1. No category

SESSIONE 2
Oral communications based on selected
abstracts
Conveners: S. Bruno, C.M. Mastroianni
HCV NS3 Quasispecies in Liver and Plasma
and Dynamics of Telaprevir-resistant Variants
Assessed by UDPS in Breakthrough Patients:
a Case Study
B. Bartolini, M. Selleri, A.R. Garbuglia, E. Giombini, G. D’Offizi, M.R. Capobianchi
National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy
Background
Extended
genetic
quasispecies
variability
•High debate on the impact of preAntiviral drug
existing variants carrying RAMs on
resistance
the outcome of antiviral treatment
•Lack of knowledge of the role of
reservoirs (liver...) in housing
resistant variants that can be
selected by drugs
???
In the liver:
DAA
resistance
In the literature……
HEPATOLOGY, 2014
no compartmentalization
between plasma and liver
compartmentalization
between plasma and liver
Blood only may not adequately
represent viruses replicating with in the liver
AIM
NS3 heterogeneity by UDPS
in two HCV GT1a patients who underwent TVR+
pINF/RBV therapy and developed treatment failure
 baseline plasma and liver biopsy samples
 plasma samples during therapy
 plasma samples after therapy suspension
The patients
Stop Treatment
8
3
7
2,5
TVR
6
5
pINF/RBV
1,84
Pt2
2
1,49
4
HCV RNA
logIU/ml
3
0,87
2
0,5
0,31
1
0
0
8
7
1
0,67
Diversity
3
HCV RNA
2,46
2,5
6
2
5
1,66
1,33
4
3
1,5
1
2
0,45
0,5
1
0
0
Baseline
1D
5D
15D
2M
3M
4M
1M after ST6M after ST
Diversity
(nt substitution/site)10-2
1,5
Pt22
No liver-plasma compartmentalization
Liver
Plasma
pt2
pt22
Intra-patients diversity and prevalence of variants
at position associated with resistance to TVR
detected by UDPS in liver and plasma at baseline.
Patient
Viral Load
(IU/ml)
sample
liver
Pt2
Variants Observed (%)
V36A
(0.46)
D168G
(0.52)
V36A
(0.33)
T54A
(0.39)
R155G
(0.31)
D168G
(0.36)
V36A
(0.38)
T54A
(0.39)
0.00478
4.31x105
plasma
liver
Pt22
Diversity
(nt substitution/site)
0.01836
0.0055
5.0 x106
plasma
0.0246
Dynamics of resistant quasispecies
(Pt2)
Variants Observed (%)
baseline
V36A
(0.33)
1day of
treatment
V36A
(0.34)
12 w after
ST
V36M,T
T54A
R155K,E
(98.04), (0.39)
(0.46)
(98.56), (0.46)
24 w after
ST
V36M
I132T
R155K
(8.47)
(0.60)
(98.94)
72 w after
ST
V36A
T54A
R155K
(0.39)
(0.44)
(7.57)
Conclusions
 No compartmentalization of HCV quasipecies between
plasma and liver
 Drug resistance patterns at baseline did not significantly
differ between plasma and liver
 De novo appearance of resistant population
 Tendency of RAM to disappear in FU
 Dynamics of disappearance to be studied with UDPS
 Longer FU and more patients to elucidate the issues
TANKS TO
Team of the Laboratory of Virology
INMI “L.Spallanzani”, Rome
Anna Rosa Garbuglia
Marina Selleri
and
Maria R. Capobianchi
you for the attention
Prevalence of variants at positions associated with
resistance to TVR detected by UDPS in plasma.
Pt2
Viral Load
(IU/ml)
Pt22
Variants Observed (%)
Viral Load
(IU/ml)
Variants Observed (%)
baseline
4.31x105
V36A
(0.33)
5.0 x106
V36A
T54A
(0.38)
(0.39)
1day of treatment
3.95 x103
V36A
(0.34)
3.40x103
V36A
D168G
(0.35)
(0.36)
x105
V36M
I132T
R155K
(93.64)
(1.01)
(98.79)
2.49 x105
V36M,T
T54A
R155K
D168A
(86.27), (0.34)
(0.35)
(87.05)
(0.47)
1 month from
therapy stop
4.69
2.58x105
V36M,T
T54A
R155K,E
(98.04), (0.39)
(0.46)
(98.56), (0.46)
6 month from
therapy stop
6.07 x104
V36M
I132T
R155K
(8.47)
(0.60)
(98.94)
18 month from
therapy stop
x105
V36A
T54A
R155K
(0.39)
(0.44)
(7.57)
3 month from
therapy stop
2.63
Intra-patients diversity and prevalence of variants at
positions associated with resistance to TVR detected by
Ultra Deep Sequencing in plasma.
Patient
Sample
N°
reads
Viral Load
(IU/ml)
Diversity
(nt substitution/site)
baseline
3634
4.31x105
0.01836
V36A
(0.33)
-
1day of treatment
4655
3.95 x103
0.0031
V36A
(0.34)
-
V36M,T
(98.04), (0.39)
T54A
(0.46)
R155K,E
(98.56), (0.46)
V36M
(8.47)
I132T
(0.60)
R155K
(98.94)
V36A
(0.39)
T54A
(0.44)
R155K
(7.57)
Variants Observed (%)
Resistance
Association (%)
V36M+R155K
3 month from
3057
2.58x105
0.0149
(96.60)
therapy stop
Pt2
6 month from
V36M+R155K
4156
6.07 x104
0.0067
therapy stop
(8.42)
18 month from
4082
2.63 x105
0.0087
therapy stop
-
Intra-patients diversity and prevalence of variants at
positions associated with resistance to TVR detected by
Ultra Deep Sequencing in plasma.
Patient
Sample
N°
reads
Viral Load
(IU/ml)
Diversity
(nt
substitution/site)
Variants Observed (%)
V36A
baseline
7707
5.0
x106
4260
3.40x103
(0.38)
-
0.0246
1day of
T54A
(0.39)
V36A
(0.35)
-
0.0045
treatment
D168G
(0.36)
V36M
(93.64)
1 month from
V36M+R155K
5059
Pt22
4.69
x105
0.0133
I132T
(1.01)
therapy stop
(92.47)
6 month from
1158
therapy stop
Resistance
Association
(%)
2.49 x105
R155K
(98.79)
V36M,T
(86.27), (0.34)
T54A
(0.35)
V36M+R155K
R155K
(87.05)
(75.82)
D168A
(0.47)
0.0166
Background
•High debate on the impact of preexisting variants carrying RAMs
on the outcome of antiviral
treatment
•Lack of knowledge of the role of
reservoirs (liver...) in housing
resistant variants that can be
selected by drugs
Extended genetic
variability
quasispecies
Antiviral drug
resistance
HEPATOLOGY, 2014
no compartmentalization
between plasma and liver
no compartmentalization
between plasma and liver
Blood only may not adequately
represent viruses replicating with in the liver
No liver-plasma compartmentalization
Liver
Plasma
pt2
pt22