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Topical Administration of Low-Dose Tenofovir Disoproxil Fumarate to Protect Infant Macaques against Multiple Oral Exposures of Low Doses of Simian Immunodeficiency Virus Koen K. A. Van Rompay,1 Kimberli A. Schmidt,1 Jonathan R. Lawson,1 Raman Singh,1 Norbert Bischofberger,2 and Marta L. Marthas1
1 California National Primate Research Center, University of California, Davis, and 2Gilead Sciences, Foster City, California
Breast-feeding among persons in developing countries continues to be a considerable risk for transmission of human immunodeficiency virus (HIV) [1, 2]. Until a vaccine is available, prolonged chemoprophylaxis throughout the period of breast-feeding may be an effective solution. To date, the development of chemoprophylactic regimens has focused on obtaining systemic levels of drug. Although this approach is directed toward maximal antiviral efficacy at all sites, the two main limitations of systemic drug levels are the considerable costs and the risk for side-effects after prolonged systemic exposure. These problems could be ameliorated if local (mucosal) levels of antiviral compounds are sufficient to protect against oral infection. For instance, oral administration of a low dose of a compound shortly before or after each breastfeeding meal may prevent infection of the nursing infant. This strategy, which is similar to the use of microbicides to reduce sexual HIV transmission, has the advantage that many compounds with strong anti-HIV activity in vitro that are not suitReceived 31 May 2002; revised 30 July 2002; electronically published 29 October 2002. Animal care complied with American Association for Accreditation of Laboratory Animal Care standards. Financial support: E. Glaser Pediatric AIDS Foundation (grant PG-51014 to K.K.A.V.R.; M.L.M. was an E. Glaser Scientist); Gilead Sciences; National Institutes of Health (grant AI-46320-01 to M.L.M). Reprints or correspondence: Dr. Koen K. A. Van Rompay, California National Primate Research Center, University of California, County Rd. 98 & Hutchison, Davis, CA 95616-8542 (
[email protected]). The Journal of Infectious Diseases 2002; 186:1508–13 䉷 2002 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2002/18610-0020$15.00
able for systemic use may still be useful for topical administration. However, antiviral efficacy must be balanced with low mucosal toxicity after prolonged use. Two groups of reversetranscriptase (RT) inhibitors are promising candidates. Certain nonnucleoside RT inhibitors can directly inactivate cell-free virions by binding tightly to HIV-1 RT [3]. These compounds are not active against HIV-2. In contrast, the nucleotide analog tenofovir (9-[2-(phosphonomethoxy)propyl]adenine [PMPA]) and its orally bioavailable prodrug tenofovirdisoproxil fumarate (DF) are active against both HIV-1 and HIV2, and systemic levels of tenofovir are highly effective for prophylaxis in animal models [4]. Although it is not directly virucidal, tenofovir DF is a good candidate for topical administration. Because of its lipophilic nature, tenofovir DF is rapidly taken up by cells in vitro, hydrolyzed to tenofovir, and rapidly converted to high levels of the active form tenofovir diphosphate, which, because of its hydrophilicity, does not exit the cell easily and has a long intracellular half-life (112–50 h) [5]. Rapid uptake by cells and a long intracellular half-life may be an advantage for an oral topical microbicide, because milk, saliva, and other fluids are likely to rapidly rinse away any compounds that reside loosely on mucosal surfaces. Simian immunodeficiency virus (SIV) infection of macaques is a useful animal model in which to screen intervention strategies aimed at reducing sexual or vertical transmission so the most promising treatments can enter human trials first. Female macaques have been used to evaluate the efficacy of topical administration of antiviral agents, and tenofovir gel protects against vaginal SIV infection (C. Miller, A. Rosenberg, N. Bischofberger,
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Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model to determine whether topical (oral) administration of antiviral compounds to the nursing infant could reduce human immunodeficiency virus transmission through breast-feeding. The reverse-transcriptase inhibitor tenofovir was selected because of previous demonstrations that systemic drug levels are effective in preventing SIV infection. To mimic the multiple exposures to virus during breast-feeding, 14 infant macaques were fed 15 low doses of SIVmac251 without chemical restraint. Six animals were treated with placebo, and 2 groups of 4 animals received oral topical doses of tenofovir disoproxil fumarate (DF; equivalent to 0.037 mg of tenofovir/day). About half the animals of each group became infected. In a subsequent study, 2 oral inoculations of 4 juvenile macaques with a mixture of tenofovir DF and SIVmac251 induced persistent infection. Topical administration of low doses of tenofovir DF did not protect against oral SIV infection.
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Materials and Methods In vitro experiments to evaluate drug cellular uptake rate. Duplicate sets of 15-mL conical tubes, each containing 3.5 ⫻ 106 CEMx174 cells in 1 mL of RPMI 1640 medium (Irvine Scientific) without any serum additives (to avoid esterase cleavage of the prodrug), were incubated for 10 min in a 37⬚C water bath. We added 10 mL of tenofovir or tenofovir DF (Gilead Sciences) to obtain a final concentration of 10 mM drug, and the tubes were incubated in a 37⬚C water bath for 15, 30, or 60 s before being chilled rapidly on wet ice to decrease membrane permeability. Duplicate sets of tubes (nonincubated) were placed on ice before the addition of the compounds. One set of 2 tubes received 50 mL of distilled water as placebo control. Cold RPMI 1640 medium was then added to a total volume of 15 mL, and cells were spun down, resuspended, and washed 2 more times. The cells were then resuspended in 1 mL of a 1:20 dilution of the SIVmac251 virus stock described below (thus, ∼5000 TCID50), diluted in complete RPMI 1640 medium (i.e., supplemented with penicillin/streptomycin; Gibco BRL), 5% heat-inactivated bovine calf serum, and 5% heatinactivated bovine fetal serum (Gemini Bio-Products), and incubated for 2.5 h in a 37⬚C, 5% CO2 humidified incubator. Cells were washed 3 times, resuspended in 5 mL of complete medium, and maintained in the incubator. Every 2 or 3 days, 3 mL of cell suspension was replaced by new complete medium, and an aliquot was saved for p27 core antigen determination by an in-house antigen capture ELISA [8] (sensitivity, ∼20 pg/mL). Animals and parameters to monitor infection. All rhesus macaques (Macaca mulatta) were from the type D retrovirus-free and SIV-free colony at the California National Primate Research Center. The newborn macaques were hand-reared in a primate nursery. For the first weeks, the main diet consisted of lactase-treated formula (Enfamil with iron; Mead Johnson). New bottles with formula were provided during the day every 2 h from 0730 h to 2130 h. Starting at age 2 weeks, animals also were offered fruit (banana and apple) and monkey chow. During the first 4 weeks of life, the infant macaques were trained daily by the investigators to drink ∼5 mL of a 1:1 RPMI 1640 medium/sucrose mixture, as described
below (i.e., the placebo vehicle). At age 4 weeks, virus or tenofovir DF was added to this mixture. For all feedings of the infant macaques, either with RPMI 1640/sucrose mixture alone (for training purposes and placebo administrations) or including tenofovir DF or virus, the infant macaques were hand held without chemical restraint and were fed the solution with pet nursing bottles (Four Paws). For blood collections, animals were immobilized with 10 mg/kg intramuscular ketamine-HCl (Parke-Davis). EDTA-anticoagulated blood samples were collected for monitoring immunologic and viral parameters and complete blood cell counts, as described elsewhere [7]. Preparation of vehicle, tenofovir, and virus. A 1:1 mixture of RPMI 1640 medium and isotonic sucrose (300 mosmol/L or 103 g/L [Sigma]; filter sterilized at 0.2 mm [Nalgene]) was used as the vehicle for the oral tenofovir DF and virus administrations. Immediately before use, tenofovir DF powder was dissolved in the RPMI 1640/sucrose mixture to a final concentration of 25 mM. This addition of tenofovir DF did not affect the pH of the RPMI 1640/sucrose solution. The virus used in this study was uncloned SIVmac251 (internal reference no. 5/98) propagated on rhesus peripheral blood mononuclear cells, with a titer of 105 TCID50, and 1.4 ⫻ 109 RNA copies/mL, as measured by SIV bDNA assay [9]. Two oral inoculations with undiluted 1-mL doses of this virus stock were previously found to induce infection of 100% of newborn (n p 11), infant (n p 9), and juvenile (n p 10) rhesus macaques (authors’ unpublished data). For the infant inoculations, a freshly thawed virus aliquot was diluted 50-fold in the RPMI 1640/sucrose mixture and 5 mL was fed per inoculation (thus, 104 TCID50/dose). A 30-min incubation at room temperature of SIVmac251 in this RPMI 1640/sucrose mixture, compared with that in RPMI 1640 medium only, followed by a limiting dilution culture assay, showed no detectable effect on viral infectivity. Juvenile macaques were inoculated orally under ketamine anesthesia with 1 mL of undiluted SIVmac251 (the same virus stock as described above) on 2 consecutive days, by dispensing the inoculum atraumatically and slowly in the mouth.
Results In vitro cellular uptake and antiviral effects of tenofovir and tenofovir DF against SIVmac251. CEMx174 cells were incubated in 10 mM of tenofovir or tenofovir DF at 37⬚C for brief periods, extensively washed, and infected with SIVmac251. As shown in figure 1A, incubation of cells with the parental compound tenofovir for up to 60 s did not result in sufficient uptake of drug to cause a detectable delay in viral replication. In contrast, in cultures exposed briefly to the lipophilic prodrug tenofovir DF, there was a delay in detection of p27 antigen. Even cultures that were incubated with tenofovir DF at 4⬚C (i.e., zero incubation at 37⬚C) had some antiviral effect (figure 1B), but incubation for 15–30 s at 37⬚C resulted, as expected, in a stronger antiviral effect. These results indicate that tenofovir DF entered the cells within seconds and induced intracellular levels that reduced the magnitude of the initial infection. Multiple low-dose exposures of infant macaques to SIV and topical administration of tenofovir DF. At age 4 weeks, infant
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unpublished data). On the basis of these results, a phase I study of topical (intravaginal) tenofovir gel has been initiated. In previous studies, we mimicked intrapartum transmission of HIV by inoculating newborn monkeys orally with 1 or 2 relatively high doses of the highly virulent isolate SIVmac251 during ketamine anesthesia. We demonstrated that systemic administration of only 1 or 2 doses of tenofovir was sufficient to prevent oral SIV infection [6, 7]. HIV transmission through breast-feeding involves prolonged daily exposure to virus in breast milk. Therefore, repeatedly feeding low doses of SIV to infant macaques is a relevant animal model, which may allow prophylactic strategies with partial efficacy to be more easily identified than when animals are exposed to a single overwhelmingly high virus inoculum. In the current study, we performed a pilot experiment to develop a multiple low-dose exposure model that we used to evaluate the efficacy of topical administration of low doses of tenofovir DF.
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macaques received 15 feedings with SIVmac251, diluted in a RPMI 1640/sucrose mixture. These feedings occurred without chemical restraint 3 times a day (at 0830, 1030, and 1430 h) for 5 consecutive days (Monday–Friday; figure 2). The goal was to have a virus dose that would infect at least 50% of the animals. We previously found that 15 inoculations with 5 mL of a 1:500 dilution of SIVmac251 induced persistent infection in only 1 of 4 infant macaques (data not shown). Therefore, in the present study, each inoculation dose consisted of 5 mL of a 1:50 dilution of SIVmac251 stock in RPMI 1640/sucrose (∼104 TCID50/dose and 1.4 ⫻ 108 viral RNA copies/dose). Two groups of 4 infant macaques (figure 2; groups A1 and A2) were fed tenofovir DF solution (25 mM in RPMI 1640/ sucrose). This concentration was selected because it is ∼5000-fold above the in vitro IC50 [5]. The daily dose was 5 mL of tenofovir DF solution and was given either as a single 5-mL dose at 0700 h (∼15–20 min before the first bottle with formula for the day to allow for cellular uptake) or divided over two 2.5-mL doses (at 0700 and 1700 h). Tenofovir DF was given on the 5 consecutive days of the virus inoculations (Monday–Friday); in addition, an extra 5-mL dose of tenofovir DF was given on the day before and the day after the virus inoculations (Sunday afternoon and Saturday morning, respectively). At these same times, the placebo-treated control groups (figure 2; groups B1 and B2) received the RPMI 1640/sucrose vehicle. As shown in figure 2, half the untreated animals became persistently infected (based on virus isolation, polymerase chain reaction, or seroconversion results). A similar rate of infection was seen among the animals given tenofovir DF. Although we tested few animals, there was no detectable difference between the tenofovir- versus placebo-treated groups with regard to onset of
viremia, peak levels of viremia, or disease course. Of the 8 infant macaques that became persistently infected, 6 were viremic within 2 weeks of the first virus inoculation; the other 2 animals (1 from group A1 and 1 from group B1) became viremic 4 weeks after the virus inoculations. Peak virus levels in these 8 infected animals were ∼2 ⫻ 10 6–169 ⫻ 106 RNA copies/mL. The 6 animals with the highest viral RNA levels developed signs of opportunistic infections (e.g., oral candidiasis) or fatal immunodeficiency by age 4 months. The uninfected animals remained healthy during the observation period (⭐1 year). Oral inoculation of juvenile macaques with a mixture of SIVmac251 and tenofovir DF. The data discussed above suggest that topical administration once or twice daily of tenofovir DF did not protect infant macaques against virus inoculations that occurred at different times of the day. An in vitro study found that simultaneous addition of SIVmac251 and tenofovir DF to CEMx174 cells exerted antiviral effects (data not shown). To test this hypothesis in vivo, a preliminary study was performed with 5 juvenile macaques (aged ∼12–18 months) that included 3 of the uninfected animals of the study described above. Juvenile animals cannot be hand held without chemical anesthesia for repeated low-dose oral virus inoculations. Therefore, these 5 juvenile macaques were inoculated orally with 1 mL of undiluted SIVmac251 (105 TCID50/mL or ∼1.4 ⫻ 109 RNA copies/mL) with ketamine anesthesia, and a second inoculation was given the next day. These 2 oral inoculations were found previously to induce infection of 100% of rhesus macaques. In the present study, the virus inoculum of 1 animal received 50 mL of distilled water as placebo. For the other 4 animals, 50 mL of 500 mM tenofovir DF solution was added to the 1-mL virus inoculum for a final concentration of 25 mM tenofovir DF (equivalent to
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Figure 1. Rate of uptake of tenofovir and tenofovir disoproxil fumarate (DF) by cells and antiviral effects. CEMx174 cells were incubated with 10 mM tenofovir (A) or tenofovir DF (B) at 37⬚C for 0–60 s, washed, and inoculated with SIVmac251. Viral replication was monitored by p27 antigen measurement in culture supernatants. All cultures were set up as duplicate sets. Data are mean of the duplicates (which gave similar results). Short-term incubations with 10 mM drug had no detectable toxicity (as determined by cell viability the next day).
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∼1016 molecules/mL). Despite the high ratio of tenofovir DF molecules to virus particles in this inoculum, all 5 animals became persistently infected. Viral RNA levels in plasma of these animals 1 and 2 weeks after SIVmac251 inoculation were within the expected range of those of historical animals (∼106–108 RNA copies/mL). Thus, mixing tenofovir DF with SIV did not prevent infection and viremia.
Discussion The first goal of this study was to develop an infant macaque model that would more closely mimic the multiple low-dose exposure that occurs during breast milk transmission of HIV and that would permit identification and more reliable comparison of effective prophylactic strategies that might be missed in the more common models that use a high viral challenge dose. The SIVmac251 stock used in this study previously infected 100% of animals when two 1-mL undiluted doses were given orally during ketamine anesthesia on 2 consecutive days. Thus, the 15 inoculations of infant macaques with 5 mL of 1:50 diluted virus had an absolute amount of virus (expressed as total TCID50 or viral RNA copies) near that of the 2 high-dose inoculation schedule but divided over multiple exposures in a much more dilute form. Although the virus concentration in this diluted inoculum (∼2.8 ⫻ 107 RNA copies/mL) is higher than the virus levels in milk of transmitting HIV-infected women (median, ∼103 RNA copies/mL; range, !200 to 14 ⫻ 104 RNA copies/ mL), the amount of milk consumed by human infants (often 1700 mL/day) could give exposures to 1107 RNA copies/day [10, 11]. Thus, the dose of SIV we used for each inoculation of the infant macaques was similar to the daily amount of HIV that many human infants are exposed to during breast-feeding. Because we achieved 50% infection of placebo-treated control animals, more inoculations and/or a higher virus concentration will be required to induce infection in a larger percentage of animals.
Our interest in topical microbicides to reduce breast milk transmission of HIV stems from the hope that such strategies, if effective, could have a very low cost. Despite the small numbers of animals in this pilot study, the results suggest that topical administration of tenofovir DF may not protect against oral SIVmac251 infection. This SIVmac251 isolate is a highly virulent isolate containing many viral variants. Thus, it is possible that the use of a more homogenous or less virulent isolate could produce a better outcome. We chose tenofovir DF for this study for several reasons. First, the absence of detectable toxicity in HIV-infected patients receiving a daily oral tablet to achieve systemic drug levels [12] suggests that topical administration of much smaller amounts is unlikely to lead to mucosal irritation. Second, the in vitro data suggest very rapid uptake of tenofovir DF by cells, which is expected to be advantageous to achieve topical antiviral activity in the upper orogastrointestinal tract. Third, studies elsewhere in macaques extensively demonstrated that systemic administration of tenofovir is highly effective in preventing oral, intravenous, or intravaginal infection [4, 6, 7, 13]. Thus, this proven record of prophylactic efficacy of systemic levels of tenofovir provides the ideal setting in which to evaluate the efficacy of topical drug levels. Accordingly, the amount of tenofovir DF we selected for topical administration was purposely one that, although its concentration exceeded the in vitro inhibitory concentration 5000-fold [5], would be too low to induce systemic antiviral drug levels. The daily amount of tenofovir DF (5 mL of 25 mM) that was used for topical administration in this infant macaque study corresponds to 0.037 mg of the parental compound tenofovir. With an oral bioavailability of tenofovir DF in humans of ∼30% [12] and considering the weight of the infant macaques at the time of drug administration (0.62–0.97 kg), this 0.037mg daily dose is expected to be equivalent to a daily parenteral tenofovir dose of 0.011–0.018 mg/kg of body weight. Thus, this dose is ∼200–2700-fold lower than the subcutaneous 4–30 mg/
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Figure 2. Experimental design and outcome summary. At age 4 weeks, infant macaques were fed diluted SIVmac251 3 times/day at indicated times for 5 consecutive days. Groups A1 and A2 received topical administration of tenofovir disoproxil fumarate (TDF) solution (25 mM) either as 5 mL once/day (group A1) or 2.5 mL twice/day (group A2); groups B1 and B2 were the respective placebo-treated control groups. During the daytime, animals received a new bottle with formula every 2 h starting at 0730 h as indicated. *, Formula feeding time.
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intravaginal but not against oral SIV infection is not known but may be a combination of factors. Even though the intravaginal challenge studies used tenofovir (not tenofovir DF), the gel had a concentration of 10 mg/mL tenofovir, ∼1000-fold higher than the concentration of tenofovir DF we used for topical oral administration. Drug is also likely to remain present in the vaginal lumen for a longer period than in the upper orogastrointestinal tract; in this regard, the physicochemical properties of the gel formulation in the intravaginal studies may also be advantageous compared with the aqueous solution of tenofovir DF that was used for the oral administration. In addition, studies in macaques suggest that after intravaginal inoculation of SIV, intraepithelial dendritic cells, which extend processes to the lumen, may be the first target cells for viral replication [19]. Breaks in the epithelial lining of the vagina or cervix may also be a portal of entry. Accordingly, the initial target cells for viral replication after intravaginal exposure are likely to be exposed to virus particles as well as to topical tenofovir. Thus, potential fundamental differences in mucosal physiology and pathogenesis of oral versus genital transmission of SIV and HIV may impose different requirements for topical microbicides to be effective. Further research is warranted in this area. The studies described further underscore the value of animal models to test antiviral intervention strategies because promising in vitro antiviral effects do not always translate into efficacy in vivo. Until a topical microbicide that is safe and effective against oral HIV infection is identified, systemic drug levels of potent antiviral compounds such as tenofovir may be the best chemoprophylactic strategy to reduce HIV transmission via breast-feeding. Acknowledgments We thank D. Bennett, T. Dearman, L. Fritts, L. Hirst, A. Spinner, W. von Morgenland, the Veterinary Staff at Colony Services and Clinical Laboratory of the California National Primate Research Center for expert technical assistance, and M. Miller (Gilead Sciences) for useful suggestions and discussions.
References 1. De Cock K, Fowler MG, Mercier E, et al. Prevention of mother-to-child HIV transmission in resource-poor countries: translating research into policy and practice. JAMA 2000; 283:1175–82. 2. Nduati R, John G, Mbori-Ngacha D, et al. Effect of breastfeeding and formula feeding on transmission of HIV-1. JAMA 2000; 283:1167–74. 3. Balzarini J, Naesens L, Verbeken E, et al. Preclinical studies on thiocarboxanilide UC-781 as a virucidal agent. AIDS 1998; 12:1129–38. 4. Tsai CC, Follis KE, Beck TW, et al. Prevention of simian immunodeficiency virus infection in macaques by 9-(2-phosphonylmethoxypropyl)adenine (PMPA). Science 1995; 270:1197–9. 5. Robbins BL, Srinivas RV, Kim C, Bischofberger N, Fridland A. Anti–human immunodeficiency virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phosphonate 9-R-(2-phosphonometh-
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kg dosage regimen that induces systemic drug levels that protect macaques against infection after inoculation with a high dose of virus [4, 6, 7, 13]. In macaques, the chemoprophylactic success of antiviral drugs becomes more pronounced when lower doses of virus are used [14]. Thus, the systemic levels of tenofovir that were effective against single high-dose exposures are expected also to be effective against multiple low-dose exposures. Accordingly, the absence of prophylaxis following topical tenofovir DF administration in the present study can be ascribed to several possible reasons. It is possible that systemic levels of tenofovir do not completely prevent the initial mucosal infection of SIV but are required to block virus dissemination. Alternatively, if mucosal levels of tenofovir play a major role in limiting the initial cycle of infection, then topical administration of tenofovir may have led to insufficient mucosal levels. Although this study was not designed to differentiate between these possible mechanisms, a number of scenarios can explain our findings. First, the very fast entry of tenofovir DF into cells in vitro may not be applicable to the mucosa of the orogastrointestinal tract. Topical administration of tenofovir only once or twice a day is also unlikely to protect against multiple exposures to virus if a significant number of target cells for SIV replication migrate in and out of the mucosa within hours [15]. Infection of juvenile macaques was also not prevented even when the virus and tenofovir DF were mixed together to maximize the chance that both SIV and tenofovir prodrug would contact the same surface cells. Because tenofovir needs to be phosphorylated to its active diphosphate derivative by cellular enzymes, it is possible that viral reverse transcription occurred before tenofovir diphosphate had reached sufficient intracellular levels. An alternative explanation for the failure of prophylaxis is that tenofovir prodrug may sufficiently enter and accumulate in the cells that directly line the mucosal surface, but that reverse transcription occurs in different cells that are not in direct contact with the lumen and therefore do not receive sufficient tenofovir levels after topical administration of small amounts of the compound. For instance, M cells, which are present in the epithelium of the tonsillar crypts, take up antigens, including virions, and transport them in vesicles across the epithelium to underlying target cells (e.g., dendritic cells, T lymphocytes, and macrophages) in which reverse transcription can occur [16, 17]. It is unclear whether other RT inhibitors (e.g., efavirenz and UC781), which are promising as microbicides because they directly inactivate virions (by tightly binding RT) and/or show intracellular accumulation [3, 18], would face similar problems. Intravaginal administration of tenofovir gel protects female macaques against a high-dose intravaginal inoculation with SIVmac251 (2 doses of 105 TCID50; C. Miller, Z. Rosenberg, N. Bischofberger, unpublished data). The reason for this apparent discrepancy of prophylactic efficacy of tenofovir against
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fumarate in HIV-1 infected adults. Antimicrob Agents Chemother 2001; 45:2733–9. 13. Otten RA, Smith DK, Adams DR, et al. Efficacy of postexposure prophylaxis after intravaginal exposure of pig-tailed macaques to a human-derived retrovirus (human immunodeficiency virus type 2). J Virol 2000;74:9771–5. ¨ berg B. Influence of the infectious dose of simian 14. Bo¨ttiger D, Vrang L, O immunodeficiency virus on the acute infection in cynomolgus monkeys and on the effect of treatment with 3-fluorothymidine. Antivir Chem Chemother 1992; 3:267–71. 15. Macatonia SE, Knight SC, Edwards AJ, Griffiths S, Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate: functional and morphological studies. J Exp Med 1987; 166:1654–67. 16. Fujimura Y. Evidence of M cells as portals of entry for antigens in the nasopharyngeal lymphoid tissue of humans. Virchows Arch 2000;436:560–6. 17. Stahl-Hennig C, Steinman RM, Tenner-Racz K, et al. Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science 1999; 285:1261–5. 18. Borkow G, Barnard J, Nguyen TM, Belmonte A, Wainbert MA, Parniak MA. Chemical barriers to human immunodeficiency virus type 1 (HIV-1) infection: retrovirucidal activity of UC781, a thiocarboxanilide nonnucleoside inhibitor of HIV-1 reverse transcriptase. J Virol 1997;71:3023–30. 19. Hu J, Gardner MB, Miller CJ. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol 2000; 74:6087–95.
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oxypropyl)adenine (PMPA), bis(isopropyloxymethylcarbonyl)PMPA.Antimicrob Agents Chemother 1998; 42:612–7. 6. Van Rompay KKA, Berardi CJ, Aguirre NL, et al. Two doses of PMPA protect newborn macaques against oral simian immunodeficiency virus infection. AIDS 1998; 12:F79–83. 7. Van Rompay KKA, McChesney MB, Aguirre NL, Schmidt KA, Bischofberger N, Marthas ML. Two low doses of tenofovir protect newborn macaques against oral simian immunodeficiency virus infection. J Infect Dis 2001; 184:429–38. 8. Lohman BL, Higgins J, Marthas ML, Marx PA, Pedersen NC. Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay. J Clin Microbiol 1991; 29:2187–92. 9. Booth J, Sawyer L, McNelley E, et al. Performance characteristics of a highly sensitive quantitative assay for SIV RNA using branched DNA technology [abstract 129]. In: Programs and abstracts of the 18th annual symposium on Nonhuman Primate Models for AIDS (Madison, Wisconsin). J Medical Primatol 2001; :242. 10. Lewis P, Nduati R, Kreiss JK, et al. Cell-free human immunodeficiency virus type 1 in breast milk. J Infect Dis 1998; 177:34–9. 11. Semba RD, Kumwenda N, Hoover DR, et al. Human immunodeficiency virus load in breast milk, mastitis, and mother-to-child transmission of human immunodeficiency virus type 1. J Infect Dis 1999; 180:93–8. 12. Barditch-Crovo P, Deeks SG, Collier A, et al. Phase I/II trial of the pharmacokinetics, safety, and antiretroviral activity of tenofovir disoproxil
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