Journal of Viral Hepatitis, 2014, 21, 633–641

doi:10.1111/jvh.12189

Hepatitis B virus-specific T-cell responses during IFN administration in a small cohort of chronic hepatitis B patients under nucleos(t)ide analogue treatment M. F. Sprinzl,1,2 C. Russo,1,3 J. Kittner,2 S. Allgayer,1,3 A. Grambihler,2 B. Bartsch,2 A. Weinmann,2 P. R. Galle,2 M. Schuchmann,2 U. Protzer1,3 and T. Bauer1,3 1Institute of Virology, Technische Universita¨t Mu¨nchen / Helmholtz Zentrum Mu¨nchen, Munich, Germany; 2Department of Medicine, Johannes Gutenberg University, unchen / Helmholtz Zentrum M€ unchen, German Mainz, Germany; and 3Clinical Cooperation Group Immune Monitoring, Technische Universit€at M€ Research Center for Environmental Health, Neuerberg, Germany Received April 2013; accepted for publication August 2013

SUMMARY. The effect of pegylated interferon-a (IFN) add-on

therapy on HBV-specific T-cell responses was evaluated in 12 patients with stable, undetectable hepatitis B virus (HBV) load under nucleos(t)ide analogue therapy. Peripheral blood mononuclear cells were isolated at week 0, 4, 8, 12, 24 and 48 of IFN add-on therapy. Quantity and quality of circulating HBV S- and core-specific CD4 and CD8 T cells were analysed ex vivo by flow cytometry. HBV S- and core-specific CD4 T-cell numbers modestly increased within 8 weeks of IFN administration (P = 0.0391 and P = 0.0195), whereas HBV-specific CD8 T cells in general showed only minor changes under IFN add-on therapy. Functionality of HBV-specific CD4 but not CD8 T cells positively correlated with serum transaminase activity. In addition, we observed an increase in CD4 T cells producing tumour necrosis factor-a (TNFa) without antigen restimu-

INTRODUCTION Antiviral nucleos(t)ide analogues prevent progression of chronic hepatitis B virus (HBV) infection but their longterm administration still remains a clinical and economic Abbreviations: HBsAg, hepatitis B surface antigen; IFN, pegylated interferon-a-2a; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; TNFa, tumour necrosis factor-a; PBMCs, peripheral blood mononuclear cells; IFNc, interferon-c; IL2, interleukin-2; MIP-1b, macrophage inflammatory protein-1b; TW, week of treatment; CD4TNF-mono, monofunctional TNFa-positive CD4 T cells; ALT, alanine transaminase; AST, aspartate transaminase; cGT, c-glutamyl-transferase; AP, alkaline phosphatase. Correspondence: Dr Martin F. Sprinzl, Institute of Virology, Technische Universita¨t Mu¨nchen / Helmholtz Zentrum Mu¨nchen, Trogerstrasse 30, 81675 Mu¨nchen, Germany. E-mail: [email protected]

© 2013 John Wiley & Sons Ltd

lation (P = 0.0039), which correlated with elevated transaminases. During IFN add-on therapy, two patients developed an anti-HBs seroconversion, only one of whom showed a relevant increase in HBV-specific T cells. In conclusion, IFN add-on therapy of chronic hepatitis B increased HBV-specific T-cell responses and affected a previously unrecognized TNFa-monofunctional CD4 T-cell population. Although the observed T-cell responses did not correlate with HBsAg seroconversion, we expect additional insights into the immunopathogenesis of hepatitis B, following the characterization of the newly identified TNF amonofunctional T-cell population. Keywords: hepatitis B surface antigen, immune response, interferon, seroconversion, tumour necrosis factor.

draw back [1–3]. Therefore, induction of immunological control by stable HBV surface antigen (HBsAg) seroconversion is a desired treatment endpoint [4]. Unfortunately, immune control is rarely achieved by HBV suppression with nucleos(t)ide analogues or by immune-stimulatory interferon-a (IFN) treatment alone. The addition of IFN to a stable nucleos(t)ide pretreatment (IFN add-on), though, showed promising HBsAg seroconversion rates of up to 16% [5]. Current reports indicate that second-generation nucleos (t)ide analogues and IFN restore immune function in patients developing HBsAg loss [6]. These immune-stimulation affected natural killer cells [7], antigen-presenting cells [8] and reduced HBV-specific T-cell exhaustion [9]. By now, information is lacking about the polyfunctional properties of HBV-specific CD4 and CD8 T-cell responses, which execute HBV clearance during antiviral treatment

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[10]. Aiming to identify mono- and polyfunctional T-cell populations during IFN add-on treatment of chronic hepatitis B, a T-cell assay was established, covering a wide range of HBV-specific responses.

PATIENTS AND METHODS

HBV-DNA was quantified using COBAS AmpliPrep/ COBAS TaqMan (Roche Diagnostics, Mannheim, Germany). Serum HBsAg levels were quantified using the ARCHITECTâ immunoassay (Abbott Diagnostics, Abbott Park, IL, USA). HBV serology and serum transaminase measurements were performed as published [5].

Patients, treatment and virological markers

Peptides

Patients (n = 12) under ongoing antiviral nucleos(t)ide therapy received additional pegylated interferon-a-2a (Pegasysâ; Roche, Basel, Switzerland) subcutaneously once weekly in a standard dose, consecutively named ‘add-on’ therapy. IFN add-on therapy was withdrawn in seven patients without HBsAg reduction after 12 weeks of treatment. In three patients, IFN add-on therapy was stopped due to therapy-related adverse events. Patients displaying HBsAg seroconversion received IFN add-on therapy for 48 weeks as recently described [5]. A comprehensive list of patient variables is provided in Table 1 and Table S1. HBV-vaccinated volunteers (n = 12), seronegative for HBV infection, served as controls. Therapy, data and blood sample acquisition were performed after patient’s informed consent, following approval of the local institutional ethics committee in accordance with the 1975 Declaration of Helsinki.

Pools covering the HBV S and core antigens (Genotype D, GenBank accession number AF121241) were generated from 15-mer peptides (>80% purity) overlapping by 11 residues (IBA, Goettingen, Germany). An influenza-, Epstein-Barrand cytomegalovirus-derived control peptide pool was obtained from the Centre for AIDS Reagents, NIBSC HPA UK, supported by the EC FP6/7 Europrise Network of Excellence, and NGIN consortia and the Bill and Melinda Gates GHRC-CAVD Project.

Sample preparation, in vitro stimulation and cell staining PBMCs were separated by Ficoll gradient within 8 h after blood acquisition and were cryopreserved in foetal calf serum (FCS, Life Technologies, Darmstadt, Germany) containing 10% DMSO (Sigma-Aldrich, Munich, Germany).

Table 1 Patient baseline characteristics at the time of treatment initiation Patient (Nr.)

Age (Years)

Sex (m/f)

HBsAg (IU/mL)

HBeAg

HBV Genotype

IFN Pretreatment

Nucleos(t)ide Treatment

Anti-HBs (Seroconversion)

1 2 3 4 5 6 7 8 9 10 11 12

56 50 54 30 25 29 44 37 54 49 45 60

m m m f f m m m m m m f

1638 8757 3106 7459 15120 16 129 1426 11 973 2740 1487 2489

Neg Neg Neg Neg Pos Neg Neg Neg Neg Neg Pos Pos

A n.d. n.d. n.d. n.d. D n.d. n.d. n.d. n.d. D A

No No No Yes Yes No Yes Yes No Yes Yes Yes

Lamivudine Lamivudine, Adefovir Lamivudine, Adefovir Entecavir Entecavir Entecavir Entecavir Entecavir Entecavir Entecavir, Adefovir Entecavir, Tenofovir Entecavir, Tenofovir

No No No No No Yes No No No No No Yes

HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; IFN, interferon-a; n.d., not determined.

Fig. 1 HBV-specific T-cell responses before and during IFN add-on therapy. CD4 and CD8 T-cell responses after ex vivo restimulation with HBV S- or core-derived peptide pools. T cells expressing at least one functional marker (IFNc, TNFa, IL2 and MIP1b) are shown as frequencies (%) of total CD4 or CD8 T cells, respectively (a). Monofunctional and polyfunctional (≥2 functions) T-cell responses are displayed as described above (b). Pie charts indicate functions of total HBV S- and corespecific CD4 or CD8 T-cell responses. Each slice corresponds to the proportion of T cells positive for a certain combination of functions. Individual marker expression is depicted by the coloured arcs. Colour codes defining the number (slices) and type (arcs) of T-cell functions are indicated in the figure legend (c, d) (*P < 0.05, Wilcoxon-matched pairs test) ns: not significant. © 2013 John Wiley & Sons Ltd

Hepatitis B virus-specific T-cell responses (a)

(b)

(c)

(d)

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(a)

(b)

(c)

(d)

© 2013 John Wiley & Sons Ltd

Hepatitis B virus-specific T-cell responses Cryopreserved PBMCs were thawed and rested (18 h at 37 °C with 5% CO2) in culture medium [RPMI 1640 (Cambrex, Taufkirchen, Germany) supplemented with 10% FCS (Biochrom AG, Berlin, Germany) and 1% Pen/Strep (Cambrex)]. After resting, PBMCs were stimulated in the presence of 2 lg/mL peptide pool and costimulatory antiCD28/anti-CD49d antibodies (BD Biosciences, Heidelberg, Germany). Peptide- and mock-stimulated samples were run in parallel to define the background for every single patient. Brefeldin A (Sigma-Aldrich, Munich, Germany) was added after 1 h during a 5-h stimulation period. Cells were labelled with the live/dead discriminator (NIR; Life technologies, Darmstadt, Germany), fixed, permeabilized (BD Cytofix/CytopermTM Kit, BD Biosciences, Heidelberg, Germany) and stained. Stained cells (CD3, CD4, CD8, CD45RA, CD154, IFNc, TNFa, IL2-APC and MIP1b) were analysed using an LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR, USA). Detailed protocol, gating strategy and representative scatter blots are provided in the supplementary material and methods (Fig. S1, S2).

Data processing and statistical analysis The expression threshold level of functional markers (IFNc, TNFa, IL2 and MIP1b) was calculated after background subtraction using the 90th percentile of negative values for each subpopulation. Values below the respective threshold level were set to 0, and all values >0 were considered as a positive response. Total antigen-specific T cells represented the cumulative frequency of each T-cell subpopulation expressing at least one functional marker. Value changes and correlations were assessed by Wilcoxon-signed rank test or Spearman’s correlation tests, respectively. Significance was tested at a confidence interval of 95%. SPICE version 4.1.5 (Mario Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health) and Prism version 5.01 (GraphPad Software Inc., San Diego, CA, USA) were used for graphical and statistical analysis.

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RESULTS Clinical outcome of patients receiving IFN add-on therapy Patients with chronic hepatitis B showing an undetectable viral load (

Hepatitis B virus-specific T-cell responses during IFN administration in a small cohort of chronic hepatitis B patients under nucleos(t)ide analogue treatment.

The effect of pegylated interferon-α (IFN) add-on therapy on HBV-specific T-cell responses was evaluated in 12 patients with stable, undetectable hepa...
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