Modeling HBV infection, pathogenesis and therapy in mice:

Targeting the HBV-Macrophage-Stellate cell axis to treat HBV-induced Liver Diseases

Lishan SuLineberger Comprehensive Cancer Center

Department of Microbiology and ImmunologySchool of Medicine

The University of North Carolina at Chapel Hill

HEP DART 2017/December 3-7, 2017/Kona, Hawaii

Today’s Topics:

• Challenges to HBV Cure Therapy (HBsAg loss/HBs Ab+)

Relevant HBV persistence models to study/evaluate HBV cure approachesHBsAg persistence maintains tolerance and develop disease in vivoHBsAg clearance: cccDNA inhibition or HBs removal (RNAi/mAb?)

• HBV immuno-pathogenesis in the human liver

Chimeric mouse models with human liver +/- human immune systemHBV 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 strategiesRemoving HBsAg to induce anti-HBs antibodies and control HBVpre-S1 targeting vaccines to induce nAb and reduce HBsAg tolerance

Outline

HBV induces liver fibrosis and cancer

Chronic HBV infection:(>350 millions)

-Chronic T cell reactions-Chronic inflammation

Liver InjuryNecrosis/ApoptosisRegeneration Fibrosis/Cirrhosis

HepatocellularCarcinoma (HCC)

20-40 years

Immune Diseases Accelerated by Cofactors: HIV-1, alcohol…

Mechanisms and therapeutic targets, and biomarkers?

Models with both human liver and immune cells are needed to study infection and pathology!

Lishan Su/University of North Carolina-Chapel Hill

Rel

evan

ceto

hum

an d

isea

ses

Easeof study

Models of Human Virus Infection & ImmunologyHuman Patients

NHPhu-Mouse

Mouse

Organsex vivo

PBMC/MEFCell Lines

Correlations!MostlyHu-Mice:

Human ImmunityHuman Targets!

Beyond correlation:Define Roles!Causal Effect!Mechanisms

Bill Maher Headline Predictions:

"Scientists Discover Mice Have Been Bullshitting

Them for Decades"

FKBPM Caspase 8FKBP

FKBPM Caspase 8FKBP

FKBPM Caspase 8FKBP

AP20187

Dimerization/Caspase 8 Activation

Dimer

AP20187

Apoptosis of target cells

B.

A.

M-FKBP FKBP Casp8 PolyAAlbumin promoter

0

20

40

60

80

100

120

140

Day -1 Day 0 Day 1 Day 3 Day 6 Day 7

ALT

(U/L

)

Control

AFC8

AFC8no drug

AP20187 AP20187

* *

>12 WeeksHCV or HBV

Lymphoid Organs and Liver Tissues

Rag-/-γC-/- AFC8-hu HSC/TEC/Hep

Human HSC+ Hepatoblasts

C.

D.

DKOhu HSC+Hep txp

AFC8hu HSC+Hep txp

AFC8No transplant

Human Albumin IHC/Liver

LNSpleenThymus

AFC8/Mock AFC8-hu/Mock AFC8-hu/HCV

Sirius Red/Liver Fibrosis

Washburn et. al., Gastroenterology 2011; Bility et. al., Nature Protocol 2013; Bility et. al., J Gastroenterol Hepatol. 2013

AFC8-hu Hep/HSC mice: human immune/liver cells vs. HCV

The NRG/FAH-AFC9 mice suppor t high levels of human liver engraf tment

NRG/FAH

NRG/AFC9 NRG-FAH-AFC9

Serum human ALB

0

0.5

1.0

1.5

2.0

2.5

Hum

an A

LB(m

g/m

l)

NRG-FAHNRG-FAH-AFC9

W6 W10 W15NRG-FAH-AFC9 with (>90%) human hepatocyte engraftment。

Anti-FAH staining

NRG/Fah-AFC9 hu Hep

NRG/FAH-/-

AF0707 (2.9 mg/ml hAlb)

Azuma, H. et al. (2007). "Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice." Nat Biotechnol 25(8): 903-910.

HBV genotype CHBV genotype B

AFC/FRG-hu Hep mice supports persistent HBV infection

HBV genotype C

Weeks post infection

HBVa ba b

MockhC

D68

hCD

3

hC

D45

H&

E

HBV infection in Hu-Hep/HSC mice induces infiltration of human immune cells in the liver

A.

HBV Peptides + hCD28 PHA0

5

10

15

20

25

30 Mock - Sp/LNHBV - Sp/LNHBV - Liver

Fol

d E

xpan

sion

of h

uman

T c

ells

Human CD8+ T cells

HB

V C

ore/

MH

C 0.19% 1.15%

HB

V E

nv/M

HC

HB

V C

ore/

MH

C

Human CD8+ T cells

Non-stimulated human HBV-Specific T Cells

HBV Peptide-stimulated human HBV-Specific T Cells

HBV - LiverHBV – Sp/LN

Mock – Sp/LN HBV – Sp/LN

B.HBV – Liver

0.95%

0.30%0.68%

Mock – Sp/LN

0.04%0.37%

3.3%

0.22%

0.70%0.22%

9.4%0.35%

0.16%0.04%

Liver–Specific Impairment of HBV-

Specific T Cells

NTP-HBV HBV1189

hGFA

Phα

SMA

MT

SR/F

G

11931169 11903Mock

1

Fibr

osis

Hep

-Ste

llate

Cel

lsHBV infection induces human liver fibrosis/human myofibroblasts

Mock HBV

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?!)

Bility et al. 2014; Li, et al. 2014, Murphy et al. 2016

Improved human Hep in humanized liver:1. Improved recipient mice (AFC9/Fah-NRG－hu)2. Improved human fetal Hep (human cytokines)

NTP - HBV Mock

hCD

68

A.

MØ

hCD

206

hCD

163

M2

MØ

hCD

86M

1 M

Ø

HBV

hCD

14/1

6

Control CHB (G1S1) CHB (G1S3) LC (HCC)

CD

68C

D20

6Si

rius

Red

B.

C.

HBV infection is associated with M2 macrophage accumulation in the liver

Bility et al. 2014;2016;2017

Hu-Mice Human Patients

M2-Like Macrophages: Targets in Treating HBV-Induced Liver Fibrosis!

M1／CAMM2／AAM

TAM/MDSC

Anti-Viral immunity? Fibrosis and HCC?

HBV induces M2-like activity in M1-polarized macrophages

Bility et al. 2014;2016;2017

IL10 (M2) IL12 (M1)0

50100150

600700800

1500200025003000

MockHBVMockHBV

Activated M∅

M2

M1

Cyt

okin

e (p

g/m

l)

E.Mock HBV

M1

M2

D.

Mock HBV0

5

10

15

20100

200

300

400N

orm

alize

d A

RG

1 F

old

In

du

ctio

nin

M1

Po

lari

ze

d M

acro

ph

ag

es

F.Arg1 mRNA

Nio et al. in prep.

HBV-stimulated macrophage supernatant activates human hepatic stellate cells

+ nAb

N T C

B MP

9

B MP

9 + n A b

T H P 1 -Mo c k

T H P 1 -HB V

T H P 1 -HB V + n A b

0 .0

0 .5

1 .0

1 .5

2 .0

a S M A

αS

MA

mR

NA

fo

ld c

han

ge **** ******* ****C D

Polyamine inhibitors suppress HBV-induced M2 macrophage

iNOS (M1) Arginase 1 (M2)0

1

2

3

4MOCKMOCK + MGBGHBVHBV + MGBG

Fold

Induc

tion

ofNo

rmali

ze (G

APDH

) Gen

e

Bility et al. in prep.

A.

B.

H&

E (H

epat

is/D

amag

e)

2736-1

2737

2730

2744

2733

2734

2736-1

2737SR

/FG

(Fib

rosis

)

2730

2744

2733

2734

Mock HBV HBV + MGC.

Humanized HSC/Hep Mice

0 2 4 6 9 12 16 wks

Sacrifice

Mock; HBV

A.

MGBG 50 ug/g (5X-1X/WK)

2736-1

2737

2730

2744

2733

2734

Mock HBV

hCD

68 (M

acro

phag

e)

HBV + MG

2736-1

2737

2730

2744

2733

2734

Mock HBV HBV + MG

hCD

163

(M2)

/hiN

OS

(M1)

B.

Polyamine inhibitors suppressHBV-induced M2 and reverse liver fibrosis

Bility et al. in prep.

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

HBV Infection, Pathogenesis & Therapy:Human Stellate Cell Activation vs. Fibrosis/HCC?

Gressner et al. Comparative Hepatology 2007 6:7

???

HBV: Virology/Viral Factors and HBV “Cure”

???HBV+

Cofactors?

TNKpDC

KupfferMf/Iron

Developmental and Cancer Biology

Modeling HBV “Cure” in Mouse ModelsCocktails of novel anti-virals and immuno-therapeutics

Suppress or Remove (cccDNA/HBsAg) & or

Immune Therap.

CureαHBs Ab!

HBVrebound

Anti-HBV NUC RTi Stop NUC RTi

Weeks After Treatment

HBsAgcccDNA

ctrl

neon

ate adult

naive

0

2000

4000

6000

anti-

HBs

(mIU

/ml)

X

XpreC/C

pol

preS1/S2/S

ITR ITR

0 1 2 4 6 8 10 12 14 16 18 20 22 240

200400600800

2000

4000

6000 1x1011vg5x1010vg2x1010vg

weeks after infection

HBsA

g (n

g/m

l)A

B D

> 60 weeks

AAV-HBV1.3x in WT Mice:

Tolerance in AAV-HBV+ Adult/Neonate Mice:

Yang et al. 2014

HBsAg:

HBs VaccineResponse:

C

“easy” to reverse

“hard” to break Anti-HBs IgG

Washburn et al. 2011

AAV8 vs. HBVInduced stable immune tolerance

to target genesAAV vectors form stable episomal circular DNA

0 7 14 28 420

300

600

900

1200 donor from EngerixBdonor from NAb+EngerixB

Days after transfer

HBsA

g (n

g/m

l)

EngerixB

NAb+EngerixB

0

1000

2000

3000

anti-

HBs

IgG2

b m

IU/m

lA

B

C

D

AAVHBV1.3 infection for 3

weeks

d0 d40

NAb

d138

HBs Vaccine

d54

0 14 28 40 54 68 82 96 110

124

138

0

1000

2000

3000

4000 NAb+EngerixBEngerixBNAb

Days after treatment

HBsA

g (n

g/m

l)

HBs mAb “reduced” HBs and reversed tolerance!

De-tolerized donor splenocytesprevented HBV persistence!

High HBV/HBs load induced high tolerance to HBs antigen

Zhu et al. 2016

HBsAg reduction itself is not enough to induce anti-HBs antibody!

PreS1 level is lower than HBsAg both in blood and in liver

A B

C

Bian et al. 2017

preS1 is not tolerized in chronic HBV carrier mice

i.V infection 5x109vg aav-HBV1.3

Days: 0 14 28 42 49 56 63

prime boost

Bleed every week for ELISA testing

Elispots

A B

C INFg ELISPOT

Bian et al. 2017

preS1 vaccination induces HBV nAbB

C D

A

HBV in HepG2-NTCP Cells

Bian et al. 2017

preS1 vaccination reduces HBsAg level/tolerance

i.V infection 5x1010vg aav-HBV1.3

Days: 0 14 28 42 49 56 63 70 84 …

preS1 vaccination

boost

Bleed every week for ELISA testing Elispots

primeHBsAg+CpG

A

B CLiver HBV

Bian et al. 2017

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

HBV “Cure” in Humanized Mice/PatientsCocktails of novel anti-virals and immuno-therapeutics

Suppress or Remove (cccDNA/HBsAg) &

Immune Therap.

CureαHBs Ab!

HBVrebound

Anti-HBV NUC RTi Stop NUC RTi

Weeks After Treatment

HBsAgcccDNA

And treat/prevent HBV-associated liver diseases!

Liang Cheng, Fumi Yasui Feng Li Guangming Li Kouki Nio Natalia Reszka-Blanco

Relevant Funding:UNC: UCRF Grants

NIH: NIAID/NIDDK/NCIRoche/Novartis/GSK/NPBio

Acknowledgements:

Collaborators:Liguo Zhang/Yangxin Fu: IBP/CAS/BeijingZheng Zhang/Fusheng Wang: Beijing 302

Junqi Niu: Jilin UniversityYves Levy/Vaccine Research Institute-VRI/Paris

Colas Tcherakian/Veronique GodotMoses Bility/Uni. Pitt.