studies

698A AASLD ABSTRACTS HEPATOLOGY, October, 2015
of HCV and the cell type specialized in the secretion of VLDL.
The known requirements for VLDL biogenesis are (i) ApoB, a
structural component essential for VLDL scaffolding, and (ii)
microsomal triglyceride transfer protein (MTP), the rate-limiting
enzyme that lipidates ApoB. However, studies with the classical
HCV culture system in hepatocarcinoma-derived Huh-7 sublines
suggested that MTP inhibitors might not efficiently block
HCV production unless high, cytotoxic concentrations are used.
Moreover, the role of ApoB remains a matter of controversy
whereas ApoE appears to be a prerequisite for HCV production
in cell line culture. Here we have reassessed the role of VLDL
biogenesis in HCV morphogenesis and secretion using a more
relevant HCV culture system in primary human adult hepatocytes
(PHH), which, contrary to Huh-7 cells, secrete authentic
VLDL and infectious particles. Methods. PHH were infected with
the HCV strain JFH1, and either treated with increasing doses
of various MTP inhibitors or depleted for ApoB or ApoE (RNA
interference technologies). Cultures were evaluated for production
of intracellular and extracellular infectious virus (focus-formation
assay), viral load (RT-qPCR), secretion of ApoB and
ApoE (ELISA), and cytotoxic effects. Results. The pharmacological
MTP inhibitor CP-346086, which efficiently inhibited
VLDL secretion in PHH, also reduced both HCV production
and infectivity at moderate, non-cytotoxic doses. The grapefruit
flavonoid naringenin, reported to inhibit MTP activity indirectly
through a PPARa-mediated mechanism, induced a significant
reduction of HCV production even at doses that did not reduce
VLDL secretion. ApoB depletion did not seem to affect HCV production
or infectivity significantly even though it inhibited VLDL
biogenesis. ApoE depletion caused only a moderate reduction
of HCV infectivity in PHH whereas it almost completely abolished
HCV production in Huh-7.5.1 cells. Conclusion. These
data in differentiated human hepatocytes show that neither
ApoB nor ApoE is essential for HCV morphogenesis whereas
MTP function is absolutely required for HCV production and
infectivity, albeit not necessarily via its role in VLDL biogenesis.
MTP inhibitors, either direct (CP-346086) or indirect (naringenin),
appear as promising host-targeting drugs to combat
HCV infection in complement with directly acting antivirals.
Disclosures:
The following authors have nothing to disclose: Veronique Pene, Maxime Villaret,
Matthieu Lemasson, Lynda Aoudjehane, Jean-François Méritet, Filomena Conti,
Yvon Calmus, Arielle R. Rosenberg
998
A miR-122/miR-224-based model to identify severe
fibrosis and cirrhosis in patients with chronic hepatitis C
Kevin Appourchaux 2 , Emilie Estrabaud 2 , Matthieu Resche-Rigon
3,1 , Martine Lapalus 2 , Michelle Martinot-Peignoux 2 , Nathalie
Boyer 4 , Michel Vidaud 5 , Pierre Bedossa 6 , Patrick Marcellin 2 , Tarik
Asselah 2,4 ; 1 INSERM, Paris, France; 2 INSERM UMR1149, Paris,
France; 3 INSERM UMR1153, Paris, France; 4 Hepatology, Beaujon
Hospital, Clichy, France; 5 INSERM UMR745, Paris, France; 6 Service
d’Anatomie Pathologique, Beaujon Hospital, Clichy, France
Background and aims Staging fibrosis is crucial in patients
with chronic hepatitis C (CHC) because it reflects the prognosis.
MiRNAs regulate the expression of up to 60% of mRNAs.
The liver-enriched miR-122 enhances HCV replication. MiRNAs
are increasingly investigated as biomarkers because of their
high stability compared to mRNAs and proteins. We aimed
to identify miRNAs associated with fibrosis in patients with
CHC. Patients and Methods A total of 202 patients with CHC
were consecutively enrolled. The inclusion criteria were having
at least one biopsy and no HCC. Serums and biopsies
samples were available for respectively 106 and 86 patients
(26 paired liver/serum). Among patients with available serum,
63.2% were male, the mean age was 48.8 years and the
mean HCV viral load was 5.81 logUI/mL. According to the
Metavir scoring system, 24.5%, 16%, 34.9%, 24.6% of the
patients were respectively F1, F2, F3 and F4. Among patients
with available biopsies, 54.2% were male, the mean age was
49.9 years and the mean HCV viral load was 5,6 logUI/mL.
Regarding the stages of fibrosis, 27,1%, 31,2%, 22,9%, and
18,8% of the patients were respectively F1, F2, F3 and F4.
MiR-20a, -27b, -29a, -92a, -122, -146a, -222, -224 were
selected for the study because of their role during liver fibrosis.
The expression of miRNAs was assessed by RT-qPCR. Results
An increased expression of hepatic miR-224 (p=0.003) was
observed in patients with mild and moderate fibrosis (F1-F2)
compared to those with severe fibrosis and cirrhosis (F3-F4).
A reduction of hepatic miR-122 (p=0.008) was observed in
patients with F3-F4 compared to those with F1-F2. For hepatic
miR-20a, -27b, -29a, -92a, -122 and -146a, no difference was
observed between patients with F3-F4 and those with F1-F2.
In the serums, none of the miRNAs tested, was significantly
deregulated between F3-F4 and F1-F2. The multivariate analysis
of clinical data (albumin, platelets count, aspartate aminotransferase
(AST), alkaline phosphatase) and hepatic miRNAs,
allowed us to build a model combining hepatic miR-122 and
miR-224, platelets count and AST. The AUC of the model was
0.90 while in the same cohort, FIB-4 and APRI had, respectively,
an AUC of 0.78 and 0.85. Conclusions Interestingly,
hepatic miR-122 and miR-224 were respectively decreased
and increased in patients with severe fibrosis and cirrhosis.
Increased expression of miR-224 might suggest a premalignant
condition to HCC. The model combining the assessment of
hepatic miR-122 and miR-224, platelets count and AST was
more accurate than FIB-4 and APRI to distinguish patients with
F3-F4 from those with F1-F2.
Disclosures:
Nathalie Boyer - Board Membership: MSD, JANSSEN, Gilead, Abbvie; Speaking
and Teaching: BMS
Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen,
MSD, Abbvie, Alios BioPharma, Idenix, Akron; Grant/Research Support: Roche,
Gilead, BMS, Novartis, Janssen, MSD, Alios BioPharma; Speaking and Teaching:
Roche, Gilead, BMS, Vertex, Novartis, Janssen, MSD, Boehringer, Pfizer,
Abbvie
Tarik Asselah - Advisory Committees or Review Panels: AbbVie, Merck, Gilead,
BMS, Roche, Janssen
The following authors have nothing to disclose: Kevin Appourchaux, Emilie
Estrabaud, Matthieu Resche-Rigon, Martine Lapalus, Michelle Martinot-Peignoux,
Michel Vidaud, Pierre Bedossa
999
Different HCV genotypes have different genetic barriers
to resistance to N5A inhibitors, explaining different
treatment outcomes
Slim FOURATI 1,2 , Stephane Chevaliez 1,2 , Alexandre Soulier 1,2 ,
Marion Lavert 1,2 , Lila Poiteau 1,2 , Jean-Michel Pawlotsky 1,2 ; 1 Virology,
Hôpital Henri Mondor, AP-HP,, National Reference Center for
Viral Hepatitis B, C and Delta,, Créteil, France; 2 INSERM U955,
Université Paris-Est, Créteil, France
The genetic barrier to resistance of an antiviral drug depends
on the number and type of nucleotide substitutions required to
overcome the drug’s selective pressure. It is a key factor for
the development of HCV drug resistance in the clinical setting.
The goal of this study was to assess whether different
HCV genotypes and subtypes have different genetic barriers
to resistance to NS5A inhibitors that could explain the clinical
results of NS5A inhibitor-containing regimens. Methods:
744 unselected sequences extracted from the European HCV
database (https://euhcvdb.ibcp.fr) were examined at 9 amino