studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 699A
acid positions located in domain I of NS5A (residues 28, 29,
30, 31, 32, 58, 62, 92 and 93); amino acid changes at these
positions are known to be responsible for reduced susceptibility
to one or several NS5A inhibitors, including ledipasvir,
daclatasvir, ombitasvir and/or elbasvir. The genetic barrier
was calculated as the sum of transitions (scored as 1) and/
or transversions (scored as 2.5) required for evolution to any
drug resistance mutation in NS5A. Comparison between genotypes
and subtypes (1a, 1b) was performed based on the most
frequent codon in the genotype/subtype. Results: The genetic
barrier was identical across all genotypes only at NS5A positions
29 and 32 (P29S and P32L scored 1 for all 6 genotypes).
In contrast, a higher barrier to resistance was found in subtype
1b than in subtype 1a at positions M/L28T (score: 3.5
vs 1, respectively), M/L28V (2.5 vs 1), M/L28A (3.5 vs 2),
Q/R30E/H/K (3.5 vs 2.5) and H/P58D (7.5 vs 2.5). Conversely,
subtype 1b displayed a lower genetic barrier than 1a
to acquire H/P58S (1 vs 3.5, respectively). Genotypes 3 and
4 displayed a lower genetic barrier to acquire A/R30S than
genotype 1 (2.5 vs 3.5, respectively). Residue Y93 was highly
conserved across genotypes 1, 2 and 3. Indeed, in contrast to
genotype 6 which displayed a high genetic barrier to acquire
93C (score 2.5) and 93H (score 5), these amino acid substitutions
required a minimal score of 1 for genotypes 1, 2 and 3.
Conclusions: HCV subtype 1a displays a lower genetic barrier
to resistance than subtype 1b at several NS5A positions at the
nucleotide level. This could account for the higher rate of failure
of NS5A inhibitor-containing regimens in patients infected with
HCV subtype 1a. In addition, genotype-specific amino acid
changes (e.g. R30S in genotype 4, P58S in subtype 1b) could
be explained by a differential genetic barrier between different
genotypes and subtypes.
Disclosures:
Slim FOURATI - Speaking and Teaching: Gilead
Stephane Chevaliez - Advisory Committees or Review Panels: Janssen; Speaking
and Teaching: Gilead, BMS
Jean-Michel Pawlotsky - Consulting: Abbvie, Achillion, Bristol-Myers Squibb, Gilead,
Janssen, Merck; Speaking and Teaching: Bristol-Myers Squibb, Gilead,
Merck, Janssen
The following authors have nothing to disclose: Alexandre Soulier, Marion Lavert,
Lila Poiteau
1000
Establishment and Characterization of a New Permissive
Cell Line for HCV Infection
Hitoshi Omura, Tetsuro Shimakami, Takayoshi Shirasaki, Kazuhisa
Murai, Masaya Funaki, Fanwei Liu, Takehiro Hayashi, Taro
Yamashita, Masao Honda, Shuichi Kaneko; Kanazawa university,
Kanazawa city, Japan
Background The in vivo HCV infection model has been needed
to clarify the mechanism of persistent infection of HCV in human
livers. Huh-7.5, which is a subline of human hepatoma cell line,
Huh-7, has been used most frequently for propagation of HCV
due to its vigorous ability to support for HCV replication. Huh-
7.5 is quite useful for the development of many direct-acting
antivirals (DAAs). Huh-7.5 is known to have the defect in its
antiviral interferon signaling, therefore, a new cell line with
intact interferon signaling is needed to mimic HCV infection
in human livers. Here, we have established and characterized
a new permissive cell line for HCV infection with intact innate
antiviral interferon signaling. Method The human hepatoma
tissue was obtained from an HCV-infected patient at a surgical
resection, and then transplanted to mice. The adherent cells to
mice were removed, followed by the MACS cell separation to
remove mice fibroblasts, and used for developing a new cell
line, designated as KH cell. We examined the permissiveness
of KH cells for HCV and characterized by comparing with
Huh-7.5 cells. Result The HCV RNA was no longer detected in
KH cells by qRT-PCR from the original patient. The karyotype
analysis for KH and Huh-7.5 cells revealed that the two cell
lines were distinct from each other. KH cells could support HCV
replication as efficiently as Huh-7.5 cells after transfection of in
vitro transcribed HCV RNA. Additionally, the infection of cell
culture-derived HCV to KH cells showed the entry of HCV into
the cells, followed by efficient replication. KH cells replicating
HCV could also produce infectious viruses, suggesting that KH
cells had the ability to support a full life cycle of HCV as could
Huh-7.5 cells. KH cells had same amount of miR-122 as did
Huh-7.5. We measured EC50 for IFN-α in KH and Huh-7.5
cells. We then compared EC50 of IFN-α between both and
found a similarity. Interestingly, the sequence analysis of RIG-I
at its 55 amino acid position for KH cells showed wild type,
T55. RIG-I from Huh-7.5 cells was mutant, T55I, which is a
representative mutation of Huh-7.5 cells. In accordance with
this result about RIG-I, the transfection of HCV RNA into KH
cells induced robust ISGs induction, such as MX1, IFN-β, and
OAS2, on the other hand, no induction was observed in Huh-
7.5 cells. Discussion We have established and characterized a
new permissive cell line for HCV infection with intact antiviral
interferon signaling. This cell line could be useful to understand
the underlying mechanism of persistent HCV infection under
pressure of host antiviral innate immunity.
Disclosures:
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Hitoshi Omura, Tetsuro Shimakami,
Takayoshi Shirasaki, Kazuhisa Murai, Masaya Funaki, Fanwei Liu, Takehiro
Hayashi, Taro Yamashita, Masao Honda
1001
PD-1 blockade enhances cytopathic control of HCV by
antiviral CD8 T cells in infectious HCV co-culture model
Keisuke Ojiro 1 , Xiaowang Qu 1,2 , Jang-June Park 1 , Hyosun Cho 1,3 ,
Kyong-Mi Chang 1 ; 1 Division of Gastroenterology, University of
Pennsylvania, Philadelphia, PA; 2 Translational Medicine Institute,
University of South China, Chenzhou, China; 3 Pharmacy, Duksung
Women’s University, Seoul, Korea (the Republic of)
Background: T-cell expression of inhibitory costimulatory
receptor PD-1 and CTLA-4 in chronic viral infection and tumor
environment is associated with functional tolerance that may
be reversed by antibody-mediated blockade in vitro and in
vivo. Thus, PD-1 blockade is in both pre-clinical and clinical
development in various settings. In this study, we examined
the effect of PD-1 blockade on T cell mediated virus control
and hepatocyte survival using the infectious HCV co-culture
system. Method: Target cells for HCV replication and antigen
presentation were established by lentiviral transduction of HLA
A2.1, PDL1 and/or GFP expression in Huh7.5 hepatoma
cells. Highly infectious JFH derived strain Jc1/Gluc2A clone
(genotype 2a) was modified by site-directed mutagenesis to
encode well-defined genotype 1a-derived NS3 1073 or NS5B
2594 epitopes. HCV-specific CD8 T cells were expanded from
peripheral lymphocytes of HCV-resolvers following 1-2 weeks
of HCV peptide stimulation or engineered by transducing CD8
T cells from uninfected blood donors by lentivirus encoding
HCV-TCR specific for HLA-A2 restricted NS3 1073- or NS5B
2594-epitopes. HCV-specific or non-specific T cells were co-cultured
with HCV-infected Huh7.5 cells at varying E/T ratio, culture
duration and inhibitory receptor blockade, with the level