Ten years ago, Cardo et al.  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 .
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.  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 .
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 . This model has been used to evaluate both preexposure and postexposure chemoprophylaxis mostly after intravenous challenge. Tsai et al.  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 . Only a few studies have evaluated the efficacy of PEP after mucosal exposure. Böttiger et al.  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.  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.
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 Médicales 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-Mérieux, 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 .
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, Université Louis Pasteur, Strasbourg, France) diluted 1: 2 in human seminal fluid using a pliable French catheter . 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 (PBMC). Briefly, freshly isolated PBMCs were activated for 3 days with 1 μg/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: 5′-AGGTTACGGCCCGGCGGAAAGAAAA and GAG B: 5′-CCTACTCCCTGACAGGCCGTCAGCATTTCT in the first-round reaction and the inner primers GAG C: 5′-AGTACATGTTAAAACATGTAGTATGGGC and GAG F: 5′-CCTTAAGCTTTTGTAGAATCTATCTACATA in the second-round reaction. In the first-round and second-round amplifications, we used 2 U of Taq polymerase (5 U/μl; Roche Diagnostic, Meylan, France), 200 pmol of each dNTP, 200 pmol of each primer in a total volume of 100 μl. The sample was denatured by heating at 94 °C for 2 min and was then subjected to 40 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. The second-round amplification was carried out using 5 μl 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. ZDV and 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 μm 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 V and 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 μg/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 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.
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 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 oral-treated 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 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).
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.
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 T max and C max 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 T max and C max 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 C max [mean: 876 nmol/l (107–2410)]. Also, T max 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 C max and T max 6.5 μmol/l (5.7–7.8) and 0.75 h (0.5–2) for 3TC, 6.7 μmol/l (5.6–8.2) and 0.5 h (0.25–2) for ZDV, 12.5 ± 4 μmol/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 T max and a clearly increased C max [mean T max and C max 0.6 h (0.5–0.75) and 9.6 μmol/l (6.5–13) for 3TC, 0.6 h (0.5–0.75) and 1.1 μmol/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.
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. , 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 . 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 . 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. , different antiretrovial drugs display very 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. , 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.
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 Hépatites 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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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
antiretroviral therapy; macaque; pharmacokinetic; postexposure prophylaxis (PEP); SIV; vaginal transmission