Last data update: Jun 03, 2024. (Total: 46935 publications since 2009)
Records 1-23 (of 23 Records) |
Query Trace: Pau CP [original query] |
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A monoclonal antibody for the detection of the antiretroviral drug emtricitabine
Youngpairoj AS , Vanderford TH , Reed MS , Granade TC , Pau CP , Pohl J , Switzer WM , Heneine W . AIDS 2022 36 (13) 1890-1893 Antibody-based testing for emtricitabine (FTC), a critical component of pre-exposure prophylaxis and antiretroviral therapy would provide low-cost detection for clinical monitoring to improve adherence. We developed a monoclonal antibody (5D2) to FTC and demonstrated its high specificity and physiologically relevant linear range of detection in a competitive enzyme immunoassay (EIA). Thus, this monoclonal antibody is a key reagent that will enable simple and low-cost lateral flow assays and EIAs for adherence monitoring. |
The Female Genital Tract Microbiome Is Associated With Vaginal Antiretroviral Drug Concentrations in Human Immunodeficiency Virus-Infected Women on Antiretroviral Therapy.
Donahue Carlson R , Sheth AN , Read TD , Frisch MB , Mehta CC , Martin A , Haaland RE , Patel AS , Pau CP , Kraft CS , Ofotokun I . J Infect Dis 2017 216 (8) 990-999 Background: The female genital tract (FGT) microbiome may affect vaginal pH and other factors that influence drug movement into the vagina. We examined the relationship between the microbiome and antiretroviral concentrations in the FGT. Methods: Over one menstrual cycle, 20 human immunodeficiency virus (HIV)-infected women virologically suppressed on tenofovir (TFV) disoproxil fumarate/emtricitabine and ritonavir-boosted atazanavir (ATV) underwent serial paired cervicovaginal and plasma sampling for antiretroviral concentrations using high-performance liquid chromatography-tandem mass spectrometry. Analysis of 16S ribosomal RNA gene sequencing of cervicovaginal lavage clustered each participant visit into a unique microbiome community type (mCT). Results: Participants were predominantly African American (95%), with a median age of 38 years. Cervicovaginal lavage sequencing (n = 109) resulted in a low-diversity mCT dominated by Lactobacillus (n = 40), and intermediate-diversity (n = 28) and high-diversity (n = 41) mCTs with abundance of anaerobic taxa. In multivariable models, geometric mean FGT:plasma ratios varied significantly by mCT for all 3 drugs. For both ATV and TFV, FGT:plasma was significantly lower in participant visits with high- and low-diversity mCT groups (all P < .02). For emtricitabine, FGT:plasma was significantly lower in participant visits with low- vs intermediate-diversity mCT groups (P = .002). Conclusions: Certain FGT mCTs are associated with decreased FGT antiretroviral concentrations. These findings are relevant for optimizing antiretrovirals used for biomedical HIV prevention in women. |
Levels of intracellular phosphorylated tenofovir and emtricitabine correlate with natural substrate concentrations in peripheral blood mononuclear cells of persons prescribed daily oral TruvadaTM for HIV pre-exposure prophylaxis
Haaland RE , Holder A , Pau CP , Swaims-Kohlmeier A , Dawson C , Smith DK , Segolodi TM , Thigpen MC , Paxton LA , Parsons TL , Hendrix CW , Hart CE . J Acquir Immune Defic Syndr 2017 75 (3) e86-e88 Successful clinical trials of antiretroviral pre-exposure prophylaxis (PrEP) to prevent HIV infection have been reported in heterosexual men and women, men who have sex with men (MSM), and injection drug users1. A daily oral drug regimen containing nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC) have been effective when participant adherence is high1. Analysis of PrEP dosing patterns estimate taking at least four TDF doses per week provides 96% protection from HIV infection and at least two doses per week provides 76% protection in MSM and transgender women2, though some estimate more frequent dosing in heterosexual women may be needed3. TDF and FTC require phosphorylation in HIV target cells to tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) to provide PrEP protection as competitive analogs of naturally occurring deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP), respectively. However, it is unclear if physiological conditions that increase dNTP concentrations can affect pharmacokinetics (PK) and pharmacodynamics (PD) of NRTIs. This may be particularly relevant when cellular deoxynucleoside triphosphate (dNTP) pools in HIV target cells increase in response to immune activation. Decreased ratios of phosphorylated NRTIs to their respective dNTPs have been associated with cell activation in vitro and in nonhuman primate studies4,5, yet this observation remains unexplored in PK and PD studies that measured intracellular drug6-8. The study presented here compared dATP and dCTP concentrations to TFV-DP and FTC-TP in peripheral blood mononuclear cell (PBMCs) of persons using daily oral Truvada™ during a successful HIV PrEP study. We further examined if variations among intracellular drug:dNTP ratios were related to lymphocyte activation. |
Antiretroviral drug activity in macaques infected during pre-exposure prophylaxis has a transient effect on cell-associated SHIV DNA reservoirs
Cong ME , Pau CP , Heneine W , Garcia-Lerma JG . PLoS One 2016 11 (11) e0164821 BACKGROUND: Pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) is a novel HIV prevention strategy. Suboptimal PrEP adherence and HIV infection creates an opportunity for continued antiretroviral drug activity during undiagnosed infection. We previously showed that macaques infected with SHIV during PrEP with FTC/TDF display reduced acute plasma viremias and limited virus diversity. We investigated the effect of PrEP on acute SHIV DNA dynamics and on the size of the persistent virus reservoir in lymphoid tissues. DESIGN: Cell-associated SHIV DNA levels in PBMCs were measured in 8 macaques infected during PrEP with FTC/TDF or single-agent TAF and was compared to those seen in untreated infections (n = 10). PrEP breakthrough infections continued treatment with 1-2 weekly drug doses to model suboptimal drug exposure during undiagnosed HIV infection in humans. SHIV DNA was also measured in lymphoid tissues collected from FTC/TDF PrEP breakthroughs after 1 year of infection. RESULTS: Compared to untreated controls, PrEP infections had reduced plasma RNA viremias both at peak and throughout weeks 1-12 (p<0.005). SHIV DNA levels were also reduced at peak and during the first 12 weeks of infection (p<0.043) but not throughout weeks 12-20. At 1 year, SHIV DNA reservoirs in lymphoid tissues were similar in size among macaques that received PrEP or placebo. CONCLUSIONS: Antiviral drug activity due to PrEP limits acute SHIV replication but has only a transient effect on cell-associated SHIV DNA levels. Our model suggests that suboptimal drug exposure in persons that are taking PrEP and become infected with HIV may not be sufficient to reduce the pool of HIV-infected cells, and that treatment intensification may be needed to sustain potential virological benefits from the PrEP regimen. |
Efficacy of topical tenofovir against transmission of a tenofovir-resistant SHIV in macaques
Dobard CW , Sharma S , Cong ME , West R , Makarova N , Holder A , Pau CP , Hanson DL , Novembre FJ , Garcia-Lerma JG , Heneine W . Retrovirology 2015 12 (1) 69 BACKGROUND: Topically delivered tenofovir (TFV) from intravaginal rings, tablets, or gels is being evaluated for HIV prevention. We previously demonstrated that TFV delivered vaginally by gel protected macaques from vaginal infection with SHIV. Here we investigated efficacy of the TFV gel against vaginal transmission of a TFV-resistant SHIV containing the K65R mutation (SHIV162P3K65R) and its relationship to drug levels in vaginal tissues. RESULTS: SHIV162P3K65R shows approximately a 5-fold reduction in susceptibility to TFV compared to wild-type SHIV. Efficacy was evaluated in pig-tailed macaques exposed vaginally twice-weekly (up to 10 weeks) to SHIV162P3K65R 30 min after receiving placebo (n = 6) or 1% TFV (n = 6) gel. Four of the six controls were infected after a median of 5 exposures. In contrast, five of six macaques that received TFV gel remained uninfected after 20 vaginal SHIV162P3K65R exposures, resulting in an estimated efficacy of 75%. The mean intracellular TFV-diphosphate (TFV-DP) concentrations in vaginal lymphocytes 4 h after a single gel dose were found to be high (1,631 fmol/10(6) cells, range 492-3,847) and within the in vitro IC75 range (1,206 fmol/10(6) cells) for SHIV162P3K65R. CONCLUSION: Both the modest resistance conferred by K65R and the high TFV-DP exposure in vaginal lymphocytes, likely explain the observed protection. The findings in this model do not predict complete loss of protection by topical TFV against vaginal exposure to HIV-1K65R viruses and provide a tissue drug target for high efficacy. These data will facilitate the development of TFV delivery platforms that have high activity on both wild-type and TFV-resistant viruses. |
Protection Against Rectal Chimeric Simian/Human Immunodeficiency Virus Transmission in Macaques by Rectal-Specific Gel Formulations of Maraviroc and Tenofovir
Dobard CW , Taylor A , Sharma S , Anderson PL , Bushman LR , Chuong D , Pau CP , Hanson D , Wang L , Garcia-Lerma JG , McGowan I , Rohan L , Heneine W . J Infect Dis 2015 212 (12) 1988-95 BACKGROUND: Rectal HIV transmission is an important driver of the HIV epidemic. Optimally formulated gels of antiretroviral drugs are under development for preventing rectally acquired HIV. We investigated in a macaque model the pharmacokinetics and efficacy of three rectal gel formulations METHODS: Single-dose pharmacokinetics of low-osmolar 1% maraviroc (MVC), 1% tenofovir (TFV), or 1%MVC/1%TFV combination gel were evaluated in blood, rectal fluids, colorectal biopsies, and rectal lymphocytes. Efficacy was evaluated over 10 twice-weekly rectal SHIV162p3 challenges in rhesus macaques that received either placebo (n=7), MVC (n=6), TFV (n=6), or MVC/TFV (n=6) gel 30 minutes before each challenge. RESULTS: MVC and TFV were detected in plasma 30 minutes after gel application and remained above IC95 levels in rectal fluids at 24h. MVC, TFV, and TFV-DP concentrations in colorectal tissues collected up to 30 cm from the anal margin were all high at 2h, demonstrating rapid and extended tissue dosing. TFV-DP concentrations in tissue homogenates and rectal lymphocytes were highly correlated (r2=0.82). All three gel formulations were highly protective (82% efficacy; log-rank test; p≤0.02). CONCLUSION: Desirable pharmacokinetic profiles and high efficacy in this macaque model support the clinical development of these gel formulations for preventing rectal HIV infection. |
Pharmacokinetic profile of raltegravir, elvitegravir and dolutegravir in plasma and mucosal secretions in rhesus macaques
Massud I , Martin A , Dinh C , Mitchell J , Jenkins L , Heneine W , Pau CP , Garcia-Lerma JG . J Antimicrob Chemother 2015 70 (5) 1473-81 OBJECTIVES: Pharmacokinetic studies in animal models are important for assessing the prophylactic potential of antiretroviral drugs for HIV prevention. This study sought to identify clinically relevant doses of the marketed integrase inhibitors raltegravir, elvitegravir and dolutegravir in macaques and investigate drug penetration and antiviral activity in mucosal secretions. METHODS: Macaques received one oral dose of raltegravir, elvitegravir or dolutegravir alone or in combination with emtricitabine and tenofovir disoproxil fumarate followed by drug level measurements in blood and rectal and vaginal secretions. Antiviral activity was investigated in TZM-bl cells exposed to SHIV162p3 in the presence of rectal secretions collected from treated animals. RESULTS: Plasma drug concentrations with 50 mg/kg raltegravir or elvitegravir were within the range seen in humans receiving 400-800 mg of raltegravir or 800 mg of unboosted elvitegravir but lower than with 150 mg of elvitegravir boosted with cobicistat. AUC0-24 values for dolutegravir increased proportionally with the dose, with a calculated human-equivalent dose of 20 mg/kg. Elvitegravir showed the highest penetration in rectal and vaginal fluids despite the absence of pharmacological boosting, followed by raltegravir and dolutegravir. Rectal secretions collected at 24 h from treated macaques blocked infection of TZM-bl cells by 50% at dilutions of 1/1000 (raltegravir), 1/800 (dolutegravir) and >1/30 000 (elvitegravir). CONCLUSIONS: We defined macaque doses of HIV integrase inhibitors that recapitulate human clinical doses, which will facilitate efficacy and dose escalation studies in macaques. High and sustained drug concentrations and activity in mucosal secretions suggest that integrase inhibitors are promising candidates for HIV prevention. |
Pharmacokinetic evaluation of tenofovir disoproxil fumarate released from an intravaginal ring in pigtailed macaques after 6 months of continuous use
Srinivasan P , Dinh C , Zhang J , Pau CP , McNicholl JM , Lo YT , Herold BC , Teller R , Kiser P , Smith JM . J Med Primatol 2014 43 (5) 364-369 BACKGROUNDS AND METHODS: A reservoir intravaginal ring (IVR) eluting tenofovir disoproxil fumarate (TDF) was evaluated for 6 months of continuous use in normally cycling female pigtailed macaques with monthly IVR exchanges to define pharmacokinetics and safety. RESULTS AND CONCLUSIONS: Tenofovir levels in vaginal secretions and tissue remained consistent for 6 months with no adverse safety concerns. |
Reference panel of cloned HIV-2 plasmid DNA for nucleic acid assay development, evaluation, and quality monitoring.
Youngpairoj AS , Curtis KA , Wells SK , Pau CP , Granade TC , Owen SM . J Clin Virol 2014 61 (2) 293-7 BACKGROUND: Currently, no FDA-approved HIV-2 nucleic acid assay is commercially available in the United States, although several laboratories have developed in-house assays to confirm HIV-2 infections. A major limitation in the development of novel HIV-2 diagnostic assays is the lack of reference materials that can be used to evaluate, optimize, and monitor assay performance. STUDY DESIGN: Eleven viral stocks of HIV-2 isolates from various West African countries, including the Ivory Coast, Senegal, and Guinea-Bissau, were used to clone the entire LTR and pol regions from each virus. RESULTS: We successfully cloned, sequenced, and group classified 22 HIV-2 DNA plasmids including 11 full length LTR ( approximately 849bp) and 11 pol ( approximately 2995bp) sequences. There were eight HIV-2 group A and three group B in both the LTR and pol regions. CONCLUSIONS: This reference panel provides a robust, quantifiable, renewable, and non-infectious set of reagents that can be used for the development and evaluation of new HIV-2 molecular diagnostic assays and quality assurance and quality control reagents for use in the clinical laboratories. |
Postexposure protection of macaques from vaginal SHIV infection by topical integrase inhibitors
Dobard C , Sharma S , Parikh UM , West R , Taylor A , Martin A , Pau CP , Hanson DL , Lipscomb J , Smith J , Novembre F , Hazuda D , Garcia-Lerma JG , Heneine W . Sci Transl Med 2014 6 (227) 227ra35 Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P < 0.05, Fisher's exact test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure. |
Lack of prophylactic efficacy of oral maraviroc in macaques despite high drug concentrations in rectal tissues
Massud I , Aung W , Martin A , Bachman S , Mitchell J , Aubert R , Tsegaye TS , Kersh E , Pau CP , Heneine W , Garcia-Lerma JG . J Virol 2013 87 (16) 8952-61 Maraviroc (MVC) is a potent CCR5 co-receptor antagonist that is in clinical testing for daily oral pre-exposure prophylaxis (PrEP) for HIV prevention. We used a macaque model consisting of weekly SHIV162p3 exposures to evaluate the efficacy of oral MVC in preventing rectal SHIV transmission. MVC dosing was informed by the pharmacokinetic profile seen in blood and rectal tissues, and consisted of a human-equivalent dose given 24h before virus exposure followed by a booster post-exposure dose. In rectal secretions, MVC peaked at 24h (10,242 ng/ml) with concentrations at 48h that were about 40 times those required to block SHIV infection of PBMCs in vitro. Median MVC concentrations in rectal tissues at 24h (1,404 ng/g) were 30 and 10 times those achieved in vaginal or lymphoid tissues, respectively. MVC significantly reduced MIP-1beta-induced CCR5 internalization in rectal mononuclear cells, an indication of efficient binding to CCR5 in rectal lymphocytes. The half-life of CCR5-bound MVC in PBMCs was 2.6 days. Despite this favorable profile, 5/6 treated macaques were infected during 5 rectal SHIV exposures as were 3/4 controls. MVC treatment was associated with a significant increase in the percentage of CD3+/CCR5+ cells in blood. We show that high and durable MVC concentrations in rectal tissues are not sufficient to prevent SHIV infection in macaques. The increases in CD3+/CCR5+ cells seen during MVC treatment point to unique immunological effects of CCR5 inhibition by MVC. The implications of these immunological effects on PrEP with MVC require further evaluation. |
Prevention of vaginal SHIV transmission in macaques by a coitally-dependent Truvada regimen
Radzio J , Aung W , Holder A , Martin A , Sweeney E , Mitchell J , Bachman S , Pau CP , Heneine W , Garcia-Lerma JG . PLoS One 2012 7 (12) e50632 BACKGROUND: Daily pre-exposure prophylaxis (PrEP) with Truvada (a combination of emtricitabine (FTC) and tenofovir (TFV) disoproxil fumarate (TDF)) is a novel HIV prevention strategy recently found to prevent HIV transmission in men who have sex with men and heterosexual couples. We previously showed that a coitally-dependent Truvada regimen protected macaques against rectal SHIV transmission. Here we examined FTC and tenofovir TFV exposure in vaginal tissues after oral dosing and assessed if peri-coital Truvada also protects macaques against vaginal SHIV infection. METHODS: The pharmacokinetic profile of emtricitabine (FTC) and tenofovir (TFV) was evaluated at first dose. FTC and TFV levels were measured in blood plasma, rectal, and vaginal secretions. Intracellular concentrations of FTC-triphosphate (FTC-TP) and TFV-diphosphate (TFV-DP) were measured in PBMCs, rectal tissues, and vaginal tissues. Efficacy of Truvada in preventing vaginal SHIV infection was assessed using a repeat-exposure vaginal SHIV transmission model consisting of weekly exposures to low doses of SHIV162p3. Six pigtail macaques with normal menstrual cycles received Truvada 24 h before and 2 h after each weekly virus exposure and six received placebo. Infection was monitored by serology and PCR amplification of SHIV RNA and DNA. RESULTS: As in humans, the concentration of FTC was higher than the concentration of TFV in vaginal secretions. Also as in humans, TFV levels in vaginal secretions were lower than in rectal secretions. Intracellular TFV-DP concentrations were also lower in vaginal tissues than in rectal tissues. Despite the low vaginal TFV exposure, all six treated macaques were protected from infection after 18 exposures or 4 full menstrual cycles. In contrast, all 6 control animals were infected. CONCLUSIONS: We modeled a peri-coital regimen with two doses of Truvada and showed that it fully protected macaques from repeated SHIV exposures. Our results open the possibility for simplified PrEP regimens to prevent vaginal HIV transmission in women. |
Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.
Pau CP , Wells SK , Granade TC . Methods Mol Biol 2012 903 263-71 This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers. |
UC781 microbicide gel retains anti-HIV activity in cervicovaginal lavages collected following twice daily vaginal application
Haaland RE , Evans-Strickfaden T , Holder A , Pau CP , McNicholl JM , Chaikummao S , Chonwattana W , Hart CE . Antimicrob Agents Chemother 2012 56 (7) 3592-6 The potent non-nucleoside reverse transcriptase inhibitor UC781 has been safety tested as a vaginal microbicide gel formulation for prevention of HIV-1 sexual transmission. To investigate whether UC781 retained anti-infective activity following exposure to the female genital tract, we conducted an ex vivo analysis of the UC781 levels and antiviral activity in cervicovaginal lavages (CVL) from 25 Thai women enrolled in a 14-day safety trial of twice-daily vaginal application of two concentrations of UC781 microbicide gel. CVL samples were collected from women in 0.1% (n=5), 0.25% (n=15) and placebo (n=5) gel arms following the first application of gel (T(15min)) and 8-24 hours after the final application (T(8-24hr)), and separated into cell-free (CVL-s) and pelletable fractions (CVL-p). As UC781 is highly hydrophobic, there were significantly higher levels of UC781 in the CVL-p samples compared to CVL-s for the UC781 gel arms. In T(8-24hr) CVL-p samples, 2/5 and 13/15 samples collected from the 0.1% and 0.25% UC781 gel arms, respectively, efficiently blocked infection with ≥4 log(10) TCID(50) of a CCR5-tropic CRF01_AE HIV-1 virus stock. Independent of arm, the 11 CVL-p samples with UC781 levels ≥5 mcg/CVL reduced infectious HIV ≥4 log(10) TCID(50). Our results suggest that the levels and anti-infective activity of UC781 gel formulations are likely to be associated with a cellular or pelletable component in CVL samples. Therefore, cellular and pelletable fractions should be assayed for drug levels and anti-infective activity in preclinical studies of candidate microbicides. |
Safe and sustained vaginal delivery of pyrimidinedione HIV-1 inhibitors from polyurethane intravaginal rings
Johnson TJ , Srinivasan P , Albright TH , Watson-Buckheit K , Rabe L , Martin A , Pau CP , Hendry RM , Otten R , McNicholl J , Buckheit R Jr , Smith J , Kiser PF . Antimicrob Agents Chemother 2012 56 (3) 1291-9 The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD1(0.5wt%)], PYD1(1wt%), PYD2(4wt%), and PYD2(14wt%)) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD2(14wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 mug/ml to vaginal fluid and 1 mug/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation. |
Durable protection from vaginal simian-human immunodeficiency virus infection in macaques by tenofovir gel and its relationship to drug levels in tissue
Dobard C , Sharma S , Martin A , Pau CP , Holder A , Kuklenyik Z , Lipscomb J , Hanson DL , Smith J , Novembre FJ , Garcia-Lerma JG , Heneine W . J Virol 2012 86 (2) 718-25 A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/10(6) cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC(95)), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC(95) in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies. |
Natural substrate concentrations can modulate the prophylactic efficacy of nucleotide HIV reverse transcriptase inhibitors
Garcia-Lerma JG , Aung W , Cong ME , Zheng Q , Youngpairoj AS , Mitchell J , Holder A , Martin A , Kuklenyik S , Luo W , Lin CY , Hanson DL , Kersh E , Pau CP , Ray AS , Rooney JF , Lee WA , Heneine W . J Virol 2011 85 (13) 6610-7 Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/10(6) cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/10(6) mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors. |
Rapid detection and differentiation of antibodies to HIV-1 and HIV-2 using multivalent antigens and magnetic immunochromatography testing (MICT)
Granade TC , Workman S , Wells SK , Holder AN , Owen SM , Pau CP . Clin Vaccine Immunol 2010 17 (6) 1034-9 A simplified lateral flow assay for the detection of antibodies to HIV using magnetic bead conjugates and multi-branched peptides from both HIV-1 and HIV-2 was developed. Magnetic immunochromatography testing (MICT) uses a standard lateral-flow platform that incorporates magnetic bead conjugates for quantitative measurement of the magnetic field distortion associated with the bound magnetic conjugate (reported as adjusted magnetic units, MAR). The results of the optimized MICT assay were compared to standard enzyme immunoassay (EIA) and Western blot (WB) results using a blinded 649-member panel of specimens from the US, Cameroon, and West Africa. The panel was comprised of samples from individuals infected with various HIV-1 subtypes (n=234), HIV-2 (n=65) and HIV-seronegative specimens (n=350). Additionally, thirteen HIV-1 sero-conversion panels (total specimens=85), a worldwide panel containing seven of the major circulating HIV-1 subtypes (n=18), an HIV-2 panel, an HIV-1/HIV-2 mixed panel, and 100 prospective specimens were tested with completely concordant results. Assay reproducibility (observed MAR) for both intra- and inter-run testing was excellent with coefficients of variation <12%. MICT can provide a rapid, low-cost method of determining HIV antibody status requiring no subjective interpretations. |
Intermittent prophylaxis with oral Truvada protects macaques from rectal SHIV infection
Garcia-Lerma JG , Cong ME , Mitchell J , Youngpairoj AS , Zheng Q , Masciotra S , Martin A , Kuklenyik Z , Holder A , Lipscomb J , Pau CP , Barr JR , Hanson DL , Otten R , Paxton L , Folks TM , Heneine W . Sci Transl Med 2010 2 (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 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. |
A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer
Pau CP , Wells SK , Rudolph DL , Owen SM , Granade TC . J Virol Methods 2009 164 55-62 In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A=2, AC=1, B=4, C=3, D=3, AE=2, F=1, BF=2, G=4) were amplified equally well with a similar threshold cycle (C(t)) number (22.9+/-1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues. |
On-line coupling of anion exchange and ion-pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors
Kuklenyik Z , Martin A , Pau CP , Holder A , Youngpairoj AS , Zheng Q , Cong ME , Garcia-Lerma JG , Heneine W , Pirkle JL , Barr JR . J Chromatogr B Analyt Technol Biomed Life Sci 2009 877 (29) 3659-66 We developed an automated on-line weak anion exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography-tandem mass spectrometry (MS/MS) detection for quantitatively measuring triphosphorylated metabolites of three reverse transcriptase inhibitors (RTI). The administered pro-drugs were Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC) and Lamivudine (3TC). Their intracellular metabolites Tenofovir-diphosphate (TFV-DP), Emtricitabine-triphosphate (FTC-TP), and Lamivudine-triphosphate (3TC-TP) were measured in peripheral blood mononuclear cells (PBMC). We coupled the WAX and IP chromatography systems using a combination of 6-port and 10-port switching valves, and we mixed the WAX elute with 1,5-dimethyl-hexyl-amine before IP chromatography separation. Multiple waste outlets allowed for eliminating potential matrix components interfering with MS/MS detection. Limits of detection were 9, 200 and 75pg per sample for TFV-DP (448/176 m/z), FTC-TP (488/130 m/z) and 3TC-TP (468/119 m/z), respectively. |
Rapid detection of HIV-1 p24 antigen using magnetic immuno-chromatography (MICT)
Workman S , Wells SK , Pau CP , Owen SM , Dong XF , LaBorde R , Granade TC . J Virol Methods 2009 160 14-21 Detection of human immunodeficiency virus (HIV) infections has been enhanced by incorporating p24 antigen detection with current HIV antibody detection using enzyme immunoassays (EIAs). However, screening for HIV antibodies has increased through the use of rapid, lateral-flow HIV antibody detection assays that currently do not have the capability to detect HIV p24 antigen. In this report, a lateral-flow based assay using super-paramagnetic particles as the detection marker was developed for the detection of HIV-1 p24 antigen. This magnetic immuno-chromatographic test (MICT) uses an inexpensive, low-maintenance instrument that detects the magnetic moment of the super-paramagnetic particles in a magnetic field. MICT is simple to perform, provides a numerical output for easier determination of reactive results and can be completed in 40 min. The lower limit of detection for HIV-1 p24 spiked into assay sample buffer and 50% plasma was 30 pg/ml for both. Detection of HIV-1 p24 antigen at 50 pg/ml was reproducible in both inter-run and intra-run assays with coefficients of variation of <13%. Furthermore, the MICT p24 assay was able to detect intact virus spiked into 50% plasma (lower detection limit of [similar to]250,000 viral RNA copies/ml). MICT detection of increasing HIV-1 p24 levels in commercially available seroconversion panels by MICT was only slightly later than that detected by much more complex EIAs. MICT could provide a simple, low-cost, and portable method for rapid HIV-1 p24 detection in a variety of testing environments. |
Complete protection from repeated vaginal simian-human immunodeficiency virus exposures in macaques by a topical gel containing tenofovir alone or with emtricitabine
Parikh UM , Dobard C , Sharma S , Cong ME , Jia H , Martin A , Pau CP , Hanson DL , Guenthner P , Smith J , Kersh E , Garcia-Lerma JG , Novembre FJ , Otten R , Folks T , Heneine W . J Virol 2009 83 (20) 10358-65 New-generation gels that deliver potent antiretroviral drugs against HIV-1 have renewed hopes in topical prophylaxis as a prevention strategy. Previous preclinical research in monkey models suggested that high concentrations and drug combinations are needed for high efficacy. We evaluated two long-acting reverse transcriptase inhibitors, tenofovir and emtricitabine (FTC), using a twice-weekly repeat-challenge macaque model and showed that a pre-exposure vaginal application of gel with 1% tenofovir alone or in combination with 5% FTC both fully protected macaques from a total of 20 exposures to SHIVSF162p3. FTC and TFV were detected in plasma 30 minutes after vaginal application suggesting rapid absorption. FTC was more frequently detected than TFV and showed higher levels reflecting the 5-fold higher concentration of this drug relative to TFV. Two of 12 repeatedly exposed but protected macaques showed limited T-cell priming which did not induce resistance to infection when macaques were re-challenged. Thus, single drugs with durable antiviral activity can provide highly effective topical prophylaxis and overcome the need for non-coital use or for drug combinations which are more complex and costly to formulate and approve. |
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