Professor Stephen Locarnini

1
ANTI-VIRALS FOR HEPATITS B DO THEY MODIFY THE
VIRAL KINETICS AS WELL AS OUTCOME?

• Professor Stephen Locarnini
• Victorian Infectious Diseases Reference
  Laboratory,
• North Melbourne, Victoria 3051,
• AUSTRALIA
• www.vidrl.org.au/publications/hep_updates.htm

2
Strategies Used by HBV to Ensure Persistence

• 1. HBeAg
• Soluble and secreted protein
• Toleragen perinatal transmission and is
  essential for PERSISTENT infection
• Possible immune regulatory function both INNATE
  and ADAPTIVE
• dampens hosts immune response to
  virus-infected hepatocytes
• Pre-core protein regulates level of HBV
  replication
• 2. HBsAg
• Excess production (?decoy) of 22 nm particles
  and filaments
• Diverts anti-HBs neutralization of virions (42nm
  forms)
• 3. HBV cccDNA
• major transcriptional template
• heterogeneous topoisomer species
• variable half-life
• resistant to nucleoside analogue therapy
• 4. HBV DNA Integration
• HBV can be generated from spliced HBV mRNA
  transcripts
• Role in latency and reactivation.

3
HBV Infection Pathogenesis

• Represents the outcome of the interplay between
  the virus, the hepatocyte and the hosts immune
  response
• under normal circumstances, HBV is not cytopathic
• in CH-B, liver damage produced as a result of
  hosts cellular immune response to HBV-infected
  hepatocytes as part of the immune clearance
  phase
• Two Therapeutic Approaches
• Direct Antiviral Agents LMV, ADV, ETV
• Immune Modulation IFN-alpha and Thymosin alpha-1

4
Therapeutic Agents Available for HBV
Infections

• Registered
• Interferon-alpha
• PEG-Interferon
• Thymosin-alpha
• Lamivudine
• Adefovir Dipivoxil
• Entecavir
• Emtricitabine (-) FTC
• Tenofovir

• Under Development
• Clevudine (L-FMAU)
• Telbivudine (L-dT)
• LB80317

5
Viral Dynamics in HBV
Hypothesis Viral dynamics maximize the use of
readily available data and allow different drugs,
dosages and regimens to be compared more
precisely. It may improve the understanding of
the mechanism of action of the drugs and help to
optimize treatment regimens. HIV / HCV proved
to be useful Estimation of the total rate of HIV
/ HCV virion production and clearance and half
life of free particles and productively infected
cells

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Viral Dynamics HIV
Viral Load
Protease Inhibitor

Steady State
SetPoint
C
2 logs
2 weeks
to
Note Assume 100 inhibition. If patient in
steady state Rate of Production Rate of
Clearance P cV c slope of PI effect V
viral level (Vge/ml)
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Standard Model of Viral Infection

• 3 differential equations describing
• The change in the number of uninfected cells
  (Target cells, T)
• dT/dt s dT -
  1- ?(t) ?VT
• The change in the number of infected cells
  (Infected cells, I)
• dI/dt 1- ?(t) ?VT - ?I
• The change of viral load (V)
• dV/dt 1-?(t) p I - cV

• s production rate of T d death rate of
  T ? infection rate of T
• clearace rate of I p production
  rate of virions
• c clearance rate of virions

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Assuming the Presence of a Steady-State Before
Therapy
Block of virion production
1st phase
2nd phase
Clearance of infected cells
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Modelling HBV Infection in CH-B
Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas
HC, McDade H Viral dynamics in HBV infection
PNAS USA 1996 934398-4402 Tsiang M, Rooney
JF, Toole J and Gibbs CS Biphasic Clearance
Kinetics of HBV from patients during ADV
therapy Hepatology 199929,61863-1869 Lewin SR,
Ribeiro RM, Walters T, Lau GK, Bowden S,
Locarnini S, Perelson AS Analysis of HBV viral
load decline under potent therapycomplex decay
profiles observed Hepatology 200134,51012-1020
Layden JE, Layden TJ, Reddy R, Levy-Drummer RS,
Poulakos J, Neumann AU First phase viral kinetics
parameters as predicitors of treatment response
and their influence on the second phase viral
decline J Viral Hep 20019340-345 Sypsa V-A,
Mimidis K, Tassopoulos NC, Chrysagis D,
Vassiliadis T A viral kinetics study using PegIFN
a2b and/or LMV in patients with CHB HBeAg
negative Hepatology 200542,177-85 Gauthier J
et al Quantitation of HBV viraemia and emergence
of YMDD variants in patients with chronic
hepatitis B treated with lamivudine J Infect Dis
1999 1801757-1762. FOR HBeAg SEROCONVERSION,
HBV VL NEEDS TO DROP BELOW 104 copies/ml
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Dynamics of HBV Infection
12
B
A
700 600 500 400 300 200 100 0
109 108 107 106 105 104 103 102 101 100
? 0.9
? 0.99
? 0.999
Viral Load (copies/ml)
? 0.9
Viral Load (copies/ml)
? 0.9999
? 0.99999
? 0.99
? 0.999
0.90 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99
1.00
? 0.9999
? 1
? 1
0 20 40 60 80 100 120 140
Time (day)
Time (day)
Tsiang, Hepatology 1999 291863
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B
Tsiang, Hepatology 1999 291863
14
Patient 4
Patient 9
HBV viral load (copies/ml)
Patient 1
Patient 14
WT HBV POL
Days post therapy
Lewin et al 2001, Hepatology
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Bio-Mathematical Modelling of Viral Infection
Dynamics During Antiviral Therapy
Triphasic decay or staircase pattern of plasma
HBV DNA
0 Drug uptake and phosphorylation

1 Initial rapid phase Clearance of HBV from
circulation
0
LogV(t)
1
2 ?
2 Second slower phase Clearance of infected
hepatocytes
3 ???
3 Important immunological aspects missing
Cytolytic vs noncytolytic clearance
mechanisms cccDNA? subviral particles??
t
Lewin Hepatology 2001, Perelson Sem Liv Dis 2004
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Randomized, Double-Blind Study Comparing Adefovir
Dipivoxil (ADV) Plus Emtricitabine (FTC)
Combination Therapy Versus ADV Alone in HBeAg ()
Chronic Hepatitis B Efficacy and Mechanisms of
Treatment Response

• GKK Lau1, H Cooksley2, R Ribeiro3, K Powers3, S
  Bowden4, H Mommeja-Marin5, J Sorbel5, E Mondou5,
  F Rousseau5, S Lewin6, A Perelson3,
  S Locarnini4, and N Naoumov2
• 1Department of Medicine, Queen Mary Hospital,
  Hong Kong SAR, China 2Institute of Hepatology,
  University College London, London, UK 3Los
  Alamos National Laboratory, Los Alamos, NM, USA
  4Victorian Infectious Diseases Reference
  Laboratory, Victoria, Australia 5Gilead Sciences
  Inc, Durham, NC, USA 6Infectious Diseases Unit,
  Alfred Hospitaland Department of Medicine,
  Monash University, Melbourne, Australia

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AIMS

• To compare the efficacy of a new combination
  therapy of ADV plus FTC versus ADV alone
• To examine early HBV kinetics and virus-specific
  T-cell reactivity to gain understanding of the
  mechanisms of successful HBV control

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Methods ? FTCB-201 Study Design
Randomized, double-blind, single center study
24 wks
48 wks
96 wks
ADV 10 mg QD FTC 200 mg QD (n 14)
ADV 10 mg QD (n 16)
Wk 0 12 (D0, 1, 3, 5, 7, 9, 11, 14 Wk 3, 4, 8,
12)

• Viral kinetics by mathematical modeling-Dr. Alan
  Perelson, Los Alamos, NM
• Digene Hybrid Capture II, LOD 4700 copies/mL
  Roche Amplicor Cobas HBV PCR,
• LOD 200 copies/mL
• Quantitative HBeAg assay at VIDRL

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Virus-specific T cell studies

• Serial PBMC prospectively collected
• Wk 0, 2, 4 and then every 4 wks till wk 96
• HBV-specific T-cell responses
• Frequency of CD4 T cells
• ELISPOT assay
• Frequency of CD8 T cells
• ELISPOT assay
• Intracellular cytokine staining (ICS) 6 HLA A2
  epitopes
• Tetramers for Core and Surface epitopes
• direct ex vivo
• in vitro expansion
• Frequency of NKT cells

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Early HBV kinetics identified two
subsetsClearance of virus (lt4700 copies/ml) by
week 12
Fast responders (n11)
Slow responders (n19)
Patients on combination treatment were more
likely to be fast responders than those on
monotherapy (8/14 vs 3/16,p0.03)
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Time course analysis of early HBV kinetic, CD4
and CD8 T-cell reactivity
Fast responder ALT flare
Increased HBV-specific CD4 T-cells
Unchanged frequency HBV-specific CD8T-cells
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Comparison between Fast Vs Slow responders during
antiviral treatment
P-value from a multivariate model of response
gt10 SFC/106, gt12 wks, overlapping W12
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HBeAg Declined Rapidly in Fast Responders
p0.02, Mann-Whitney U-test
Slow responders
Fast responders
HBeAg threshold was defined at wk 12 Fast
responders lt 300 PE IU/ml (p0.007 Vs slow
responders)
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Conclusions

• The combination of ADV FTC demonstrated
  significantly greater antiviral activity compared
  to ADV monotherapy
• Different patterns of virus-specific T-cell
  reactivity have been identified during antiviral
  treatment
• Assessment of early virological response and
  T-cell reactivity may provide a basis for
  individualized approach and optimization of mono
  or combination therapies in CHB
• Quantitative HBeAg testing confirms Virological
  Response (Fast versus Slow Responder) by Week 12
  (lt300 PE IU/ml) and may predict HBeAg
  Seroconversion

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Kinetics Of Acute Versus Chronic HBV Infection

• Whalley et al(2001)J Exp
  Med 193847
• After peak VL in serum gt1010 copies/ml
• Clearance of HBV DNA followed a two or three
  phase decay pattern
• P1 mean t½ HBV DNA 3.7? 1.2 days (similar to
  non-cytolytic clearance of cccDNA)
• P2 mean t½ HBV DNA 4.8 284 days (rate of
  loss of infected hepatocytes)
• Free virus mean t½ 1.2 ? 0.6 days
• Peak HBV production 1013 virions/day
• Infected hepatocyte 200-1,000 virions/day
• 90 of virus-infected hepatocytes were cleared of
  HBV by antiviral cytokines (IL-2, IFN-gamma,
  TNF?, IFN?/ß) in a non-cytolytic process
• CONCLUSION
• P-1 (first phase elimination) due to
  non-cytolytic removal/inhibition of HBV
  cccDNA replenishment

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Dynamics of Hepatitis B Virus Clearance in
Chimpanzees JM Murray, SF Wieland, RH Purcell and
FV Chisari, PNAS 2005 1024817780-85
Mathematical modeling was performed to test the
extent to which cytopathic or noncytopathic T
cell effector functions contribute to the
resolution of primary HBV infection. Simulation
based exclusively on cytopathic mechanisms show a
poor fit to the data and would require the
destruction and regeneration of 11 livers for
clearance. Simulation based on a combination
of cytopathic and non cytopathic mechanisms
show a better fitting to the data (Plt0.001) and
required as much as 5 fold less Destruction to
clear the virus. The best fit simulation
supports the notion that during the early phase
of HBV clearance, non cytopathic T cell effector
mechanisms inhibit viral replication And greatly
shorten the half-life of cccDNA
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cccDNA in Chronic Hepatitis B

• cccDNA is produced by repair of double stranded
  DNA and incorporated in the nucleosome as a
  stable, episomal minichromosome
• The source of cccDNA is incoming virus and the
  recycling of nucleocapsids from the hepatocyte
  cytoplasm
• cccDNA serves as the transcriptional template for
  viral mRNAs
• (that is pregenomic RNA and mRNA for surface,
  core, and polymerase)

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cccDNA in Chronic Hepatitis B

• cccDNA is the viral reservoir responsible for
  persistent infection of hepatocytes during CHB,
  believed to be responsible for relapse after
  antiviral therapy
• Stability related to long half-life of
  hepatocytes
• cccDNA represents an genetic archive for a rapid
• reselection of resistant mutants
• Existing antiviral therapies have no direct
  inhibitory
• effect on cccDNA

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Nucleos(t)ide Analogues (NA)HBeAg Infected
Hepatocyte Load
Virus Attachment
Uncoating
Nuclear transport
ccc DNA
Minichromosome
5
?
3
?
3.5kb
precore
mRNA
mRNA Transcription
(RNA Genome)
5
?
3
?
X
AAA
Translation
3.5kb
pgRNA
Reverse
Transcription
Precore
(p25)
DNA ()
synthesis
DNA (-)
Post-translational
Nucleos
(t)
ide
modification
analogues
DNA ()
Envelope and
HBeAg (p14-17)
Secretion
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A Lok 2005
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Hepatic HBV cccDNA Levels in Different Patient
Populations
Quantitative real-time PCR used to measure cccDNA
from liver biopsies
Werle, Petersen, Locarnini, Zoulim
Gastroenterology 2004
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ADV Associated Serum HBsAg Reductions are Similar
in Magnitude to cccDNA Reductions
Serum HBV DNA
Total Intracellular DNA
cccDNA DNA
Serum HBsAg

• Change in cccDNA -0.8 Log10 copies/cell (84
  reduction)
• Change in serum HBsAg titer -0.6 Log10 ng/mL
  (73 reduction)

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Changes in Serum HBsAg Levels are Positively
Correlated with Changes in cccDNA Levels
plt0.01
ADVPEG
0
copies/cell)
-1
Change in cccDNA
10
-2
(log
-3
-3
-2
-1
0
1
Change in Serum HBsAg
(log
ng/mL)
10
Petersen EASL 2005
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HBV Clearance from Hepatocytes Appears to be
Primarily Non-Cytolytic
Baseline
Week 48

• Decline in cccDNA (84) and serum HBsAg (73) is
  not paralleled by a similar decline in the number
  of HBcAg cells
• Suggests cccDNA depleted primarily by
  non-cytolytic mechanisms

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Is cccDNA Clearance Achievable?

• Possibly, in a small percentage of patients using
  antiviral therapies that are more potent and have
  a negligible rate of resistance for long
  durations
• Since cccDNA can be detected in inactive carriers
  and people with resolved infections, long-term
  suppression to low concentrations may be
  sufficient in preventing progressive liver damage
  
• Need to Activate Host Immune Responses against HBV

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Therapeutic Agents Available for HBV
Infections

• Registered
• Interferon-alpha
• PEG-Interferon
• Thymosin-alpha
• Lamivudine
• Adefovir Dipivoxil
• Entecavir
• Emtricitabine (-) FTC
• Tenofovir

• Under Development
• Clevudine (L-FMAU)
• Telbivudine (L-dT)
• LB 80317

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Conclusion

• HBV kinetics are not adequately described by
  biphasic curves
• First challenge is that the antiviral efficacy
  (e) is not reducing virus loads lt104 copies/ml
• Second challenge is in second phase where
  additional antiviral agents or immune modulators
  required to promote both cytolytic and
  non-cytolytic clearance of infected cell loss
  (and thus the pool of intra hepatic HBV cccDNA)
• need to design and tailor therapeutic regimen
  most likely to be associated with sustained viral
  clearance
• need to define the duration of therapy with an
  end-point (HBeAg Seroconversion or HBsAg loss)
• to optimize existing treatments and to guide
  combination therapy protocol development

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Perspectives

• Further studies are warranted to
• Refine the model for cccDNA clearance in
  chronically
• HBV infected patients
• Demonstrate that monitoring of cccDNA may
  provide
• an independent predictor of outcome
• Further investigate if HBsAg and HBeAg
  quantification might act as noninvasive
  surrogate parameters for changes in
• cccDNA levels
• Target cccDNA function beyond cccDNA
  quantification

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• SPARE

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Combination Antiviral Therapies

• Principal of rapid maximal suppression
• Superiority of combinations requires testing
• Proof of principle exists from HIV treatment
• Viral kinetics during treatment may facilitate
  understanding

S. Lewin et al (2001) Hepatology 341012-20
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Conclusions Viral Dynamics

• HBV kinetics might help to improve understanding
  of mechanisms of action of antiviral drugs and
  improve the management of patients with CH-B
• The results of HBV kinetics studies should be
  considered preliminary (pending larger trials)
• Various patterns of response have been reported,
  the significance of which remains in parts
  unclear
• Recent reports to quantify cccDNA in the liver
  might improve interpretation of viral kinetics
  analyses in patients with CHB receiving antiviral
  therapy
• For HBeAg seroconversion HBV VL needs to be lt104
  copies/ml

Gauthier et al 1999. J. Inf. Dis 1801757
42
Conclusions cccDNA

• Hepatitis B Virus cccDNA is a new marker in the
  evaluation of response to chronic hepatitis B
  therapy
• cccDNA persists through all phases of the natural
  history of CH-B
• 48 weeks of ADV and PEG IFN therapy results in a
• -2.4 log decrease in hepatic cccDNA compared to
• -0.8 log decrease after ADV mono therapy
• HBsAg titers are positively correlated with
  cccDNA levels and confirm that antiviral
  therapies reduced the reservoir of
  transcriptionally active viral cccDNA

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Basic Model of Viral Dynamics

Neumann et al, Science 1998
44
Baseline Characteristics
45
Immune System Activity
Infected Cells Loss
Infected cells to
Infected cells from virions conversion
virions
conversion
Viral Load
Virions production rate
Free virions clearance rate
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Immune System Activity
Infected Cells Loss
Infected cells to
Infected cells from virions conversion
virions
conversion
Viral Load
Virions production rate
Free virions clearance rate
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Table 1 Comparative Dynamics Among the Three
Viral Infections Caused by HBV, HIV and HCV
(Modified from Tsiang et al. 1999).

• HBV HBV HIV HCV HBV6
• ADV1 LMV2 Ritonavir3 IFN-a4,5 LMV or
  LMVFCV
• Plasma Virus
• Half-life 26.4 h 24 h 5.8 h 2.7-7.2
  h 18.9 h
• Mean lifespan 36.9 h 34.6 h 8.4 h 3.9-10.4
  h 27.0 h
• Mean viral
• generation time 24.8 d 24.7 d 2.7 d
  3.8-7.3 d 20.5 d
• Daily turnover 48 50 95
  94-99.8 56
• Daily production (plasma) 2.1x1012 1011
  1010 (1.1-12.7)x10 11 9.8x10 11
• Total load 1.9x1012 2x1011 1.2x109
  (3.8-5.6)x1010 1.4x10 12
• Infected Cells
• Half life 11-30 d 10-100 d 1.6 d 2.4-4.9
  d 2.4 - gt120 d
• Mean lifespan 23.3 d 23.3 d 2.3
  d 3.5-7.1 d 19.4 d
• Daily turnover 2.3-6.2 1-7
  38 13-25 4.7
• 1 The HBV ADV data is from Tsiang et al.
  Hepatology 1999 291863.
• 2 The HBV LMV data is from Nowak et al. PNAS,
  1996 93226
• 3 The HIV data are from Perelson et al.
  Science, 19962711582

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Enhanced Efficacy of Lamivudine/Famciclovir Compar
ed With Lamivudine Alone
lamivudine
lamivudine/famciclovir
Lau et al. Hepatology, 2000
52
Issues To Resolve

• Validation that technique is specific for cccDNA
• Sampling error
• Non-invasive surrogate parameter

53
T target cells, hepatocytes
54
HBV Dynamics Computed Parameters
Studies treatment patients
weeks t1/2 virus t1/2 I
antiviral efficacy (h)
(d) Nowak et al LMV 20-600
HBeAg 4 24
10-100 87-99 Lewin et al
LMV 150 HBeAg 12
28.5 2.4gt120 95
LMVFCV 99 Tsiang et al ADV
HBeAg 12
26.4 11-30 99 Wolters et
al ETV HBeAg /- 4
16 5.2-31.8 87-98 Wolters
et al LMV150 HBeAg 4
13 lt0-331 92-96
Sypsa et al Peg IFN1/200 HBeAg -
4 12.7 2.7-75
83 Peg
IFNLMV 93
LMV 100 96
Different sampling schedule during the first
48 hours
from Sypsa et Hepatology 2005, modified
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Molecular Pathogenesis of Viral Hepatitis
Hepatocyte
Immune Response
Hepatitis Virus
Extra-Hepatocyte Reservoir
Virus replication
Host antiviral immune response
Infection controlled?
Persistent infection?
High virus dose Intravenous delivery Viral
tropism for antigen presenting cells
Rapid viral replication rate Viral resistance to
host defences Immunoincompetent host
CTL exhaustion
Heavy virus replication
Persistent infection