1. Fabien ZOULIM

2. Fabien Zoulim INSERM U871 & Liver Department Lyon, France
How to use virological tools for the optimal management of chronic hepatitis B
Fabien Zoulim
INSERM U871
& Liver Department
Lyon, France

3. Pathobiology and Natural History of the Disease

4. Immunopathology of HBV Infection
Viral
replication
Immune
response
CD8+
HBV
Immune tolerance
CD8+
HBV
Clairance phase
Chronic hepatitis
CD8+
HBV
Seroconversion
Remission
Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001

5. 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

6. Natural history of hepatitis B
Acute infection
Lee, N Engl J Med 1997
Lok, Hepatology 2001
Ganem, NEJM 2004
Recovery
Chronic infection
Chronic hepatitis
Wild type virus (HBeAg+)
Pre-core mutant (HBeAg-)
Inactive carrier
Immune tolerance
Reactivation
Cirrhosis
30-50 years
Hepatocellular carcinoma

7. Virological monitoring
Viral genome heterogeneity
Viral load
Liver damage
Reactivation
Drug resistance
Viral persistence
Treatment response
Drug resistance

8. Monitoring of Viral Load

9. HBsAg 9- 8- 7- 6- 5- 4- 3- 2- 1- 1000 100 10 1 0,1 0,01 0,001 HBeAg +
UI/ml
pg/ml
HBeAg +
anti-HBe Ab +
ALT
1000 9- 8- 7- 6- 5- 4- 3- 2- 1-
HBV-
DNA
100 10 1
hybridsiation
0,1
0,01
PCR
0,001
Tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

10. Evolution of Intrahepatic cccDNA During the Natural History
Median
cccDNA (copies/cell)
Total HBV DNA
(copies/cell)
HBeAg+ (63)
HBeAg- (18)
HBSAg- (7)
HBeAg+ (63)
HBeAg- (18)
HBSAg- (7)
Inact. Carriers (10)
Inact. Carriers (10)
Werle et al, Gastroenterology 2004

11. Serum titre Histology (inflammation) < 104 31/37 had HAI < 3
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:

12. Pre-core mutants

13. HBeAg and Precore Mutation
ATG
ATG
Basic
Core Promoter
Precore Core
region region
HBcAg
Virion
HBeAg
Serum
HBeAg
Serum

15. Outcome of Chronic HBeAg Negative Hepatitis B
Biochemical patterns in 164 untreated patients
after 23 months (range 12-36) monthly monitoring
With flares and normalization
73 pts
( 44.5% )
Asymptomatic
flare-up:
90% of cases
Without flares A L T
59 pts
( 36.0% )
Flare-up yearly
frequency:
once 57.1%
twice 20%
< once 22.8%
With flares but without normalization
32 pts
( 19.5% )
months
Brunetto MR et al, J Hepatol 2002

16. Diagnosis of inactive carrier versus HBeAg negative chronic hepatitis
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

17. HBV genotypes

18. 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

19. IFN response (HBeAg loss)
Viral genotypes and
IFN response (HBeAg loss)
47% 50
44% % 40
28% 30
25% 20 10 A
n=90 B
n=23 C
n=39 D
n=103
Jansen et al, Lancet, 2005

20. Monitoring of Antiviral Therapy

21. 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

22. NKT CD4 CD8 B Infected liver Blood circulation Viral load
Infected hepatocytes
Infected liver
Blood circulation
Viral load
NKT
CD8
CD4 B
cccDNA

23. Werle et al, Gastroenterology 2004
Infected hepatocytes
Infected liver
Blood circulation
Viral load
NKT
Antivirals
CD8
CD4 B
cccDNA
Werle et al, Gastroenterology 2004

24. (Log10 copies/mL Log10copies/cell)
Reductions in Serum HBV DNA, Total Intrahepatic HBV DNA and cccDNA During ADV Therapy
Werle et al, Gastroenterology 2004
(Log10 copies/mL Log10copies/cell)
Median
48 weeks of ADV resulted in significant reductions in :
serum HBV DNA > total intrahepatic HBV DNA > cccDNA
-> 14 years of therapy to clear completely viral cccDNA

26. 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: ; Delmas et al Antimicrob Agents Chemother 2002; 46: ; Kock et al Hepatology2003; 38: ; Richman Hepatology 2000;32:

27. Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy (Telbivudine vs. Lamivudine trial)
HBeAg Positive, n=921
HBeAg Negative, n=446
Telbivudine
Lamivudine
Di Bisceglie et al., Abstract #112, AASLD 2006

28. 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

29. HBV drug resistance mutations
Spacer
Pol/RT
RNaseH
Terminal protein 1
183
349
692
845 a.a.
(rt1)
(rt 344)
GVGLSPFLLA
YMDD
I(G)
II(F) A B C D E
V173L
L180M M204I/V
LAM / FTC
ADV
A181V
N236T
I233V
ETV
I169T T184G S202G/I M250V
LdT
M204I
TDF
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 ; Schildgen et al NEJM 2006; Villet et al J Hepatol 2007

30. Line Probe Assay Versus Sequencing for the Detection of HBV Drug Resistance20 40 60 80
100
HBV DNA
(10E+6 genome eq/ml)
ALT(U/L)
140
180 1
595
200
300
400
Codon 528
LiPA
Seq
Codon 552
Codon 555 L M V 39
290
L/M
M/V
Day T A G C
Sequencing of PCR products
Can detect any new mutation
Line probe assay
Very sensitive (minor species and low viremia)
Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002

31. HBV DNA Quantification Dynamic Range of HBV DNA Detection
log copies/ml
Graph adapted from J. Hepatol., 39, S3-S25, 2003

33. 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

34. Approaches to Management Depend on Cross-Resistance Data
Resistance mutations

35. 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

37. Incidence of Resistance in Nucleoside Naive Patients
resistance mutations
% of patients with
Lai et al CID 2003; Hadzyiannis et al Gastroenterology 2006; Colonno et al AASLD 2006; Di Bisceglie et al AASLD 2006

38. Incidence of Resistance in Lamivudine Refractory Patients
resistance mutations
% of patients with
Lampertico et al AASLD 2006; Colonno et al AASLD 2006

39. Management of HBV drug resistance
~20% resistance/2 years
16% resistance/3 years
Adefovir switch
Lamivudine
70% resistance
at 5 years
Adefovir add-on
0% resistance
at 3 years
Entecavir Switch
38% resistance
at 3 years
Lamivudine add-on
Entecavir add-on
Telbivudine add-on
Adefovir ?
30% resistance
at 5 years
Adefovir add-on
Tenofovir add-on*
Entecavir ?
resistance
at 5 years ?
Adefovir add-on
Tenofovir add-on*
Telbivudine ?
resistance
at 5 years ?
* Not yet approved for HBV therapy

40. 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

41. 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 ?