RESUMEN
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 mAb (5D2) to FTC and demonstrated its high specificity and physiologically relevant linear range of detection in a competitive enzyme immunoassay. Thus, this mAb is a key reagent that will enable simple and low-cost lateral flow assays and enzyme immunoassays for adherence monitoring.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Profilaxis Pre-Exposición , Fármacos Anti-VIH/uso terapéutico , Antirretrovirales/uso terapéutico , Emtricitabina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Humanos , Tenofovir/uso terapéuticoRESUMEN
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.
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Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Microbiota , Adenina/uso terapéutico , Adolescente , Adulto , Estudios de Cohortes , Desoxicitidina/uso terapéutico , Emtricitabina/uso terapéutico , Femenino , Fumaratos/uso terapéutico , Genitales Femeninos/microbiología , Humanos , Persona de Mediana Edad , Organofosfonatos/uso terapéutico , Estudios Prospectivos , Tenofovir/uso terapéutico , Adulto JovenAsunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Combinación Emtricitabina y Fumarato de Tenofovir Disoproxil/farmacocinética , Infecciones por VIH/prevención & control , Profilaxis Pre-Exposición , Inhibidores de la Transcriptasa Inversa/farmacocinética , Adulto , Femenino , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Fosforilación , Especificidad por Sustrato , Carga ViralRESUMEN
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.
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Fármacos Anti-VIH/farmacología , Profilaxis Pre-Exposición , Provirus/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Fármacos Anti-VIH/administración & dosificación , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/virología , Macaca mulatta , Provirus/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Factores de Tiempo , Carga ViralRESUMEN
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.
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Administración Intravaginal , VIH/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Tenofovir/administración & dosificación , Vagina/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Femenino , Geles , Infecciones por VIH/virología , Humanos , Macaca radiata , Profilaxis Pre-Exposición , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Vagina/virologíaRESUMEN
BACKGROUND: Rectal human immunodeficiency virus (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 3 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 biopsy specimens, 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 95% inhibitory concentrations in rectal fluids at 24 hours. MVC, TFV, and TFV diphosphate (TFV-DP) concentrations in colorectal tissues collected up to 30 cm from the anal margin were all high at 2 hours, demonstrating rapid and extended tissue dosing. TFV-DP concentrations in tissue homogenates and rectal lymphocytes were highly correlated (r(2) = 0.82). All 3 gel formulations were highly protective (82% efficacy; P ≤ .02 by the log-rank test). CONCLUSIONS: Desirable pharmacokinetic profiles and high efficacy in this macaque model support the clinical development of these gel formulations for preventing rectal HIV infection.
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Fármacos Anti-VIH/administración & dosificación , Ciclohexanos/administración & dosificación , Transmisión de Enfermedad Infecciosa/prevención & control , Geles/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Tenofovir/administración & dosificación , Triazoles/administración & dosificación , Administración Tópica , Animales , Fármacos Anti-VIH/farmacocinética , Estudios Cruzados , Ciclohexanos/farmacocinética , Modelos Animales de Enfermedad , Macaca , Maraviroc , Placebos/administración & dosificación , Tenofovir/farmacocinética , Resultado del Tratamiento , Triazoles/farmacocinéticaRESUMEN
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 >1/30â000 (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.
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Fármacos Anti-VIH/farmacocinética , Secreciones Corporales/química , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Membrana Mucosa/química , Plasma/química , Quinolonas/farmacocinética , Raltegravir Potásico/farmacocinética , Administración Oral , Animales , Fármacos Anti-VIH/administración & dosificación , Cromatografía Líquida de Alta Presión , Femenino , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Macaca mulatta , Oxazinas , Piperazinas , Piridonas , Quinolonas/administración & dosificación , Raltegravir Potásico/administración & dosificación , Recto/química , Espectrometría de Masas en Tándem , Vagina/químicaRESUMEN
BACKGROUND 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.
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Adenina/análogos & derivados , Fármacos Anti-VIH/farmacocinética , VIH/efectos de los fármacos , Infecciones por Lentivirus/prevención & control , Macaca nemestrina , Organofosfonatos/farmacología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Adenina/farmacocinética , Adenina/farmacología , Administración Intravaginal , Animales , Sistemas de Liberación de Medicamentos , Femenino , Organofosfonatos/administración & dosificación , Organofosfonatos/farmacocinética , Tenofovir , Factores de Tiempo , Vagina/metabolismoRESUMEN
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 (â¼849 bp) and 11 pol (â¼2995 bp) 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.
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Infecciones por VIH/diagnóstico , VIH-2/aislamiento & purificación , Técnicas de Diagnóstico Molecular/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , ADN Viral/genética , Duplicado del Terminal Largo de VIH/genética , VIH-2/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Plásmidos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estados Unidos , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
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.
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Inhibidores de Integrasa/uso terapéutico , Macaca/virología , Profilaxis Posexposición , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/fisiología , Vagina/virología , Administración Intravaginal , Administración Tópica , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Líquidos Corporales , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral/efectos de los fármacos , Femenino , Geles , Inhibidores de Integrasa/farmacología , Cinética , Macaca/sangre , Progesterona/sangre , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Factores de Tiempo , Vagina/efectos de los fármacos , Vagina/patología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: It is not known if fluctuations in genital tract antiretroviral drug concentrations correlate with genital virus shedding in human immunodeficiency virus (HIV)-infected women on antiretroviral therapy (ART). METHODS: Among 20 HIV-infected women on ART (tenofovir [TFV], emtricitabine [FTC], and ritonavir-boosted atazanavir [ATV]) with suppressed plasma virus loads, blood and cervicovaginal samples collected twice weekly for 3 weeks were tested for antiretroviral concentrations, HIV-1 RNA, and proviral DNA. RESULTS: Cervicovaginal:plasma antiretroviral concentration ratios were highest for FTC (11.9, 95% confidence interval [CI], 8.66-16.3), then TFV (3.52, 95% CI, 2.27-5.48), and ATV (2.39, 95% CI, 1.69-3.38). Within- and between-person variations in plasma and genital antiretroviral concentrations were observed. Low amounts of genital HIV-1 RNA (<50 copies/mL) were detected in 45% of women at 16% of visits. Genital HIV-1 DNA was detected in 70% of women at 35% of visits. Genital virus detection was associated with higher concentrations of mucosal leukocytes but not with genital antiretroviral concentrations, menstrual cycle phase, bacterial vaginosis, genital bleeding, or plasma virus detection. CONCLUSIONS: Standard doses of ART achieved higher genital than plasma concentrations across the menstrual cycle. Therapeutic ART suppresses genital virus shedding throughout the menstrual cycle, even in the presence of factors reported to increase virus shedding.
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Antirretrovirales/administración & dosificación , Genitales Femeninos/química , Genitales Femeninos/virología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Ciclo Menstrual , Esparcimiento de Virus , Adenina/administración & dosificación , Adenina/análogos & derivados , Adenina/farmacocinética , Adulto , Antirretrovirales/farmacocinética , ADN Viral/genética , ADN Viral/aislamiento & purificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Emtricitabina , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Persona de Mediana Edad , Organofosfonatos/administración & dosificación , Organofosfonatos/farmacocinética , Plasma/química , Plasma/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Ritonavir/administración & dosificación , Ritonavir/farmacocinética , Tenofovir , Carga ViralRESUMEN
Maraviroc (MVC) is a potent CCR5 coreceptor 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 24 h before virus exposure, followed by a booster postexposure dose. In rectal secretions, MVC peaked at 24 h (10,242 ng/ml) with concentrations at 48 h that were about 40 times those required to block SHIV infection of peripheral blood mononuclear cells (PBMCs) in vitro. Median MVC concentrations in rectal tissues at 24 h (1,404 ng/g) were 30 and 10 times those achieved in vaginal or lymphoid tissues, respectively. MVC significantly reduced macrophage inflammatory protein 1ß-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 five 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.
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Antirretrovirales/administración & dosificación , Antirretrovirales/farmacocinética , Quimioprevención/métodos , Ciclohexanos/administración & dosificación , Ciclohexanos/farmacocinética , Recto/química , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Triazoles/administración & dosificación , Triazoles/farmacocinética , Animales , Femenino , Mucosa Intestinal/química , Macaca , Masculino , Maraviroc , Plasma/química , Insuficiencia del TratamientoRESUMEN
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.
Asunto(s)
Coito , Desoxicitidina/análogos & derivados , Infecciones por VIH/prevención & control , Compuestos Organofosforados/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vagina , Animales , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapéutico , Combinación de Medicamentos , Combinación Emtricitabina y Fumarato de Tenofovir Disoproxil , Femenino , Macaca nemestrina , Compuestos Organofosforados/farmacocinética , Progesterona/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisiónRESUMEN
Antiretroviral-based microbicides applied topically to the vagina may play an important role in protecting women from HIV infection. Incorporation of the nucleoside reverse transcriptase inhibitor tenofovir (TFV) into intravaginal rings (IVRs) for sustained mucosal delivery may lead to increased microbicide product adherence and efficacy compared with those of conventional vaginal formulations. Formulations of a novel "pod IVR" platform spanning a range of IVR drug loadings and daily release rates of TFV were evaluated in a pig-tailed macaque model. The rings were safe and exhibited sustained release at controlled rates over 28 days. Vaginal secretion TFV levels were independent of IVR drug loading and were able to be varied over 1.5 log units by changing the ring configuration. Mean TFV levels in vaginal secretions were 72.4 ± 109 µg ml(-1) (slow releasing) and 1.84 ± 1.97 mg ml(-1) (fast releasing). The mean TFV vaginal tissue concentration from the slow-releasing IVRs was 76.4 ± 54.8 µg g(-1) and remained at steady state 7 days after IVR removal, consistent with the long intracellular half-life of TFV. Intracellular tenofovir diphosphate (TFV-DP), the active moiety in defining efficacy, was measured in vaginal lymphocytes collected in the study using the fast-releasing IVR formulation. Mean intracellular TFV-DP levels of 446 ± 150 fmol/10(6) cells fall within a range that may be protective of simian-human immunodeficiency virus strain SF162p3 (SHIV(SF162p3)) infection in nonhuman primates. These data suggest that TFV-releasing IVRs based on the pod design have potential for the prevention of transmission of human immunodeficiency virus type 1 (HIV-1) and merit further clinical investigation.
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Adenina/análogos & derivados , Dispositivos Anticonceptivos Femeninos/veterinaria , Preparaciones de Acción Retardada/farmacocinética , Organofosfonatos/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacocinética , Vagina/metabolismo , Adenina/farmacocinética , Administración Intravaginal , Animales , Citocinas/metabolismo , Femenino , Semivida , Linfocitos/química , Macaca nemestrina , Tenofovir , Vagina/citología , Vagina/efectos de los fármacosRESUMEN
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.
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Cartilla de ADN/genética , ADN Viral/sangre , ADN Viral/genética , VIH-1/genética , Técnicas de Diagnóstico Molecular/métodos , Provirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sangre/virología , ADN Viral/aislamiento & purificación , Humanos , Secuencias Repetidas Terminales/genética , Factores de TiempoRESUMEN
The potent nonnucleoside 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 lavage (CVL) fluids from 25 Thai women enrolled in a 14-day safety trial of twice-daily vaginal application of two concentrations of the UC781 microbicide gel. CVL samples were collected from women in the 0.1% (n = 5), 0.25% (n = 15), and placebo (n = 5) gel arms following the first application of gel (T(15 min)) and 8 to 24 h after the final application (T(8-24 h)) and separated into cell-free (CVL-s) and pelletable (CVL-p) fractions. As UC781 is highly hydrophobic, there were significantly higher levels of UC781 in the CVL-p samples than in the CVL-s samples for the UC781 gel arms. In T(8-24 h) 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) 50% tissue culture infective dose (TCID(50)) of a CCR5-tropic CRF01_AE HIV-1 virus stock. Independent of the arm, the 11 CVL-p samples with UC781 levels of ≥ 5 µg/CVL sample reduced infectious HIV by ≥ 4 log(10) TCID(50). Our results suggest that the levels and anti-infective activities 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.
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Anilidas/administración & dosificación , Anilidas/farmacocinética , Antiinfecciosos/administración & dosificación , Antiinfecciosos/farmacocinética , Antivirales/administración & dosificación , Antivirales/farmacocinética , Líquidos Corporales/metabolismo , Furanos/administración & dosificación , Furanos/farmacocinética , Vagina/metabolismo , Adolescente , Adulto , Femenino , Humanos , Persona de Mediana Edad , Tioamidas , Cremas, Espumas y Geles Vaginales/administración & dosificación , Cremas, Espumas y Geles Vaginales/farmacocinética , Adulto JovenRESUMEN
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.
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Adenina/análogos & derivados , Fármacos Anti-VIH/administración & dosificación , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Organofosfonatos/administración & dosificación , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Vagina/efectos de los fármacos , Cremas, Espumas y Geles Vaginales/administración & dosificación , Adenina/administración & dosificación , Adenina/química , Animales , Fármacos Anti-VIH/química , Células Cultivadas , Estabilidad de Medicamentos , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Semivida , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Estudios Longitudinales , Organofosfonatos/química , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Tenofovir , Vagina/inmunología , Vagina/virología , Cremas, Espumas y Geles Vaginales/químicaRESUMEN
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(0.5 wt%)], PYD1(1 wt%), PYD2(4 wt%), and PYD2(14 wt%)) 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(14 wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 µg/ml to vaginal fluid and 1 µg/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.
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Fármacos Anti-VIH/farmacocinética , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Pirimidinonas/farmacocinética , Vagina/efectos de los fármacos , Administración Intravaginal , Animales , Fármacos Anti-VIH/uso terapéutico , Línea Celular , Dispositivos Anticonceptivos Femeninos , Citocinas/biosíntesis , Citocinas/inmunología , Estabilidad de Medicamentos , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Concentración 50 Inhibidora , Macaca nemestrina , Poliuretanos , Pirimidinonas/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Distribución Tisular , Vagina/inmunología , Vagina/virología , Replicación Viral/efectos de los fármacosRESUMEN
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.
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Adenina/análogos & derivados , Fármacos Anti-VIH/uso terapéutico , Nucleótidos de Desoxiadenina/metabolismo , Infecciones por VIH/prevención & control , Organofosfonatos/uso terapéutico , Profármacos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Adenina/administración & dosificación , Adenina/farmacocinética , Adenina/uso terapéutico , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Quimioprevención , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Humanos , Linfocitos/metabolismo , Tejido Linfoide/metabolismo , Macaca , Organofosfonatos/administración & dosificación , Organofosfonatos/farmacocinética , Profármacos/administración & dosificación , Profármacos/farmacocinética , Recto/virología , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/farmacocinética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Especificidad por Sustrato , TenofovirRESUMEN
A simplified lateral-flow assay for the detection of antibodies to HIV using magnetic-bead conjugates and multibranched 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 relative magnetic units [MAR]). The results of the optimized MICT assay were compared to standard enzyme immunoassay (EIA) and Western blotting (WB) results using a blinded 649-member panel of specimens from the United States, Cameroon, and West Africa. The panel was comprised of samples from individuals infected with various HIV-1 subtypes (n = 234) or HIV-2 (n = 65) and HIV-seronegative specimens (n = 350). Additionally, 13 HIV-1 seroconversion 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 interrun testing was excellent, with coefficients of variation of <12%. MICT can provide a rapid, low-cost method of determining HIV antibody status requiring no subjective interpretations.