Prevention of vaginal simian immunodeficiency virus transmission in

Prevention of vaginal simian immunodeficiency virus
transmission in macaques by postexposure
prophylaxis with zidovudine, lamivudine
and indinavir
Olivier Bourrya,c,f, Patricia Brochardc,f, Sandrine Souquiereb,
Maria Makuwab, Julien Calvoc,f, Nathalie Dereudre-Bosquetc,d,
Fre´de´ric Martinonc,f, Henri Beneche, Mirdad Kazanjib,g and
Roger Le Grandc,f
Objective: To evaluate the efficacy of postexposure prophylaxis with a combination of
zidovudine (ZDV), lamivudine (3TC) and indinavir (IDV), after vaginal exposure to HIV.
Design: Experimental intravaginal exposure of female cynomolgus macaques to SIVmac251.
Methods: ZDV/3TC/IDV treatment was initiated 4 h after exposure and continued for
28 days. Groups of six animals received a placebo or a combination of oral ZDV
(4.5 mg/kg), 3TC (2.5 mg/kg) and IDV (20 mg/kg) twice daily or subcutaneous ZDV
(4.5 mg/kg) and 3TC (2.5 mg/kg) twice daily, and a higher dose of IDV (60 mg/kg)
administered orally twice daily.
Results: In the placebo group, all animals were infected. Antiretroviral association
protected one of the six animals if all drugs were administered orally and four of the six
animals if ZDV and 3TC were administered subcutaneously and IDV was given orally at
triple dose. In infected animals, viremia was significantly delayed and lowered in
treated animals than in animals given placebo, and high CD4 cell counts were
maintained in the treated animals, at least in the medium term. Antiretroviral dosages
made in macaques receiving the same treatments showed that protection efficacy could
be linked to antiretroviral plasmatic concentration.
Conclusion: This study shows, for the first time in macaques, that the postexposure
prophylaxis recommended for humans may be effective after vaginal exposure.
Improvements in pharmacokinetic parameters significantly increased treatment
ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
efficiency.
AIDS 2009, 23:447–454
Keywords: antiretroviral therapy, macaque, pharmacokinetic, postexposure
prophylaxis (PEP), SIV, vaginal transmission
a
Centre de Primatologie, Centre International de Recherches Me´dicales de Franceville, Franceville, bService de Re´trovirologie,
Centre International de Recherches Me´dicales de Franceville, Franceville, Gabon, cCEA, Division of Immunovirology, Institute of
Emerging Diseases and Innovative Therapies (IMETI), DSV, Fontenay aux Roses, dSPIBIO, Montigny-le, Bretonneux, eCEA, Service
de Pharmacologie et d’Immunoanalyse, DSV/iBiTecS, CEA/Saclay, Gif-sur-Yvette, fUniv Paris-Sud 11, UMR E01, Orsay, and
g
Re´seau International des Instituts Pasteur, Institut Pasteur, Paris, France.
Correspondence to Olivier Bourry, Service d’immuno-virologie, Commissariat a` l’Energie Atomique, DSV/iMETI, 18 route du
Panorama, 92265 Fontenay-aux-Roses Cedex, France.
E-mail: [email protected]
Received: 3 June 2008; revised: 3 November 2008; accepted: 7 November 2008.
DOI:10.1097/QAD.0b013e328321302d
ISSN 0269-9370 Q 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
447
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AIDS
2009, Vol 23 No 4
Introduction
Ten years ago, Cardo et al. [1] showed for the first time
that zidovudine (ZDV) treatment could reduce the risk of
HIV infection after percutaneous exposure by 80%
among healthcare workers. However, the results of this
case–control study have never been confirmed, and
studies of ZDV postexposure prophylaxis (PEP) in animal
models have only established a reduction of plasma viral
load (PVL) but no full protection from infection [2–4].
Despite the few formal evidences of PEP efficiency in
humans and cases of treatment failure reported among
individuals treated within 2 h of exposure to contaminated fluids [5,6], the use of PEP is nowadays largely
widespread; consisting currently in highly active antiretroviral associations. In several countries, the use of PEP
is not limited to occupational exposure to HIV, but is also
recommended after sexual exposure to HIV [7].
As truly effective vaccines and topical microbicides are
probably years away from commercial availability, the use
of antiviral therapy is now also considered for preexposure
prophylaxis (PrEP) in populations at high risk of
transmission. Very recently, Garcia-Lerma et al. [8]
showed the prevention of rectal simian/human immunodeficiency virus (SHIV) transmission in macaques by
daily or intermittent prophylaxis with emtricitabine and
tenofovir. Several trials based on the use of tenofovir
administered as a once-daily pill alone or in combination
for PrEP are also underway in various highly exposed
populations [9].
The use of animal models to evaluate HIV-prevention
strategies is indispensable, because of the difficulties
involved in carrying out trials in humans. Macaques
infected with pathogenic strains of simian immunodeficiency virus (SIV) or related chimeras are currently the
most relevant models of human HIV infection and AIDS
[10]. This model has been used to evaluate both
preexposure and postexposure chemoprophylaxis mostly
after intravenous challenge. Tsai et al. [11] demonstrated
the efficacy of tenofovir for preventing SIV infection in
macaques after intravenous challenge, provided that
treatment was initiated between 48 h before inoculation
and 24 h after inoculation and continued for 28 days. A
related study on a closely related compound (BEA-005)
showed that PEP was not as effective if initiated 48 or 72 h
after exposure or continued for only 3 days, demonstrating the importance of treatment timing and duration [12].
Only a few studies have evaluated the efficacy of PEP after
mucosal exposure. Bo¨ttiger et al. [12] showed that both of
the two macaques given BEA-005 1 h after exposure were
protected against rectal challenge with SIV. To date, only
Otten et al. [13] have studied PEP after vaginal exposure.
They showed that tenofovir could protect pig-tailed
macaques against HIV-2 infection if treatment was
initiated within 36 h of viral inoculation.
Many of the molecules tested in animal models may not
be suitable for PEP in humans, and very few studies have
focused on the antiretroviral drugs routinely used in
HAART, although combinations of nonnucleoside and
nucleoside analogues and protease inhibitors are frequently used after occupational or sexual exposure to
HIV. We previously demonstrated that the combination
of ZDV/lamivudine (3TC) and indinavir (IDV), initiated
within 4 h of intravenous inoculation with SHIV89.6P or
SIVmac251 and continued for 28 days, could not prevent
infection in macaques but may have a significant impact
on disease progression [10,14]. Here we demonstrate that
complete prevention from infection could be nevertheless
achieved with a similar treatment against the vaginal
transmission of pathogenic SIVmac251 probably because
of the specificity of viral dissemination after mucosal
exposure. In addition, pharmacokinetics of the compounds appeared as a key factor for prevention efficiency.
Methods
Animals
Eighteen adult cynomolgus female macaques (Macaca
fascicularis), each weighing 4–6 kg, were imported from
Mauritius. All animals were confirmed negative for SIV,
simian T-lymphotropic virus (STLV), herpes B virus,
filovirus, SRV-1, SRV-2 and measles before study
initiation and were housed in single cages within level
3 biosafety facilities at the Centre International de
Recherches Me´dicales de Franceville (Franceville,
Gabon). Studies were conducted in accordance with
European guidelines for animal care and were approved
by the CIRMF ethics committee for animal experimentation. The animals were sedated with ketamine
chlorhydrate (10–15 mg/kg; Rhone-Me´rieux, Lyon,
France), before inoculation with the virus, blood sample
collection and treatment. Female macaques were treated
with a single 30 mg intramuscular injection of medroxyprogesterone acetate (Depo-provera; Pharmacia&Upjohn, St Quentin-en-Yvelines, France) 30 days before
virus inoculation, to synchronize their menstrual cycles
and thin the vaginal mucosa [15].
Virus inoculation
The vaginal vault of the animals was inoculated with
50 intravaginal AID50 (corresponding to 6 107 vRNA
copies) of a cell-free stock of pathogenic SIVmac251
(kindly provided by A.M. Aubertin, Universite´ Louis
Pasteur, Strasbourg, France) diluted 1 : 2 in human
seminal fluid using a pliable French catheter [16]. This
inoculation caused no trauma.
In-vitro anti-simian immunodeficiency virus
activity
Anti-SIV activities of ZDV, 3TC and IDV were evaluated
in vitro on human peripheral blood mononuclear cell
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
PEP in macaques after SIV vaginal challenge Bourry et al.
(PBMC). Briefly, freshly isolated PBMCs were activated
for 3 days with 1 mg/ml phytohaemaglutin-P (Difco
Laboratories, Detroit, Michigan, USA) and then cultivated
in cell culture medium supplemented with 20 IU/ml
human recombinant interleukin-2 (Roche, Meylan,
France). PBMCs were treated with ZDV (0.1, 1, 10,
100 and 400 nmol/l), 3TC (0.1, 1, 10, 100 and
1000 nnmol/l), IDV (0.1, 1, 10, 100 and 1000 nnmol/l)
alone or in combination (1, 10 and 100 nnmol/l of each
molecule) and infected 30 min later with 360 TCID50 of
SIVmac251 per 150 000 PBMC. Viral replication was
assessed by quantifying RT activity in cell culture
supernatants harvested 7 days after infection (RT Retrosys
kit; Innovagen, Lund, Sweden). The 50, 70 and 90%
effective doses (ED50, ED70 and ED90) were calculated
using SoftmaxPro 4.6 software (Molecular Devices,
Sunnyvale, California, USA). ED50 and ED90 of ZDV
were 39 and more than 400 nmol/l, respectively, that of
3TC were 25 and 360 nmol/l, respectively, that of IDV
were 3 and 81 nmol/l, respectively, and that of ZDV/3TC/
IDV combination were 1.4 and 6.9 nmol/l, respectively.
Treatment of the animals
Six animals received oral ZDV (4.5 mg/kg body weight),
3TC (2.5 mg/kg) and IDV (20 mg/kg) twice daily, through
a nasogastric catheter (oral group). Six macaques were
given ZDV (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice daily and a higher dose of IDV (60 mg/kg),
orally twice daily (subcutaneous group). Treatment was
initiated 4 h after virus exposure and was continued for
4 weeks. Six macaques were treated with orally and
subcutaneously administered placebo (placebo group).
Clinical, biological and virological evaluations
Plasma and cell-associated viral loads and T-lymphocyte
subsets were determined as previously described [17,18].
A 1 : 10 dilution of each plasma sample in calf fetal serum
was assayed for the presence of SIV-specific antibodies,
using a commercially available ELISA kit (Genscreen HIV
1/2 version 2; Bio-Rad Laboratories, France).
Nested PCR on lymph node mononuclear cells
Inguinal lymph nodes were collected when the animals
were killed, for the detection of SIV infection. Genomic
DNA was extracted from lysates of 5–10 106 lymphoid
cells, using the DNEASY Tissue kit (Qiagen, Courtaboeuf, France) and analyzed for the presence of viral
DNA. Nested PCR amplification was carried out with
primers specific for SIV gag, using the outer primers GAG
A: 50 -AGGTTACGGCCCGGCGGAAAGAAAA and
GAG B: 50 -CCTACTCCCTGACAGGCCGTCAGCATTTCT in the first-round reaction and the inner
primers GAG C: 50 -AGTACATGTTAAAACATGTAGTATGGGC and GAG F: 50 -CCTTAAGCTTTTGTAGAATCTATCTACATA in the second-round reaction.
In the first-round and second-round amplifications, we
used 2 U of Taq polymerase (5 U/ml; Roche Diagnostic,
Meylan, France), 200 pmol of each dNTP, 200 pmol of
each primer in a total volume of 100 ml. The sample was
denatured by heating at 94 8C for 2 min and was then
subjected to 40 cycles of 94 8C for 30 s, 55 8C for 30 s and
72 8C for 1 min. The second-round amplification was
carried out using 5 ml of products from the first-round
PCR and a similar cycling profile. The amplified products
were analyzed by electrophoresis in 1% agarose gels.
Determination of antiretroviral drug levels
Pharmacokinetic studies were carried out on another set of
animals. Four animals receiving the oral or the subcutaneous combinations were bled 15, 30, 45, 60, 120, 180
and 240 min after the first administration of the treatment.
ZDVand 3TC concentrations were determined in monkey
plasma using the previously described LC–MS/MS assay
method [19,20]. Plasma IDV concentration was determined with a newly developed UPLC–MS/MS method.
Plasma proteins were precipitated in ethanol, and
UPLC (Waters) was carried out on a C18-Shield
2.1 mm 100 mm 1.7 mm column with a mobile phase
consisting of a gradient of phase A (2 mmol/l ammonium
acetate/0.1% formic acid) and phase B (0.1% formic acid in
acetonitrile). The mobile phase was delivered at a rate of
0.5 ml/min, as follows: from T0 to T0.2 min, 75% A/25%
B; from 0.2 to 2 min, linear gradient to 30% A; from 2 to
2.3 min, linear gradient to 0% A; from 2.3 to 2.7 min, linear
gradient to 75% A; 75% A/25% B maintained until
4.5 min. Quantification was carried out by electrospray
positive ionization, followed by triple quadruple MS/MS
in a Quattro Premier XE (Waters). Capillary voltage
reached 4 kV, cone voltage 40 and 35 Vand collision energy
36 and 35 eV for methyl-IDV (internal standard) and IDV,
respectively. The transitions followed were (in m/z)
628.5 ! 421, 614.5 ! 421.3 for methyl-IDV and IDV,
respectively. With a quantification limit of 0.012 mg/ml,
validation experiments showed that precision and accuracy
were within the recommended ranges of about 15% (20%
at the lower limit of quantification).
Statistical analysis
Statistical analysis was carried out using Stat View
software (SAS Institute Inc., Cary, North Carolina, USA).
In a first step, the three treatment groups were compared
for the day of PVL peak, the value for PVL peak and the
area under the curve of PVL (between days 28 and 170),
using a nonparametric Kruskall–Wallis test for multiple
group analysis. When significant, the three groups were
compared one by one using a nonparametric Mann–
Whitney test.
Results
HAART protects macaques against SIVmac251
vaginal challenge
In the placebo group, all six macaques became infected
with peak of viremia at day 14 after infection (median
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
449
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AIDS
2009, Vol 23 No 4
Fig. 1. Plasma viral load and CD4 cell count in intravaginally exposed macaques treated with a placebo or ZDV/3TC/IDV.
Numbers of copies of viral RNA in the plasma of macaques treated with the placebo (a) or with ZDV/3TC/IDV association per os
(oral group) (b) or with ZDV and 3TC subcutaneous and IDV at triple dose (subcutaneous group) (c). Solid line represents the
median plasma viral load for infected animals in each group (6/6 in placebo, 5/6 in oral group and 2/6 in subcutaneous group). The
grey area indicates the quantitative threshold of our qRT-PCR assay (<60 RNA copies/ml). Numbers of CD4 circulating T cells
(relative to baseline) in the blood of macaques treated with placebo (d), or with ZDV/3TC/IDV association per os (oral group) (e), or
with ZDV and 3TC subcutaneous and IDV at triple dose (subcutaneous group) (f). Solid line represents the median CD3þCD4þ
cells number related to baseline for infected animals and dotted line the median for protected animals (0/6 in placebo, 1/6 in oral
group and 4/6 in subcutaneous group).
peak PVL 6.2 106 vRNA copies/ml) (Fig. 1a). The
animals showed a rapid, severe and persistent decline in
CD4 cell counts associated with AIDS. Among placebo
macaques, two animals were sacrificed 125 days after
infection following an acute diarrhea episode and two
others were euthanized 175 days after infection
subsequent to AIDS symptoms (Fig. 1d and Table 1).
In the group treated orally with ZDV/3TC/IDV
combination, viral RNA remained under the quantification threshold (<60 vRNA copies/ml) and CD4 cell
counts were stable in one of the six female animals (S339)
(Fig. 1b and e). This animal remained seronegative and
virus could not be detected by PCR in the PBMC or
lymph node mononuclear cell (LNMC) (day 230 after
infection) (Table 1), confirming complete protection
against virus transmission. In the remaining animals of
the same group, PVL peaks were significantly lower
(median 2.7 104 vRNA copies/ml; P < 0.05) and
delayed (median 21 days after infection; P < 0.05) when
compared with that in controls, and a smaller decrease in
CD4 cell counts was observed (nadir median at day 28
after infection 58 and 34% of baseline for the oral and
placebo groups, respectively) (Fig. 1b and e). After
treatment had ended, the infected animals of the oraltreated group had CD4 cell counts close to normal values
and better control of viremia than the placebo group, as
shown by the lower area under the curve (AUC28–170d)
for PVL (oral group median 3.6 106 copies-day/ml
versus 27.4 106 for placebo; P < 0.05). Corroborating
the better preservation of CD4 cell count in the oral
group, only two macaques of this group evolved lately
toward AIDS.
In the group treated subcutaneously with ZDV/3TC and
which received a triple dosage of oral IDV, four of the six
animals were protected (7963, 9693, A936 and G281) as
shown by persistent viremia under the quantification
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
b
b
b
b
threshold and undetectable virus in PBMC and LNMC.
No modification was observed in the CD4 cell counts and
none seroconverted (Fig. 1c and f, and Table 1). The two
macaques infected after intravaginal challenge and receiving subcutaneous treatment showed no detectable viremia
until the end of treatment, subsequently presenting a
delayed, reduced PVL peak (5.5 105 copies/ml at day
35 after infection and 1.7 105 copies/ml at day 49 after
infection) (Fig. 1c). Like animals treated orally, the two
infected animals treated subcutaneously presented better
control of viremia than did the placebo animals (median
PVL AUC28–170d 4.5 106 and 27.4 106 copies-day/ml
for treated and control animals, respectively).
b
b
Pharmacokinetics of antiviral drugs could
explicit difference of postexposure prophylaxis
efficacy between oral and subcutaneous groups
In order to elucidate the efficiency differences of oral and
subcutaneous treatment, we further determined the
plasmatic drug levels in two groups of four macaques
receiving the same ZDV/3TC/IDV regimens orally
or subcutaneously.
c
LNMC, lymph node mononuclear cell; SIV, simian immunodeficiency virus.
Acute diarrhoea 125 days after infection.
b
Sacrificed in good health condition at 230 day after infection.
c
Metabolic disorder 75 days after infection.
d
Stomach dilatation 175 days after infection.
Nested PCR on LNMC
at sacrifice
Clinical outcome/cause
of death
a
AIDS 175 days
after infection
a
AIDS 175 days
after infection
b
a
b
AIDS 230 days
after infection
b
b
AIDS 230 days
after infection
d
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þ
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2
28
35
49
70
98
140
230
Anti-SIV antibody detection
at day postinoculation
E216 9693 E353 B121 G281 A936 7963
C243
F990 E021
Q845
L784 L786
C043
N310 L541 S339
E245
Subcutaneous
Oral
Placebo
Day after
infection
Treatment group
Table 1. Antibody responses to simian immunodeficiency virus: viral DNA in lymph node mononuclear cell and clinical outcome of macaques after vaginal exposure to simian immunodeficiency
virus.
PEP in macaques after SIV vaginal challenge Bourry et al.
Oral administration in macaques of the three-drug
combination (2.5, 4.5 and 20 mg/kg for 3TC, ZDV
and IDV, respectively) results in slow, weak and variable
absorption (Fig. 2): mean Tmax and Cmax were 3.3 h (3–4)
and 144 nmol/l (17–318) for 3TC, 2.2 h (0.7–3) and
97 nmol/l (17–195) for ZDV. Regarding IDV, two
animals had plasmatic concentrations constantly under
the detection threshold and two other macaques had Tmax
and Cmax as 2 and 3 h and 495 and 542 nmol/l,
respectively. The administration of a triple oral dose
of IDV (60 mg/kg) resulted in increased Cmax
[mean: 876 nmol/l (107–2410)]. Also, Tmax remained
unchanged [mean 2.7 h (0.75–4)], efficient antiviral
concentrations were reached as early as 30 min after
treatment in 4/4 animals.
Compared with the pharmacokinetic data in humans
receiving the same drugs at the same doses [mean Cmax
and Tmax 6.5 mmol/l (5.7 –7.8) and 0.75 h (0.5–2)
for 3TC, 6.7 mmol/l (5.6–8.2) and 0.5 h (0.25–2) for
ZDV, 12.5 4 mmol/l and 0.8 0.3 h for IDV], the
absorption of antiretroviral drugs after oral administration
seemed rather slow and weak in our macaque model
[21–23].
Interestingly, the subcutaneous administration of 3TC
and ZDV at the same dose (2.5 and 4.5 mg/kg,
respectively) resulted in a better and more reproducible
absorption with an early Tmax and a clearly increased
Cmax [mean Tmax and Cmax 0.6 h (0.5–0.75) and
9.6 mmol/l (6.5–13) for 3TC, 0.6 h (0.5–0.75) and
1.1 mmol/l (0.8–1.4) for ZDV]. After subcutaneous
injection, the plasmatic concentration of 3TC in macaque
was similar to that in humans receiving the same oral
dose.
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451
AIDS
2009, Vol 23 No 4
(a)
(b)
Zidovudine
100000
ZDV/Oral
ZDV/SC
10000
(c)
Lamivudine
Indinavir
3TC/Oral
3TC/SC
IDV 20 mg/kg
IDV 60 mg/kg
1000
nM
452
100
10
1
0
50
100
150
200
250 0
50
Time (mn)
100
150
Time (mn)
200
250 0
50
100
150
200
250
Time (mn)
Fig. 2. ZDV, 3TC and IDV plasmatic concentrations in macaques after a single administration of drug. (a) Plasma concentration
(nmol/l) of zidovudine (ZDV) over a 6-h period after a single administration of 4.5 mg/kg subcutaneous (solid line, mean of four
macaques) or per os (dotted line, mean of four macaques); symbols, individual animals. (b) Plasma concentration (nmol/l) of
lamivudine (3TC) over a 6-h interval after a single administration of 2.5 mg/kg subcutaneous (solid line, mean of four macaques) or
per os (dotted line, mean of four macaques); symbols, individual animals. (c) Plasma concentration (nmol/l) of indinavir (IDV) over
a 6-h interval after a single administration of 60 mg/kg (solid line, mean of four macaques) or 20 mg/kg (dotted line, mean of
four macaques) per os; symbols, individual animals.
Discussion
Our study shows for the first time, in the macaque/SIV
model of HIV infection and AIDS, that the ZDV/3TC/
IDV combination may prevent infection in animals after
experimental vaginal exposure to SIVmac251.
The ZDV/3TC/IDV combination protected five of the
12 animals treated after vaginal exposure, whereas the
same treatment gave no protection after intravenous
inoculation of the same virus [14,18]. The difference in
efficacy against intravenous and intravaginal exposure is
consistent with the other studies in the SIV/macaque
model of HIV transmission. As shown by Zhang et al.
[24], after the intravenous infusion of radiolabeled SIV in
macaques, the circulating virus had a half-life of only
4 min and was rapidly transported to the liver, lungs and
lymph nodes. It is possible that, following intravenous
exposure, even if the antiretroviral drugs used have optimal
pharmacokinetics, the virus may penetrate the target cells
too rapidly to be blocked by the treatment. Alternatively,
the initial target cells may be located in tissues inaccessible
to antiviral drugs. It should also be borne in mind that ZDV
and 3TC are prodrugs that require three phosphorylation
steps to render them active against the viral reverse
transcriptase. Defected or delayed phosphorylation in
target cells may also affect the efficacy of treatment.
Although the virus can reach the target cells within a
few minutes of intravenous inoculation, it must first
interact with immune cells of the cervicovaginal mucosa
following atraumatic vaginal inoculation, subsequently
disseminating in the draining lymph nodes within 2 days
and becoming detectable in the bloodstream by day 5
[25]. These data are in favor for a larger window of
opportunity for prophylaxis in individuals exposed to the
virus during sexual intercourse than in individuals
exposed to the virus through the blood. During this
period, it may be possible to stop either the initial
infection of cells or viral dissemination, by administering
antiretroviral treatment.
ZDV/3TC/IDV administered orally protected one in six
intravaginally inoculated macaques, whereas an optimized version of the same treatment (NRTIs administered subcutaneously and the IP administered at a triple
dose) was around two-thirds effective. These results are in
accordance with our pharmacokinetic data showing that,
after oral administration of the ZDV/3TC/IDV combination, plasma antiretroviral drug concentrations in
macaques are lower than those in human patients on
the day of treatment initiation [21–23]. After subcutaneous administration in the macaque, ZDV and 3TC
showed a rapid absorption and higher plasmatic
concentrations associated with a much better efficiency
to prevent vaginal infection. In a very recent study,
Garcia-Lerma et al. showed in the same way that
subcutaneous injection of tenofovir (22 mg/kg) and
emtricitabine (20 mg/kg) was more effective to prevent
rectal SHIV transmission than the same association
administrated orally. Although the subcutaneous route is
inappropriate for preexposure chemoprophylaxis or
postexposure chemoprophylaxis in humans, all these
data suggest to carefully consider dosage and pharmacokinetics when selecting drug combinations for PEP.
The biodistribution of antiviral drugs is also probably
critical for protection [11]. After vaginal exposure to
VIH/SIV virus, we could assume that the antiretroviral
drugs act mainly on the target cells of the vaginal mucosa
and the adjacent tissues. As recently shown by Dumond
et al. [26], different antiretrovial drugs display very
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
PEP in macaques after SIV vaginal challenge Bourry et al.
different diffusion properties in the female genital
tract. ZDV, 3TC and tenofovir concentrate in vaginal
secretions, whereas protease inhibitors show genital tract
concentrations lower than in plasma. Hypothesizing that
antiviral drugs secreted in the vaginal lumen with high
levels have also high concentrations in the surrounding
cervico-vaginal tissues; ZDV, 3TC and tenofovir would
be of particular interest as PrEP/PEP candidates [13,27].
In all the animals infected despite antiretroviral therapy
(ART), a strong effect on viral load was observed
regardless of the mode of treatment administration. In
macaques treated orally, viremia remained low and peak
viremia was delayed during antiviral treatment. In animals
treated subcutaneously, viral load remained almost
undetectable until the end of treatment. Once ART
was stopped, viral load increased rapidly, suggesting that
virus levels had already begun to increase before the end
of treatment. In all treated animals becoming infected, the
partial control of viral replication seems to be associated
with the maintenance of high CD4 cell counts at least in
the medium term and a lowered evolution toward AIDS.
So, even if the treatment was unable to prevent infection
in some animals, it had nonetheless a positive effect on the
issue of the infection.
Although we have not tested resistance emergence at the
end of treatment, our previous unpublished monotherapy
PEP trials indicated that nonsynonymous nucleotide
mutations increased in pol sequence of virus isolated
from treated animals. Nevertheless, as also reported by
Fournier et al. [28], no resistance mutations could be
detected in transmitted virus. Considering these data and
those of pharmacokinetics, we could suppose that the
treatment failure that occurred in some animals is rather
related to insufficient or late drug concentration in the
initial sites of viral replication than to transmission of
viruses resistant to drugs.
Postexposure prophylaxis studies in macaques are very
useful for designing preventive strategies for HIV
transmission in humans. Our study shows, for the first
time, that one of the classical PEP treatments currently
recommended in humans may prevent SIV infection after
vaginal exposure, although it may not be able to prevent
intravenous transmission. This study also highlights the
importance for optimizing drug pharmacokinetics
and the need for cautious design of prophylactic
treatments.
Acknowledgements
We thank Sandrine Burton and Paul Bamba for excellent
technical assistance. We also thank Christophe Joubert
and the technical staff of the CEA and CIRMF for
animal care.
Author’s contributions: R.L.G. and O.B. conceived and
designed the experiments. O.B., P.B., S.S., M.M., J.C.,
and F.M. performed the experiments. O.B., N.D.B.,
H.B., and R.L.G. analyzed the data. M.K. and H.B.
contributed reagents/materials/analysis tools. O.B. and
R.L.G. wrote the paper.
This work was supported by the French national
AIDS agency, Agence Nationale de Recherche sur le
SIDA et les He´patites Virales (ANRS, Paris France),
EMPRO (LSH-2002-2.3.0-2), EUROPRISE European
network of excellence (LSHP-CT-2006-037611) and
DORMEUR foundation.
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