targeting the hbv-macrophage-stellate cell axis to treat...
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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.