RESUMO
Background: Herpes simplex virus type 2 (HSV-2; herpes) exacerbates human immunodeficiency virus type 1 (HIV) by unclear mechanisms. These studies tested the impact of HSV-2 on systemic T-cells and HIV reservoirs. Methods: Peripheral blood mononuclear cells from HIV-infected women on antiretroviral therapy who were HSV-2 seropositive or seronegative and HIV-uninfected controls were analyzed by flow cytometry. Cell-associated HIV DNA and RNA were quantified in the absence or presence of activating stimuli, recombinant interleukin 32γ (IL-32γ), and a RUNX1 inhibitor. RNA was assessed by nanostring. Results: CD4, but not CD8, T-cell phenotypes differed in HIV+/HSV-2+ versus HIV+/HSV-2- (overall P = .002) with increased frequency of CCR5+, CXCR4+, PD-1+, and CD69+ and decreased frequency of CCR10+ and CCR6+ T-cells. The changes were associated with higher HIV DNA. Paradoxically, IL-32, a proinflammatory cytokine, was lower in subpopulations of CD4+ T-cells in HSV-2+ versus HSV-2- women. Recombinant IL-32γ blocked HIV reactivation in CD4+ T-cells and was associated with an increase in RUNX1 expression; the blockade was overcome by a RUNX1 inhibitor. Conclusions: Herpes is associated with phenotypic changes in CD4+ T-cells, including a decrease in IL-32, which may contribute to increased HIV reservoirs. Blocking IL-32 may facilitate HIV reactivation to improve shock and kill strategies.
Assuntos
Infecções por HIV/imunologia , HIV-1/fisiologia , Herpes Genital/imunologia , Herpesvirus Humano 2/fisiologia , Interleucinas/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Coinfecção/virologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Estudos Transversais , DNA Viral/isolamento & purificação , Feminino , Humanos , Interleucinas/antagonistas & inibidores , Leucócitos Mononucleares/imunologia , Pessoa de Meia-Idade , RNA Viral/isolamento & purificação , Proteínas Recombinantes/metabolismo , Carga Viral , Adulto JovemRESUMO
Topical preexposure prophylaxis interrupts HIV transmission at the site of mucosal exposure. Intermittently dosed vaginal gels containing the HIV-1 reverse transcriptase inhibitor tenofovir protected pigtailed macaques depending on the timing of viral challenge relative to gel application. However, modest or no protection was observed in clinical trials. Intravaginal rings (IVRs) may improve efficacy by providing long-term sustained drug delivery leading to constant mucosal antiretroviral concentrations and enhancing adherence. Although a few IVRs have entered the clinical pipeline, 100% efficacy in a repeated macaque vaginal challenge model has not been achieved. Here we describe a reservoir IVR technology that delivers the tenofovir prodrug tenofovir disoproxil fumarate (TDF) continuously over 28 d. With four monthly ring changes in this repeated challenge model, TDF IVRs generated reproducible and protective drug levels. All TDF IVR-treated macaques (n = 6) remained seronegative and simian-HIV RNA negative after 16 weekly vaginal exposures to 50 tissue culture infectious dose SHIV162p3. In contrast, 11/12 control macaques became infected, with a median of four exposures assuming an eclipse of 7 d from infection to virus RNA detection. Protection was associated with tenofovir levels in vaginal fluid [mean 1.8 × 10(5) ng/mL (range 1.1 × 10(4) to 6.6 × 10(5) ng/mL)] and ex vivo antiviral activity of cervicovaginal lavage samples. These observations support further advancement of TDF IVRs as well as the concept that extended duration drug delivery devices delivering topical antiretrovirals could be effective tools in preventing the sexual transmission of HIV in humans.
Assuntos
Adenina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , HIV/efeitos dos fármacos , Infecções por Lentivirus/prevenção & controle , Organofosfonatos/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Adenina/administração & dosagem , Adenina/farmacologia , Administração Intravaginal , Animais , Preparações de Ação Retardada , Feminino , Macaca mulatta , Organofosfonatos/administração & dosagem , TenofovirRESUMO
We evaluated the in vivo pharmacokinetics and used a complementary ex vivo coculture assay to determine the pharmacodynamics of IQB3002 gel containing 1% IQP-0528, a nonnucleoside reverse transcriptase inhibitor (NNRTI), in rhesus macaques (RM). The gel (1.5 ml) was applied vaginally to 6 simian-human immunodeficiency (SHIV)-positive female RM. Blood, vaginal fluids, and rectal fluids were collected at 0, 1, 2, and 4 h. RM were euthanized at 4 h, and vaginal, cervical, rectal, and regional lymph node tissues were harvested. Anti-human immunodeficiency virus (HIV) activity was evaluated ex vivo by coculturing fresh or frozen vaginal tissues with activated human peripheral blood mononuclear cells (PBMCs) and measuring the p24 levels for 10 days after an HIV-1Ba-L challenge. The median levels of IQP-0528, determined using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) methods, were between 10(4) and 10(5) ng/g in vaginal and cervical tissue, between 10(3) and 10(4) ng/g in rectal tissues, and between 10(5) and 10(7) ng/ml in vaginal fluids over the 4-h period. The vaginal tissues protected the cocultured PBMCs from HIV-1 infection ex vivo, with a viral inhibition range of 81 to 100% in fresh and frozen tissues that were proximal, medial, and distal relative to the cervix. No viral inhibition was detected in untreated baseline tissues. Collectively, the median drug levels observed were 5 to 7 logs higher than the in vitro 50% effective concentration (EC50) range (0.21 ng/ml to 1.29 ng/ml), suggesting that 1.5 ml of the gel delivers IQP-0528 throughout the RM vaginal compartment at levels that are highly inhibitory to HIV-1. Importantly, antiviral activity was observed in both fresh and frozen vaginal tissues, broadening the scope of the ex vivo coculture model for future NNRTI efficacy studies.
Assuntos
Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Pirimidinonas/farmacocinética , Inibidores da Transcriptase Reversa/farmacocinética , Administração Intravaginal , Animais , Técnicas de Cocultura , Criopreservação , Feminino , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macaca mulatta , Pirimidinonas/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológicoRESUMO
Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in â¼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.
Assuntos
Transporte Biológico/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Herpes Genital/prevenção & controle , Herpesvirus Humano 2/efeitos dos fármacos , Tenofovir/farmacologia , Administração Intravaginal , Fármacos Anti-HIV/uso terapêutico , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Endocitose/efeitos dos fármacos , Feminino , Infecções por HIV/tratamento farmacológico , Herpes Genital/tratamento farmacológico , Humanos , Nitrofenóis/farmacologia , Compostos Organofosforados/farmacologia , Linfócitos T/efeitos dos fármacos , Espectrometria de Massas em Tandem , Tenofovir/administração & dosagemRESUMO
Griffithsin, which binds N-linked glycans on gp120 to prevent HIV entry, has the most potent HIV-1 inhibitory activity described for any antiviral lectin and is being developed for topical preexposure prophylaxis. The current studies were designed to further assess its potential by exploring its activity against herpes simplex virus 2 (HSV-2), a cofactor for HIV acquisition, in vitro and in a murine model. Safety was evaluated by examining its impact on epithelial barrier integrity in polarized cultures and testing whether repeated intravaginal dosing potentiates the susceptibility of mice to genital herpes. Griffithsin displayed modest inhibitory activity against HSV-2 if present during viral entry but completely blocked plaque formation if present postentry, reduced plaque size, and prevented cell-to-cell spread. These in vitro findings translated to significant protection against genital herpes in mice treated with 0.1% griffithsin gel. Griffithsin, but not placebo gel, prevented viral spread (visualized with a luciferase-expressing virus), significantly reduced disease scores, and resulted in greater survival (P < 0.05, log rank test). Protection persisted when HSV-2 was introduced in seminal plasma. Although griffithsin triggered a small decline in transepithelial electrical resistance in polarized cultures, this did not translate to any significant increase in the ability of HIV to migrate from the apical to the basolateral chamber nor to an increase in susceptibility to HSV-2 in mice treated with griffithsin gel for 7 days. These findings demonstrate that griffithsin inhibits HSV-2 by a unique mechanism of blocking cell-to-cell spread and support its further development for HIV and HSV-2 prevention.
Assuntos
Antivirais/administração & dosagem , Herpes Genital/prevenção & controle , Herpes Genital/virologia , Herpesvirus Humano 2/efeitos dos fármacos , Lectinas de Plantas/administração & dosagem , Animais , Feminino , Herpesvirus Humano 2/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: The limited success of recent HIV topical pre-exposure prophylaxis clinical trials highlights the need for more predictive models of drug efficacy that better simulate what may happen during sexual exposure. To address this gap, we developed complementary in vitro models to evaluate the ability of drugs to retain anti-HIV activity if cells were washed with seminal plasma (simulating what may happen following exposure to ejaculate), and to protect drug-naive T cells (representing newly recruited immune cells) co-cultured with explants that had been pretreated with drug. We focused on tenofovir disoproxil fumarate (TDF), the non-nucleoside reverse transcriptase inhibitors dapivirine (DPV) and IQP-0528, and the entry inhibitors maraviroc (MVC) and the D-peptide chol-PIE-12 trimer (PIE12). Studies were extended to macaques and the ability of cervical biopsies obtained from animals treated with an intravaginal ring formulation of IQP-0528 to protect ex vivo co-cultured T cells was determined. The antiviral activity of cervicovaginal lavage samples against a primary Clade C isolate was also measured and correlated with drug levels. RESULTS: Cells exposed to TDF were equally protected from HIV whether or not the drug-treated cells were washed with medium or seminal plasma prior to challenge. In contrast, several-fold higher concentrations of NNRTIs and entry inhibitors were needed to attain similar levels of HIV inhibition following a wash with seminal plasma. Conversely, the NNRTIs and PIE12, but not TDF or MVC, were effectively transferred from ex vivo treated explants and protected co-cultured T cells. Biopsies obtained from IQP-0528 ring-treated macaques also protected co-cultured T cells with viral inhibition ranging from 42-72%. Antiviral activity correlated with the concentration of drug recovered. Combinations of TDF with IQP-0528 protected in both in vitro models. CONCLUSIONS: Together, these models suggest that intracellularly retained drugs such as TDF may protect resident immune cells following coitus but sustained delivery may be required to protect immune cells subsequently recruited into the genital tract. Sustained delivery may also be critical for NNRTIs, which are rapidly transported out of cells and could be lost following sexual intercourse. An ideal approach may be a combination of drugs with complementary bioavailability profiles formulated for sustained delivery.
Assuntos
Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/farmacocinética , Quimioprevenção/métodos , Transmissão de Doença Infecciosa/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Infecções por HIV/prevenção & controle , Linfócitos T/virologia , Animais , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Feminino , Infecções por HIV/transmissão , Macaca , Técnicas de Cultura de Órgãos/métodosRESUMO
OBJECTIVES: A safe and effective topical prevention strategy will likely require sustained delivery of potent antiviral drugs and a delivery system that simultaneously maximizes drug distribution and overcomes the behavioural challenges related to adherence. Activity against HIV and herpes simplex virus (HSV) would be advantageous, given the epidemiological link between the two pathogens. We hypothesize that tenofovir disoproxil fumarate (tenofovir DF), a prodrug of tenofovir, may be more potent than tenofovir and ideal for sustained intravaginal ring (IVR) delivery. METHODS: The anti-HIV and anti-HSV activity of tenofovir and tenofovir DF were assessed in cell and explant models. Cumulative tenofovir DF release and stability from polyether urethane (PEU), ethylene-co-vinyl acetate (EVA) and silicone IVRs were compared, and the activity and safety of drug released were evaluated in cervical explants and in a polarized dual-chamber model. RESULTS: Tenofovir DF inhibited HIV and HSV at ≈ 100-fold lower concentrations than tenofovir and retained activity in the presence of semen. PEU rings delivered >1 mg/day of tenofovir DF for 30 days. Pre-treatment of cervical explants with 10 µg/mL tenofovir DF or eluants from PEU minirings resulted in >90% inhibition of HIV and reduced HSV-2 yields by 2.5 log. Tenofovir DF and eluants did not prevent cell growth or polarization, or have any deleterious effects on an epithelial barrier. CONCLUSIONS: The findings support the development of a PEU tenofovir DF ring, which may provide potent and sustained protection against HIV and HSV.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Dispositivos Anticoncepcionais Femininos , HIV/efeitos dos fármacos , Organofosfonatos/farmacologia , Simplexvirus/efeitos dos fármacos , Adenina/farmacologia , Técnicas de Cultura de Células , Quimioprevenção/métodos , Feminino , Infecções por HIV/prevenção & controle , Herpes Genital/prevenção & controle , Humanos , Técnicas de Cultura de Órgãos , TenofovirRESUMO
A structurally novel candidate microbicide, PPCM, which is formed from the reaction of D,L-mandelic acid with sulfuric acid, provides activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) and is not cytotoxic. The objectives of the current studies were to comprehensively evaluate the activity of PPCM in cell and explant cultures, explore the possibility of combining PPCM with HIV-specific reverse transcriptase inhibitors, and evaluate the efficacy of a formulated gel against genital herpes in a murine model. PPCM inhibited infection by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical and endocervical tissue explants exposed to HIV-1(BaL) in the presence of PPCM were protected (50% inhibitory concentrations [IC(50)] of 3.9 microg/ml for ectocervix and 3.1 microg/ml for endocervix), and transfer of virus to target T cells via migratory cells was significantly impaired (IC(50) of 35.7 microg/ml for ectocervix and 54.6 microg/ml for endocervix). The drug also blocked infection by cell-associated virus. Combinations of PPCM with UC-781 or PMPA in vitro exhibited additive anti-HIV activity. PPCM was incorporated into stable, low-pH gel formulations at concentrations of 0.4% and 4%. Both gels prevented genital herpesvirus infection in mice, even when virus was introduced in human seminal plasma. The abilities of PPCM to inhibit primary HIV isolates, reduce infection by cell-associated virus, and transfer of HIV from migratory to T cells, combined with the complete protection provided by formulated gel against genital herpes, indicate that this drug is an excellent candidate for inclusion in a combination microbicide and would provide protection against both HIV and HSV.
Assuntos
Anti-Infecciosos/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Herpes Genital/prevenção & controle , Simplexvirus/efeitos dos fármacos , Animais , Anti-Infecciosos/química , Géis/farmacologia , Infecções por HIV/virologia , Células HeLa , Herpes Genital/virologia , Humanos , Concentração Inibidora 50 , Luciferases , Ácidos Mandélicos/química , Camundongos , Ácidos Sulfúricos/químicaRESUMO
Secretory leukocyte protease inhibitor (SLPI), an anti-inflammatory mediator of mucosal immunity, inhibits human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cell culture. Epidemiological studies demonstrate that higher concentrations of SLPI in mucosal secretions are associated with a reduced risk of HIV transmission. The current studies were designed to test the hypothesis that HSV triggers a loss of SLPI to evade innate immunity and that this response may contribute to the increased risk of HIV infection in the setting of HSV infection. Exposure of human cervical epithelial cells to HSV-1 or HSV-2, but not HIV or vesicular stomatitis virus, triggered a significant and sustained reduction in SLPI levels. The reduction persisted when cells were infected in the presence of acyclovir but not following infection with UV-inactivated virus, indicating that viral gene expression, but not replication, is required. Reverse transcriptase PCR studies demonstrated that the loss of SLPI is mediated by downregulation of gene expression. SLPI downregulation was associated with activation of NF-kappaB signaling pathways and upregulation of proinflammatory cytokines, consistent with the known inhibitor effects of SLPI on NF-kappaB pathways. The downregulation mapped to viral early-gene expression, as variants impaired in expression of the ICP4 or ICP0 immediate-early gene failed to downregulate SLPI or activate NF-kappaB. Together, these results identify a novel role for HSV immediate-early-gene expression in regulating mucosal immune responses.
Assuntos
Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 2/enzimologia , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Citocinas/metabolismo , Infecções por HIV , Humanos , Sistema Imunitário , Inflamação , NF-kappa B/metabolismo , Risco , Transdução de Sinais , Células VeroRESUMO
The objective of this study was to characterize cervicovaginal (CV) mucosal factors modulating susceptibility to human immunodeficiency virus (HIV) acquisition in healthy premenopausal (PRE) and postmenopausal (POST) women before and after treatment with estradiol (E2). We compared CV mucosal epithelial histology and immune cells, vaginal microbiota, antimicrobial activity of and soluble mucosal protein concentrations in the CV fluid lavage (CVL), and p24 antigen production after ex vivo infection of ectocervical tissues with HIV-1BaL among PRE women (n = 20) in the follicular and luteal phases of the menstrual cycle and POST women (n = 17) at baseline and after â¼1 month of treatment with 0.01% vaginal E2 cream. Compared to PRE women, we measured higher levels of p24 antigen after ex vivo infection in tissues from POST women. POST women had a significantly thinner vaginal epithelium with decreased tight junction proteins and a higher density of mucosal immune T cells and lower levels of CD1a antigen-presenting cells, antimicrobial peptides, and inflammatory cytokines in the CVL (p values <.05). POST women had higher vaginal pH and lower vaginal Lactobacilli (p values <.05) than PRE women. After vaginal E2 therapy, CV endpoints and ex vivo HIV replication in POST tissues were similar to those observed in PRE tissues. The CV mucosa in POST women is thinned and compromised, with increased HIV-target immune cells and decreased antimicrobial factors, being more susceptible to HIV infection. After POST women receive topical E2 treatment, mucosal endpoints are similar to PRE levels.
Assuntos
Suscetibilidade a Doenças , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Infecções por HIV/imunologia , HIV/imunologia , Imunidade nas Mucosas , Administração Intravaginal , Adulto , Idoso , Colo do Útero/virologia , Feminino , HIV/crescimento & desenvolvimento , Proteína do Núcleo p24 do HIV/análise , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Heterosexual intercourse remains the major route of HIV-1 transmission worldwide, with almost 5 million new infections occurring each year. Women increasingly bear a disproportionate burden of the pandemic, thus there is an urgent need to develop new strategies to reduce HIV-1 transmission that could be controlled by women themselves. The potential of topical microbicides to reduce HIV transmission across mucosal surfaces has been clearly identified, and some agents are currently under evaluation in clinical trials. Many of these "first generation" microbicides consist of polyanionic compounds designed to interfere with viral attachment. Here we have evaluated two candidate polyanion compounds in clinical trials, PRO 2000 and dextrin sulphate (DxS) to determine their safety and efficacy against in vitro HIV-1 and HSV-2 infection using cellular and tissue explant models. RESULTS: PRO 2000 and DxS potently inhibited infection by HIV-1 X4 and R5 isolates when present during viral exposure. However PRO 2000 required 10-fold and DxS 2000-fold more compound to block infection with R5 virus than X4. While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus. PRO 2000 efficiently inhibited infection of cervical explants and dissemination of virus by migratory DC. DxS was less active, able to completely inhibit cervical explant infection, but providing only partial reduction of virus dissemination by DC. PRO 2000, but not DxS, also inhibited HIV-1 binding to DC-SIGN+ cells and trans infection of co-cultured target cells. The inflammatory potential of both compounds was screened by measurement of cytokine production from cervical explants, and statistically significant increases were only observed for IL-1beta and RANTES following treatment with PRO 2000. Both compounds also demonstrated potent activity against HSV-2 infection of cervical epithelial cells. CONCLUSION: Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants. DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways. PRO 2000 has now entered human phase III efficacy trials.
Assuntos
Colo do Útero/virologia , Infecções por HIV/transmissão , HIV-1/genética , Animais , Fármacos Anti-HIV/farmacologia , Moléculas de Adesão Celular/química , Chlorocebus aethiops , Citocinas/metabolismo , Dextrinas/farmacologia , Feminino , Humanos , Lectinas Tipo C/química , Naftalenossulfonatos/farmacologia , Polímeros/farmacologia , Receptores de Superfície Celular/química , Receptores de HIV/química , Sulfatos/farmacologia , Células VeroRESUMO
The purpose of this study was to evaluate differences in vaginal immune cell populations, vaginal tissue gene expression, antimicrobial activity of the cervicovaginal (CV) lavage (CVL), vaginal flora, and p24 antigen production from CV tissues after ex vivo human immunodeficiency virus (HIV) infection between follicular (FOL) and luteal (LUT) phases of the menstrual cycle. CV tissue biopsies, CV secretions, and blood samples were obtained as part of two longitudinal clinical trials of healthy women (CONRAD D11-119 and A12-124 studies). Participants (n = 39) were HIV-seronegative women not using exogenous hormone supplementation, with normal menstrual cycles, who were screened to exclude sexually transmitted and reproductive tract infections. Serum levels of estradiol and progesterone were significantly higher in the LUT versus the FOL phase of the menstrual cycle. Controlling for race, reported contraceptive use/sexual practices, and clinical trial, we found no differences in vaginal tissue immune cell populations and activation status, transcriptomes, inhibition of HIV, herpes simplex virus type 2 and Escherichia coli by the CVL, vaginal pH or Nugent score, or production of p24 antigen after ex vivo infection by HIV-1BaL between CV samples obtained in the FOL phase versus the LUT phase of the menstrual cycle. There were no significant correlations between serum estradiol and progesterone levels and CV endpoints. The hypothesis that the LUT phase of the menstrual cycle represents a more vulnerable stage for mucosal infection with HIV was not supported by data from samples obtained from the lower genital tract (ectocervix and vagina) from these two clinical trials.
Assuntos
Suscetibilidade a Doenças , Fase Folicular/imunologia , Infecções por HIV/imunologia , Fase Luteal/imunologia , Vagina/imunologia , Adulto , Biópsia , Análise Química do Sangue , Secreções Corporais , Escherichia coli/imunologia , Feminino , HIV-1/imunologia , Voluntários Saudáveis , Herpesvirus Humano 2/imunologia , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir (TFV), may be ideal for topical HIV preexposure prophylaxis because it has higher tissue and cell permeability than TFV; is not adversely impacted by seminal proteins; and its active metabolite, TFV-diphosphate (TFV-DP), has a long intracellular half-life. We engineered a TDF eluting polyurethane reservoir intravaginal ring (IVR) to provide near constant mucosal antiretroviral concentrations. METHODS: A first-in-human randomized placebo-controlled trial was conducted to assess the safety and pharmacokinetics of the TDF IVR in healthy, sexually abstinent women (15 TDF and 15 placebo). Drug concentrations were measured in cervicovaginal fluid (CVF) obtained by swab, cervical tissue, plasma, and dried blood spots (DBS) over 14 days of continuous ring use. RESULTS: There were 43 total, 23 reproductive tract, and eight product-related grade 1 adverse events. Steady-state CVF TFV concentrations were achieved proximal (vagina, ectocervix) and distal (introitus) to the TDF IVR 1 day after ring insertion. Median tissue TFV-DP concentrations 14 days after TDF IVR placement were 120âfmol/mg (interquartile range 90, 550). CVF collected from the cervix 1 week and 2 weeks after TDF IVR insertion provided significant protection against ex-vivo HIV challenge. Eleven of 14 (78%) participants had detectable TFV-DP DBS concentrations 14 days after TDF IVR placement, suggesting that DBS may provide a surrogate marker of adherence in future clinical trials. CONCLUSION: A TDF IVR is safe, well tolerated, and results in mucosal TFV concentrations that exceed those associated with HIV protection. The findings support further clinical evaluation of this TDF IVR.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/farmacocinética , Dispositivos Anticoncepcionais Femininos/efeitos adversos , Tenofovir/efeitos adversos , Tenofovir/farmacocinética , Adolescente , Adulto , Fármacos Anti-HIV/administração & dosagem , Líquidos Corporais/química , Quimioprevenção/métodos , Transmissão de Doença Infecciosa/prevenção & controle , Feminino , Infecções por HIV/prevenção & controle , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Placebos/administração & dosagem , Tenofovir/administração & dosagem , Adulto JovemRESUMO
HIV may induce gastrointestinal (GI) mucosal immune dysregulation similar to inflammation observed in ulcerative colitis (UC). Colorectal biopsies from healthy controls (N=12) and from participants with HIV (N=20) or UC (N=9) were subjected to real time (RT)-PCR for selected cytokines, chemokines, antimicrobial peptides, Toll-like receptors, and inflammatory signaling and epithelial barrier proteins. HIV long terminal repeat relative copy number (RCN) in HIV participant biopsies was quantified by RT-PCR. Mean interleukin (IL)-6 mRNA levels did not differ significantly between HIV and UC participants (p=0.48) but were significantly higher relative to control mRNA levels only for HIV participants (p=0.03). Mean IL-8 and human defensin (HD) 5 mRNA levels were similar between HIV and UC participants (p=1.0 and p=0.35, respectively) and were significantly greater in both groups relative to controls (p<0.05 for all). Human beta-defensin (HBD)-2 mRNA levels were higher in UC relative to HIV and control participants (p<0.01 for both). Conversely, HBD-1 mRNA levels were downregulated in UC vs. HIV participants (p=0.01). Mediator gene expression did not differ significantly between HIV participants with detectable (N=10) or nondetectable (N=10) plasma viral loads. Tissue HIV relative copy number (RCN) correlated with plasma viral load (r=0.88, p<0.01) but not with mediator mRNA levels. The results of this study indicate that both chronic HIV infection and UC are associated with similar patterns of IL-6, IL- 8, and HD5 expression in colorectal biopsy tissue. These findings suggest overlapping mechanisms for GI mucosal inflammation in these two illnesses and merit further investigation in larger studies.
Assuntos
Colo/patologia , Citocinas/biossíntese , Expressão Gênica , Infecções por HIV/patologia , Mucosa Intestinal/patologia , Reto/patologia , alfa-Defensinas/biossíntese , Adulto , Biópsia , Doença Crônica , Citocinas/genética , Feminino , Perfilação da Expressão Gênica , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima , alfa-Defensinas/genéticaRESUMO
Bacterial vaginosis (BV) has been linked to an increased risk of human immunodeficiency virus (HIV) acquisition and transmission in observational studies, but the underlying biological mechanisms are unknown. We measured biomarkers of subclinical vaginal inflammation, endogenous antimicrobial activity, and vaginal flora in women with BV and repeated sampling 1 week and 1 month after completion of metronidazole therapy. We also compared this cohort of women with BV to a healthy control cohort without BV. A longitudinal, open label study of 33 women with a Nugent score of 4 or higher was conducted. All women had genital swabs, cervicovaginal lavage (CVL) fluid, and cervicovaginal biopsies obtained at enrollment and received 7 days of metronidazole treatment. Repeat sampling was performed approximately 1 week and 1 month after completion of therapy. Participant's baseline samples were compared to a healthy, racially matched control group (n=13) without BV. The CVL from women with resolved BV (Nugent 0-3) had significantly higher anti-HIV activity, secretory leukocyte protease inhibitor (SLPI), and growth-related oncogene alpha (GRO-α) levels and their ectocervical tissues had significantly more CD8 cells in the epithelium. Women with persistent BV after treatment had significantly higher levels of interleukin-1ß, tumor necrosis factor alpha (TNF-α), and intercellular adhesion molecule 1 (ICAM-1) in the CVL. At study entry, participants had significantly greater numbers of CCR5(+) immune cells and a higher CD4/CD8 ratio in ectocervical tissues prior to metronidazole treatment, compared to a racially matched cohort of women with a Nugent score of 0-3. These data indicate that BV is associated with changes in select soluble immune mediators, an increase in HIV target cells, and a reduction in endogenous antimicrobial activity, which may contribute to the increased risk of HIV acquisition.
Assuntos
Biomarcadores/análise , Imunidade nas Mucosas , Inflamação/patologia , Infecções do Sistema Genital/diagnóstico , Infecções do Sistema Genital/patologia , Vaginose Bacteriana/diagnóstico , Adulto , Anti-Infecciosos/administração & dosagem , Infecções Assintomáticas , Biópsia , Colo do Útero/microbiologia , Feminino , Humanos , Estudos Longitudinais , Metronidazol/administração & dosagem , Infecções do Sistema Genital/tratamento farmacológico , Vagina/microbiologia , Ducha Vaginal , Vaginose Bacteriana/tratamento farmacológicoRESUMO
BACKGROUND: Surrogate markers of HIV-1 pre-exposure prophylaxis and microbicide efficacy are needed. One potential surrogate is the antiviral activity in cervicovaginal lavage (CVL) after exposure to candidate products. We measured CVL antiviral activity in women using oral or vaginal tenofovir-based pre-exposure prophylaxis and correlated activity with drug and immune mediator levels. METHODS: Inhibitory activity against HIV-1 and herpes simplex virus (HSV)-2 and concentrations of interleukin (IL)-1ß, IL-6, IL-8, interferon-γ, induced protein 10 (IP-10), macrophage inflammatory protein (MIP)-1α, MIP-3a, lactoferrin, secretory leukocyte protease inhibitor, and defensins were measured in CVL obtained from 60 women at baseline and after 6 weeks of a randomized sequence of oral and topical tenofovir. CVL tenofovir concentrations were measured by mass spectrometry. RESULTS: The number of women with CVL anti-HIV activity ≥ 90% increased significantly from 5.0% at baseline to 89.1% after daily use of 1% tenofovir gel (relative risk = 17.85, P < 0.001), but there was no increase after daily oral tenofovir. The CVL anti-HIV activity correlated with drug levels (Spearman correlation coefficient 0.64 after tenofovir gel; P < 0.001) but not with the concentrations of mucosal immune mediators. No increase in CVL anti-HSV activity was observed after either drug regimen, an observation consistent with the higher concentrations of tenofovir needed to inhibit HSV-2 infection. The CVL anti-HSV activity correlated with lactoferrin, defensins, IP-10, IL-8, and detectable levels of MIP-1α but not with drug levels. CONCLUSIONS: CVL may provide a surrogate for local but not systemic drug efficacy and a tool to better understand mucosal factors that modulate antiviral activity in genital tract secretions.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacocinética , Secreções Corporais/química , Quimioprevenção/métodos , Genitália Feminina/química , Organofosfonatos/administração & dosagem , Organofosfonatos/farmacocinética , Adenina/administração & dosagem , Adenina/análise , Adenina/farmacocinética , Administração Intravaginal , Administração Oral , Adulto , Fármacos Anti-HIV/análise , Feminino , HIV-1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Organofosfonatos/análise , TenofovirRESUMO
Vaginal pre-exposure prophylaxis has focused heavily on gel formulations. Low adherence linked with frequent dosing and short therapeutic duration has emerged as the major reason for inconsistent efficacy outcomes with gels in clinical trials. Osmotic pumps can achieve versatile drug release profiles however, have not been explored for vaginal delivery. In this report, we describe an osmotic pump tablet (OPT) that can deliver antiretrovirals for several days. We also describe configuring the OPT for pH sensitive delivery where the drug delivery system consistently delivers an antiretroviral at vaginal pH and then gives a burst release triggered by a coitally associated pH increase. We have investigated the vaginal OPT for multiple day delivery of a potent antiretroviral, IQP-0528 in a sheep model. To effectively register spatial drug distribution we also engineered a tool to precisely collect multiple vaginal fluid samples. In a 10-day duration post single application, high micromolar mucosal levels were obtained with peak concentration more than 6 logs higher than the EC50 of IQP-0528. Overall, our results show successful implementation of the osmotic pump technology for vaginal antiretroviral delivery.
Assuntos
Fármacos Anti-HIV/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Infecções por HIV/tratamento farmacológico , Mucosa/efeitos dos fármacos , Pirimidinonas/farmacocinética , Vagina/efeitos dos fármacos , Animais , Fármacos Anti-HIV/química , Química Farmacêutica , Sistemas de Liberação de Medicamentos/instrumentação , Feminino , Humanos , Concentração de Íons de Hidrogênio , Bombas de Infusão Implantáveis , Mucosa/química , Osmose , Pirimidinonas/química , Ovinos , Comprimidos/química , Comprimidos/farmacocinética , Vagina/químicaRESUMO
In the past few years, the transdisciplinary field of HIV prevention has reached several milestones. Topically applied tenofovir gel provided significant protection from sexual transmission of HIV in a large-scale clinical trial and oral Truvada (emtricitabine/tenofovir disoproxil fumarate) was recently approved for preexposure prophylaxis (PrEP) following two successful clinical trials in men and women. These achievements are tempered by the disappointing results of other clinical trials, which highlight the complexities of prevention research. In this perspective, we discuss scientific and developmental gaps for topical chemoprophylaxis of the sexual transmission of HIV, which depends on the complex interactions between the pharmacokinetics and pharmacodynamics of drugs, formulation and delivery systems, anatomic site of transmission, and host mucosal immune defenses. Despite the considerable time and resources devoted to unraveling the initial steps in sexual transmission of HIV, current knowledge is based on animal models and human explanted tissue, which may not fully recapitulate what happens clinically. Understanding these events, including the role that sex hormones, semen, and mucosal secretions play in transmission, and the interplay between innate immunity, the mucosal environment, and drug efficacy is paramount. This drives some of the most pressing questions in the field.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/administração & dosagem , Quimioprevenção , Desoxicitidina/análogos & derivados , Infecções por HIV/prevenção & controle , Imunidade Inata , Mucosa/virologia , Organofosfonatos/administração & dosagem , Compostos Organofosforados/administração & dosagem , Adenina/administração & dosagem , Adenina/farmacologia , Administração Oral , Administração Tópica , Canal Anal/efeitos dos fármacos , Canal Anal/imunologia , Canal Anal/virologia , Fármacos Anti-HIV/farmacologia , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Combinação de Medicamentos , Combinação Emtricitabina e Fumarato de Tenofovir Desoproxila , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Humanos , Masculino , Adesão à Medicação , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/imunologia , Mucosa Bucal/virologia , Mucosa/efeitos dos fármacos , Mucosa/imunologia , Organofosfonatos/farmacologia , Compostos Organofosforados/farmacologia , Parceiros Sexuais , Tenofovir , Vagina/efeitos dos fármacos , Vagina/imunologia , Vagina/virologia , Cremes, Espumas e Géis VaginaisRESUMO
Pharmacologic strategies for the prevention of HIV include vaccines, post-exposure prophylaxis with antiretroviral therapy, and topical microbicides. Vaginal microbicides have the potential to augment innate defenses in the genital tract but may also disrupt endogenous protection and increase HIV acquisition risk, as observed in clinical trials of nonoxynol-9. The initially disappointing results of microbicide clinical trials stimulated the development of more sensitive and comprehensive pre-clinical safety studies, which include dual-chamber culture systems to model the epithelial barrier and post-coital studies to evaluate the effects of semen and sexual intercourse on microbicide efficacy. This review discusses the key factors that contribute to a healthy female genital tract environment, the impact of semen on mucosal defense, and how our understanding of these mediators informs the development of effective vaginal microbicides.
Assuntos
Anti-Infecciosos Locais/farmacologia , Genitália Feminina/metabolismo , Infecções por HIV/prevenção & controle , Sêmen , Infecções Sexualmente Transmissíveis/prevenção & controle , Anti-Infecciosos Locais/uso terapêutico , Feminino , Infecções por HIV/imunologia , Humanos , Imunidade nas Mucosas , Nonoxinol/farmacologia , Nonoxinol/uso terapêutico , Infecções Sexualmente Transmissíveis/imunologiaRESUMO
BACKGROUND: Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models' utility for evaluating future products. METHODS: Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control. RESULTS: CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture. CONCLUSIONS: CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically.