Modeling HBV infection, pathogenesis and therapy in mice: Targeting the HBV-Macrophage-Stellate cell axis to treat HBV-induced Liver Diseases

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1 Modeling HBV infection, pathogenesis and therapy in mice: Targeting the HBV-Macrophage-Stellate cell axis to treat HBV-induced Liver Diseases Lishan Su Lineberger Comprehensive Cancer Center Department of Microbiology and Immunology School of Medicine The University of North Carolina at Chapel Hill HEP DART 2017/December 3-7, 2017/Kona, Hawaii

2 Today s Topics: Outline Challenges to HBV Cure Therapy (HBsAg loss/hbs Ab+) Relevant HBV persistence models to study/evaluate HBV cure approaches HBsAg persistence maintains tolerance and develop disease in vivo HBsAg clearance: cccdna inhibition or HBs removal (RNAi/mAb?) HBV immuno-pathogenesis in the human liver Chimeric mouse models with human liver +/- human immune system HBV v M2-like macrophages in the liver (HCV, NASH ) Targeting M2-like macrophages to treat HBV-associated liver diseases Modeling CHB functional cure in AAV8-HBV1.3/B6 mice AAV8-HBV1.3 in wt mice as a robust model to test HBV cure strategies Removing HBsAg to induce anti-hbs antibodies and control HBV pre-s1 targeting vaccines to induce nab and reduce HBsAg tolerance

3 HBV induces liver fibrosis and cancer Lishan Su/University of North Carolina-Chapel Hill Immune Diseases Accelerated by Cofactors: HIV-1, alcohol Chronic HBV infection: (>350 millions) -Chronic T cell reactions -Chronic inflammation Liver Injury Necrosis/Apoptosis Regeneration Fibrosis/Cirrhosis years Hepatocellular Carcinoma (HCC) Mechanisms and therapeutic targets, and biomarkers? Models with both human liver and immune cells are needed to study infection and pathology!

4 Models of Human Virus Infection & Immunology Hu-Mice: Human Immunity Human Targets! Beyond correlation: Define Roles! Causal Effect! Mechanisms Bill Maher Headline Predictions: "Scientists Discover Mice Have Been Bullshitting Them for Decades" Relevance to human diseases Human Patients Correlations! Mostly NHP Organs ex vivo Mouse hu-mouse PBMC/MEF Cell Lines Ease of study

5 A. AFC8-hu Hep/HSC mice: human immune/liver cells vs. HCV M FKBP FKBP Caspase 8 M FKBP FKBP Caspase 8 AP20187 Dimer AP20187 M FKBP FKBP Caspase 8 Dimerization/Caspase 8 Activation Apoptosis of target cells C. D. Human HSC+ Hepatoblasts Thymus Rag -/- γ -/- C AFC8-hu HSC/TEC/Hep >12 Weeks HCV or HBV Lymphoid Organs and Liver Tissues Spleen LN B. Albumin promoter M-FKBP FKBP Casp8 PolyA Control AFC8 AFC8 no drug * * AFC8 No transplant Human Albumin IHC/Liver DKO hu HSC+Hep txp AFC8 hu HSC+Hep txp ALT (U/L) Sirius Red/Liver Fibrosis 20 AP20187 AP Day -1 Day 0 Day 1 Day 3 Day 6 Day 7 AFC8/Mock AFC8-hu/Mock AFC8-hu/HCV Washburn et. al., Gastroenterology 2011; Bility et. al., Nature Protocol 2013; Bility et. al., J Gastroenterol Hepatol. 2013

6 The NRG/FAH-AFC9 mice suppor t high levels of human liver engraf tment NRG/FAH Azuma, H. et al. (2007). "Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice." Nat Biotechnol 25(8): NRG/AFC9 NRG-FAH-AFC9 2.5 Serum human ALB NRG-FAH-AFC9 NRG-FAH Anti-FAH staining NRG/FAH-/- Human ALB (mg/ml) NRG/Fah-AFC9 hu Hep 0 W6 W10 W15 NRG-FAH-AFC9 with (>90%) human hepatocyte engraftment AF0707 (2.9 mg/ml halb)

7 AFC/FRG-hu Hep mice supports persistent HBV infection HBV genotype B HBV genotype C HBV genotype C Weeks post infection

8 HBV infection in Hu-Hep/HSC mice induces infiltration of human immune cells in the liver Mock HBV a b a b hcd68 hcd3 hcd45 H&E

9 Human CD8+ T cells Liver Specific Impairment of HBV- Specific T Cells B. HBV Core/MHC Non-stimulated human HBV-Specific T Cells Mock Sp/LN HBV Sp/LN 0.19% 1.15% HBV Liver 0.95% A. Fold Expansion of human T cells Mock - Sp/LN HBV - Sp/LN HBV - Liver HBV Peptides + hcd28 PHA HBV Env/MHC HBV Core/MHC Human CD8+ T cells HBV Peptide-stimulated human HBV-Specific T Cells Mock Sp/LN HBV Sp/LN HBV - Liver 9.4% 0.35% 3.3% 0.68% 0.30% 0.22% 0.37% 0.04% 0.04% 0.16% 0.22% 0.70%

10 HBV infection induces human liver fibrosis/human myofibroblasts Mock HBV Fibrosis Hep-Stellate Cells hgfap hαsma MT SR/FG NTP-HBV 1 3 Mock HBV 1193

11 HBV infection in humanized HSC/Hep mice Hu-HSC/Hep mice efficiently repopulate human immune cells but only low human liver cells (5~10%) Hu-HSC/Hep mice can be persistently infected with patient/cloned HBV isolates (biologic or molecular clones) HBV infection induces specific human immune responses, including T and B cell responses (liver-specific tolerance?) Hu-HSC/Hep mice develop liver fibrosis after HBV infection: human Stellate cell activation (human only?!) Improved human Hep in humanized liver: 1. Improved recipient mice (AFC9/Fah-NRG-hu) 2. Improved human fetal Hep (human cytokines) Bility et al. 2014; Li, et al. 2014, Murphy et al. 2016

12 HBV infection is associated with M2 macrophage accumulation in the liver A. NTP - HBV Hu-Mice Mock HBV B. Human Patients hcd68 Control CHB (G1S1) CHB (G1S3) LC (HCC) M1 MØ hcd86 M2 MØ hcd206 hcd16 3 MØ hcd14/16 CD206 CD68 C. Sirius Red Bility et al. 2014;2016;2017

13 M2-Like Macrophages: Targets in Treating HBV-Induced Liver Fibrosis! M1/CAM M2/AAM TAM/MDSC Anti-Viral immunity? Fibrosis and HCC?

14 HBV induces M2-like activity in M1-polarized macrophages M1 D. M2 Mock HBV E. Normalized ARG1 Fold Induction in M1 Polarized Macrophages Arg1 mrna Mock HBV F. Cytokine (pg/ml) Activated M Mock HBV Mock HBV M2 M1 IL10 (M2) IL12 (M1) Bility et al. 2014;2016;2017

15 HBV-stimulated macrophage supernatant activates human hepatic stellate cells C + nab D α SMA mrna fold change **** **** asma **** *** NTC BMP9 BMP9+nAb TH P1-M ock TH P1-H B V TH P1-H B V+nA b Nio et al. in prep.

16 Polyamine inhibitors suppress HBV-induced M2 macrophage B. Fold Induction of Normalize (GAPDH) Gene MOCK MOCK + MGBG HBV HBV + MGBG 0 inos (M1) Arginase 1 (M2) A. Bility et al. in prep.

17 Polyamine inhibitors suppress HBV-induced M2 and reverse liver fibrosis A. Humanized HSC/Hep Mice Sacrifice wks C. Mock HBV HBV + MG B. Mock; HBV hcd68 (Macrophage) hcd163 (M2)/hiNOS (M1) MGBG 50 ug/g (5X-1X/WK) Mock HBV HBV + MG Mock HBV HBV + MG H&E (Hepatis/Damage) SR/FG (Fibrosis) Bility et al. in prep.

18 Summary Hu-HSC/Hep mice efficiently repopulate human immune cells and human liver cells (5-10%?) Hu-HSC/Hep mice can be persistently infected with patient/cloned HBV/HCV isolates (biologic or molecular clones) HBV/HCV infection induces specific human immune responses, including T and B cell responses (liver-specific tolerance?) Hu-HSC/Hep mice develop liver fibrosis after HBV infection: human Stellate cell activation (human HepSC activation only?) HBV/HCV infection induces M2-like macrophages, fibrosis and Oxidative Stress/DNA damage (HCC development and cofactors?) Similar pathology is observed in HBV/HCV-infected patients HBV/HCV are able to reprogram/enhance M1/M2 to path. Mac. M2 inhibitors can reverse HBV-associated liver diseases

19 HBV Infection, Pathogenesis & Therapy: Human Stellate Cell Activation vs. Fibrosis/HCC? HBV: Virology/Viral Factors and HBV Cure T NK pdc??? Kupffer Mf/Iron HBV+ Cofactors???? Developmental and Cancer Biology Gressner et al. Comparative Hepatology :7

20 Modeling HBV Cure in Mouse Models Cocktails of novel anti-virals and immuno-therapeutics Anti-HBV NUC RTi Stop NUC RTi Suppress or Remove (cccdna/hbsag) & or Immune Therap. HBsAg cccdna HBV rebound Cure αhbs Ab! Weeks After Treatment

21 AAV-HBV1.3x in WT Mice: A prec/c pres1/s2/s X ITR X pol ITR B HBsAg (ng/ml) HBsAg: 4 6 weeks after infection 1x10 11 vg 5x10 10 vg 2x10 10 vg hard to break easy to reverse > 60 weeks D Anti-HBs IgG C anti-hbs (miu/ml) Tolerance in AAV-HBV+ Adult/Neonate Mice: HBs Vaccine Response: AAV8 vs. HBV Induced stable immune tolerance to target genes AAV vectors form stable episomal circular DNA ctrl neonate adult naive Washburn et al Yang et al. 2014

22 HBs mab reduced HBs and reversed tolerance! A d0 C NAb anti-hbs IgG2b miu/ml High HBV/HBs load induced high tolerance to HBs antigen AAVHBV1.3 infection for 3 weeks d40 EngerixB d54 HBs Vaccine NAb+EngerixB d138 HBsAg reduction itself is not enough to induce anti-hbs antibody! B D HBsAg (ng/ml) HBsAg (ng/ml) donor from EngerixB donor from NAb+EngerixB Days after treatment Days after transfer NAb+EngerixB EngerixB NAb De-tolerized donor splenocytes prevented HBV persistence! 42 Zhu et al. 2016

23 PreS1 level is lower than HBsAg both in blood and in liver A B C Bian et al. 2017

24 pres1 is not tolerized in chronic HBV carrier mice A B i.v infection 5x10 9 vg aav-hbv1.3 prime boost Elispots Days: Bleed every week for ELISA testing C INFg ELISPOT Bian et al. 2017

25 A pres1 vaccination induces HBV nab B C HBV in HepG2-NTCP Cells D Bian et al. 2017

26 pres1 vaccination reduces HBsAg level/tolerance A i.v infection 5x10 10 vg aav-hbv1.3 pres1 vaccination prime boost HBsAg+CpG Days: Bleed every week for ELISA testing Elispots B C Liver HBV Bian et al. 2017

27 Summary AAV8-HBV establishes persistent HBV production in WT mice AAV8-HBV induces immune tolerance with no liver injury CpG can reverse T/B tolerance to low persistent HBV to control AAV8-HBV, with no apparent liver injury Plasma HBsAg and hepatic HBV HBsAg levels determine response to therapeutic interventions PreS1-based therapeutic vaccines show great promise to induce nab and to reduce HBsAg tolerance. Cocktail combination therapeutics (NUCi, cccdna/hbs inhibitors and immune modulators) will be needed to treat hosts with high levels of HBV/HBsAg. Washburn, 2011,Yang et al. 2014, Zhu et al. 2016, Bian et al. 2017

28 HBV Cure in Humanized Mice/Patients Cocktails of novel anti-virals and immuno-therapeutics Anti-HBV NUC RTi Stop NUC RTi Suppress or Remove (cccdna/hbsag) & Immune Therap. HBsAg cccdna HBV rebound Cure αhbs Ab! Weeks After Treatment And treat/prevent HBV-associated liver diseases!

29 Acknowledgements: Liang Cheng, Fumi Yasui Feng Li Guangming Li Kouki Nio Natalia Reszka-Blanco Moses Bility/Uni. Pitt. Collaborators: Liguo Zhang/Yangxin Fu: IBP/CAS/Beijing Zheng Zhang/Fusheng Wang: Beijing 302 Junqi Niu: Jilin University Yves Levy/Vaccine Research Institute-VRI/Paris Colas Tcherakian/Veronique Godot Relevant Funding: UNC: UCRF Grants NIH: NIAID/NIDDK/NCI Roche/Novartis/GSK/NPBio