Hepatitis C virus entry – molecular mechanisms and antiviral targets
Thomas Baumert, MD Professor of Medicine
Inserm Virology Unit 1110 Center for Digestive Disease and Hepatology
University of Strasbourg France
HEPCam
Cambridge, June 27, 2013
HCV entry – a key step within the viral life cycle
Georgel / Schuster / Baumert Trends in Molecular Medicine 2010
First step of virus-host interactions
Required for initiation, spread and maintenance of infection
Mediated by viral envelope glycoproteins and host factors
Target of first line host immune responses – neutralizing antibodies
Important for pathogenesis of HCV infection and liver disease
Important target for antiviral strategies
Model of the HCV Life Cycle
HCV Cell Culture Model • Recombinant viral proteins and virus-like particles (Kato et al. Proc. Natl. Acad. Sci. 1991, Rosa et al. Proc. Natl. Acad. Sci. 1996, Baumert et al. J. Virol. 1998)
• Subgenomic replicons (Lohmann et al. Science 1999, Blight et al. Science 2000)
• Retroviral HCV pseudotypes (HCVpp) (Bartosch et al. J. Exp. Med. 2003, Hsu et al. Proc. Natl. Acad. Sci 2003)
• Recombinant infectious JFH-1 (HCVcc) (Wakita et al. Nat Med. 2005, Lindenbach et al. Science 2005, Zhong et al. PNAS 2005)
• Chimeric reporter viruses and cell-based reporters (Pietschmann et al. PNAS 2006, Ploss et al. PNAS 2010, Jones et al. Nat. Biotechnol. 2010; Gottwein et al. Gastroenterology 2011; Schwartz et al. PNAS 2012)
Milestones: Development of cell culture model systems for the study of HCV life cycle
HCV entry: host factors
Pileri et al. Science 1998 Scarselli et al. EMBO J. 2002 Barth et al. J. Biol. Chem. 2003 Evans et al. Nature 2007
Ploss et al. Nature 2009
Tetraspanin CD81
Scavenger receptor class B type I
Claudin-1 Occludin
Highly sulfated heparan sulfate
(hsHS)
Concepts in molecular virology of HCV entry
Virus-associated lipoproteins and cholesterol are relevant for HCV entry
Cell entry is regulated by host cell kinases
Viral glycoprotein
Capsid
Viral RNA
Envelope VLDL
apoE
apoC
apolipoproteins
Virion-associated lipoproteins and HCV entry
Apolipoproteins are associated with infectious virions in patients and HCVcc (André et al. J. Virol. 2002, Gastaminza et al. J. Virol. 2006, Merz et al. J. Biol. Chem. 2011)
Enveloped subviral particles containing apoB (Scholtes et al. Hepatology 2012)
ApoE is required for viral production and virion infectivity (Chang et al. J. Virol. 2007, Benga et al. Hepatology 2010, Gang et al. Gastroenterology 2011; Lefèvre EASL 2013)
ApoE-syndecan interactions mediate viral entry (Shi et al. J. Virol. 2012; Lefèvre et al. EASL 2013)
HCV-apoE-lipid interactions mediate viral evasion from host neutralizing responses (Felmlee et al. EASL 2013)
Niemann-Pick C1-like cholesterol absorption receptor (NPC1L1) is a co-factor for HCV entry
NPC1L1 - cholesterol-sensing receptor, cholesterol absorption and whole-body cholesterol homeostasis
Re-absorbs cholesterol from bile
Role of NPC1L1 in HCV entry shown by siRNA and antibody studies
NPC1L1 inhibitor Ezetimibe inhibits HCV infection in vitro and in vivo
NPC1L1-mediated HCV entry appears cholesterol-dependent and occurs postbinding
Potential indirect effect by altering membrane cholesterol content
A.
Sainz Jr et al., Nat. Med. 2012
B.
Cartoon from Lupberger / Baumert J. Hepatol. 2012
Concepts in molecular virology of HCV entry
Virus-associated lipoproteins and cholesterol are relevant for HCV entry
Cell entry is regulated by host cell kinases
Identification of host kinases required for HCV entry and infection using a functional siRNA screen
• Functional siRNA HCV entry screen in Huh7 liver-derived cells
• Identification of a functional network of host cell kinases as HCV co-factors
• Identification of a novel class of antivirals
• Investigator-initiated clinical trial
• Among these kinases was EGFR, a well characterized drug target in cancer therapy
Functional genomics for target discovery: siRNA screen identifies HCV entry factors as antiviral targets
Lupberger and Zeisel et al. Nature Medicine 2011
Huh7.5.1 serum-starved cells sucrose-cushion purified HCVpp
EGFR expression and function are relevant for HCV entry
Silencing of EGFR expression inhibits HCV entry in human hepatocytes and viral entry is rescued by exogenous EGFR expression
A
Clinical EGFR inhibitor Erlotinib inhibits dose-dependently HCV entry and infection
EGF increases HCV entry and Erlotinib reverses this increase
B C
Lupberger*, Zeisel* et al., Nat. Med. 2011
PHH
Molecular mechanism: EGFR/HRas signaling promotes formation of the HCV host cell receptor complex
Zona , Lupberger et al. Cell Host & Microbe 2013
• HCV entry requires HRas activation downstream of receptor tyrosine kinase signaling
• HRas associates with membrane microdomains containing entry factors CD81 and claudin-1
• CD81-associated proteins Rap2B and integrin beta1 are additional HCV entry co-factors
• HRas triggers lateral membrane diffusion of CD81 and host entry factor complex formation
Model of HCV entry
Adapted from Gerold & Rice,
Cholesterol modulated by NPC1L1
transferrin receptor 1
Clinical impact of HCV entry: Liver transplantation
HCV-related cirrhosis and hepatocellular carcinoma
Major indications for liver transplantation (LTx)
Universal re-infection of the graft
Absent strategy for prevention of re-infection
Accelerated progression of disease to cirrhosis
Low efficacy and poor tolerance of antiviral therapy
Recurrent liver disease with poor outcome
HCV re-infection after liver transplantation
Early - immediately following transplantation Rapid - viral spread within days following engraftment Efficient - high viral load despite presence of antibodies « Genetic bottleneck »
LTx
Vira
l loa
d
Time
Feliu et al. Liver Transpl. 2004, Schwoerer et al. J. Infect. Dis. 2007
Plasma 7 days after LT
Cloning and sequencing E1-E2 25 clones / time point / patient
Identification of selected and non selected variants
Methods: study of HCV entry and neutralization using the retroviral HCV pseudotype model system
Plasma Before LT
Selected variants
Non selected variants
E1E2 (HCV)
Plasmid 1
Production of HCVpp (E1E2)
Gag-Pol (MLV)
Plasmid 2 Plasmid 3
Luciferase
+
Methods: Bartosch / Cosset J Exp Med 2003; Pestka / Baumert PNAS 2007
Enhanced viral entry and escape from neutralization a key determinants determinants for selection of HCV variants during liver transplantation
B.
A. H
CVp
p en
try(
Log 1
0 RLU
)
C.
Neu
tral
izat
ion
titer
Fafi-Kremer / Baumert J. Exp. Med. 2010 Fofana / Baumert Gastroenterology 2012
• Evolution of viral quasispecies changes following transplantation
• Variants re-infecting the liver graft are characterized • most efficient viral entry • poor neutralization by patient
antibodies • Genetic variability allows the virus to
rapidly adapt, infect the graft and evade neutralizing responses
• Viral entry is a target for prevention and therapy of liver graft infection
Transplant Patient
VL
VA
VL
VC
Before LT After LT
E1 E2
HVR1 HVR2 CD81 Binding Domains
---- A -- S
---- G -- G
VL VC
---- G -- VA
------ R ----
C ----
Q
H
R ---
---
---
T
A
V --------------
------
----
----
----------------- L
------ F aa 39
3 39
7
404
447
458
478
Mapping of mutations mediating enhanced entry and escape from neutralization
Altered cell entry factor use determines viral evasion of escape variants
P01 VL/JFH1
P01 VLVA447 /JFH1
P01 VLVC458+478 /JFH1
A. B.
C. D.
Fofana et al. Gastroenterology 2012 Collaboration R. Bartenschlager, Heidelberg
Viral entry and escape from neutralization are key determinants of HCV liver graft infection
(Fafi-Kremer et al., J Exp Med 2010)
Enhanced entry and neutralization escape through altered cell host receptor use
(Fofana, Fafi-Kremer et al., Gastroenterology 2012)
A novel and clinically important mechanism of viral evasion: co-evolution between receptor usage and escape from neutralization
Viral entry is a promising target for the
development of antiviral strategies
Escape variants
HSLDL-R SR-BI
CD81
CLDN1
OCLN
H+H+
Rab5a
HSLDL-R SR-BI
CD81
CLDN1
OCLN
H+H+
Rab5a
Nonselected variants
HSLDL-R SR-BI
CD81 CLDN1
Viral entry and pathogenesis of acute and chronic infection
Target Examples of compounds Stage References
Heparan sulfate Heparin, SALPs Cell culture Barth et al. J. Virol. 2006, Krepstakis et al. JID 2012
SR-BI
Anti-SR-BI Ab Cell culture
Mouse model
Zeisel et al. Hepatology 2007, Grove et al. J. Virol.
2007, Catanese et al. J. Virol. 2007; Zahid et al. Hepatology 2012, Dorner et al. Nature 2011, Meuleman et al. Hepatology 2011, Lacek et al. J. Hepatol. 2012
Itherx ITX5061 Phase I Syder et al. J. Hepatol. 2011, RCT ongoing
Serum amyloid A Cell culture Lavie et al. Hepatology 2006
CD81 Anti-CD81 Ab
Imidazole based compounds
Mouse model
Cell culture
Vanwolleghem et al. Hepatology 2008
Van Compernolle et al. Virology 2003
CLDN1 Anti-CLDN1 Ab Cell culture
Mouse model
Krieger et al. Hepatology 2010 Fofana et al. Gastroenterology 2010 Mailly et al. 2013 in revision
EGFR Erlotinib Cell culture, Mouse
model, Phase I/IIa
Lupberger, Zeisel et al. Nat. Med. 2011, Diao et al. J. Virol. 2012, Zona et al. Cell Host & Microbe 2013 Investigator-initiated RCT U Hospital Strasbourg
NPC1L1 Anti-NPC1L1 Ab, Ezitimibe Mouse model Sainz Jr et al. Nat. Med. 2012
Internalization /fusion
PS-ON Mouse model Matsumura et al. Gastroenterology 2009
Chloroquine Cell culture Tscherne et al. J. Virol. 2006 Blanchard et al., J. Virol 2006
Silymarin/ Silibinin
Cell culture Phase II
Wagoner et al. Hepatology 2010 and PloS One 2011 Ferenci et al. Gastroenterology 2008, RCT ongoing
Host entry factors as targets for antiviral therapy
Adapted from Zeisel MB, Fofana I, Fafi-Kremer S, Baumert TF J. Hepatol. 2011
Inhibition of HCV infection using antibodies targeting cell entry factor Claudin-1 (CLDN1)
Krieger et al. Hepatology 2010, Fofana et al. Gastroenterology 2010
HCV entry via CD81/CLDN1 co-receptor complex
CLDN1-specific antibody blocks viral infection
Anti-CLDN1 antibodies inhibit infection of HCVpp bearing envelope glycoproteins in a pangenotypic manner
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
0
40
80
120
% H
CVp
p in
fect
ion
% H
CVp
p in
fect
ion
Genotype 6 Genotype 5
Genotype 1a Genotype 1b Genotype 3a
Genotype 4
Genotype 2a
VSV
0
40
80
120
0
40
80
120
0
40
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120
0
40
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120
0
40
80
120
0
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80
120
0
40
80
120
0
40
80
120
0
40
80
120
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
mAb (μg/ml)0.01 0.1 1 10 100
CTRL IgGOM-6E1-B5OM-8A9-A3 OM-6D9-A6OM-7C8-A8OM-4A4-D4OM-7D3-B3
CTRL IgGOM-6E1-B5OM-8A9-A3 OM-6D9-A6OM-7C8-A8OM-4A4-D4OM-7D3-B3
Huh7.5.1 cells
Fofana et al. Gastroenterology 2010
Entry inhibitors inhibit infection of HCV escape variants that are resistant to host neutralizing antibodies
Anti-HCV autologous serum Anti-CLDN1 mAb
0
20
40
60
80
100
+ CTRL serum
+ + transplant
serum
% H
VCpp
ent
ry
autologous anti-HCV transplant serum
P01V
L P0
1VL
P02V
I
P03V
C
P04V
E
P05V
F
P06V
I
P02V
J
P02V
H
P04V
D
P04V
C
P05V
E P0
5VD
P06V
G
P06V
H
HC
V-J
anti-CLDN1 mAb (OM-7D3-B3) +
CTRL mAb
+
% H
CVp
p en
try
P01V
L P0
1VL
P02V
I
P03V
C
P04V
E
P05V
F
P06V
I
P02V
J
P02V
H
P04V
D
P04V
C
P05V
E P0
5VD
P06V
G
P06V
H
HC
V-J
0
20
40
60
80
100
Transplant-derived escape variants
HCVpp
Human Hepatocytes
Anti-CLDN1 mAb
+
Method: Pestka et al. PNAS 2007, Fafi-Kremer et al. JEM 2010
A.
B.
A. daclatasvir + anti-CLDN1 mAb B. sofosbuvir + erlotinib
0 10 20 30 40 50 60 70 80 90
100 110 120 130 140
0.0001 0.001 0.01 0.1 1 10
concentration (nM or µg/ml)
% H
CVc
c in
fect
ion
daclatasvir anti-CLDN1 daclatasvir + 0.01 µg/ml anti-CLDN1
CTRL sofosbuvir erlotinib
sofosbuvir + 0.1 µM erlotinib
CTRL
concentration (µM)
% H
CVc
c in
fect
ion
0 10 20 30 40 50 60 70 80 90
100 110 120 130 140
0.0001 0.001 0.01 0.1 1 10
Synergistic effect of entry inhibitors and DAAs on inhibition of HCV infection
-20-0 0-20
20-40 40-60
% in
hibi
tion
abov
e or
bel
ow e
xpec
ted
0.0001 0.001 0.01 0.1 1 10 10
0.1 0.001
-20
0
20
40
60
0.0001 0.01
1
% in
hibi
tion
abov
e or
bel
ow e
xpec
ted
0.0001 0.001 0.01 0.1 1 10 10
0.1 0.001
-20
0
20
40
60
0.0001 0.01
1
-20-0 0-20
20-40 40-60
Fofana I*, Xiao F* et al. EASL 2013
Combination of DAA with HTEIs prevent antiviral resistance and result in sustained viral clearance in cell culture models
LOQ
Small animal models for the study of HCV infection
• Chimeric human liver uPA-SCID mice (Kneteman et al. Nat. Med. 2001, Meuleman et al. Hepatology 2005, Fafi-Kremer et al. J. Exp. Med. 2010, Lupberger, Zeisel et al. Nat. Med. 2011)
• Transgenic mice expressing HCV entry factors – partial life cycle, immunocompetent (Dorner et al. Nature 2011)
Small animal models for HCV infection
Primary HumanHepatocytes (PHH)
HCVHuman liver
Cartoon modified from Robinet and Baumert J. Hepatol. 2010
uPA-SCID mice grafted with human hepatocytes
Human chimeric mouse liver architecture is similar to human liver
Chimeric human mouse liver Human liver
huCD10 huCLDN1
uninfected
HCV infected
Collaboration: Jane A. McKeating, University of Birmingham
CLDN1-specific mAb completely prevents of HCV infection in human liver chimeric mice
Time (day)
3
4
5
6
7
8
-7 0 7 14 21 28 35 42
HCV
RNA
(Log
10 c
opie
s/m
L)
LOQ
isotype CLDN1 mAb
Mailly et al. 2013, submitted
500µg mAb
inoculation
Genotype 1b Genotype 4
A. B.
3
4
5
6
7
8
9
-7 0 7 14 21 28 35 42 49
HCV
RNA
(Log
10 c
opie
s/m
L)
LOQ
Time (day)
Clearance of persistent HCV infection using a CLDN1-specific antibodies in human liver chimeric mice
3
4
5
6
7
8
-42 -28 -14 0 14 28 42 56 70
HCV
RNA
(Log
10 c
opie
s/m
L)
LOQ
Time (day)
isotype CLDN1 mAb
500µg mAb
Viral load A.
3
4
5
6
7
8
-7 0 7 14 21 28 35 42 49 56 63 70 77 Time (day)
HCV
RNA
(Log
10 c
opie
s/m
L)
LOQ
B.
Genotype 2a Genotype 1b
Mailly et al. 2013, submitted
Inoculum (HCV Jc1) Anti-CLDN1 mAb
VA (1)
VC (2)
VB (10)
Post-treatment serum
Jc1 (7)
V1 (1) V2 (1)
AA 207 240 297 355 358 390 391 394 410 412 449 460 534 626 651 683 712 724HCV JC1 C I H V G S Q K Q S R N V T Y A IVA EVB AVC D AVD P A PVE R AVF L AVG DEL A A HVH F A VVI H AVJ R AVK Y AVL K AVM L AVN F AVO M S A 0
25
50
75
100
125
VSVG Jc1 VA VB VC
Vira
l pp
entr
y in
the
pres
ence
ofan
ti--C
LDN
1 m
Ab
(% o
f con
trol
mA
b)0
25
50
75
100
125
VSVG Jc1 VA VB VC
Vira
l pp
entr
y in
the
pres
ence
ofan
ti--C
LDN
1 m
Ab
(% o
f con
trol
mA
b)
A.
B. C.
Absence of detectable antiviral resistance in HCV infected mice
isotype Anti-CLDN1
mAb
Cell-cell transmission assay Anti-CLDN1 inhibit cell-cell transmission
Mechanism of action 1: anti-CLDN1 mAb dose-dependently inhibits HCV cell-cell transmission and viral spread
Method: Witteveldt et al. J Gen Virol 2009, Lupberger et al. Nat. Med. 2011
0
5
10
15
20
25
30
35
mAb conc. (µg/mL)
Infe
cted
targ
et c
ells
(%)
Control mAb
Anti-CLDN1 (µg/mL)
0.1 1 10 100
3
4
5
6
7
8
0 2 4 6 8 10 12 14 Post-infection time (day)
HCVc
c in
fect
ion
(Log
10 R
LU)
0
Luc-Jc1 infection
Anti-CLDN1 mAb
Anti-CLDN1 inhibit cell spreading
A. B.
C.
F. Xiao, unpublished 2013
Mechanism of action 2: anti-CLDN1 modulates virus-induced signaling in HCV infected cells
MAPK signaling is induced in HCV-infected cells Anti-CLDN1 inhibits ERK1/2 phosphorylation suggesting interference with
virus-induced signaling J. Lupberger, unpublished 2013
Cartoon from Zeisel / Baumert J. Hepatol. 2013
Entry inhibitors - early stage of development (preclinical, early clinical)
Proof-of-concept for prevention and treatment of HCV infection
Pan-genotypic efficacy, effective against escape variants and resistant virus
Efficient and simple antiviral strategy for prevention of liver graft infection
Prevention/treatment of antiviral resistance in chronic infection
Unraveling the mechanisms of HCV entry identifies a novel class of antivirals for prevention of liver graft infection
Acknowledgements Inserm Unit 1110, Laboratory of Excellence HepSys University of Strasbourg, France Joachim Lupberger Laetitia Zona Isabel Fofana Dan Felmlee Fei Xiao Mathieu Lefèvre Catherine Fauvelle Mohammed-Lamine Hafirassou Nauman Zahid Marine Turek Marie Parnot Christine Thumann Laurent Mailly Quentin Lepellier Samira Fafi-Kremer Françoise Stoll-Keller Michel Doffoël François Habersetzer Patrick Pessaux Heidi Barth Mirjam B. Zeisel Catherine Schuster
Molecular Virology, U Heidelberg, Germany Marie-Sophie Huet, Gang Long, Ralf Bartenschlager Inserm U758, Human Virology, ENS Lyon, France Els Verhoeyen, Dimitri Lavillette, François-Loic Cosset Department of Medicine, University Hospital Hamburg Marc Lutgehetmann, Maura Dandri Division of Immunity and Infection, U Birmingham, UK Garrick K. Wilson, Jane A. McKeating
Twincore, Medizinische Hochschule Hannover, Germany S. Haid, E. Steinmann, T. Pietschmann
Center for Vaccinology, Ghent University, Ghent, Belgium Philip Meuleman, Geert Leroux-Roels
Institute of Virology, University Hospital Essen, Germany Michael Roggendorf
Dept. of Biomedicine, Hepatology, University of Basel, CH François Duong, Markus Heim
The Rockefeller University, New York, NY, USA Charles M. Rice
