Transcript Fabien ZOULIM How to use virological tools for the optimal management of chronic hepatitis B Fabien Zoulim INSERM U871 & Liver Department Lyon, France Pathobiology and Natural History of.

Fabien ZOULIM
How to use virological tools for
the optimal management of
chronic hepatitis B
Fabien Zoulim
INSERM U871
& Liver Department
Lyon, France
Pathobiology and Natural
History of the Disease
Immunopathology of HBV Infection
Immune
response
Viral
replication
CD8+
HBV
Immune tolerance
CD8+
HBV
Clairance phase
Chronic hepatitis
Seroconversion
Remission
CD8+
Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001
HBV
Phases of the disease
•Immunotolerance phase
- High viral load and normal ALT levels
•Immunoactive phase / chronic hepatitis
- Viral replication and elevation of ALT levels
•Inactive carrier state
- Low viral load and normal ALT levels
•Reactivation
- Wild type virus or pre-core mutant
•Resolved Infection
- Clearance of HBsAg
Fattovich, J Hepatol 2003
Natural history of hepatitis B
Acute infection
Recovery
Lee, N Engl J Med 1997
Lok, Hepatology 2001
Ganem, NEJM 2004
Chronic infection
Chronic hepatitis
Immune tolerance
Wild type virus (HBeAg+)
Pre-core mutant (HBeAg-)
Inactive carrier
Reactivation
Cirrhosis
30-50 years
Hepatocellular carcinoma
Virological monitoring
Viral load
Liver damage
Reactivation
Drug resistance
Viral genome
heterogeneity
Viral persistence
Treatment response
Drug resistance
Monitoring of Viral Load
HBsAg
UI/ml
pg/ml
HBeAg +
anti-HBe Ab +
ALT
1000
9-
HBVDNA
100
8-
7-
10
6-
hybridsiation
5-1
40,1
30,01
2-
PCR
10,001
Tolerance
chronic hepatitis inactive carrier pre-core mt
occult HBV
Median
10 2
10 1
10
0
10
-1
10
-2
10
-3
10 4
(copies/cell)
10 3
Total HBV DNA
cccDNA (copies/cell)
Evolution of Intrahepatic cccDNA
During the Natural History
10 3
10 2
10 1
10 0
10 -1
10 -2
10 -3
Werle et al, Gastroenterology 2004
Serum Viral Load in Chronic Hepatitis
Titre vs histology in HBeAg-negative patients
Serum titre
Histology
(inflammation)
< 104
31/37 had HAI < 3
> 2  105
15/22 had HAI > 4
> 107
5/6 had HAI > 7
Lindh et al J Viral Hepatitis 2000;7:258-67.
Pre-core mutants
HBeAg and Precore Mutation
ATG
1762-1764 1814
Basic
Core Promoter
Precore
region
ATG
1896 1901
Core
region
HBcAg
HBeAg
HBeAg
Virion
Serum
Serum
Outcome of Chronic HBeAg Negative Hepatitis B
Biochemical patterns in 164 untreated patients
after 23 months (range 12-36) monthly monitoring
400
With flares and normalization
73 pts
( 44.5% )
300
200
Asymptomatic
flare-up:
90% of cases
100
0
400
A
L
T
Without flares
59 pts
( 36.0% )
300
200
Flare-up yearly
frequency:
once 57.1%
twice 20%
< once 22.8%
100
0
With flares but without normalization
400
32 pts
( 19.5% )
300
200
100
0
0
12
months
24
Brunetto MR et al, J Hepatol 2002
Diagnosis of inactive carrier versus
HBeAg negative chronic hepatitis
• Inactive Carrier
– Persistently normal ALT levels
– Persistently low levels of serum HBV DNA
• Threshold : 103 or 104 copies / mL ?
– Wild type genome; sometimes pre-core mutations
– The key : careful monitoring !
• HBeAg negative chronic hepatitis
– Fluctuation / exacerbation of ALT
– Fluctuations of HBV DNA levels usually below 106
copies / mL
– Presence of pre-core / core promoter mutations
HBV genotypes
HBV genotypes
• Influence on the type of pre-core or BCP mutation
• Impact on the outcome of infection and severity of
liver disease (HCC)
• Impact on IFN response
• No clear impact on response to nucleoside analogs
Zhang J Med Virol 1996, Orito Hepatology 2001, Mayerat J Viral Hepat 1999;
Wai Hepatology 2002, Jansen Lancet 2005 Pichoud et al, Hepatology 1999;
Grandjacques J Hepatol 2000; Si Ahmed et al, Hepatology 2000; Yang et al,
Gastroenterology 2004
Viral genotypes and
IFN response (HBeAg loss)
50
47%
%
44%
40
28%
30
25%
20
10
0
A
B
C
D
n=90
n=23
n=39
n=103
Jansen et al, Lancet, 2005
Monitoring of Antiviral Therapy
Goals and types of response
Virological response
-HBV DNA < 104 copies/mL: decreased liver damage
- HBV DNA < 103 copies/mL: decreased risk of resistance
Biochemical response
- normalization of ALT levels
Histological response
- improvement in HAI or Metavir score
Combined response / Complete response
Timing during therapy
Initial response / Maintained response
End of treatment response / Sustained reponse
Hoofnagle, J Hepatol 2003
Infected liver
Blood circulation
Viral load
NKT
CD8
CD4
B
cccDNA
Infected hepatocytes
Infected liver
Blood circulation
Viral load
NKT
CD8
Antivirals
CD4
B
cccDNA
Infected hepatocytes
Werle et al, Gastroenterology 2004
Change in HBV DNA from Baseline
(log10 copies mL (cell))
Reductions in Serum HBV DNA, Total Intrahepatic
HBV DNA 2and cccDNA During ADV Therapy
1
Werle et al, Gastroenterology
2004
0
-1
-2
-3
-4
-5
Serum HBV DNA
Total Intrahepatic HBV DNA
Intrahepatic cccDNA
-6
ADV
PBO
 48 weeks of ADV resulted
in significant reductions in :
Treatment Group
serum HBV DNA > total intrahepatic HBV DNA > cccDNA
-> 14 years of therapy to clear completely viral cccDNA
Virologic Consequences of
Persistent Viremia
1) Infection of new hepatocytes
 slower kinetics of clearance of infected cells
and cccDNA
2) Increases the risk of occurrence and selection
of HBV mutations responsible for drug
resistance
3) On-treatment prediction of HBV drug
resistance
Le Guerhier et al Antimicrob Agents Chemoter 2000;44:111-122; Delmas et al Antimicrob
Agents Chemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman
Hepatology 2000;32:866-867
Viral Load at Week 24 is a Predictor of
Resistance at Week 104 of Therapy
(Telbivudine vs. Lamivudine trial)
HBeAg Positive,
n=921
100%
80%
60%
45%
41%
40%
29%
30%
25% 24%
20%
% of patients with resistance
80%
% of patients with resistance
HBeAg Negative,
n=446
100%
60%
56%
60%
50%
40%
20%
20%
12%
9%
4%
2%
0%
5%
6%
0%
< QL,
n=203,146
QL - 3,
n=57,63
3 to 4,
n=83,79
> 4,
n=115,175
Telbivudine
< QL,
n=178,157
QL - 3,
n=18,20
3 to 4,
n=16,24
> 4,
n=10,23
Lamivudine
Di Bisceglie et al., Abstract #112, AASLD 2006
Clinical
Clinical Definition of HBV
Resistance to Antivirals
•
Genotypic Resistance: Detection of mutations in the HBV genome, known to
confer resistance, which develop during anti-viral therapy
•
Virologic Breakthrough: Rebound in serum HBV DNA levels following the
development of genotypic resistance
•
Clinical Breakthrough: Virologic breakthrough with increased ALT levels or
worsening histology
Laboratory Investigations
•
Phenotypic Resistance: Decreased susceptibility (in vitro testing) to inhibition
by anti-viral drugs associated with genotypic resistance.
•
Cross Resistance: Mutants selected by one agent that also confer resistance to
other antiviral agents
Zoulim et al; Future Virology 2006
HBV drug resistance mutations
Terminal protein
1
Spacer
II(F)
RNaseH
349
(rt1)
183
I(G)
Pol/RT
A
LAM / FTC
ADV
ETV
LdT
TDF
692
(rt 344)
GVGLSPFLLA
YMDD
B
C
D
845 a.a.
E
V173L
L180M
A181V
M204I/V
I169T T184G
S202G/I
M204I
N236T
I233V
M250V
A194T ?
* All ETV resistance requires background YMDD mutations
Allen et al. Hepatology 1998;27:1670–7; Gish et al. J Hepatol 2005;43:60–6; Qi et al. J Hepatol 2004;40(Suppl 1):20–1;
Tenney et al. AAC 2004;48:3498–507; Lai et al. Gastroenterology 2005;129:528–36; Sheldon et al. Antivir Ther
2005;10:727–34; Delaney et al. AAC 2006 ; Schildgen et al NEJM 2006; Villet et al J Hepatol 2007
180
100
140
80
100
60
60
40
20
0
20
1
100
200
300
400
595
Day
1
39
290
400
595
Codon 528 LiPA
Seq
L
L
L
L
L/M
L/M
M
M
M
M
Codon 552 LiPA
Seq
M
M
M
M
M/V
M
V
V
V
V
Codon 555 LiPA
Seq
V
V
V
V
V
V
V
V
V
V
HBV DNA
(10E+6 genome eq/ml)
ALT(U/L)
Line Probe Assay Versus Sequencing for the
Detection of HBV Drug Resistance
0
Sequencing of PCR products
C T C M T G G C T
Can detect any new mutation
T A T A T G G A T
Line probe assay
Very sensitive (minor species and low viremia)
Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002
HBV DNA Quantification
Dynamic Range of HBV DNA Detection
11
Graph adapted from J. Hepatol., 39, S3-S25, 2003
10
9
log copies/ml
8
7
6
5
4
3
2
1
Digene HBV
Digene HBV Digene UltraRoche
Roche Cobas Roche TaqMan Bayer Versant Bayer Versant
Hybrid Capture Hybrid Capture
Sensitive
Amplicor HBV Amplicor HBV
HBV
HBV DNA 1.0 HBV DNA 3.0
I
II
Hybrid Capture
Monitor
Monitor
II
Abbott HBV
PCR Kit
Strategies for Monitoring Treatment Response
and Detecting HBV Drug Resistance
Viraemia levels and ALT every 3 months
- Antiviral response and potency
- Persisting viraemia
- Early detection of drug resistance
Serologic assays
- HBeAg/Anti-HBeAb: every 6 months in HBeAg+ patients
- HBsAg/Anti-HBsAb: when HBV DNA < limit of detection
Genotypic assays
- In multidrug experienced patients
- At the time of virologic breakthrough
- When viral load is not suppressed for long period of time
Approaches to Management Depend
on Cross-Resistance Data
Resistance mutations
L180M + M 204V
Drugs with
reduced activity
Drugs with
intermediate
activity
Drugs that remain
active
Lamivudine
Emtricitabine
Telbivudine
Elvucitabine
Clevudine
Entecavir
M204I
Lamivudine
Emtricitabine
Telbivudine
Entecavir
Adefovir
Tenofovir
N236T
A181V
Adefovir
Adefovir
Tenofovir
Lamivudine
Lamivudine
Emtricitabine
Entecavir
Telbivudine
Tenofovir
Entecavir
*Ź: in the presence of primary resistance mutations (L180M + M204V)
S202G/I*
M250I*
Entecavir
Lamivudine
Telbivudine
Adefovir
Tenofovir
Conclusions
Requirement for the most sensitive / quantitative assays
• Management of chronic HBV infection
– Low levels of replication : inactive carriers / occult infection
– Early detection / prediction of reactivation
– Treatment eligibility
• Monitoring of antiviral therapy
–
–
–
–
Early virological response
Viral breakthrough / drug resistance
Genotypic assays
Individualized treatment adaptation for 2nd or 3rd line
treatment to avoid multidrug resistance
Incidence of Resistance in
Nucleoside Naive Patients
80
70
60
50
year 5
40
year 4
30
year 3
20
year 2
10
0
year 1
Lamivudine
Adefovir
Entecavir
Telbivudine
Lai et al CID 2003; Hadzyiannis et al Gastroenterology 2006; Colonno et al AASLD 2006; Di Bisceglie et al AASLD 2006
Incidence of Resistance in
Lamivudine Refractory Patients
40
30
Year 3
Year 2
Year 1
baseline
20
10
0
Adfefovir
switch
Adevofir
add-on
Entecavir
switch
Lampertico et al AASLD 2006; Colonno et al AASLD 2006
Management of HBV drug resistance
Adefovir switch
~20% resistance/2 year
16% resistance/3 years
Adefovir add-on
0% resistance
at 3 years
Lamivudine
70% resistance
at 5 years
Entecavir Switch
Lamivudine add-on
Adefovir
Entecavir add-on
30% resistance
at 5 years
38% resistance
at 3 years
?
Telbivudine add-on
Adefovir add-on
Entecavir
resistance
at 5 years ?
Telbivudine
Tenofovir add-on*
?
Adefovir add-on
resistance
at 5 years ?
* Not yet approved for HBV therapy
Tenofovir add-on*
?
Mechanisms of HBV Drug Resistance
Virus
Virus
Hepatocytes
Viral polymerase
spontaneous error rate
cccDNA
Long half-life
Infected cells
Long half-life
Defective
immune
response
Impairment of
innate
response
Viral quasi-species
Viral persistence
Host
Selection of
escape mutants
Selective pressure
Antivirals or others
Replication fitness
Replication space
Immune response
Drug PK
Treatment failure
Zoulim Antivir Res 2004;64:1–15
The Hepadnavirus Genome and its Variability
« a » determinant
vaccine/HBIg
8 genotypes
A to H
RT domain
antivirals
core mt
CTL response
pre-core mt
anti-e response
?