Caring Ambassadors Program Hepatitis C Literature Review © 2015 Caring Ambassadors

27 Pages·2015·0.37 MB·English

by  Lorren Sandt

Caring Ambassadors Program
Hepatitis C Newsletter
www.HepCChallenge.org

May 2015

CLINICAL TRIALS, COHORT STUDIES, PILOT STUDIES 1 - 6
BASIC AND APPLIED SCIENCE, PRE-CLINICAL STUDIES 6 - 11
HIV/HCV COINFECTION 11 - 18
EPIDEMIOLOGY, DIAGNOSTICS & MISCELLANEOUS WORKS 18 - 23
LIVER CANCER 23 - 27

CLINICAL TRIALS, COHORT STUDIES, PILOT STUDIES

Patterns of Hepatitis C Virus RNA Levels during Acute Infection: The InC3 Study.
Hajarizadeh B1, Grady B2, Page K3, et al. PLoS One. 2015 Apr 2;10(4):e0122232. doi:
10.1371/journal.pone.0122232. eCollection 2015.
BACKGROUND: Understanding the patterns of HCV RNA levels during acute hepatitis C
virus (HCV) infection provides insights into immunopathogenesis and is important for vaccine
design. This study evaluated patterns of HCV RNA levels and associated factors among
individuals with acute infection. METHODS: Data were from an international collaboration of
nine prospective cohorts of acute HCV (InC3 Study). Participants with well-characterized acute
HCV infection (detected within three months post-infection and interval between the peak and
subsequent HCV RNA levels≤120 days) were categorised by a priori-defined patterns of HCV
RNA levels: i) spontaneous clearance, ii) partial viral control with persistence (≥1 log IU/mL
decline in HCV RNA levels following peak) and iii) viral plateau with persistence (increase or
<1 log IU/mL decline in HCV RNA levels following peak). Factors associated with HCV RNA
patterns were assessed using multinomial logistic regression. RESULTS: Among 643
individuals with acute HCV, 162 with well-characterized acute HCV were identified:
spontaneous clearance (32%), partial viral control with persistence (27%), and viral plateau with
persistence (41%). HCV RNA levels reached a high viraemic phase within two months following
infection, with higher levels in the spontaneous clearance and partial viral control groups,
compared to the viral plateau group (median: 6.0, 6.2, 5.3 log IU/mL, respectively; P=0.018). In
the two groups with persistence, Interferon lambda 3 (IFNL3) CC genotype was independently
associated with partial viral control compared to viral plateau (adjusted odds ratio [AOR]: 2.75;
95%CI: 1.08, 7.02). In the two groups with viral control, female sex was independently
associated with spontaneous clearance compared to partial viral control (AOR: 2.86; 95%CI:
1.04, 7.83). CONCLUSIONS: Among individuals with acute HCV, a spectrum of HCV RNA
patterns is evident. IFNL3 CC genotype is associated with initial viral control, while female sex
is associated with ultimate spontaneous clearance.

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Predicting the impact of adverse events and treatment duration on medical resource
utilization-related costs in hepatitis C genotype 1 treatment-naïve patients receiving
antiviral therapy. Akpo EI1, Cerri K, Kleintjens J. Pharmacoeconomics. 2015 Apr;33(4):409-
22. doi: 10.1007/s40273-014-0249-4.
OBJECTIVES: Studies on medical resource utilization (MRU) and related costs are important
for evaluating the potential patient management and cost-effectiveness implications of antiviral
treatments for hepatitis C virus (HCV) infection. The objectives of this study were (i) to compare
the MRU and related costs for two treatment approaches; (ii) to identify the main drivers of
resource use and costs; and (iii) to assess the effects of various treatment regimen attributes on
MRU-related costs in a UK clinical setting. METHODS: The analysis used data collected
alongside the simeprevir (SMV) phase III trials for treatment-naïve genotype 1 HCV-infected
patients; these data covered outpatient consultations with specialists, emergency room visits and
hospital admissions. Logistic regressions were constructed to estimate the predictors of resource
utilization, and a two-part multivariable analysis model was used to determine the total costs of
treatment in the UK. RESULTS: Data on 731 patients receiving SMV plus pegylated interferon
and ribavirin (SMV/PegIFN/R) or PegIFN/R were included in the analysis. While MRU was
similar between the SMV and PegIFN/R groups, MRU-related costs were significantly lower in
the SMV group than in the PegIFN/R group (P < 0.05). High body mass index (P < 0.05), severe
fibrosis (P < 0.05), shortened treatment duration to 24 weeks (P < 0.05), and anaemia and rash
during treatment (P < 0.001) were identified as predictors of hospitalization and outpatient visits
and as drivers of total costs. Univariate sensitivity analyses suggested that shortened treatment
duration and lower occurrence of rash lead to large cost savings. CONCLUSION: This study
identified both baseline and on-treatment antiviral therapy characteristics as drivers of MRU-
related costs for HCV patients following antiviral therapy. The shortened treatment duration and
reduction in rash due to treatment with SMV triple therapy lead to substantial non-drug cost
savings, compared with PegIFN/R treatment. This suggests that there are potential patient
management and cost-effectiveness implications associated with the choice of specific antiviral
treatments.

Direct-acting antiviral-based triple therapy on alpha-fetoprotein level in chronic hepatitis
C patients. Takayama K1, Furusyo N1, Ogawa E1, et al. World J Gastroenterol. 2015 Apr
21;21(15):4696-706. doi: 10.3748/wjg.v21.i15.4696.
AIM: To investigate the impact of telaprevir-based triple therapy on the serum alpha-fetoprotein
(AFP) level of chronic hepatitis C patients. METHODS: A total of 210 patients with chronic
hepatitis C genotype 1 of high viral load (baseline serum hepatitis C virus RNA > 5.0 log10
IU/mL) were divided into two groups by type of treatment: triple therapy with telaprevir,
pegylated-interferon-α (PEG-IFNα), and ribavirin (RBV) for 24 wk (n = 88), or dual therapy
with PEG-IFNα and RBV for 48 wk (n = 122). The relationship between virological response
and the change in the serum AFP level from baseline to 24 wk after the end of treatment was
examined. RESULTS: No significant difference in mean baseline AFP level was found between
the triple and dual therapy groups (8.8 ng/mL vs 7.8 ng/mL). Triple therapy produced significant
declines in the AFP level in sustained virological response (SVR) and non-SVR patients (7.8
ng/mL at baseline to 3.5 ng/mL at 24 wk after the end of treatment, P < 0.001 and 14.3 ng/mL to
9.5 ng/mL, P = 0.004, respectively). In contrast, dual therapy resulted in a significant decline in
AFP level only in SVR patients (4.7 ng/mL to 2.8 ng/mL, P < 0.001), but not in non-SVR
patients (10.2 ng/mL to 10.1 ng/mL). Among patients with a high-baseline AFP level (≥ 10
Caring Ambassadors Program Hepatitis C Literature Review © 2015
ng/mL), the decline in the AFP level was significantly higher in the triple therapy than in the
dual therapy group (15.9 ng/mL vs 1.6 ng/mL, P = 0.037). CONCLUSION: Regardless of
virological response, telaprevir-based triple therapy reduced the serum AFP level.

Sustained virologic response rates with telaprevir-based therapy in treatment-naive
patients evaluated by race or ethnicity. Flamm SL1, Muir AJ, Fried MW, et al. J Clin
Gastroenterol. 2015 Apr;49(4):336-44. doi: 10.1097/MCG.0000000000000150.
BACKGROUND: The phase 3 studies of telaprevir (T) in combination with peginterferon α-2a
and ribavirin (PR) in treatment-naive genotype 1 chronic hepatitis C virus-infected patients
(ADVANCE/ILLUMINATE) were not designed a priori to assess the effect of race and ethnicity
on treatment response. However, these factors are important given the lower sustained virologic
response (SVR) rates observed in black and Hispanic/Latino patients treated with PR. GOALS:
This retrospective pooled analysis evaluated the effect of race or ethnicity on treatment-naive
patient response to telaprevir-based therapy and assessed resistant variant profiles.
MATERIALS AND METHODS: This analysis comprised patients enrolled in ADVANCE
(N=363) and ILLUMINATE (N=540) who received 12 weeks of telaprevir in combination with
PR followed by 12 or 36 weeks of PR alone and patients in ADVANCE (N=361) who received
48 weeks of PR alone. Race and ethnicity were self-reported and not mutually exclusive.
RESULTS: Higher SVR rates were observed with telaprevir-based therapy compared with PR in
blacks [n=99 (62%) vs. n=28 (29%), respectively] and in Hispanics/Latinos [n=89 (72%) vs.
n=38 (39%)]. The SVR was lower in telaprevir-treated blacks [n=99 (62%)] compared with
nonblacks [n=791 (78%)] and in Hispanic/Latinos compared with non-Hispanics/Latinos [n=89
(72%) vs. n=801 (76%)]. Low discontinuation rates due to adverse events, including rash and
anemia, were observed across subgroups. Resistance profiles were similar among the subgroups.
CONCLUSIONS: Treatment-naive black and Hispanic/Latino patients with genotype 1 chronic
hepatitis C virus infection may benefit from telaprevir-based therapy, an important finding given
the lower SVR rates observed in these patients when they are treated with PR alone.

The clinical features of patients with a Y93H variant of hepatitis C virus detected by a PCR
invader assay. Kan T1, Hashimoto S, Kawabe N, et al. Gastroenterol. 2015 Apr 24. [Epub ahead
of print]
BACKGROUND: Resistance-associated variants (RAVs) reduce the efficacy of interferon
(IFN)-free therapy with asunaprevir and daclatasvir for patients infected with hepatitis C virus
(HCV) genotype 1b. The characteristics of patients with an L31 or a Y93 variant in the
nonstructural 5A region detected by a polymerase chain reaction invader assay were
investigated. METHODS: In total, 201 patients with HCV genotype 1b were examined for
L31F/M/V variants or a Y93H variant by the polymerase chain reaction invader assay.
RESULTS: L31M and Y93H variants were detected in 4.6 and 21.4 % of patients, respectively.
Patients with an L31M variant had no significant characteristics. Patients with a Y93H variant
had significantly higher HCV RNA levels (6.5 ± 0.5 log copies per milliliter vs 6.1 ± 0.7 log
copies per milliliter, p = 0.0002), higher frequency of mutant type of the IFN-sensitivity-
determining region (88.4 % vs 71.7 %, p = 0.0251), and higher frequency of TT genotype at
rs8099917 of IL28B (91.7 % vs 54.3 %, p < 0.0001) than those with Y93 wild-type strains.
Multivariate analysis identified HCV RNA levels [odds ratio (OR) 3.72, 95 % confidence
interval (CI) 1.71-8.06, p = 0.0009] and TT genotype at rs8099917 (OR 7.45, 95 % CI 2.11-26.4,
p = 0.0018) as factors associated with the presence of a Y93H variant. CONCLUSION: The
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presence of a Y93H variant was associated with higher HCV RNA levels and TT genotype at
rs8099917 of IL28B. Thus, patients with a Y93H variant may be ideal candidates for IFN-based
therapy rather than IFN-free therapy, although the high viral load of these patients may reduce
the response rate of IFN-based therapy.

Grazoprevir-Elbasvir Combination Therapy for Treatment-Naive Cirrhotic and
Noncirrhotic Patients With Chronic HCV Genotype 1, 4, or 6 Infection: A Randomized
Trial. Zeuzem S, Ghalib R, Reddy KR, et al. Ann Intern Med. 2015 Apr 24. doi: 10.7326/M15-
0785. [Epub ahead of print]
BACKGROUND: Novel interferon- and ribavirin-free regimens are needed to treat hepatitis C
virus (HCV) infection. OBJECTIVE: To evaluate the safety and efficacy of grazoprevir
(NS3/4A protease inhibitor) and elbasvir (NS5A inhibitor) in treatment-naive patients.
DESIGN: Randomized, blinded, placebo-controlled trial. (ClinicalTrials.gov: NCT02105467).
SETTING: 60 centers in the United States, Europe, Australia, Scandinavia, and Asia.
PATIENTS: Cirrhotic and noncirrhotic treatment-naive adults with genotype 1, 4, or 6
infection. INTERVENTION: Oral, once-daily, fixed-dose grazoprevir 100 mg/elbasvir 50 mg
for 12 weeks, stratified by fibrosis and genotype. Patients were randomly assigned 3:1 to
immediate or deferred therapy. MEASUREMENTS: Proportion of patients in immediate-
treatment group achieving unquantifiable HCV RNA 12 weeks after treatment (SVR12); adverse
events in both groups. RESULTS: Among 421 participants, 194 (46%) were women, 157 (37%)
were nonwhite, 382 (91%) had genotype 1 infection, and 92 (22%) had cirrhosis. Of 316 patients
receiving immediate treatment, 299 of 316 (95% [95% CI, 92% to 97%]) achieved SVR12,
including 144 of 157 (92% [CI, 86% to 96%]) with genotype 1a, 129 of 131 (99% [CI, 95% to
100%]) with genotype 1b, 18 of 18 (100% [CI, 82% to 100%]) with genotype 4, 8 of 10 (80%
[CI, 44% to 98%]) with genotype 6, 68 of 70 (97% [CI, 90% to 100%]) with cirrhosis, and 231
of 246 (94% [CI, 90% to 97%]) without cirrhosis. Virologic failure occurred in 13 patients (4%),
including 1 case of breakthrough infection and 12 relapses, and was associated with baseline
NS5A polymorphisms and emergent NS3 or NS5A variants or both. Serious adverse events
occurred in 9 (2.8%) and 3 (2.9%) patients in the active and placebo groups, respectively
(difference <0.05 percentage point [CI, -5.4 to 3.1 percentage points]); none were considered
drug related. The most common adverse events in the active group were headache (17%), fatigue
(16%), and nausea (9%). LIMITATION: The study lacked an active-comparator control group
and included relatively few genotype 4 and 6 infections. CONCLUSION: Grazoprevir-elbasvir
achieved high SVR12 rates in treatment-naive cirrhotic and noncirrhotic patients with genotype
1, 4, or 6 infections. This once-daily, all-oral, fixed-combination regimen represents a potent new
therapeutic option for chronic HCV infection.

Baseline prevalence and emergence of protease inhibitor resistance mutations following
treatment in chronic HCV genotype 1-infected individuals. Nguyen LT1, Gray E, Dean J,
Carr M, et al. Antivir Ther. 2015 Apr 29. doi: 10.3851/IMP2964. [Epub ahead of print]
BACKGROUND: The hepatitis C virus (HCV) NS3/4A serine protease inhibitors (PIs)
boceprevir (BOC), telaprevir (TVR) and simeprevir (SMV) are approved for treatment of
chronic hepatitis C virus infection in combination with pegylated interferon and ribavirin. The
present study investigated the prevalence of HCV NS3 drug resistance mutations (DRMs)
associated with HCV genotype 1-infected individuals at baseline and in viral breakthrough
following BOC and TVR treatment. METHODS: HCV genotype 1-infected individuals were
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enrolled in a multi-center, prospective outcomes study. The HCV NS3 viral protease was
analyzed for DRMs at baseline (n=164) and at viral breakthrough (n=18) following BOC/TVR
treatment. RESULTS: Viral NS3 protease subtype analysis showed 65.2% (107/164) were HCV
subtype-1a and 34.8% (57/164) were HCV subtype-1b infections. Naturally-occurring PIs DRMs
in NS3 (V36L, T54S, V55A, Q80K/R and I132V) were identified in 57.3% (94/164) cases at
baseline. The NS3 Q80K polymorphism was found in 43/107 (40.2%) of HCV subtype-1a and
exclusively in clade 1 (43/82; 52.4%) versus clade 2 viruses (0/25; 0%, P<10-6). The pre-
treatment I132V variant was found in 78.9% (45/57) of subtype-1b. Of 18 patients who had viral
breakthrough, the majority was subtype-1a (77.8%, 14/18). BOC/TVR-associated DRMs were
detected in 94.4% (17/18), of which 64.7% (11/17) emerged on treatment. CONCLUSIONS: To
ensure the most appropriate DAA-based treatment regimen is employed, baseline reporting of
clade and resistance mutations for HCV subtype-1a using nucleotide sequence-based analysis is
warranted prior to commencement of therapy.

Efficacy and safety of pegylated interferon base treatment in patients with chronic hepatitis
C on dialysis. Ahn SB1, Jun DW2, Kim SG3, et al. Eur J Intern Med. 2015 May;26(4):292-6.
doi: 10.1016/j.ejim.2015.03.011. Epub 2015 Apr 13.
INTRODUCTION: Patients with chronic hepatitis C (CHC) and end-stage renal disease
(ESRD) on dialysis are difficult to treat and show higher dropout rates during treatment. The aim
of this study was to analyze the treatment outcomes in patients with CHC and underlying end-
stage renal disease on dialysis in Korea. METHODS: A retrospective multi-center study of 35
patients with CHC and underlying ESRD on regular dialysis from 13 centers were analyzed. We
investigated the tolerability and efficacy of pegylated interferon therapy with or without ribavirin
on dialysis patients. RESULTS: Twenty patients (57%) were genotype 1. Sixteen patients (46%)
were treated with pegylated interferon monotherapy. Nineteen patients (54%) were treated with
pegylated interferon and ribavirin. The overall sustained virological response (SVR) rate was
65.7% in all subjects. Thirteen patients (37%) dropped out before completion of treatment, and
six patients (46.2%) showed SVR despite premature termination of treatment. Twenty patients
(90.9%) achieved SVR among the 22 patients who completed the scheduled course. The most
common side effects were anemia and neutropenia. The patients receiving ribavirin treatment
showed a higher dropout rate (52.6% vs. 18.8%, p=0.04) and higher SVR rate (68.4% vs. 62.5%,
p=0.07) compared to the pegylated interferon mono-treatment group. CONCLUSIONS: The
difficulty in treating HCV patients with ESRD was attributed to higher dropout rate. However,
despite the high dropout rate (37%), the SVR rate in genotype 1 was 65% and in genotypes 2 and
3 was 66%. Patients who completed the treatment showed a high SVR rate of 89.5%.

HCV-specific lymphocyte responses in individuals with positive anti-HCV but negative
HCV-RNA. Sili U1, Kaya A2, Aydin S2, et al. J Clin Virol. 2015 Jun;67:73-7. doi:
10.1016/j.jcv.2015.04.014. Epub 2015 Apr 15.
BACKGROUND: Hepatitis C virus (HCV) status cannot be reliably predicted in anti-HCV
positive/HCV-RNA negative individuals who may either have recovered spontaneously or have
a false-positive test due to antibody cross-reaction. Investigating T lymphocyte responses in
individuals with different HCV status may help understand the cellular immune mechanisms
underlying spontaneous recovery, treatment response, and chronicity. OBJECTIVE: We aimed
to determine whether anti-HCV positive, HCV-RNA negative individuals are truly spontaneous
recoverers from acute HCV infection. STUDY DESIGN: We used enzyme-linked
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immunosorbent spot (ELISPOT) assay to compare HCV-specific lymphocyte response among
anti-HCV positive/HCV-RNA negative individuals, patients with sustained virological response
to interferon-γ/ribavirin treatment, and patients with chronic HCV infection. RESULTS:
We found that 83% of anti-HCV positive/HCV-RNA negative individuals without a past medical
history of acute icteric hepatitis had an HCV-specific T lymphocyte response in peripheral blood.
Lymphocyte responses in these individuals were similar in magnitude to treatment responders
unlike patients with chronic HCV whose virus-directed immunity was significantly suppressed.
CONCLUSIONS: Detection of HCV-specific T lymphocyte responses using ELISPOT is a
feasible method to ascertain past asymptomatic acute HCV infection.

Short and long-term effects of telaprevir on kidney function in patients with hepatitis C
virus infection: a retrospective cohort study. Sise ME1, Backman ES2, Wenger JB1, et al.
PLoS One. 2015 Apr 29;10(4):e0124139. doi: 10.1371/journal.pone.0124139. eCollection 2015.
BACKGROUND: Recent reports suggest that telaprevir, a protease inhibitor used to treat
hepatitis C infection, is associated with decline in kidney function during therapy, particularly in
patients with baseline renal impairment. METHODS: Patients treated with telaprevir in a single
healthcare network were retrospectively reviewed. Kidney function was determined at baseline,
during therapy, and twelve weeks and twelve months after telaprevir discontinuation. Significant
creatinine rise during therapy was defined as an increase in serum creatinine ≥ 0.3mg/dL from
baseline during treatment with telaprevir. RESULTS: Between July 2011 to January
2013,seventy-eight patients began treatment. The majority completed the prescribed twelve
weeks of telaprevir therapy; 32% discontinued due to side effects. The average rise in serum
creatinine during therapy was 0.22mg/dL (standard deviation 0.22mg/dL). Thirty-one percent
experienced a significant creatinine rise during therapy. Decline in estimated glomerular
filtration rate (eGFR) was lower in those with baseline eGFR < 90 mL/min/1.73m2 compared to
the group with baseline eGFR ≥ 90 mL/min/1.73m2 (12 vs. 18 mL/min/1.73m2, P = 0.047).
Serum creatinine fully normalized by twelve weeks after cessation of telaprevir in 83% of
patients, however experiencing a significant creatinine rise during telaprevir use was associated
with a 6.6mL/min/1.73m2 decrease in estimated glomerular filtration rate at twelve months in an
adjusted model. CONCLUSIONS: Decline in kidney function during therapy with telaprevir is
common and is not associated with baseline eGFR < 90mL/min/1.73m2 as previously reported.

BASIC AND APPLIED SCIENCE, PRE-CLINICAL STUDIES

Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires
Epidermal Growth Factor Receptor (EGFR). Trussoni CE1, Tabibian JH1, Splinter PL1,
O'Hara SP1. PLoS One. 2015 Apr 27;10(4):e0125793. doi: 10.1371/journal.pone.0125793.
eCollection 2015.
Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory
responses in the liver and contribute to inflammatory and infectious cholangiopathies. We
previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to
proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of
NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage
normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of
EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays,
quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without
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inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on
LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis,
primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association
between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR
phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated
with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced
IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-
treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased
phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was
inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that
EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a
robust proinflammatory cholangiocyte response. These findings have implications not only for
revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of
cholangiocyte TLR-induced proinflammatory processes.

Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of
viral nonstructural proteins. Kazakov T1, Yang F1, Ramanathan HN1, et al. PLoS Pathog.
2015 Apr 13;11(4):e1004817. doi: 10.1371/journal.ppat.1004817. eCollection 2015.
Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes
are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences
are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an
important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes
five nonstructural (NS) genes that are required for RNA replication. To date, only two of these
genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other
replicase genes work only in cis. We describe a new quantitative system to measure the cis- and
trans-requirements for HCV NS gene function in RNA replication and identify several lethal
mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans,
alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA.
Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein
expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly
distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities
of the NS3 helicase domain were required in cis, suggesting that these activities play an essential
role in RNA template selection. A comprehensive complementation group analysis revealed
functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A
genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity,
which could explain the synergy of this antiviral compound with nucleoside analogs in patients.
Together, these studies define several new aspects of HCV replicase structure-function, help to
explain the potency of HCV-specific combination therapies, and provide an experimental
framework for the study of cis- and trans-acting activities in positive-strand RNA virus
replication more generally.

EFTUD2 is a novel innate immune regulator restricting hepatitis C virus infection through
the RIG-I/MDA5 pathway. Zhu C1, Xiao F2, Hong J3, et al. J Virol. 2015 Apr 15. pii:
JVI.00364-15. [Epub ahead of print]
The Elongation factor Tu GTP binding domain containing 2 (EFTUD2) was identified as an anti-
HCV host factor in our recent genome-wide siRNA screen. In this study, we sought to further
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determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2
and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and
JAK/STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and
the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver
tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2
expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by
inducing expression of the interferon-stimulated genes (ISGs) in Huh7 cells. However, its impact
on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I defective
Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I.
Furthermore, EFTUD2 upregulated the expression of the RIG-I like receptors (RLR) RIG-I and
MDA5 to enhance innate immune response by gene splicing. Functional experiments revealed
that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2
downstream regulators RIG-I, MDA5, TBK1 and IRF3. Interestingly, the EFTUD2-induced
antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data
demonstrate that EFTUD2 restricts HCV infection mainly through a RIG-I/MDA5-mediated,
JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel
innate immune regulator and suggesting a potentially targetable antiviral pathway.
IMPORTANCE: Innate immunity is the first line defense against HCV and determines the
outcome of HCV infection. Based on a recent high throughput whole-genome siRNA library
screen revealing a network of host factors mediating antiviral effects against HCV, we identified
EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture
model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV
infection. Furthermore, we elucidated that EFTUD2 exerts its antiviral activity mainly through
governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and
induce classical ISGs expression independent of JAT-STAT signaling pathway. This study
broadens our understanding of the HCV innate immune response and provides a possible new
antiviral strategy targeting this novel regulator of the innate response.

Anti-E1E2 antibodies do predict response to triple therapy in treatment-experienced
Hepatitis C Virus-cirrhosis cases. Petit MA1, Berthillon P2, Pradat P3, et al. Clin Res Hepatol
Gastroenterol. 2015 Apr 17. pii: S2210-7401(15)00075-3. doi: 10.1016/j.clinre.2015.03.002.
[Epub ahead of print]
BACKGROUND AND AIMS: We previously showed that pre-treatment serum anti-E1E2
predicted hepatitis C virus (HCV) RNA viral kinetics (VKs) and treatment outcome in patients
with chronic hepatitis C receiving pegylated interferon/ribavirin (Peg-IFN/RBV) double therapy.
Here, we determined whether baseline anti-E1E2 was correlated with the on-treatment VK and
could predict virological outcome in treatment-experienced HCV-infected cirrhotic patients
receiving protease inhibitor-based triple therapy. METHODS: Sera from 19 patients with HCV
genotype 1 infection and compensated cirrhosis who failed to respond to a prior course of Peg-
IFN/RBV were selected at time 0 before starting triple therapy with boceprevir or telaprevir. We
assessed patients with sustained viral response 12weeks after the end of triple therapy (SVR12)
by analyzing VKs at weeks 4, 12, 24, 36, 48 (end of treatment) and 60. RESULTS: Patients
baseline characteristics were similar to the well-defined CUPIC cohort (age, HCV subtype,
baseline viremia, and treatment history). Among the 19 patients, 11 achieved an SVR12. Fifteen
patients were positive for pre-treatment anti-E1E2 and all of them achieved SVR12. Moreover,
anti-E1E2 and SVR12 correlated with prior response to IFN/RBV therapy (relapse, partial or null
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response). CONCLUSIONS: Baseline anti-E1E2 could be considered as a new biomarker to
predict SVR12 after triple therapy in this most difficult-to-treat population. These results warrant
further validation on larger cohorts including patients receiving highly effective direct-acting
antivirals to explore whether this test could help in better defining treatment duration for these
very costly molecules.

Control of temporal activation of hepatitis C virus-induced interferon response by domain
2 of nonstructural protein 5A. Hiet MS1, Bauhofer O1, Zayas M1, et al. J Hepatol. 2015 Apr
20. pii: S0168-8278(15)00298-6. doi: 10.1016/j.jhep.2015.04.015. [Epub ahead of print]
BACKGROUND & AIMS: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a
multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and
perturbing multiple host cell pathways. We showed previously that removal of a region in
domain 2 (D2) of NS5A (mutant NS5AD2Δ) is dispensable for viral replication in hepatoma cell
lines. By using a mouse model and immune-competent cell systems, here we studied the role of
D2 in controlling the innate immune response. METHODS: In vivo replication competence of
NS5AD2Δ was studied in transgenic mice with human liver xenografts. Results were validated
using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in
engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. RESULTS:
Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-
region, mutant NS5AD2Δ was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but
severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to
higher activation of the IFN response, concomitant with reduced viral replication and virus
production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential
activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma
differentiation associated factor 5), respectively, that was significantly higher in case of the
mutant lacking most of NS5A D2. CONCLUSIONS: Our study reveals an important role of
NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in
a sequential manner.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core
protein. Li M1, Wang Q1, Liu SA1, et al. World J Gastroenterol. 2015 Apr 21;21(15):4517-25.
doi: 10.3748/wjg.v21.i15.4517.
AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by
hepatitis C virus (HCV) core protein in HepG2 cells. METHODS: HCV genotype 1b core
protein was cloned and expressed in HepG2 cells. The cholesterol content was determined after
transfection. The expression of sterol regulatory element binding protein 2 (SREBP2) and the
rate-limiting enzyme in cholesterol synthesis (HMGCR) was measured by quantitative real-time
PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter
and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive
algorithms, microRNA (miRNA) mimics/inhibitors, and site-directed mutation to identify a
putative target of a particular miRNA. RESULTS: HCV core protein expression in HepG2 cells
increased the total intracellular cholesterol level (4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this
increase corresponded to an increase in SREBP2 and HMGCR mRNA levels (P = 0.009 and
0.037, respectively) and protein expression. The molecular mechanism study revealed that the
HCV core protein increased the expression of SREBP2 by enhancing its promoter activity (P =
0.004). In addition, miR-185-5p expression was tightly regulated by the HCV core protein (P =
Caring Ambassadors Program Hepatitis C Literature Review © 2015
0.041). Moreover, overexpression of miR-185-5p repressed the SREBP2 mRNA level (P =
0.022) and protein expression. In contrast, inhibition of miR-185-5p caused upregulation of
SREBP2 protein expression. miR-185-5p was involved in the regulation of SREBP2 expression
by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis
in HepG2 cells via the SREBP2 pathway; miR-185-5p is involved in the regulation of SREBP2
by the core protein.

Advances in experimental systems to study hepatitis C virus in vitro and in vivo. Catanese
MT1, Dorner M2. Virology. 2015 May;479-480C:221-233. doi: 10.1016/j.virol.2015.03.014.
Epub 2015 Apr 4.
Hepatitis C virus (HCV) represents a global health concern affecting over 185 million people
worldwide. Chronic HCV infection causes liver fibrosis and cirrhosis and is the leading
indication for liver transplantation. Recent advances in the field of direct-acting antiviral drugs
(DAAs) promise a cure for HCV in over 90% of cases that will get access to these expensive
treatments. Nevertheless, the lack of a protective vaccine and likely emergence of drug-resistant
viral variants call for further studies of HCV biology. With chimpanzees being for a long time
the only non-human in vivo model of HCV infection, strong efforts were put into establishing in
vitro experimental systems. The initial models only enabled to study specific aspects of the HCV
life cycle, such as viral replication with the subgenomic replicon and entry using HCV
pseudotyped particles (HCVpp). Subsequent development of protocols to grow infectious HCV
particles in cell-culture (HCVcc) ignited investigations on the full cycle of HCV infection and
the virus-host interactions required for virus propagation. More recently, small animal models
permissive to HCV were generated that allowed in vivo testing of novel antiviral therapies as
well as vaccine candidates. This review provides an overview of the currently available in vitro
and in vivo experimental systems to study HCV biology. Particular emphasis is given to how
these model systems furthered our understanding of virus-host interactions, viral pathogenesis
and immunological responses to HCV infection, as well as drug and vaccine development.

F protein increases CD4+CD25+ T cell population in patients with chronic hepatitis C.
Hashempour T1, Bamdad T2, Bergamini A3, et al. Pathog Dis. 2015 Jun;73(4). pii: ftv022. doi:
10.1093/femspd/ftv022. Epub 2015 Apr 9.
HCV is a global health problem with an estimated 230 million chronically infected people
worldwide. It has been reported that a 17-kd protein translated from core-encoding genomic
region can contribute to immune-mediated mechanisms associated with the development of the
chronic disease. Also, Treg cells can be contributed to an inadequate response against the
viruses, leading to chronic infection. Here we evaluated the ability of protein F to modulate the
frequency of CD4+CD25+FoxP3+T and IL-10+T cells in patients with chronic HCV infection. F
gene was amplified and cloned in the expression vector. The protein was purified and used for
stimulation of PBMCs in the HCV chronic patients and the control groups. The frequency of
CD4+CD25+FoxP3+ T cell-like populations and IL-10-producing CD4+CD25+ T cells was
assessed in the HCV-infected patients and in the healthy controls by flow cytometry, which
showed an increase of both CD4+CD25+FoxP3+ T cell-like population and IL-10-producing
CD4+CD25+ T cells in the HCV-infected patients positive for anti-F antibody. Our results
suggest the potential involvement of F and core antigens in increasing the frequency of
CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells which
may be associated with HCV-persistent infection.
Caring Ambassadors Program Hepatitis C Literature Review © 2015