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

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 11 genes Wieland S et al. PNAS 24

5 HBV is a poor inducer of innate responses Cytokine and chemokine production in acute HBV infection is significantly more Fold changes in group mean cytokine level modest and delayed compared with acute HIV infection (Stacey AR J. Virol. 29) Time (days since T) Time (days since T) HIV infection HBV infection Low production of type I IFN, IL-15 and IFN-λ1, associated with high serum IL-1 levels, at the early stages of HBV infection (Dunn C. et al Gastroenterology 29)

6 Is HBV able to inhibit innate responses? HBsAg HBeAg HBx HBVp ol

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 T cell inhibition to avoid excessive clearance of HBV activation damage Dunn C et al Gastroenterology 29 1x18 1 HBV-DNA HBV-DNA 1 NK cells 2ALT HBV-specific HBV T cell responses 15 5x , 2 2, 25 %IFNg+/NK cells 1 6 Weeks from infection Weeks Poor induction of Efficient and timely early intracellularinduction of adaptive innate responses responses ALT IU/L HBV-DNA copies x 1 6 / ml Clinically overt infections

9 Maturation of long-lasting memory T cell responses in selflimited HBV infections HBV INFECTION Strong, multi-specific, T1 oriented T cell responses % CD8-mediated cytotoxicity Self-limited 4 Long-lasting protective responses 3 ACUTE PHASE RECOVERY PHASE (2 years from recovery) 2 1 POLYMERASE X CORE HLA-A2 restricted HBV peptides ENVELOPE 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 21) LIVER BLOOD Mean %TET+/CD8+ intrahepatic cells p= p=.6 p=.3 > 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=.78 p=.1 p=.2 1. BLOOD.5 LIVER TNF-α /IL2 HBV LIVER % CD4+CD8 contr CD4 AND CD8 T CELLS (Fisicaro P. et al. Gastroenterology 212 and personal comunication) r = -.72 p =.14 1x1 1x12 1x14 1x16 HBV-DNA IU/ml 1x18 1x11

14 NK cells in chronic infection

15 NK cell functional dichotomy in chronic HBV infection Impaired production with normal cytotoxicity (I) P<.1 % IFNγ production NK Total 25 CD17a % CD17 production NK Total Healthy CHB N=29 N=46 Healthy CHB N=21 N=33 Peppa D. et al Plos Pathogens 21

16 NK cell functional dichotomy in chronic HBV infection Impaired production with normal cytotoxicity (II) IL2+IL12 18h %IFNγ p= HD CD17a %CD17a+ NK cells 1 anti-nkp3 HBV anti-nkp46 anti-nkg2d HD HBV HD HBV HD HBV Oliviero B et al Gastroenterology 29

17 NK cell functional dichotomy in chronic HBV infection Impaired production with normal cytotoxicity (III) IL12+IL18 24h P<.1 Tjwa E. et al. Journal of Hepatology 211

18 NK cell functional dichotomy in chronic HBV infection Impaired production with normal cytotoxicity (IV).233 %IFNg+/CD56 bright iv e na C H B H ea lth y NK cells seem to be2more pathogenic than protective in chronic HBV infection 4 2 CH B na ive H ea lth y %CD17a/NKdim 6 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 T cell dysfunction chronic HBV -Mechanisms Mechanisms of of T cell dysfunction in chronicinhbv infection infection - 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 IL-2 TNF-α 3 25 Mean %, IL-2 and TNF-α +/T cells 2 CD4+ T cell responses CD8+ T cell responses Naïve HBeAg negative CHB Inactive carriers Boni C, Laccabue D et al. Gastroenterology 212;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) Proliferation Acute infection Naïve CD8 cell en e tig anc n A ar cle Memory CD8 cell A pe ntige rsi ste n nc e Effector CD8 cell +++ Proliferation Functional T cell Partial Exhaustion I Partial Exhaustion II Tolerizing liver environment Full Exhaustion Death deletion TNF-α TNF-α IL IL-2 Cytotoxicity ++ Cytotoxicity / Antigen load Sinusoid - - Space of Disse High Wherry EJ et al J.Virol. 77: ;23 Hepatocyte

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 Effector CD8 cell +Ag d re en tig n A pe Ant rs ige is te n nc e Antigen decline Rapid Proliferation / Differentiation a cle 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 1 % of HBV dextramer+ CD8 T cells producing IL CD17a 4 2 Naive CHB NUC treated HBsAg neg NUC treated HBsAg neg NUC treated Acute hepatitis B HBsAg pos (follow-up) medium peptide medium % % 1% 72% peptide % 88.7% FLU CD8 CD8 Dext core Acute hepatitis B (follow-up) In vitro % Dext FLU 29% CMV Dext core Dext CMV Boni C. et al. Gastroenterology 212

25 T cell restoration following long-term NUC treatment is partial ex vivo 1 % of HBV dextramer+ CD8 T cells producing Ex vivo In vitro IL-2 Restoration of the T cell function is efficient in vitro but only partial ex vivo8 even following complete control of virus 6 replication and decline of antigen CD17a 4 2 Naive CHB NUC treated HBsAg neg NUC treated HBsAg neg NUC treated Acute hepatitis B HBsAg pos (follow-up) medium peptide medium % % 1% 72% % 88.7% FLU CD8 CD8 Dext core Acute hepatitis B (follow-up) peptide % Dext FLU 29% CMV Dext core Dext CMV Boni C. et al. Gastroenterology 212

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 Modified from U. Protzer et al. Nature Reviews in Immunology 212

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. 28

29 INTRAHEPATIC INHIBITORY MECHANISMS Modified from U. Protzer et al. Nature Reviews in Immunology 212

30 MECHANISMS OF HEPATIC TOLERANCE: IMMUNOSUPPRESSIVE CYTOKINE MILIEU TGF-β Hepatocytes StellateCell SPECE OF DISSE Dendritic cell Kuppfer Cell Dendritic cell HEPATIC SINUSOID Sinusoidal endothelial cells IL-1

31 THE IMMUNOSUPPRESSIVE CYTOKINE MILIEU CAN IMPAIR PRODUCTION BY NK CELLS LIMITING THEIR ANTI-VIRAL ACTIVITY HBV infected hepatocytes VIRAL PERSISTENCE AND LIVER DAMAGE TRAIL-R2 TRAIL NK cell NK cell NK cell IL-1 and TGFβ-mediated suppression of production by NK cells NK cell Preserved cytolytic activity Dunn et al J.Exp.Med 27 Peppa et al PloS Pathogens 21

32 NK CELL MEDIATED DELETION OF HBV-SPECIFIC T CELLS HBV infected hepatocytes VIRAL PERSISTENCE AND LIVER DAMAGE TRAIL-R2 TRAIL NK cell NK cell TRAILmediated T cell deletion TRAIL-R2 NK cell NK cell T cell Peppa et al. J.Exp.Med. 212

33 INTRAHEPATIC INHIBITORY MECHANISMS Bim mediates deletion of antigen-specific CD8 cells in patients unable to control infection (Lopes et al. J.Clin.Invest. 28) Bim mediates premature death of CD8 T cells following intrahepatic antigen presentation (Holtz et al Gastroenterology 28) Modified from U. Protzer et al. Nature Reviews in Immunology 212

34 INTRAHEPATIC INHIBITORY MECHANISMS Modified from U. Protzer et al. Nature Reviews in Immunology 212

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 OX4L CTLA-4 CD28 ICOS INHIBITION CD7 CD4 4-1BBL OX4 CD27 CD4L 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 PDL1 Hepatocytes StellateCell 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 StellateCell SPECE OF DISSE HBVspecific T cell Kupffer Cell Dendritic cell Dendritic cell HEPATIC SINUSOID Sinusoidal endothelial cells

38 Expression of various inhibitory receptors on circulating and T cell restoration by Tim-3 blockade intrahepatic virus-specific CD8 cells of patients with chronic HBV infection Control Anti-TIM-3 cell restoration CTLA-4 blockade T cellt restoration by by PD-1/PD-L1 blockade Bengsch et al J.Hepatol. 214 Liver Blood Tp=.12 cell restoration by 2B4 blockade p= p =.3 9 p = p=.29 PD-1 2B4 CONTROL Anti-PD-L1 1 p =.156 LIVER.5 p=.2 LIVER BLOOD CD16 CTLA-4 LAG-3 CD A et al Hepatology 211 Schurich Fisicaro P.1et al. Gastroenterology Anti-PD-L1 CONTROL fold increase 1 %+/CD4+CD3+ 8 %COSTIM.+/TET+ %+/CD8+CD BLOOD Raziorrouh B et al, Hepatology 21 p=.1 Fisicaro P. et al. Gastroenterology 21, 212 Nebbia G. et al. Plos One 212

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 atient H1 2 H2 3 H3 P A A A A A ction E E E E E infe LT ACUT 785 ACUT 98 ACUT 59 ACUT 118 ACUT 11 R RESO LVED HEP B 6 R RESO LVED HEP B 7 R RESO LVED HEP B R CONTROLS RESO LVED HEP B SPECIFICITY CELL 2 H C H C H C THY NIC THY NIC THY NIC HEAL CHRO HEAL CHRO HEAL CHRO LU LU 6 LU 8 A Pre-sorting Isolation of HBV/FLU-specific CD8+ T cells by cell sorting.4% RNA extraction and amplification Gene expression by microarray analysis (4x44K Agilent) F 4 VALIDATION AND DISCOVERY OF NEW TARGETS F F 9 6 Post-sorting 98.7%

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

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

43 Efficient control of HBV replication by NUC therapy Clinical practice ETV (n=418) 1 85% 95% 96% 99% Patients % Patients % % 91% 9% 91% 65% TDF (n=32) % Slow HBsAg decline during NUC therapy: need of life-long NUC administration Months Reijnders JGP et al J-Hepatol. 211 Clinical needs in HBV therapy for CH-B: to shorten NUC therapy by accelerating HBsAg clearance

44 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 CD4 CD8 CTL IL-2 Proliferation CD4 CD8 -/+ -/+ -/+ -/+ Modified from: Ferrari C. Gastroenterology 28 CTL IL-2 Proliferation HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

45 EFFECT OF ANTI-PD-1 THERAPY ON HCV INFECTED CHIMPANZEES Effect on viral load Fuller MJ et al. PNAS 213 Effect on magnitude of T cell responses

46 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.3mg/kg -1mg/kg 3 subjects w/ > 4 log HCV RNA decline: All 3 received 1mg/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:1.1371/journal.pone

47 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 CD4 CD8 CTL IL-2 Proliferation CD4 CD8 -/+ -/+ -/+ -/+ Modified from: Ferrari C. Gastroenterology 28 CTL IL-2 Proliferation HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

48 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 CD4 CD8 CTL IL-2 Proliferation CD4 CD8 -/+ -/+ -/+ -/+ Modified from: Ferrari C. Gastroenterology 28 CTL IL-2 Proliferation T CELL VACCINES Recombinant (Gilead) DNA (Transgene) Peptides (Immune Targeting System) HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

49 Synergistic effect of PD-L1 blockade and therapeutic vaccination on T cell responses and viral control (Liu J. et al. PLOS Pathogens 214) Vaccine + anti-pd-l1 T cell immunity Vaccine Control Entecavir Entecavir + DNA vaccination Entecavir + DNA vaccination + anti-pdl1 WHA replication

50 Synergistic effect of PD-L1 blockade and therapeutic vaccination on T cell responses and viral control (Ha S-J. et al. J. Exp. Med. 28) + α PD-L1 DbGP33-41-specific CD8+T cells/16 PBMC 15 Vaccine + anti-pd-l1 14 Vaccine Days post-infection + α PD-L1 15 Serum virus (pfu/ml) VV/WT VV/GP33 VV/WT+ αpd-l1 VV/GP33+ αpd-l Vaccine Days post-infection 6 Vaccine + anti-pd-l1

51 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 CD4 CD8 CTL IL-2 Proliferation CD4 CD8 -/+ -/+ -/+ -/+ Modified from: Ferrari C. Gastroenterology 28 CTL IL-2 Proliferation TLR-7 AGONISTS GS-962 preclinical studies: HBsAg and HBV-DNA reduction in woodchucks and chimpanzees Favorable safety profile in healthy donors HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

52 TLR8 agonists can trigger potent activation of innate immune cells in human liver TLR agonists TLR7 TLR8 Hepatic monocytes IL18 IL12 CD161bright MAIT NKbright J.Jo et al. PLOS Pathogens 214

53 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 CD4 CD8 CTL IL-2 Proliferation CD4 CD8 -/+ -/+ -/+ -/+ Modified from: Ferrari C. Gastroenterology 28 CTL IL-2 Proliferation HBsAg CLEARANCE ANTI-HBs SEROCONVERSION

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

55 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 P. Lampertico M. Colombo Singapore Institute for Clinical University of Milano, Italy Sciences, A*STAR, Singapore M. Levrero La Sapienza University Rome, Italy