Pr. Christian TREPO
INSERM- U1052-LYON-FRANCE
HOPITAL DE LA CROIX-ROUSSEHEPATOGASTROENTEROLOGIE-LYON-FRANCE
HBV infection
450 Million
Chronic
carriers
Cirrhosis
Hepatocellular carcinoma
> 2 millions
deaths/an
May we improve actual HBV treatments?
• Virostatic
Immunotherapy
•Expensive/life lasting
•Not affordable for all
Ghada
KHAWAJA
- Stimulate the immune system
- Induce immunotolerance break
anti HBe/ Anti-HBs
23 mars 2012
2
30 Juin 2011
Chronic HBV Infection
HBV
Strong T response,
polyclonal, multi-spécific
Acute infection
Acquired Immune R: weakT
Innate response: IFN alpha =
0, alteration mDc, pDc
No anti-HBs
Chronic
Hepatitis B
Resolved
hepatitis
Strong T response,
Th1 ++
Viral persistence
Immunotolerance break may happen
observed in chronic carriers with spontaneous resolution or following IFN-α or
lamivudine treatments (Boni, 1998; Boni, 2001)
Very first immunotherapeutic treatments of HBV carriers disapointings
New alternatives?
Ghada
KHAWAJA
23 mars 2012
3
30 Juin 2011
New anti HBV strategy?
(1) a targeted immuno-therapeutic strategy aimed at
mobilizing a patient’s own biodefense system de novo
to prevent diseases / eliminate persistent infection.
(2) applicable to enhancing Ag-specific immunity.
Primary
infection
Prophylactic
Vaccine
Immuno
Therapeutic
approach
persistent
infection
Therapeutic
Vaccine +…?
Disease
30 Juin 2011
HEPADNAVIRUSES
Orthohepadnavirus
HBV - Humans
WHV - Woodchucks
GSHV- ground squirrel
TSHV – Tree squirrel
ASHBV – Artic squirrel
Avihepadnavirus
DHBV - Duck
HHBV - Heron
SGHBV - Goose
STHBV - Stork
Old world monkeys :
- Gibbon (GiHBV)
- Gorilla (GoHBV)
- Orang-outang (OuHV)
- Chimpanzee (ChHBV)
New world monkeys :
- Woolly Monkey (WMHBV)
30 Juin 2011
Models for HBV infection and novel therapeutic approaches study
•
HBV – extremely narrow host range
 Animal models
• HBV / Chimpanzee
• WHV / woodchuck
•
HBV/ humans
Woodchuck
Hepatitis Virus
(WHV)
Host : american
woodchuck
DHBV / duck
Chimpanzee
Duck Hepatitis Virus
(DHBV)
Host : domestic
Pekin duck
30 Juin 2011
ANIMAL MODELS FOR HBV :
AIMING AT NEW IMMUNOTHERAPEUTIC
APPROACHES
1) Woodchucks
2) Chimpanzees and apes
3) Towards a small primate model (macaque)
for hepatitis B
WHV/Woodchuck (Marmota monax)
• WHV and HBV have high similarities in morphology, genome and replication
• WHV and HBV infections resemble in
• natural history of infection: chronicity, HCC
• host immune respones at B- and T-cell levels
• Woodchuck has been used to study:
• Pathogenesis of hepadnavirus infection
• New vaccines and therapeutic vaccination
• Antiviral drugs
What did we learn from the woodchuck model for the understanding of
hepadnaviral infection
1. Early T cell immune response after infection prevents chronic outcome
(Menne, Roggendorf: J. Virol. 2003; Webster et al. Hepatology 2000)
2. High rate of HCC in woodchuck is caused by integration of WHV DNA
near c-myc of n-my (Möröy T........C, Tiollais P, Buendia MA.Nature,1986.)
3. Immunization with core protein or DNA vaccines expressing WHV core
prevents infection with WHV
(Schödel, Roggendorf: Vaccine 1993, Siegel,Lu Roggendorf,J Virol 2001
Kosinska Talk 123)
4. Intra hepatic expression of wIFN-α and less efficient wIFN-γ reduces WHV
replication in
chronic carriers using adenoviral vectors
(Fiedler, Roggendorf: J. Virol. 2004,Zhu Y, Mason WS, Jilbert AR Virology 2004
5. Combination of antiviral treatment and therapeutic vaccination (WHcAg, WHsAg)
prolongs down-regulation of WHV replication after stopping antiviral therapy
(Lu, Roggendorf, Menne et al. J. Virol. 2002, J. Virol. 2003)
Chronic Outcome
Resolved Outcome
Serum Markers
Serum Markers
Serum
Viral load
WHV7
Acute
Liver Injury
0
4
WHV8
8
12
16 20 24
0
Age (weeks)
Menne, Roggendorf et al 2007
4
8
12
16 20
24
Woodchuck Model
Serum WHV DNA Levels
(% of control)
10000
LVD 5 mg/kg/day
Treatment Period
1000
Placebo
100
10
1
0,1
ETV 0.5 mg/kg/wk
0,01
0,001
ETV 5mg /Kg
0,0001
ETV 0.1 mg/kg/day
0,00001
0
5
10
15
Weeks
20
25
The woodchuck as a preclinical model for pathogenesis
and therapy of chronic hepatitis B
•
Woodchuck hepatits virus (WHV)
the most closely related to HBV
•
Similarity in:
– virion structure, genomic organization
and mechanism of replication
– course of
chronic
infection:
self-limiting
and
– pathogenesis and profiles of immune
response, HCC development
•
Limitations:
– expensive model – limited number of
animals ,no inbread animals
– lack of sophisticated immunological tools
to examine virus-specific T cell response
Menne S, et al. J. Virol. 1997
Lu M, J Virol. 1999
Siegel F, Lu M, Roggendorf M.2001
Menne S, et al. J. Virol. 2002
Lu M, et al. J. Virol. 2005
Frank I, et al. J Virol. 2007
E. Zhang E. et al PLoS One 2011;
New concept of combination therapy
Treatment with
nucleosid analoga
Viral load
Control group
Vaccine group
time
HBV DNA vaccine
Booster injection
Anti- PDL1
preclinical studies on therapeutic vaccination performed in
the woodchuck(Summary)
Study
#1
#2
#3
#4
Total
Number of
treated
animals
Antiviral
treatment
9
ETV
0,5mg/kg
6
ETV
0.2mg
5
ETV
0,2mg/kg
3
ETV
0,2mg/kg
Duration
month
Vaccine
Number
of shots
6
DNA vaccine
WhsAg
WhcAg
Delayed
rebound
WHV DNA neg
in follow up
6
9/9
1/7 (14,3%)
12
DNA vaccin
WhsAg
WhcAg
12
6/6
2/6 (33,3%)
6
DNA vaccine
WhcAg
Adenov.
10
5/5
2/5 (40,0%)
6
DNA vaccine
WhsAg
WhcAg
Anti- PDL1
12
3/3
2/3 (66,7%)
23/23
0/10
7/21 (33,3%)
0/10
23 vaccinated animals
10 control animals 4 studies
#1. M Lu, et al. J Virol 2007.
#3. A. Kosinska,el al PLOS Patho. 2013
Outcome
#2M.Lu et.a.t unpubl.
#4. Jia Liu et al, PLOS Pathogens 2014
ANIMAL MODELS FOR HBV :
AIMING AT NEW IMMUNOTHERAPEUTIC
APPROACHES
1) Woodchucks
2) Chimpanzees and apes
3) Towards a small primate model (macaque)
for hepatitis B
HBV prevalence among primates
HBV DNA detection
n= 16
Wooly
monkey
Singe
laineux
n= 347
Gibbon (Nomascus spp.)
n= 9
Gibbon (Hylobates spp.)
Orang-outan
Bonobo
Chimpanzé
Gorille
0,00%
n= 531
n= 27
n= 735
n= 85
10,00%
20,00%
30,00%
40,00%
50,00%
30 Juin 2011
Hepadnavirus in great Apes
ChHBV
Asymptomatic
Low vireamia
HBV vaccine
development
11 %
divergence/HBV
OuHV
Asymptomatic
Low vireamia
>8 %/HBV
GiHBV
Asymptomatic
Low vireamia
10 %/HBV
GoHBV
Asymptomatic
Low vireamia
>8 %/HBV
WMHBV
Asymptomatic
Low vireamia
1 fulminant
hepatitis
14 %
divergence/HBV
30 Juin 2011
Phylogeny of HBV
Wooly monkey
Norder et al,2004
30 Juin 2011
Innate responses and HBV infection
The reasons of the controversy
In vivo models
Cell culture models
Hepatoma
cell lines
HepaRG cells
Human progenitor cells
susceptible to HBV (PNAS
2002, Gastroenterology 2004)
Acute versus chronic infection study models
PHH
Is HBV a stealth virus ?
Innate immune
response genes
Adaptive immune
response genes
Wieland S et al, PNAS 2004
HBV a poorly sensed virus
• Weak induction of early innate responses
– Wieland et al, PNAS 2004
– Stacey et al, J Virol 2009
– Dunn et al, Gastroenterology 2009
• IL10 induction
• Transient inhibition of NK and T cell responses
HBV can induce innate responses
• HBV can be sensed by infected cells or cells of
innate immunity
– In vivo
• In patients: early induction of NK and NT cell responses
- Fisicaro et al, Gut 2009
• In woodchucks: Guy et al, J Virol 2009
– In hepatocyte culture
• Role of Kuppfer cells: Hosel et al, Hepatology 2009
• Role of Hepatocytes: Lucifora et al, Hepatology 2010
ANIMAL MODELS FOR HBV :
AIMING AT NEW IMMUNOTHERAPEUTIC
APPROACHES
1) Woodchucks
2) Chimpanzees and apes
3) Towards a small primate model (macaque)
for hepatitis B
Searching HBV infections
among macaques
Hominoidea
Catarhinae
• 20 sylvanus
Macaques/Morocco
Hominidae
Ponginae
Haplorhini
C.cephus
Cercopithecidae
Cercopithecidae
Primates
Macaque
Platyrhinae
Colobinae
M.sylvanus
120
cynomolgus
M.cynomolgus(M.fasicularis)
•
macaques/Asia
Tarsioidea
Lemuroidea
• 120 cynomolgus
Macaques/ Mauritius
Lorisoidea
Passage de la barrière d’espèce, Lucifora, Vincent et al., Hepatology,
2010, Gheit et al, 2002, Vincent et al, En préparation
30 Juin 2011
HEPATOLOGY IN PRESS
HBV in Macaques
• Editorial – Hepatology (May 23, 2013)
Persistent Human Hepatitis B Virus Infection in Cynomolgus Monkeys: A
Novel Animal Model in The Search for a Cure?
J Bukh, R.E. Lanford, and R.H. Purcell
• Comment – nature/middleeast (May 15, 2013)
Macaques-new animal models to test anti-HBV drugs and vaccines. B.
Das
30 Juin 2011
Serum HBV among Macaques
of different origins
120 cynomolgus
M. from Asia
20 sylvanus M
from Morocco
All remained
negative for
HBV DNA by
PCR
30 Juin 2011
HBV among Macaques
from Mauritius
50 Liver biopsies
42 % (21/50)
ADN +
HBsAg+
HBcAg + by IF
120 sera
25.8 % (31/120)
ADN +
Vireamia for 8
months
102-106 copies/ml
120 Cynomolgus
from Mauritius
42.8 %
HBsAg +
30 Juin 2011
HBV DNA follow up
for 8 months in the serum of 6 animals
10000000
1000000
VGE/ml
100000
10000
month 1
1000
month 2
month 8
100
10
1
OBGD6
OBHJ6
OBJG6
OBPR6
OBRF6
OBRG6
Macaque
30 Juin 2011
HBV protein expression in Macaques
liver sections
HBcAg
HBsAg
Negative
Control
Macaque
HBV +
30 Juin 2011
Phylogenic analysis
HBV sequences cluster
with HBV genotype D3
(whole HBV)
D1
D5
D2
Similar results with gene S
and C
D6
D3
D4
30 Juin 2011
HBV transmission from cynomolgus
to sylvanus Macaques
250
200
BL12
ALT peak
3/3 animals
150
100
Days
50
50
HBsAg+ week 4/7 PI
100
150
3,5
3
2,5
2
1,5
HBsAg+ /HBcAg
1
Days
0,5
+ by IF on liver sections
BL14
0
0
50
100
150
200
BL13
PCR HBV DNA (+) X 12 weeks
About 103 copies HBV DNA/ ml
at week 9 pi
30 Juin 2011
250
HBV genome Eq/ml for BL14
200
150
BL12 ALT
100
BL13 ALT
BL14 ALT
50
0
1600
1400
1200
1000
800
600
400
200
0
HBV genome (Eq/ml)
Confirmed by bDNA and
Amplicor Monitor Test
D0
D2
D7
D9
D16
D21
D23
D51
D63
D66
D71
D79
D84
D93
D151
D177
D206
D212
Serum Transaminase Activity (IU/L)
A
HBV transmission from cynomolgus
to sylvanus Macaques
Days
B
4
3,5
2,5
T+ 0 1 2 3 4
2
BL12 HBsAg
1,5
BL13 HBsAg
1
BL14 HBsAg
5
6
7
8
9 10
Weeks post infection
0,5
0
D0
D2
D7
D9
D16
D21
D23
D51
D63
D66
D71
D79
D84
D93
D151
D177
D206
D212
HBsAG DO
3
Days
30 Juin 2011
CONCLUSION
First demonstration of natural HBV infection in Macaques
High prevalence (43%) of HBV in M. cynomolgus in Mauritius
island
Chronic HBV infection : HBV DNA detected at least for 8 months,
low vireamia (102 - 106 copy/ml)
HBsAg and HBcAg detection in the liver (IF)
Possible interspecies transmission of the Mauritius isolate to
Sylvanus Macaques
30 Juin 2011
Perspectives
Develop
Immunotherapeutic
approaches
HBV mutants
replication
Acute and chronic HBV infections
Epigenetic
modulations
•HBV DCyno infectiosity for humans
(UpA-mice humanized)
• HBV receptor study (NTCP*)
• How this strain adapted to its host?
Anrs grant
•TLR9 in Macaque/HBV + : PBMCs, pDcs, Bcells.
30 Juin 2011
Acknowledgements
INSERM U1052
Lyon, France
T Dupinay
T Gheit
L Cova
I Vincent
P Berthillon
M Michelet
I Chemin
Swedish Institute of
Infectious disease
Solna, Suède
CEA
Fontenay aux Roses,
France
H Norder
P Roques
R Legrand
Histopathology and IF
Lyon, France
M. Chevallier
P. Chevallier
Davis University
CA, USA
N Lerche
B Chomel
Institut Pasteur
Casablanca
Maroc
S. Sekkat
S Benjelloun
30 Juin 2011