In vitro susceptibility of adefovir-associated hepatitis B virus polymerase mutations to other antiviral agents.

Abstract

Adefovir dipivoxil is a nucleotide prodrug approved for the treatment of chronic hepatitis B. During clinical trials, ADV-associated mutations were observed in 0, 3, 11, 18 and 29% of patients after 48, 96, 144, 192 and 240 weeks of therapy, respectively. Hepatitis B virus (HBV) polymerase mutations associated with virological breakthrough to ADV include rtA181V and rtN236T, which occur alone or in combination. The rtA181T mutation has also been observed at low frequency, alone or in combination with rtN236T.
To investigate the in vitro activity of adefovir and other anti-HBV agents against these mutants, we generated five stable cell lines that each expressed one of the following HBV mutants: rtN236T, rtA181V, rtA181V + rtN236T, rtA181T + rtN236T and rtA181T. Using these cell lines, we quantified in vitro changes in drug susceptibility for eight nucleotide/nucleoside analogues.
The rtN236T mutant had 7-fold resistance to adefovir but remained sensitive to entecavir, telbivudine and torcitabine (53.2-fold reduced susceptibility). The A181V mutant had 4.3-fold resistance to adefovir and had reduced susceptibility to multiple other agents ranging from 3.2-fold (tenofovir) to >191-fold (clevudine). The A181V + rtN236T double mutant was the most highly resistant showing 18-fold resistance to adefovir and higher levels of resistance to other tested drugs with the exception of tenofovir (10-fold reduced susceptibility). Our results and preliminary clinical data suggest that patients with rtN236T or rtA181V remain susceptible to tenofovir, entecavir and lamivudine. Further clinical data are necessary to precisely define in vitro cutoffs indicative of clinically-relevant resistance, particularly for drugs in development such as emtricitabine, telbivudine, torcitabine and clevudine.

Full text

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Background: Adefovir dipivoxil is a nucleotide prodrug
approved for the treatment of chronic hepatitis B. During
clinical trials, ADV-associated mutations were observed
in 0, 3, 11, 18 and 29% of patients after 48, 96, 144, 192
and 240 weeks of therapy, respectively. Hepatitis B virus
(HBV) polymerase mutations associated with virological
breakthrough to ADV include rtA181V and rtN236T,
which occur alone or in combination. The rtA181T muta-
tion has also been observed at low frequency, alone or in
combination with rtN236T.
Methods: To investigate the in vitro activity of adefovir
and other anti-HBV agents against these mutants, we
generated five stable cell lines that each expressed one
of the following HBV mutants: rtN236T, rtA181V,
rtA181V+rtN236T, rtA181T+rtN236T and rtA181T. Using
these cell lines, we quantified in vitro changes in drug
susceptibility for eight nucleotide/nucleoside analogues.
Results: The rtN236T mutant had 7-fold resistance to
adefovir but remained sensitive to entecavir, telbivudine
and torcitabine (≤3.2-fold reduced susceptibility). The
A181V mutant had 4.3-fold resistance to adefovir and
had reduced susceptibility to multiple other agents
ranging from 3.2-fold (tenofovir) to >191-fold (clevu-
dine). The A181V+rtN236T double mutant was the most
highly resistant showing 18-fold resistance to adefovir
and higher levels of resistance to other tested drugs with
the exception of tenofovir (10-fold reduced suscepti-
bility). Our results and preliminary clinical data suggest
that patients with rtN236T or rtA181V remain susceptible
to tenofovir, entecavir and lamivudine. Further clinical
data are necessary to precisely define in vitro cutoffs
indicative of clinically-relevant resistance, particularly for
drugs in development such as emtricitabine, telbivudine,
torcitabine and clevudine.
In vitro susceptibility of adefovir-associated
hepatitis B virus polymerase mutations to other
antiviral agents
Xiaoping Qi, Shelly Xiong, Huiling Yang, Michael Miller and William E Delaney IV*
Department of Clinical Virology, Gilead Sciences, Foster City, CA, USA
*Corresponding author: Tel: +1 650 522 5598; Fax: +1 650 522 5890; Email: william.delaney@gilead.com
Antiviral Therapy 12:355–362
Significant advances have been made in the treatment
of chronic hepatitis B in the past decade with the
approval of three oral therapies. Lamivudine (3TC), an
L-deoxycytidine analogue, was the first approved oral
drug for treatment of chronic hepatitis B, followed by
adefovir dipivoxil (ADV), a pro-drug of the nucleotide
adefovir (AFV), and, more recently, the deoxyguano-
sine analogue entecavir (ETV). Although nucleoside/
nucleotide analogues have proven to be safe and effec-
tive treatments for hepatitis B virus (HBV), resistance
is a frequent consequence of prolonged use of this class
of antiviral drugs. Development of resistance to 3TC
monotherapy is rapid and frequent, as primary 3TC
resistance mutations including rtM204V and rtM204I,
located in the YMDD motif of HBV reverse transcrip-
tase, emerge in approximately 20% of patients per
year, reaching 70% after 4 years of therapy [1]. In
comparison with 3TC, development of ADV resistance
is significantly slower, with no resistant viruses
detected in patients after 1 year of therapy and in 29%
of patients after 5 years of treatment in Phase III trials
[2]. Development of resistance to ETV is also infre-
quent in treatment-naive patients (0% reported at
1 year) [3]. However, 7% of patients who had previ-
ously failed 3TC therapy because of viral resistance
developed ETV-resistant viruses after 1 year of ETV
therapy. An additional 6% of patients infected with
3TC-resistant viruses have ETV resistance mutations
prior to ETV exposure [4,5].
Two mutations in HBV polymerase have been identi-
fied as primary ADV resistance mutations: rtN236T and
rtA181V. These mutations were observed in clinical
isolates from patients either alone or in combination
after >1 year of ADV monotherapy. In some patients,
the emergence of these two mutations was also associ-
ated with viral breakthrough and loss of clinical benefit.
In vitro phenotypic testing of rtN236T and rtA181V
mutant HBV isolated from patient sera or generated by
mutagenesis of laboratory stains has shown that the
susceptibility of rtN236T and rtA181V to AFV is
Introduction
© 2007 International Medical Press 1359-6535
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reduced by 4–13-fold and 3–4-fold, respectively [6,7]. A
mutation in codon rt181 that causes substitution of
threonine for alanine (rtA181T) has also been observed
at low frequency in ADV-treated patients, either alone or
in combination with rtN236T. Initial phenotypic evalua-
tion of the rtA181T mutant indicated that it confers only
<2-fold reduced susceptibility to AFV in vitro, and there
is no association of emergence of the rtA181T mutation
with serum HBV DNA rebound in clinical trial patients
(unpublished data). The frequencies of the five ADV-
associated mutational patterns observed among patients
failing ADV therapy during Phase III clinical trials were
rtN236T (47%), rtA181V (30%), rtA181V+rtN236T
(17%), rtA181T+rtN236T (3%) and rtA181T (3%) [2].
Despite the short-term success of nucleoside/
nucleotide analogues in treating chronic hepatitis B,
new treatment options are still needed to enable physi-
cians to manage resistance and tailor therapy to suit
individual patients. Several additional nucleoside/
nucleotide analogues are currently under clinical
development for chronic HBV. Compounds in Phase III
clinical trials include tenofovir disoproxil fumarate
(TDF), a nucleotide prodrug approved for the treat-
ment of HIV, the L-deoxycytidine analogue emtric-
itabine (FTC), also approved for HIV therapy, and the
L-thymidine analogues telbivudine (L-dT) and clevudine
(L-FMAU). The L-deoxycytidine analogue torcitabine
(L-dC) is currently in Phase II studies as the valine ester
prodrug valtorcitabine.
In this report, we characterize the in vitro cross-
resistance conferred by the five major patterns of ADV-
associated mutations to
developmental anti-HBV agents. A clear understanding
of cross-resistance is crucial to the optimal manage-
ment of drug-resistant HBV infection, and provides a
guide to the rational design of combination therapies.
Combination therapy using two or more drugs with
distinct resistance profiles has been successfully
employed in HIV-infected patients to maximize HIV
suppression and to reduce the incidence of drug resis-
tance compared with monotherapy. This approach is
also being explored for HBV treatment in multiple clin-
ical trials, for instance ADV+3TC [8] and L-dT+3TC
[9]. The results presented in this report provide insights
into the suitability of agents for treatment of ADV-
resistant HBV or for use in combination with ADV to
minimize the emergence of resistance.
seven approved or
Materials and methods
Antiviral compounds (Figure 1)
AFV, tenofovir (TFV), FTC, L-FMAU and ETV were
synthesized by Gilead Sciences (Foster City, CA, USA).
3TC, L-dT and L-dC were purchased from Moravek
Biochemicals (Brea, CA, USA).
Generation of plasmids expressing wild-type and
ADV-resistant HBV
Plasmid pHY108 encodes a wild-type 1.1× unit length
HBV genome (genotype A, serotype adw2) under the
transcriptional control of the CMV promoter as well
as a neomycin resistance gene under the control of the
SV40 promoter in a pBR322 backbone. AFV resis-
tance mutations were introduced into pHY108 by site-
directed mutagenesis using a QuikChange kit
(Stratagene, La Jolla, CA, USA). Mutagenesis primers
used to generate the rtA181T, rtA181V and rtN236T
mutations are listed
rtA181T+rtN236T and rtA181V+rtN236T double
mutants were created from the rtA181T and rtA181V
single mutants by second rounds of mutagenesis. To
eliminate the possibility of unwanted mutations else-
where in the HBV genome, the region of the poly-
merase gene containing the mutations was excised
from mutagenized pHY108 using ApaI and HpaI and
sub-cloned back into an unmanipulated stock of
pHY108. The sub-cloned region, including the restric-
tion junctions, was sequenced to confirm that only the
intended mutations were present in the final HBV
expression constructs.
in Table 1. The
Cell culture
HepG2 cells were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and
maintained in humidified incubators at 37°C under 5%
CO2. HepG2 cells were grown in minimal essential
medium (ATCC) supplemented with 100 units/ml peni-
cillin, 10 μg/ml streptomycin, and 10% fetal bovine
serum (Irvine Scientific, Santa Ana, CA, USA). Novel
stable cell lines (described below) were maintained
under conditions identical to HepG2 cells.
Generation of stable cell lines
HepG2 cells were transfected with plasmids encoding
wild-type or mutant HBV and a neomycin resistance
gene (described above) using Fugene 6 (Roche,
Indianapolis, IN, USA). Three days after transfection,
cells were selected in media supplemented with
800 μg/ml of G418 (Invitrogen) for 2–3 weeks. G418-
resistant colonies were transferred to 48-well plates
and screened for hepatitis B e antigen (HBeAg) secre-
tion using an ETI-EBK+ immunoassay kit (DiaSorin
Stillwater, MN, USA). HBeAg+
expanded and screened for other markers of HBV
replication including extracellular HBV DNA (by PCR
analysis of conditioned media according to the method
of Gunther et al. [10]) and intracellular viral replica-
tive intermediates (by Southern blotting). PCR-ampli-
fied extracellular HBV DNA was also sequenced using
the primer HBV-Seq-10 (5′-CTG GAT GTG TCT
GCG GCG-3′) to confirm that each cell line expressed
colonies were
X Qi et al.
© 2007 International Medical Press
356

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HBV with the intended reverse transcriptase muta-
tions. Cell lines positive for all markers of viral repli-
cation were further expanded and stored frozen in
liquid nitrogen.
Comparison of HBV expression in selected cell lines
To compare HBV expression levels in novel cell lines,
confluent monolayers were trypsinized and 2×105cells
were aliquoted into microcentrifuge tubes. Cells were
Antiviral Therapy 12:3
357
Cross-resistance profile of HBV-encoding, adefovir-associated polymerase mutations
HO
O
P
O
N
N
Lamivudine
Telbivudine
Emtricitabine
Tenofovir AdefovirEntecavir
N
N
NH2
HO
O
OH
HO
N
N
N
N
NH2
NH2
N
N
N
NH
O
CH3
O
N
N
NH2
O
OH
S
O
N
N
NH2
F
O
OH
S
Clevudine
O
F
N
HN
O
CH3
O
OH
OH
O
N
HN
O
CH3
O
OH
OH
Torcitabine
O
N
HN
CH3
NH2
O
OH
OH
HO
O
P
HO
Figure 1. Chemical structures of selected nucleoside and nucleotide analogues that inhibit HBV
MutationPrimer sequence (5′→3′)
A181T
A181V
N236T
AGT CCG TTT CTC TTG ACT CAG TTT ACT AGT GCC
AGT CCG TTT CTC TTG GTT CAG TTT ACT AGT GCC
TCT CTG GGT ATA CAT TTA ACC CCT AAC AAA ACA AAA AGA TGG
Bold sequences indicate the mutated polymerase codon.
Table 1. Primer sequences for introduction of the rtA181T, rtA181V, and rtN236T mutations

Page 4

pelleted by spinning at 750 g for 1 min, resuspended in
200 μl of PBS, and adjusted to 0.3% Nonidet P40 for
lysis. Lysates were centrifuged at 14,500 g for 1 min to
pellet nuclei. Each supernatant (35 μl) was transferred
to a 96-well PCR plate (MJ Research, South San
Francisco, CA, USA), and mixed with 65 μl of
BuccalAmp extraction buffer (Epicentre, Madison, WI,
USA). HBV DNA was released from the cytoplasmic
core particles by heat extraction in an MJ Research
PTC-100 thermocycler
programme: 30 min at 65ºC, followed by 17 min at
94ºC. Extracted DNA was quantified by real-time PCR
as described below.
using the following
Antiviral assays
Cells were seeded in 96-well plates at a density of 2×104
cells/well and allowed to attach overnight. The
following day (day 1) the cell culture medium was aspi-
rated using a 12-well vacuum manifold and cells were
treated with fresh medium containing drug. Serial drug
dilutions were prepared in medium in 96-well plates
using a Precision 2000 Automated Pipetting System
(Bio-tek Instruments Inc., Winooski, VT, USA). Cells
were treated with fresh medium containing drug on
days 3 and 5. Wild-type and ADV-resistant cell lines
were assayed in parallel using the same dilutions for
each drug. On day 7, medium was aspirated and cells
were washed once with PBS then lysed in 100 μl of
0.3% Nonidet P40 in PBS. The lysates were transferred
into 96-well V-bottom plates and spun at 500 g for
5 min to pellet cellular nuclei. Supernatant (35 μl) from
each well were transferred to 96-well PCR plates and
mixed with 65 μl BucAmp extraction buffer. HBV
DNA was released from cytoplasmic core particles by
heat extraction as described above. Extracts were
stored frozen at -20ºC until real-time PCR quanti-
fication of HBV DNA was performed (see below). The
resulting data were fitted to the 3-parameter logistic
dose–response equation y=a/(1+(b/x)^c) using Prism
v4.0 (Graphpad Software Inc., San Diego, CA, USA).
Fold resistance was calculated as the ratio of mutant
effective concentration to inhibit 50% of HBV replica-
tion (EC50) to wild-type EC50. To determine whether
mutant EC50s were significantly different from those of
wild type, EC50 data were log transformed and
analysed by paired, two-tailed t-tests. P-values of <0.05
were considered significant. GraphPad Prism 4.0 was
used to calculate P-values.
Real-time PCR quantification of HBV DNA
Cytoplasmic extracts were analysed by real-time quan-
titative PCR (qPCR) using an ABI Prism 7900HT
Sequence Detection System (Applied Biosystems,
Foster City, CA, USA). PCR primers HBV-Taq1
(forward) and HBV-Taq2 (reverse) and the
FAM/TAMRA labelled TaqMan probe BS-1 were used
for HBV quantification as described previously [11].
PCR reactions were performed in a 20 μl volume using
10
μl of 2×
TaqMan Master Mix (Applied
Biosystems), 200 nM BS-1 probe, 300 nM HBV-Taq1
primer, 300 nM HBV-Taq2 primer, and 2 μl of
extracted DNA. Plasmid pHBVEcoRI, which encodes
a single genome of HBV DNA, was run as a standard.
A linear dynamic range from 101to 107copies was
observed, in agreement with previous reports for this
primer/probe set [11].
Results
Generation of stable cell lines expressing major patterns
of ADV-associated mutant HBV and quantification of
HBV expression by qPCR
The method of using real-time qPCR for high
throughput anti-HBV testing with stable cell lines has
been developed and validated previously in our labora-
tory [12]. To evaluate the in vitro susceptibility of
ADV-associated HBV mutants to other anti-HBV
compounds, we generated five stable cell lines that each
expressed one of the ADV-associated mutations
rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+
rtN236T and rtA181T. Multiple positive clonal cell
lines, which ranged in expression levels, were identified
for each viral mutant. One clonal cell line for each
mutant was selected for further studies based on HBV
expression levels and confirmation of intracellular
single- and double-stranded replication intermediates
by Southern blot (data not shown). The expression
levels of cytoplasmic HBV DNA in these cell lines
ranged from 9 to 123 copies/cell as quantified by real-
time qPCR (Table 2). Each cell line was confirmed to
encode the intended HBV mutation by DNA
sequencing. WT-42, a previously reported cell line
expressing wild-type HBV [12], was tested in parallel
with the mutant HBV cell lines. Each of the cell lines
produced a signal within the linear range of the qPCR
assay (101to 107copies) (Figure 2).
X Qi et al.
© 2007 International Medical Press
358
Mutational pattern Cell line Cytoplasmic HBV*, copies/cell
rtN236T
rtA181V
rtA181V+rtN236T
rtA181T+rtN236T
rtA181T
Wild type
NT-4-15
AV-81
AVNT-3-39
ATNT-2-1
AT-1-15
WT-42
52.3 ±3.2
41.2 ±2.7
9.7 ±1.1
19.5 ±5.0
123.5 ±16.4
8.9 ±0.9
*Cytoplasmic hepatitis B virus (HBV) was measured by qPCR as described in
Materials and methods. Values represent the mean ±SD.
Table 2. Quantification of cytoplasmic HBV copies in stable
cell lines

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Susceptibility of ADV-associated mutations to AFV
We first used the five characterized cell lines to assess
the sensitivity of each ADV-associated mutant to AFV.
The rtN236T mutant had approximately 7-fold
reduced susceptibility to AFV (Table 3). The rtA181V
mutant was slightly less resistant to AFV than rtN236T
and increased the EC50 by 4.3-fold. However, the
rtA181T mutant showed near wild-type sensitivity to
AFV with only a 1.2-fold change in EC50, which was
not statistically significant (P=0.4) . When rtA181T
was added to the rtN236T mutant, the resulting double
mutant displayed only a 5.2-fold increase in AFV EC50
compared with the 7-fold increase observed for the
rtN236T single mutant, suggesting the possibility of a
resensitization effect. By contrast, the rtA181V+
rtN236T double mutant showed an increased level of
resistance to AFV (18-fold increase in EC50) compared
with the single rtN236T mutant.
Cross-resistance testing of other anti-HBV compounds
We next selected seven approved or developmental
anti-HBV compounds to test against ADV-associated
mutants. These included TFV, ETV, 3TC, FTC, L-
FMAU, L-dT and L-dC. TFV is an acyclic nucleotide
analogue structurally similar to AFV (Figure 1). The
rtN236T and rtA181V mutations reduced TFV suscep-
tibility by 4- and 3.2-fold, respectively; these suscepti-
bility shifts were smaller than those observed for AFV.
Like AFV, TFV susceptibility did not change signi-
ficantly for the rtA181T mutant (1.5-fold increase in
EC50; P=0.12). The double mutant rtA181T+rtN236T
was also more susceptible to TFV than rtN236T alone
(3-fold vs 4-fold respectively). The double mutant
rtA181V+rtN236T showed the greatest shift in suscep-
tibility to TFV (10-fold), but again this was less than
the 18-fold shift observed for AFV.
ETV had an EC50of 3.3 nM for wild-type HBV,
consistent with previous results. ETV demonstrated
similar inhibitory activity against wild-type HBV and
the rtN236T mutant with an EC50changed by 2.1-fold
(P=0.18; Table 3). However, ETV’s in vitro activity
decreased 8.4- and 12-fold, respectively, against the
rtA181T and rtA181V mutants. Interestingly, the
rtA181T+rtN236T double mutant was more suscep-
tible to ETV than the rtA181T single mutant (3-fold vs
8.4-fold), similar to our observations for AFV and TFV
with this double mutant and rtN236T. The
rtA181V+rtN236T double mutant had a greater reduc-
tion in susceptibility to ETV than either single mutant
(21-fold increase in EC50).
Five L-nucleoside analogues (3TC, FTC, L-FMAU,
L-dT and L-dC) were assayed for activity against
Antiviral Therapy 12:3
359
Cross-resistance profile of HBV-encoding, adefovir-associated polymerase mutations
Fold resistance†,‡
A181V+N236TCompoundWT EC50*, μM N236TA181V A181T+N236T A181T
Adefovir
Tenofovir
Entecavir
Lamivudine
Emtricitabine
Clevudine
Telbivudine
Torcitabine
1.17 ±0.43
0.92 ±0.23
0.003 ±0.001
0.11 ±0.02
0.14 ±0.01
0.77 ±0.32
3.41 ±0.71
0.36 ±0.03
7.0 ±4.1§
4.0 ±2.7#
2.1 ±1.2
13 ±9#
13 ±5§
7.4 ±2.4#
3.2 ±1.3**
2.3 ±2.0
4.3 ±1.2¶
3.2 ±0.9¶
12 ±1¶
15 ±7¶
15 ±6¶
>191 ±94§
>27 ±9§
82 ±45¶
18 ±2¶
10 ±3¶
21 ±12¶
35 ±13¶
70 ±34¶
144 ±29¶
>40 ±17**
>307 ±45#
5.2 ±1.7#
3.0 ±0.4¶
3.0 ±1.1§
34 ±10¶
47 ±13¶
149 ±26*
19 ±2#
105 ±8¶
1.2 ±0.5
1.5 ±0.5
8.4 ±7.4
12 ±6§
15 ±9#
117 ±65§
15 ±6#
118 ±93¶
*Wild type (WT) value represents the mean ±SD of at least three independent experiments performed in triplicate. †Fold resistance calculated as the mutant 50%
effective concentration (EC50)/WT EC50. The indicated values are the mean fold difference ±SD for three or more experiments performed in triplicate where WT was
assayed in parallel with the mutants. ‡Statistically significant changes in EC50between the mutants and WT hepatitis B virus (HBV) are indicated based on two-tailed,
paired t-tests: §P<0.01. ¶P<0.001. #P<0.05. **P=0.05 for WT vs mutant for telbivudine susceptibility.
Table 3. Fold resistance of nucleoside and nucleotide analogues to HBV encoding the major AFV resistance mutations
8
6
5
7
4
3
2
1
0
C(T) cycle
1015
20 25
a: AT-1-15
b: NT-4-15
c: AV-81
d: ATNT-2-1
e: AVNT-3-39
f: WT-42
abcdef
30 3540
Log10 HBV copies
Figure 2. Copies of cytoplasmic HBV in one real-time qPCR
reaction
2 μl of hepatitis B virus (HBV) DNA extracted from one well of a 96-well plate
was added to each reaction. Plasmid pHBVEcoR1 serially diluted 10-fold from
107to 101copies was run in parallel with the samples to generate the standard
curve. Under these assay conditions the copy number of HBV amplified from
each well lies in the middle of the dynamic range of the qPCR assay. The labels
in the figure legend correspond to cell lines specified in Table 2. C(T) cycle,
threshold cycle at which the sample signal is greater than background and is
defined as PCR-positive.