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1.
Gene Ther ; 29(10-11): 616-623, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-34759330

RESUMO

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.


Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Hepatite C/terapia , Linfócitos T , Receptores de Antígenos de Linfócitos T , Peptídeos
2.
J Immunother ; 45(3): 139-149, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34802014

RESUMO

Chimeric antigen receptor expressing T cells (CAR-T cells) have shown remarkable efficacy against some blood cancers and have potential to treat many other human diseases. During CAR-T cell manufacturing, T cells are activated via engagement of the T-cell receptor (TCR); however, persistent TCR engagement can induce unchecked activation, differentiation, and exhaustion, which can negatively affect CAR-T cell product quality and in vivo potency. In addition, T cells may not uniformly respond to TCR-dependent activation (TCRD) contributing to lot-to-lot variability, poor expansion, and manufacturing failures. TCRD also presents challenges during manufacturing of allogeneic CAR-T cells when endogenous TCR is deleted to prevent graft-versus-host disease. Thus, novel strategies to activate T cells may help improve CAR-T cell product attributes and reduce manufacturing failures. In this study, we compared the effect of TCRD and TCR-independent activation (TCRI) on CAR-T cell product attributes. We found that TCRI in presence of a Src-kinase inhibitor significantly improved CAR-T cell expansion and yield without affecting viability and CD4/CD8 ratio. Markers of T-cell activation, exhaustion and differentiation were also reduced in these CAR-T cells compared with CAR-T cells manufactured by TCRD. TCRI did not affect CAR-T cell in vitro potency; however, following co-culture with target cells, CAR-T cells manufactured by TCRI released significantly less inflammatory cytokines compared with CAR-T cells manufactured by TCRD. Together, these data suggest that manufacturing CAR-T cells by TCRI activation in the presence of a Src-kinase inhibitor improves product quality attributes and may help reduce manufacturing failures and improve CAR-T cell safety and efficacy in vivo.


Assuntos
Imunoterapia Adotiva , Quinases da Família src , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T
3.
Front Immunol ; 12: 693016, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220853

RESUMO

Engineered T cell therapies such as chimeric antigen receptor (CAR) expressing T cells (CAR-T cells) have great potential to treat many human diseases; however, inflammatory toxicities associated with these therapies present safety risks and can greatly limit its widespread use. This article briefly reviews our current understanding of mechanisms for inflammatory toxicities during CAR T-cell therapy, current strategies for management and mitigation of these risks and highlights key areas of knowledge gap for future research.


Assuntos
Imunoterapia Adotiva/efeitos adversos , Inflamação/prevenção & controle , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Animais , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/genética , Medição de Risco , Fatores de Risco , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Resultado do Tratamento
4.
Sci Rep ; 8(1): 10910, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026610

RESUMO

Immune responses against gene therapy products limit its therapeutic efficacy and present a safety risk. Identification of agents that blunt immune reactions may aid in developing novel immunomodulatory therapies. Fingolimod (FTY720) is an FDA approved immunomodulatory drug for treating multiple sclerosis that inhibits lymphocyte egress from lymphoid tissues by down regulating sphingosine-1 phosphate receptor (S1PR). Recent studies found that FTY720 inhibits T cell activation (TCA) in a S1PR-independent manner; however, the mechanism is incompletely understood. Here we characterized the effects of FTY720 on human T cell receptor (TCR) signaling pathways. FTY720 inhibited both the TCR-dependent and independent activation of primary human T cells. FTY720 did not affect proximal TCR signaling events as measured by phosphorylation of Lck, ZAP-70 and LAT; however, inhibited PMA/Ionomycin induced distal TCR signaling as measured by IL-2, IFN-γ release and CD25 expression. FTY720 induced aberrant NFAT1, AP1 and NFκB activation which were associated with increased acetylation of histone (H3K9). Phosphorylated FTY720 did not inhibit TCA, and arachidonic acid did not rescue FTY720 mediated inhibition of TCA. These data suggest that FTY720 mediated inhibition of TCA is due to inhibition of distal TCR signaling. Understanding FTY720-mediated inhibition of TCA may aid in developing novel FTY720-based immunomodulatory agents.


Assuntos
Cloridrato de Fingolimode/farmacologia , Fatores Imunológicos/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linhagem Celular , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células THP-1
5.
PLoS One ; 12(10): e0187123, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29073235

RESUMO

T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR) T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs) are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.


Assuntos
Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Quinases da Família src/metabolismo , Inibidores de Calcineurina/farmacologia , Humanos , Células Jurkat , Linfócitos T/efeitos dos fármacos
6.
J Infect Dis ; 216(9): 1164-1175, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28968905

RESUMO

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.


Assuntos
Vírus da Dengue/genética , RNA/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Zika virus/genética , Vírus da Dengue/patogenicidade , Humanos , Vírus da Febre Amarela/patogenicidade , Zika virus/patogenicidade
7.
PLoS Pathog ; 13(2): e1006232, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28235043

RESUMO

Among human RNA viruses, hepatitis C virus (HCV) is unusual in that it causes persistent infection in the majority of infected people. To establish persistence, HCV evades host innate and adaptive immune responses by multiple mechanisms. Recent studies identified virus genome-derived small RNAs (vsRNAs) in HCV-infected cells; however, their biological significance during human HCV infection is unknown. One such vsRNA arising from the hepatitis C virus (HCV) E2 coding region impairs T cell receptor (TCR) signaling by reducing expression of a Src-kinase regulatory phosphatase (PTPRE) in vitro. Since TCR signaling is a critical first step in T cell activation, differentiation, and effector function, its inhibition may contribute towards HCV persistence in vivo. The effect of HCV infection on PTPRE expression in vivo has not been examined. Here, we found that PTPRE levels were significantly reduced in liver tissue and peripheral blood mononuclear cells (PBMCs) obtained from HCV-infected humans compared to uninfected controls. Loss of PTPRE expression impaired antigen-specific TCR signaling, and curative HCV therapy restored PTPRE expression in PBMCs; restoring antigen-specific TCR signaling defects. The extent of PTPRE expression correlated with the amount of sequence complementarity between the HCV E2 vsRNA and the PTPRE 3' UTR target sites. Transfection of a hepatocyte cell line with full-length HCV RNA or with synthetic HCV vsRNA duplexes inhibited PTPRE expression, recapitulating the in vivo observation. Together, these data demonstrate that HCV infection reduces PTPRE expression in the liver and PBMCs of infected humans, and suggest that the HCV E2 vsRNA is a novel viral factor that may contribute towards viral persistence.


Assuntos
Hepatite C/imunologia , Evasão da Resposta Imune/imunologia , Ativação Linfocitária/imunologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/imunologia , Linfócitos T/imunologia , Ensaio de Imunoadsorção Enzimática , Hepacivirus/imunologia , Humanos , Immunoblotting , RNA Viral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção
8.
PLoS Pathog ; 11(9): e1005183, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26421924

RESUMO

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp's contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.


Assuntos
Hepatite C/imunologia , Ativação Linfocitária/imunologia , RNA Viral/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Bases , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hepacivirus , Humanos , Immunoblotting , Imunoprecipitação , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/imunologia , Vírion/imunologia
9.
Virology ; 485: 116-27, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26245365

RESUMO

Human Pegivirus (HPgV, formally GB virus C) infects lymphocytes and NK cells in vivo, and infection is associated with reduced T cell and NK cell activation in HIV-infected individuals. The mechanism by which HPgV inhibits NK cell activation has not been assessed. Following IL-12 stimulation, IFNγ expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects (p=0.02). In addition, HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited NK cell IL-12-mediated IFNγ release. E2 protein also inhibited Tyk2 activation following IL-12 stimulation. In contrast, cytolytic NK cell function was not altered by HPgV. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV persistence and reduced immune activation during HIV-coinfection. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.


Assuntos
Infecções por Flaviviridae/virologia , Flavivirus/fisiologia , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , Interleucina-12/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Adulto , Sequência de Aminoácidos , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Coinfecção , Feminino , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/patologia , Flavivirus/patogenicidade , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Interações Hospedeiro-Patógeno , Humanos , Interferon gama , Células K562 , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral , Replicação Viral
10.
Trans Am Clin Climatol Assoc ; 125: 14-24; discussion 24-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25125715

RESUMO

Hepatitis C virus (HCV) and GB virus type C (GBV-C) are associated with impaired T cell function despite the fact that HCV replicates in hepatocytes and GBV-C in a small proportion of lymphocytes. Recently, we showed that HCV and GBV-C E2-envelope proteins reduce T cell activation via the T cell receptor (TCR) by competing for phosphorylation with a critical kinase in the TCR signaling cascade (Lck). E2 interfered with TCR signaling in E2 expressing cells and in bystander cells. The bystander effect was mediated by virus particles and extracellular microvesicular particles (exosomes). Multiple kinase substrate sites are predicted to reside on viral structural proteins and based on bioinformatic predictions, many RNA virus pathogens may interfere with TCR signaling via a similar mechanism. Identification of T cell inhibitory effects of virus structural proteins may provide novel approaches to enhance the immunogenicity and memory of viral vaccines.


Assuntos
Vírus GB C/imunologia , Hepacivirus/imunologia , Hepatite/imunologia , Evasão da Resposta Imune , Ativação Linfocitária , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Vírus GB C/metabolismo , Vírus GB C/patogenicidade , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Hepatite/epidemiologia , Hepatite/história , Hepatite/metabolismo , Hepatite/virologia , História do Século XX , História do Século XXI , Interações Hospedeiro-Patógeno , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/virologia , Proteínas do Envelope Viral/metabolismo
11.
J Gen Virol ; 95(Pt 6): 1307-1319, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24668525

RESUMO

Human pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity, despite causing persistent infection, and is associated with prolonged survival in human immunodeficiency virus-infected individuals. Although HPgV RNA is found in and produced by T- and B-lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4(+) and CD8(+) T-cells, including naïve, central memory and effector memory populations, and in B-cells (CD19(+)), NK cells (CD56(+)) and monocytes (CD14(+)) using real-time reverse transcription-PCR. Single-genome sequencing was performed on viruses within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4(+) and CD8(+) T-lymphocytes (nine of nine subjects), B-lymphocytes (seven of ten subjects), NK cells and monocytes (both four of five). HPgV RNA levels were higher in naïve (CD45RA(+)) CD4(+) cells than in central memory and effector memory cells (P<0.01). HPgV sequences were highly conserved among subjects (0.117±0.02 substitutions per site; range 0.58-0.14) and within subjects (0.006±0.003 substitutions per site; range 0.006-0.010). The non-synonymous/synonymous substitution ratio was 0.07, suggesting a low selective pressure. Carboxyfluorescein succinimidyl ester (CFSE)-labelled HPgV RNA-containing particles precipitated by a commercial exosome isolation reagent delivered CSFE to uninfected monocytes, NK cells and T- and B-lymphocytes, and HPgV RNA was transferred to PBMCs with evidence of subsequent virus replication. Thus, HPgV RNA-containing serum particles including microvesicles may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.


Assuntos
Vírus GB C/isolamento & purificação , Vírus GB C/patogenicidade , Leucócitos Mononucleares/virologia , RNA Viral/sangue , Adulto , Sequência de Aminoácidos , Linfócitos B/virologia , Sequência de Bases , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Sequência Conservada , Feminino , Infecções por Flaviviridae/complicações , Infecções por Flaviviridae/virologia , Vírus GB C/genética , Infecções por HIV/complicações , Hepatite Viral Humana/complicações , Hepatite Viral Humana/virologia , Humanos , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monócitos/virologia , Filogenia , RNA Helicases/genética , RNA Viral/genética , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/genética , Virulência , Adulto Jovem
12.
J Immunol ; 190(12): 6351-9, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23686495

RESUMO

Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4(+) and CD8(+) T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.


Assuntos
Vírus GB C/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vírus GB C/metabolismo , Hepatite Viral Humana/imunologia , Hepatite Viral Humana/metabolismo , Humanos , Immunoblotting , Células Jurkat , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo
13.
J Gen Virol ; 94(Pt 4): 774-782, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23288422

RESUMO

GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4(+) Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus-HIV interactions.


Assuntos
Linfócitos T CD4-Positivos/virologia , Vírus GB A/fisiologia , Vírus GB C/fisiologia , HIV-1/fisiologia , Proteínas do Envelope Viral/metabolismo , Interferência Viral , Replicação Viral , Animais , Vírus GB A/isolamento & purificação , Vírus GB C/isolamento & purificação , Humanos , Células Jurkat , Dados de Sequência Molecular , Pan troglodytes , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética
14.
Antivir Ther ; 17(7): 1271-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22951385

RESUMO

BACKGROUND: GB virus C (GBV-C) coinfection is associated with reduced immune activation and a block in CD4(+) T-cell proliferation following interleukin-2 (IL-2) therapy in HIV-infected individuals. We examined peripheral blood mononuclear cells (PBMCs) from HIV-infected subjects with and without GBV-C viraemia to determine if GBV-C correlated with reactivation of latent HIV, T-cell proliferation or T-cell survival following in vitro activation with phytohaemagglutinin A and IL-2 (PHA/IL-2). METHODS: HIV-infected subjects whose HIV viral load was suppressed on combination antiretroviral therapy (cART) for >6 months were studied. PBMCs were cultured with and without PHA/IL-2 and monitored for HIV reactivation, proliferation and survival. GBV-C viraemia and in vitro replication were detected by real-time RT-PCR. HIV reactivation was determined by measuring HIV p24 antigen in culture supernatants. Proliferation was measured by counting viable cells and survival measured by flow cytometry. RESULTS: Of 49 HIV-infected individuals, 26 had GBV-C viraemia. Significantly less HIV reactivation and PBMC proliferation following in vitro activation with PHA/IL-2 was observed in samples from GBV-C viraemic subjects compared with non-viraemic controls. Following 5 weeks in culture, GBV-C replication was associated with preservation of CD4(+) and CD8(+) T-cells compared with non-viraemic controls. CONCLUSIONS: GBV-C appears to inhibit immune activation and IL-2 signalling pathways, which might contribute to a reduction in reactivation of latent HIV from cellular reservoirs. In addition, GBV-C viraemia was associated with a reduction in activation-induced T-cell death. GBV-C-associated T-cell effects could contribute to the observed protective effect of GBV-C coinfection in HIV-infected individuals.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus GB C/patogenicidade , Infecções por HIV/imunologia , Ativação Linfocitária , Ativação Viral , Latência Viral , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Casos e Controles , Morte Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Coinfecção/virologia , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/virologia , Citometria de Fluxo , Vírus GB C/fisiologia , Proteína do Núcleo p24 do HIV/análise , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Hepatite Viral Humana/imunologia , Hepatite Viral Humana/virologia , Humanos , Interleucina-2/imunologia , Interleucina-2/farmacologia , Fito-Hemaglutininas/imunologia , Fito-Hemaglutininas/farmacologia , Transdução de Sinais , Carga Viral , Viremia/virologia
15.
J Infect Dis ; 206(9): 1469-72, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22927453

RESUMO

Double-negative T cells (DNTCs; ie, CD3(+)CD4(-)CD8(-) T cells) play a role in limiting chronic immune activation. GB virus C (GBV-C) infection is associated with reduced T-cell activation in human immunodeficiency virus (HIV)-infected individuals. T-cell activation and DNTCs were measured in HIV-infected subjects with a nondetectable HIV load. GBV-C-viremic subjects had significantly reduced CD4(+) and CD8(+) T-cell activation (P = .003 and .034, respectively) and significantly increased DNTCs (P = .038), compared with nonviremic subjects. GBV-C load correlated with DNTC percentage (P = .004). Thus, GBV-C infection is associated with an increase in DNTCs, which may contribute to reduced immune activation during HIV infection.


Assuntos
Terapia Antirretroviral de Alta Atividade/métodos , Vírus GB C/isolamento & purificação , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Hepatite Viral Humana/complicações , Subpopulações de Linfócitos T/imunologia , Viremia/complicações , Complexo CD3/análise , Antígenos CD4/análise , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/análise , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Vírus GB C/patogenicidade , Infecções por HIV/imunologia , Hepatite Viral Humana/imunologia , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/química , Viremia/imunologia
16.
J Immunol ; 189(5): 2211-6, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22844114

RESUMO

GB virus type C (GBV-C) viremia is associated with reduced CD4+ T cell expansion following IL-2 therapy and with a reduction in T cell activation in HIV-infected individuals. The mechanism(s) by which GBV-C might alter T cell activation or IL-2 signaling have not been studied. In this study, we assess IL-2 release, IL-2R expression, IL-2 signaling, and cell proliferation in tet-off Jurkat cells expressing the GBV-C envelope glycoprotein (E2) following activation through the TCR. TCR activation was induced by incubation in anti-CD3/CD28 Abs. IL-2 release was measured by ELISA, STAT5 phosphorylation was assessed by immunoblot, and IL-2Rα (CD25) expression and cell proliferation were determined by flow cytometry. IL-2 and IL-2Rα steady-state mRNA levels were measured by real-time PCR. GBV-C E2 expression significantly inhibited IL-2 release, CD25 expression, STAT5 phosphorylation, and cellular proliferation in Jurkat cells following activation through the TCR compared with control cell lines. Reducing E2 expression by doxycycline reversed the inhibitory effects observed in the E2-expressing cells. The N-terminal 219 aa of E2 was sufficient to inhibit IL-2 signaling. Addition of purified recombinant GBV-C E2 protein to primary human CD4+ and CD8+ T cells inhibited TCR activation-induced IL-2 release and upregulation of IL-2Rα expression. These data provide evidence that the GBV-C E2 protein may contribute to the block in CD4+ T cell expansion following IL-2 therapy in HIV-infected individuals. Furthermore, the effects of GBV-C on IL-2 and IL-2-signaling pathways may contribute to the reduction in chronic immune activation observed in GBV-C/HIV-coinfected individuals.


Assuntos
Vírus GB C/imunologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Proteínas do Envelope Viral/fisiologia , Internalização do Vírus , Vírus GB C/genética , Humanos , Interleucina-2/biossíntese , Células Jurkat
18.
Virology ; 430(1): 53-62, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22608061

RESUMO

GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV.


Assuntos
Fármacos Anti-HIV/farmacologia , Vírus GB C/metabolismo , HIV/efeitos dos fármacos , HIV/fisiologia , Interações Microbianas , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus GB C/genética , HIV/crescimento & desenvolvimento , Humanos , Células Jurkat , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética
19.
Trends Microbiol ; 20(3): 124-30, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22325031

RESUMO

GB virus C (GBV-C) is a lymphotropic human virus discovered in 1995 that is related to hepatitis C virus (HCV). GBV-C infection has not been convincingly associated with any disease; however, several studies found an association between persistent GBV-C infection and improved survival in HIV-positive individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. In vitro studies confirm these clinical observations and demonstrate an anti-HIV replication effect of GBV-C. This review summarizes existing data on potential mechanisms by which GBV-C interferes with HIV, and the research needed to capitalize on this epidemiological observation.


Assuntos
Infecções por Flaviviridae/virologia , Vírus GB C/fisiologia , Infecções por HIV/virologia , Animais , Infecções por Flaviviridae/imunologia , Vírus GB C/genética , Vírus GB C/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Humanos , Linfócitos T/imunologia , Linfócitos T/virologia , Replicação Viral
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