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1.
Antimicrob Agents Chemother ; 59(7): 4190-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25941222

RESUMO

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Compostos de Diazônio/uso terapêutico , Farneseno Álcool/análogos & derivados , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Fármacos Anti-HIV/administração & dosagem , Barreira Hematoencefálica , Compostos de Diazônio/administração & dosagem , Compostos de Diazônio/farmacocinética , Farneseno Álcool/administração & dosagem , Farneseno Álcool/farmacocinética , Farneseno Álcool/uso terapêutico , Citometria de Fluxo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Meia-Vida , Humanos , Técnicas In Vitro , Macaca mulatta , Camundongos , Camundongos SCID , Monócitos/efeitos dos fármacos , Monócitos/virologia , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/farmacocinética , Vírus da Imunodeficiência Símia , Viremia/tratamento farmacológico , Viremia/virologia
2.
Virology ; 462-463: 115-25, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24971704

RESUMO

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/terapia , HIV-1/imunologia , Animais , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/imunologia , Masculino , Camundongos , Camundongos SCID , Resultado do Tratamento
3.
Virology ; 436(1): 162-72, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23200770

RESUMO

We previously showed that reduced infectivity of HIV with incompletely processed capsid-spacer protein 1 (CA-SP1) is rescued by cellular activation or increased expression of HSP90AB1, a member of the cytosolic heat shock protein 90 family. Here we show that HSP90AB1 is present in HIV virions and that HSP90AB1, but not nonfunctional mutated HSP90AB1(E42A+D88A), restores infectivity to HIV with mutations in CA that alter core stability. Further, the CA mutants were hypersensitive to pharmacological inhibition of HSP90AB1. In agreement with Roesch et al. (2012), we found that culturing HIV at 39.5°C enhanced viral infectivity up to 30-fold in human peripheral blood mononuclear cells (p=0.002) and rescued CA-mutant infectivity in nonactivated cells, concurrent with elevated expression of HSP90AB1 during hyperthermia. In sum, the transdominant effect of HSP90AB1 on CA-mutant HIV infectivity suggests a potential role for this class of cellular chaperones in HIV core stability and uncoating.


Assuntos
Proteínas do Capsídeo/genética , HIV/genética , HIV/patogenicidade , Proteínas de Choque Térmico HSP90/metabolismo , Linhagem Celular , Células HEK293 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Temperatura Alta , Humanos , Células Jurkat , Leucócitos Mononucleares/virologia , Mutação , Linfócitos T/virologia , Proteínas do Core Viral/genética
4.
Virology ; 417(1): 154-60, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21684569

RESUMO

Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34(+) hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ(-/-)) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4(+) T cells per µl 12 weeks after CD34(+) injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.


Assuntos
Infecções por HIV/transmissão , HIV/imunologia , Subunidade gama Comum de Receptores de Interleucina/fisiologia , Leucócitos/fisiologia , Animais , Antígenos CD34 , Suscetibilidade a Doenças , Feminino , HIV/fisiologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante Heterólogo , Vagina
5.
PLoS One ; 2(11): e1251, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18043758

RESUMO

BACKGROUND: The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation. METHODS AND FINDINGS: Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1. CONCLUSIONS: These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Succinatos/farmacologia , Triterpenos/farmacologia , Administração Oral , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/sangue , Western Blotting , Citometria de Fluxo , HIV-1/fisiologia , Humanos , Depleção Linfocítica , Masculino , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Succinatos/administração & dosagem , Succinatos/sangue , Timo/virologia , Triterpenos/administração & dosagem , Triterpenos/sangue , Replicação Viral/efeitos dos fármacos
6.
PLoS One ; 2(7): e655, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17668043

RESUMO

BACKGROUND: The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals. METHODOLOGY/PRINCIPAL FINDINGS: Endpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions. CONCLUSION: Given the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Camundongos SCID/genética , Imunodeficiência Combinada Severa/genética , Síndrome da Imunodeficiência Adquirida/genética , Animais , Antirretrovirais/uso terapêutico , DNA Viral/genética , Modelos Animais de Doenças , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , RNA Viral/genética , Imunodeficiência Combinada Severa/imunologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
7.
Vaccine ; 25(3): 481-9, 2007 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-17052811

RESUMO

Parainfluenza virus type 3 (PIV3) infections continue to be a significant health risk for infants, young children, and immunocompromised adults. We describe a gene-based vaccine strategy against PIV3 using replication-defective alphavirus vectors. These RNA replicon vectors, delivered as virus-like particles and expressing the PIV3 hemagglutinin-neuraminidase glycoprotein, were shown to be highly immunogenic in mice and hamsters, inducing PIV3-specific neutralizing antibody responses. Importantly, the replicon particle-based vaccine administered intramuscularly or intranasally protected against mucosal PIV3 challenge in hamsters, preventing virus replication in both nasal turbinates and lungs. These data suggest that the alphavirus replicon platform can be useful for a PIV3 vaccine and possibly other respiratory viruses.


Assuntos
Alphavirus/genética , Vacinas contra Parainfluenza/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/prevenção & controle , RNA Viral/genética , RNA Viral/imunologia , Replicon/genética , Replicon/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Cricetinae , Vírus da Encefalite Equina Venezuelana/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Inibição da Hemaglutinação , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Vírus da Parainfluenza 3 Humana/crescimento & desenvolvimento , Sindbis virus/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
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