Characterization of the Specific CD4+ T Cell Response against the F Protein during Chronic Hepatitis C Virus Infection
et al. (2010) Characterization of the Specific CD4+ T Cell Response against the F Protein during Chronic
Hepatitis C Virus Infection. PLoS ONE 5(12): e14237. doi:10.1371/journal.pone.0014237
+ Characterization of the Specific CD4 T Cell Response against the F Protein during Chronic Hepatitis C Virus Infection
De-Yong Gao 0
Gen-Di Jin 0
Bi-Lian Yao 0
Dong-Hua Zhang 0
Lei-Lei Gu 0
Zhi-Meng Lu 0
Qiming Gong 0
Yu-Chun Lone 0
Qiang Deng 0
Xin-Xin Zhang 0
Mario A. Ostrowski, University of Toronto, Canada
0 1 Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine , Shanghai , China , 2 Songjiang Hospital Affiliated to Shanghai First People's Hospital, Shanghai Jiaotong University , Shanghai , China , 3 INSERM U 542, Ho pital Paul Brousse , Villejuif , France , 4 Unit of Tumor Virology, Institut Pasteur of Shanghai, Chinese Academy of Sciences , Shanghai , China
Background: The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4+ T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4+ T cell responses in HCV chronically infected patients. Methodology: DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- c intracellular staining. Principal Findings: At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4+ T cell responses against HCV F protein as well in patients chronically infected with HCV. Conclusion: The current study provided the evidence for the first time that HCV F protein could elicit specific CD4+ T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.
Funding: This study was supported by National Nature Science Grant (30471523 and 30671839), National Special Key Research Grant (2008ZX10005-009), and
Major Program of Knowledge Innovation, CAS (KSCX1-YW-10). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Over 170 million people worldwide are chronically infected
with HCV. The chronic hepatitis C often results in cirrhosis of the
liver and increases the probability of developing hepatocellular
carcinoma [1,2]. There is no HCV vaccine available so far 
despite the fact that the combination of PEG-IFN-a and ribavirin
is at present a standard regimen used for treating hepatitis C
Cellular immune responses, involving both CD8+ cytotoxic T
lymphocytes (CTLs) and CD4+ T-helper lymphocytes (HTLs),
play an essential role in the control of HCV infection, as they do in
other persistent viral diseases. Whereas CTLs are traditionally
thought to be the main effector cells that eliminate HCV-infected
cells , it is clear that HCV-specific CD4+ T cells also play a
critical role. A growing body of evidence indicates that
spontaneous clearance of HCV is associated with a strong
HCV-specific proliferative CD4+ Th cell response. A number of
studies on persistent murine and human viral infections indicate
that virus specific CD4+ T cells play a critical role in the outcome
of viral infections [6,7,8,9,10], and are required to maintain
effective cytotoxic T cell responses  and neutralizing
antibodies . Notably, incomplete control of HCV replication
due to inadequate CD4+ T cell help is usually associated with the
emergence of viral escape mutation epitopes.
HCV alternate reading frame protein (ARFP/F) of the 1b
genotype is a double-frame shift product of the HCV core gene
[13,14,15]. It has been demonstrated that HCV F protein could
elicit a specific antibody response other than the anti-core protein
response [16,17]. The presence and the level of anti-F antibody
response could be induced by interferon plus ribavirin treatment
and associated with sustained virological response (SVR) in
hepatitis C patients . The current study was designed to
comprehensively determine the specific CD4+ T cell responses in a
cohort of patients with diverse HLA backgrounds, in order to
understand the potential helper T cell response against HCV F
protein during chronic HCV infection.
Expression and Identification of the HCV F proteins in
cultured cell line
HCV F protein is composed of a central frameshift F domain
(amino acids [aa] 43144, genotype 1b) flanked by N-terminal and
C-terminal fragments from HCV core protein. Expression of the F
protein was studied with gWiz-F, a plasmid bearing the chimeric F
gene under the control of cytomegalovirus early gene promoter.
After transient transfection of gWiz-F to human hepatoma cell line
Huh 7, the expression of HCV F protein was identified in cell
lysates with its expected size (25 KDa) by western blot using
specific anti-HCV core and anti- HCV F antibodies (Fig. 1).
HCV F protein can also be recognized by anti-HCV core
antibody, but with less intensity (Fig. 1B).
HCV F protein activates specific CD4+ T cell response in
HLA transgenic mice
We first investigated whether the MHC class II binding
determinants of HCV F protein could specifically stimulate
CD4+ T cell response by DNA vaccination in humanized mouse
models. The transgenic mice expressing the human HLA-DR1 or
HLA-DP4 molecules  were intramuscularly immunized twice
with gWiz-F. 7 days after the second injection, splenocytes from
immunized mice were isolated and subjected to in vitro
proliferation assays co-cultured with F protein-derived 15-mer peptides
individually (Table 1). As shown in Fig. 2, after the stimulation
with some F protein derived peptides, the splenocytes experienced
a significant in vitro expansion (p65 for HLA-DR1 mice, and p57,
p69 for HLA-DP4 mice). p65 was frequently recognized (7/8) by
HLA-DR1 mice, while 5/9 and all the tested HLA-DP4 mice
elicited p69- or p57-specific immune response respectively. p58,
the overlapping peptide adjacent to p57, could also be reactive to
the PBMCs from several HLA-DP4 mice although the
proliferative responses were less significant compared to p57 stimulation
The peptide p79 (LAHGVRVLEDGVNYA), located at the
common C-terminal region of HCV F and the parental core
protein, was shown as well in our study as a potential epitope to
stimulate a significant proliferation response in HLA-DR1 mice
(Fig. 2A). By intracellular and surface staining, however, the p79
activated cells were mostly identified as CD8+ lymphocytes,
whereas p65 induced IFN-c-producing cells were stained as the
CD4+ population. As shown in Fig. 2C, the IFN-c-producing cells
even represented as 9.41% of CD4+ lymphocytes following p65
restimulation. Although p57 and p69 elicited significant proliferative
responses of splenocytes from HLA-DP4 mice following DNA
immunization, the peptides failed to stimulate significant IFN-c
production in the ex vivo intracellular staining assay (data not
shown) which might be due to a low frequency of the specific T
cells presented in the mice.
Taken together, our study in HLA-DR1 and -DP4 transgenic
mice revealed that HCV F protein could elicit HLA-class II
restricted CD4+ T cell response in vivo. Several stimulating peptides
within HCV F protein were identified as HLA-DR or HLA-DP
F protein elicited specific CD4+ T cell response in chronic
In order to identify HCV F protein-specific HTL responses
during HCV natural infection, we firstly recruited a cohort of 47
patients with HCV chronic infection and a control group of 22
healthy subjects in our study. HLA-DR and -DP genotyping were
assayed by sequence-specific-primer PCR (Polymerase chain
reaction) using extracted genomic DNA as templates. 5 patients
and 3 control subjects were finally identified as HLA-DP*0401,
whereas only one patient and one healthy control expressed the
MHC class II allele of HLA-DRB1*01 (Table 2).
Freshly isolated PBMCs from HLA-DR1 and -DP4 subjects
were further incubated in the presence of a mixed peptide pool
(p57, p65, and p69). After 10 days of in vitro expansion, cells
were harvested to test IFN-c production following the
restimulation of the cognate peptides (p65 for HLA-DR1, p57 and
p69 for HLA-DP4 subjects). In the patient of HLA-DR1
genotype, HCV-F specific CD4+ T cell response against p65
(TLGPSMAMRAWGGQD) was exhibited by intracellular
cytokine staining as shown in Fig. 3A. The percentage of
IFN-c-producing cells represented up to 9.36% of CD4+ T cells
in the presence of p65 stimulation, compared with 8.37% by
SEB and 1% in the absence of stimulation respectively. In the 5
patients of HLA-DP4 genotype, p57 stimulated CD4+ T cell
activation only in the PBMCs of two subjects (3.02% and 1.38%
of CD4+ T cells respectively, Fig. 3B, C). p69 induced-specific
production of IFN-c was also observed in the two subjects, but
to a less extent (1.34% and 1.24% respectively). In the healthy
control with HLA-DR1 or -DP4 genotype, the three peptides
did not stimulate obvious IFN-c production by the PBMCs after
in vitro expansion.
We further analyzed the 3 HCV patients who exhibited the F
protein specific T cell response. According to the clinical
a, values of the Elisa assay are presented as the number of positive patients
versus patients tested, with calculated percentages in the parentheses;
b, a synthetic 99 aa F peptide spanning the central frameshift F domain;
c, the HLA class II haplotypes including HLA-DRB1*01, 03, 04, 07, 08, 09, 10, 11,
12, 13, 14, 15, 16, and HLA-DP*0401 negative or positive subtypes.
investigation, the three patients displayed lower viral copies
(3.626104, 5.386105, and 2.816104 copies/ml) and mild alanine
transaminase levels (96, 102, and 132 IU/ml) in the sera before
antiviral treatments, compared with the mean viral load of
3.326106 copies/ml and transaminase level of 115 IU/ml
respectively of the total recruited patients. We thus proposed that
the F protein specific HTL response, even only at a detectable
level, might reflect a favorable state for the antiviral immunity of
the hosts. Nevertheless, no obvious preference for the antiviral
treatments was observed in the three patients. Actually, most
recruited patients had a remarkably improved viral control
following antiviral treatments or even a spontaneous resolution.
In addition to our first try in 47 HCV patients, a new cohort of
15 more patients has been recruited for testing the F
proteinspecific HTL response. Considering the low incidence of
HLADR1 and -DP4 haplotypes in HCV patients, three pools of mixed
peptides spanning the central F frameshift domain (pool A,
p57p62; pool B, p63p68; and pool C, p69p75) were used directly for
the in vitro expansion of freshly isolated PBMCs from these
patients, regardless of the HLA class II subtypes. After
restimulation with the mixed peptides, although some activation
could be observed in several PBMC samples, they were not strictly
significant according to the assay of IFN-c intracellular staining
(data not shown). Moreover, the humoral immune response
against HCV was examined in the recruited patients (Table 2).
Interestingly, the three subjects who exhibited F protein specific
CD4+ T cell response were all anti-F antibody positive by the
ELISA assays. It is also noteworthy that an overall 22 of tested
patients (35%) displayed anti-F antibodies in the sera although
most of them did not show the HLA-DR1 or -DP4 restricted T cell
responses. Taken together, the current study provided the
evidence that HCV F protein specific CD4+ T cell response could
be induced in HCV chronically infected patients, although the
potency and width of the HTL response were relatively low. The
hyporesponsiveness to HCV F protein might reflect a progressive
dysfunction of specific T cell responses during the chronic viral
Bain and colleagues previously identified a specific CD8+ T cell
response against the HCV F protein which was expressed from
an alternate reading frame of the viral genome during HCV
natural infection . Recently, we and others have further
demonstrated that the specific antibodies against the F protein
presented in HCV chronically infected patients [16,17].
However, the specific CD4+ T cell responses against the F protein during
HCV infection and the pathological implications remain unclear.
CD4+ T cells can differentiate into Th1, Th2, Th17, and
regulatory T cell subsets, whose different immunological
functions are associated with the production of particular
cytokines that play important roles during HCV infection.
Interestingly, Drouin C et al  reported more recently that
cell-mediated immune responses directed against HCV F protein
were undetectable during the acute viral infection. In the current
study, comparatively, we screened the MHC class II-presenting
epitopes of the F protein through HLA-transgenic mouse models
and eventually validated the specific CD4+ T cell responses in
HCV chronically infected patients.
The antigen-processing machinery is highly conserved in mouse
and human cells . HLA-transgenic mice with knockouts of
murine MHC molecules have proven to be an excellent preclinical
model for the characterization of epitopes relevant to T cell
recognition in humans. In this study, we constructed the gWiz-F as
a fusion gene vector which could produce the F protein in
eukaryotic cells as indicated by western blot assay. gWiz-F was
further applied by DNA vaccination which made it possible to
assess the immune activity of F protein in HLA-transgenic mice in
vivo. Historically, the proliferative response of T cells to mitogenic
challenge has been a good standard for assessing T cell function in
both clinical and investigative immunology. In our study, three
potential HLA-DR1 or -DP4 presenting epitopes were eventually
defined by the splenocyte proliferation assay with humanized
We next tried to identify HLA-matched subjects in order to
characterize the specific CD4+ T cell response against the F
protein during HCV natural infection. The HLA class II
restricted T cell response to hepatitis C virus (HCV) antigens is
believed to influence the final outcome of HCV infection .
HLA-DRB1*01 allele appears to be related to a better HCV
specific T cell response according to the previous report by Barrett
S et al , and is thus with a rare incidence in the patients with
HCV chronic infection. In our study, we successfully recruited
some HCV patients with HLA-DP*0401 or HLA-DRB1*01
alleles. With the PBMCs from those patients, we eventually
validated the epitopes and the CD4+ T cell response defined by the
experiments with transgenic mice.
Tsai et al  previously demonstrated that viral clearance was
more likely to occur when PBMCs of acute hepatitis C patients
displayed a Th1 profile (IFN-c and IL-2) upon stimulation with
HCV antigens than in patients developing a Th2 phenotype (IL-4
and IL-10). Even in the chronic infection, a strong Th1 response
seems to be associated with a less inflammatory course of the
disease . Therefore, a Th1 profile may be considered as
generating more protective immune responses in HCV infection
and in contrast a failure to induce this phenotype may contribute
to the evolution to chronicity. In our study, the overlapping
15mer peptide pools spanning the HCV F sequence were used for
the PBMCs expansion in vitro to characterize the IFN-c production
by peptide re-stimulation individually. The three HCV F peptides
activated INF-c producing response in some patients with
wellmatched HLA class II subtype respectively. It is noteworthy that,
although eventually five HCV patients were identified with the
proper HLA-DP4 haplotype, they did not commonly exhibit the
CD4+ T cell response against the defined peptides. This might
reflect the difference between the immune competent nave state
in mouse models and the specific immune hyporesponsiveness
during the natural HCV persistent infection.
Analysis of HCV-specific CD4+ T cell responses in chronic
HCV infection using Elispot or intracellular staining showed
responses at low frequency in blood and only targeted a limited
number of epitopes [18,26,27,28]. Recent data also suggest that, in
the presence of viraemia, HCV-specific CD4+ T cell populations
do exist but lack proliferative capacity . These may explain the
hyporesponsiveness of PBMCs upon F peptide stimulation
observed in patients with chronic hepatitis C. Additionally, the
geographical regions, races of the patients, HCV genotypes, and
sample sizes could have impacts on the investigation. The very
limited proportion of Chinese HCV patients with HLA-DP*0401
and HLA-DRB1*01 genotypes is a significant restriction factor of
the current study.
In summary, HCV F protein, the alternative reading product of
the viral genome, is expressed during the chronic viral infection
that stimulates specific immune responses in vivo. Our study
directly evidenced the specific CD4+ T cell responses against HCV
F protein in transgenic mice following DNA immunization, and
also in patients chronically infected with HCV. Although the F
protein induced potent CD4+ T cell responses with defined
epitopes in HLA-transgenic mice, these specific T cell responses
were less obvious in HCV chronically infected patients. The
hyporesponsiveness to HCV F protein might reflect a progressive
dysfunction of specific T cell responses during the chronic viral
infection. However, the study of the HCV F protein is still at an
early stage. Clearly, more investigations of the relationship
between anti-F immune response and the pathogenesis of
Hepatitis C virus infection are warranted.
Materials and Methods
The current research involving human participants has been
approved by the institutional review board of Shanghai Ruijin
Hospital with written consents. All experiments involving mice
were performed according to approved protocols and guidelines of
the animal facility of Hopital Paul Brousse (Villejuif, France) with
the agreement number 94-076-32, and the permit number 94241
from Direction des Services Veterinaires.
The HLA-A2/-DR1, and HLA-A2/-DP4 transgenic mice (H-2
class II knockout, HLA-A*0201 transgenic, and HLA-DRB1*0101
or -DP*0401 transgenic)  were bred in the animal facility of
Hopital Paul Brousse. Groups of 610 weeks old female mice
received two vaccinations, 2-week apart, of gWiz-core or gWiz-F.
Intramuscular DNA immunizations were performed under
anesthesia by injecting 100 mg (1 mg/ml) of plasmid DNA into
regenerating (i.e. cardiotoxin-treated) tibialis anteriormuscles .
A total of 37 peptides covering the entire 191 aa of HCV F
protein were synthesized by Gelson Chemical (Shanghai, China).
These peptides were 15 aa in length, overlapping adjacent
peptides by 10 aa. The sequence of individual peptide from the
central frameshift F domain (aa43-144) of the HCV F protein is
listed in Table 1. Stock solutions of all synthetic peptides were
produced at 1 mg/ml in either water or 20% DMSO, according
to the suppliers instruction. The peptides were used for the in vitro
co-culture and the re-stimulation of splenocytes. All the peptides
spanning the chimeric HCV F protein (Table 1) were tested by
the splenocytes proliferation assay. Peptides with the potential
avidity were further verified by a second round of screening.
HCV core gene was PCR amplified from the viral cDNA after
reverse transcription using the serum sample from genotype 1b
HCV infected patients. The HCV F protein-coding sequence was
achieved by the +1/21 frame-shifting between codon 43 and
codon 144 of the core gene by overlapping PCR. The resulting
HCV F cDNA was subcloned into the gWiz expression vector
(Genlantis, San Diego, CA) to have gWiz-F. The plasmid of
gWizcore encoded HCV core gene (genotype 1b) driven by the
cytomegalovirus (CMV) early gene promoter.
In vitro transfection assay
Human hepatoma Huh 7 cells  were maintained in DMEM
containing 10% fetal calf serum. Cells were harvested 48 h after
Lipofectamine 2000 transfection (Invitrogen). Cell lysates were
analyzed by Western-blot according to standard procedures, with
anti-core and anti-HCV F monoclonal antibodies (CNRS,
France). Signals were visualized using the enhanced
chemiluminescence method with a horseradish peroxidase-labeled rabbit
anti-mouse immunoglobulin (Dako, Carpenteria, CA).
Proliferation assay of mouse splenocytes
7 to 10 days after the second DNA injection, mouse spleen was
smashed with a syringe plunger in a 70 mm cell strainer (100 mm
Nylon, BD). Red blood cells were removed by Ficoll gradient
centrifugation. Isolated splenocytes (106 per well) were incubated
with 10 mg/ml peptide for 3 days in supplemented HL1
serumfree medium and pulsed for the final 16 h with 1 mCi
(3H)thymidine per well . All the peptides spanning the chimeric
HCV F protein (Table 1) were measured by the splenocytes
proliferation assay. Peptides with the potential avidity were further
verified by a second round of screening.
Totally sixty two subjects of chronic HCV infection with written
informed consent were enrolled in this study from the Department
of Infectious Diseases of Shanghai Ruijin Hospital. No subject had
been treated with antiviral therapy before this study. The diagnosis
of chronic hepatitis C was based on internationally accepted
criteria . All subjects were anti-HCV positive as measured by
Third-Generation Enzyme Immunoassay. The Mean HCV load
(copies/ml) was around 3.326106 (range from 2.36104 to
2.26108) quantified in serum samples by real-time PCR after
reverse transcription of the 59noncoding region of the HCV
genome (PJ Co. Ltd., China). HCV genotypes were determined
using Realchip Kit (Realchip Biotechnology, China) by which
66% of the samples were of HCV-1b subtype. The average
Genotyping of HLA class II alleles
The genomic DNA was extracted from patients PBMCs using
the Puregene DNA isolation kit for blood (Qiagen). HLA-DR and
HLA-DP genotyping were carried out by primer specific PCR
using Olerup SSP Genovision kit (Sweden).
In Vitro Expansion of the PBMCs
PBMCs were isolated from fresh heparinized blood by Ficoll
density gradient centrifugation and seeded at 56106 cells/well in
24-well plates with RPMI 1640 complete medium containing
2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino
acids, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10%
FCS (Gibico). Cells were stimulated by incubation with HCV F
peptide pools (1 mg/ml of each peptide). Half the medium was
replaced every 3 days with complete medium supplemented with
recombinant IL-2 (50 IU/ml) (Roche). After 10 days of culture,
HCV F-specific IFN-c-producing cells were quantified by
intracellular staining and flow cytometry analysis.
Intracellular staining assay
Freshly isolated PBMCs were incubated overnight either with
500 ng/ml staphylococcal enterotoxin B (SEB, Sigma) as a
positive control, or with individual HCV F-derived peptides
(1 mg/ml) in the presence of 2 mg/ml brefeldin A (BFA, Sigma).
Cells were suspended in PBS containing 1% BSA and labeled with
anti-CD4-APC (clone HIT3a) and anti-CD8-FITC (clone
RPAT4) for 20 minutes at 4uC. After fixation and permeabilization,
cells were further stained with anti-human IFN-c-PE (clone 4S.B3)
for 30 minutes at 4uC. At least 50,000 lymphocyte-gated events
were acquired on a FACSCalibur flow cytometer (BD Biosciences)
and analyzed using Flowjo software (version 8.7.3, Tree Star).
Conjugated antibodies were purchased from BD Biosciences.
Background staining was assessed with an isotype-matched control
monoclonal antibody. Single peptides that elicited distinct
responses of more than two times background IFN-c production
were considered positive .
Enzyme-linked immunosorbent assay (ELISA)
The ELSIA assay testing the humoral immune response in sera
of HCV patients was described elsewhere . A synthetic 99 aa F
peptide encoding the central frameshift F domain was synthesized
by Gelson Chemical. The recombinant 66His tagged HCV core
protein were prokaryotically expressed and purified according to
our previous work. In addition, antibody to hepatitis C virus in
these patients were detected as controls by using hepatitis C Virus
encoded Antigen (Recombinant c22-3, c200 and NS5) with
ORTHO HCV Version 3.0 ELISA Test System (Ortho-Clinical
Diagnostics) according to the manufacturers recommendations.
We thank Dr. Genevieve Inchauspe (Lyon ENS, France) for kindly
providing us the monoclonal anti-F and anti-C. We thank Dr. Xiaoming
Zhang for the FACS analysis and constant supports during this study. We
also thank Dr. Chaoyang Deng for the English correction and helpful
Conceived and designed the experiments: YCL QD XXZ. Performed the
experiments: DYG GDJ BLY DHZ LLG QD. Analyzed the data: DYG
ZML QD XXZ. Contributed reagents/materials/analysis tools: GDJ BLY
DHZ QG YCL. Wrote the paper: DYG QD XXZ.
1. Cohen J ( 1999 ) The scientific challenge of hepatitis C . Science 285 : 26 - 30 .
2. Lauer GM , Walker BD ( 2001 ) Hepatitis C virus infection . N Engl J Med 345 : 41 - 52 .
3. Berzofsky JA , Ahlers JD , Janik J , Morris J , Oh S , et al. ( 2004 ) Progress on new vaccine strategies against chronic viral infections . J Clin Invest 114 : 450 - 462 .
4. Liang TJ , Rehermann B , Seeff LB , Hoofnagle JH ( 2000 ) Pathogenesis, natural history , treatment, and prevention of hepatitis C. Ann Intern Med 132 : 296 - 305 .
5. Gremion C , Cerny A ( 2005 ) Hepatitis C virus and the immune system: a concise review . Rev Med Virol 15 : 235 - 268 .
6. Day CL , Walker BD ( 2003 ) Progress in defining CD4 helper cell responses in chronic viral infections . J Exp Med 198 : 1773 - 1777 .
7. Missale G , Bertoni R , Lamonaca V , Valli A , Massari M , et al. ( 1996 ) Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response . J Clin Invest 98 : 706 - 714 .
8. Diepolder HM , Zachoval R , Hoffmann RM , Wierenga EA , Santantonio T , et al. ( 1995 ) Possible mechanism involving T-lymphocyte response to nonstructural protein 3 in viral clearance in acute hepatitis C virus infection . Lancet 346 : 1006 - 1007 .
9. Eckels DD , Wang H , Bian TH , Tabatabai N , Gill JC ( 2000 ) Immunobiology of hepatitis C virus (HCV) infection: the role of CD4 T cells in HCV infection . Immunol Rev 174 : 90 - 97 .
10. Shoukry NH , Cawthon AG , Walker CM ( 2004 ) Cell-mediated immunity and the outcome of hepatitis C virus infection . Annu Rev Microbiol 58 : 391 - 424 .
11. Cardin RD , Brooks JW , Sarawar SR , Doherty PC ( 1996 ) Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells . J Exp Med 184 : 863 - 871 .
12. Planz O , Ehl S , Furrer E , Horvath E , Brundler MA , et al. ( 1997 ) A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers . Proc Natl Acad Sci U S A 94 : 6874 - 6879 .
13. Boulant S , Becchi M , Penin F , Lavergne JP ( 2003 ) Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b . J Biol Chem 278 : 45785 - 45792 .
14. Branch AD , Stump DD , Gutierrez JA , Eng F , Walewski JL ( 2005 ) The hepatitis C virus alternate reading frame (ARF) and its family of novel products: the alternate reading frame protein/F-protein, the double-frameshift protein, and others . Semin Liver Dis 25 : 105 - 117 .
15. Vassilaki N , Mavromara P ( 2009 ) The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product . IUBMB Life 61 : 739 - 752 .
16. Komurian-Pradel F , Rajoharison A , Berland JL , Khouri V , Perret M , et al. ( 2004 ) Antigenic relevance of F protein in chronic hepatitis C virus infection . Hepatology 40 : 900 - 909 .
17. Gao DY , Zhang XX , Hou G , Jin GD , Deng Q , et al. ( 2008 ) Assessment of specific antibodies to F protein in serum samples from Chinese hepatitis C patients treated with interferon plus ribavarin . J Clin Microbiol 46 : 3746 - 3751 .
18. Mancini-Bourgine M , Bayard F , Soussan P , Deng Q , Lone YC , et al. ( 2007 ) Hepatitis B virus splice-generated protein induces T-cell responses in HLAtransgenic mice and hepatitis B virus-infected patients . J Virol 81 : 4963 - 4972 .
19. Bain C , Parroche P , Lavergne JP , Duverger B , Vieux C , et al. ( 2004 ) Memory Tcell-mediated immune responses specific to an alternative core protein in hepatitis C virus infection . J Virol 78 : 10460 - 10469 .
20. Drouin C , Lamarche S , Bruneau J , Soudeyns H , Shoukry NH ( 2010 ) Cellmediated immune responses directed against hepatitis C virus (HCV) alternate reading frame protein (ARFP) are undetectable during acute infection . J Clin Virol 47 : 102 - 103 .
21. Pascolo S ( 2005 ) HLA class I transgenic mice: development, utilisation and improvement . Expert Opin Biol Ther 5 : 919 - 938 .
22. Singh R , Kaul R , Kaul A , Khan K ( 2007 ) A comparative review of HLA associations with hepatitis B and C viral infections across global populations . World J Gastroenterol 13 : 1770 - 1787 .
23. Barrett S , Ryan E , Crowe J ( 1999 ) Association of the HLA-DRB1*01 allele with spontaneous viral clearance in an Irish cohort infected with hepatitis C virus via contaminated anti-D immunoglobulin . J Hepatol 30 : 979 - 983 .
24. Tsai SL , Liaw YF , Chen MH , Huang CY , Kuo GC ( 1997 ) Detection of type 2- like T-helper cells in hepatitis C virus infection: implications for hepatitis C virus chronicity . Hepatology 25 : 449 - 458 .
25. Woitas RP , Lechmann M , Jung G , Kaiser R , Sauerbruch T , et al. ( 1997 ) CD30 induction and cytokine profiles in hepatitis C virus core-specific peripheral blood T lymphocytes . J Immunol 159 : 1012 - 1018 .
26. Ulsenheimer A , Gerlach JT , Gruener NH , Jung MC , Schirren CA , et al. ( 2003 ) Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic hepatitis C . Hepatology 37 : 1189 - 1198 .
27. Rosen HR , Miner C , Sasaki AW , Lewinsohn DM , Conrad AJ , et al. ( 2002 ) Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: correlation with clinical disease stages . Hepatology 35 : 190 - 198 .
28. Wertheimer AM , Miner C , Lewinsohn DM , Sasaki AW , Kaufman E , et al. ( 2003 ) Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection . Hepatology 37 : 577 - 589 .
29. Semmo N , Krashias G , Willberg C , Klenerman P ( 2007 ) Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection . J Viral Hepat 14 : 492 - 502 .
30. Mancini M , Davis H , Tiollais P , Michel ML ( 1996 ) DNA-based immunization against the envelope proteins of the hepatitis B virus . J Biotechnol 44 : 47 - 57 .
31. Nakabayashi H , Taketa K , Yamane T , Miyazaki M , Miyano K , et al. ( 1984 ) Phenotypical stability of a human hepatoma cell line, HuH-7, in long-term culture with chemically defined medium . Gann 75 : 151 - 158 .
32. Bedossa P , Poynard T ( 1996 ) An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group . Hepatology 24 : 289 - 293 .
33. Day CL , Lauer GM , Robbins GK , McGovern B , Wurcel AG , et al. ( 2002 ) Broad specificity of virus-specific CD4+ T-helper-cell responses in resolved hepatitis C virus infection . J Virol 76 : 12584 - 12595 .