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GB virus type C infection modulates T-cell activation independently of HIV-1 viral load

Maidana-Giret, Maria Teresaa; Silva, Tânia Ma; Sauer, Mariana Ma; Tomiyama, Helenaa; Levi, José Eduardob; Bassichetto, Katia Cc; Nishiya, Annad; Diaz, Ricardo Sa; Sabino, Ester Cd; Palacios, Ricardoa; Kallas, Esper Georgesa,e

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doi: 10.1097/QAD.0b013e32832d7a11
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Abstract

Introduction

GB virus type C [GBV-C, also previously called hepatitis G virus (HGV)] is a common human flavivirus closely related to hepatitis C virus (HCV) [1–3]. GBV-C is not associated with any disease; however, persistent infection is associated with a lower mortality rate among HIV-1-infected patients, slower progression to AIDS, a longer survival period once AIDS has developed, and better response to antiretrovirals [4–8]. Further evidence of this coinfection protective effect is that the course of HIV-1 disease is adversely affected by the clearance of GBV-C viremia [9].

GBV-C is lymphotropic and replicates in T lymphocytes (both CD4+ and CD8+) and B lymphocytes [10–12]. GBV-C infection is very common in HIV-1-infected patients, and up to 43% of HIV-1-infected patients are viremic with GBV-C in cross-sectional studies [13,14]. In addition, concurrent transmission of HIV and GBV-C appears to be relatively common in HIV-1 seroconvertors [15].

The mechanisms of such a protective effect remain elusive. The fact that both HIV-1 and GBV-C can infect and replicate within peripheral blood mononuclear cells (PBMCs) suggests that these two viruses interact either directly or indirectly throughout the cell cycle. One potential mechanism of the GBV-C protective effect may involve downregulation of the expression of chemokine (C–C motif) receptor 5 (CCR5) on infected cells, a coreceptor for HIV-1, that influences HIV-1 transmission and progression to immunodeficiency [16]. Because CCR5 is downregulated in GBV-C-infected cells [17,18], it has been hypothesized that GBV-C might also use this HIV-1 coreceptor as a receptor. Jurkat cells, however, do not have detectable CCR5 on their surface yet they are permissive to envelope glycoprotein E2 cell binding. Alternatively, an earlier study suggested that CD81, a member of a family of tetraspanins that presumably acts as a receptor for HCV as it binds to HCV E2 envelope glycoprotein, would be a cellular receptor for GBV-C E2. However, a recent study [19] demonstrated that E2 bound to cells independently of CD81 expression, suggesting that the virus could use more than one receptor. Taken together, it is still unclear whether the effect of GBV-C on HIV-1 progression could be a result of an HIV-1 cellular receptor interference.

As in many viral infections, chronic immune activation is a hallmark of progressive HIV disease. Indeed, polyclonal B-cell activation was one of the first immunological abnormalities described in HIV-1-infected patients [20]. Subsequently, increased T-cell turnover [21], increased frequencies of T cells with an activated phenotype [22], and increased serum levels of proinflammatory cytokines and chemokines [23] were also described. Notably, the degree of immune activation can be a better predictor of disease progression than plasma viral load [24]. Therefore, the basis for cellular activation upregulation might be a result of the dynamic interactions occurring in vivo between HIV-1 and the immune response [25]. Among the different surrogate markers for cellular activation, CD38 is not only an important prognostic marker but also an active player in HIV-1 pathogenesis. Previous studies [26,27] have shown a positive correlation between the level of viral replication and the activation status of HIV-1-specific CD8+ T cells. In contrast to viremic controls, CD38 expression by HIV-1-specific CD8+ T cells from HIV-1 controllers was very low [28].

As cellular immune responses play a critical role in the control of viral replication, dissecting the effect of coinfection with GBV-C on T-cell responses directed against HIV-1 will be important to understanding disease progression. Therefore, we decided to explore whether GBV-C could impact cellular activation in the context of HIV-1 infection.

Patients and methods

Study design and patients

A total of 48 recently HIV-1-infected (23 GBV-C viremic), antiretroviral-naive patients were included in the study. All patients were enrolled as a part of the prospective cohort of recently HIV-1-infected patients, after providing Institutional Review Board-approved written informed consent. All recently HIV-1-infected patients were identified by the serologic testing algorithm for recent HIV seroconversion (STARHS) [29].

To investigate the putative protective effect of GBV-C viral infection among HIV-seropositive patients and the effect on T-cell activation, plasma was separated from blood obtained from the patients and PBMCs were obtained from leukapheresis with Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Age, sex, race, transmission mode, and laboratory data were obtained as previously described [29]. T lymphocyte counts, and expression of CD38 and CCR5 as well as expanded immunophenotyping were determined by flow cytometry, and HIV plasma viral load was determined using an Amplicor Monitor (Roche Diagnostics Systems, Branchburg, New Jersey, USA), with a lower detection limit of 400 copies/ml.

All baseline plasma samples were also tested for the presence of E2 antibodies against GBV-C envelope glycoprotein E2 and the presence of GBV-C RNA in plasma was determined by reverse-transcription PCR (RT-PCR) after HIV seroconversion in order to establish the prevalence of GBV-C infection near the time of HIV seroconversion. Positive samples were confirmed by a GBV-C-specific nested RT-PCR assay, and GBV-C viral load was determined by real-time PCR.

Detection and quantification of GB V-C RNA

Viral RNA was extracted from 140 μl plasma samples using QIAamp Viral RNA Mini Kit (QIAGEN Inc., California, USA), according to the manufacturer's instructions. A 5 μl aliquot of the RNA extracted was diluted in a mix containing 150 ng of random primer (Random Primer, Pharmacia Biotech, Sweden) and 10 mmol/l deoxyribonucleoside triphosphate (dNTP, Invitrogen Inc., Carlsbad, California, USA); the solution was kept at 65°C for 5 min. Complementary DNA (cDNA) synthesis was carried out by the addition of 200 U of Super Script III Reverse transcriptase (Invitrogen Inc.) in a buffer solution with 10 U of ribonuclease inhibitor (Invitrogen Inc.) at 25°C for 5 min, 50°C for 60 min, and 70°C for 15 min at a final volume of 20 μl.

A fragment of the nonstructural 5a region (NS5a) was amplified in a reaction mixture containing 5 μl of the cDNA solution, 1 μl of the primer-mix solution with 10 pmol/μl of NS5a 1–5′-CTCTTTGTGGTAGTAGCCGAGAGAT-3′ and NS5a 2–5′-CGAATGAGTCAGAGGACGGGGTAT-3′ [30], 0.3 μl of Taq-DNA Polymerase 5 U/μl (Invitrogen Inc.), and 1.5 mmol/l MgCl2 (Invitrogen Inc.) to a final volume of 50 μl. The reaction was performed as follows: an initial step at 95°C for 5 min, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension period of 10 min at 72°C for the final 156 bp amplified fragment. After amplification, 5 μl of the PCR product was analyzed by electrophoresis on a 2% agarose gel. The positive and negative samples were corroborated with nested RT-PCR that amplified a fragment of 344 bp of the 5′ noncoding region using the followings primers located at positions 108 (5′-AGGTGGTGGATGGGTGAT-3′; sense, outer), 134 (5′-TGGTAGGTCGTAAATCCCGGT-3′; sense, inner), 476 (5′-GGAGCTGGGTGGCCCCATGCAT-3′; antisense, inner), and 531 (5′-TGCCACCCGCCCTCACCCGAA-3′; antisense, outer) [31,32]. Amplification was over 40 cycles for both first and second rounds of PCR, with the following times and temperatures: 94°C 30 s, 50°C 30 s, and 72°C 30 s for the first round and 94°C 30 s, 60°C 30 s, and 72°C 30 s for the second round. After amplification, 5 μl of the PCR product was analyzed by electrophoresis on a 2% agarose gel.

The GBV-C load was quantified in all GBV-C RNA-positive samples in triplicate by real-time PCR using a TaqMan PCR detection kit (Perkin-Elmer Applied Biosystems, Foster City, California, USA). The following oligonucleotides were used in the real-time PCR located at positions 111–130 (5′-GTGGTGGATGGGTGATGACA-3′; sense), 192–171(5′-GACCCACCTATAGTGGCTACCA-3′, antisense). The HGV-specific probe tagged with fluorescence FAM CCGGGATTTACGACCTACC NFQ (MGB, Minor Groove Binder; NFQ, nonfluorescent quencher) antisense 154-136 numbered according to Accession NC_001710.1 [1] synthesized by Applied Biosystems. A strongly positive GBV-C RNA plasma bag from an HIV negative blood donor was obtained and serial dilutions of it were used to estimate the assay endpoint sensitivity. This corresponded to a 10 000× dilution of the original plasma. On that basis, this standard plasma bag was estimated to contain 10 000 detectable units of HGV-RNA and was therefore used in real-time assays to quantify viral load in HIV patients. Results are provided in relation to this ‘standard’. The lower limit of detection was one arbitrary unit/ml.

Detection of E2 antibody

As markers of GBV-C RNA clearance and prior exposure [33], plasma E2 antibodies were detected using an immunoassay using recombinant E2 (mPlate Anti-Hgenv test; Roche Diagnostics, kindly provided by Dietmar Zdunek) in accordance with the manufacturer's instructions. Plates were incubated with diluted (1: 20) serum, and E2 antibodies were detected using anti-human immunoglobulin G peroxidase conjugate and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate. In accordance with the manufacturer's cutoff, an optical density (OD) of less than 0.10 was considered to be negative, and an OD of at least 0.10 was considered to be positive.

Monoclonal antibodies and sample preparation

The following monoclonal antibodies (mAbs) were used in cell surface staining assays: tube 1 – CD38 fluorescein isothiocyanate (FITC) clone HI T2, CCR5 phycoerythrin clone 2D7, CD3 peridin chlorophyll protein (PerCP) clone SK7, CD4 R-phycoerythrin cyanine dye Cy7 (phycoerythrin Cy7) clone SK3, CD8-allophycocyanin (APC) clone SK1; tube 2 – CD25-FITC clone 2A3, human leukocyte antigen DR-1 (HLA-DR)-PerCP clone L243, CD69-phycoerythrin clone L78, CD3-PECy7 clone SK7, CD4-APC clone SK3, CD8 APC carbocyanin 7 (APC-Cy7) clone SK1. mAbs were obtained from Becton Dickinson Immunocytometry Systems (San Jose, California, USA), and CCR5-phycoerythrin (clone 2D7) was obtained from PharMingen (San Diego, California, USA).

In all experiments, thawed PBMCs were transferred into 96 V bottom well plates (Nunc, Denmark) with 100 μl of MACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) [phosphate buffered saline (PBS) with 500 mmol/l EDTA and 0.5% bovine serum albumin] according to the manufacturer's instructions. Cells were washed and centrifuged at 1500 rpm for 5 min, stained with the surface mAb panel and incubated at 4°C in darkness for 30 min. After, incubation cells were washed in MACS buffer, centrifuged, and resuspended in 200 μl of fixation buffer 1% paraformaldehyde (Polysciences, Warrington, Pennsylvania, USA in PBS, pH 7.4–7.6) for flow cytometry analysis. Samples were acquired on a FACSCanto, using FACSDiva software (BD Biosciences) and analyzed with FlowJo software (Tree Star, San Carlo, California, USA). Fluorescence voltages were determined using matched unstained cells. Compensation was carried out with CompBeads (BD Biosciences) single stained with CD3-PerCP, CD4-FITC, CD8-APC-Cy7, CD4-phycoerythrin-Cy7, CD3-phycoerythrin, and CD3-APC. Samples were acquired until at least 100 000 events in a live lymphocyte gate were obtained.

Statistical analysis

Percentage of CD38+ and CCR5+ in CD4+ and CD8+ T cells were used as outcomes in statistical analyses. Comparisons between groups were carried out using two-sided Student's t tests or Mann–Whitney nonparametric tests. Correlation between outcomes and independent variables was analyzed in linear regression analysis, Spearman nonparametric test, and ANOVA univariate and multivariate models. All the analyses were performed in SPSS 15.0 (SPSS, Inc., Chicago, Illinois, USA). Values of P less than 0.05 were considered statistically significant.

Results

Study population

A total of 175 patients who acquired HIV infection in the previous 170 days (by STARHS) were evaluated for GBV-C infection. Active GBV-C infection (defined by the presence of GBV-C RNA without E2 antibody) was detected in 42 out of 175 patients (24%), and past GBV-C infection (defined by the presence of E2 antibody without GBV-C RNA) was detected in 31 out of 123 patients (25%). Only one of the RNA-positive patients was also positive for E2 antibody. To study the impact of GBV-C viremia on cellular activation, we randomly selected 48 patients: 23 patients with persistent GBV-C viremia (the persistent GBV-C viremia group), 10 anti-E2-positive patients (the GBV-C clearance group), and 15 patients without GBV-C viremia or anti-E2 antibody (the HIV-1 monoinfected group) were compared. The baseline demographics and clinical characteristics of the patients are summarized in Table 1. Overall, 46 of the 48 patients (96%) were young men and the median age was 32 years (P25–75, 24.6–35.6). The median CD4+ T-cell count was 519 cells/μl (P25–75, 433–626) and the median viral load was 24 100 HIV-RNA copies/ml (P25–75, 6350–53 200), reflecting their recent HIV-1 infection status.

Table 1
Table 1:
Profiles of HIV-1 and HIV-1/GB virus type C coinfected patients.

Correlation between the GB V-C viral load, HIV viral load, and CD4+ T-cell counts

There were no significant differences regarding age, sex, baseline CD4 cell count, and HIV-1 viral load. However, in a bivariate analysis, we found a weak positive correlation between the GBV-C load and CD4+ cell count (r = 0.23, P = 0.2953) (Fig. 1a), and also an inverse, but not statistically significant, correlation was observed between GBV-C viral load and HIV viral load (r = −0.17, P = 0.4533) (Fig. 1b). The HIV-1-infected patients that were also infected with GBV-C had lower mean HIV viral loads: 4.57 ± 0.3 log10 copies/ml as compared with 4.99 ± 0.5 log10 copies/ml for the GBV-C negative/anti-E2-positive patients and 5.02 ± 0.5 log10 copies/ml for patients solely infected by HIV-1.

Fig. 1
Fig. 1:
Correlation between GB V-C viral loads and CD4+T-cell counts or HIV viral loads. The correlation between the GBV-C viral load and the CD4+ T-cell count revealed a trend to a positive correlation between the two variables (a), whereas that between the HIV load and the GBV-C load revealed a trend to an inverse correlation between the two variables (b). The P values were determined by the Spearman rank correlation test. GBV-C, GB virus type C.

HIV-1-infected patients with active GB V-C infection possess an unusual activation profile

We examined the activation status of the CD4+ and CD8+ T cells by evaluating cell surface expression of CD38 and CCR5 in HIV-1/GBV-C coinfected (active infection) and HIV-1 monoinfected patients. We observed that patients with HIV-1/GBV-C coinfection had lower expression of CD38 on their CD4+ T cells (median 32.9%; P25–75, 13.3–45.3 versus median 48.5%; P25–75, 39.4–58.4) (P = 0.031) and lower expression of CD38 on their CD8+ T cells (median 40.1%; P25–75, 17.5–48.5 versus median 66%; P25–75, 48.1–73.8) (P = 0.007) than those HIV-1 monoinfected patients (Fig. 2a and b). When we examined the expression of CCR5 on the CD4+ T cells, no statistical difference was observed (median 18.9%; P25–75, 16.1–24.4 versus median 23.6%; P25–75, 18.3–30.7) (P = 0.3461). However, the expression of this marker on the CD8+ T cells was also significantly lower in the group of coinfected patients (median 41.5%; P25–75, 29.3–48.4 versus median 59.18%; P25–75, 51.1–65.3) (P < 0.000) (Fig. 2c and d), reflecting also the lower activation status in the GBV-C-infected patients. In multiple comparisons Tukey's honestly significant difference test analysis, we examined the same markers, categorizing them now into three groups, according to their GBV-C status: GBV-C nonviremic-anti-E2-negative, GBV-C viremic, and GBV-C nonviremic-anti-E2-positive individuals. When we analyzed expression of CD38 on CD8+ T cells, we found a statistically significant difference between the GBV-C nonviremic anti-E2-negative patients versus the GBV-C viremic patients (P = 0.042), and also between the GBV-C viremic versus the GBV-C nonviremic-anti-E2-positive patients (P = 0.012) (Fig. 2e). The expression of CD38 on CD4+ T cells showed a statistically significant difference between the groups of GBV viremic versus GBV-C nonviremic-anti-E2-positive patients (P = 0.038) (Fig. 2f). Multivariate regression analyses revealed that the downregulation of these markers was likely caused by the replication of the GBV-C virus rather than the loss of GBV-C.

Fig. 2
Fig. 2:
CD38 and CCR5 expression in GB V-C nonviremic and viremic patients. CD38 expression on CD4+T cells (a) and CD8+T cells (b) and CCR5 expression on CD4+T cells (c) and CD8+T cells (d). P values were determined by one-way ANOVA. CD38 and CCR5 expression in GBV-C nonviremic, GBV-C viremic, and GBV-C nonviremic-anti-E2-positive patients on CD8+ T cells (e) and CD4+ T cells (f). The level of CD38 and CCR5 expression was determined by flow cytometry on CD3+CD8+ T cells and CD3+CD4+ T cells as depicted in the panels above. P values were determined by one-way ANOVA and the Tukey's honestly significant difference multiple comparisons test. CD38 expression on CD8+ T cells and GBV-C viral load (g). P values were determined by the Spearman rank correlation. Correlation between CD38 expression on CD8+ T cells and HIV-1 viral load (h). The open symbols represent the HIV-1/GBV-C-nonviremic patients (r = 0.5780, P = 0.0025), and the solid symbols represent the HIV/GBV-C-viremic patients (r = 0.2047, P = 0.3733). P values were determined by the Spearman rank correlation. CCR5, chemokine (C–C motif) receptor 5; GBV-C, GB virus type C; ns, not significant.
Fig. 2
Fig. 2:
(Continued)

Relationship between CD38 expression on CD8+ T cells and HIV-1 plasma viremia

We sought to investigate the correlation between the proportion of CD8+ T cells expressing CD38 and the level of HIV-1 plasma viremia in 48 HIV-1-infected patients (including a number of HIV-1/GBV-C coinfected patients). We first confirmed the positive correlation between HIV-1 viral load and CD38 expression on CD8+ T cells (r = 0.35, P < 0.05) (data not shown). By contrast, GBV-C viral load showed a negative correlation with CD38 expression on CD8+ T cells (r = −0.1609, P > 0.05) (Fig. 2g). To assess the influence of GBV-C status on CD38 expression on CD8+ T cells and HIV-1 viral load, we further investigated the correlation between HIV-1 viral load and CD38 expression on CD8+ T cells. The significant correlation between CD38 on CD8+ T cells and HIV-1 viral load in the group of patients solely infected by HIV-1 (r = 0.5780, P = 0.0025) was lost if we considered just HIV-1/GBV-C coinfected patients (r = 0.2047, P = 0.3733) (Fig. 2h). Additionally, in a regression model, we observed that the association between the GBV-C productive infection and the downregulation of CD38 and CCR5 on CD8+ T cells and CD38 on CD4+ T cells was independent of HIV-1 viral load, CCR5Δ32 polymorphism, CD4+ T cell counts, and CD8+ T cell counts, which have been recognized as predictors of HIV-1 disease progression (Table 2). In addition, the downregulation of the activation markers observed during GBV-C infection may play a role in containment of viral replication in HIV-1-infected patients.

Table 2
Table 2:
Regression model for CD4+ T cells, CD8+ T cells, HIV-1 viral load, CCR5 Δ 32 heterozygosis, and GB V-C infection with chemokine (C–C motif) receptor 5 and CD38 on CD4+ and CD8+ T cells as dependent variables.

Other activation markers

To confirm the previous results, we performed additional immunophenotyping on a subset of patients to identify activated CD4 and CD8 lymphocytes by looking at the other activation markers such as HLA-DR, CD25 (a component of the receptor for interleukin 2), and CD69 in the early phase of the immune response. When the same analyses were restricted to the groups of viremic and nonviremic GBV-C patients, we demonstrated that CD69 is also downregulated in HIV-1/GBV-C-positive patients as compared with those HIV-1 singly infected patients on the basis of CD4+ T cells (median 2.1%; P25–75, 1.4–2.9 versus median 4.6%; P25–75, 3.4–9.8) (P = 0.004) (Fig. 3a) and on CD8+ T cells (median 4.2%; P25–75, 3.4–7.1 versus median 7.6%; P25–75, 6.6–12.6) (P = 0.022) (Fig. 3b). When we examined the expression of CD25, we also demonstrated the downregulation of this marker on CD4+ T cells (median 2.7%; P25–75, 2.1–3.3 versus median 10.8%; P25–75, 8.7–12.5) (P < 0.001) (Fig. 3c); nevertheless, no statistical difference was observed in CD25-expressing CD8+ T cells (median 0.6%; P25–75, 0.3–1.1 versus median 1.0%; P25–75, 0.8–1.5) (P = 0.192) (Fig. 3d). Frequencies of activated CD4+ and CD8+ lymphocytes were comparable between the two study groups when we analyzed the expression of HLA-DR on CD4+ T cells (median 6.7%; P25–75, 4.9–12.2 versus median 8.7%; P25–75, 5.4–10.3) (P > 0.546) and HLA-DR on CD8+ T cells (median 16.0%; P25–75, 12.9–32.8 versus median 21.0%; P25–75, 16.6–28.2) (P = 0.886) (data not shown). Failure to show the same downregulation by HLA-DR staining may be due to the fact that we worked with frozen samples, which may result in significant changes of its expression on CD4+ and CD8+ T lymphocytes [34].

Fig. 3
Fig. 3:
CD69 and CD25 expression in GB V-C nonviremic and viremic patients. (a) CD69 expression on CD4+ T cells and (b) CD8+ T cells. CD25 expression on (c) CD4+ T cells and (d) CD8+ T cells. The level of CD69 and CD25 expression was determined by flow cytometry, as depicted in the upper panels. P values were determined by Mann–Whitney test. GBV-C, GB virus type C.

Discussion

Although immune activation in HIV infection is a subject of increasing interest, no data on activation status are available in HIV-1/GBV-C coinfected patients. Using CD38 expression on T cells as a measure of T-cell activation, together with other activation markers, we assessed the influence of GBV-C viremia during HIV-1 infection. First, we confirmed the positive correlation between HIV-1 viral load and CD38 expression on CD8+ T cells, as previously demonstrated [35,36]. Second, GBV-C viral load was negatively correlated with CD38 expression on CD8+ T cells. Third, the significant correlation between CD38 expression on CD8+ T cells and HIV-1 viral load seen in the patients solely infected by HIV-1 was lost in the HIV-1/GBV-C coinfected patients. Fourth, GBV-C active infection was associated with less CD4+ and CD8+ T-cell activation, measured by different cellular surface markers in HIV-1-infected patients. Fifth, the association between GBV-C active infection and the downregulation of CD38 and CCR5 on CD8+ T cells and CD38 on CD4+ T cells was independent of HIV-1 viral load, CCR5Δ32 polymorphism, CD4+ T-cell count, and CD8+ T-cell count. Taken together, these finding suggest that the GBV-C viremia might, in fact, reduce the levels of T-cell activation in HIV-1-infected patients.

Persistent immune activation has been recognized as one of the main factors driving disease progression in HIV-1 infection and is predictive of disease development and treatment outcome [22,27,35,37,38]. The causes of immune activation observed during the acute phase of infection, in contrast to the chronic phase, may include factors in addition to translocation of microbial products [39]. Our results suggest that GBV-C may also interfere in cell activation. We observed that the positive correlation between CD38 expression on CD8+ T cells and HIV-1 viral load was present in patients infected solely by HIV-1, but not in those coinfected with GBV-C. Moreover, a trend towards a negative correlation was observed in the association between HIV-1 viral load and GBV-C viral load. To explore a link between GBV-C infection and cellular activation, we measured the activation profile, through CD38 and CCR5 expression, in three groups categorized according to their GBV-C status. In the multivariate analysis, surprisingly, the low expression of CD38 and CCR5 on CD8+ T cells and CD38 on CD4+ T cells was observed in the group of GBV-C viremic patients, but not in controls or GBV-C nonviremic anti-E2-seropositive individuals. These results suggest that active GBV-C replication is necessary to downmodulate cellular activation, independently of previous exposure to GBV-C. In regression models, we confirmed that this effect was independent of CD4+T-cell count CD8+ T-cell count, HIV-1 viral load, and CCR5Δ32 polymorphism. One could also argue that the observed effect might be restricted to CD38 and CCR5 expression. However, when we analyzed the other activation markers, downregulation was also seen in CD69 and CD25 expression. In all cases, the GBV-C/HIV-1 coinfected patients exhibited lower levels of these markers with statistically difference in CD69+CD4+, CD69+CD8+, and CD25+CD4+ T cell expression. These observations confirm the role of GBV-C virus in reducing immune activation.

GBV-C infection has been shown to counteract the high levels of activation and the rapid destructive events in gut-associated lymphoid tissue seen during the acute phase of HIV-1 infection [40]. Considering that relatively slow progression in the chronic phase of HIV-1 infection is dependent on the degree of immune activation, our findings suggest that this downregulation can also be influenced by GBV-C viremia.

It has been demonstrated that a substantial proportion of CD8+ T cells expressing high levels of CD38 is highly susceptible to spontaneous and Fas-mediated apoptosis, resulting in a population of cells with poor antiviral effector function [41]. The T-cell activation levels, measured by CD38 expression on CD8 T cells, appear to be established early in HIV-1 infection. Activation levels decline after successful antiretroviral therapy, but remain elevated when compared with HIV-1-uninfected individuals [35]. This study focused on HIV-1-infected patients before therapy intervention, excluding the influence that might be observed after HAART.

We failed to demonstrate lower expression of CCR5 on CD4 T cells. A fluctuation in the frequency of CCR5 expressing CD4+ T cells during primary infection could be explained in part by the developing hypothesis that CD4+ T cells expressing CCR5 may be short-lived. These cells are constantly undergoing rapid turnover in vivo compared with a static population that persists throughout life [42]. Therefore, CCR5 expression can be highly labile and these cells may redistribute throughout the lymphatic system, limiting interpretations of the loss of these cells, as measured in the peripheral blood [43].

There remain many questions to be answered before a full understanding of the impact of GBV-C coinfection with HIV and the influence of other viruses can be determined. Little work has been done in relation to HBV and GBVC coinfection. What has been shown is that there was no significant effect of GBV-C on HBV DNA levels [44]. Reduction in hepatitis C-related liver disease associated with GBV-C in HIV coinfection was described recently by Berzsenyi et al.[45]. The presence of other major coinfections (i.e. HBV, HCV, HTLV-2, HSV-2) has been reported previously by our group [46] and investigated as a potentially confounding variable. Therefore, no individual coinfected with other virus beside GBV-C and HIV-1 was included in our study.

The previously described survival advantage conferred by the GBV-C virus among HIV-infected people was dependent on the persistence of GBV-C viremia. Our results are consistent with these observations, suggesting that the increase in activation levels after GBV-C clearance may result in the loss of the protective effect of GBV-C and support the notion that GBV-C clearance may also be a contributor to HIV-1 disease progression. Longitudinal studies will be necessary to support the conclusion that GBV-C infection modulates activation of CD8+ T cells as an important factor for the control of HIV-1 disease progression, reducing the loss of CD4+ cells and development of AIDS.

In summary, we demonstrated that GBV-C infection is associated with a lower T-cell activation independently of HIV-1 viral load. We recognize that it is not yet possible to discriminate whether the GBV-C replication is a cause or a consequence of the lower T-cell activation, but our findings may reflect the impact of the natural history of GBV-C on HIV-1 disease. Nevertheless, the results suggest that GBV-C infection may be one important mechanism involved in the protection against HIV-1 disease progression and could represent an important topic for future studies, including the development of new therapeutic approaches for HIV infection.

Acknowledgements

This study was supported with funding from the Brazilian Program for STD and AIDS, Ministry of Health (914/BRA/3014-UNESCO/Kallas), the São Paulo City Health Department (2004-0.168.922-7/Kallas), and the Fundação de Amparo a Pesquisa do Estado de São Paulo (04/15856-9/Diaz, Sabino & Kallas; 05/01072-9/Levi). M.T.M.G. and M.M.S. were supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Ministry of Education.

We thank Professor David Watkins for the critical review of the manuscript and Maria Cecilia Sucupira, Priscilla Costa, Fernanda Bruno, Leandro Tarosso, and Debora Rocha for the laboratory support.

M.T.M.G. developed the project, performed the experiments, analyzed the results, and wrote the manuscript; T.M.S. performed the experiments and also analyzed the flow cytometry results; M.M.S. followed all the volunteers in the cohort and obtained the clinical data; H.T. organized the sample repository, supported the conduction of experiments, and was in charge of the main database; J.E.L. obtained funds for the study, coordinated the conduction of GBV-C assays, and wrote the manuscript; K.C.B. worked in the set up of the cohort and participated in the volunteers' enrollment; A.N. performed the GBV-C sequence experiments and reviewed the manuscript; R.S.D. obtained funds for the study, coordinated the HIV assays, and reviewed the manuscript; E.C.S. obtained funds for the study, coordinated the GBV-C virus sequencing, and reviewed the manuscript; R.P. did the statistical analyses and reviewed the manuscript; E.G.K. developed the project, obtained funds for the study, reviewed the results, reviewed the statistical analyses, wrote, and reviewed the manuscript.

There are no conflicts of interest.

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Keywords:

activation; CD38; coinfection; GB virus type C; HIV-1; T lymphocyte

© 2009 Lippincott Williams & Wilkins, Inc.