Benefit of interferon-free, nucleic acid polymer-based
combination therapy for chronic hepatitis B
Jonathan Quinet1, Catherine Jamard1, Andrew Vaillant2, Lucyna Cova1
1.INSERM U1052, University Lyon 1, CRCL, Lyon, France, 2. Replicor Inc. Montreal, Canada
BACKGROUND & AIMS
•
MATERIAL & METHODS
Nucleic acid polymers (NAPs) are a promising new
therapy for chronic hepatitis B treatment since
they inhibit HBsAg release from infected
hepatocytes.
•
The clinical NAP compound, REP 2139, was shown
to achieve the elimination of circulating HBsAg in
human subjects with chronic HBV infection.
 The goal of this preclinical study was to examine
the in vivo antiviral effect of combining REP 2139
with tenofovir disoproxil fumarate (TDF) and
entecavir (ETV) in the chronic DHBV infection
model.
•
Three-day-old Pekin ducklings were infected with 2x1011 VGE/ml of DHBV from infectious duck serum
•
Antiviral treatment was started in 28 days-old animals and lasted for 28 days using the following dosing regimens:
• REP 2139-Ca: daily via 10mg/kg IP injection (REP 2139 formulated as a calcium chelate complex).
• ETV: 1 mg daily/ oral gavage
• TDF: 15mg daily / oral gavage
•
Treatment groups (n=10) consisted of NS (IP) control, REP 2139-Ca, TDF, REP 2139-Ca + TDF and REP 2139-Ca + TDF + ETV.
•
Antiviral activity was assessed by monitoring serum DHBsAg and anti-DHBpreS (anti-DHBsAg) antibodies by ELISA and serum and liver
DHBV DNA and cccDNA by quantitative PCR.
•
Viremia was assessed by qPCR at day 1, 14 and 28 of treatment and 4 and 8 weeks after the end of treatment (follow up). Liver DHBV DNA
and cccDNA were assessed by qPCR at autopsy.
•
Immunostaining of surface antigen (DHBsAg) was performed using primary 1H1 Mab and secondary HRP-conjugated sheep anti-mouse
IgG.
RESULTS
TDF
Normal saline
Treatment
(4 weeks)
Serum
DHBsAg
Serum DHBsAg (OD 450 nm)
1.6
1.4
1.2
1.0
Treatment
(4 weeks)
Follow up
(8 weeks)
926
927
929
931
932
933
934
936
965
935
937
938
939
940
941
942
944
949
953
926
927
929
931
932
933
934
965
935
937
938
939
940
941
942
944
949
953
REP 2139-Ca
Follow up
(8 weeks)
Treatment
(4 weeks)
REP 2139-Ca + TDF + ETV
REP 2139-Ca + TDF
Treatment
(4 weeks)
Follow up
(8 weeks)
Follow up
(8 weeks)
Treatment
(4 weeks)
964
968
974
981
986
991
956
957
958
959
962
971
Follow up
(8 weeks)
947
979
980
983
988
989
992
994
0.8
0.6
0.4
0.2
0.0
Serum
DHBV DNA
Serum DHBV DNA (x103 vge/mL)
1.E+07
1.E+06
1.E+05
1.E+04
7
6
5
947
979
980
983
988
989
992
994
964
968
974
981
986
991
4
3
1.E+03
956
957
958
959
962
971
1.E+02
1.E+01
Day 1
1
LLOQ
LLOQ
LLOQ
1.E+00
2
Day 14 Day 28 Week 4 Week8
(autopsy)
Day 1
Day 14 Day 28 Week 4 Week 8
(autopsy)
Day 1
Day 14 Day 28 Week 4 Week 8
(autopsy)
LLOQ
LLOQ
0
Day 1
Day 14 Day 28 Week 4 Week 8
(autopsy)
Day 1
Day 14 Day 28 Week 4 Week 8
(autopsy)
Figure 1. Effect of NAP-based combination therapy on viremia and DHBsAg in DHBV-infected Pekin ducks. Serum DHBV DNA and DHBsAg analysis during 28 days of treatment and 8 weeks follow-up
after end of treatment. Sampling points are indicated on X-axis (not to scale). A rebound of viral replication was observed in all ducks after TDF monotherapy cessation. In contrast, combined therapy
with REP 2139-Ca+TDF or REP 2139-Ca+TDF+ETV produced a more rapid antiviral effect on treatment and elicited a sustained virologic responses off-treatment in a majority of animals.
A
B
Normal saline
TDF
REP 2139-Ca
REP 2139-Ca + TDF
REP 2139-Ca +
TDF + ETV
1.E+10
1.E+09
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
927
929
932
933
934
936
965
Duck number
935
937
938
939
940
941
942
944
949
953
956
957
958
959
962
964
971
968
974
986
947
991
979
980
983
988
989
992
1.E+10
1.E+09
994
Duck number
Duck number
Duck number
Duck number
981
Controls
TDF
1.E+09
REP 2139
REP 2139 + REP 2139 +
TDF
TDF + ETV
*p=0.01
*p=0.02
Liver DHBV cccDNA
(copies/mg total DNA)
Liver DHBV cccDNA
(copies/mg total DNA)
931
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
p=0.21
1.E+08
926
1.E+09
Liver
ccc DNA
*p=0.04
p=0.22
Liver DHBV total DNA
(vge/mg total DNA)
Liver DHBV total DNA
(vge/mg total DNA)
Liver
DHBV DNA
*p=0.02
1.E+11
1.E+11
926
927
929
931
932
933
934
936
965
Duck number
935
937
938
939
940
941
942
944
949
953
956
957
Duck number
958
959
962
964
971
968
974
981
986
991
947
Duck number
Duck number
979
980
983
988
989
992
*p=0.05
p=0.16
1.E+08
1.E+07
994
Controls
TDF
REP 2139 REP 2139 + REP 2139 +
TDF
TDF + ETV
Duck number
Figure 2. Effect of NAP-based combination therapy on DHBV in the liver of infected Pekin Ducks. Analysis of liver DHBV DNA and cccDNA at end of follow up. Reductions in liver DHBV DNA and
cccDNA in individual animals (A) and among treatment groups (B) were observed in REP 2139-Ca treated ducks, but not in TDF-monotherapy group, and were only found in animals who experienced a
sustained virologic response after treatment (SVR). In (B) statistically significant differences between group means (total and cccDNA) are indicated (*) as determined by Mann-Whitney U test.
Normal saline
926
927
REP 2139-Ca + TDF + ETV
TDF
929
935
937
938
947
979
980
3500
983
931
932
934
936
933
965
939
942
REP 2139-Ca
958
959
956
962
940
944
941
949
988
953
971
964
981
968
986
974
991
992
994
Summary : semi-quantitative score. % of DHBsAg-positive / total
hepatocytes
REP 2139-Ca + TDF
957
989
Normal
saline
TDF
REP2139
REP2139 +
TDF
REP2139 +
TDF+ ETV
0%
+
1-37%
+/++
38-50%
++
51-60%
++/+++
+++
61-80% 85-100%
Total
1
2
4
-
1
1
9
4
2
-
5
-
3
-
2
-
10
6
5
-
1
-
-
-
6
7
-
1
-
-
-
8
Figure 3. Detection of DHBsAg positive hepatocytes by immunostaining of autopsy liver samples. All animals from TDF-monotherapy group
exhibited detectable DHBsAg by immunostaing. By contrast SVR from REP 2139-Ca treated groups had no liver DHBsAg
(40x magnification).
Duck weight (g)
3000


On-treatment antiviral performance of REP 2139 can be improved in the presence of TDF or ETV.
Importantly after 2 months off combination therapy (REP 2139 with TDF or TDF and ETV) a marked decrease in viral
replication was still observed in large majority of animals indicating sustained virologic response (SVR).
Interestingly ducks with SVR exhibiting low total and cccDNA at the end of follow-up had also undetectable liver
DHBsAg as assessed by immunostaining.
An interferon-free regimen of REP 2139 with TDF or ETV could lead to improved antiviral outcomes or allow the
shortening of antiviral regimens in patients with chronic HBV infection.
2000
normal saline
G1
G2
TDF
G3
REP 2139-Ca
G4
REP 2139-Ca + TDF
G5
REP 2139-Ca + TDF + ETV
1500
1000
500
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28
Days of treatment
Figure 4. Mean group body weights during
treatment.
No weight loss was observed in any treatment
group, indicating absence of adverse effect
CONCLUSIONS & PERSPECTIVE


2500
REFERENCES
1. Noordeen et al., 2013 Antimicrob Agents Chemother. 57: 5291-5298.
2. Noordeen et al., 2013 Antimicrob Agents Chemother. 57: 5299-5406.
3. Noordeen et al., 2015 PLoS ONE 10(11): e0140909.
Contact Information:
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