HBV and the immune response

Transcription

1 HBV and the immune response C. Ferrari Unit of Infectious Diseases and Hepatology Laboratory of Viral Immunopathology Azienda Ospedaliero-Universitaria di Parma Italy

2 List of topics - Kinetics of immune responses: from the early stages of infection to HBV control or persistence - Features of T cell and NK responses in chronic infection - Mechanisms of T cell dysfunction in chronic HBV infection - Effect of virus control on T and NK cell responses in chronic patients - Potential strategies to reconstitute the anti-viral T cell function

3 List of topics - Kinetics of immune responses: from the early stages of infection to HBV control or persistence - Features of T cell and NK responses in chronic infection - Mechanisms of T cell dysfunction in chronic HBV infection - Effect of virus control on T and NK cell responses in chronic patients - Potential strategies to reconstitute the anti-viral T cell function

4 HBV is a stealth virus poorly sensed by the innate immune system INNATE IMMUNE RESPONSE GENES No gene ADAPTIVE IMMUNE RESPONSE GENES 110 genes Wieland S et al. PNAS 2004

5 HBV is a poor inducer of innate responses Cytokine and chemokine production in acute HBV infection is significantly more modest and delayed compared with acute HIV infection (Stacey AR J. Fold changes in group mean cytokine level Virol. 2009) Time (days since T0) Time (days since T0) HIV infection HBV infection Low production of type I IFN, IL-15 and IFN-λ1, associated with high serum IL-10 levels, at the early stages of HBV infection (Dunn C. et al Gastroenterology 2009) IFN-alpha A IL-15 B p=0.02 p=0.03 p= p= p= p= pg/ml 8 pg/ml pg/ml p= IFN-λ1 C Healthy n.27 DNA Resolution Acute high phase HAV n.11 n.17 n.13 Healthy n.10 DNA Resolution Acute phase HAV high n.11 n.16 n.13 Healthy n.25 DNA Resolution Acute phase HAV high n.13 n.18 n.11

6 Is HBV able to inhibit innate responses? Extracellular sensing (TLR) Intracellular sensing (RIG-1) HBsAg HBeAg TLR2 TLR4 TLR3 TRIF RIG-1 NF-kB HBx P P IRF-3 HBVp ol P IRF-3 P IRF-7 IFN-β PRD NF-kB target genes

7 Is HBV able to inhibit innate responses? Interferon loop IFN-α IFN-β IFN - α IFN-β -β IFN IFN IFNreceptor receptor TYK2 JAK1 SOCS1 STAT2 ISGF3 STAT1 SOCS3 HBVpol IRF-9 ISG expression ISRE PKR, ADAR1, 2-5OAS, IFIT1, Viperin, ISG6-16, MxA

8 Summary of the early events in HBV infection Early non cytolytic Delayed NK cells clearance of HBV activation T cell inhibition to avoid excessive damage Dunn C et al Gastroenterology x108 NK cells IFN-γ 20 ALT HBV-specific HBV T cell responses 15 5x ,000 6 Weeks from infection Weeks Poor induction of Efficient and timely early intracellularinduction of adaptive innate responses responses ALT IU/L HBV-DNA 10 2, %IFNg+/NK cells 100 HBV-DNA HBV-DNA copies x 10 6 / ml Clinically overt infections

9 Maturation of long-lasting memory T cell responses in self-limited HBV infections HBV INFECTION Strong, multi-specific, T1 oriented T cell responses % CD8-mediated cytotoxicity Self-limited 40 Long-lasting protective responses 30 ACUTE PHASE RECOVERY PHASE (20 years from recovery) POLYMERASE X CORE ENVELOPE HLA-A2 restricted HBV peptides Virus control / occult infection

10 Progressive T cell functional impairment in chronically evolving acute HBV infections HBV INFECTION Weak and narrowly focused T cell responses Chronic evolution Persistent and progressive impairment of protective responses CD4 RESPONSES (to core peptides) HBV POL HBV X HBV CORE CD8 RESPONSES (to HBV peptides) HBV peptides Virus persistence HBV ENV

11 HBV-specific T cells in chronic infection

12 HBV-SPECIFIC CD8 CELLS ARE PREFERENTIALLY CONCENTRATED WITHIN THE LIVER IN PATIENTS WITH CHRONIC HBV INFECTION %HBV-TET+/CD8+ (Fisicaro P. et al. Gastroenterology 2010) LIVER BLOOD Mean %TET+/CD8+ intrahepatic cells p= p=0.006 p=0.03 > HBV-DNA TITER IU/ml

13 INTRAHEPATIC HBV-SPECIFIC T CELLS ARE MORE DEEPLY EXHAUSTED THAN THEIR PERIPHERAL BLOOD COUNTERPARTS IN CHRONIC HBV INFECTION 1.5 p= p=0.01 p= BLOOD 0.5 LIVER 0 TNF-α IFN-γ IFN-γ/IL2 HBV LIVER %IFN-γ CD4+CD8 contr CD4 AND CD8 T CELLS (Fisicaro P. et al. Gastroenterology 2012 and personal comunication) r = p = x100 1x102 1x104 1x106 HBV-DNA IU/ml 1x108 1x1010

14 NK cells in chronic infection

15 NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (I) % IFNγ production NK Total P< CD107a % CD107 production NK Total IFN-γ Healthy N= Healthy N=29 CHB N=46 CHB N=33 Peppa D. et al Plos Pathogens 2010

16 NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (II) IL2+IL12 18h %IFNγ IFN-γ 80 p= HD CD107a %CD107a+ NK cells 100 anti-nkp30 HBV anti-nkp46 anti-nkg2d HD HBV HD HBV HD HBV Oliviero B et al Gastroenterology 2009

17 NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (III) IL12+IL18 24h P<0.01 Tjwa E. et al. Journal of Hepatology 2011

18 NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (IV) %IFNg+/CD56 bright na iv e C H B H ea lth y NK cells seem to be20 more pathogenic than protective 0 in chronic HBV infection CH B na iv e 0 He al th y %CD107a/NKdim 60 Boni C. et al. Unpublished data

19 List of topics - Kinetics of immune responses: from the early stages of infection to HBV control or persistence - Feature of T cell and NK responses in chronic infection - Mechanismsof of TTcell dysfunction in chronicin HBV infection HBV infection Mechanisms cell dysfunction chronic - Effect of virus control on T and NK cell responses in chronic patients - Potential strategies to reconstitute the anti-viral T cell function

20 Different levels of T cell functional efficiency in different conditions of HBV control IFN-γ IL-2 TNF-α Mean % IFN-γ, IL-2 and TNF-α +/T cells 20 CD4+ T cell responses CD8+ T cell responses Naïve HBeAg negative CHB Inactive carriers Boni C, Laccabue D et al. Gastroenterology 2012;143: Occult infections Acute hepatitis B (resolution phase)

21 PUTATIVE MECHANISMS OF T CELL EXHAUSTION IN HBV INFECTION MODEL FOR HIERARCHICAL LOSS OF CD8 FUNCTIONS DURING CHRONIC VIRAL INFECTIONS High viral load with massive production of secretory proteins (HBsAg, HBeAg) Acute infection Naïve CD8 cell Memory CD8 cell Effector CD8 cell Functional T cell Partial Exhaustion I Partial Exhaustion II Tolerizing liver environment Full Exhaustion Death deletion Proliferation IFN-γ TNF-α IL-2 Cytotoxicity Proliferation IFN-γ TNF-α IL / Cytotoxicity Antigen load Sinusoid - - High Wherry EJ et al J.Virol. 77: ;2003 Hepatocyte Space of Disse

22 Are the virus-specifi c T cell defects of chronic HBV infection reversible? Effect of antigen decline Acute Self-limited Infection Effi cient T cell function/differentiation Effector memory Naïve CD8 cell +Ag Rapid Proliferation / Differentiation en tig ed n A ear cl pe Ant rs ige is te n nc e Antigen decline Effector CD8 cell Central memory Chronic infection Ineffi cient T cell function/differentiation? Restored T cell function/differentiation

23 Effect of long-term NUC therapy on T cell responses

24 T cell restoration following long-term NUC treatment is efficient in vitro 100 % of HBV dextramer+ CD8 T cells producing IFN-γ IL-2 In vitro CD107a Naive CHB NUC treated HBsAg neg medium NUC treated HBsAg neg 0% NUC treated Acute hepatitis B HBsAg pos (follow-up) peptide medium peptide 0% 1% 72% 0% 88.7% FLU CD8 Acute hepatitis B (followup) CD8 Dext core 0% Dext FLU 29% CMV Dext core Dext CMV IFN-γ IFN-γ Boni C. et al. Gastroenterology 2012

25 T cell restoration following long-term NUC treatment is partial ex vivo 100 % of HBV dextramer+ CD8 T cells producing IFN-γ IL-2 Ex vivo In vitro Restoration of the T cell function is efficient in vitro 80 but only partial ex vivo even following complete 60 control of virus replication and decline CD107aof antigen Naive CHB NUC treated HBsAg neg medium NUC treated HBsAg neg 0% NUC treated Acute hepatitis B HBsAg pos (follow-up) peptide medium peptide 0% 1% 72% 0% 88.7% FLU CD8 Acute hepatitis B (followup) CD8 Dext core 0% Dext FLU 29% CMV Dext core Dext CMV IFN-γ IFN-γ Boni C. et al. Gastroenterology 2012

26 List of topics - Kinetics of T cell responses: from the early stages of infection to HBV control or persistence - Additional mechanisms of T cell dysfunction in chronic HBV infection - Effect of virus control on T cell responses in chronic patients - Potential strategies to reconstitute the anti-viral T cell function - Implications for future therapies

27 INTRAHEPATIC INHIBITORY MECHANISMS Modifi ed from U. Protzer et al. Nature Reviews in Immunology 2012

28 THE INTRAHEPATIC MILIEU IMPAIRS IL-2 PRODUCTION BY T CELLS CD3ζ downregulation Myeloid suppressor cells + T cell L-arginine depletion ARGINASE T cell T cell + CD3ζ dependent impairment of IL2 production by intrahepatic T cells Hepatocytes Das et al J.Exp.Med. 2008

29 INTRAHEPATIC INHIBITORY MECHANISMS Modifi ed from U. Protzer et al. Nature Reviews in Immunology 2012

30 MECHANISMS OF HEPATIC TOLERANCE: IMMUNOSUPPRESSIVE CYTOKINE MILIEU TGF-β Hepatocytes Stellate Cell SPECE OF DISSE Dendritic cell Kuppfer Cell Dendritic cell HEPATIC SINUSOID Sinusoidal endothelial cells IL-10

31 THE IMMUNOSUPPRESSIVE CYTOKINE MILIEU CAN IMPAIR IFN-γ PRODUCTION BY NK CELLS LIMITING THEIR ANTI-VIRAL ACTIVITY HBV infected hepatocytes VIRAL PERSISTENCE AND LIVER DAMAGE TRAIL-R2 TRAIL IFN-γ IFN-γ IFN-γ NK cell NK cell NK cell IL-10 and TGFβ-mediated suppression of IFN-γ production by NK cells NK cell Preserved cytolytic activity Dunn et al J.Exp.Med 2007 Peppa et al PloS Pathogens 2010

32 NK CELL MEDIATED DELETION OF HBV-SPECIFIC T CELLS HBV infected hepatocytes VIRAL PERSISTENCE AND LIVER DAMAGE TRAIL-R2 IFN-γ TRAIL IFN-γ IFN-γ NK cell NK cell TRAIL-R2 TRAILmediated T cell deletion NK cell NK cell T cell Peppa et al. J.Exp.Med. 2012

33 INTRAHEPATIC INHIBITORY MECHANISMS Bim mediates deletion of antigen-specific CD8 cells in patients unable to control infection (Lopes et al. J.Clin.Invest. 2008) Bim mediates premature death of CD8 T cells following intrahepatic antigen presentation (Holtz et al Gastroenterology 2008) Modifi ed from U. Protzer et al. Nature Reviews in Immunology 2012

34 INTRAHEPATIC INHIBITORY MECHANISMS Modifi ed from U. Protzer et al. Nature Reviews in Immunology 2012

35 Expression of various inhibitory receptors on circulating and intrahepatic virus-specific CD8 cells of patients with chronic HBV infection APC TNF SUPERFAMILY B7 FAMILY PD-L1 PD-L2 B7.1 or B7.2 ICOSL PD-1 OX40L CTLA-4 CD28 ICOS INHIBITION CD70 CD40 4-1BBL OX40 CD27 CD40L 4-1BB COSTIMULATORY SIGNALS T CELL

36 T CELL CO-INHIBITORY MOLECULES IN THE LIVER PD-1 is up-regulated on HBV-specific T cells Kuppfer, LSEC and stellate cells express PD-L1 Hepatocytes Stellate Cell SPECE OF DISSE HBVspecific T cell Dendritic cell Kuppfer Cell Dendritic cell HEPATIC SINUSOID Sinusoidal endothelial cells

37 T CELL CO-INHIBITORY MOLECULES IN THE LIVER TIM-3 is up-regulated on HBV-specific T cells Kupffer cells express galactine-9 Hepatocytes Stellate Cell SPECE OF DISSE HBVspecific T cell Dendritic cell Kupffer Cell Dendritic cell HEPATIC SINUSOID Sinusoidal endothelial cells

38 Expression of various inhibitory receptors on circulating T cell restoration by Tim-3 blockade and intrahepatic virus-specific CD8 cells of patients with chronic HBV infection Control cell restoration CTLA-4 Anti-TIM-3 blockade T cellt restoration by by PD-1/PD-L1 blockade Bengsch et al J.Hepatol Liver Blood T cell restoration by 2B4 blockade p=0.012 p= p = p = p=0.029 PD-1 2B4 0 CONTROL Anti-PD-L1 1 p = LIVER p=0.02 LIVER BLOOD CD160 CTLA-4 LAG-3 CD Schurich A et al Hepatology Fisicaro 0 P. et al. Gastroenterology Anti-PD-L1 CONTROL 0 fold increase 0.5 %IFN-γ+/CD4+CD %COSTIM.+/TET+ %IFN-γ+/CD8+CD BLOOD Raziorrouh B et al, Hepatology 2010 p=0.01 Fisicaro P. et al. Gastroenterology 2010, 2012 Nebbia G. et al. Plos One 2012

39 HBV-specific T cells Genome-wide expression profiling Mysregulated genes and pathways associated with T cell exhaustion Correction strategies to restore anti-viral T cell functions

40 TRANSCRIPTOME STUDY IN ACUTE AND CHRONIC HBV INFECTION Patient infection ALT A1 ACUTE 1785 A2 ACUTE 998 A3 ACUTE 659 A4 ACUTE 1118 A5 ACUTE 211 R1 RESOLVED HEP B 16 R2 RESOLVED HEP B 17 R3 RESOLVED HEP B 20 R4 RESOLVED HEP B 12 CH1 CHRONIC 40 CH2 CHRONIC 96 CH3 CHRONIC 68 CH4 CHRONIC 63 CONTROLS CELL SPECIFICITY H1 HEALTHY FLU H2 HEALTHY FLU H3 HEALTHY FLU H4 HEALTHY FLU H5 HEALTHY FLU Pre-sorting Isolation of HBV/FLU-specific CD8+ T cells by cell sorting 0.4% RNA extraction and amplification Gene expression by microarray analysis (4x44K Agilent) VALIDATION AND DISCOVERY OF NEW TARGETS Post-sorting 98.7%

41 TRANSCRIPTOME STUDY IN ACUTE AND CHRONIC HBV INFECTION Data analysis Visualization of overall sample distribution and distances between groups of patients by principal component analysis (PCA) Resolved B FLU-specific Comparisons of gene expression profiles cells in different groups of patients to determine from FLU-specific cells differentially expressed genes by healthy t test, by ANOVA, by291 hierarchical clustering and by from Chronic B Venn diagrams differentially expressed Gene ontology and pathway enrichment analysis starting from the lists of genes identified differentially expressed genes (ANOVA test, corrected, Acute B p<0.05) Chronic B Gene Set Enrichment Analysis Acute Res Chronic

42 PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation Heme biosynthesis Huntington s disease Mitochondrial Gene Expression Translation factors Proteasome degradation Oxidative phosphorylation Metabolic pathways DOWN-REGULATED CELLULAR METABOLISM Myometrial Relaxation and Contraction Pathways Mucin type O-Glycan biosynthesis Glycine, serine and threonine metabolism Vascolar smooth muscle contraction Porphyrin and chlorophyll metabolism Lysosome Purine metabolism Protein processing in endoplasmic reticulum Parkin-Ubiquitin Proteasomal System pathway

43 PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation Heme biosynthesis Huntington s disease Mitochondrial Gene Expression Translation factors Proteasome degradation Oxidative phosphorylation Metabolic pathways Myometrial Relaxation and Contraction Pathways Mucin type O-Glycan biosynthesis Glycine, serine and threonine metabolism Vascolar smooth muscle contraction Porphyrin and chlorophyll metabolism Lysosome Purine metabolism Protein processing in endoplasmic reticulum Parkin-Ubiquitin Proteasomal System pathway DOWN-REGULATED PROTEASOMAL FUNCTION

44 PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation Heme biosynthesis Huntington s disease Mitochondrial Gene Expression Translation factors Proteasome degradation Oxidative phosphorylation Metabolic pathways Myometrial Relaxation and Contraction Pathways Mucin type O-Glycan biosynthesis Glycine, serine and threonine metabolism Vascolar smooth muscle contraction Porphyrin and chlorophyll metabolism Lysosome Purine metabolism Protein processing in endoplasmic reticulum Parkin-Ubiquitin Proteasomal System pathway DOWN-REGULATED MITOCHONDRIAL FUNCTION

45 Main message Evidence Question A deep metabolic and energetic impairment is Multiple levels of correction will certainly be needed to Is restoration of an efficient anti-viral T cell typical of exhausted T cells restore an efficient anti-viral T cell function function an achievable objective?

46 Potential strategies to reconstitute the anti-viral T cell function and implications for future therapies

47 Efficient control of HBV replication by NUC therapy Clinical practice ETV (n=418) 80 85% 95% 96% 99% 68% % 91% 90% 91% 65% TDF (n=302) 100 Patients % Patients % 100 Slow HBsAg decline during NUC therapy: need of life-long NUC administration Months Reijnders JGP et al J-Hepatol Clinical needs in HBV therapy for CH-B: to shorten NUC therapy by accelerating HBsAg clearance

48 SEQUENCIAL NUC/IFN-α THERAPY Potential strategy to to shorten NUC therapies NUC treatment Decline of antigen load Inhibition of negative costimulatory pathways Several months Antigen load DC CD8 T cell stimulation Blocking antibodies T CELL DYSFUNCTION CD4 RECOVERY OF T CELL RESPONSIVENESS CD4 CD8 CTL -/+ IFN-γ -/+ IL-2 -/+ Proliferation CD4 CD4 CD8 -/+ Modifi ed from: Ferrari C. Gastroenterology 2008 CTL ++ IFN-γ ++ IL-2 ++ Proliferation ++ HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

49 EFFECT OF ANTI-PD-1 THERAPY ON HCV INFECTED CHIMPANZEES Effect on viral load Fuller MJ et al. PNAS 2013 Effect on magnitude of T cell responses

50 PD-1 PATHWAY BLOCKADE Proof of concept of α-pd-1 in Chronic HCV Blinded, PBO controlled, SAD study α-pd-1 in 54 HCV infected patients, IFN failures and treatment naive 0.03mg/kg -10mg/kg 3 subjects w/ > 4 log HCV RNA decline: All 3 received 10mg/kg dose 1 subject (A) had isolated, transient Grade 4 ALT increase to ~17x ULN 1 subject (B) undetectable > 1 year post treatment Gardiner et al PLoS ONE 8(5): e doi: /journal.pone

51 SEQUENCIAL NUC/IFN-α THERAPY Potential strategy to optimize IFN-α efficacy and to shorten NUC therapies Antigen load NUC treatment Decline of antigen load T CELL DYSFUNCTION CD4 RECOVERY OF T CELL RESPONSIVENESS CD4 CD8 CTL -/+ IFN-γ -/+ IL-2 -/+ Proliferation CD4 CD4 CD8 -/+ Modifi ed from: Ferrari C. Gastroenterology 2008 CTL ++ IFN-γ ++ IL-2 ++ Proliferation ++ HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

52 SEQUENCIAL NUC/IFN-α THERAPY Potential strategy to optimize IFN-α efficacy and to shorten NUC therapies NUC treatment Decline of antigen load Antigen load SPECIFIC VACCINES T CELL DYSFUNCTION CD4 RECOVERY OF T CELL RESPONSIVENESS CD4 CD8 CTL -/+ IFN-γ -/+ IL-2 -/+ Proliferation CD4 CD4 CD8 -/+ Modifi ed from: Ferrari C. Gastroenterology 2008 CTL ++ IFN-γ ++ IL-2 ++ Proliferation ++ T CELL VACCINES Recombinant (Gilead) DNA (Transgene) Peptides (Immune Targeting System) HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

53 Synergistic effect of PD-L1 blockade and therapeutic vaccination on T cell responses and viral control (Liu J. et al. PLOS Pathogens 2014) Vaccine + anti-pd-l1 T cell immunity Vaccine Control Entecavir Entecavir + DNA vaccination Entecavir + DNA vaccination + anti-pdl1 WHA replication

54 Synergistic effect of PD-L1 blockade and therapeutic vaccination on T cell responses and viral control DbGP33-41-specific CD8+T cells/106pbmc (Ha S-J. et al. J. Exp. Med. 2008) + α PD-L1 105 Vaccine + anti-pd-l1 104 Vaccine VV/WT VV/GP33 VV/WT+ αpd-l1 VV/GP33+ αpd-l1 Days post-infection Serum virus (pfu/ml) α PD-L Vaccine Days post-infection 60 Vaccine + anti-pd-l1

55 SEQUENCIAL NUC/IFN-α THERAPY Potential strategy to optimize IFN-α efficacy and to shorten NUC therapies NUC treatment Decline of antigen load Antigen load TLR AGONISTS T CELL DYSFUNCTION CD4 RECOVERY OF T CELL RESPONSIVENESS CD4 CD8 CTL -/+ IFN-γ -/+ IL-2 -/+ Proliferation CD4 CD4 CD8 -/+ Modifi ed from: Ferrari C. Gastroenterology 2008 CTL ++ IFN-γ ++ IL-2 ++ Proliferation ++ TLR-7 AGONISTS GS-9620 preclinical studies: HBsAg and HBV-DNA reduction in woodchucks and chimpanzees Favorable safety profile in healthy donors HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

56 TLR8 agonists can trigger potent activation of innate immune cells in human liver TLR agonists TLR7 TLR8 Hepatic monocytes IL18 IL12 CD161bright MAIT NKbrig ht J.Jo et al. PLOS Pathogens 2014 IFN-γ

57 SEQUENCIAL NUC/IFN-α THERAPY Potential strategy to optimize IFN-α efficacy and to shorten NUC therapies NUC treatment Decline of antigen load Antigen load IFNα, IFNγ, IFNλ T CELL DYSFUNCTION CD4 RECOVERY OF T CELL RESPONSIVENESS CD4 CD8 CTL -/+ IFN-γ -/+ IL-2 -/+ Proliferation CD4 CD4 CD8 -/+ Modifi ed from: Ferrari C. Gastroenterology 2008 CTL ++ IFN-γ ++ IL-2 ++ Proliferation ++ HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

58 FUTURE POTENTIAL IMMUNE MODOLATORY STRATEGIES TO TREAT HBV INFECTION NUC therapy Virus/antigen load Decline of antigen load Blockade of inhibitory pathways T cell stimulation T cell functional effi ciency Full T cell exhaustion Partial T cell exhaustion Partial T cell restoration

59 Acknowledgments C. Boni V. Barili D. Laccabue L. Talamona M. Rossi C. Cavallo P. Fisicaro A. Penna M. Pilli A. Orlandini T. Giuberti C. Mori G. Missale Laboratory Viral Immunopathology Unit Infectious Diseases and Hepatology Azienda Ospedaliero-Universitaria di Parma, Italy A. Bertoletti Singapore Institute for Clinical Sciences, A*STAR, Singapore P. Lampertico M. Colombo University of Milano, Italy M. Levrero La Sapienza University Rome, Italy