Professor Stephen Locarnini

1
Molecular Resistance Pathways of Hepatitis B
Virus

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

2
Annual Prevalent Resistance Rates for Lamivudine,
Adefovir, Entecavir, Emtricitabine and Telbivudine
amodified and updated from CL Lai et al 2003 and
NW Leung et al 2001bfrom S Locarnini et al 2005
Hadziyannis S et al 2005. NEJM 3522673.
cfrom Lee, Y-S et al 2006 Hepatology431385
dfrom RP Perrillo et al 2005 and Colonno et al
2006 eIn the LAM comparator arm, the percentage
was only 8 based on a complex case definition of
antiviral drug resistance/treatment failure. One
would thus expect a comparable relative level
fof 10-12 based on genotypic resistance compared
with Lamivudine (25 per annum).e Lok A
McMahon B 2007 Hepatology 45 507 .
3
Hepatitis B The Problem of Drug Resistance

• Why is HBV Resistance a bigger issue than in HIV?

• One Viral Target - the HBV Pol
• Only one class of drug (Nucleos(t)ide Analogues)
• But Multiple Groups of Antiviral Agents
• Lamivudine / Telbivudine (L-NUCLEOSIDES)
• Adefovir / Tenofovir (ACYCLIC PHOSPHONATES)
• Entecavir (CYCLOPENTENE RING GROUP)

4
Indications of Emergence of Drug-Resistant Virus

• 1. Increasing viral load (?1.0 log IU/ml)
• 2. Increasing serum ALT level
• 3. Clinical deterioration
• Identification of known genotypic markers of drug
  resistance within viral polymerase
• primary resistance mutations
  (rtM204V/I)
• secondary/compensatory mutations
  (rtV173L)

5
Primary Resistance Mutations
LMV Resistance
rtA181T/V rtM204V/I L-dT Resistance rtA181T/V
rtM204I ADV Resistance rtA181T/V
rtN236T TDF Resistance rtA181T/V
rtA194T/rtM204V/I ETV Resistance rtI169T
rtL180M rtT184S/A/I/L/G/C/M rtS202C/G/I
rtM204V rtM250I/V
6
Antiviral Drug-Resistance Associated Mutations

• Primary Resistance Mutations
• The Important Codons
• B-domain 169 C-domain 202
• 180 204
• 181
• 184
• D-domain 236 E-domain 250

TOTAL 8 169 180 181
184 202 204 236 250

• Proposed Key Pathways
• rtM204V/I
• rtN236T
• rtA181T/V
• rtL180MrtM204VT184 or S202 or M250
• rtA181TrtI233VrtN236TrtM250L

L-NUCLEOSIDES (LMV, LdT) ETV in LMV-Experienced
ACYCLIC PHOSPHONATES (ADV, TFV)
SHARED
ETV (naïve)
MDR
7
LMV MonoTherapy Major Issues

• In HIV
• Compensatory Mutations Fix The Primary Drug
  Resistance Mutations

8
LMV MonoTherapy Major Issues
9
ADV MonoTherapy Major Issues
?
10
Entecavir MonoTherapy Possible Issues
?
ETV MonoTherapy
L180M M204V
X
(I169T) T184GS202I
LMV / TLV
X
(I169T) M250V
LMV / TLV
X
LMV / TLV
T184GM250V
X
LMV / TLV
(I169T) T184I/P/L
11
Entecavir Resistance
Suzuki et al 2007. J Clin Virol39149-52
12
Telbivudine MonoTherapy Possible Issues
?
TLV MonoTherapy
rtM204I
LMV
LMV
rtL80I/V
LMV / ADV / TFV
rtA181T
LMV
rtL180M
LMV
rtL229W/V
13
Multi-Drug Resistance
?
ADV
LMV
ADVLMV
ADVETV
L180M T181V M204V
A181T I233V N236T M250L
?
14
In vitro Phenotypic Data Set
NA Not Available
1Chin et al. Antimicrob Agents Chemother 2001
452495-2501. 2Delaney et al. Antivir Chem
Chemother 2001 121-35. 3Angus et al.
Gastroenterology 2003 125292-297. 4Chin et al.
Antimicrob Agents Chemother 2001 452495-2501.
5Ono-Nita et al. Antimicrob Agents Chemother
2003 462602-2605. 6Levine et al. Antimicrob
Agents Chemother 2002 462525-2532. 7Delaney et
al. Antivir Chem Chemother 2001 121-35. 8Angus
et al. Antivir Ther 2004 9145-148.
rtL180MM204V
15
Polymerase-HBsAg Overlap

• The HBV surface gene overlaps completely with the
  reverse transcriptase (RT) gene

Reverse transcriptase
Polymerase
Terminal Protein
Spacer
RNAse H
A
B
C
D
E
F
G
Pre S2
PreS1
S
Surface
a determinant
Mutations selected in the RT during antiviral
therapy can result in changes in the surface gene
16
Polymerase-HBsAg Link
Telbivudine
Entecavir
Adefovir
Lamivudine
X
X
X
X
X
Terminal Protein
Spacer
Polymerase
A
B
C
D
E
F
G
RNAse H
Pre S2
PreS1
S
Surface
X
X
X
17
Loop 1 of a determinant
121
124
s-s-
126
Vaccine Escape Mutants T126S T131N
M133L K141E D144E G145R
Y
158
131
F
99
133
Diagnostic Escape Mutants P120T/A/Q D144A T131K G
145R
--s--s-
149
137
164
E
-s-s-
107
D
138
s-s-
139
147
145
195
141
G
R
144
I
D
M
E
W
S
Loop 2 of a determinant
M
196
210
198
I
(Carman, W. J Viral Hepatitis 1997 4 11-20)
18
HBsAg Mutation Selected by LMV/ADV/ETV
LMV/L-dT ADV ETV
T
K/R
120
C
C
P
121 -s-s-
T/M
124
G
T/I/A
T/V
Loop 1 of a determinant
P/C
S
A/V
161 L
T
A/G
F/L
Q
K/R
A
T
196 L/S/Stop
W
F/Y
G
S/T
S
L
N/T
99
S/T
S
164 D
S
W
G
D
P
M
W
M
P
Y
I
F/Y
M/I
I
W
I/L
Q
P
P
A/V
L
G
A
I
S
194 F
M
S/L
P
S
C
C
-s-s-
L
137
149
107
C
P
V
V
T
-s-s-
C
138
R
C
139
-s-s-
C
147
F
173 F
S/T
N
S
K
G
P
D
L
L
226
S/T
Loop 2 of a determinant
172 Stop
176 V
19
POL and ENV Link
Torresi et al. 2002. Virology 293305
rtL180M (L526M) in Polymerase causes NO change
in envelope
20
Frequency of 3TC Resistance in Patients with
Detectable HBV Viremia
Mathews et al. 2006. AIDS20863.
21
Selection of HBV Resistant Mutants in HIV/HBV
Coinfected Patients Failing Antiretrovirals with
Anti-HBV Activity Implications for Diagnostics
Vaccine Escape

Julie Sheldon, Belen Ramos, Javier
Garcia-Samaniego, Pilar Rios, Angeline
Bartholomeusz, Miriam Romero, Stephen Locarnini
and Vincent Soriano
Hospital Carlos III, Madrid, Spain Victorian
Infectious Diseases Reference Laboratory, North
Melbourne, Australia
22
Mutations in the HBV RT and HBsAg
RT HBsAg T128N P120T V142I M133I no
change D144E W153Q G145R
Spontaneous mutations
23
Implications

• Resistance changes in HBV selected by LAM, FTC,
  ETV, TBV,ADV and TFV may affect HBsAg
  antigenicity, mainly in genotype A patients,
  while pre-existing changes in genotype D may
  produce vaccine-escape mutants.
• HBV genotyping and polymerase sequencing should
  be considered to manage patients with chronic
  hepatitis B undergoing antiviral therapy,
  particularly in HIV coinfected patients.

24
Transmission of LMV-Resistant HBV

• Features
• rtL180M plus rtM204V
• lower viral replication level during acute phase
• incubation period of 2-3 months
• typical acute hepatitis
• not previously vaccinated against HBV

Thibault et al. 2002 AIDS 16131
25
Adefovir Associated Mutations
N236T
A181V
A181T
Reverse transcriptase
Terminal Protein
Spacer
RNAse H
Polymerase
A
B
C
D
E
F
G
Pre S2
Envelope
PreS1
S
W172
L173F
a determinant
N
Virion surface
163
X
100
D
Membrane
COOH
Virion interior
30
79
Warner N. 2006. VIDRL
26
Subcellular Localisation
WT
rtA181T
HBV encoding rtA181T is defective in virion
secretion acts as a dominant
negative mutant for WT HBV secretion is
retained in the cell (Warner,N and Locarnini,S
2007)
27
Table 1
4 patients had rtM204I, 1 patient had rM204V.
28
Figure 2
29
Summary

• Current emerging patterns of antiviral drug
  resistance to HBV Pol are complex
• Primary resistance mutations across groups
  A181T/V and M204I pathways
• Broad clusters of compensatory mutations during
  Lamivudine therapy (rtV214A, rtQ215S Vs
  rtI169TrtV173L Vs rtT184S) compromising future
  salvage therapy options (Adefovir, Tenofovir,
  Entecavir)
• Requirement for HBV Pol sequencing to determine
  profile of antiviral drug resistance
• need for interactive database programs to guide
  rescue therapy
• Public Health Relevance of Pol-Env Link

30
Future Needs for the Management of HBV Drug
Resistance

• Algorithm for the use of viral load genomic
  assays in the monitoring of antiviral therapy
• Management of drug resistance best strategies to
  rescue drug resistance with long-term treatment
  end-points
• Prevention of drug resistance
• Treatment strategy trials de novo combination
  versus early add-on therapy
• Drugs without cross-resistance
• Long-term endpoints

From Zoulim, F. 2006
31
SPARE
32
Viral Replication and Mutational Frequency

• High virion production 1012-13 virions per day
• Wild-type HBV Pol lacks proof-reading function
• High mutational rate10-5 substitution/base/cycle
• 1010-11 point mutations produced per day
• All possible single base changes can be produced
  per day
• Single / double mutations pre-exist in HBV from
  patients prior to therapy WHY MOST MONOTHERAPIES
  FAIL
• Triple / quadruple mutations require replication
  in the presence of selection pressure and rarely
  pre-exist WHY COMBINATION TREATMENT WORKS
  (Colgrone Japour. 1999. AVR. 4145)

33
Tenofovir Resistance

• 43 HIV-HBV coinfected patients from 5 European
  centres on TDF gt 6 months with HBV DNA gt 200 c/ml
• Duration TDF mean 11.2m
• Sequence HBV pol
• Two patients selected novel mutations
• A194T (L180M M204V).
• detected at 48 and 77 weeks of TDF

Sheldon et.al. 2005. Antiviral Therapy10727-734
34
Tenofovir Resistance
Susceptibility to TDF
Sheldon et.al. 2005. Antiviral Therapy10727-734
35
Three Groups of Nucleoside/Nucleotide Analogues
used in Chronic Hepatitis B

• L-Nucleoside Group
• lamivudine (3TC)
• emtricitabine (FTC)
• telbivudine (L-dT)
• clevudine (L-FMAU)
• Acyclic Phosphonate Group
• adefovir (PMEA)
• tenofovir (PMPA)
• Cyclopentane/pentene Ring Group
• entecavir (ETV)
• abacavir/carbovir (ABC)

36
A
B
a determinant
a determinant
(M)N4-glyco.
(M)R47-tryps.
N146-glyco.
N146-glyco.
N-
ER lumen/virion surface
100
163
172
172
Membrane
226
-COOH
-COOH
30
79
Cytosol/virion interior
C
D
a determinant
a determinant
.
N146-glyco.
N146-glyco.
ER lumen/virion surface
172
Myr
172
Membrane
Myr
-COOH
-COOH
Cytosol/virion interior
37
Indications of Emergence of Drug-Resistant Virus

• 1. Increasing viral load (?1.0 log IU/ml)
• 2. Increasing serum ALT level
• 3. Clinical deterioration
• Identification of known genotypic markers of drug
  resistance within viral polymerase
• primary resistance mutations
  (rtM204V/I)
• secondary/compensatory mutations
  (rtV173L)

38
The Hepatitis B Virus Polymerase Mutation rtV173L
is Selected During Lamivudine Therapy and
Enhances Viral Replication in vitro

• William E. Delaney IV,1 Huiling Yang,1
  Christopher E. Westland,1 Kalyan Das,2,3 Eddy
  Arnold,2,3 Craig S. Gibbs,1 Michael D. Miller,1
  and Shelly Xiong1

Triple Pol Mutant (rtV173LrtL180MrtM204V) found
in 9-22 of LMV-resistant patients with CH-B.
J. Virol. 2003. 771183
39
Results
40
Mutations in the HBV RT and HBsAg
Number of patients (Genotype)
RT HBsAg HIV/HBV HBV LAM selected V173L
M204V E164D I195M 3 (A) M204V
I195M 16 (15A, 1D) 1 (A) M204I W196S/L 3
(A) 1 (D) V191I W182stop 2 (A) M204I
V207I I195M M198I W199stop 1 (A) M204I
V207I I195M M198I 3 (A) V207L M198I 1
(F) Spontaneous T128N P120T 1
(D) V142I M133I 1(D) 1 (D) No
change D144E 1 (E) Mixed T128N
M204I P120T W196L 1 (D)
41
Results
A n44
D n23
RT mutations
p 0.001
Lamivudine associated Spontaneous vaccine
escape Lamivudine - selected
28 0 28
3 4 3
Envelope mutations
p 0.011
p 0.001
42
WHO Global Program for Hepatitis B
ACUTE /CHRONIC HEPATITIS B
CIRRHOSIS ESLD
HEPATOCELLULAR CARCINOMA
HBV
PRIMARY PREVENTION
CHEMO PREVENTION
CANCER SCREENING
Antivirals Cytokines
Tumour Marker Ultrasound Transplantation
Vaccination
43
Occult/Cryptic HBV Infection

• Case Definition
• the detection of HBV DNA by PCR or other
  amplification assays in HBsAg-negative
  individuals
• typically low level of HBV DNA (lt103 copies/ml)
• Prevalence
• common in endemic areas of HBV infection and
  amongst seropositive individuals anti-HBc ? /-
  anti-HBs ?
• Diagnosis
• serum, PBMCs and liver tissue
• PCR for HBV DNA must be validated
• performance must be meticulous to prevent
  contamination of samples, inclusion of negative
  controls and performance of assays in duplicate
  using two independent sets of HBV primers.

Conjeevaran, HS and Lok AS (2001) Hepatology
34204-206
44
What Causes Antiviral Drug Resistance?

• Antiviral drug resistance reflects reduced
  susceptibility of a virus to the inhibitory
  effect of a drug
• Antiviral drug resistance results from a process
  of adaptive mutations under therapy
• High replication rates
• Low fidelity of the viral polymerase
• Selective pressure of the drug
• Genetic Barrier of the Drug
• Role of replication space (liver turnover)
• Fitness of mutant

45
Antiviral Selection Pressure
CONCLUSION No Replication No Resistance
46
Selective Pressure Influences the Virus
Population
In the absence of selective pressure, one strain
dominates the population
Sheldon J, et al. J Viral Hep 2006 13427434
Locarnini S. J Hepatol 2003 39S124S132
47
Maximal Suppression of Virus Replication
Dominate Strain
Treatment Initiates
Naturally Occurring Variants
HBV Replication

• Maximal Suppression Minimal Resistance

Detection Level
Time
Locarnini S, et al. Antivir Ther 2004 9679693
48
Incomplete Suppression of Virus Replication
Dominate Strain
Naturally Occurring Variants
Treatment Initiates
Drug Resistant Variant

• Incomplete Suppression
• - Inadequate Potency/Drug Levels
• - Inadequate Adherence
• - Pre-Existing Resistance Variants

HBV Replication
Detection Level
Time
Fung SK Lok ASF. Antivir Ther 2004
910131026 Locarnini S, et al. Antivir Ther
2004 9679693
49
"Potency and High Resistance Barrier - A
Successful Approach to Avoiding Resistance"
Richard Colonno
Bristol-Myers Squibb Pharmaceutical Research
Institute
50
Genetic Barriers to Antiviral Drug Resistance
In patients without lamivudine resistance
mutations, emergent resistance with virologic
rebound has been observed in one patient through
3 years of treatment (simultaneous emergence of
204180202)
US Prescribing Information for Epivir-HBV
(2004), Hepsera (Aug 2006), Baraclude (July
2006) Colonno R, et al. Hepatology
200644(Suppl. 1)Abstract 110
51
Primary Factors in Preventing Resistance

• Potency - Degree to which viral replication is
  suppressed to avoid generation of new mutations

52
Relative Potency of HBV Antivirals
100
Ratio TP at Clinical Cave/IC50
Strong
10

• WT HBV
• LVDr HBV

1.0
Relative Potency
0.1
Weak
0.01
ETV 0.5mg
ETV 1.0mg
LVD 100mg
ADV 10mg
LdT 600mg
Treatment4
Baraclude (entecavir) Summary of Product
Characteristics. Bristol-Myers Squibb Pharma
EEIG. June 2006. Seifer M, et al. Antimicrob
Agents Chemother. 1998423200-3208 Yamanaka G,
et al. Antimicrob Agents Chemother.
199943190-193 Tenney D, et al. Antimicrob
Agents Chemother. 200751902-911
53
SeqHepB A Predictive Tool for Physicians
Treating Patients with Hepatitis B
www.evivar.com
54
What is SeqHepB

• A fast and efficient system to analyse the
  Hepatitis B viral genome utilising standardised
  methods to correlate clinical, virological,
  biochemical and genomic data
• SeqHepB comprises
• SeqHepB Analysis Engine
• Database
• Patient Management Tool

www.evivar.com
55
SeqHepB
viral genome sequence data
clinical and routine pathology test data
in-vitro phenotypic data
www.evivar.com
56
Ways to Prevent Resistance (D. Richman)

• Maximize antiviral activity
• increase maximum tolerated dose
• select most effective regimen (combination)
• nucleoside analogue potentiation
• Maximize genetic barriers to resistance
• avoid sequential monotherapy
• choose drugs requiring multiple resistance
  mutations (1 or 2 mutations pre-exist vs 3 or 4
  require ongoing selection)
• choose drugs where patient is naïve
• Increase pharmacologic barriers
• patient compliance
• raising trough levels
• Prior drug experience
• pharmacodynamic issues (eg, cirrhosis)