Fabien ZOULIM

How to use virological tools for How to use virological tools for the optimal management of the optimal management of

chronic hepatitis Bchronic hepatitis B

Fabien ZoulimFabien Zoulim

INSERM U871 INSERM U871

& Liver Department& Liver Department

Lyon, FranceLyon, France

Pathobiology and Natural Pathobiology and Natural History of the DiseaseHistory of the Disease

Immunopathology of HBV InfectionImmunopathology of HBV Infection

Immune toleranceImmune tolerance

Clairance phaseClairance phaseChronic hepatitisChronic hepatitis

SeroconversionSeroconversionRemission Remission

CD8+CD8+

HBVHBV

CD8+CD8+ HBVHBV

CD8+CD8+HBVHBV

ImmuneImmuneresponseresponse

ViralViralreplicationreplication

Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001 Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001

Phases of the diseasePhases of the disease•Immunotolerance phaseImmunotolerance phase

- High viral load and normal ALT levels- High viral load and normal ALT levels

•Immunoactive phase / chronic hepatitisImmunoactive phase / chronic hepatitis

- Viral replication and elevation of ALT levels- Viral replication and elevation of ALT levels

•Inactive carrier stateInactive carrier state

- Low viral load and normal ALT levels- Low viral load and normal ALT levels

•ReactivationReactivation

- Wild type virus or pre-core mutant- Wild type virus or pre-core mutant

•Resolved InfectionResolved Infection

- Clearance of HBsAg- Clearance of HBsAgFattovich, J Hepatol 2003Fattovich, J Hepatol 2003

Natural history of hepatitis BAcute infectionAcute infection

Chronic infectionChronic infection

Immune toleranceImmune tolerance

Chronic hepatitisChronic hepatitis

Inactive carrierInactive carrier

Recovery Recovery

Wild type virus (HBeAg+) Wild type virus (HBeAg+) Pre-core mutant (HBeAg-)Pre-core mutant (HBeAg-)

Cirrhosis Cirrhosis

Hepatocellular carcinomaHepatocellular carcinoma

Reactivation Reactivation

30-50 years30-50 years

Lee, N Engl J Med 1997Lok, Hepatology 2001Ganem, NEJM 2004

Virological monitoring

Viral loadViral loadViral genome Viral genome heterogeneityheterogeneity

Liver damageLiver damageReactivationReactivation

Drug resistanceDrug resistance

Viral persistenceViral persistenceTreatment responseTreatment response

Drug resistanceDrug resistance

Monitoring of Viral LoadMonitoring of Viral Load

0,001

0,01

0,1

1

10

100

1000ALT

HBV-DNA

HBeAg +HBeAg + anti-HBe Ab +anti-HBe Ab +UI/mlUI/mlpg/mlpg/ml

HBsAgHBsAg

Tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

hybridsiationhybridsiation

PCRPCR

9-9-

8-8-

7-7-

6-6-

5-5-

4-4-

3-3-

2-2-

1-1-

ccc

DN

A

ccc

DN

A (c

op

ies/

cell

)(c

op

ies/

cell

)

To

tal H

BV

DN

A

To

tal H

BV

DN

A

(co

pie

s/ce

ll)

(co

pie

s/ce

ll)

Evolution of Intrahepatic cccDNA Evolution of Intrahepatic cccDNA During the Natural HistoryDuring the Natural History

10 -3

10 -2

10 -1

10 0

10 1

10 2

10 3

10 4

10 -3

10 -2

10 -1

10 0

10 1

10 2

10 3

HBeAg+ (63)

HBeAg+ (63)

Inact.

Carriers

(10)

Inact.

Carriers

(10)

HBSAg- (7)

HBSAg- (7)

HBeAg- (18)

HBeAg- (18)

HBeAg+ (63)

HBeAg+ (63)

Inact.

Carriers

(10)

Inact.

Carriers

(10)

HBSAg- (7)

HBSAg- (7)

HBeAg- (18)

HBeAg- (18)

MedianMedian

Werle et al, Gastroenterology 2004Werle et al, Gastroenterology 2004

Serum Viral Load in Chronic HepatitisSerum Viral Load in Chronic HepatitisTitre vs histology in HBeAg-negative patientsTitre vs histology in HBeAg-negative patients

Serum titreSerum titreHistologyHistology

(inflammation)(inflammation)

< 10< 1044 31/37 had HAI 31/37 had HAI << 3 3

> 2 > 2 10 1055 15/22 had HAI 15/22 had HAI >> 4 4

> 10> 1077 5/6 had HAI 5/6 had HAI >> 7 7

Lindh et al J Viral Hepatitis 2000;7:258-67.Lindh et al J Viral Hepatitis 2000;7:258-67.

Pre-core mutantsPre-core mutants

HBeAg and Precore Mutation

1814 1896 1901

Precore Coreregion region

HBcAg

HBeAg

ATG ATG

Virion

Serum

Basic Core Promoter

HBeAg Serum

1762-1764

Outcome of Chronic HBeAg Negative Hepatitis B

0

100

200

300

400

0

100

200

300

400

0

100

200

300

400

Biochemical patterns in 164 untreated patientsBiochemical patterns in 164 untreated patientsafter 23 months (range 12-36) monthly monitoringafter 23 months (range 12-36) monthly monitoring

00 1212 2424monthsmonths

With flares and normalizationWith flares and normalization

Without flaresWithout flares

With flares but without normalizationWith flares but without normalization

73 pts 73 pts ( 44.5% )( 44.5% )

59 pts 59 pts ( 36.0% )( 36.0% )

32 pts 32 pts ( 19.5% )( 19.5% )

Asymptomatic Asymptomatic flare-up: flare-up:

90% of cases90% of cases

AALLTT Flare-up yearlyFlare-up yearly

frequency:frequency:once 57.1%once 57.1%twice 20%twice 20%

< once 22.8%< once 22.8%

Brunetto MR et al, J Hepatol 2002Brunetto MR et al, J Hepatol 2002

Diagnosis of inactive carrier versus Diagnosis of inactive carrier versus HBeAg negative chronic hepatitisHBeAg negative chronic hepatitis

• Inactive CarrierInactive Carrier– Persistently normal ALT levelsPersistently normal ALT levels– Persistently low levels of serum HBV DNAPersistently low levels of serum HBV DNA

• Threshold : 10Threshold : 1033 or 10 or 1044 copies / mL ? copies / mL ?

– Wild type genome; sometimes pre-core mutationsWild type genome; sometimes pre-core mutations

– The key : careful monitoring !The key : careful monitoring !• HBeAg negative chronic hepatitisHBeAg negative chronic hepatitis

– Fluctuation / exacerbation of ALTFluctuation / exacerbation of ALT– Fluctuations of HBV DNA levels usually below 10Fluctuations of HBV DNA levels usually below 1066 copies copies

/ mL/ mL– Presence of pre-core / core promoter mutationsPresence of pre-core / core promoter mutations

HBV genotypesHBV genotypes

HBV genotypesHBV genotypes

• Influence on the type of pre-core or BCP mutationInfluence on the type of pre-core or BCP mutation

• Impact on the outcome of infection and severity of Impact on the outcome of infection and severity of liver disease (HCC)liver disease (HCC)

• Impact on IFN responseImpact on IFN response

• No clear impact on response to nucleoside analogsNo clear impact on response to nucleoside analogs

Zhang J Med Virol 1996, Orito Hepatology 2001, Mayerat J Viral Hepat 1999; Zhang J Med Virol 1996, Orito Hepatology 2001, Mayerat J Viral Hepat 1999; Wai Hepatology 2002, Jansen Lancet 2005 Pichoud et al, Hepatology 1999Wai Hepatology 2002, Jansen Lancet 2005 Pichoud et al, Hepatology 1999 ; ; Grandjacques J Hepatol 2000Grandjacques J Hepatol 2000; ; Si Ahmed et al, Hepatology 2000Si Ahmed et al, Hepatology 2000; ; Yang et al, Yang et al, Gastroenterology 2004Gastroenterology 2004

0

10

20

30

40

50

A n=90

%

28%

47%44%

25%

Viral genotypes and IFN response (HBeAg loss)

Bn=23

C n=39

D n=103

Jansen et al, Lancet, 2005Jansen et al, Lancet, 2005

Monitoring of Antiviral TherapyMonitoring of Antiviral Therapy

Goals and types of responseGoals and types of responseVirological responseVirological response-HBV DNA < 10HBV DNA < 1044 copies/mL: decreased liver damage copies/mL: decreased liver damage- HBV DNA < 10- HBV DNA < 1033 copies/mL: decreased risk of resistance copies/mL: decreased risk of resistanceBiochemical responseBiochemical response- normalization of ALT levels- normalization of ALT levelsHistological responseHistological response- improvement in HAI or Metavir score- improvement in HAI or Metavir scoreCombined response / Complete responseCombined response / Complete response

Timing during therapyTiming during therapyInitial response / Maintained responseInitial response / Maintained responseEnd of treatment response / Sustained reponseEnd of treatment response / Sustained reponse

Hoofnagle, J Hepatol 2003Hoofnagle, J Hepatol 2003

Blood circulationViral load

Infected hepatocytes

Infected liver

CD8

NKT

CD4

B

cccDNA

Blood circulationViral load

Infected hepatocytes

Infected liver

Antivirals

CD8

NKT

CD4

B

Werle et al, Gastroenterology 2004

cccDNA

Med

ian

Med

ian

(Lo

g(L

og

1010 c

op

ies/

mL

c

op

ies/

mL

Lo

gL

og

1010c

op

ies/

cell)

cop

ies/

cell)

Reductions in Serum HBV DNA, Total Reductions in Serum HBV DNA, Total Intrahepatic HBV DNA and cccDNA During Intrahepatic HBV DNA and cccDNA During ADV TherapyADV Therapy

48 weeks of ADV resulted in significant reductions in : 48 weeks of ADV resulted in significant reductions in :

serum HBV DNA > total intrahepatic HBV DNA > cccDNA serum HBV DNA > total intrahepatic HBV DNA > cccDNA

-> 14 years of therapy to clear completely viral cccDNA-> 14 years of therapy to clear completely viral cccDNA

Werle et al, Gastroenterology 2004Werle et al, Gastroenterology 2004

Virologic Consequences of Virologic Consequences of Persistent ViremiaPersistent 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 Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy Resistance at Week 104 of Therapy (Telbivudine vs. Lamivudine trial)(Telbivudine vs. Lamivudine trial)

4%

25%29%

30%

9%

24%

41%45%

0%

20%

40%

60%

80%

100%

< QL,n=203,146

QL - 3,n=57,63

3 to 4,n=83,79

> 4,n=115,175

% o

f p

atie

nts

wit

h r

esis

tan

ce

2%

12%

20%

60%

5% 6%

50%

56%

0%

20%

40%

60%

80%

100%

< QL,n=178,157

QL - 3,n=18,20

3 to 4,n=16,24

> 4,n=10,23

% o

f p

atie

nts

wit

h r

esis

tan

ce

Telbivudine Lamivudine

HBeAg Positive, HBeAg Positive, n=921n=921

HBeAg Negative, HBeAg Negative, n=446n=446

Di Bisceglie et al., Abstract #112, AASLD 2006

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

HBV drug resistance mutationsHBV drug resistance 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

RNaseH

845 a.a.

Terminal protein Spacer Pol/RT

A B C ED

1 183 349 692

YMDD

V173L

L180M M204I/V

GVGLSPFLLA

I(G) II(F)

(rt1) (rt 344)

LAM / FTC

ETV I169T T184G S202G/I M250V

ADV A181V N236TI233V

LdT M204I

* All ETV resistance requires background YMDD mutations

TDF A194T ?

Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002

Line Probe Assay Versus Sequencing for the Line Probe Assay Versus Sequencing for the Detection of HBV Drug ResistanceDetection of HBV Drug Resistance

Can detect any new mutation

Very sensitive (minor species and low viremia)

Line probe assayLine probe assay

Sequencing of PCR productsSequencing of PCR products

0

20

40

60

80

100

HB

V D

NA

(10E

+6

ge

nom

e e

q/m

l)

ALT

(U/L

)

020

60

100

140

180

1 595100 200 300 400

Codon 528 LiPASeq

Codon 552 LiPASeq

Codon 555 LiPASeq

1

LL

MM

VV

39

LL

MM

VV

290

L/ML/M

M/VM

VV

400

MM

VV

VV

595

MM

VV

VV

Day

T A T A T G

C T C M T G

G A T

G C T

1

2

3

4

5

6

7

8

9

10

11

Digene HBVHybrid Capture

I

Digene HBVHybrid Capture

II

Digene Ultra-Sensitive

Hybrid CaptureII

RocheAmplicor HBV

Monitor

Roche CobasAmplicor HBV

Monitor

Roche TaqManHBV

Bayer VersantHBV DNA 1.0

Bayer VersantHBV DNA 3.0

Abbott HBVPCR Kit

log

cop

ies/

ml

log

cop

ies/

ml

Graph adapted from J. Hepatol., 39, S3-S25, 2003

HBV DNA Quantification HBV DNA Quantification Dynamic Range of HBV DNA DetectionDynamic Range of HBV DNA Detection

Strategies for Monitoring Treatment Response Strategies for Monitoring Treatment Response and Detecting HBV Drug Resistanceand 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

L180M + M204V M204I N236T A181V S202G/I* M250I*

Drugs withreduced activity

Lamivudine Lamivudine Emtricitabine Emtricitabine Telbivudine Telbivudine Elvucitabine Clevudine

Adefovir Adefovir EntecavirLamivudineTelbivudine

Drugs withintermediate

activity

Entecavir Entecavir Tenofovir Lamivudine

Drugs that remainactive

AdefovirTenofovir

LamivudineEmtricitabine

EntecavirTelbivudine

TenofovirEntecavir

AdefovirTenofovir

*Ź: in the presence of primary resistance mutations (L180M + M204V)

Approaches to Management Depend Approaches to Management Depend on Cross-Resistance Dataon Cross-Resistance Data

Resistance mutations

ConclusionsConclusions

• Management of chronic HBV infectionManagement of chronic HBV infection– Low levels of replication : inactive carriers / occult infectionLow levels of replication : inactive carriers / occult infection

– Early detection / prediction of reactivation Early detection / prediction of reactivation

– Treatment eligibilityTreatment eligibility

• Monitoring of antiviral therapyMonitoring of antiviral therapy– Early virological responseEarly virological response

– Viral breakthrough / drug resistanceViral breakthrough / drug resistance

– Genotypic assays Genotypic assays

– Individualized treatment adaptation for 2nd or 3rd line Individualized treatment adaptation for 2nd or 3rd line treatment to avoid multidrug resistancetreatment to avoid multidrug resistance

Requirement for the most sensitive / quantitative assaysRequirement for the most sensitive / quantitative assays

0

10

20

30

40

50

60

70

80

Lamivudine Adefovir Entecavir Telbivudine

year 1

year 2

year 3

year 4

year 5

Incidence of Resistance in Nucleoside Naive Patients

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

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

0

10

20

30

40

Adfefovirswitch

Adevofiradd-on

Entecavirswitch

baselineYear 1

Year 2Year 3

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

Lampertico et al AASLD 2006; Colonno et al AASLD 2006

Management of HBV drug resistance

Lamivudine

Adefovir switch

Adefovir add-on

Entecavir Switch

Adefovir

Entecavir

~20% resistance/2 years16% resistance/3 years

0% resistance at 3 years

38% resistance at 3 years

Lamivudine add-on

Entecavir add-on

Telbivudine add-on

?

Adefovir add-on

Tenofovir add-on* ?

70% resistance at 5 years

30% resistance at 5 years

resistance at 5 years ?

TelbivudineAdefovir add-on

Tenofovir add-on*?

resistance at 5 years ?

* Not yet approved for HBV therapy

Mechanisms of HBV Drug Resistance

Viral persistence

cccDNA Long half-life

Infected cells Long half-life

Defective Defective immune immune

responseresponse

VirusVirus HepatocytesHepatocytes

Impairment of Impairment of innate innate

responseresponse

HostHost

Selective pressure Antivirals or others

Viral polymerasespontaneous error rate

VirusVirus

Selection of Selection of escape mutantsescape mutants

Treatment failureTreatment failure

Replication fitnessReplication space

Viral quasi-species

Immune responseDrug PK

Zoulim Antivir Res 2004;64:1–15

The Hepadnavirus Genome and its The Hepadnavirus Genome and its VariabilityVariability

« a » determinant« a » determinantvaccine/HBIgvaccine/HBIg

RT domainRT domainantiviralsantivirals

pre-core mtpre-core mtanti-e responseanti-e response ??

core mtcore mtCTL responseCTL response

8 genotypes8 genotypesA to HA to H