Fabien ZOULIM

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Fabien ZOULIM
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How to use virological tools for the optimal
management of chronic hepatitis B

• Fabien Zoulim
• INSERM U871
• Liver Department
• Lyon, France

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Pathobiology and Natural History of the Disease
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Immunopathology of HBV Infection
Viral replication
Immune response
Immune tolerance
Clairance phase Chronic hepatitis
Seroconversion Remission
Guidotti, Science 1999 Guo, J. Virol 2000
Kakimi J Exp Med 2000 Zhu J Virol 2001
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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
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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
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Virological monitoring
Viral genome heterogeneity
Viral load
Liver damage Reactivation Drug resistance
Viral persistence Treatment response Drug
resistance
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Monitoring of Viral Load
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HBsAg
UI/ml pg/ml
HBeAg
anti-HBe Ab
ALT
1000
9- 8- 7- 6- 5- 4- 3- 2- 1-
HBV-
DNA
100
10
hybridsiation
1
0,1
0,01
PCR
0,001
Tolerance chronic hepatitis inactive
carrier pre-core mt occult HBV
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Evolution of Intrahepatic cccDNA During the
Natural History
Median
cccDNA (copies/cell)
Total HBV DNA (copies/cell)
HBSAg- (7)
HBeAg- (18)
HBSAg- (7)
HBeAg (63)
HBeAg- (18)
HBeAg (63)
Inact. Carriers (10)
Inact. Carriers (10)
Werle et al, Gastroenterology 2004
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Serum Viral Load in Chronic Hepatitis Titre vs
histology in HBeAg-negative patients
Serum titre Histology (inflammation)
lt 104 31/37 had HAI lt 3
gt 2 ? 105 15/22 had HAI gt 4
gt 107 5/6 had HAI gt 7
Lindh et al J Viral Hepatitis 20007258-67.
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Pre-core mutants
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HBeAg and Precore Mutation
ATG
ATG
1814 1896 1901
1762-1764
Basic Core Promoter
Precore Core region region
HBcAg
Virion
HBeAg
Serum
HBeAg
Serum
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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 lt once 22.8
With flares but without normalization
32 pts ( 19.5 )
0 12 24
months
Brunetto MR et al, J Hepatol 2002
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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

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HBV genotypes
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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
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Viral genotypes and IFN response (HBeAg loss)
47
50
44

40
28
30
25
20
10
0
B n23
C n39
D n103
A n90
Jansen et al, Lancet, 2005
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Monitoring of Antiviral Therapy
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Goals and types of response

• Virological response
• HBV DNA lt 104 copies/mL decreased liver damage
• - HBV DNA lt 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
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Blood circulation Viral load
NKT
CD8
CD4
B
cccDNA
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Blood circulation Viral load
NKT
CD8
CD4
B
cccDNA
Werle et al, Gastroenterology 2004
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Reductions in Serum HBV DNA, Total Intrahepatic
HBV DNA and cccDNA During ADV Therapy
Werle et al, Gastroenterology 2004
Median (Log10 copies/mL Log10copies/cell)

• 48 weeks of ADV resulted in significant
  reductions in
• serum HBV DNA gt total intrahepatic HBV DNA gt
  cccDNA
• -gt 14 years of therapy to clear completely viral
  cccDNA

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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
200044111-122 Delmas et al Antimicrob Agents
Chemother 2002 46425-433 Kock et al
Hepatology2003 381410-1418 Richman Hepatology
200032866-867
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Viral Load at Week 24 is a Predictor of
Resistance at Week 104 of Therapy (Telbivudine
vs. Lamivudine trial)
HBeAg Positive, n921
HBeAg Negative, n446
Di Bisceglie et al., Abstract 112, AASLD 2006
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Clinical Definition of HBV Resistance to
Antivirals

• Clinical
• 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
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HBV drug resistance mutations
Spacer
Pol/RT
RNaseH
Terminal protein
845 a.a.
349
692
1
183
(rt1)
(rt 344)
YMDD
GVGLSPFLLA
A
B
C
E
D
I(G)
II(F)
V173L L180M M204I/V
LAM / FTC
A181V
N236T I233V
ADV
ETV
I169T T184G S202G/I M250V
LdT
M204I
TDF
A194T ?
All ETV resistance requires background YMDD
mutations
Allen et al. Hepatology 19982716707 Gish et
al. J Hepatol 200543606 Qi et al. J Hepatol
200440(Suppl 1)201 Tenney et al. AAC
2004483498507 Lai et al. Gastroenterology
200512952836 Sheldon et al. Antivir Ther
20051072734 Delaney et al. AAC 2006
Schildgen et al NEJM 2006 Villet et al J Hepatol
2007
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Line Probe Assay Versus Sequencing for the
Detection of HBV Drug Resistance
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
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HBV DNA Quantification Dynamic Range of HBV DNA
Detection
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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 lt 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

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Approaches to Management Depend on
Cross-Resistance Data
Resistance mutations
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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

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Incidence of Resistance in Nucleoside Naive
Patients
of patients with resistance mutations
Lai et al CID 2003 Hadzyiannis et al
Gastroenterology 2006 Colonno et al AASLD 2006
Di Bisceglie et al AASLD 2006
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Incidence of Resistance in Lamivudine Refractory
Patients
of patients with resistance mutations
Lampertico et al AASLD 2006 Colonno et al AASLD
2006
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Management of HBV drug resistance
20 resistance/2 years 16 resistance/3 years
Adefovir switch
Lamivudine
Adefovir add-on
0 resistance at 3 years
70 resistance at 5 years
38 resistance at 3 years
Entecavir Switch
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
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Mechanisms of HBV Drug Resistance
Virus
Hepatocytes
Virus
cccDNA Long half-life
Infected cells Long half-life
Viral polymerase spontaneous error rate
Defective immune response
Impairment of innate response
Viral persistence
Viral quasi-species
Host
Selection of escape mutants
Selective pressure Antivirals or others
Replication fitness Replication space
Immune response Drug PK
Treatment failure
Zoulim Antivir Res 200464115
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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
?