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Influence of amantadine on CD81 expression on lymphocytes in chronic hepatitis C
Digestive and Liver Disease 42 (2010) 735–740
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Influence of amantadine on CD81 expression on lymphocytes in chronic hepatitis C Martin-Walter Welker a , Michael von Wagner a , Dana Ochs b , Vincent Zimmer c , Wolf Peter Hofmann a , Albrecht Piiper a , Rolf W. Hartmann d , Eva Herrmann e , Stefan Zeuzem a , Bernd Kronenberger a,∗ a
Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Kinderklinik, Vinzentius Krankenhaus, Cornichonstraße 4, 76829 Landau in der Pfalz, Germany c Klinik für Innere Medizin II, Universitätsklinikum des Saarlandes, Kirrberger Str., 66421 Homburg/Saar, Germany d Pharmazeutische und Medizinische Chemie, Universität des Saarlandes, Postfach 151150, 66041 Saarbrücken, Germany e Institut für Biostatistik und mathematische Modellierung, Fachbereich Medizin, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany b
a r t i c l e
i n f o
Article history: Received 18 September 2009 Accepted 17 March 2010 Available online 24 April 2010 Keywords: Amantadine CD81 HCV PBMC
a b s t r a c t Introduction: Interferon alpha (IFN) down regulates CD81 expression on peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis C virus (HCV) infection. Aim of our study was to investigate whether amantadine alters IFN associated down regulation of CD81 expression on PBMC in patients with chronic hepatitis C. Methods: Nineteen patients with chronic HCV infection received peginterferon alpha-2a/ribavirin (SOC) for 48 weeks. Patients were randomised to 12 weeks amantadine therapy (n = 12) or no additional treatment (n = 7). FACS analysis of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was performed at baseline, week (TW) 4, TW12, and TW24 of antiviral therapy. Results: A significant decline of CD81 expression was observed on CD4(+), CD8(+), and CD56(+) cells (p = 0.011, p < 0.001, p = 0.015, respectively) but not on CD19(+) cells (p > 0.2). CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was not different between patients treated with SOC plus amantadine and patients treated with SOC alone. Conclusion: The current study confirms that CD81 expression is down regulated by SOC on CD4(+), CD8(+) and CD56(+) cells. Amantadine treatment was not associated with CD81 expression. Interaction between amantadine and CD81 is unlikely to be involved in potential antiviral activity of amantadine in chronic HCV infection. © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis C virus (HCV) primary infects hepatocytes and is a major cause of chronic hepatitis and its sequelae, including liver cirrhosis and hepatocellular carcinoma [1,2]. Standard of care (SOC) antiviral therapy is burdened with severe side effects and leads to sustained virologic response in only 48–52% in HCV genotype 1 infected patients, the most common genotype in Western countries [3–6]. The 26-kDa cell surface protein CD81 is a tetraspanin composed of 4 transmembrane and 2 extracellular loops and is expressed on hepatocytes as well as on peripheral blood mononuclear cells (PBMC’s) [7]. Interaction of the second extracellular loop of CD81 with the HCV envelope protein 2 is an essential step in HCV cell entry [8,9]. Interferon alpha was shown to down regulate CD81
∗ Corresponding author. Tel.: +49 69 6301 87680; fax: +49 69 6301 87676. E-mail address: [email protected] (B. Kronenberger).
expression on PBMC’s in vitro, while HCV infection per se alters CD81 expression to higher levels [10]. Furthermore, CD81 expression on CD8(+) and CD56(+) PBMC’s decreases during IFN-based antiviral therapy in vivo and down regulation of CD81 is correlated with initial virologic response [10–13]. In general, CD81 is involved in cellular activation [14], adhesion [9], proliferation [15], and differentiation processes [16]. Expressed on rat astrocytes, CD81 activation has antiproliferative effects. Wagner et al. tested boraadamantaneamine derivates in a rat astrocyte proliferation assay in combination with immunoblotting [17]. Herein, 1-boraadamataneamine derivatives were found to bind and activate CD81 [17]. Amantadine (1-adamantanamine), a chemical analogue to 1boraadamataneamine, has been shown to slightly improve sustained virologic rates in patients treated with interferonbased antiviral therapy, but not when used in treatment regimes comprising pegylated interferon alpha and ribavirin [18–25]. Aim of our study was to investigate the effect of amantadine on CD81 expression during peginterferon-based antiviral therapy.
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Table 1 Demographic and baseline characteristics of the patients and treatment outcome.
Baseline characteristics Demography Gender Age Body mass index Caucasian Biochemistry ALT AST GGT Virology HCV RNA
AMA 200 mg q.d. plus SOC n=6
AMA 200 mg b.i.d. plus SOC n=6
SOC alone n=7
(Female/male) (Years, mean ± SD) (kg/m2 , mean ± SD)
4/2 41 ± 14 25.8 ± 3.2 6
5/1 52 ± 6 28.2 ± 2.3 6
5/2 52 ± 15 24.7 ± 3.1 7
(n.s.) (n.s.) (n.s.) (n.s.)
(×ULN, mean ± SD) (×ULN, mean ± SD) (×ULN, mean ± SD)
3.6 ± 2.8 1.9 ± 0.9 1.3 ± 0.9
2.3 ± 0.9 1.5 ± 0.3 1.8 ± 0.6
2.9 ± 1.5 1.6 ± 0.7 1.9 ± 1.4
(n.s.) (n.s.) (n.s.)
(log IU/mL, mean ± SD)
5.9 ± 0.4
6.1 ± 0.6
5.6 ± 0.4
(n.s.)
1/1/4
3/1/2
0/1/6
(n.s.)
Treatment outcome NR/relapse/SVR
AMA, amantadine; NR, non-response; SD, standard deviation; SOC, standard of care; SVR, sustained virologic response; ULN, upper limit of normal.
2. Patients and methods
2.4. Study endpoints
2.1. Patients and treatment
Primary endpoint of our study was CD81 expression at TW 4 and TW 12 in patients treated with amantadine plus SOC in comparison to patients with SOC only. Secondary endpoint was influence of amantadine treatment on CD81 kinetics during antiviral therapy.
Nineteen treatment naive patients with chronic hepatitis C virus genotype 1 infection were enrolled in the present study. Co-infection with hepatitis B virus or human immunodeficiency virus was an exclusion criterion. All patients received peginterferon alpha-2a 180 g once weekly and ribavirin 800–1200 mg/die according to body weight for 48 weeks. Before treatment patients were randomized to 12 weeks amantadine therapy (200 mg q.d., n = 6; 200 mg b.i.d., n = 6) or no amantadine (n = 7). All patients had given written informed consent according to the Declaration of Helsinki. The study was approved by the local Ethics Committee. 2.2. Quantitative measurement of HCV RNA and HCV genotyping Quantitative assessment of HCV RNA concentration was performed by AmplicorTM HCV Monitor HCV test version 2.0 (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 615 IU/mL. Genotyping of HCV, according to the classification of Simmonds et al. [26], was done by reverse hybridisation assay (INNO LiPA HCV-II; Innogenetics, Ghent, Belgium). Serum HCV concentration was measured at baseline, treatment weeks (TW) 4, 12, 24, 48, and 24 weeks after withdrawal of therapy. 2.3. Immunocytochemistry and FACS analysis Blood was collected in sterile ethylene-diamine-tetra-acetate filled blood collection tubes. Whole blood (100 L) was incubated with R-phycoerythrin-conjugated anti-CD81-antibodies (20 L; Clone JS-81, mouse anti-human IgG, Pharmingen, Heidelberg, Germany). For labelling of lymphocyte subsets, peripheral blood mononuclear cells were costained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (Clone RPA-T4, mouse anti-human IgG, Pharmingen), anti-CD8 (Clone HIT8a, mouse anti-human IgG, Pharmingen), anti-CD19 (Clone 4G7, mouse anti-human, Pharmingen), and anti-CD56 antibodies (Clone NCAM 16.2, mouse anti-human IgG, Pharmingen) for 30 min at 4 ◦ C. Cells were subsequently fixed and erythrocytes lysed using cell lysis buffer (10 min, 4 ◦ C). Fluorescence was analyzed with a fluorescence activated cell sorter (FACS; FACScan, Becton Dickinson, Heidelberg, Germany). FITC-, PE-conjugated mouse IgG antibodies (Pharmingen) were used as isotype controls for the quantification of background fluorescence. FACS analysis of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was performed at baseline, TW 4, and TW 12 during antiviral therapy. Fluorescence was quantified in relative fluorescence units (RFU).
2.5. Statistical analyses Chi-squared test (gender), Kruskal–Wallis test (age, BMI, GOT, GPT, GGT serum levels), and ANOVA test (log10 HCV RNA serum concentration) were applied to compare baseline characteristics of the 3 treatment groups. All tests were two-tailed and p values < 0.05 were considered significant. Clinical and biochemical characteristics of patients were expressed as mean ± standard deviation (SD). Changes of CD81 expression during treatment were tested by the Friedman Chi2 test for multiple comparisons. If Friedman Chi2 test showed a significant change of CD81 expression during treatment, CD81 expression at baseline was compared with CD81 expression at TW 4 and 12 by multiple Wilcoxon–Wilcox comparisons. Differences in CD81 expression between the different treatment arms were investigated by Kruskal–Wallis and Mann–Whitney Utest. Sample size was sufficient to guarantee a power above 80% if there is a probability of about 90% that CD81 expression in patients treated with amantadine plus SOC is less than CD81 expression in patients treated with SOC in single comparisons. 3. Results 3.1. Patients characteristics and treatment outcome All patients included in the present study were infected with hepatitis C virus genotype 1. Epidemiological, medical, and virologic characteristics are given in Table 1, as well as treatment outcome. The 3 treatment groups did not differ between gender, age, body mass index, race, as well as baseline serum ALT, AST, GGT levels or HCV RNA serum concentration and treatment outcome, respectively (Table 1). 3.2. CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells at baseline and during antiviral therapy CD81 expression on the different lymphocyte subtypes was quantified by FACS analysis (Fig. 1). Mean CD81 expression at baseline was 613 ± 131 RFU, 787 ± 237 RFU, 546 ± 118 RFU, and 527 ± 206 RFU on CD4(+), CD8(+), CD19(+), and CD56(+) lymphocytes, respectively. The changes of CD81 expression during
M.-W. Welker et al. / Digestive and Liver Disease 42 (2010) 735–740
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Fig. 1. FACS analysis of CD81 expression on (A) CD4(+), (B) CD8(+), (C) CD19(+), and (D) CD56(+) cells after gating for lymphocytes. CD81 was labeled with PE-conjugated mouse anti-human CD81-antibodies; CD4, CD8, CD19, CD56 were labeled with respective FITC-conjugated mouse anti-human antibodies. The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies, respectively. Cells in the upper right quadrant were considered as double positive for CD81 and CD4/CD8/CD19/CD56 and used for quantification of CD81 expression on the different lymphocyte subtypes.
treatment are shown in Fig. 2. During treatment a significant decline of CD81 expression was observed on CD4(+), CD8(+), and CD56(+) cells (p = 0.011, p < 0.001, and p = 0.015 for all time points, respectively) but not on CD19(+) cells (p > 0.2 for all time points, Fig. 2). We next analyzed whether the lowest level of CD81 (nadir) was different between the lymphocyte subtypes. The CD81 expression nadir was similar on CD4(+), CD19(+), and CD8(+) cells (Fig. 3). On CD56(+) cells the CD81 expression nadir was lower compared with the other subtypes (p < 0.01 for all; Fig. 3). Furthermore, the decline of CD81 expression during treatment compared with baseline was analyzed. The maximum decline of CD81 expression in relation to baseline on CD8(+) and CD56(+) was greater than the maximum decline of CD81 expression on CD4(+)
cells (0.55 vs. 0.41 vs. 0.70-fold, p < 0.05 for CD4 vs. CD8 and CD4 vs. CD56). The CD81 expression level may be associated with virologic response. However, in the present study, no correlation between CD81 expression on the different lymphocyte subtypes with viral load was observed (p > 0.2 for all subtypes and time points, data not shown). The percentage of CD4(+), CD8(+), CD19(+), and CD56(+) cells at baseline was 38.8 ± 9.9%, 20.0 ± 6.2%, 7.3 ± 3.3%, and 7.6 ± 5.4%, respectively. During treatment no changes in the percentage of lymphocyte subtypes were observed (p = 0.17, p > 0.2, p = 0.07, p = 0.17 for CD4(+), CD8(+), CD19(+), CD56(+), respectively; data not shown). The percentage of lymphocyte subtypes could be correlated with CD81 expression. In the present study, however, no correlation between CD81 expression and the percentage of lymphocyte subtypes was observed (p > 0.05 for all; data not shown).
3.3. Association of amantadine treatment with CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells
Fig. 2. Dynamics of CD81 expression on the different lymphocyte subtypes for all patients during the first 12 weeks of antiviral therapy. Symbols indicate lymphocyte subtypes (), CD4(+) cells; (), CD8(+) cells; (), CD19(+) cells; (×), CD56(+) cells. Significant differences in CD81 expression during treatment compared with baseline are indicated, * for p < 0.05, **for p < 0.01.
CD81 expression during treatment was compared between the amantadine and the control group. During treatment CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was not different between patients treated with SOC plus amantadine and patients treated with SOC alone (p > 0.05 for all subtypes and TW 4 and 12; data no shown). The comparison between baseline CD81 and the CD81 expression nadir is shown in Fig. 3a–d. On all lymphocyte subgroups a significant reduction between CD81 expression at baseline and the CD81 expression nadir was observed in the amantadine and the control group (Fig. 3). However, no difference was noted between the CD81 expression nadir between the amantadine and the control group. Furthermore, we observed no difference in CD81
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Fig. 3. Bar graph of baseline (BL) and nadir CD81 expression for amantadine plus SOC treated patients (AMA) and only SOC treated patients (SOC) for (A) CD4(+) cells, (B) CD8(+) cells, (C) CD19(+) cells, and (D) CD56(+) cells. Although a decrease of CD81 expression occurred in each lymphocyte subset population, no differences between AMA and SOC groups were observed. RFU, relative fluorescence units.
expression on the different cell types between the end of amantadine treatment (TW12) and after amantadine treatment (TW24; data not shown). The decline of CD81 during treatment was also compared between the amantadine and the control group. However, no difference in CD81 decline during treatment was observed between the amantadine and the control group (p > 0.05 for all time points and subtypes; data not shown). The percentage of CD4(+), CD8(+), CD19(+), and CD56(+) lymphocytes was not different between the amantadine and the control group (p > 0.2 for all; data not shown). Neither in the amantadine nor in the control group a significant correlation between CD81 expression and the percentage of lymphocyte subtypes was observed (p > 0.1; data not shown). 4. Discussion HCV primarily infects hepatocytes, but genomic and antigenomic HCV RNA was detected in PBMC, – mainly CD8(+) and CD19(+) cells – , also, suggesting putative lymphotropism of HCV [27–31]. Lymphotropism of HCV may be responsible for impaired, specific immune response and development of autoimmune diseases in patients with chronic hepatitis C [32–35]. We have shown previously, that CD81 expression is down regulated on CD8(+) and CD56(+) PBMC’s by interferon alpha in vitro and in vivo [10,36]. Down regulation of CD81 expression on lymphocyte subtypes during antiviral treatment could reduce the interaction between HCV and lymphocytes and could restore HCV associated immune dysfunction or reduce de novo infection [33]. Thus, down regulation of CD81 could be an antiviral mechanism. This may have consequences for immune function and potential HCV infection. Indeed, previous studies showed an association of CD81 expression on CD8(+) PBMC’s with antiviral response. In the current
study, we confirmed down regulation of CD81 by interferon alphabased therapy as a significant decrease in CD81 expression was shown on CD4(+), CD8(+), and CD56(+) cells during antiviral therapy. However, in the present study we observed no association between virologic response and CD81 expression. In previous studies, the difference in CD81 expression between patients who a achieved a sustained virologic response and those without sustained virologic response was found after treatment [13,36]. In the present study, CD81 expression was measured after 4 and 12 weeks because amantadine treatment was performed for the first 12 weeks only. These time points may be too early to detect a significant association between CD81 expression and virologic response. Nevertheless, as early down regulation of CD81 expression may be merely drug induced [10,36], it can be anticipated that a potential amantadine effect on CD81 should be detectable within the 12 weeks amantadine was administered. Amantadine therapy has been shown to increase sustained virologic rates to a minor extent in combination with standard interferon alpha and ribavirin, but not in patients treated with peginterferon alpha and ribavirin [18–25]. The weak potential therapeutic mechanism of amantadine therapy in patients with chronic HCV infection and interferon/ribavirin therapy is not clarified, so far. It was shown that amantadine may interfere with the HCV p7 ion channel and it was assumed that the interference of amantadine with HCV p7 may be an antiviral mechanism [37]. Substitution of L20F within HCV p7 was associated with non-response to combination therapy specifically with amantadine in HCV genotype 1b infected patients [38]. The potential antiviral activity of amantadine on the HCV p7 ion channel was further explored using a system for production of infectious HCV particles in cell culture [39,40]. In this model, however, the HCV p7 ion channel activity was not affected by amantadine suggesting that interference of amantadine with HCV p7 is not a relevant antiviral mechanism [40]. Further-
M.-W. Welker et al. / Digestive and Liver Disease 42 (2010) 735–740
more, amantadine affected neither RNA replication nor the release or infectivity of HCV particles in the infectious HCV particle cell culture model [40]. A chemical analogue to amantadine, 1-boraadamataneamine, was found to bind and activate CD81 [17]. If amantadine binds to CD81, CD81 expression might change during treatment and alter HCV/cell interaction and HCV de novo infection. Furthermore, the interaction between CD81 and amantadine may alter the immune response against HCV as CD81 is an important costimulatory molecule on lymphocytes. Reduction of HCV/CD81 interaction and modulation of CD81 expression could be further antiviral mechanisms of amantadine. In the present study, we therefore investigated whether IFN-induced down regulation of CD81 may be potentiated by amantadine treatment. Our findings show, that amantadine therapy during 12 weeks of antiviral therapy with peginterferon alpha-2a and ribavirin does not alter CD81 expression on PBMC subsets. Furthermore, we did not find any correlation between HCV RNA and CD81 expression neither in the amantadine nor in the control group. Overall, our results show, that direct interaction of amantadine with CD81 is unlikely to be involved in antiviral activity of amantadine in chronic HCV infection. However, due to the limited number of patients investigated, it is unlikely but not completely excluded that a marginal influence of amantadine to CD81 expression may exist. Anyhow, the results of the present study support the concept that amantadine is not an HCV-selective antiviral. In conclusion, we have confirmed that CD81 expression is down regulated during peginterferon alpha-based antiviral therapy on CD4(+), CD8(+), and CD56(+) cells. Additional amantadine treatment does not influence CD81 expression on these PBMC subsets. Overall, amantadine is not a CD81 selective antiviral. Conflict of interest None.
List of abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; E2, envelope protein 2; GGT, gammaglutamyltransferase; HCV, hepatitis C virus; IFN, interferon alfa; PBMC(’s), peripheral blood mononuclear cell(s); RFU, relative fluorescence units; SOC, standard of care; SVR, sustained virologic response; TW, treatment week(s)
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Influence of amantadine on CD81 expression on lymphocytes in chronic hepatitis C Martin-Walter Welker a , Michael von Wagner a , Dana Ochs b , Vincent Zimmer c , Wolf Peter Hofmann a , Albrecht Piiper a , Rolf W. Hartmann d , Eva Herrmann e , Stefan Zeuzem a , Bernd Kronenberger a,∗ a
Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Kinderklinik, Vinzentius Krankenhaus, Cornichonstraße 4, 76829 Landau in der Pfalz, Germany c Klinik für Innere Medizin II, Universitätsklinikum des Saarlandes, Kirrberger Str., 66421 Homburg/Saar, Germany d Pharmazeutische und Medizinische Chemie, Universität des Saarlandes, Postfach 151150, 66041 Saarbrücken, Germany e Institut für Biostatistik und mathematische Modellierung, Fachbereich Medizin, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany b
a r t i c l e
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Article history: Received 18 September 2009 Accepted 17 March 2010 Available online 24 April 2010 Keywords: Amantadine CD81 HCV PBMC
a b s t r a c t Introduction: Interferon alpha (IFN) down regulates CD81 expression on peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis C virus (HCV) infection. Aim of our study was to investigate whether amantadine alters IFN associated down regulation of CD81 expression on PBMC in patients with chronic hepatitis C. Methods: Nineteen patients with chronic HCV infection received peginterferon alpha-2a/ribavirin (SOC) for 48 weeks. Patients were randomised to 12 weeks amantadine therapy (n = 12) or no additional treatment (n = 7). FACS analysis of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was performed at baseline, week (TW) 4, TW12, and TW24 of antiviral therapy. Results: A significant decline of CD81 expression was observed on CD4(+), CD8(+), and CD56(+) cells (p = 0.011, p < 0.001, p = 0.015, respectively) but not on CD19(+) cells (p > 0.2). CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was not different between patients treated with SOC plus amantadine and patients treated with SOC alone. Conclusion: The current study confirms that CD81 expression is down regulated by SOC on CD4(+), CD8(+) and CD56(+) cells. Amantadine treatment was not associated with CD81 expression. Interaction between amantadine and CD81 is unlikely to be involved in potential antiviral activity of amantadine in chronic HCV infection. © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis C virus (HCV) primary infects hepatocytes and is a major cause of chronic hepatitis and its sequelae, including liver cirrhosis and hepatocellular carcinoma [1,2]. Standard of care (SOC) antiviral therapy is burdened with severe side effects and leads to sustained virologic response in only 48–52% in HCV genotype 1 infected patients, the most common genotype in Western countries [3–6]. The 26-kDa cell surface protein CD81 is a tetraspanin composed of 4 transmembrane and 2 extracellular loops and is expressed on hepatocytes as well as on peripheral blood mononuclear cells (PBMC’s) [7]. Interaction of the second extracellular loop of CD81 with the HCV envelope protein 2 is an essential step in HCV cell entry [8,9]. Interferon alpha was shown to down regulate CD81
∗ Corresponding author. Tel.: +49 69 6301 87680; fax: +49 69 6301 87676. E-mail address: [email protected] (B. Kronenberger).
expression on PBMC’s in vitro, while HCV infection per se alters CD81 expression to higher levels [10]. Furthermore, CD81 expression on CD8(+) and CD56(+) PBMC’s decreases during IFN-based antiviral therapy in vivo and down regulation of CD81 is correlated with initial virologic response [10–13]. In general, CD81 is involved in cellular activation [14], adhesion [9], proliferation [15], and differentiation processes [16]. Expressed on rat astrocytes, CD81 activation has antiproliferative effects. Wagner et al. tested boraadamantaneamine derivates in a rat astrocyte proliferation assay in combination with immunoblotting [17]. Herein, 1-boraadamataneamine derivatives were found to bind and activate CD81 [17]. Amantadine (1-adamantanamine), a chemical analogue to 1boraadamataneamine, has been shown to slightly improve sustained virologic rates in patients treated with interferonbased antiviral therapy, but not when used in treatment regimes comprising pegylated interferon alpha and ribavirin [18–25]. Aim of our study was to investigate the effect of amantadine on CD81 expression during peginterferon-based antiviral therapy.
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Table 1 Demographic and baseline characteristics of the patients and treatment outcome.
Baseline characteristics Demography Gender Age Body mass index Caucasian Biochemistry ALT AST GGT Virology HCV RNA
AMA 200 mg q.d. plus SOC n=6
AMA 200 mg b.i.d. plus SOC n=6
SOC alone n=7
(Female/male) (Years, mean ± SD) (kg/m2 , mean ± SD)
4/2 41 ± 14 25.8 ± 3.2 6
5/1 52 ± 6 28.2 ± 2.3 6
5/2 52 ± 15 24.7 ± 3.1 7
(n.s.) (n.s.) (n.s.) (n.s.)
(×ULN, mean ± SD) (×ULN, mean ± SD) (×ULN, mean ± SD)
3.6 ± 2.8 1.9 ± 0.9 1.3 ± 0.9
2.3 ± 0.9 1.5 ± 0.3 1.8 ± 0.6
2.9 ± 1.5 1.6 ± 0.7 1.9 ± 1.4
(n.s.) (n.s.) (n.s.)
(log IU/mL, mean ± SD)
5.9 ± 0.4
6.1 ± 0.6
5.6 ± 0.4
(n.s.)
1/1/4
3/1/2
0/1/6
(n.s.)
Treatment outcome NR/relapse/SVR
AMA, amantadine; NR, non-response; SD, standard deviation; SOC, standard of care; SVR, sustained virologic response; ULN, upper limit of normal.
2. Patients and methods
2.4. Study endpoints
2.1. Patients and treatment
Primary endpoint of our study was CD81 expression at TW 4 and TW 12 in patients treated with amantadine plus SOC in comparison to patients with SOC only. Secondary endpoint was influence of amantadine treatment on CD81 kinetics during antiviral therapy.
Nineteen treatment naive patients with chronic hepatitis C virus genotype 1 infection were enrolled in the present study. Co-infection with hepatitis B virus or human immunodeficiency virus was an exclusion criterion. All patients received peginterferon alpha-2a 180 g once weekly and ribavirin 800–1200 mg/die according to body weight for 48 weeks. Before treatment patients were randomized to 12 weeks amantadine therapy (200 mg q.d., n = 6; 200 mg b.i.d., n = 6) or no amantadine (n = 7). All patients had given written informed consent according to the Declaration of Helsinki. The study was approved by the local Ethics Committee. 2.2. Quantitative measurement of HCV RNA and HCV genotyping Quantitative assessment of HCV RNA concentration was performed by AmplicorTM HCV Monitor HCV test version 2.0 (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 615 IU/mL. Genotyping of HCV, according to the classification of Simmonds et al. [26], was done by reverse hybridisation assay (INNO LiPA HCV-II; Innogenetics, Ghent, Belgium). Serum HCV concentration was measured at baseline, treatment weeks (TW) 4, 12, 24, 48, and 24 weeks after withdrawal of therapy. 2.3. Immunocytochemistry and FACS analysis Blood was collected in sterile ethylene-diamine-tetra-acetate filled blood collection tubes. Whole blood (100 L) was incubated with R-phycoerythrin-conjugated anti-CD81-antibodies (20 L; Clone JS-81, mouse anti-human IgG, Pharmingen, Heidelberg, Germany). For labelling of lymphocyte subsets, peripheral blood mononuclear cells were costained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (Clone RPA-T4, mouse anti-human IgG, Pharmingen), anti-CD8 (Clone HIT8a, mouse anti-human IgG, Pharmingen), anti-CD19 (Clone 4G7, mouse anti-human, Pharmingen), and anti-CD56 antibodies (Clone NCAM 16.2, mouse anti-human IgG, Pharmingen) for 30 min at 4 ◦ C. Cells were subsequently fixed and erythrocytes lysed using cell lysis buffer (10 min, 4 ◦ C). Fluorescence was analyzed with a fluorescence activated cell sorter (FACS; FACScan, Becton Dickinson, Heidelberg, Germany). FITC-, PE-conjugated mouse IgG antibodies (Pharmingen) were used as isotype controls for the quantification of background fluorescence. FACS analysis of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was performed at baseline, TW 4, and TW 12 during antiviral therapy. Fluorescence was quantified in relative fluorescence units (RFU).
2.5. Statistical analyses Chi-squared test (gender), Kruskal–Wallis test (age, BMI, GOT, GPT, GGT serum levels), and ANOVA test (log10 HCV RNA serum concentration) were applied to compare baseline characteristics of the 3 treatment groups. All tests were two-tailed and p values < 0.05 were considered significant. Clinical and biochemical characteristics of patients were expressed as mean ± standard deviation (SD). Changes of CD81 expression during treatment were tested by the Friedman Chi2 test for multiple comparisons. If Friedman Chi2 test showed a significant change of CD81 expression during treatment, CD81 expression at baseline was compared with CD81 expression at TW 4 and 12 by multiple Wilcoxon–Wilcox comparisons. Differences in CD81 expression between the different treatment arms were investigated by Kruskal–Wallis and Mann–Whitney Utest. Sample size was sufficient to guarantee a power above 80% if there is a probability of about 90% that CD81 expression in patients treated with amantadine plus SOC is less than CD81 expression in patients treated with SOC in single comparisons. 3. Results 3.1. Patients characteristics and treatment outcome All patients included in the present study were infected with hepatitis C virus genotype 1. Epidemiological, medical, and virologic characteristics are given in Table 1, as well as treatment outcome. The 3 treatment groups did not differ between gender, age, body mass index, race, as well as baseline serum ALT, AST, GGT levels or HCV RNA serum concentration and treatment outcome, respectively (Table 1). 3.2. CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells at baseline and during antiviral therapy CD81 expression on the different lymphocyte subtypes was quantified by FACS analysis (Fig. 1). Mean CD81 expression at baseline was 613 ± 131 RFU, 787 ± 237 RFU, 546 ± 118 RFU, and 527 ± 206 RFU on CD4(+), CD8(+), CD19(+), and CD56(+) lymphocytes, respectively. The changes of CD81 expression during
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Fig. 1. FACS analysis of CD81 expression on (A) CD4(+), (B) CD8(+), (C) CD19(+), and (D) CD56(+) cells after gating for lymphocytes. CD81 was labeled with PE-conjugated mouse anti-human CD81-antibodies; CD4, CD8, CD19, CD56 were labeled with respective FITC-conjugated mouse anti-human antibodies. The lower-left quadrant indicates the range of the isotype control for PE- and FITC-conjugated antibodies, respectively. Cells in the upper right quadrant were considered as double positive for CD81 and CD4/CD8/CD19/CD56 and used for quantification of CD81 expression on the different lymphocyte subtypes.
treatment are shown in Fig. 2. During treatment a significant decline of CD81 expression was observed on CD4(+), CD8(+), and CD56(+) cells (p = 0.011, p < 0.001, and p = 0.015 for all time points, respectively) but not on CD19(+) cells (p > 0.2 for all time points, Fig. 2). We next analyzed whether the lowest level of CD81 (nadir) was different between the lymphocyte subtypes. The CD81 expression nadir was similar on CD4(+), CD19(+), and CD8(+) cells (Fig. 3). On CD56(+) cells the CD81 expression nadir was lower compared with the other subtypes (p < 0.01 for all; Fig. 3). Furthermore, the decline of CD81 expression during treatment compared with baseline was analyzed. The maximum decline of CD81 expression in relation to baseline on CD8(+) and CD56(+) was greater than the maximum decline of CD81 expression on CD4(+)
cells (0.55 vs. 0.41 vs. 0.70-fold, p < 0.05 for CD4 vs. CD8 and CD4 vs. CD56). The CD81 expression level may be associated with virologic response. However, in the present study, no correlation between CD81 expression on the different lymphocyte subtypes with viral load was observed (p > 0.2 for all subtypes and time points, data not shown). The percentage of CD4(+), CD8(+), CD19(+), and CD56(+) cells at baseline was 38.8 ± 9.9%, 20.0 ± 6.2%, 7.3 ± 3.3%, and 7.6 ± 5.4%, respectively. During treatment no changes in the percentage of lymphocyte subtypes were observed (p = 0.17, p > 0.2, p = 0.07, p = 0.17 for CD4(+), CD8(+), CD19(+), CD56(+), respectively; data not shown). The percentage of lymphocyte subtypes could be correlated with CD81 expression. In the present study, however, no correlation between CD81 expression and the percentage of lymphocyte subtypes was observed (p > 0.05 for all; data not shown).
3.3. Association of amantadine treatment with CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells
Fig. 2. Dynamics of CD81 expression on the different lymphocyte subtypes for all patients during the first 12 weeks of antiviral therapy. Symbols indicate lymphocyte subtypes (), CD4(+) cells; (), CD8(+) cells; (), CD19(+) cells; (×), CD56(+) cells. Significant differences in CD81 expression during treatment compared with baseline are indicated, * for p < 0.05, **for p < 0.01.
CD81 expression during treatment was compared between the amantadine and the control group. During treatment CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was not different between patients treated with SOC plus amantadine and patients treated with SOC alone (p > 0.05 for all subtypes and TW 4 and 12; data no shown). The comparison between baseline CD81 and the CD81 expression nadir is shown in Fig. 3a–d. On all lymphocyte subgroups a significant reduction between CD81 expression at baseline and the CD81 expression nadir was observed in the amantadine and the control group (Fig. 3). However, no difference was noted between the CD81 expression nadir between the amantadine and the control group. Furthermore, we observed no difference in CD81
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Fig. 3. Bar graph of baseline (BL) and nadir CD81 expression for amantadine plus SOC treated patients (AMA) and only SOC treated patients (SOC) for (A) CD4(+) cells, (B) CD8(+) cells, (C) CD19(+) cells, and (D) CD56(+) cells. Although a decrease of CD81 expression occurred in each lymphocyte subset population, no differences between AMA and SOC groups were observed. RFU, relative fluorescence units.
expression on the different cell types between the end of amantadine treatment (TW12) and after amantadine treatment (TW24; data not shown). The decline of CD81 during treatment was also compared between the amantadine and the control group. However, no difference in CD81 decline during treatment was observed between the amantadine and the control group (p > 0.05 for all time points and subtypes; data not shown). The percentage of CD4(+), CD8(+), CD19(+), and CD56(+) lymphocytes was not different between the amantadine and the control group (p > 0.2 for all; data not shown). Neither in the amantadine nor in the control group a significant correlation between CD81 expression and the percentage of lymphocyte subtypes was observed (p > 0.1; data not shown). 4. Discussion HCV primarily infects hepatocytes, but genomic and antigenomic HCV RNA was detected in PBMC, – mainly CD8(+) and CD19(+) cells – , also, suggesting putative lymphotropism of HCV [27–31]. Lymphotropism of HCV may be responsible for impaired, specific immune response and development of autoimmune diseases in patients with chronic hepatitis C [32–35]. We have shown previously, that CD81 expression is down regulated on CD8(+) and CD56(+) PBMC’s by interferon alpha in vitro and in vivo [10,36]. Down regulation of CD81 expression on lymphocyte subtypes during antiviral treatment could reduce the interaction between HCV and lymphocytes and could restore HCV associated immune dysfunction or reduce de novo infection [33]. Thus, down regulation of CD81 could be an antiviral mechanism. This may have consequences for immune function and potential HCV infection. Indeed, previous studies showed an association of CD81 expression on CD8(+) PBMC’s with antiviral response. In the current
study, we confirmed down regulation of CD81 by interferon alphabased therapy as a significant decrease in CD81 expression was shown on CD4(+), CD8(+), and CD56(+) cells during antiviral therapy. However, in the present study we observed no association between virologic response and CD81 expression. In previous studies, the difference in CD81 expression between patients who a achieved a sustained virologic response and those without sustained virologic response was found after treatment [13,36]. In the present study, CD81 expression was measured after 4 and 12 weeks because amantadine treatment was performed for the first 12 weeks only. These time points may be too early to detect a significant association between CD81 expression and virologic response. Nevertheless, as early down regulation of CD81 expression may be merely drug induced [10,36], it can be anticipated that a potential amantadine effect on CD81 should be detectable within the 12 weeks amantadine was administered. Amantadine therapy has been shown to increase sustained virologic rates to a minor extent in combination with standard interferon alpha and ribavirin, but not in patients treated with peginterferon alpha and ribavirin [18–25]. The weak potential therapeutic mechanism of amantadine therapy in patients with chronic HCV infection and interferon/ribavirin therapy is not clarified, so far. It was shown that amantadine may interfere with the HCV p7 ion channel and it was assumed that the interference of amantadine with HCV p7 may be an antiviral mechanism [37]. Substitution of L20F within HCV p7 was associated with non-response to combination therapy specifically with amantadine in HCV genotype 1b infected patients [38]. The potential antiviral activity of amantadine on the HCV p7 ion channel was further explored using a system for production of infectious HCV particles in cell culture [39,40]. In this model, however, the HCV p7 ion channel activity was not affected by amantadine suggesting that interference of amantadine with HCV p7 is not a relevant antiviral mechanism [40]. Further-
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more, amantadine affected neither RNA replication nor the release or infectivity of HCV particles in the infectious HCV particle cell culture model [40]. A chemical analogue to amantadine, 1-boraadamataneamine, was found to bind and activate CD81 [17]. If amantadine binds to CD81, CD81 expression might change during treatment and alter HCV/cell interaction and HCV de novo infection. Furthermore, the interaction between CD81 and amantadine may alter the immune response against HCV as CD81 is an important costimulatory molecule on lymphocytes. Reduction of HCV/CD81 interaction and modulation of CD81 expression could be further antiviral mechanisms of amantadine. In the present study, we therefore investigated whether IFN-induced down regulation of CD81 may be potentiated by amantadine treatment. Our findings show, that amantadine therapy during 12 weeks of antiviral therapy with peginterferon alpha-2a and ribavirin does not alter CD81 expression on PBMC subsets. Furthermore, we did not find any correlation between HCV RNA and CD81 expression neither in the amantadine nor in the control group. Overall, our results show, that direct interaction of amantadine with CD81 is unlikely to be involved in antiviral activity of amantadine in chronic HCV infection. However, due to the limited number of patients investigated, it is unlikely but not completely excluded that a marginal influence of amantadine to CD81 expression may exist. Anyhow, the results of the present study support the concept that amantadine is not an HCV-selective antiviral. In conclusion, we have confirmed that CD81 expression is down regulated during peginterferon alpha-based antiviral therapy on CD4(+), CD8(+), and CD56(+) cells. Additional amantadine treatment does not influence CD81 expression on these PBMC subsets. Overall, amantadine is not a CD81 selective antiviral. Conflict of interest None.
List of abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; E2, envelope protein 2; GGT, gammaglutamyltransferase; HCV, hepatitis C virus; IFN, interferon alfa; PBMC(’s), peripheral blood mononuclear cell(s); RFU, relative fluorescence units; SOC, standard of care; SVR, sustained virologic response; TW, treatment week(s)
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