Il virus dell`epatite B La patogenesi dell`epatite B

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Il virus dell`epatite B La patogenesi dell`epatite B
Il virus dell’epatite B
Giovanni Raimondo
Epatologia Clinica e Biomolecolare
Policlinico Universitario di Messina
La patogenesi dell’epatite B
Carlo Ferrari
Unità Operativa di Malattie Infettive ed Epatologia
Azienda Ospedaliero-Universitaria di Parma
Il virus dell’epatite B
Giovanni Raimondo
Epatologia Clinica e Biomolecolare
Policlinico Universitario di Messina
Modulo 1
VIROLOGY OF HEPATITIS B VIRUS (HBV)
Virology of Hepatitis B Virus (HBV)
Family: Hepadnaviridae
Mature virus (Dane particle) 42 nm diameter
Genus: Hepadnavirus
Orthohepadnavirus (Mammalian)
Human Hepatitis B-Virus (HBV)
Chimpanzee Hepatitis B Virus (ChHBV)
Gibbon Hepatitis B-Virus (GiHBV)
Orang Utan Hepatitis B Virus (OuHBV)
Gorilla Hepatitis B-Virus (GoHBV)
Woolly Monkey Hepatitis B-Virus (WMHBV)
Woodchuck Hepatitis-Virus (WHV)
Ground squirrel Hepatitis-Virus (GSHV)
Arctic Ground squirrel Hepatitis-Virus (AGSHV)
Avihepadnavirus
Duck Hepatitis B-Virus (DHBV)
Heron Hepatitis-Virus (HHV)
Ross Goose Hepatitis BVirus (RGHBV)
Snow Goose Hepatitis BVirus (SGHBV)
Maned Duck Hepatitis BVirus (MDHBV)
Grey Teal Hepatitis B Virus (GTHBV)
Stork Hepatitis B-Virus (STHBV)
• DNA virus
• 3.2 kilobases
• partial-double-stranded
• enveloped
Target cells: hepatocytes
Receptor: unknown
Ligand: preS1-protein
HEPATITIS B VIRUS
HBV GENOME
S gene: pre-S1 (large S), pre-S2 (middle S), S (small S)
Core gene: core, e
Pol gene:
P
X gene:
X
HEPATITIS B VIRUS
B
B
C
C
A
A
HEPATITIS B VIRUS Genome Organization
• 3.2 kb pdDNA
• 3 promoters = Core, S-, and X
• 4 ORF = preC/Core, pre-S1/pre-S2/S,
Polymerase, X-gene
HBsAg and Reverse Transcriptase (RT) Protein Synthesis
GENOMIC REGIONS
RT
ATG..... AGC CCG TTT CTC CTG GCT CAG TTT ACT
S
ATG ..... GCC CGT TTC TCC TGG CTC AGT TTA CTA
Transcription
mRNA
…… … … … … … … … … … … … … … … … …
mRNA
… … … … … … … … … … … … … … … … …
Translation
RT
HBsAg
Met- ………… Pro - Phe - Leu - Leu - Ala Gln - Phe - Thr
Met- ……… Ala - Arg - Phe - Ser - Trp - Leu - Ser - Leu - Leu
HBsAg and RT Protein Syntheses in HBV Strains with A181V aa Change in RT
GENOMIC REGIONS
RT
ATG..... AGC CCG TTT CTC CTG ACT CAG TTT ACT
S
ATG ..... GCC CGT TTC TCC TGA CTC AGT TTA CTA
Transcription
mRNA
…… … … … … … … … … … … … … … … … …
mRNA
… … … … … … … … … … … … … … … … …
Translation
RT
HBsAg
Met- ………… Pro - Phe - Leu - Leu - Thr Gln - Phe - Thr
Met- ……… Ala - Arg - Phe - Ser - STOP
HBV Life Cycle
HBV Life Cycle
RECYCLING
Modulo 1
HBV GENETIC HETEROGENEITY
Geographic Distribution of HBV Genotypes
Genotype A
Worldwide
“ “
B
Asia
“ “
C
Asia
“ “
D
South Europe, Americas, Australia
“ “
E
Africa
“ “
F
Native Americans and Polynesians
“ “
G
Europe, USA
“ “
H
Native Americans
HBV Genomic Variants…
• Naturally occurring
• Therapeutically induced
…When Are They Clinically Relevant?
Relative Replication Yield (RC) of HBV Mutants
V173L+L180M+M204V*
I169T+V173L+L180M+M204V*
A181T
L180M+A181V
A181T+N236T
WT
L180M+M204V*
N236T
I169T+V173L+L180M+M204V+M250V*
L180M+A181V+M204V
M204I
M250V*
A181V
L180M+T184G+S202I+M204V*
L180M
A181V+N236T
L180M+A181V+N236T
0
1
2
3
4
5
6
7
8
9
10
Modified from Edwards R, et al. Global Antiviral Journal 2005;1(Suppl2):77
Secondary Mutations at the RT Level in HBV Isolates from Naïve Patients
Single Mutation
Multiple Mutations
aa substitution
No. of cases
N94R
1
V173M
aa substitutions
No. of Cases
V173M
1
1
F221Y
1
A181D *
1
P237T
2
T207I
1
N238H
1
V214A/E
2
L217R
3
Q215S/H/P
9
N238T
2
R217L
1
V214E
1
S219A
3
N94R
1
F221Y
3
S185I
1
I233V
3
P237T
N238H
Q215S/H/P
+
F221Y
+
N238H
S219A
+
+
F221Y + I233V + N238D
1
2
Q215S + R217L + F221Y
1
4
S202T +L217R + S219A + F221Y
1
*Previously unreported aa substitution in a position critical for primary
resistance to Lamivudine and Adefovir.
Pollicino T, et al. Antivir Ther 2009;14:649-654
Modulo 1
MOST COMMON, NATURALLY
OCCURING HBV GENETIC VARIANTS
PreC/C-gene
np 1814-6
ATG
np 1901-3
ATG
np 1896
TGG
HBcAg
Predicted precore protein
HBeAg
np = nucleotide position
PreC/C-gene
np 1814
ATG
np 1901
ATG
np 1896
TGG
TAG stop codon
HBcAg
Predicted precore protein
HBeAg
np = nucleotide position
Basal-Core Promoter Mutations
A
T G
np 1762
A
1764
Enhancer II/basal core promoter
P
Pre-C C
X
Hepatitis B Virus Genome Organization
PreS/S Gene Transcription and Envelope Proteins (ORFs: Pre-S1/Pre-S2/S)
Pre-S1 RNA (2.4kb)
LHBs
A
T
G
A
T
G
Pre-S1
Pre-S2
A
T
G
S
S
p r e-S1 p r o t e i n
Pre-S2/S RNA (2.1kb)
MHBs
A
T
G
Pre-S2
A
T
G
S
p r e-S2 p r o t e i n
Pre-S2/S RNA (2.1kb)
SHBs
A
T
G
S
S protein
Envelope Gene and Proteins
S-gene
Large
protein
(390-399 aa)
Middle protein (281 aa)
Small protein (226 aa)
Natural Occurring Pre-S1 HBV Variant
A
T
G
A
T
G
PRE-S1
183-bp
deletion
Gerken G, et al. Virology 1991;183:555-565
PRE-S2
A
T
G
S
Pre-S2 Minus HBV Variants
A
T
G
PRE-S1
A
G
G
A
T
G
S
HBV Variants Carrying Deletion in Pre-S2 Region
A
T
G
PRE-S1
A
T
G
PRE-S2
In frame
deletion
A
T
G
S
PreS2-Defective HBV Variants: Evidence of Clinical Impact
-
Fulminant hepatitis
-
Fibrosing cholestatic hepatitis
-
Cirrhosis and HCC
Infection with HBV PreS2-Defective Variants is associated with HCC
Distribution of pre-S2-defective
variants in HBsAg+ patients (pts) with
different clinical pictures
Chen BF, et al. Gastroenterology 2006;130:1153-1168
Raimondo G, et al. J Hepatol 2004;40:515-519
Diagnosis
No. pts
No. pS2 variants
Inactive carriers°
15
2
Chronic hepatitis°*
50
25
Cirrhosis°*
26
13
HCC°*
19
16
°p=0.002
*p=0.02
All pts infected with genotype D.
Prevalence of pre-S mutants in patients
infected with genotype B, C,
or mixed genotypes
cc : chronic (inactive) carriers
CH : chronic hepatitis
LC : liver cirrhosis
Deletion
Wild-type + deletion
Wild-type
HBV Surface Antigen “a” Determinant
Clinically Relevant Mutations in the “a” Determinant of HBsAg
Tyr
IIe,Glu
Asn,
Thr
Met
Thr
Arg
Tyr
Thr
Ile
8 amino acid
insertion)
Lys
Cys
Cys
124
137
Gly 145
Ser
Cys
139
Cys
147
Hepatits B Virus
…leads to an infection that lasts indefinitely and is referred to as “occult
infection” when it is mantained without HBsAg
…replication process takes place through a reverse transcription phase like
retroviruses, and like retroviruses it may be integrated into the host genome
…has a compact genomic structure, with partial overlapping of its different
genomic regions
…cannot eliminate the viral mutants that develop during each replicative cycle, so
that they may accumulate leading to the formation of a complex quasispecies,
often influencing the clinical evolution of the liver disease
La patogenesi dell’epatite B
Carlo Ferrari
Unità Operativa di Malattie Infettive ed Epatologia
Azienda Ospedaliero-Universitaria di Parma
Modulo 1
INNATE IMMUNE RESPONSES
IN HBV INFECTION
Role of Innate Immunity in Acute Viral Infections
Early viral containment
Priming and maturation of
adaptive immune responses
Viral load
Innate
response
T cell response
Antibody response
1
2
3
4
Time (months) after infection
5
6
Kinetics of Virus Replication and Liver Damage in HBV Infection
Majority of viral clearance prior to peak of liver inflammation
non-cytolytic mechanisms likely implicated
Webster GR, et al. Hepatology 2000;32:1395-1406
Thimme R, et al. J Virol 2003;77:68-76
Guidotti L, et al. Science 1999;284:825
Chimpanzee infection
Human infection
100
2,000
1,000
1
HBV
2
4
6
8
10
12
Weeks after infection
14
16
0
18
400
Liver
HBV DNA
10
ALT
300
200
1
100
0
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Weeks after infection
ALT (U/L)
10
Liver HBV-DNA (%)
ALT
500
100
Intrahepatic CTL Frequency, HBV Replication, and Liver Damage in Acute Hepatitis B
Intrahepatic HBV-specific CD8 T cells in chimps
10
HBVDNA
400
sALT (U/L)
ALT
80
60
200
40
100
0
20
0
0
Thimme R, et al. J Virol 2003;77:68
2
4
6
8
10
12
14
16
18
20
22
24
% HBV-DNA liver
300
5
100
HBVspecific
CD8 cells
In Vivo Chimpanzee Data
Lack of IFN-inducible genes in chimpanzee model of HBV infection
HBV seems unable to induce innate responses: a ‘stealth virus’
Genes associated with entry and expansion
of the virus, reflecting activation and effector
function of the innate immune response
No gene
Genes associated with viral clearance
(brought into the liver by cells of the adaptive
immune response)
110 genes
Wieland S, et al. PNAS 2004;101:6669-6674
HBV Is a Good Inducer of Innate Responses: Proofs in Favor
• Intrahepatic expression of innate immune response genes immediately after
infection in woodchucks infected with WHV (Guy CS. J Virol 2008;82:8579-8591)
• Induction of IFN-I in HepaRG hepatocytes infected with baculovirus carrying HBV
genome results in non-cytolytic control of HBV (Lucifora J. Hepatology 2009;51:63-72)
• Primary human hepatocytes infected with HBV stimulate Kupffer cells to produce
IL-6 with anti-HBV activity (Hösel J. Hepatology 2009;50:1773-1782)
Flaws of These Studies
• In woodchuck infection, activation of innate responses is transient and it is
stimulated by inocula with very high viral concentrations; moreover, this animal
model may only partially reproduce human infection
• IFN-I production by HepaRG cells is stimulated by high intracellular HBV
replication levels which likely do not reproduce the early kinetics of HBV
replication in natural infection
HBV Is a Poor Inducer of Innate Responses: Proofs in Favor
Cytokine production
in acute HBV infection
is significantly more modest
and delayed compared with
acute HIV infection
(Stacey AR, et al. J Virol
2009;83:3719-3733)
(Dunn C, et al. Gastroenterology
2009;137:1289-1300)
A
Time (days since T0)
Time (days since T0)
HIV infection
HBV infection
IFN-α
B
p=0.05
8
300
6
pg/ml
400
200
IL-15
DN Resolution Acute
Healthy
phase HAV
A
high
n.27
n.13
n.17
n.11
IFN-1
p=0.0001
p=0.0002
p=0.0001
800
4
2
100
C
p=0.03
p=0.05
p=0.02
pg/ml
Low production of type 1 IFN,
IL-15 and IFN-1, associated
with high serum IL-10 levels,
at the early stages
of HBV infection
-10 -5 0 5 10 15 20
pg/ml
Fold changes in
group mean
cytokine level
-10 -5 0 5 10 15 20
400
400
200
Healthy DNA Resolution Acute
phase
HAV
high
n.10
n.13
n.11
n.16
Healthy DNA Resolution Acute
phase HAV
high
n.11
n.25
n.13
n.18
Can the lack of IFN-I detection in acute HBV infection be
attributable to active suppression rather than a complete
failure of induction?
Regulation of Toll-like Expression by HBV
• HBeAg can suppress TLR-2 expression in chronic hepatitis B (Visvanathan K, et al.
Hepatology 2007;45:102-110)
• Low levels of TLR-1, 2, 4, and 6 messenger RNA transcripts in PBMC of CAH
patients (Chen Z, et al. Clin Immunol 2008;128:400-408)
• Hepatitis B virus suppresses Toll-like receptor mediated innate immune
responses in murine parenchimal and nonparenchimal liver cells
Hepatology 2009;49:1132-1140)
(Wu J, et al.
Intracellular Host Innate Immune Defences
Extracellular sensing (TLR)
Intracellular sensing (RIG-1)
Interferon loop
IFN-
TLR2
IFN-
IFN-
TLR3
TLR4
IFN
IFNreceptor
receptor
TYK2
JAK1
SOCS1
TRIF
SOCS3
Virions
HBsAg
HBeAg
ISGF3
STAT2
NF-kB
STAT1
RIG-1
IRF-9
P
P
IRF-3
P
P
IRF-3
IRF-7
ISG expression
IFN-
PRD
NF-kB target genes
PKR, ADAR1, 2-5OAS,
IFIT1, Viperin, ISG6-16,
MxA
What Role Do NK Cells Play in Acute HBV Infection?
Hepatocytes express very low levels of MHC Class I, such that any upregulation of cellular stress ligands able to engage NK activatory
receptors should be able to induce local NK cell effector function
NK cells are extremely abundant in the liver, constituting 30-40% of
intrahepatic lymphocytes
Kinetics of NK Cell Responses
Early induction after infection
(Fisicaro P, et al. Gut 2009;58:974-982)
250,000
200,000
150,000
100,000
50,000
0
HBV-DNA
copies/mL
10
70
12.5
35
0
0
5
7
10
4
15
3
2
1
10
5
0
0
0
2
5
7
10
13
Weeks from HBsAg detection
Normalized
release
51Cr
IFN
NKG2D MFI
3.5
3
2.5
2
1.5
1
0.5
0
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
7
10
13
Weeks from HBsAg detection
CD8 IFN
CD4 IFN
NK IFN
% CD8/4+IFNg+
2
% CD69
% CD56+CD3-IFNg+
% IFN
NK cells
CD56+CD3-
7
25
0
FUNCTION
5
NKG2D MFI
2
release
PHENOTYPE
NK cells
CD56+CD3-
% CD69+
0
51Cr
HBV-DNA
Kinetics of NK Cell Responses
Delayed induction after infection
0.02
HBV-DNA
25
NK cells
20
15
10
5
0
0
5
10
5
20
Percent CD69+ NK cells
Percent CD69+ NK cells
(Dunn C, et al. Gastroenterology 2009;137:1289-1300)
0.009
15
5
0
Healthy
Weeks
n.15
Percent IFN+ NK cells
Percent IFN+ NK cells
HBV-DNA
NK cells
25
20
15
10
5
0
5
Weeks
0.003
10
10
5
DNA Resolution
phase
high
n.10
n.6
P=0.03
15
10
5
0
Healthy
20
n.27
DNA Resolution
phase
high
n.17
n.11
Modulo 1
ADAPTIVE IMMUNE RESPONSES
IN HBV INFECTION
Kinetics of Virus Replication and HBV-specific Responses During Acute Infection
HBsAg
- - - - +
+
+
+ + + + +
100
0.8
CD8+ T cells
% tet.+
HBV-DNA
10
1
0.4
nd
40
100
CD4+ T cells
HBV-DNA
20
10
1
nd
2
0
4
6
8
10 12 14 16 18 20
Weeks after infection
Webster GR, et al.
Hepatology 2000;32:1395-1406
CD8 Cells Are the Main Effectors of Viral Clearance and Disease in Acute Hepatitis B
(Thimme R, et al. J Virol 2003;77:68-76)
Control Ab
400
10
300
Liver
HBV-DNA
sALT
1
200
100
0
0
2
4
6
8 10 12 14 16 18
Weeks after infection
20
22 24
400
10
sALT
300
Liver
HBV-DNA
200
1
100
0
CD8 depletion
500
100
CD8 Ab
Liver
HBV-DNA
400
10
300
200
1
sALT
100
0
0
0
2
4
6
8 10 12 14 16 18
Weeks after infection
20
22 24
sALT (U/L)
0
CD4 Ab
0
Liver HBV-DNA (%)
500
100
sALT (U/L)
Liver HBV-DNA (%)
No depletion
500
100
sALT (U/L)
Liver HBV-DNA (%)
CD4 depletion
Role of HBV-specific CTL in Liver Damage and Virus Control
HBV is not directly cythopatic; HBV-specific CD8 cells not only protect, but also damage
CYTOPATHIC
NON-CYTOPATHIC
CD8 CELL
CD8 CELL
CLEARANCE BY LYSIS
OF INFECTED CELLS
CLEARANCE BY CYTOKINES
IFNγ/TNFα
HBV Pathogenesis
Cured liver cells
Cytokines
Chemokines
Amplification of liver
damage
Liver cell
killing
IFN/TNFα
Infected liver
cell
FasL
Perforins
HBV-specific
CTL
HBV Infection
Self-limited
Vigorous, T1-oriented,
multispecific acute phase
responses
Long-lasting protective
responses
40
30
20
ACUTE PHASE
10
%
Lysis
0
30
RECOVERY PHASE
(20 years from recovery)
20
10
0
POLIMERASE
X
CORE
ENVELOPE
CD4-mediated Proliferative Response to HBV Core in Acute HBV Infection
50
Stimulation Index
45
40
35
30
25
20
15
10
5
0
1
Chronic
Urbani S, et al. Hepatology 2005;41:826-831
1
2
3
4
Self-limited
Evolution of infection
5
6
HBV Infection
Chronic evolution
Inefficient acute phase
immune responses
Progressive impairment of
protective responses
CD4 RESPONSES
HBV HBV HBV HBV
POL
X CORE ENV
CD8 RESPONSES
Summary of the Early Immune Events in HBV Infection
NK cells
activation
Early HBV-DNA
clearance by noncytolytic
mechanisms
2
Liver damage and
elimination of infected
cells by cythopatic
mechanisms
5
4
100
2,000
ALT
HBV
10
1,000
1
0
0
2
4
1
Poor induction
of early
intracellular
innate responses
6
8
10
3
Efficient and
timely induction
of adaptive
responses
12
14
16
18
ANTIBODY PRODUCTION IN ACUTE HBV INFECTION
Acute HBV infection
EARLY
LATE
Antigen excess
Partial neutralization
Anti-HBs negative
Antibody excess
Complete neutralization
Anti-HBs positive
Th1
Helper T cells
Expansion of
immune response
B cells
Ab production
Blocking cell to cell spread
Neutralization of
circulating virus
Th1
Th2
B
B
Th2
B
B
Inefficient control
Chronic active infection
mean % IFN+/tot
Partial control
Chronic inactive infection
mean % IFN/tot
Efficient control
Occult infection
mean % IFN/tot
Hierarchy of HBV Control
10
5
0
1 2 3 4 5 6 7 8 9 10 111213 14 1516
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 1011 12 13 14 15 16
10
5
0
1 2 3 4 5 6 7 8 9 10 111213 14 15 16
HBV-specific T Cell Responses are Stronger in Acute than in Chronic Hepatitis B
Ex Vivo Elispot Analysis
Chronic patients
Acute patients
Pt. A2
Pt.
100
A3 50
0
Pt. A4
100
50
0
Pt.
100
A5 50
0
Pt.
250
A6 50
0
1 2 3 4 5 6 7 8 9 10111213 1415 16
Pt. C2
1 2 3 4 5 6 7 8 9 1011121314 1516
1 2 3 4 5 6 7 8 9 10111213141516
1 2 3 4 5 6 7 8 9 10111213141516
SFU/2 x 105 PBMC
0
100
50
0
100
100
50
50
0
0
12 34 5 6 7 8 910111213141516
123 4 5 67 8 9 10111213141516
12 34 5 6 7 8910111213141516
100
100
100
50
50
50
0
0
0
1 2 34 5 6 7 8 91011 213141516
1 23 4 5 6 7 89 10111213141516
1 23 45 6 7 8 9 10111213141516
100
50
0
Pt. C3 100
50
0
Pt. C4
100
50
0
100
100
50
50
0
0
1 23 45 6 7 8 9 10111213141516
12 3 4 5 6 78 910111213141516
1 23 4 5 6 7 89 10111213141516
100
100
50
50
0
0
1 2 34 5 6 7 89 10111213141516 1 23 4 5 67 8 910111213141516
1 234 5 67 8 9 10111213141516
SFU/2 x 105 PBMC
Pt. C1
350
Pt. A1 50
100
100
50
50
0
0
12 34 5 67 89 10111213141516 12 3 45 67 8 910111213141516
12 34 5 67 8 9 1011 213141516
100
100
100
Pt. C6 50
50
50
0
0
0
1 2 34 5 6 7 89 10111213141516 1 23 45 6 78 910111213141516
1234 5 67 8 910111213141516
100
100
100
50
Pt. C7 50
50
0
0
0
1 2 3 4 56 7 8 9 1011 213141516 12 3 45 67 8 910111213141516
12 34 5 6 78 910111213141516
x core env
pol
x core env
pol
x core env
pol
Pt. C5 100
50
0
1 2 3 4 5 6 7 8 9 10111213141516
1 2 3 4 5 6 7 8 9 1011 121314 1516
pol
x core env
Boni C, et al. J Virol 2007;81:4215-4225
Effect of Antigen Stimulation on HBV-specific T Cell Responses and Correlation with Viral Load
In vitro
0
100
50
0
100
50
0
100
50
0
Time point 1
SFU/ 2 x 105 PBMC
0
100
Pt. C2
50
Time point 3
100
50
0
500
250
0
500
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
500
250
0
Pt. C2250
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
Pt. C5250
100
Pt. C6
50
0
100
50
0
100
50
0
Pt. C6250
100
Pt. C7
50
0
100
50
0
100
50
0
0
100
Pt. C4
50
0
100
Pt. C5
50
0
x core env pol
x
core env pol
ALT
200
150
100
50
% IFNγ + cells/CD4
250
500
Pt. C3
250
0
500
Pt. C4250
0
500
0
500
0
500
Pt. C7250
x core env pol
0
x coreenv pol
x coreenv pol
1.4
6,000,000
1.2
5,000,000
1
4,000,000
0.8
3,000,000
0.6
2,000,000
0.4
1,000,000
0.2
0
0
17-01-2003
Boni C, et al. J Virol 2007;81:4215-4225
Time point 3
500
250
0
100
50
0
100
Pt. C3
50
Time point 2
500
Pt. C1250
0
25-03-2003
12-05-2003
x core env pol
HBV-DNA
Time point 2
Time point 1
100
Pt. C1
50
SFU/ 2 x 105 PBMC
Ex vivo
% HBV-TET+/CD8+
Frequency of Intrahepatic and Circulating HBV-specific CD8 Cells in Chronic HBV Infection
10
8
6
4
2
1
0.1
0.08
0.06
0.04
0.02
0
BLOOD
LIVER
Mean % TET+/CD8+
intrahepatic cells
p=0.0002
6
p=0.006
5
p=0.03
4
3
2
1
0
0-104
104-105
HBV-DNA TITER IU/mL
Fisicaro P, et al. Gastroenterology 2010;138:682-93, 693.e1-4
>105
HBV-specific T Cell Dysfunction
Possible causes
Persistent exposure to
high Ag doses
(HBeAg-HBsAg)
Dysfunctional
HBV-T cells
Dendritic cell
impairment
T reg
suppression
Defective innate
responses
Hyper-production of
regulatory cytokines
HBV-specific T Cell Dysfunction
Possible causes
Persistent exposure to high Ag doses
Persistent inflammation
Amplification of negative
costimulatory pathways
Impairment of TCR
signaling by  -chain
down-regulation
Dysfunctional
HBV-specific T cells
Enhanced T cell apoptosis
caused by Bim upregulation
Costimulatory Pathways in T Cell Activation
APC
TNF SUPERFAMILY
B7 FAMILY
PD-L1
PD-L2
B7.1 or B7.2
ICOSL
4-1BBL
OX40L
CD70 CD40
PD-1 CTLA-4
INHIBITION
CD28
OX40 CD27
ICOS
CD40L 4-1BB
COSTIMULATORY SIGNALS
T CELL
PD-1/PD-L1 Pathway
PD-L1
Dendritic cells
Liver cells
PD-1
T lymphocyte
Up-regulation
- activated T cells
- high antigen doses
Up-regulation
in viral infections
Anti-PD-L1
T cell inactivation and
functional exhaustion
T cell functional restoration
Barber DL, et al. Nature 2006;439:682-687
Effect of Anti-PD-L1 Treatment on HBV-specific CD8 T Cell Function
Anti-PD-L1
T cell functional restoration
Barber DL, et al. Nature 2006;439:682-687
EXPANSION
1.5
40
20
30
1
0.5
15
20
10
10
5
0
0
0
1
2
5
10
IL-2 PRODUCTION
IFN PRODUCTION
0
0
1
2
g/mL anti-PD-L1
10
Boni C, et al. J Virol 2007;81:4215-4225
0
2
5
10
CONCLUSIONS
Immune
subjects
Asymptomatic
carriers
Chronic
patients
T cell efficiency
+++
++
+
Antigen load
HBV-DNA negative
HBV-DNA <104
HBV-DNA >104
Chronic HBV infection comprises a wide spectrum of different virological and immunological
situations which are the expression of a complex natural history, where levels of virus
replication and levels of T cell reactivity appear to be inversely correlated and where HBV
persistence is likely caused by the synergistic effect of different mechanisms, including
exhaustion by high antigen concentrations.
FUTURE PERSPECTIVES OF ANTI-HBV IMMUNE THERAPY
(Ferrari C. Gastroenterology 2008;134:1601-1604)
Virus/antigen load
Antiviral treatment
Decline of antigen load
Inhibition of negative
costimulatory pathways
DC
CD8
Blocking
antibodies
T CELL
DYSFUNCTION
VACCINATION
RECOVERY OF T CELL
RESPONSIVENESS
CONTROL OF
INFECTION