GB virus C infection is associated with a reduced rate of reactivation of latent HIV and protection against activation-induced T-cell death.
ABSTRACT: 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).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.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.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.
Project description:GB virus C (GBV-C) is a common, non-pathogenic human virus that infects lymphocytes. Persistent GBV-C infection of humans with coexistent human immunodeficiency virus (HIV) infection is associated with prolonged survival, and GBV-C replication inhibits HIV replication in vitro. A GBV-C virus variant was identified in chimpanzees in 1998 and was named GBV-C(trog) or GBV-C(cpz). The prevalence and natural history of GBV-C in chimpanzees remains uncharacterized. We examined the sera from 235 captive chimpanzees for the presence of GBV-C viraemia, viral persistence and clearance, E2 antibody kinetics and RNA sequence diversity. Sequences from six isolates shared more sequence identity with GBV-C(cpz) than with human GBV-C. The prevalence of GBV-C(cpz) viraemia and E2 antibody in chimpanzees (2.5 and 11?%, respectively) was similar to human GBV-C prevalence in healthy human blood donors (1.8 and 9?%, respectively). Persistent GBV-C(cpz) infection occurred in two of the six viraemic animals and was documented for 19 years in one animal. Host subspecies troglodyte GBV-C isolates and published verus GBV-C isolates shared a high degree of sequence identity, suggesting that GBV-C in chimpanzees should be identified with a chimpanzee designation (GBV-C(cpz)). The prevalence and natural history of chimpanzee GBV-C variant (GBV-C(cpz)) appears to be similar to human GBV-C infection. The chimpanzee could serve as an animal model to study HIV-GBV-C co-infection.
Project description:BACKGROUND:The success of increasing access to antiretroviral therapy (ART) in paediatric HIV infection prompts the question of the potential for eradication of HIV infection in this age group. 'Shock-and-kill' HIV cure approaches, currently in development, may depend upon an effective antiviral T-cell response to eradicate virus-infected cells. METHOD:We here investigate the ability of HIV-infected children receiving ART from early childhood (median 24 months' age) to generate effective HIV-specific CD4 and CD8 T-cell immune responses that would facilitate future immune-based cure therapies. RESULTS:Initial analysis of ART-naive HIV-infected children demonstrated that maintenance of normal-for-age absolute CD4 T-cell counts was strongly linked to high IL-2 production and polyfunctional HIV-specific CD4 T-cell responses (P?<?0.0001 in each case). Low viral load was, similarly, strongly associated with markedly low IFN-? and high IL-2 HIV-specific CD4 T-cell responses (P?<?0.0001). In children receiving ART, establishment of this immune profile (high IL-2 and low IFN-? HIV-specific T-cell production) was strongly related to the duration of viraemic suppression. Failure to suppress viraemia on ART, and even the successful suppression of viraemia interrupted by the occurrence of transient viraemia of more than 1000 HIV copies/ml, was associated with an immune profile of high IFN-? and low IL-2 HIV-specific T-cell responses and low polyfunctionality. CONCLUSION:These data are consistent with recovery of functional CD4 T-cell responses in ART-treated children, in contrast to relative lack of CD4 T-cell function recovery described in ART-treated adults. However, the challenges of achieving long-term suppression of viraemia in ART-treated children through adolescence remain daunting.
Project description:The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.
Project description:Homeostatic proliferation ensures the longevity of central memory T-cells by inducing cell proliferation in the absence of cellular differentiation or activation. This process is governed mainly by IL-7. Central memory T-cells can also be stimulated via engagement of the T-cell receptor, leading to cell proliferation but also activation and differentiation. Using an in vitro model of HIV-1 latency, we have examined in detail the effects of homeostatic proliferation on latently infected central memory T cells. We have also used antigenic stimulation via anti-CD3/anti-CD28 antibodies and established a comparison with a homeostatic proliferation stimulus, to evaluate potential differences in how either treatment affects the dynamics of latent virus populations. First, we show that homeostatic proliferation, as induced by a combination of IL-2 plus IL-7, leads to partial reactivation of latent HIV-1 but is unable to reduce the size of the reservoir in vitro. Second, latently infected cells are able to homeostatically proliferate in the absence of viral reactivation or cell differentiation. These results indicate that IL-2 plus IL-7 may induce a detrimental effect by favoring the maintenance of the latent HIV-1 reservoir. On the other hand, antigenic stimulation efficiently reactivated latent HIV-1 in cultured central memory cells and led to depletion of the latently infected cells via virus-induced cell death.
Project description:Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor ? (TNF?) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.
Project description:CD32 has been shown to be preferentially expressed in latently HIV-1-infected cells in an in vitro model of quiescent CD4 T cells. Here we show that stimulation of CD4+ T cells with IL-2, IL-7, PHA, and anti-CD3/CD28 antibodies induces T-cell proliferation, co-expression of CD32 and the activation of the markers HLA-DR and CD69. HIV-1 infection increases CD32 expression. 79.2% of the CD32+/CD4+ T cells from HIV+ individuals under antiretroviral treatment were HLA-DR+. Resting CD4+ T cells infected in vitro generally results in higher integration of provirus. We observe no difference in provirus integration or replication-competent inducible latent HIV-1 in CD32+ or CD32- CD4+ T cells from HIV+ individuals. Our results demonstrate that CD32 expression is a marker of CD4+ T cell activation in HIV+ individuals and raises questions regarding the immune resting status of CD32+ cells harboring HIV-1 proviruses.
Project description: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.
Project description:A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.
Project description:Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.
Project description:Cytolytic CD4+ T cells play a prominent role in chronic viral infection. CD4+ CTLs clones specific for HIV-1 Nef and Gag are capable of killing HIV-1 infected CD4+ T cells and macrophages. Additionally, HIV-specific cytolytic CD4+ T cell responses in acute HIV infection are predictive of disease progression. CD57 expression on CD4s identifies cytolytic cells. These cells were dramatically increased in chronic HIV infection. CD57 expression correlated with cytolytic granules, granzyme B and perforin expression. They express lower CCR5 compared to CD57- cells, have less HIV total DNA, and were a minor component of the HIV reservoir. A small percentage of CD57+ CD4+ CTLs from EC were HIV-specific, could upregulate IFN? with Gag peptide stimulation, express cytolytic granule markers and maintain TbethighEomes+ transcription factor phenotype. This was not observed in viraemic controllers. The maintenance of HIV-specific CD4 cytolytic function in Elite controllers together with CD8 CTLs may be important for the control of HIV viraemia and of potential relevance to cure strategies.