Clinical Science: Concise Communication
GBV-C viremia is associated with reduced CD4 expansion in HIV-infected people receiving HAART and interleukin-2 therapy
Stapleton, Jack Ta,d; Chaloner, Kathrynb,c; Zhang, Jingyangb; Klinzman, Donnad; Souza, Inara Ea; Xiang, Jinhuaa,d; Landay, Alane; Fahey, Johnf; Pollard, Richardg; Mitsuyasu, Ronaldf
aThe University of Iowa, Departments of Internal Medicine, USA
cStatistics & Actuarial Science, USA
dIowa City VA Medical Center, Iowa City, Iowa, USA
eRush Medical College, USA
fUniversity of California, Los Angeles, David Geffen School of Medicine, Los Angeles, USA
gUniversity of California, Davis, School of Medicine, Sacramento, California, USA.
Received 16 June, 2008
Revised 22 October, 2008
Accepted 23 October, 2008
Correspondence to Jack T. Stapleton, MD, Department of Internal Medicine, The University of Iowa College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242, USA. Tel: +1 319 356 3168; fax: +1 319 356 4600; e-mail: firstname.lastname@example.org
Objective: Interleukin-2 (IL-2) is a cytokine with multiple effects on lymphocytes including induction of CD4+ T-cell proliferation. IL-2 administration has been shown to increase CD4 cell counts in HIV-infected people receiving antiretroviral therapy. GB virus C (GBV-C) is an apparently nonpathogenic flavivirus that replicates in CD4+ T cells and inhibits HIV replication in vitro by mechanisms including downregulation of HIV entry coreceptors (CCR5 and CXCR4) and induction of chemokines (RANTES, MIP-1α, MIP-1 β, and SDF-1). GBV-C replication is significantly inhibited in vitro by activation of primary CD4+ cell cultures with IL-2 and phytohemagglutinin. We sought to determine if there is an interaction between GBV-C and IL-2 in vivo.
Methods: GBV-C viremia status was characterized in 92 HIV-infected individuals participating in a randomized trial of IL-2 and antiretroviral therapy [AIDS Clinical Trials Group Study (ACTG) 328]. Changes in CD4 cell counts and HIV RNA levels in individuals assigned IL-2 were compared with those in individuals assigned antiretroviral therapy alone.
Results: Individuals lacking GBV-C viremia had a significantly greater rise in CD4 cell count with IL-2, compared with GBV-C viremic individuals (by 511 cells/μl at week 84; interaction P = 0.02): GBV-C viremic individuals assigned IL-2 did not demonstrate a significant increase in CD4 cell count compared with individuals not assigned to receive IL-2 (95% CI for difference −255 to 397 cells/μl).
Conclusion: GBV-C viremia was associated with a block in CD4 cell expansion following IL-2 therapy in the ACTG 328 study, and GBV-C status may be an important factor in IL-2 treatment response.
Interleukin-2 (IL-2) is a cytokine that is a key regulatory molecule in T-cell biology. It is critical for proper regulation of T-cell proliferation, is a key contributor to the generation and function of CD4+CD25+ regulatory T cells, and mediates a process termed ‘activation-induced cell death’ or AICD [1,2]. IL-2 also exerts pleiotropic effects on other cells in the immune system including B-lymphocytes, natural killer (NK) cells, monocytes, macrophages, and dendritic cells (reviewed in ).
The idea of enhancing immune function in HIV-infected people by administration of IL-2 is attractive, although this approach was initially controversial as IL-2 enhances HIV replication in primary CD4+ T lymphocytes [1,4]. These concerns have been reduced due to the effectiveness of combination antiretroviral therapy (ART), and IL-2 has been administered to many HIV-infected people in an attempt to increase CD4+ T-cell number, decrease HIV-induced cytokine dysregulation, enhance innate and adaptive immune responses to opportunistic infections, and to decrease the latent HIV-infected pool of cells as a result of activating resting CD4+ T cells [5,6].
GB virus C (GBV-C, also known as hepatitis G virus) is a lymphotropic member of the family Flaviviridae that was discovered in 1995 (reviewed in [7,8]). The virus is transmitted by sexual and blood exposure, and is quite common in humans with viremia detected in approximately 1 to 3% of healthy US blood donors. Approximately 10% of donors also have antibodies that indicate prior GBV-C infection (reviewed in ). Although GBV-C viremia is typically cleared within a few years in most immune competent individuals [9,10], it persists longer in most HIV-infected people, and 80% of individuals with HIV-GBV-C coinfection maintained GBV-C viremia for more than 5 years in one longitudinal study . Epidemiological studies have not identified an association between GBV-C infection and any known human disease. In contrast, several studies prior to the advent of effective ART found a statistically significant association between persistent GBV-C infection and prolonged survival among HIV-infected individuals (reviewed in [12–14]). Supporting a potential role for GBV-C viremia in this association, serum GBV-C RNA levels are inversely related to HIV RNA levels in vivo [15,16]; and coinfection of human CD4+ T cells with GBV-C and HIV results in inhibition of HIV replication in vitro [17–19].
GBV-C replicates in primary human T and B-lymphocytes in vitro [20–22], and incubation of infected lymphocyte cultures with IL-2 and phytohaemogglutinin resulted in decreased GBV-C replication in vitro . To examine the possibility that GBV-C might influence the IL-2 response of HIV-infected individuals, the GBV-C viremia status and change in CD4 following assignment to IL-2 therapy or no IL-2 therapy in individuals in the AIDS Clinical Trials Group 328 study  were characterized.
The ACTG 328 study, an open label, randomized trial of intermittent recombinant human interleukin-2 (IL-2) by intravenous (IV) or subcutaneous administration in individuals with HIV infection receiving combination antiretroviral therapy (ART) compared with ART alone, has been described previously . Briefly, participants in the ACTG 328 study were HIV-infected individuals with baseline CD4 cell counts between 50 and 350 cells/μl on one occasion within 30 days prior to study entry, and who had never received protease-inhibitor or IL-2 therapy. Individuals did not have active HIV-related complications at the time of enrollment and all individuals initiated one of three ART regimens (indinavir combined with either ZDV + 3TC, ZDV + ddI, or d4T + ddI, of which at least one nucleoside reverse transcriptase inhibitor was new for the individual). One hundred and fifty-nine individuals whose HIV RNA concentration decreased to 5000 copies/ml or less after 11 weeks of therapy were randomly assigned at 12 weeks to receive no IL-2 (n = 52), 9 million international units (MIU) IV IL-2 daily for 5 days every 8 weeks for 72 weeks (n = 53), or 7.5 MIU IL-2 SC twice daily for 5 days every 8 weeks for 72 weeks (n = 54). Per protocol, thirty individuals in the IV IL-2 group switched to the subcutaneous IL-2 regimen after three or six cycles if their CD4 cell counts increased by 25% or at least 100 cells/μl compared with their baseline values . Individuals were monitored and the increase in CD4 cell count at weeks 60 (primary endpoint) and 84 (secondary endpoint) of over 50% of week 12 CD4 cell count were assessed as well as change in HIV RNA at weeks 60 and 84.
Available samples from ACTG 328 at the time of randomization (week 12) were analyzed for GBV-C RNA by real time RT-PCR (limit of detection 105 copies/ml)  by laboratory personnel who were not aware of any clinical data. GBV-C antibody testing was not done, as the commercial assay is no longer available (Georg Hess, Roche Diagnostics, personal communication). Change in CD4 cell count and HIV RNA following IL-2 therapy (week 12 to week 60, and week 12 to week 84), was analyzed by treatment group and 12 week GBV-C classification. The proportion of individuals in each group with an increase in CD4 cell count at weeks 60 and 84 of over 50% of week 12 count was also analyzed. In addition, the proportion of individuals at weeks 60 and 84 with an HIV RNA increase of greater than or equal to 0.7 log10 compared with week 12 was analyzed by logistic regression (a secondary endpoint). To address whether the subset of individuals with samples for GBV-C testing responded to IL-2 therapy differently than the entire study population, the proportion of individuals with a CD4 cell response of 50% or more at week 60 was compared, by treatment assignment, between those with samples tested for GBV-C and those without. The same analysis was performed for the week 84 responses. All analyses were performed in the statistical environment R .
Administration of IL-2 (IV and/or subcutaneous) in combination with ART was shown to significantly increase CD4 cell counts in the ACTG 328 study population when compared with individuals who received ART without IL-2, and there was no change in plasma HIV RNA . Fifty-eight percent of the individuals in ACTG study 328 (92 of 159) had samples available from the week 12 visit for GBV-C RNA testing. These 92 individuals did not have significant differences in baseline characteristics or changes in CD4 cell responses and HIV RNA responses following randomization observed when compared with the total study cohort (data not shown). GBV-C viremia was detected in 41% of individuals (38/92), similar to that observed in other studies of HIV-infected people .
Individuals without GBV-C viremia who were assigned to receive IL-2 had greater increases in CD4 cell counts than those with GBV-C viremia at week 60 (Fig. 1a), by 679 and 193 cells/μl for those assigned to receive IV and subcutaneous IL2 respectively. These differences in change in CD4 based on GBV-C viremia status persisted throughout the 84 weeks of the study (Fig. 1b). The interaction was significant for individuals receiving IL-2 at weeks 60 and 84 [P = 0.01, 95% confidence interval (CI) for difference 98 to 692 cells/μl [Table 1]; week 84 interaction P = 0.02, 95% CI for difference 97 to 926; Table 1]. Individuals who did not have GBV-C viremia demonstrated a significant increase in CD4 cell counts with assignment of IL-2 (Table 1, 95% CI 257–617, P < 10−5) whereas individuals with GBV-C viremia did not demonstrate a significant increase in CD4 cell counts with assignment to IL-2 (Table 1, 95% CI −194 to 278).
Among those without GBV-C viremia, CD4 cell responses were significantly greater among those assigned to receive IV IL-2 when compared with those assigned to receive subcutaneous IL-2 at both week 60 and week 84 (Table 1, P < 10−6 and P < 10−7, respectively). In contrast, individuals with GBV-C viremia assigned to IV IL-2 did not demonstrate a significant increase over those assigned subcutaneous (P = 0.50 at week 60, P = 0.89 at week 84, respectively). The comparison of CD4 cell response in the IV versus subcutaneous comparison for those with GBV-C viremia to those nonviremic was significant (interaction P = 0.002 at week 60 and P < 10−4 at week 84).
There were no differences in HIV RNA levels between GBV-C viremic and nonviremic patients, irrespective of IL-2 treatment group, and GBV-C viral load did not correlate with change in CD4 cell counts (data not shown). IL-2 administration was not associated with significant differences in GBV-C viral load at any time point, nor did CD4 cell count changes correlate with GBV-C viral load (data not shown).
In this substudy of ACTG 328, the GBV-C viremia status was characterized at the time individuals were randomly assigned to IL-2 or no IL-2 in combination with ART. Among individuals with GBV-C viremia, there was no significant difference in CD4 cell count increases in subjects assigned to receive IL-2 compared with individuals who were not assigned IL-2. Thus, IL-2 failed to stimulate CD4 cell expansion in people with GBV-C viremia. In contrast, CD4 cell counts increased significantly among those without GBV-C viremia assigned to IL-2 (437 cells/μl greater than those not assigned IL-2 at week 60). Treatment interaction was significant based on GBV-C viremia status at week 60 (P = 0.01) and week 84 (P = 0.02), providing strong statistical credibility to the interaction of GBV-C viremia with IL-2 [26,27].
IL-2 upregulates CCR5 and CXCR4 expression on T cells [28,29] whereas GBV-C interactions with CD4 cells results in decreased CCR5 surface density [18,19,30,31], suggesting that GBV-C dampens T-cell activation. Furthermore, addition of IL-2 and phytohemagglutinin (PHA) to peripheral blood mononuclear cells (PBMCs) obtained from HIV-GBV-C coinfected individuals resulted in significant reduction in GBV-C replication . Taken together with the results observed in the ACTG cohort, these data suggest a specific interaction between GBV-C and IL-2-mediated proliferation and potentially activation, both of which would have a beneficial effect on HIV disease .
Limitations of this analysis include the fact that samples were not available for GBV-C testing in 42% of the randomized individuals in ACTG 328, and that this study was designed retrospectively. Despite these limitations, significant differences in CD4 cell count responses were observed between GBV-C positive and GBV-C negative individuals following both 60 and 84 weeks of therapy. In addition, individuals with samples studied for GBV-C were not significantly different from the entire ACTG 328 cohort either at baseline or in CD4 cell change and HIV change following IL-2 within the treatment groups (data not shown). Thus, further investigations of GBV-C status in randomized studies of IL-2 administration in HIV-infected people are warranted. If the results observed in this study were validated, the measurement of GBV-C viremia status would be important prior to administration of IL-2 therapy, particularly if the goal is to increase CD4 cell number.
In this study GBV-C viremia was associated with a block in IL-2-related T-cell proliferation. This, in combination with the in vitro evidence that IL-2 decreases GBV-C replication suggests that there is an interaction between GBV-C and IL-2 signaling pathways. Further study of mechanisms by which GBV-C influences IL-2 response is warranted.
This work was supported in part by Merit Review Grants from the Veterans Administration (J.T.S. and J.X.), and by an NIH RO1 grant (AI-58740, J.T.S.), ACTU grants, (AI-69424, AI-27660, R.M. and J.F.). We are grateful to Rebecca Gelman PhD and Deborah Wang Cheng MS for providing the ACTG data and to Suhong Zhang PhD for an analysis of an earlier data set, and Jennifer Nowack for assistance with specimens. Supported in part by the AIDS Clinical Trials Group funded by the National Institute of Allergy and Infectious Diseases.
J.S. proposed the idea to the ACTG, and oversaw all aspects of the protocol design, testing, data evaluation, and writing of the manuscript. K.C. assisted in preparing the proposal, in study design, data analysis and interpretation, and in writing the manuscript. J.Z. assisted in the data analysis, interpretation, and writing the manuscript. D.K. and I.S. oversaw GBV-C testing, data analysis and interpretation, and writing the manuscript. J.X. developed and validated the real-time PCR method for GBV-C quantification used in these studies, and assisted in testing samples, interpreting results and writing the manuscript. A.L., J.F., R.P., and R.M. served as co-chairs for the ACTG protocols, coordinated sample shipping, data transferal, study interpretation and writing the manuscript.
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