Therapeutic Vaccination against Chronic Hepatitis:
precinical Studies and perspectives for the
treatment of Patients
05.06.2014 Berlin
Michael Roggendorf
Institute for Viroloy
Universitäts-Klinkum Essen/Klinikum Rechts der Isar München
NSFC
TRR60
Immunological control of HBV infection
Clearance by
cytokines
IFNγ / TNFα
Lysis of
infected cells
Inhibition of virus
spreading
anti-HBs
Resolution of infection
1) Transgenetic mouse expressing
HBV proteins (E. Chisari, L.Giudotti, review 2001)
2) Chimpanzee L. Giudotti et al. Science (1999) 284, 825
3)Thimme, JVI (2003), 77(1), p.68-76
Immunological control of HBV infection
Clearance by
cytokines
IFNγ / TNFα
Lysis of
infected cells
Inhibition of virus
spreading
anti-HBs
Prevention
„B cell Vaccine“:Classical
HBsAg Vaccine
New Pres1 containing
Vaccine
„T cell Vaccine“: e.g.DNA
Vaccine containing HBcAg
Lu M, Hilken G, Kruppenbacher J, Kemper T,
Schirmbeck R, Reimann J, Roggendorf M.
J Virol. 1999 Jan;73(1):281-9..
Resolution of infection
1) Transgenetic mouse expressing
HBV proteins (E. Chisari, L.Giudotti, review 2001)
2) Chimpanzee L. Giudotti et al. Science (1999) 284, 825
3)Thimme, JVI (2003), 77(1), p.68-76
Immunological control of HBV infection
Clearance by
cytokines
IFNγ / TNFα
Lysis of
infected cells
Inhibition of virus
spreading
anti-HBs
Resolution of infection
1) Transgenetic mouse expressing
HBV proteins (E. Chisari, L.Giudotti, review 2001)
2) Chimpanzee L. Giudotti et al. Science (1999) 284, 825
No cytokine
production
No inhibition of
virus spreading
No lysis of
infected cells
No anti-HBs
Chronic HBV infection
3)Thimme, JVI (2003), 77(1), p.68-76
Immunological control of HBV infection
Clearance by
cytokines
IFNγ / TNFα
Lysis of
infected cells
Inhibition of virus
spreading
anti-HBs
No cytokine
production
No inhibition of
virus spreading
No lysis of
infected cells
No anti-HBs
Therapeutic
vaccination
Resolution of infection
1) Transgenetic mouse expressing
HBV proteins (E. Chisari, L.Giudotti, review 2001)
2) Chimpanzee L. Giudotti et al. Science (1999) 284, 825
Chronic HBV infection
3)Thimme, JVI (2003), 77(1), p.68-76
Summary
1.Control of HBV is achived
by specific immune responses
on B-and T-Cell level (Thimme et al 2003 J.Virol)
2.Absence of T-cell response
in the early phase of infection
results in viral persistence
(Menne et al. J.Virol 2003,Webster et al. Hepatology 2000)
3.Antibodies (anti-HBs)prevent
reinfection of hepatocytes
Currently available treatments for chronic HBV:
(1) pegylated interferon α (PEG-IFNα)


leads to a sustained antiviral response in about 30% patient
side effects
(2) nucleot(s)ide analogues (entecavir, tenofovir, etc.)

emergence of drug resistance mutations
rebounding viremia after cessation of therapy
Alternative strategies are needed
Problem 1
Problem 1
Science. 2014 Mar 14;343(6176):1221-8.
Specific and nonhepatotoxic degradation of nuclear
hepatitis B virus cccDNA.
Lucifora J......, Protzer U.
Chronic hepatitis B
infected
hepatocyte
Blood
HBsAg= 10-50 g/ml
Defective particles
103- 106 fold excess
over virions
HBV-DNA
Problem 2
= 105- 109 copies/ml
= 2-300 pg/ml
Chronic hepatitis B
infected
hepatocyte
Blood
HBsAg= 10-50 g/ml
Defective particles
103- 106 fold excess
over virions
HBV-DNA
Nucleos(t)ide analogues:
Inhibition of HBV replication
= 105- 109 copies/ml
= 2-300 pg/ml
Chronic hepatitis B
infected
hepatocyte
Nucleos(t)ide analogues:
Inhibition of HBV replication
Blood
HBsAg= 10-50 g/ml
Defective particles
103- 106 fold excess
over virions
HBV-DNA
= 105- 109 copies/ml
= 2-300 pg/ml
Chronic hepatitis B
infected
hepatocyte
Nucleos(t)ide analogues:
Inhibition of HBV replication
Blood
HBsAg= 10-50 g/ml
Defective particles
103- 106 fold excess
over virions
HBV-DNA
= 105- 109 copies/ml
= 2-300 pg/ml
HBeAg Loss :30%
The Aim of Therapeutic Immunization
in chronic Hepatitis B:
achieve long term control of HBV by stimulating
specific immune responses on B-and T-Cell level
in chronic HBV carriers
NUC
treatment
1.Seroconversion from HBeAg to anti-HBe (about 30%)
2. Long term Reduction of HBV DNA
__________________________________________
Therapeutic
Vaccine
3.Loss of HBV DNA in plasma and cccHBV DNA liver
4.Loss of HBsAg
Therapeutic Vaccination (HBsAg):
published clinical trials
Conventional HBsAg vaccines
• Pol S. et al., Acta Gastroenterol. Belg., 1998, J. Hepatol.,
2001
• Couline et al., J. Infect. Dis., 2001
• Jung C. et al., Vaccine, 2002
•
• Ren F., et al., J. Med. Virol., 2003
• Yalcin et al., J. Clin. Gastroenterol., 2003
• Safadi R. et al., American J. Gastroenterol., 2003
• Dikici et al., J Gastroenterol. Hepatol., 2003
• Vandepapeliere et al Vaccine 2007
Therapeutic Vaccines (HBsAg/HBcAg)):
published clinical trials
T cell vaccines/Combination Therapy
– CTL-peptide(HBcAg)
Heathcote et al., Hepatology, 1999
– DNA vaccine
M. Sällberg et al Human GeneTherapy 1998( Chimpanzee,HBcAg)
Mancini-Bourgine et al., Hepatology, 2004
O.Godon et al Molecular Therapy 2013
Fontaine H. et al Gut. 2014 Feb 2014
_ HBsAg vaccine+lamivudine
A.Dahmen et al;J.med Virol,2002 ( i.d.) simultanious
Horiike N., J Clin. Virol., 2005
Vandepapeliere et al Vaccine 2007
Hoa P.T.L.et al Antimicrobal Agents and Chemotherapy 2009
– HBsAg-anti-HBs immune complexes
Wen Y. et al., Lancet, 1995,
Xu D. Z. et al., Vaccine, 2005
Xin Yao et al.Vaccine 2007
Xu D.Z. et al 2008 PloS One
Xu D.Z. et al 2013 J Hepatol
Therapeutic DNA-Vaccination in combination with nucleoside
analogs in Patients with chronic hepatitis B (double blind)
Inclusion criteria:
• Chronic hepatitis B without cirrhosis (n=36 and controls n= 36)
•Treatment by nucleos(t)idic(s) for at least three years
•HBV-DNA<12 IU/ml in the last 12 months
Vaccination: DNA-Vaccine (HBsAg,5 times)
Treatment discontinuation at week 48:
• if HBV-DNA<12 IU/ml, without HBV progression, and ALT < 5N until
week 48
•
Godon1, H Fontaine2, S Kahi3, JF Meritet4, D Scott Algara5, S Pol2, ML Michel1 and M Bourgine1; for
the [Q1] ANRS HB02 study
Molecular Therapy 2013
Anti-HBV DNA vaccination does not prevent relapse after discontinuation of analogues in the treatment of
chronic hepatitis B: a randomised trial--ANRS HB02 VAC-ADN.
Fontaine H et al
Gut. 2014 Feb
Incomplete Tolerance : Immunological Response and restoration of
during Nucleoside Therapy
O.Godon et al Molecular Therapy 2013
CD8 Tcell response to HBV core in passed
and chronic Hepatitis B*
*antiviral treatment < 2000IU
Boni C. et al., Hepatology, 2001
Gastroenterology 2012
H.Kephalakis/J.Timm et al unpublished results
New concept of combination therapy
Treatment with
nucleosid analoga
Viral load
Control group
Vaccine group
time
HBV DNA vaccine
Booster injection
Anti- PDL1
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;
Prime Boost Vaccination
DNA
Prime
Adenoviral
Boost
antiviral treatment ( e.g.Entecavir)
0
6
months
12
Modification of the DNA and AdV vaccines used in the study:
insertion of a β-globin intron
:
CMV-IE
WHcAg
polyA
∆E1
LTR
CMV-IE WHcAg polyA
∆E3
AdV DNA
LTR
pWHcIm
 improved constructs:
CMV-IE
intron
WHcAg
polyA
∆E1
pCGWHc
LTR
CMV-IE intron WHcAg polyA
∆E3
AdV DNA
LTR
Comparison of heterologous DNA – Ad5 regimen with DNA
only immunization
A.Kosinska et al. J.Virol 2012
Evaluation of CD8+ T cell response: Intracellular IFNγ staining
2x pCG – Ad5GFP
2x DNA – Ad5WHc
2x DNA - DNA
CD8
80
% IFN+ CD8+ T cells
IFNγ
c13-21
60
2 x pCG - Ad5GFP (n=5)
2 x pCGWHc - pCGWHc (n=7)
2 x pCGWHc - Ad5WHc (n=7)
***
**
40
***
20
ns
***
ns
0
e
lat
u
m
d
sti
un
u
ted
a
l
nre
e
d
pti
pe
3-2
c1
1
6-9
c8
4
A.Kosinska et al. J.Virol 2012
In vivo cytotoxicity assay
*
Unloaded cells
CFSE low labelling
modified from: Barber et al., 2003
Cells loaded with epitope
CFSE high labelling
c13-21
Immunized mouse
c13-21
analysis of target cell
elimination
A.Kosinska et al. J.Virol 2012
Evaluation of CD8+ T cell cytotoxic potential in vivo:
in vivo cytotoxicity assay
2x pCGWHc – pCGWHc
2x pCGWHc – Ad5WHc
# Cells
2x pCG – Ad5GFP
CFSE
CFSE low: unloaded
CFSE high: loaded with c13-21
% killing [spleen]
60
*
**
*
40
20
0
A.Kosinska et al. J.Virol 2012
2xpCG-Ad5GFP (n=4)
2 x pCGWHc - pCGWHc (n=6)
2 x pCGWHc - Ad5WHc (n=6)
Thrapeutic Vaccination of WHV Tg mice
A.Kosinska et al. PLOS Pathogens 2013
IFNγ
Therapeutic Vaccination of WHV Tg mice
2x pCG – Ad5GFP
2x pCGWHc - pCGWHc
2x pCGWHc – Ad5WHc
2x pCGWHc – Ad5 Ad35WHc
0,2%
2,2%
9,2%
7,9%
99,8%
97,8%
90,8%
92,1%
% IFN+ CD8+ T cells
8
**
***
6
4
*
2
0
2 x pCG - Ad5GFP (n=10)
2 x pCGWHc - pCGWHc (n=12)
2 x pCGWHc - Ad5WHc (n=18)
2 xpCGWHc-Ad5-Ad35WHc (n=12)
% CD107a + CD8+ T cells
CD8
10.0
**
***
7.5
5.0
*
2.5
0.0
2 x pCG - Ad5GFP (n=10)
2 x pCGWHc - pCGWHc (n=12)
2 x pCGWHc - Ad5WHc (n=12)
2 xpCGWHc-Ad5-Ad35WHc (n=9)
A.Kosinska et al. PLOS Pathogens 2013
Therapeutic immunization significantly supress WHV replication
in WHV Tg mice
Males
9
**
2x pCG - Ad5GFP
9
2x pCGWHc - Ad5WHc
ns
9
6
5
4
3
***
8
7
detection
limit
2
7
13/17
3/8
7
6
5
4
detection
limit
3
6
2
before
week 2 after
immunizations
the last
(week -1)
immunization
(week 8)
5
4
detection
limit
3
2
Females
before
week 2 after
immunizations
the last
(week -1)
immunization
(week 8)
9
***
8
Viral load
[log GE/ml]
before
week 2 after
immunizations
the last
(week -1)
immunization
(week 8)
Viral load
[log GE/ml]
Viral load
[log GE/ml]
8
Viral load
[log GE/ml]
8
7
1/9
6
5
4
3
detection
limit
2
A.Kosinska et al. PLOS Pathogens 2013
A.Kosinska et al unpubl
before
week 2 after
immunizations
the last
(week -1)
immunization
(week 8)
WHcAg- specific CD8+ T cell response:
CD107a degranulation assay
CD107a+ CD3+ CD4T cells
c96-110
2.5
W -1
W8
W 22 W 25 W 27 W 29
2.0
1.5
1.0
mean
background
value
0.5
0.0
ETV + vaccine
ETV only
A.Kosinska et al. PLOS Pathogens 2013
WHsAg- specific CD8+ T cell response:
CD107a degranulation assay
CD107a+ CD3+ CD4T cells
s220-234
2.5
W -1
W8
W 22 W 25 W 27 W 29
2.0
1.5
1.0
mean
background
value
0.5
0.0
ETV + vaccine
ETV only
A.Kosinska et al. PLOS Pathogens 2013
Monitoring of viral loads and sAg loads
WHsAg
12
11
10
9
8
7
6
5
4
3
2
W0
W8
W12-19
W 25
W31-33
detection
limit
ETV + vaccine
ETV only
WHsAg [% of control]
Viral load [log GE/ml]
Viral load
200
W0
W8
W 22
W31-33
150
100
50
0
ETV + vaccine
ETV only
A.Kosinska et al. PLOS Pathogens 2013
WHV chronic: viral load, sAg and anti-WHs
viral load
WHsAg
61792
61793
ETV
anti-WHs + + + + + + + + +
300
100
+/- (-)(-)
0
0 2 4 6 8 10 12 14 16 19 22 25 27 29 31 33 35 38 40 46 52 64
12
11
10
9
8
7
6
5
4
3
400
anti-WHs + + + + + +
200
100
-1
0
2
4
6
week of therapy
61786
viral load
WHsAg
61789
100
n.d.
4
6
8 10 12 14 16 19 22 25 27 29 31 33
week of therapy
0
Viral load
[log GE/ml]
Viral load
[log GE/ml]
200
2
viral load
WHsAg
12
11
10
9
8
7
6
5
4
3
400
300
200
100
n.d.
-1
0
2
4
6
8 10 12 14 16 19 22 25 27 29 31 33
0
week of therapy
A.Kosinska et al. PLOS Pathogens 2013
WHsAg
[g/ml]
300
WHsAg
[g/ml]
400
400
0
0
ETV
800
-1
8 10 12 14 16 19 22 25 27 29 31 33
week of therapy
ETV
12
11
10
9
8
7
6
5
4
3
300
WHsAg
[g/ml]
200
Viral load
[log GE/ml]
400
WHsAg
[g/ml]
Viral load
[log GE/ml]
ETV
12
11
10
9
8
7
6
5
4
3
viral load
WHsAg
Development of anti-WHs antibodies
A.Kosinska et al. PLOS Pathogens 2013
3,0
78
9
61
kb
61
78
6
(w
38
)
(
w
61
79 35)
2
(w
61
79 43)
3
61 (w
79 31
)
1
61 (w
79 36
)
5
(w
35
)
WHV replication in the liver
RC DNA
1,5
ssDNA
A.Kosinska et al. PLOS Pathogens 2013
Steps in Loss of T cell function
G. J. Freeman, E. J. Wherry, R. Ahmed, A. H. Sharpe, The
Journal of experimental medicine 203, 2223–7 (2006).
Reverse T cell exhaustion by PD-1/PD-L1 pathway blockade
PD-L1 Ab
PD-1 Ab
Exhausted
Proliferation
Cytokine secretion
Cytotoxicity
Functional
Proliferation
IFN-γ, TNF-α, IL-2
Cytotoxicity
Immunotherapy of chronic hepatitis C with antibodies against programmed cell death-1 (PD-1) Ahmed R, Walker CM.PNAS 2013
New concept of combination therapy
Treatment with
nucleosid analoga
Viral load
Control group
Vaccine group
time
HBV DNA vaccine
Booster injection
Anti- PDL1
Characterization of woodchuck PD-1 and PD-L1
Structure prediction
Homology of PD-1
Species
Woodchuck
Human
Mouse
Woodchuck
100
-
-
Human
74.71
( 62.98 )
100
-
Mouse
71.95
( 58.82 )
70.93
( 59.72 )
100
PD-1
Homology tree
Homology of PD-L1
Species
Woodchuck
Human
Mouse
Woodchuck
100
-
-
Human
84.4
( 75.9 )
100
-
Mouse
75.3
( 66.2 )
76.6
( 69.9 )
100
E. Zhang E. et al PLoS One
2011;6:e26196
PD-L1
Elevated wPD-1 and wPD-L1 expression in PBMCs of chronically
WHV-infected woodchucks
wPD-1
wPD-L1
***
14
10
8
6
4
2
ch
ro
ni
c
0
na
iv
e
% of PD1+ cells
of CD8+ T Cells
12
Zhang E. et al PLoS One 2011;6:e26196
Enhanced functions of WHV-specific T cells by blockage of wPD1/wPD-L interaction in vitro
PD-L1 blocking
PD-1 blocking
*
2.0
*
2.0
1.6
1.2
1.2
0.0
0.0
l
A
1
-L
PD
co
nt
ro
Zhang E. et al PLoS One 2011;6:e26196
A
b
0.4
PD
-1
0.4
co
nt
0.8
b
0.8
ro
l
SI
SI
1.6
Reduction of PD-1 expression on CD8 T cell during antiviral
treatment in chronic WHV infection.
Jia Liu et al, PLOS Pathogens 2014
Combination therapy strategy
C:
E:
ED:
EDA:
Control
Entecavir (ETV)
ETV + DNA vaccination
ETV + DNA vaccination + αPD-L1
(n=3)
(n=3)
(n=3)
(n=3)
Jia Liu et al, PLOS Pathogens 2014
In vivo PD-L1 blockade synergizes with therapeutic vaccination to enhance
WHcAg-specific T cell immunity
Jia Liu et al, PLOS Pathogens 2014
In vivo PD-L1 blockade synergizes with therapeutic vaccination to control
WHV replication.
DNA
WHsAg
Jia Liu et al, PLOS Pathogens 2014
In vivo PD-L1 blockade synergizes with therapeutic vaccination to induce
WHsAb in treated woodchucks.
No WHsAb was detectable in C, E and ED groups.
Jia Liu et al, PLOS Pathogens2014
Combine therapy inhibits WHV replication in the lives (Southern blot and PCR)
16 weeks after stopping ETV treatment
Jia Liu et al, PLOS Pathogens2014
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
Duration
month
Vaccine
Number
of shots
6
DNA vaccine
WhsAg
WhcAg
ETV
0,2mg/kg
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
Summary
Part 1
all chonically WHV-infected woodchucks that received ETV treatment andDNAprime
Ad5/35 boost vaccinations demonstrated prolonged suppression of WHV replication
in comparison to ETV controls
2 out of 4 chronic WHV carriers from combination therapy group achieved sustained
antiviral response (undetectable viral load and development of anti-WHs)
one of them completely eradicated WHV from the liver
Part 2
Expression of PD-1 on CD8 T cells (CD3+ CD4-) during acute WHV infection
correlates with viremia.
PD-1 expression on CD8 T cells elevated in woodchucks with chronic WHV infection,
and can be strongly down-regulated by antiviral treatment.
Combination therapy of antiviral treatment, DNA vaccination and in vivo PD-1/PD-L1
pathway blockade can restore WHV specific CD8 T cell functions, and lead to a
potent and sustained suppression of viral replication in chronic WHV infection
woodchucks.