animals (P < 0.0001).
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iPrEP effectiveness. High drug concentrations in rectal secretions at 24
hours may increase intracellular drug concentrations in rectal tissues
and explain the higher (by factors of 3 to 5) intracellular TFV-DP con-
centrations at 24 hours in rectal lymphocytes when compared to PBMCs
and the presence of TFV-DP in tissues 2 to 7 days after administration of
TDF. We also show that FTC was more rapidly detected than TFV in
rectal secretions, indicating more rapid absorption and tissue distribution.
Together, these findings suggest that the rapid FTC distribution in tissues
andthelongintracellularTFVpersistencecomplementeachothertomax-
imize iPrEP effectiveness.
We recently found that macaques failing daily PrEP with FTC or
Truvada had lower acute viremias than untreated macaques (8). We
also show here attenuated viremias in animals infected during iPrEP
with only two doses of Truvada per week, likely reflecting the extended
antiviral activity of FTC and TDF resulting from their long intracellular
half-life. Such decreased acute viremias may have clinical and public
health implications. Acutely infected persons with very high viral loads
may play a key role in the epidemic spread of HIV-1 because they are
more infectious than chronically infected persons who have lower virus
loads (25–27). Therefore, a reduction in acute viremia during iPrEP
treatment may contribute to decreases in HIV-1 transmission in the
population and could add to the overall effectiveness of PrEP. Substan-
tial reductions in virus loads during acute viremia could also reduce
CD4+T cell depletion, help preserve immune function, and attenuate
the course of HIV infection (28).
Notably, none of the animals that failed iPrEP had detectable re-
sistance even with sensitive testing for minority M184V or K65R
mutants associated with FTC or TFV resistance, a finding that diff-
ered from our earlier observations in macaques that failed daily PrEP
with FTC or Truvada in which two of six failures acquired M184V/I
(8). The lack of resistance seen in all seven macaques failing iPrEP
is encouraging and suggests that two weekly doses of Truvada may
minimize selection of drug-resistant viruses. However, our data need
to be interpreted cautiously because it is not known whether the risks
of resistance emergence would increase with more frequent exposure
to drug resulting from more frequent administration of Truvada (29).
In addition, our study cannot test the emergence of resistance after
prolonged (>12 weeks) intermittent drug exposure, because viremias
in animals infected with SHIV162p3 tend to decrease to low or un-
detectable values over time.
Our study is subject to several limitations. First, all viral chal-
lenges were nontraumatic and were done in the absence of semen
or semen-derived factors that may enhance HIV infection in vitro
or other cofactors that may increase transmission risks, such as sexually
transmitted infections. Our use of the less pathogenic SHIV162p3 iso-
late might also potentially overestimate efficacy. However, SHIV162p3
is easily transmissible to macaques at low [10 TCID50(tissue culture
infectious dose)] infectious doses both rectally and vaginally (8, 9, 16)
and, thus, is well-suited for transmission studies. Second, we used a
wild-type virus that is fully susceptible to both TFV and FTC. Because
circulating drug-resistant viruses may be common, more work should
be done to define PrEP efficacy on drug-resistant viruses. Third, our
study was not powered to evaluate statistical differences between the
different iPrEP modalities or between daily PrEP and iPrEP because
of the limited number of animals per group. Finally, it is important to
confirm the efficacy of these PrEP regimens against vaginal transmis-
sion in appropriate macaque models. Although many biologic simi-
larities exist between rectal and vaginal HIV transmission, some
differences in the early events of mucosal infection or changes in
susceptibilities associated with the menstrual cycle and thinning
of the epithelium are possible (13, 30, 31). The pharmacokinetic
profiles of FTC and TFV in the female genital tract may also be
different from those in rectal tissues and could potentially affect
iPrEP effectiveness.
In macaques repeatedly exposed rectally to SHIV, intermittent
prophylaxis with Truvada may be highly effective for preventing
infection with a wide window of protection. All current PrEP trials
are based on daily drug-dosing intervals that were selected because
they are effective for treatment. The protection seen in our ma-
caques suggests that Truvada might prevent transmission in humans
if taken on the basis of exposure events or after a fixed weekly
schedule of one to two doses with a booster dose after any exposure
to virus. Less frequent drug administration would reduce cost and
might decrease drug toxicities and foster adherence by reducing
unnecessary drug exposure and frequency of mild side effects.
Ongoing clinical trials with daily PrEP will shed light on the effi-
cacy of PrEP as a prevention strategy. If the human daily PrEP
trials prove effective, additional trials would be needed to evaluate
whether iPrEP modalities may be ultimately sufficient to prevent
HIV transmission.
MATERIALS AND METHODS
Drug preparation and administration
For oral administration, TDF was first suspended in phosphate-
buffered saline (PBS) and dissolved with NaOH followed by the
addition of FTC. Drugs were given orally by gavage to anesthetized
macaques via a gastric feeding tube (8). For subcutaneous injec-
tions, stock solutions of TFV and FTC were prepared in deionized
water or PBS, respectively (8). TDF, TFV, and FTC were provided
by Gilead Sciences.
Repeat-exposure macaque model
The efficacy of different iPrEP modalities was evaluated with a repeat-
exposure macaque model of rectal transmission previously described
(8, 9, 16). Rhesus macaques were exposed rectally once weekly or every
2 weeks (group 5 only) to a SHIVSF162P3chimeric virus that contains
the tat, rev, and env coding regions of HIV-1SF162in a background of
SIVmac239 [National Institutes of Health AIDS Research and
Reference Reagent Program (32)]. The SHIV162p3 challenge dose
was 10 TCID50or 7.6 × 105RNA copies, which is within the range
of HIV-1 RNA concentrations in semen (103to 106copies per milli-
liter) during acute infection in humans and higher than the concentra-
tions (102to 104copies per milliliter) seen after primary viremia (33).
Virus exposures (up to 14) were done by nontraumatic inoculation of
1 ml of SHIVSF162P3into the rectal vault via a sterile gastric feeding tube
of adjusted length (16). Macaques were anesthetized with standard
doses of ketamine hydrochloride. Anesthetized macaques remained re-
cumbent for at least 15 min after each intrarectal inoculation. Virus ex-
posures were stopped when a macaque became SHIV RNA–positive.
Macaques infected during PrEP continued treatment for a median of
13.5 weeks (range, 9 to 16). All experiments were done under highly
controlled conditions by the same personnel using the same virus stock,
inoculum dose, and inoculation method. The animal handlers who
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administered drug or performed the virus challenges were not blinded.
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
Plasma SHIV RNA was quantified with a real-time reverse transcrip-
tion polymerase chain reaction (PCR) assay as previously described (9).
This assay format has a sensitivity of 50 RNA copies per milliliter. De-
tection of low-frequency K65R and M184V mutants in plasma was per-
formed with sensitive allele-specific real-time PCR methods as
previouslydescribed (8, 34). Virus-specific serologic responses (immuno-
globulins G and M) were measured with a synthetic peptide enzyme
immunoassay (Genetic Systems HIV-1/HIV-2, Bio-Rad). Animals in
the iPrEP arms were considered protected from systemic SHIV in-
fection if they remained seronegative and negative for SHIV plasma
RNA and SHIV DNA in PBMCs during PrEP and during the follow-
ing 70 days of washout in the absence of any drug treatment (35).
Measurement of drug concentrations in plasma, PBMCs,
and tissues
FTC and TFV concentrations in plasma and rectal secretions were
measured by high-performance liquid chromatography–tandem mass
spectrometry (MS/MS) (36). Intracellular FTC-TP and TFV-DP concen-
trations were measured with an automated online weak anion exchange
solid-phase extraction method coupled with ion-pair chromatography–
MS/MS. Procedures are detailed in the Supplementary Material.
Statistical analysis
The Cox proportional hazards model was used to estimate instanta-
neous risk for infection, as a HR, in controls relative to treated ani-
mals, assuming constant risk at all inoculations. Graphical methods of
model assessment supported the use of Cox proportional hazards re-
gression. The two-sided Fisher’s exact test was used for a categorical
analysis of number of infections per total exposures in each group rel-
ative to controls. Plasma FTC and TFV concentrations were compared
with a mixed-effect model, with a random intercept and unstructured
covariance to account for within-subject correlated measurements.
Mean area under the curve (AUC24 hours) and plasma terminal elimi-
nation half-life (HL lz) values among infected and protected animals
were compared with the Wilcoxon-Mann-Whitney test (two-sided).
The WinNonlin software (version 5.2; Pharsight) was used to calculate
AUC24 hoursand HL lz values. The Wilcoxon rank-sum statistic was
again implemented to test for group differences in magnitude of peak
virus load; mixed-effects regression was used to assess differences be-
tween treatment and control groups in the rate of decline after peak
virus load. All statistical analyses were performed with SAS software
(version 9.1; SAS Institute).
SUPPLEMENTARY MATERIAL
www.sciencetranslationalmedicine.org/cgi/content/full/2/14/14ra4/DC1
Materials and Methods
Fig. S1. Plasma FTC and TFV concentrations in macaques treated with Truvada 2 hours before
and 24 hours after virus exposure.
Fig. S2. Longitudinal FTC and TFV concentrations in plasma from protected and infected macaques.
Fig. S3. Efficacy of iPrEP with a double dose of Truvada and plasma drug concentrations.
Fig. S4. Dynamics of infection in macaques infected during iPrEP.
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monkey specimens and performing virus load testing, Dr. C. Hendrix for providing us with
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Funding: Centers for Disease Control and Prevention.
Author contributions: J.G.G.-L., R.O., L.P., T.M.F., and W.H. designed the research. M.C., J.M., A.S.J.,
Q.Z., S.M., and J.L. performed the experiments. A.M., Z.K., A.H., C.-P.P., and J.R.B. developed new
analytical tools for drugmeasurements and quantified drugconcentrations in blood, secretions,
and tissues. D.L.H. performed the statistical analyses. J.G.G.-L. and W.H. wrote the paper.
Competing interests: J.G.G.-L., R.O., T.M.F., and W.H. are named in a U.S. Government patent
application related to methods for HIV prophylaxis. The findings and conclusions in this
study are those of the authors and do not necessarily represent the views of the Centers
for Disease Control and Prevention.
Submitted 16 September 2009
Accepted 18 December 2009
Published 13 January 2010
10.1126/scitranslmed.3000391
Citation: J. G. García-Lerma, M. Cong, J. Mitchell, A. S. Youngpairoj, Q. Zheng, S. Masciotra, A. Martin,
Z. Kuklenyik, A. Holder, J. Lipscomb, C.-P. Pau, J. R. Barr, D. L. Hanson, R. Otten, L. Paxton, T. M. Folks,
W. Heneine, Intermittent prophylaxis with oral Truvada protects macaques from rectal SHIV infection.
Sci. Transl. Med. 2, 14ra4 (2010).
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