HBV and the immune response - PDF Free Download

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

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

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

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) - 10-5 - 10 0 5 10 15 20-5 0 5 10 15 20 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=0.05 300 6 200 4 2 100 p=0.0001 p=0.0002 p=0.0001 800 pg/ml 8 pg/ml pg/ml p=0.05 400 IFN-λ1 C 400 400 200 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

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

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

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 2009 1x108 NK cells IFN-γ 20 ALT HBV-specific HBV T cell responses 15 5x107 10 1 5 0 0 2 4 1,000 6 Weeks from infection 8 10 12 Weeks Poor induction of Efficient and timely early intracellularinduction of adaptive innate responses responses 14 16 0 18 ALT IU/L HBV-DNA 10 2,000 25 %IFNg+/NK cells 100 HBV-DNA HBV-DNA copies x 10 6 / ml Clinically overt infections

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 20 10 0 30 RECOVERY PHASE (20 years from recovery) 20 10 0 POLYMERASE X CORE ENVELOPE HLA-A2 restricted HBV peptides Virus control / occult infection

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

HBV-specific T cells in chronic infection

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) 10 8 6 4 2 1 0.1 0.08 0.06 0.04 0.02 0 LIVER BLOOD Mean %TET+/CD8+ intrahepatic cells p=0.0002 6 5 4 3 2 1 0 p=0.006 p=0.03 >105 0-104 104-105 HBV-DNA TITER IU/ml

INTRAHEPATIC HBV-SPECIFIC T CELLS ARE MORE DEEPLY EXHAUSTED THAN THEIR PERIPHERAL BLOOD COUNTERPARTS IN CHRONIC HBV INFECTION 1.5 p=0.0078 p=0.01 p=0.02 1.0 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) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 r = - 0.72 p = 0.014 1x100 1x102 1x104 1x106 HBV-DNA IU/ml 1x108 1x1010

NK cells in chronic infection

NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (I) % IFNγ production NK Total P<0.0001 CD107a % CD107 production NK Total IFN-γ Healthy N=21 25 20 15 10 5 0 Healthy N=29 CHB N=46 CHB N=33 Peppa D. et al Plos Pathogens 2010

NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (II) IL2+IL12 18h %IFNγ + 100 IFN-γ 80 p=0.0045 60 40 20 0 HD CD107a %CD107a+ NK cells 100 anti-nkp30 HBV anti-nkp46 anti-nkg2d 75 50 25 0 HD HBV HD HBV HD HBV Oliviero B et al Gastroenterology 2009

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

NK cell functional dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity (IV) 0.0233 %IFNg+/CD56 bright 100 80 60 40 na iv e C H B H ea lth y NK cells seem to be20 more pathogenic than protective 0 in chronic HBV infection 40 20 CH B na iv e 0 He al th y %CD107a/NKdim 60 Boni C. et al. Unpublished data

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

Different levels of T cell functional efficiency in different conditions of HBV control IFN-γ IL-2 TNF-α 30 25 Mean % IFN-γ, IL-2 and TNF-α +/T cells 20 CD4+ T cell responses 15 10 5 0 30 25 20 CD8+ T cell responses 15 10 5 0 Naïve HBeAg negative CHB Inactive carriers Boni C, Laccabue D et al. Gastroenterology 2012;143:963 973 Occult infections Acute hepatitis B (resolution phase)

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-2 +++ +++ +++ +++ +++ +++ ++ +/- - - + + - - - - - - - - - - - Cytotoxicity Antigen load Sinusoid - - High Wherry EJ et al J.Virol. 77:4911-27;2003 Hepatocyte Space of Disse

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

Effect of long-term NUC therapy on T cell responses

T cell restoration following long-term NUC treatment is efficient in vitro 100 % of HBV dextramer+ CD8 T cells producing 80 60 IFN-γ 40 20 0 60 5 4 3 2 1 0 100 IL-2 In vitro 80 60 CD107a 40 20 0 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

T cell restoration following long-term NUC treatment is partial ex vivo 100 % of HBV dextramer+ CD8 T cells producing 80 60 IFN-γ 40 20 0 60 5 4 3 2 1 0 100 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 40 20 0 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

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

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

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

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

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

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

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

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

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

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

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

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

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. 2014 Liver Blood T cell restoration by 2B4 blockade p=0.012 p=0.04 100 3 p = 0.0003 3 90 p = 0.011 0 70 60 50 40 30 3 20 2.5 10 1 0 0.5 p=0.029 PD-1 2B4 0 CONTROL Anti-PD-L1 1 p = 0.0156 LIVER 0.5 0 p=0.02 LIVER BLOOD 3 50 45 2.5 40 35 1 30 25 CD160 CTLA-4 LAG-3 CD137 20 0.5 15 Schurich A et al Hepatology 2011 10 Fisicaro 0 P. et al. Gastroenterology 2012 5 Anti-PD-L1 CONTROL 0 fold increase 0.5 %IFN-γ+/CD4+CD3+ 1 2.5 80 %COSTIM.+/TET+ %IFN-γ+/CD8+CD3+ 2.5 BLOOD Raziorrouh B et al, Hepatology 2010 p=0.01 Fisicaro P. et al. Gastroenterology 2010, 2012 Nebbia G. et al. Plos One 2012

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

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%

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

PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation 0.0003 Heme biosynthesis 0.0006 Huntington s disease 0.0012 Mitochondrial Gene Expression 0.0012 Translation factors 0.0014 Proteasome degradation 0.0028 Oxidative phosphorylation 0.0306 Metabolic pathways 0.0037 DOWN-REGULATED CELLULAR METABOLISM Myometrial Relaxation and Contraction Pathways 0.0054 Mucin type O-Glycan biosynthesis 0.0074 Glycine, serine and threonine metabolism 0.0083 Vascolar smooth muscle contraction 0.0138 Porphyrin and chlorophyll metabolism 0.0147 Lysosome 0.0155 Purine metabolism 0.0329 Protein processing in endoplasmic reticulum 0.0344 Parkin-Ubiquitin Proteasomal System pathway 0.0375

PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation 0.0003 Heme biosynthesis 0.0006 Huntington s disease 0.0012 Mitochondrial Gene Expression 0.0012 Translation factors 0.0014 Proteasome degradation 0.0028 Oxidative phosphorylation 0.0306 Metabolic pathways 0.0037 Myometrial Relaxation and Contraction Pathways 0.0054 Mucin type O-Glycan biosynthesis 0.0074 Glycine, serine and threonine metabolism 0.0083 Vascolar smooth muscle contraction 0.0138 Porphyrin and chlorophyll metabolism 0.0147 Lysosome 0.0155 Purine metabolism 0.0329 Protein processing in endoplasmic reticulum 0.0344 Parkin-Ubiquitin Proteasomal System pathway 0.0375 DOWN-REGULATED PROTEASOMAL FUNCTION

PATHWAY ANALYSIS PATHWAY p value Antigen processing and presentation 0.0003 Heme biosynthesis 0.0006 Huntington s disease 0.0012 Mitochondrial Gene Expression 0.0012 Translation factors 0.0014 Proteasome degradation 0.0028 Oxidative phosphorylation 0.0306 Metabolic pathways 0.0037 Myometrial Relaxation and Contraction Pathways 0.0054 Mucin type O-Glycan biosynthesis 0.0074 Glycine, serine and threonine metabolism 0.0083 Vascolar smooth muscle contraction 0.0138 Porphyrin and chlorophyll metabolism 0.0147 Lysosome 0.0155 Purine metabolism 0.0329 Protein processing in endoplasmic reticulum 0.0344 Parkin-Ubiquitin Proteasomal System pathway 0.0375 DOWN-REGULATED MITOCHONDRIAL FUNCTION

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?

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

Efficient control of HBV replication by NUC therapy Clinical practice ETV (n=418) 80 85% 95% 96% 99% 68% 60 40 80 84% 91% 90% 91% 65% 60 40 20 20 0 TDF (n=302) 100 Patients % Patients % 100 Slow HBsAg decline during NUC therapy: need of life-long NUC administration 6 12 24 36 0 48 Months 6 12 18 24 30 Reijnders JGP et al J-Hepatol. 2011 Clinical needs in HBV therapy for CH-B: to shorten NUC therapy by accelerating HBsAg clearance

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

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

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. 2013. PLoS ONE 8(5): e63818. doi:10.1371/journal.pone.0063818.

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

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

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

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 103 10 30 35 45 50 60 VV/WT VV/GP33 VV/WT+ αpd-l1 VV/GP33+ αpd-l1 Days post-infection Serum virus (pfu/ml) 105 + α PD-L1 104 103 Vaccine 102 101 10 30 35 45 50 Days post-infection 60 Vaccine + anti-pd-l1

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

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

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

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

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