Gastroenterology 2019;156:1820–1833 Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8D T Cells and Evolve at the Population Level Hadi Karimzadeh,1,2,20,* Muthamia M. Kiraithe,3,* Valerie Oberhardt,3,4,* Elahe Salimi Alizei,3,5 Jan Bockmann,6,7 Julian Schulze zur Wiesch,6,7 Bettina Budeus,8 Daniel Hoffmann,8 Heiner Wedemeyer,7,9,10 Markus Cornberg,7,9 Adalbert Krawczyk,2,11 Jassin Rashidi-Alavijeh,10 Francisco Rodríguez-Frías,12 Rosario Casillas,12 Maria Buti,12 Antonina Smedile,13 Seyed Moayed Alavian,14 Andreas Heinold,15 Florian Emmerich,16 Marcus Panning,17 Emma Gostick,18 David A. Price,18 Jörg Timm,19 Maike Hofmann,3 Bijan Raziorrouh,20 Robert Thimme,3 Ulrike Protzer,1,7 Michael Roggendorf,1,2,7,§ and Christoph Neumann-Haefelin3,§ 1 Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany; 2 Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany; 3Department of Medicine II, University Hospital Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 4Faculty of Biology, University of Freiburg, Freiburg, Germany; 5Faculty of Chemistry and Pharmacy, University of Freiburg, Freiburg, Germany; 6Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 7German Center for Infection Research (DZIF), Sites Hamburg-Lübeck-Borstel-Riems, Hannover-Braunschweig and Munich, Germany; 8Department of Bioinformatics, University of Duisburg-Essen, Essen, Germany; 9Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 10Department of Gastroenterology and Hepatology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany; 11Department of Infectious Diseases, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany; 12CIBERehd and Departments of Biochemistry/Microbiology and Hepatology, Vall d’Hebron Hospital, University Autónoma de Barcelona (UAB), Barcelona, Spain; 13Department of Medical Sciences, University of Turin, Turin, Italy; 14Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran; 15Institute of Transfusion Medicine, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany; 16Institute for Transfusion Medicine and Gene Therapy, University Hospital Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 17Institute of Virology, University Hospital Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 18Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom; 19 Institute of Virology, Heinrich-Heine-University, University Hospital, Duesseldorf, Germany; and 20Department of Internal TRANSLATIONAL LIVER Medicine II, University Hospital Munich-Grosshadern, Munich, Germany BASIC AND BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection virus, little is known about which HDV epitopes are targeted by in patients with hepatitis B virus (HBV) is associated with rapid virus-specific CD8þ T cells or why these cells ultimately fail to progression to liver cirrhosis and hepatocellular carcinoma. control the infection. We aimed to define how HDV escapes the Treatment options are limited, and no vaccine is available. CD8þ T-cell–mediated response. METHODS: We collected Although HDV-specific CD8þ T cells are thought to control the plasma and DNA samples from 104 patients with chronic HDV May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1821 and HBV infection at medical centers in Europe and the Middle WHAT YOU NEED TO KNOW East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in BACKGROUND AND CONTEXT HDV RNA associated with specific HLA class I alleles. We pre- HDV infection is associated with severe liver disease, but dicted epitopes in HDV that would be recognized by CD8þ T little is known about the corresponding virus-specific cells and corresponded with the identified virus poly- CD8þ T cells, which are thought to mediate immune morphisms in patients with resolved (n ¼ 12) or chronic (n ¼ protection against HDV. 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class NEW FINDINGS I alleles (P < .005). Five of these polymorphisms were found to In persistent infection, HDV mutates to escape from the correspond to epitopes in HDV that are recognized by CD8þ T virus-specific CD8þ T cell response. At the population- cells; we confirmed that CD8þ T cells in culture targeted these level, HDV has evolved to evade from recognition in the HDV epitopes. HDV variant peptides were only partially cross- context of common HLA class I alleles. recognized by CD8þ T cells isolated from patients, indicating LIMITATIONS that the virus had escaped detection by these cells. These newly The observational nature of this study precludes formal identified HDV epitopes were restricted by relatively infrequent identification of a causal association between viral HLA class I alleles, and they bound most frequently to HLA-B. In escape and CD8þ T-cell failure in the context of contrast, frequent HLA class I alleles were not associated with infection with HDV. HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present IMPACT epitope peptides to CD8þ T cells in patients with persistent HDV These results suggest that viral escape should be infection. We identified polymorphisms in the HDV proteome considered as a key parameter in the design of immune- that associate with HLA class I alleles. Some variant peptides in based interventions to prevent and treat liver disease epitopes from HDV were only partially recognized by CD8þ T associated with HDV. cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated between these 2 proteins is a 19–amino acid extension at the with uncommon HLA class I alleles, indicating that HDV evolves, C-terminus of L-HDAg. An initial report described 2 HDV- at the population level, to evade recognition by common HLA specific CD8þ T-cell epitopes in human leukocyte antigen class I alleles. (HLA)-A*02 transgenic mice and patients with resolved HDV infection, but these findings were not confirmed in patients with persistent HDV infection.4 A subsequent report sug- Keywords: Cytotoxic T Cell; MHC Class I; TCR; Antigen gested that immune exhaustion may impair the efficacy of Presentation. HDV-specific CD8þ T cells in vivo.5 In a recent study, we identified 2 HLA-B*27–restricted HDV-specific CD8þ T-cell TRANSLATIONAL LIVER epitopes, both of which were subject to viral escape by mu- BASIC AND tation.6 Of note, HLA-B*27 is associated with spontaneous H epatitis D virus (HDV) has infected approximately 10% of hepatitis B virus (HBV)–seropositive individuals, affecting 15–20 million people worldwide.1 clearance of hepatitis C virus (HCV) and elite control of hu- man immunodeficiency virus (HIV), and HLA-B*27– Patients with simultaneous HDV/HBV infection often expe- restricted CD8þ T-cell responses drive viral evolution in rience severe acute hepatitis, with an enhanced risk of patients infected with HCV or HIV.7 It nonetheless remains fulminant disease, but frequently clear both viruses. In unclear if viral escape is a generalizable phenomenon that contrast, HBV-seropositive patients who become super- underlies immune failure in patients infected with HDV. infected with HDV generally develop chronic HDV/HBV In the present study, we analyzed HLA class I–associated infection. These patients have a high risk of rapid progres- viral sequence polymorphisms across the entire HDV sion to liver cirrhosis and hepatocellular carcinoma. At genome as surrogate markers of CD8þ T-cell–driven viral present, the sole treatment option for chronic HDV/HBV infection is pegylated interferon (IFN) alfa, which is usually *Authors share co-first authorship; § Authors share co-senior authorship. administered for 48 weeks. However, only approximately 30% of patients respond to treatment, and many subse- Abbreviations used in this paper: HDAg, hepatitis delta antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HIV, human quently develop viral relapse.2 immunodeficiency virus; HLA, human leukocyte antigen; IC50, half Virus-specific CD8þ T cells are thought to play a key role maximal inhibitory concentration; IFN, interferon; IL-2, interleukin 2; L- in the outcome of HDV/HBV infection. This contention has HDAg, large variant of the hepatitis delta antigen; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PCR, polymerase been supported by vaccination studies in mice and wood- chain reaction; PD-1, programmed cell death protein 1; S-HDAg, small chucks.3 However, little is known about naturally occurring variant of the hepatitis delta antigen; TCF1, T-cell factor 1. HDV-specific CD8þ T-cell responses, despite the urgent Most current article need for novel prophylactic and therapeutic interventions. © 2019 by the AGA Institute. Published by Elsevier Inc. This is an open The HDV genome encodes a single viral protein, hepatitis access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). delta antigen (HDAg), translated as large and small variants 0016-5085 (L-HDAg and S-HDAg, respectively). The only difference https://doi.org/10.1053/j.gastro.2019.02.003 1822 Karimzadeh et al Gastroenterology Vol. 156, No. 6 escape. These “HLA footprints” were used to identify CD8þ Peptides, Antibodies, and Tetramers T-cell epitopes, confirm predicted escape mutations, and Peptides were synthesized with a free amine NH2 terminus explore features of the host–virus interactome, providing and a free acid COOH terminus with standard Fmoc chemistry mechanistic insights into adaptive immunity against HDV. (Genaxxon Bioscience, Ulm, Germany). Anti-CD8 (RPA-T8, 1:100; SK1, 1:300), anti-CD38 (Hb7, 1:200), anti-CD45RA (HI100, 1:50), anti-IFN-g (4S.B3, 1:50), and anti–programmed cell death protein Materials and Methods 1 (PD-1) (EH12.1, 1:33) were purchased from BD Biosciences (Germany). Anti-Bcl-2 (Bcl-2/100, 1:200), anti-CCR7 (G043H7, Patients and Samples 1:33), anti-CD8 (RPA-T8, 1:400), and anti-CD127 (A019D5, 1:33) Patients with chronic HDV/HBV infection (n ¼ 104) were were purchased from BioLegend (San Diego, CA). Anti-CD14 recruited from 8 medical centers located in Germany (Bonn, (61D3, 1:100), anti-CD19 (HIB19, 1:100), anti-Eomes (WD1928, Düsseldorf, Essen, Hannover, and Munich), Spain, Italy, and Iran.6 1:50), anti-killer cell lectin-like receptor G1 (KLRG1) (13F12F2, Viral sequences and HLA class I genotypes were determined 1:33), and anti-T-bet (4B10, 1:200) were purchased from Thermo according to standard protocols.6 CD8þ T-cell assays were per- Scientific. Anti-T-cell factor (1TCF1) (C63D9, 1:100) was pur- formed with additional samples obtained from 12 patients with chased from Cell Signaling Technology (Danvers, MA). Tetrameric resolved HDV infection and 13 patients with chronic HDV/HBV complexes of HLA-B*15:01/L-HDAg170–179 SMQGVPESPF were infection, recruited from Freiburg, Hamburg, and Munich (Ger- generated as described previously.8 many). All patients were infected with HDV genotype 1. Patient characteristics are summarized in Supplementary Tables 1 and 2. Ethical approval was granted by the Ethik-Kommission der Analysis of HDV Sequences and HLA Class Albert-Ludwigs-Universität Freiburg (no. 369/15). Written I-Associated Viral Polymorphisms informed consent was obtained in all cases according to federal Total RNA was extracted from serum samples and reverse guidelines and the Declaration of Helsinki. Venous blood samples transcribed into cDNA with Moloney Murine Leukemia (50 mL per draw) were collected in EDTA-anticoagulated tubes. Virus Reverse Transcriptase (Promega, Madison, WI) and the Peripheral blood mononuclear cells (PBMCs) were isolated HDV-specific primer 771R (50 -CGGTCCCCTCGGAATGTTG-30 ). with lymphocyte separation medium density gradients (Pancoll The L-HDAg-encoding region was amplified from cDNA separation medium, PAN Biotech GmbH; Aidenbach, Germany) with Pfu DNA Polymerase (Promega) in a 2-step nested poly- and resuspended in RPMI 1640 medium supplemented with merase chain reaction (PCR) incorporating the HDV-specific 10% fetal calf serum, 1% penicillin/streptomycin, and 1.5% primers 891-F (50 -AGGTCGGACCGCGAGGAGGT-30 ), 339-R HEPES buffer 1 mol/L (complete medium; all additives from (50 -GCTGAAGGGGTCCTCTGGAGGTG-30 ), 912-F (50 -GAGATGC Thermo Scientific (Waltham, MA). CATGCCGACCCGAAGAG-30 ), and 1674-R (50 -AGAAAAGAGTAA Table 1.HLA Class I–Associated Viral Sequence Polymorphisms and HDV-Specific CD8þ T Cell Epitopes TRANSLATIONAL LIVER AA sequence Positive responses/ No. HLA AA position Mutation P value epitope candidate AA range number of patients tested BASIC AND 1 B*15 170 S170N 2.9 3 10–8 SMQGVPESPF 170–179 10/14 2 B*13 33 D33E .0001 DLRKVKKKI 33–41 0/5 3 B*37 101 D101E .0002 QDHRRRKAL 100–108 1/1 4 A*29 63 K63R .0002 — — — 5 A*30 47 D47E .0010 KVKKKIKK 36–43 0/3 6 B*37 89 P89T/I .0011 VDSGPRKRPL 81–90 1/1 7 B*49 37 V37A/T .0015 EELERDLRKV 28–37 ND 8 B*13 100 Q100K .0018 RQDHRRRKAL 99–108 0/5 9 B*51 81 V81I .0019 — — — 10 B*13 43 K43R .0021 DLRKVKKKI 33–41 0/5 11 B*41 158 G158A/D/M .0023 — — — 12 B*18 47 E47D .0027 DENPWLGNI 46–54 1/5 13 A*33 37 V37T/A .0028 EELERDLR 28–35 ND 14 B*14 107 A107T .0028 DHRRRKAL 101–108 0/1 15 A*30 49 P49L/S .0031 KVKKKIKK 36–43 0/3 16 B*41 139 R139K .0034 RERRVAGPPV 140–149 1/1 17 B*13 96 D96E .0035 RQDHRRRKAL 99–108 0/5 18 B*27 105 R105K .0039 RRKALENKK 104–112 Previously described6 RRDHRRRKAL 99–108 Previously described6 19 B*38 131 K131G .0039 — — — 20 B*13 113 K113R .0043 RQDHRRRKAL 99–108 0/5 KQLSAGGKNL 113–122 0/5 21 A*68 134 T134A .0045 LTEEDERR 133–140 0/5 NOTE. Confirmed epitopes are displayed in bold. AA, amino acid; ND, not done. May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1823 Figure 1. HLA-B*15–restricted CD8þ T-cell responses specific for L-HDAg170–179 drive viral escape. (A) P values for the as- TRANSLATIONAL LIVER sociations between HDV sequence polymorphisms and the presence of HLA-B*15, plotted for each amino acid residue in the L-HDAg protein. The cutoff for significance was set at P ¼ .005 (dotted red line). (B) Predicted HLA-B*15 binding affinity of BASIC AND candidate peptide epitopes in L-HDAg. IC50 values were predicted for 8mers, 9mers, and 10mers with the ANN 3.4 method (www.iedb.org). The best hit corresponding to the confirmed HLA-B*15–restricted epitope L-HDAg170–179 is highlighted in green. (C) Viral sequences from individual HLA-B*15þ and HLA-B*15– patients compared with the consensus sequence derived from all 104 patients (all HLA-B*15þ patients are shown above the line, and the first 20 HLA-B*15– patients are shown below the line). Dots indicate agreement with consensus, and single-letter amino acid codes indicate variation from consensus. The HLA-B*15–associated polymorphism S170N is highlighted in dark grey, and the confirmed HLA-B*15– restricted epitope L-HDAg170–179 is highlighted in grey. (D) PBMCs from HLA-B*15þ patient 13 (2 years after IFN treatment) were expanded in the presence of the wild-type or variant peptide corresponding to the L-HDAg170–179 epitope. After 14 days, cells were restimulated in parallel with each peptide and tested for IFN gamma production. Negative controls without peptide restimulation and positive controls stimulated with phorbol myristate acetate/ionomycin are also shown. (E) Equivalent results after restimulation with each peptide in serial dilution assays. PBMCs from patient 13 (2 and 7 years after IFN treatment) and HLA-B*15þ patient 21 were processed and tested as in D. M, mol/L; P, patient; PMA, phorbol myristate acetate. GAGYACTGAGG-30 ). The following thermal profile was used for determine significant associations in a 2  2 contingency table all PCRs: (i) 94 C for 10 minutes; (ii) 94 C for 30 seconds, 54 C format with counts for observed combinations of amino acids for 45 seconds, and 72 C for 90 seconds (35 cycles); and (iii) and HLA class I allotypes at each alignment position. Further 72 C for 7 minutes. PCR products were purified with a QIA- analyses were based on a cutoff P value of <.005. quick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced on an ABI 3730xl DNA Analyzer (Eurofins Genomics Germany GmbH; Ebersberg, Germany) with the internal Prediction of HDV-Specific CD8þ T-Cell Epitopes primers 912-F and 1674-R. All sequences were submitted to Viral amino acid sequences, 15 residues N-terminal and 15 GenBank (accession nos. MF175257–MF175360). HDV se- residues C-terminal, of the identified HLA class I–associated quences were aligned with ClustalX2 software.9 HLA class I viral sequence polymorphisms were analyzed for the corre- associations with amino acid residues at each alignment posi- sponding binding motifs with 4 online prediction tools: ANN 3.4 tion were tested using the R package SeqFeatR (https:// and netMHCpan 2.8 on the Immune Epitope Database seqfeatr.zmb.uni-due.de).10 The Fisher exact test was used to website,11,12 SYFPEITHI,13 and BIMAS.14 The 8mer, 9mer, and 1824 Karimzadeh et al Gastroenterology Vol. 156, No. 6 Figure 2. Viral sequence polymorphisms are associated with infrequent HLA class I alleles and spare the N- and C-termini of L- HDAg. (A) Distribution of identified HLA footprints across L-HDAg. Red arrows indicate HLA footprints corresponding to confirmed (predescribed or newly identified) epitopes. Filled black arrows indicate HLA footprints corresponding to predicted epitopes. Empty black arrows indicate HLA footprints without defined or predicted epitopes. Newly identified epitopes are shown as red bars, and pre-described epitopes are shown as blue bars. (B) HLA-A and HLA-B allele frequencies in the study cohort compared with reference populations from Germany, Italy, and Iran. HLA class I alleles identified in at least 2 patients in the study cohort are shown (HLA class I alleles present in 1 patient cannot reach statistical power for the identification of HLA class I–associated viral sequence polymorphisms). Footprint-linked HLA class I alleles are shown in bold and marked with an arrowhead. TRANSLATIONAL LIVER BASIC AND 10mer peptides were tested if available via the respective and anti-CD8, fixed/permeabilized with Cytofix/Cytoperm, prediction tool. A half maximal inhibitory concentration (IC50) and stained with anti-IFN gamma (all reagents from BD Bio- of 1000 nmol/L, a SYFPEITHI score of 20, and a BIMAS sciences). Stained cells were fixed in phosphate-buffered sa- score of 20 were used as cutoffs. Candidate epitopes were line (PBS) containing 2% paraformaldehyde. Data were ranked against epitopes predicted across the entire sequence of acquired with an BD FACSCanto II flow cytometer (BD Bio- L-HDAg. sciences) and analyzed with FlowJo software, version 10 (FlowJo, Ashland, OR). Peptide-Specific CD8þ T-Cell Lines PBMCs were activated with peptides, as described previ- HLA Class I Tetramer-Based Analysis and Cell ously.15 Briefly, 4  106 PBMCs were stimulated once with 10 Enrichment mg/mL peptide and 0.5 mg/mL anti-CD28 (BD Biosciences) and Tetramer staining procedures were carried out as described fed every 3 days with complete medium containing 20 U/mL previously.17 Briefly, 1  106 PBMCs per well were incubated recombinant interleukin (IL)-2 (Miltenyi Biotec, Bergisch in a 96-well plate with the relevant HLA class I tetramer for 15 Gladbach, Germany). Peptide-specific CD8þ T-cell lines were minutes at 37 C. Cells were then washed 3 times with PBS used for experimental purposes after 14 days. containing 1% fetal calf serum and stained with the indicated surface and/or intracellular antibodies. Dead cells were excluded from the analysis with the fixable viability dye Intracellular IFN Gamma Staining eFluor780 (1:5000, eBioscience, Germany). Cytoplasmic and Procedures were carried out as described previously.16 nuclear molecules were shown with a FoxP3/Transcription Briefly, expanded CD8þ T cells or peptide-specific CD8þ T-cell Factor Staining Buffer Set (eBioscience). Stained cells were lines (0.2  106 cells per well in a 96-well plate) were stim- fixed in PBS containing 2% paraformaldehyde. Tetramer-based ulated with peptides (10 mg/mL) in the presence of 50 U/mL enrichment was performed as described by Alanio et al.18 recombinant IL-2 and 1 mL/mL brefeldin A (BD Biosciences). Briefly, 10–15  106 PBMCs were labeled for 30 minutes at After 5 hours, cells were stained with 7-aminoactinomycin D room temperature with the HLA-B*15:01/L-HDAg170–179 May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1825 TRANSLATIONAL LIVER Figure 3. HLA-B*18–restricted CD8þ T-cell responses specific for L-HDAg46–54 drive viral escape and population-level evo- lution of HDV. (A–E) Details as per the corresponding panels in Figure 1. In this case, the ancestral peptide is denoted as the BASIC AND prototype, and the variant peptide is denoted as the consensus, reflecting a negative association between HLA-B*18 and the viral polymorphism D47E. Representative data from patient 3 are shown in (D) and (E). M, mol/L; PMA, phorbol myristate acetate. SMQGVPESPF tetramer coupled to phycoerythrin and enriched The strongest association linked residue 170 in L-HDAg using anti-PE beads with MACS technology (Miltenyi Biotec, with HLA-B*15. All 8 HLA-B*15þ patients harbored viruses Germany). Frequencies of virus-specific CD8þ T cells were with a serine (S) to asparagine (N) substitution at this calculated as described previously.18 Data were acquired using position (S170N) compared with only 16 of 96 HLA-B*15– an LSRFortessa flow cytometer and analyzed with FlowJo patients (P ¼ 2.9  10–8) (Figure 1A and C). software version 10 (FlowJo, Ashland, OR). Only 5 of 21 significant polymorphisms (24%) were associated with HLA-A alleles, whereas 16 of 21 significant Results polymorphisms (76%) were associated with HLA-B alleles (Table 1). Moreover, these HLA footprints were distributed Identification of HLA Class I–Associated Viral quite evenly across the central region of HDAg, sparing the Sequence Polymorphisms in L-HDAg N-terminal 30 amino acids of S/L-HDAg, the C-terminal 25 To assess the impact of HDV-specific CD8þ T-cell– amino acids of S-HDAg, and the entire C-terminal extension mediated selection pressure in vivo, we analyzed a cohort of unique to L-HDAg (Figure 2A). 104 patients with chronic HDV/HBV infection for HLA class I–associated viral sequence polymorphisms (HLA foot- prints) in L-HDAg. Using a highly significant cutoff value Validation of CD8þ T-Cell Epitopes and Viral (P < .005) based on studies of other persistent viral in- Escape Mutations in L-HDAg fections,19,20 we identified 21 viral sequence poly- To confirm the relevance of these associations, we used morphisms that were associated with specific HLA class I in silico prediction tools to analyze viral amino acid alleles (Table 1). sequences for HLA class I binding motifs, guided by the 1826 Karimzadeh et al Gastroenterology Vol. 156, No. 6 Figure 4. The rare allotype HLA-B*37 selects for viral escape in the CD8þ T-cell epitopes L-HDAg81–90 and L-HDAg100–108. (A–E) Details as per the corresponding panels in Figure 1. (B) the BIMAS score (higher scores indicate higher binding affinities) TRANSLATIONAL LIVER was used in place of the ANN 3.4 method, which does not include a prediction algorithm for HLA-B*37 binders. (D, E) BASIC AND Representative data from patient 1 (epitope L-HDAg100–108). PMA, phorbol myristate acetate. corresponding footprint data (see Materials and Methods). generation of epitopes derived from HCV and HIV In addition to 8mer, 9mer, and 10mer peptides, an (Supplementary Table 4). extended window incorporating 15 residues either side of We then evaluated the immunogenicity of candidate the HLA footprint was tested to allow the identification of epitopes in functional assays using peptide-stimulated CD8þ CD8þ T-cell epitopes associated with flanking mutations, T-cell lines generated from HLA class I–matched patients which can disrupt antigen processing and facilitate viral with resolved HDV infection (n ¼ 12) or chronic HDV/HBV escape.21–25 Using this approach with defined cutoffs (an infection (n ¼ 13) (Supplementary Table 2). Five novel IC50  1000 nmol/L, a SYFPEITHI score  20, or a BIMAS HDV-specific CD8þ T-cell epitopes were identified with this score  20), we identified 18 candidate CD8þ T-cell epi- approach (Table 1), including 1 restricted by HLA-B*15 topes spanning 17 of the 21 HLA footprints (Figures 1B, 3B, (Figure 1), 1 restricted by HLA-B*18 (Figure 3), 2 and 4B; Table 1; and Supplementary Table 3). As expected, restricted by HLA-B*37 (Figure 4), and 1 restricted by HLA- these candidates included 2 HLA-B*27-restricted epitopes B*41 (data not shown). In further experiments, we gener- identified previously with the same sequence data set, ated peptide-specific CD8þ T-cell lines targeting either the validating the overall strategy.6 Of note, 12 of 18 HLA prototype or variant epitopes and tested the impact of viral footprints were located within the respective candidate sequence variation using IFN gamma production assays. As epitopes, whereas 6 of 18 HLA footprints were located in shown in Figures 1D, 3D, and 4D, and more clearly in serial the regions flanking the respective candidate epitopes. dilution assays (Figures 1E, 3E, and 4E), the variant peptides These latter footprints did not affect antigen processing in were recognized suboptimally by some, but not all, silico, as predicted with an online algorithm (www.iedb. prototype-specific CD8þ T cells. Conversely, variant-specific org),26 but negative results were also obtained in similar CD8þ T cells responded poorly to the corresponding pro- analyses of flanking mutations known to impair the totype and variant peptides without exception, consistent May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1827 Figure 5. Viral escape in the HLA-B*15–restricted epitope L-HDAg170–179 after superinfection with HDV. (A) Clinical course of HDV/HBV superinfection in patient 18 (HLA-A*24:02þ, -B*15:01þ, -B*35:02þ). (B) Viral sequences in patient 18 at early (week 0 and week 2) and late time points (week 71 and week 200) after superinfection with HDV. All longitudinal sequence variants are shown (n ¼ 4). Dots indicate agreement with consensus across the study cohort. Residues with de novo mutations are shown in red, and residues that revert to wild type are shown in green. The de novo mutation S170N is located in the confirmed HLA-B*15–restricted epitope L-HDAg170–179 and corresponds to the linked footprint (Table 1). Also, the F27L reversion colocalizes with an anchor residue for the pre-described HLA-A*02–restricted epitope L-HDAg26–34. ALT, alanine amino- transferase; T, time. TRANSLATIONAL LIVER BASIC AND with population-level viral escape (Figures 1D and E, 3D and aminotransferase levels approaching 2000 U/L, which E, 4D and E). declined to <500 U/L over a period of 1 week, and serum An association of special interest was detected at residue HDV RNA levels approaching 107 IU/mL, which declined 47 (Figure 3A and C). Here, the consensus aspartate (D) was by approximately 2 orders of magnitude in the first week conserved in 13 of 14 HLA-B*18þ patients, whereas the and subsequently relapsed to plateau at >107 IU/mL variant glutamate (E) was found in 42 of 90 HLA-B*18– (Figure 5A). Over a period of >200 weeks before initiation patients (P ¼ .0027). This observation is compatible with of antiviral therapy with pegylated IFN alfa, viral sequence the notion that HLA-B*18 drives population-level evolution analysis showed a total of 4 amino acid substitutions in from glutamate, which likely represents the ancestral res- L-HDAg. Two of these mutations were reversions toward idue, to aspartate and further suggests that the E47D sub- the consensus sequence, and 2 mutations encoded de novo stitution does not markedly impair viral fitness, given the substitutions away from the consensus sequence relative paucity of sequence revertants in the absence of (Figure 5B). One of the reversion mutations occurred at an HLA-B*18. In line with these interpretations, the variant HLA-A*02 anchor residue, suggesting primary acquisition peptide (47E) induced a small but reproducible IFN gamma of an escape virus from an HLA-A*02þ individual (patient response, whereas the consensus peptide (47D) was not P18 lacked HLA-A*02). More importantly, 1 of the de novo recognized in parallel assays (Figure 3D and E). mutations (S170N) was a bone fide escape variant located in L-HDAg170–179 SMQGVPESPF, matching the HLA-B*15 Viral Evolution During Superinfection With HDV footprint identified in our cohort of patients with To probe the biological implications of these findings, chronic HDV/HBV infection (Figure 1 and Table 1). These we characterized the emergence of viral escape mutations observations clearly show the longitudinal accumulation of in longitudinal samples from an HLA-B*15þ patient with viral escape mutations in L-HDAg and tentatively link this acute HDV/HBV superinfection (P18 in Supplementary phenomenon with CD8þ T-cell failure in the context of Table 2). This patient presented with alanine superinfection with HDV. 1828 Karimzadeh et al Gastroenterology Vol. 156, No. 6 TRANSLATIONAL LIVER BASIC AND May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1829 HDV Sequence Polymorphisms Are Associated These data can be explained by the concept of viral With Infrequent HLA Class I Alleles evolution at the population level. Accordingly, relatively As noted, most of the HLA class I footprints identified in infrequent HLA class I allotypes continue to drive the our cohort were associated with HLA-B alleles (Table 1). In accumulation of escape variants in vivo, whereas more further analyses, we realized that common HLA class I al- common HLA class I allotypes shape the circulating quasi- leles, such as HLA-A*01, -A*02, -A*03, -B*07, -B*08, -B*35, species and therefore no longer select for de novo muta- and -B*44, all of which occurred at frequencies >10% in the tions in HDV. German reference population, were not associated with viral sequence polymorphisms (Figure 2B). Conversely, signifi- cant footprints in the HDV genome were almost exclusively HDV-Specific CD8þ T Cells Are Maintained at associated with relatively uncommon HLA class I alleles, Very Low Frequencies After Viral Escape namely HLA-A*29, -A*30, -B*13, -B*14, -B*15, -B*18, -B*27, In line with previous studies,4,6 we were unable to detect -B*37, -B*38, -B*41, -B*49, and -B*51 (Figure 2B and HDV-specific CD8þ T cells directly ex vivo, even with the aid Table 1). of fluorochrome-labeled tetramers corresponding to the To extend these observations, we compared HLA class HLA-B*15-restricted epitope L-HDAg170–179 SMQGVPESPF I allele frequencies in our cohort of patients with chronic (Figure 6A, upper panels). We therefore characterized these HDV/HBV infection with the corresponding HLA class I cells using a tetramer-based enrichment strategy.27,28 allele frequencies in Germany, Italy, and Iran, where the Distinct populations of tetramerþ CD8þ T cells were primary medical centers were located for recruitment detected after enrichment in all 7 HLA-B*15þ patients with purposes (Figure 2B). Additional patients were recruited chronic HDV/HBV infection (patients 13, 14, 16, 17, 18, 21, from a medical center in Barcelona, but HLA class I allele and 23) (Figure 6A, lower panels, and B). The frequencies of frequency data were not available for the general popu- these HLA-B*15–restricted HDV-specific CD8þ T cells were lation in Spain. The overall distribution of HLA class I low, however, subordinate even to rarely detectable virus- allele frequencies in our cohort of patients with chronic specific CD8þ T cells in patients with chronic HCV infec- HDV/HBV infection was similar to the overall distribution tion (Figure 6B). In patients 13, 14, 17, 18, 21, and 23, of HLA class I allele frequencies in Germany, Italy, and SMQGVPESPF-specific CD8þ T cells displayed a predomi- Iran (Figure 2B). However, the footprint-linked allele HLA- nant effector-memory phenotype, whereas in patient 16, B*51 was relatively common in our study cohort and in the SMQGVPESPF-specific CD8þ T cells displayed a predomi- general populations of Italy and Iran (10%–11%), and we nant central-memory phenotype (Figure 6C). These HDV- were unable to identify an HLA-B*51–restricted candidate specific CD8þ T cells expressed intermediate to high levels epitope in HDV (Table 1 and Supplementary Table 3). of PD-1 (Figure 6D), intermediate levels of the inhibitory Other immune selection pressures, such as interactions receptor KLRG1 (Figure 6D), and intermediate levels of the between the Bw4 motif and killer cell immunoglobulin-like transcription factors T-bet and Eomes (Figure 6E). In line TRANSLATIONAL LIVER receptors, may therefore drive viral mutation in the with these characteristics, which contrast with the typical phenotype of terminally exhausted cells (PD-1hiKLRG1þT- BASIC AND context of HLA-B*51. Similarly, the footprint-linked allele HLA-B*18 was relatively common in our study cohort betdimEomeshi), SMQGVPESPF-specific CD8þ T cells also (6.7%) and in the general population of Italy (9.7%). This expressed low levels of the activation marker CD38, akin to observation is compatible with the negative association HCV-specific CD8þ T cells targeting escape variants between HLA-B*18 and sequence variation at residue 47 in (Figure 6F). Moreover, SMQGVPESPF-specific CD8þ T cells L-HDAg. preferentially displayed a PD-1þCD127þ phenotype = Figure 6. Characterization of HLA-B*15–restricted HDV-specific CD8þ T cells in patients with resolved or persistent HDV infection. (A) HLA-B*15–restricted CD8þ T cells specific for L-HDAg170–179 were tetramer-enriched from PBMCs isolated from patients with chronic HDV/HBV infection. Representative tetramer stainings from patient 13 (relatively large tetramerþ pop- ulation) and patient 17 (relatively small tetramerþ population) are shown before (upper panels) and after enrichment (lower panels). (B) Frequency of tetramerþ cells among total CD8þ T cells. HDV-specific CD8þ T cells from 7 patients with chronic HDV/HBV infection and 1 patient with resolved HDV infection (patient 5, highlighted in red) were compared with HCV-specific CD8þ T cells targeting epitopes with conserved or escaped viral sequences in chronic HCV infection. Autologous viral se- quences corresponding to the L-HDAg170–179 epitope are displayed next to the patient codes. S170N indicates the presence of the viral escape sequence, and S170S/N indicates presence of the prototype and the viral escape sequence, respectively. ND indicates that sequences were not obtained from patients with low levels of HDV RNA. (C–H) Enriched HDV-specific CD8þ T cells were characterized by flow cytometry. Gray histograms indicate non-naive bulk CD8þ T cells. Representative plots are derived from patient 21. (C) Distribution among naive (CD45RAþCD27þCCR7þ), central memory (CD45ROþCCR7þ), effector memory (CD45ROþCCR7–), and terminally differentiated effector memory subsets (CD45RO–CCR7–). (D) Expression levels of the exhaustion markers PD-1 and KLRG1. (E) Expression levels of the transcriptions factors T-bet and Eomes. (F) Expression of the activation marker CD38 compared with HCV-specific CD8þ T cells targeting epitopes with conserved (wildtype) or escaped viral sequences in chronic HCV infection. (G) Distribution among subsets defined by expression levels of PD-1 and CD127. (H) Expression of the transcription factors TCF1 and BCL2. Dotted lines indicates fluorescence-minus-1 controls. MFI, median fluorescence intensity; ND, not done; P, patient; TCM, central memory; TEM, effector memory; TEMRA, terminally differentiated effector-memory subset; wt, wild type. 1830 Karimzadeh et al Gastroenterology Vol. 156, No. 6 (Figure 6G) and expressed relatively high levels of TCF1 allotypes were not present in the HDV proteome (L-HDAg).6 (Figure 6H), a transcription factor that defines memory-like However, these findings need to be confirmed with direct cells with proliferative capacity.29 Similar levels of TCF1 immunogenicity assays, because prediction algorithms are were expressed by SMQGVPESPF-specific CD8þ T cells in a potentially fallible, and certain epitopes may not be subject patient (patient 5) with resolved HDV infection (Figure 6H). to viral escape, particularly if they are located in biologically Expression of the pro-survival factor BCL2 was more vari- constrained regions of L-HDAg. In this context, our experi- able, however, with the highest levels expressed by mental strategy was not designed to identify CD8þ T-cell SMQGVPESPF-specific CD8þ T cells in patient 5 (Figure 6H). epitopes that are not subject to viral escape. Moreover, our These phenotypic characteristics broadly mirror those study cohort did not include patients with acute HDV reported previously for HCV-specific CD8þ T cells in the infection, potentially limiting the detection of novel CD8þ T- absence of antigenic stimulation, reflecting viral escape or cell specificities. An unbiased approach will therefore be viral clearance.17,29 Although further studies are required to required to define the full range of immunogenic epitopes establish such parallels across different viral infections, it is derived from HDV.30 The association between immunoge- notable that SMQGVPESPF-specific CD8þ T cells in patient nicity and infrequent allotypes reported here may also 14, who retained a subpopulation of prototype viruses, be confounded by HLA class I polymorphisms. For example, expressed relatively low levels of TCF1 and BCL2 in the viral mutation S170N was found in all patients conjunction with an atypical CD38þPD-1þCD127– pheno- with HLA-B*15:01 but was not found in patients type (Figure 6F and G). with HLA-B*15:10 or HLA-B*15:18 (data not shown). Similarly, common polymorphisms in HLA-B*27 can lead to subtype-specific patterns of epitope targeting in the context Discussion of other persistent viral infections, including HCV and HDV/HBV infection is one of the few remaining difficult- HIV.31,32 to-treat conditions in viral hepatitis, leading to liver Viral escape has direct implications for immune efficacy cirrhosis, liver failure, and hepatocellular carcinoma in and the development of immunotherapeutic strategies in many patients.1,2 Despite the urgent need for novel pro- the setting of chronic HDV/HBV infection. Recent vaccine phylactic and therapeutic interventions, remarkably little is studies in chimpanzees and humans with chronic HCV known about the natural repertoire of virus-specific CD8þ T infection showed good immunogenicity against non- cells, which are thought to play a key role in immune pro- conserved viral epitopes, but the corresponding vaccine- tection against HDV. In this study, we analyzed HLA class boosted CD8þ T cells were ineffective, because they failed I–associated viral sequence polymorphisms in L-HDAg, to recognize the circulating virus.33,34 Effective therapeutic giving new insights into the immunobiology of HDV infec- vaccines will therefore likely need to prime and/or boost tion. First, we extended the repertoire of defined HDV- virus-specific CD8þ T cells that target conserved epitopes. specific CD8þ T-cell epitopes from 4, encompassing 2 The most significant footprint-linked allele in the pre- TRANSLATIONAL LIVER restricted by HLA-A*024 and 2 restricted by HLA-B*27,6 to sent study was HLA-B*15, which has also been shown to 9, now also encompassing 1 restricted by HLA-B*15, 1 select for viral escape mutations during persistent HCV BASIC AND restricted by HLA-B*18, 2 restricted by HLA-B*37, and infection.35 In addition, genetic association studies have 1 restricted by HLA-B*41. Second, we showed that viral linked HLA-B*15 and/or its serological equivalent (B62) escape occurs during persistent HDV infection. Third, we with better outcomes after infection with HCV.36–38 How- uncovered a link between viral escape and infrequent HLA ever, it remains unclear if HLA-B*15 is associated with class I alleles, indicating population-level adaptation of HDV. spontaneous clearance of HDV, and, moreover, any such Finally, we showed that HDV-specific CD8þ T cells in pa- associations will be extremely difficult to identify, given the tients with resolved or persistent HDV infection display clinical heterogeneity of HDV-infected patients and the similar non–terminally exhausted phenotypes, consistent requirement for co-infection with HBV. In our study cohort, with viral escape in the absence of clearance. In line with the presence of HDV-specific CD8þ T-cell responses did not this interpretation, HLA-B*15–restricted HDV-specific CD8þ correlate with the outcome of infection (clearance vs T cells that reproducibly select for viral escape could be persistence). However, the presence of HDV amino acid expanded from patients with chronic HDV/HBV infection, variations corresponding to polymorphisms associated with whereas HDV-specific CD8þ T cells restricted by HLA class I the patient’s HLA alleles (Supplementary Table 1) displayed allotypes that less commonly select for viral escape could a trend for association with both lower HDV RNA titers not be expanded from patients with chronic HDV/HBV (median, 20,638 IU/mL [range, 6 –20,098,000,000 IU/mL] infection. in patients with 1 variation vs median, 61,930 IU/mL The phenomenon of viral adaptation at the population [range, 10–9,804,000 IU/mL] in patients without any vari- level may lead to a relative paucity of CD8þ T-cell epitopes ation; P ¼ .054 [Mann–Whitney test]) and lower alanine restricted by common HLA class I allotypes, which in turn aminotransferase levels (median, 73 U/L [range, 19–281 U/ may impede the development of an effective vaccine against L] in patients with 1 variation vs median 86 U/L [range, HDV. In line with this prediction, viral sequence poly- 31–507 U/L] in patients without any variation; P ¼ .070 morphisms were not associated with the common HLA [Mann–Whitney test]), respectively. These results suggest alleles A*01, A*02, A*03, A*24, B*07, B*08, B*35, and B*44, that HLA class I footprint mutations indicate efficient CD8þ and suitable binding motifs for many of the corresponding T cell pressure, leading to partial control of viremia and May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1831 liver inflammation. This interpretation is based on the 6. Karimzadeh H, Kiraithe MM, Kosinska AD, et al. Amino assumption, however, that partial cross-recognition of acid substitutions within HLA-B*27-restricted T cell epi- escape variants occurs and is sufficient to exert pressure on topes prevent recognition by hepatitis delta virus- the virus. specific CD8þ T cells. J Virol 2018;92:e01891–17. It is particularly notable that our findings considerably 7. Neumann-Haefelin C. HLA-B27-mediated protection in overlap with those reported previously in the setting of HIV and hepatitis C virus infection and pathogenesis persistent infection with HCV or HIV. For example, in all 3 in spondyloarthritis: two sides of the same coin? Curr infections, virus-specific CD8þ T-cell pressure can select Opin Rheumatol 2013;25:426–433. escape variants to fixation (HDV: HLA-B*15–associated 8. Price DA, Brenchley JM, Ruff LE, et al. Avidity for antigen polymorphism S170N; HCV: HLA-A*01–associated poly- shapes clonal dominance in CD8þ T cell populations morphism Y1444F)39 and drive viral evolution at the pop- specific for persistent DNA viruses. J Exp Med 2005; ulation level to extinguish commonly restricted epitopes 202:1349–1361. (HDV: HLA-B*18–restricted epitope L-HDAg47–54; HCV: 9. Larkin MA, Blackshields G, Brown NP, et al. Clustal W HLA-A*01–restricted epitope NS31436–1444;39 HIV: HLA- and clustal X version 2.0. Bioinformatics 2007;23: B*51–-restricted epitope RT128–135).40 Similarly, in all 3 in- 2947–2948. fections, virus-specific CD8þ T cells are preferentially 10. Budeus B, Timm J, Hoffmann D. SeqFeatR for the dis- restricted by HLA-B,41–44 rare allotypes confer a particular covery of feature-sequence associations. PLoS One 2016;11:e0146409. advantage,45 and viral escape mutations undergo reversion in the absence of selection pressure after transmission.46,47 11. Hoof I, Peters B, Sidney J, et al. NetMHCpan, a method for MHC class I binding prediction beyond humans. Im- Finally, in all 3 infections, CD8þ T cells targeting escape munogenetics 2009;61:1–13. variants display a memory-like phenotype, indicating a loss 12. Nielsen M, Lundegaard C, Worning P, et al. Reliable pre- of antigenic drive.17,25,29 diction of T-cell epitopes using neural networks with novel Despite these striking parallels across distinct viruses sequence representations. Protein Sci 2003;12:1007–1017. with respect to adaptive host–pathogen interactions, the 13. Rammensee H, Bachmann J, Emmerich NP, et al. SYF- immunobiology of HDV infection is complicated by the PEITHI: database for MHC ligands and peptide motifs. mandatory requirement for co-infection with HBV. Much Immunogenetics 1999;50:213–219. work will therefore be required to unravel the parameters 14. Parker KC, Bednarek MA, Coligan JE. Scheme for that govern clinical outcome in the midst of constantly ranking potential HLA-A2 binding peptides based on shifting immune responses that simultaneously target independent binding of individual peptide side-chains. rapidly evolving antigens derived from 2 different viruses in J Immunol 1994;152:163–175. a genetically diverse host population. The findings reported 15. 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Persis- Reprint requests tent hepatitis C viral replication despite priming of Address requests for reprints to: Michael Roggendorf, MD, Institute of Virology, functional CD8þ T cells by combined therapy with a Technical University of Munich, Schneckenburger Strasse 8, 81675 Munich, Germany. e-mail: michael.roggendorf@tum.de; fax: þ49/8941407444. vaccine and a direct-acting antiviral. Hepatology 2016; Christoph Neumann-Haefelin, MD, Department of Medicine II, University 63:1442–1454. Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. e-mail: 34. Kelly C, Swadling L, Capone S, et al. Chronic hepatitis christoph.neumann-haefelin@uniklinik-freiburg.de; fax: þ49/76127032800. C viral infection subverts vaccine-induced T-cell immu- Acknowledgments nity in humans. Hepatology 2016;63:1455–1470. We thank the individuals who donated blood for this study, the physicians and nurses involved in patient recruitment and sample acquisition, and the 35. Ruhl M, Knuschke T, Schewior K, et al. CD8þ T-cell technicians who isolated PBMCs. We are especially grateful to Christin response promotes evolution of hepatitis C virus Ackermann, Thomas Schirdewahn, and Ulrich Spengler for providing nonstructural proteins. Gastroenterology 2011; information from patients. Author contributions: Hadi Karimzadeh, Muthamia M. Kiraithe, Michael 140:2064–2073. Roggendorf, and Christoph Neumann-Haefelin designed the study and 36. Fitzmaurice K, Hurst J, Dring M, et al. Additive interpreted the data; Hadi Karimzadeh performed viral sequence and footprint analyses; Muthamia M. Kiraithe, Valerie Oberhardt, and Elahe Salimi effects of HLA alleles and innate immune genes deter- Alizei performed immunological experiments; Jan Bockmann, Julian Schulze mine viral outcome in HCV infection. Gut 2015;64: zur Wiesch, Heiner Wedemeyer, Markus Cornberg, Adalbert Krawczyk, 813–819. Jassin Rashidi-Alavijeh, Francisco Rodríguez-Frías, Rosario Casillas, Maria Buti, Antonina Smedile, Seyed Moayed Alavian, Marcus Panning, Bijan 37. Hraber P, Kuiken C, Yusim K. Evidence for Raziorrouh, and Christoph Neumann-Haefelin enrolled patients and human leukocyte antigen heterozygote advantage performed clinical evaluations; Bettina Budeus and Daniel Hoffmann assisted with statistical analyses; Andreas Heinold, and Florian Emmerich performed against hepatitis C virus infection. Hepatology 2007; HLA class I genotyping; Emma Gostick, and David A. Price generated HLA 46:1713–1721. class I tetramers; Jörg Timm, Maike Hofmann, Robert Thimme, and Ulrike May 2019 Viral Escape From HDV-Specific CD8+ T Cells 1833 Protzer helped design and interpret the experiments; Hadi Karimzadeh, Schulze zur Wiesch (SFB841, SFB1328), Daniel Hoffmann (TRR60/B1), Jörg Muthamia M. Kiraithe, Valerie Oberhardt, David A. Price, Michael Timm (TI323/4-1), Maike Hofmann, Robert Thimme (SFB-TRR-179/TP1), Roggendorf, and Christoph Neumann-Haefelin wrote the manuscript; all Ulrike Protzer (SFB-TR179/TP14 and TRR36/B13), and Christoph Neumann- authors read and approved the final manuscript. Haefelin (SFB-TRR-179/TP2). Additional funding was provided by the Medical Faculty of the University of Duisburg-Essen/Technical University of Conflicts of interest Munich and by a registry grant from the European Association for the Study The authors disclose no conflicts. of the Liver. Hadi Karimzadeh was supported by a grant from the German Academic Exchange Service. David A. Price was supported by a Wellcome Funding Trust Senior Investigator Award (100326Z/12/Z). Julian Schulze zur Wiesch, This work was supported in part by the Deutsche Forschungsgemeinschaft via Heiner Wedemeyer, Markus Cornberg, Ulrike Protzer, and Michael grants to Bijan Raziorrouh (SFB-TR179/TP3 project number 272983813), Julian Roggendorf were supported by the German Center for Infection Research. TRANSLATIONAL LIVER BASIC AND