Hepatitis B

Jay H. Hoofnagle, M.D.Division of Digestive Diseases

and Nutrition

National Institute of Diabetes and Digestive and Kidney Diseases

National Institutes of Health

FDA Advisory Panel Meeting: August 7, 2002

Hepatitis B

• HBV, small double stranded DNA virus

• Hepadnaviridae

• Infection restricted to humans and higher apes

• High levels in blood (102 to 1010 copies/ml)

• Causes both acute and chronic hepatitis

• Parenteral, sexual and maternal-infant spread

• Marked geographic variation in incidence

• Common in Asia & Africa, uncommon in the United States and Western Europe

Hepatitis B Virus

HBsAg

HBsAg

HBsAgHBcAg

Dane Particle Tubule

Sphere

HBeAg

AAAA

AAAA

AAAA

AAAA

AAAA

AAAA

2.1kb RNA2.1kb RNA

2.4kb RNA2.4kb RNA

3.5kb RNA3.5kb RNA

0.7kb RNA0.7kb RNA

Pre-S1Pre-S1 Pre-S2Pre-S2

ORF-SORF-S

ORF-PORF-P

ORF-XORF-X

ORF-CORF-C DR1DR1

5’5’

5’5’

+strand+strand

-strand-strand

Pre-CPre-C

DR2DR2

Hepatitis B Virus RNAsHepatitis B Virus RNAs

Hepatitis B Viral Genome

• Circular, partially doubled-stranded DNA • Four open reading frames

– HBsAg (pre-S1, pre-S2 and S)– HBcAg (pre-core & core) – Polymerase (multifunctional)– HBxAg (transactivating factor)

• Replicates largely in liver• Through RNA intermediate and reverse

transcription

Infectious cycle of hepatitis B virus

degradation- antigen-specific- non-specificY

Y

Y

Y

Y

Y Y

Ycell death

Virus - half-life: 1-2 days-production: 1011-1013/daymutation rate: 1-3 x 10-5/site/yr

Zeuzem et al: 2000

Hepatitis B Virus Mutants

• Variations in nucleotide sequence in one of the HBV genes can result in change in the virological and, in some cases, clinical features of the infection.

• S gene: vaccine or HBIG escape mutants

• C gene: can affect disease severity or serological and clinical manifestations

• P gene: can effect replicative efficiency and resistance to antiviral therapy

Hepatitis B Core Antigen Mutants

Nucleocapsid region: Pre-core and Core

• Pre-core: May result in inability to produce HBeAg. HBeAg-negative mutants. Most frequently, GA at nt 1896.

• Core: Substitutions in core region are frequent among pts with severe disease or resistance to interferon, in areas of major B cell and T cell epitopes, thus important in T cell cytotoxicity and viral clearance

HBeAg-negative Variants

• GA at nt 1896 creates a stop codon in the pre-core region that therefore blocks the synthesis of HBeAg.

• nt 1896 is in the highly structured stem-loop encapsidation signal region of HBV RNA and base-pairs with nt 1858

• If nt 1858 is a T (ayw, adr, some adw), stem loop of is maintained by either G or A; if it is a C (adw), stem loop is disrupted by A and replication is stopped.

• Thus HBeAg-negative variants are more common with genotypes B, C and D than genotype A

Outcome of Hepatitis B Virus Infection

Acute Hepatitis B

Asymptomatic subclinical infection

Fulminant Hepatitis

Chronic Hepatitis B

Inactive Carrier State Cirrhosis

Liver Cancer?

65%

35%

<1%

5%

30%50%

Typical Acute Hepatitis B

0100200300400500600700800900

1000

0 1 2 3 4 5 6 12 24 36 48 60

ALT

HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

AL

T a

nd

HB

V D

NA

IU/L

an

d m

illio

n c

op

ies/

ml

Symptoms

Typical Chronic Hepatitis B

0

100

200

300

400

500

600

700

800

0 1 2 3 4 5 6 12 24 36 48 60

ALT

HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

AL

T a

nd

HB

V D

NA

IU/L

or

mill

ion

co

pie

s/m

l

Chronic Hepatitis B: Transition to Inactive Carrier State

0

100

200

300

400

500

600

700

800

0 1 2 3 4 5 6 12 24 36 48 60 72 80

ALT

``HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

AL

T a

nd

HB

V D

NA

IU/L

an

d m

illio

n c

op

ies/

ml

Anti-HBe

Evolution of HBeAg Negative Mutant

0

50

100

150

200

250

300

350

400

450

0 3 6 9 12 15 18 21 24 27 30 33 36

ALT

HBsAg

HBV DNA

Normal ALT levels

Months

AL

T a

nd

HB

V D

NA

IU/L

an

d m

illio

n c

op

ies/

ml

Anti-HBeHBeAg

Chronic Hepatitis B: Three Clinical Forms:

• HBeAg Positive Chronic Hepatitis B

• HBeAg, raised ALT, HBV DNA in serum and chronic hepatitis on biopsy

• HBeAg Negative Chronic Hepatitis B

• Anti-HBe, raised ALT and HBV DNA in serum, chronic hepatitis on biopsy

• Inactive HBsAg Carrier State

• Anti-HBe, normal ALT & no HBV DNA, minimal nonspecific changes on biopsy

Chronic Hepatitis B: Clinical Forms: HBV DNA levels

HBeAg Positive Chronic Hepatitis B

107 to 1011 copies per mlHBeAg Negative Chronic Hepatitis B

104 to 108 copies per mlInactive HBsAg Carrier State

< 101 to 104 copies per ml

qualitative PCR quantitative PCR(Amplicor)

bDNA (Versant) Hybridization(Abbott)

Hybridization(Digene)

350

3.5

.035

Lo

g1

0 co

pie

s/m

L pg

/mL

8

6

4

2

10 35,000

Dynamic Range of Detection of HBV DNA: 5 Assays

HBV DNA Detection

.0035

Genotypes of Hepatitis B Virus

A adw, adw2, ayw1 US, Northern Europe, Africa

B adw2, ayw1 China, Indonesia, Vietnam

C adr, ayr China, Korea, Japan, Vietnam

D ayw2, ayw3 Mediterranian, Middle East, India

E ayw4 West Africa

F adw4 Polynesia, US (rare)

G Europe, US (rare)

Type Subtype Geographical Distribution

Acute Hepatitis BSentinel County Study: 1982-98

• Currently, HBV causes 34% of viral hepatitis• Decline in incidence by 76% between 1987-98• 20% hospitalized, 1% fatal• Gradual rise in median age (27 to 32 yrs)• More common in men than women• African-Americans > Hispanic whites > whites• Current proportions with risk factors

Injection drug use: 14% Men who has sex with men: 15% Heterosexual activity: 40% Occupational exposure: 2%

Goldstein et al: 2002

0

50

100

150

200

78 80 82 84 86 88 90 92 94 96 98

Vaccinelicensed

HBsAg screeningof pregnant

women Routine infantimmunization

OSHA RuleRoutine

adolescent immunization

Decline in MSM & HCW

Decline in injecting drug users

Decline in high-riskheterosexuals

Year

Inf e

c tio

ns

pe r

10 0

, 00 0

Acute Hepatitis B Incidence in the U.S.: 1978-1998

Alter et al: CDC

Chronic Liver Disease: United States 1999

Hepatitis C 57%Alcohol

25%

Hepatitis B

Oth

er

NASH 10%

Bell et al 2001

Hepatitis B accounted for only 4.4% of newly-diagnosed chronic liver disease

Chronic Hepatitis BLong-Term Complications

• Cirrhosis

• Hepatocellular carcinoma

• Glomerulonephritis

• Polyarteritis Nodosa

Chronic Hepatitis BHistology

• Necroinflammatory Changes (Grade)• Periportal inflammation and necrosis

(piecemeal necrosis, interface hepatitis)• Lobular inflammation and single cell necrosis• Portal inflammation

• Fibrosis (Stage)• Portal• Septa formation• Bridging fibrosis• Cirrhosis

Chronic Hepatitis BHistology Scoring Systems

• Histology Activity Index (Knodell) :• Periportal necrosis & inflammation (0-10)• Lobular necrosis & inflammation (0-4)• Portal inflammation (0-4)

• Fibrosis• None = 0 • Portal fibrosis = 1• Bridging fibrosis = 3• Cirrhosis = 4

Chronic Hepatitis BHistology Scoring Systems

• Histology Activity Index (Ishak) :• Periportal necrosis & inflammation (0-4)• Bridging necrosis (0-6)• Lobular necrosis & inflammation (0-4)• Portal inflammation (0-4)

• Fibrosis• None = 0 • Portal fibrosis = 1 or 2• Bridging fibrosis = 3 or 4• Cirrhosis = 5 or 6

Therapy of

Hepatitis B

Chronic Hepatitis BGoals of Therapy

• Improve symptoms and quality of life

• Decrease infectivity

• Prevent progression of disease

• Hepatic Decompensation

• Death from liver disease

What surrogate end-points

correlate with these outcomes ?

Therapy of Chronic Hepatitis B: Major Issues

What are appropriate end-points? Are they the same for different forms of HBV?

Loss of HBeAg Loss of HBsAg Loss of HBV DNA (fall below 105 copies/ml) Normalization of ALT Improvement in histology

What amount of follow up is appropriate in assessing benefit of therapy?

Definition of Responses to Therapy in Chronic Hepatitis B

Type:

Virological: Loss of HBeAg and/or HBV DNA Biochemical: Normal ALTHistological: Improvement in histology scoresComplete: All of above & loss of HBsAg

Timing: Initial: within first 6 mo of therapyEnd-of-therapy: when therapy is stoppedSustained: 6 or 12 mo after stoppingMaintained: present while continuing therapy

Virological Response in Chronic Hepatitis B

Loss of HBeAg and fall of HBV DNA levels to below 105 copies/mL

Occurs in 25-48% of patients given a 4-5 month course of alpha interferon

Occurs in 20-32% of patients given a 12 month course of lamivudine

Occurs in 8-12% of patients on no therapyIs this response durable and does it result in

long-term improvement in disease and lack of progression to cirrhosis and HCC?

Virological Response in Chronic Hepatitis B

Loss of HBeAg cannot be used as an endpoint in patients with HBeAg-negative disease

Generally rely upon decrease in HBV DNA to below 105 copies/ml

HBV DNA levels, however, can fluctuate widely, and with nucleoside therapy will rapidly return to baseline when treatment is stopped.

How durable is decrease in HBV DNA without other changes in viral status?

Virological Response in Chronic Hepatitis B

Loss of HBsAg and development of anti-HBsOccurs in 8% of patients given a 4-5 month

course of alpha interferonOccurs in 1-2% of patients given a 12 month

course of lamivudineOccurs in <1% of patients on no therapyExtremely rare in treatment trials of HBeAg-

negative chronic hepatitis BThis response is durable and associated

with resolution of liver disease

Biochemical Response in Chronic Hepatitis B

Fall of ALT levels into the Normal RangeOften accompanies loss of HBeAg or

decrease in HBV DNA to below 105 copies/mLNot durable unless the decrease in HBV DNA

is durable.Surrogate, indirect marker for decrease in

necroinflammatory disease

Histological Response in Chronic Hepatitis B

Improvements in HistologyUsed in virtually all studies of antiviral therapyTypically, improvement is called a > 2 point

improvement in HAI score (0-22) compared to baseline

However, necroinflammatory scores can change rapidly and improve and worsen

Fibrosis scores represent best evidence for progression of disease and are unlikely to improve with treatment

Alpha Interferon Human cytokine made by lymphocytes

in response to viral infectionActs through cell-surface receptorsActivates Jak/Stat systemInduces transcription of proteins with

antiviral activity (2-5 OAS, PKR, eIF2)Recombinant human alpha interferonPegylated forms now available

Chronic Hepatitis BLong-term Response to Interferon

0

100

200

300

400

500

-2 0 1 2 3 4 5 6 9 12 18 24

AL

T (

U/L

)

HBeAg

HBsAg

Anti-HBe

Anti-HBs

Alpha Interferon

ALT

Months After Start of TherapyPatient A

HBV DNA (dot blot)

Chronic Hepatitis BResponse to Interferon & Relapse

0

200

400

600

800

1000

1200

1400

-4 -2 0 1 2 3 4 5 6 9 12 18 24

AL

T (

U/L

)

HBeAg

HBsAg

Alpha Interferon

ALT

Months After Start of Therapy

HBeAgAnti-HBe

Patient B

HBeAg-Negative Chronic Hepatitis BResponse to Interferon and Relapse

0

100

200

300

400

500

600

700

800

-12 -8 -4 -2 -1 0 1 2 3 4 5 6 9 12 18 24

AL

T (

U/L

)

HBsAg

Alpha Interferon

ALT

HBV DNA

Months After Start of Therapy

Patient C

Anti-HBe

+ + + +- --

Interferon for Chronic Hepatitis B: Problems

• Effective in only 1/3rd of cases

• Expensive

• Side effects are common and can be severe

• Not appropriate for many categories of pts:

– Immune suppressed

– Renal failure or dialysis

– Solid organ transplant

– Decompensated liver disease

Lamivudine Negative enantiomer of 3-thiacytidineBoth an unnatural nucleoside and

chain terminator Highly active against HBV in vitroDose: 100 mg, once daily by mouthApproved for use in chronic hepatitis B

as one year course of therapyContinuous, long-term use is common

but must be considered experimental

Lamivudine TherapyMaintained Response

0

100

200

300

400

500

600

-2 0 2 4 6 8 10 12 18 24 30 36 42 48

Months After Starting Therapy

AL

T L

evel

s (U

/L)

HBV DNA

ALT

102

104

106

108

HB

V D

NA

Levels

Therapy with Lamivudine (100 mg/d)

HAI Scores:Pre: 14Yr 1: 4Yr 4: 1

HBeAg

HBsAg

Patient B

HBeAg-Negative Chronic Hepatitis B

0

100

200

300

400

500

600

-4 -2 0 3 6 9 12 15 18 21 24 30 36 42 48 54

AL

T (

U/L

)

HBsAg and Anti-HBe

Lamivudine

ALT

Months After Start of Therapy

Patient C

HAI = 4200 copies/ml

HAI = 1353.1 million copies/ml

HAI = 1<100 copies/ml

Liver BiopsyHBV DNA

Lamivudine Therapy:Viral Resistance

0

100

200

300

400

500

600

Pre 0 2 4 6 8 10 12 18 24 30 36 42 48

Months After Starting Therapy

AL

T L

evel

s (U

/L)

HBV DNA

ALT

102

104

106

108

HB

V D

NA

Levels

Normal

Therapy with Lamivudine (100 mg)

HAI Scores:Pre: 14Yr 1: 10Yr 4: 17 (Cirrhosis)

wt

YVDD

1010

Patient D

0

2

4

6

8

10

12

Maintained Resistance

Pre 1 year 4 years

9.8 9.9

2.5

8.4

1.3

7.1

139

134

99Tot

al H

AI

Sco

res

(0 t

o 18

)Lamivudine for Chronic Hepatitis B

Histology Activity Index Scores

0

1

2

3

4

5

6

Maintained Resistance

Pre 1 year 4 years

4.2

2.9

1.3

3.93.3

4.3

13 134

9Fib

rosi

s S

core

(0

to 6

)Lamivudine for Chronic Hepatitis B:

Fibrosis Scores

9 9

Lamivudine TherapyLate Relapse

0

100

200

300

400

500

600

-2 0 2 4 6 8 10 12 18 24 30 36 42 48 54 60

Months After Starting Therapy

AL

T L

evel

s (U

/L)

HBV DNA

ALT

102

104

106

108

HB

V D

NA

Levels

Therapy with Lamivudine (100 mg/d)

HAI Scores:Pre: 14Yr 1: 4Yr 4: 1HBeAg

HBsAg

Patient B

0

20

40

60

80

100

0 6 12 18 24 30 36 42 48

Months of Therapy

Pe

rce

nt

wit

h R

es

ista

nc

e (

%)

HBeAg +

HBeAg -

p = 0.00185%

50%

Lamivudine The major shortcoming of long-term

lamivudine therapy for hepatitis B is emergence of lamivudine resistance

Occurs in 20%-30% of patients per yr, approaching 90% by 5 yrs

Loss of HBsAg, but not loss of HBeAg, appears to reliably predict long-term benefit & ability to stop lamivudine

Future studies should focus on combinations that might prevent resistance

Optimal Therapy of Hepatitis B?

Monotherapy or combination therapy? For a defined period (48 wks) or continuous? For all pts or only those with mod-severe disease? If monotherapy, which agent? If combination, which combination?

Standard interferon and lamivudine Pegylated interferon and lamivudine Lamivudine and adefovir Lamivudine and entecavir

Management of Hepatitis B

Initial evaluation: Routine liver tests, HBsAg, HBeAg & anti-HBe, HBV DNA, anti-HDV, abdominal US

If ALT elevated & HBV DNA present: liver biopsy and assess for therapy

Reserve therapy for patients with significant underlying liver disease

Therapy?

Lok, Heathcote & Hoofnagle: 2001

Therapy of Chronic Hepatitis B: Future Directions

Focus must be on combination therapyLong term outcomes with histological

verification of long-term benefitLoss of HBsAg might be a gold standardAppropriate directions:

Combinations of alpha interferon & lamivudine Nucleoside combinations without cross-reactive

resistance (lamivudine & adefovir or entecavir) Novel approaches: immunological or molecular