Until recently, the standard treatment for hepatitis C virus (HCV) infection consisted mainly of a combination of interferon α (IFN-α) and ribavirin (RBV),1,2 nowadays other treatment approaches have been proposed.3 This combination is effective in a significant proportion of patients including HIV-1-infected patients, where the frequency of sustained virologic response (SVR) to treatment is lower than in HCV-monoinfected patients and is complicated by toxicities.2 IFN-α belongs to a family of type 1 interferons produced mainly by dendritic and other cells as part of the host's toll-like receptor-mediated antiviral response.4
It has been reported that IFN-α is also effective in reducing HIV-1 RNA loads in naive patients and in patients receiving antiretroviral treatment/therapy (ART).5–11 Also, a reduction of integrated HIV-1 DNA in CD4 T cells has been reported in 2 different works. One analyzed HIV-1-infected patients (not infected with HCV) who interrupted suppressive ART to receive treatment with IFN-α,12 and the other analyzed HIV-1/HCV-coinfected patients under ART who received IFN-α and RBV as HCV treatment.13 Both works supported a role for immunomediated approaches for functional cure and HIV-1 eradication.
However, no ex vivo studies of the effect of IFN-α on human T-lymphotropic virus type 2 (HTLV-2)-infected patients have been reported yet, although HTLV-2 infection is highly prevalent in European active/former injecting drug users coinfected with HIV-1.14–19 The absence of direct clinical symptoms clearly associated to HTLV-2 infection may partially explain an underestimate of the real HTLV-2 prevalence rate and its effects in patients concurrently infected with HIV-1 and HCV. However, it has recently been reported that IFN-α can restrict HTLV-1 and 2 in vitro infection through the activation of protein kinase RNA–activated or its inhibition.20,21
Here, we analyzed the level of cell-associated HTLV-2 DNA in patients with HCV infection and patients with no HCV infection, either after treatment with IFN-α and RBV or after spontaneous HCV RNA clearance or IFN-free treatment, in HCV–HIV-1-coinfected patients receiving ART. We also analyzed other immune factors including immune activation and interleukin 6 (IL-6) levels.
MATERIALS AND METHODS
We conducted a survey for the presence of HTLV-1/2 antibodies in our cohort of HIV-1-infected patients, followed in the University Hospital Ramón y Cajal, Madrid, Spain, who were active or former injecting drug users. Of 695 HIV-1-infected patients with a history of injecting drug use, 67 were coinfected with HTLV-2 (9.64%). All of them showed antibodies anti-HCV at the time of HIV-1 diagnosis. Among them, 57 patients had at least 1 year of suppressive ART and were included in this study into 3 different groups: (1) 37 patients with HCV infection who had never received pegylated IFN-α–based HCV treatment (HCV patients); (2) 15 patients who received IFN-α 2a or 2b combined with weight-based RBV, 10 of them with SVR, that is, patients with undetectable HCV RNA at 24 weeks after treatment completion, 2 patients who relapsed, that is, patients with undetectable viremia during treatment but with subsequent viremia after treatment cessation, and 3 patients who did not respond, that is, patients with detectable circulating HCV RNA throughout treatment (IFN patients); and (3) 9 patients who had spontaneous HCV RNA clearance (SHC patients). Baseline characteristics of the patients are shown in (Table 1) (for duration of the IFN-based treatment, treatment response, and the exact time of sample collection of the 15 IFN patients, see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A663). The same sample was used to perform cell-associated HTLV-2 quantification, T-cell immune activation, and plasma IL-6 quantification.
This study was performed according to the recommendations of the Declaration of Helsinki and current Spanish legislation. All patients provided written informed consent for participation, sample collection, and laboratory determinations. Whole blood was collected in Vacutainer EDTA tubes (Becton-Dickinson, Madrid, Spain).
Cell-Associated HTLV-2 DNA Quantification
Total cell-associated HTLV-2 DNA was quantified by in-house real-time polymerase chain reaction. DNA was extracted from the 106 cryopreserved peripheral blood mononuclear cells using QIAamp DNA kit (Qiagen GmbH, Hilden, Germany). A standard curve was generated in each run using 5-fold dilution of 106 copies of a recombinant plasmid (TOPO TA cloning; Invitrogen, Grand Island, NY) containing 1 copy of HTLV-2 tax gene fragment from a patient with HTLV-2 subtype b, using primers common for subtypes a and b (Mo and NRA isolates, respectively, for reference, with 171 bp amplification).17 Besides, another standard curve was generated using 5-fold dilution of 106 copies of a recombinant plasmid containing 1 copy of human genomic GAPDH gene fragment (162 bp amplification).
The amplification reaction was performed in triplicate using LightCycler 2.0 (Roche Diagnostics, Pleasanton, CA). All reaction mixtures were performed in a total volume of 20 μL containing LightCycler FastStart DNA Master PLUS HybProbe 5X (Roche Diagnostics), 200 ng genomic DNA, 50 pmol each primer, Tax-F (5′ACTCCTGTCTCCCCCAAG) and Tax-R (5′TACGGTTTTTCCCCAGG) for tax gene, GADH-F (5′ CTGACCTGCCGTCTAGA) and GADH-R (5′GTCGTTGAGGGCAATGC) for GADH gene, along with 2 pmol of the following fluorescent probes: 5′-FAM-CACCCGCCTTCTTCCAATCAATGCGAAAG-TAMRA and 5′-CAGGTGGTCTCCTCTGACTTCAAC-[Flc]/5′-LC705 CGACACCCACTCCTCCACCTTTG-[Phos]-3′ for tax gene and GADH gene, respectively. These primers amplify either subtype a or b within HTLV-2.
The cycling parameters for both amplifications began with hot start at 95°C for 10 minutes and continued with 40 cycles of denaturation at 95°C for 10 seconds, annealing at 62°C for 15 seconds, and extension at 72°C for 20 seconds for tax gene, and 40 cycles of denaturation at 95°C for 5 seconds, annealing at 62°C for 10 seconds, and extension at 72°C for 20 seconds for GADH gene. The results were expressed as HTLV-2 DNA copies per million peripheral blood mononuclear cells.
T-Cell Immune Activation
Flow cytometry using fresh EDTA-anticoagulated whole blood was used to analyze CD4+ and CD8+ T-cell activation with the following antibody combination: CD3-V500, CD4-peridinin–chlorophyll–protein complex, CD8-phycoerythrin-Cy7, and the coexpression of CD38-phycoerythrin and HLA-DR-allophycocyanin. Briefly, 100 μL of blood was lyzed with the FACS Lysing solution (Becton-Dickinson) for 30 minutes at room temperature, incubated with the antibodies during 20 minutes at 4°C, which were washed and resuspended in phosphate-buffered saline containing 1% azida. Cells were analyzed in a Gallios flow cytometer (Beckman-Coulter, Brea, CA). At least 30,000 CD3+ T cells were collected for each sample and analyzed with Kaluza software (Beckman-Coulter) initially gating lymphocytes according to morphological parameters.
Plasma IL-6 Level
Plasma IL-6 quantification, an immune inflammation marker, was measured in duplicate according to manufacturer's instructions (Human IL-6 Quantikine High Sensitivity ELISA Kit; R&D Systems, Minneapolis, MN).
Continuous variables were expressed as the median and interquartile range, and discrete variables as percentages. The Student t test for independent samples was used to compare normally distributed continuous variables, and the Mann–Whitney test to compare non-normally distributed continuous variables. Comparison of continuous variables between the 3 groups was performed using analysis of variance test. Categorical variables were described as proportions. The association between categorical variables was evaluated using the χ2 test. Bivariate correlations by Spearman's rank coefficient were obtained between different factors. A linear regression model was created using cell-associated HTLV-2 DNA as a dependent variable. The overall fit of the model was assessed, and standardized beta coefficients were reviewed for statistical significance and contribution to the model, together with statistically significant variables in the univariate analysis. A P value less than 0.05 denoted the presence of statistical significance. Statistical analysis was performed using SPSS software 16.0 (Chicago, IL).
Baseline Characteristics of the Patients
All the baseline variables were similar comparing the 3 groups of patients studied, as analyzed by analysis of variance test (P > 0.05) and shown in Table 1. It is very important to note that age, time of HIV-1 diagnosis, time on ART, and time of suppressive ART were similar in these 3 groups of patients, because these parameters might influence the level of cell-associated HTLV-2 DNA. Hence, these groups of patients are well comparable.
Lower Cell-Associated HTLV-2 DNA in IFN and SHC Patients
Either IFN patients or SHC patients had lower level of cell-associated HTLV-2 DNA compared with HCV patients (P = 0.022 and P = 0.040, respectively). Interestingly, IFN patients had similar levels compared with SVR patients (P = 0.973), as shown in Figure 1.
CD8 Percentage and Had Received IFN-Based Treatment or Had HCV Clearance Are Independently Associated to Cell-Associated HTLV-2 DNA
Cell-associated HTLV-2 DNA positively correlated with CD8 T-cell count (P = 0.001, r = 0.641) and CD8 T-cell percentage (P < 0.001, r = 0.703), and negatively correlated with CD4 T-cell percentage (P = 0.001, r = −0.635) and CD4/CD8 ratio (P < 0.001, r = −0.694). Also, cell-associated HTLV-2 DNA positively correlated with a variable that categorized HCV patients versus patients who either received IFN-based treatment or had spontaneous HCV clearance (IFN + SHC) (P = 0.006, r = 0.591). Cell-associated HTLV-2 DNA did not correlate either with immune activation markers or IL-6 levels (see Table, Supplemental Digital Content 2, http://links.lww.com/QAI/A663). Multivariate analysis (linear regression model) showed that only CD8 percentage and IFN + SHC were independently associated with cell-associated HTLV-2 DNA (P < 0.001, β coefficient 0.435; 95% confidence interval: 0.018 to 0.057 and P = 0.038, β coefficient −0.243; 95% confidence interval: −1.119 to −0.032) (see Table, Supplemental Digital Content 2, http://links.lww.com/QAI/A663).
Immune Activation and IL-6 Level are Higher in HCV Patients
HCV patients had higher levels of CD8 T-cell activation compared with either IFN patients (P = 0.048) and SHC patients (P = 0.026), and higher IL-6 levels compared with SHC patients (P = 0.024) (see Figure, Supplemental Digital Content 3, http://links.lww.com/QAI/A663).
Among HCV patients, 31 (84%) were genotyped for HCV and distributed as follows: genotype 1, 19 patients (61.3%); genotype 2, 2 patients (6.5%); genotype 3, 7 patients (22.6%); and genotype 4, 3 patients (9.6%). However, 13 IFN patients were genotyped for HCV (86.6%) and distributed as follows: genotype 1, 6 patients (46.1%); genotype 2, 2 patients (15.4%); and genotype 3, 5 patients (38.4%).
This work represents the first analysis of the effect of the therapy with IFN-α and RBV in cell-associated HTLV-2 DNA in HTLV-2–HIV-1–HCV-coinfected patients under ART. Our data indicate that patients treated with IFN-α and RBV have lower total cell-associated HTLV-2 DNA. On one hand, this observed effect reflects changes after HCV treatment and does not reflect a general decline of HTLV-2 DNA because HTLV proviral load is very stable over time.22–26 On the other hand, this work pointed out the importance of HCV infection on the level of cell-associated HTLV-2 DNA, because we report here that patients with no HCV infection due to spontaneous HCV RNA clearance also had lower level of cell-associated HTLV-2 DNA.
The mechanism of IFN-α-induced decline of cell-associated retrovirus (HIV-1 and HTLV-2) DNA in ART-treated patients is uncertain. It has been proposed that reductions of HIV-1 DNA during IFN-α/RBV therapy may simply result from unspecific lymphocellular toxicity of this treatment regimen because this treatment is associated with decreasing CD4 T-cell counts.27,28 This is not applicable to the decline shown in cell-associated HTLV-2 DNA because the main cell target for this virus is the CD8 T cell that is not modified by this treatment.
Because cell-associated HTLV-2 DNA positively correlated with CD8 count (and percentage) and negatively with CD4 T-cell percentage, CD4/CD8 ratio was consequently altered. In this way, HCV patients had lower CD4/CD8 ratio compared with HCV-uninfected patients. Cell-associated HTLV-2 DNA also positively correlated with a variable that categorized HCV patients versus patients who either received IFN-α-based treatment or had SHC (variable IFN + SHC). Regardless of the negative correlation between cell-associated HTLV-2 DNA and CD4/CD8 ratio, no correlation with immune activation was found although a higher level of activation was found in HCV patients. Multivariate analysis showed that only CD8 percentage and variable IFN + SHC were independently associated to cell-associated HTLV-2 DNA. This analysis demonstrates that IFN-α can reduce the level of infection of virus other than HCV during treatment and however suggests that patients with an immune system (mainly through cytotoxic response) strong enough to clear HCV infection can also lower the infection of other viruses such as HTLV-2.
One limitation of this study regarding the reduced cell-associated HTLV-2 DNA found in patients who received HCV treatment is the fact that it was measured in 2 different groups of patients. This analysis would have been more accurate if performed in the same group of patients after and before IFN-based treatment. However, the groups of patients compared in this study are well comparable because variables, such as age, time of HIV-1 diagnosis, time on ART, and time on suppressive ART, are similar between them.
Further investigation of the mechanisms underlying the decline in cell-associated retrovirus DNA during IFN-α/RBV therapy may be helpful to reduce retrovirus (HIV/HTLV) reservoirs, in designing improved therapeutic strategies, and to contribute to functional cure or eradication. Besides, the absence of HCV infection, at least in patients with spontaneous HCV RNA clearance, influences lowering cell-associated HTLV-2 DNA. Both facts, IFN-α treatment and HCV RNA clearance, might be important in keeping HTLV-2 infection in a low level.
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