Content uploaded by Elisabetta Cariani
Author content
All content in this area was uploaded by Elisabetta Cariani
Content may be subject to copyright.
The Impairment of CD8 Responses Limits the Selection of
Escape Mutations in Acute Hepatitis C Virus Infection
1
Simona Urbani, Barbara Amadei, Elisabetta Cariani, Paola Fisicaro, Alessandra Orlandini,
Gabriele Missale, and Carlo Ferrari
2
Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV
persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was
conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of
overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining.
Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic
sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients,
mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells
than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were
targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8
responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations
represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved. The Journal of
Immunology, 2005, 175: 7519 –7529.
Hepatitis C virus is a common cause of liver disease, and
the majority of infected individuals develop persistent
infection. Among the different mechanisms of HCV per-
sistence, the emergence of mutations within immunodominant
CD8 epitopes represents a possible strategy that HCV can exploit
to evade immune control (1– 8). In general, the selection of CD8
escape mutations is more likely to occur if the mutation rate of the
virus is high, if the protective CD8 responses to evade are targeted
against variable viral regions, which can tolerate mutations without
loss of vital viral functions, and if the CD8 function is sufficient to
exert the immunological pressure needed to induce the selection of
escape mutations (9). Moreover, the likelihood of escape by this
mechanism is expected to be greater if the CD8 response is nar-
rowly focused on individual or a few strongly dominant epitopes.
Although HCV has a very high mutation rate due to the lack of
proofreading capacity of its polymerase (10), the overall repertoire
of HCV epitopes recognized by CD8 cells and their hierarchy of
immunodominance in individual infected subjects has been so far
difficult to define owing to technical limitations in analyzing the
global HCV-specific CD8 response in its entirety. Moreover, var-
ious types of functional CD8 cell defects at the early stages of
HCV infection have been reported, which may affect the overall
antiviral function of the CD8 response, thus limiting its capacity to
exert selective pressure on HCV (11–16). This makes difficult to
predict the actual role that the mutational escape of HCV from
CD8 surveillance can have as a mechanism of persistence.
The role of virus evasion from CD8 surveillance by escape mu-
tations has been demonstrated in different infections, such as HIV
and CMV (17, 18). Proof of this concept in human HCV infection
has been limited by different factors, including the asymptomatic
nature of HCV infection that makes the longitudinal analysis of
viral evolution and host immune responses from the early stages of
infection a difficult task. Moreover, the infecting inoculum is gen-
erally unknown in the majority of patients with acute infection.
Because of these limitations, until very recently, amino acid vari-
ations within HLA class I-restricted epitopes or their flanking re-
gions were reported, but no definitive proof of selection by the
CD8 immune pressure was given (1– 8, 19 –21). Only more recent
studies have provided convincing evidence of HCV escape in pa-
tients with acute HCV infection by the identification of escape
mutations within multiple CTL epitopes (22–26), two of whom
infected from a single viral source (22, 23), and in patients with
chronic hepatitis C by the analysis of viral evolution from a com-
mon infectious inoculum conducted several years after the time of
infection (24). These studies indicate that mutations can occur
within epitopic regions not only to allow HCV escape from CD8
surveillance but also to allow reversion of the virus to a more fit
pre-existing state (25). This extends to the human infection previ-
ous evidence of escape derived from the chimpanzee model of
infection, where abrogation of immunodominant CD8 responses
caused by the emergence of mutations was directly linked to HCV
persistence (2).
The relevance of this mechanism in the pathogenesis of HCV
persistence still remains partially understood. To further clarify
this issue, in this study, we first looked for immunodominant CD8
epitopes in six patients with acute hepatitis C followed by chronic
evolution of infection. The behavior of CD8 responses against
Laboratory of Viral Immunopathology, Department of Infectious Diseases and Hepa-
tology, Azienda Ospedaliera di Parma, Parma, Italy
Received for publication April 19, 2005. Accepted for publication September
15, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The study was supported by ViRgil European Commission No. LSHM-CT-2004-
503359, Fondo per gli Investimenti della Ricerca di Base-Ministry of Education,
University and Research Grant RBNE013PMJ_006, Progetto di Ricerca Finalizzato
Ministry of Health Grant 120 (Italy), European Union Grant QLK2-CT-2002-00700,
by a grant from Schering-Plough S.p.A. (Milan, Italy), and by a grant from Cassa
Risparmio di Parma (Parma, Italy).
2
Address correspondence and reprint requests to Dr. Carlo Ferrari, Laboratorio Im-
munopatologia Virale, Unit of Infectious Diseases and Hepatology, Azienda Osped-
aliero Universitaria di Parma, Via Gramsci 14, 43100 Parma, Italy. E-mail address:
cafer@tin.it
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
them was subsequently monitored over time in relation to the evo-
lution of the sequence of the corresponding epitopes. The results
indicate that only a fraction of the immunodominant epitopes iden-
tified at the onset of the disease underwent a strong selective pres-
sure by CD8 responses, leading to the emergence of escape mu-
tations. CD8 responses to four of the seven immunodominant
epitopes studied were functionally impaired from the earliest time
points analyzed and therefore unable to perform selection. Our
study confirms that escape mutations can contribute to HCV per-
sistence, although other mechanisms of CD8 cell inhibition can
silence immunodominant responses early after infection before the
selection of escape mutations can take place.
Materials and Methods
Patients and virological assessment
Six patients with acute hepatitis C were enrolled at the Unit of Infectious
Diseases and Hepatology of the University Hospital of Parma. The diag-
nosis of acute HCV infection was based on the following criteria: docu-
mented seroconversion to anti-HCV Abs by Recombinant ImmunoBlot
assay, levels of serum alanine aminotransferase (ALT)
3
at least 10 times
the upper limit of normal (50 U/L), detection of HCV RNA, exclusion of
other possible causes of acute hepatitis (i.e., viruses, toxins, alcohol, au-
toimmunity, metabolic factors). Anti-HCV Abs were determined by com-
mercial enzyme immunoassay kits (Ortho Diagnostic Systems) and by
Recombinant ImmunoBlot (RIBA II; Ortho Diagnostic Systems). Serum
HCV-RNA was quantified by branched DNA assay and was expressed as
copies per milliliter of serum (Bayer System 340bDNA Analyzer; Bayer);
the lower limit of detection by this method is 2500 copies/ml. The study
was approved by the Ethical Committee of the Azienda Ospedaliero Uni-
versitaria di Parma, and all subjects gave written informed consent.
Synthetic peptides, recombinant proteins, peptide-HLA class I
tetramers, and Abs
Synthetic peptides representing HLA-A2-restricted epitopes corresponding
to HCV NS3 1073–1081 (CINGVCWTV), NS3 1406 –1415 (KLVALG
INAV), NS4 1812–1820 (LLFNILGGWV), NS4B 1992–2000 (VLSDFK
TWL), and NS5 2594 –2602 (ALYDVVTKL), the mutated sequences of
the HCV regions NS3 1073–1081 and NS4 1992–2000 detected in patients
2, 3, and 6, influenza virus Matrix 58 –66 (GILGFVFTL), CMV pp65
(NLVPMVATV), EBV BMLF-1 (GLCTLVAML), and a panel of 601 15-
mer peptides based on a genotype 1a sequence (HCV-1) covering all struc-
tural (core, E1, E2) and nonstructural (NS3, NS4, NS5) HCV regions and
overlapping by 10 residues were purchased from Chiron Mimotopes. The
HCV Ags E1, E2, core, NS3, NS4, and NS5 were expressed as C-terminal
fusion proteins with human superoxide dismutase in yeast and were pro-
vided by Chiron. Purity of the Ag preparation ranged from 85 to 95%.
PE-labeled tetrameric peptide-HLA class I complexes were purchased
from Proimmune. HLA A2 tetramers contained the HCV peptides NS3
1073–1081, NS3 1406 –1415, NS4 1812–1820, NS4B 1992–2000, and
NS5 2594 –2602. Anti-IFN-
␥
FITC (conjugated FITC-FITC) was pur-
chased from Sigma-Aldrich; anti-CD8 allophycocyanin and anti-CD 107
FITC were purchased from BD Pharmingen.
Isolation of PBMC and in vitro expansion of HCV-specific CTL
PBMC were isolated from fresh heparinized blood by Ficoll-Hypaque den-
sity gradient centrifugation and resuspended in RPMI 1640 supplemented
with 25 mM HEPES, 2 mM L-glutamine, 50
g/ml gentamicin, and 8%
human serum (complete medium). For CTL expansion, PBMC were re-
suspended in 96-well plates at a concentration 2 ⫻10
6
/ml in complete
medium and stimulated with HCV peptides at 1
M final concentration.
Recombinant IL-2 was added on day 4 of culture (50 UI/ml), and the
immunological assays were performed on day 10.
Cell surface and intracellular staining
Staining with tetramers and other surface markers. A total of 1 ⫻10
6
PBMC either freshly isolated or following in vitro expansion for 10 days
was incubated for 30 min at 37°C with the PE-labeled tetrameric complex
in RPMI 1640 and 8% human serum. After washing, staining was per-
formed for 15 min in the dark, using an anti-CD8 APC-conjugated Ab. The
cells were then washed and analyzed immediately on a BD Biosciences
flow cytometry (FACSCalibur) using the CellQuest software.
IFN-
␥
staining. Tetramer stained cells were incubated in medium alone
(control) or with viral peptides (1
M) for 1 h; brefeldin A (10
g/ml) was
added for an additional4hofincubation. After washing, the cells were
stained with an anti-CD8 quantum red mAb for 20 min at 4°C and then
fixed and permeabilized with a permeabilization buffer (Caltag
Laboratories). Cells were finally stained with anti-IFN-
␥
-FITC for 15 min
at room temperature, washed again, and analyzed by flow cytometry.
ELISPOT assay
The 601 15-mer peptides overlapping by 10 residues and covering the
overall HCV-1 sequence were pooled in 60 mixtures, each containing 10
synthetic peptides. HCV-specific T cell responses were analyzed upon
overnight stimulation with individual peptide mixtures (1
M of each pep-
tide). Briefly, 96-well plates (Multiscreen-I; Millipore) were coated over-
night at 4°C as recommended by the manufacturer with 5
g/ml capture
mouse anti-human IFN-
␥
mAb (1 DIK; Mabtech). Plates were then washed
seven times with PBS/0.05% Tween 20, and then blocked with RPMI
1640/10% FCS for2hat37°C. A total of 2 ⫻10
5
PBMC was seeded per
well. The plate was incubated for 18 h at 37°C in the presence or in the
absence of peptides. After washing with PBS/0.05% Tween 20, a biotin-
ylated secondary mouse anti-human IFN-
␥
mAb (1
g/ml, 7B6-1;
Mabtech) was added. After3hofincubation at room temperature, plates
were washed four times, and 100
l of goat alkaline phosphatase anti-
biotin Ab (Vector Laboratories) was added to wells, and the plates were
incubated for an additional2hatroom temperature. Plates were then
washed four times, and 75
l of alkaline phosphatase conjugate substrate
(5-bromo-4-chloro-3-indolyl phosphate; Bio-Rad) was added. After 4 –7
min, the colorimetric reaction was stopped by washing with distilled water.
Plates were air-dried, and spots were counted using an automated
ELISPOT reader (AID Elispot Reader System; Autoimmune Diagnostika).
IFN-
␥
-producing cells were expressed as spot-forming cells (SFC) per 1 ⫻
10
6
cells. The number of specific IFN-
␥
-secreting cells was calculated by
subtracting the unstimulated control value from the stimulated sample. Un-
stimulated wells never exceeded five to seven spots per well. Positive con-
trols consisted of PBMC stimulated with PHA. Wells were considered
positive if they were at least twice above background.
RNA extraction, amplification by the PCR, direct sequencing,
and sequencing of molecular clones
Viral RNA was extracted from 600
l of serum using a kit RNAfast iso-
lation system (RNA fast; Molecular System). The RNA was eluted in 30
l
of RNase-free water. The extracted RNA (3
l) was reverse transcribed at
24°C for 10 min, 42°C for 25 min, and 99°C for 5 min in a 20-
l reaction
mixture with 50
M random hexamers and2Uofmurine leukemia virus
reverse transcriptase (GeneAmp RNA PCR; Applied Biosystems). Nested
primer sets were constructed to amplify each region of the HCV genome
that contained HLA class I-restricted epitopes. The sequences of the prim-
ers are indicated in Table I. PCR conditions were as follows: 1 cycle
(95°C-5 min, 56°C-2 min, 72°C-1 min); 35 cycles (95°C-45 s, 56°C-45 s,
72°C-45 s); and 1 cycle (72°C-10 min). Five microliters of the first PCR
product was then reamplified with inner primers for 35 cycles under the
same reaction conditions as in the first round of PCR. As the template for
the amplification, 5
l of the cDNA was used. PCR was performed in a
total volume of 50
l in a reaction mixture containing 50 pmol of each of
the respective primers and5UofTaqDNA polymerase (AmpliTaq Gold;
Applied Biosystems). A product of the predicted size was observed after
electrophoresis on a 1.5% agarose gel when visualized under UV light after
ethidium bromide staining. This cDNA band was excised from the gel and
the cDNA was purified using a cDNA purification kit (QIAquick gel ex-
traction kit; Qiagen). PCR products were cloned and sequenced. The ex-
tracted products were ligated into the plasmid pGEM-Teasy vector (Pro-
mega). Transformation of recombinant plasmid DNA into Escherichia coli
competent cells was performed as specified by the manufacturer and trans-
formants were grown on Luria-Bertani/Ampicillin/isopropyl

-D-thiogal-
actoside/5-bromo-4-chloro-3-indolyl

-D-galactoside plates. Plasmid
DNA was isolated by the miniprep method. For each sample, 15–25 clones
were amplified, and DNA sequences were determined using the ABI Prism
377 DNA Sequencing System (PerkinElmer).
3
Abbreviations used in this paper: ALT, alanine aminotransferase; SFC, spot-forming
cell; ICS, intracellular cytokine staining.
7520 CD8 FUNCTION AND EPITOPE MUTATIONS IN ACUTE HCV INFECTION
Results
Identification of immunodominant T cell responses in genotype 1
infected patients
The global profile of the HCV-specific T cell response was analyzed
in three HLA-A2
⫹
patients with acute hepatitis C infected by geno-
type 1 (genotype 1b, patients 1 and 3; genotype 1a, patient 2). In these
patients the entire HCV genome was comprehensively screened by
ELISPOT using a panel of 601 15-mer peptides overlapping by 10
residues and spanning the entire HCV sequence of genotype 1. The
analysis of the breath and the magnitude of the T cell response during
the acute phase of infection at the peak of ALT, revealed that in
patients 1 and 2 virus-specific responses were generally weak and
directed against a limited number of epitopes. Of the 60 mixtures of
synthetic peptides tested, 5 in patient 1 and 4 in patient 2 were able to
induce IFN-
␥
production with different intensity ranging from 50 to
500 SFC/10
6
PBMC (Fig. 1A). A response of similar intensity was
detected in patient 3 but a larger number of peptide pools (17 pools)
were positive. The breadth of the T cell response was confirmed 2 wk
after the time point represented in Fig. 1; the same positive pools were
still detectable in patients 1 and 2 and 9 of the 14 responses were still
positive in patient 3 (data not shown). Further analysis performed by
intracellular cytokine staining (ICS) with the peptides contained in the
stimulatory pools allowed to identify in each patient the epitopes cor-
responding to the best ELISPOT responses. The only peptides respon-
sible for cytokine production within pool 22 in patient 1 and within
pools 22 and 40 in patient 2 were the 15 mers containing the HLA-
A2-restricted epitopes HCV NS3 1073–1081 and NS4 1992–2000
(data not shown). Responses against these epitopes were subsequently
analyzed by ex vivo tetramer staining. Elevated frequencies of HCV-
specific CD8 cells were detected, confirming the immunodominance
of these epitopes in these two patients (Fig. 1, Aand B). In patient 3,
the peptides contained in each responsive pool could not be analyzed
extensively because of cell limitations. Only pool 22, which was able
to stimulate the strongest ELISPOT response, was screened in ICS
with the individual component peptides. A detectable response was
elicited only by the 15 mer peptide containing the sequence 1073–
1081, as confirmed by tetramer staining detecting elevated frequen-
cies of CD8 cells specific for the NS3 1073–1081 epitope (Figs. 1, A
and B, and 2).
Analysis of the CD8 response against preselected peptides in
genotype non-1 (genotype 3) infected patients
Three more patients with acute hepatitis, infected by genotype 3
HCV were studied. Because the panel of peptides spanning the
Table I. HCV primers for amplification and sequencing
Sense Antisense
CVNGVCWTV 1B (ES) 5⬘-ATCACGGCCTACTCCCAACA-3⬘3426 –3445 (EA) 5⬘-ATCAGCATGTCTCGTGACCA-3⬘3755–3736
(IS) 5⬘-AGCCTCACAGGCCGGGACAA-3⬘3477–3496 (IA) 5⬘-TCCGAGCTGCCGCAGGTGCA-3⬘3727–3708
CINGVCWTV 1A (ES) 5⬘-GTACGCCCAGCAGACAAG-3⬘3434 –3451 (EA) 5⬘-TGCAGGGTGTCAATGAGC-3⬘3711–3694
(IS) 5⬘-GTACGCCCAGCAGACAAG-3⬘3434 –3451 (IA) 5⬘-TCTGGATGACAGGACCCTT-3⬘3639 –3621
VLSDFKSWL 3A (ES) 5⬘-TGCTGAGTTCTCTAACTGTC-3⬘6198 – 6217 (EA) 5⬘-GTACAGGGGCCCGTGGTGTA-3⬘6569– 6550
(IS) 5⬘-CTTGTAGCGACGATTGGCTAC-3⬘6269 – 6289 (IA) 5⬘-CCCTGCAAGCCGCAGGACCC-3⬘6500– 6481
FIGURE 1. Identification of immu-
nodominant CD8-mediated responses
in 6 patients with acute hepatitis C. A,
IFN-
␥
production by direct ex vivo
ELISPOT analysis. PBMC from three
patients (patients 1, 2, and 3) acutely
infected by HCV of genotype 1 were
stimulated overnight with 60 pools of
overlapping 15-mer peptides covering
the whole HCV sequence (Mix1– 4,
HCV core; Mix 5– 8, HCV E1; Mix
9 –15, HCV E2; Mix 16–17, HCV p7;
Mix 18 –21, HCV NS2; Mix 22–33,
HCV NS3; Mix 34 –40, HCV NS4;
Mix 41– 49, HCV NS5A; Mix 50–60,
HCV NS5B). findicates the peptide
pools able to induce the strongest T cell
responses, which were then analyzed
with individual peptides to identify the
stimulatory sequences. The results are
expressed as
␦
SFC per 10
6
PBMC. B
and C, Peripheral blood frequency of
HCV-specific CD8 cells by ex vivo tet-
ramer staining in HLA-A2
⫹
patients
infected either by HCV of genotype 1
(patients 1, 2, and 3; B)orbyHCVof
genotype 3 (patients 4, 5, and 6; C).
ELISPOT and dot plot analyses were
performed during the acute phase of in-
fection at the ALT peak.
7521The Journal of Immunology
entire virus corresponded to a sequence of genotype 1 (HCV-1), it
was not possible to perform a comprehensive analysis of the HCV-
specific T cell repertoire in these patients (patients 4, 5, and 6). For
this reason, T cell analysis was limited to five CD8 epitopes,
known to be frequently recognized by HLA-A2 positive patients in
acute HCV infection corresponding to NS3 1073–1081, NS3
1406 –1415, NS4 1812–1821, NS4 1992–2000 and NS5
2627–2635. Sequencing of the HCV regions NS3 1073–1081 and
NS3 1406 –1415 was performed in patients 5 and 6 and the pep-
tides corresponding to the autologous sequences (Fig. 3) were syn-
thesized. With these peptides matching the corresponding se-
quence of the infecting virus, ELISPOT and ICS analyses were
performed in patients 5 and 6 as well as in patient 4. Moreover, a
panel of peptides containing the NS4 1812 (MFFNILGGWV),
NS4 1992 (VLSDFKSWL), and NS5 2594 (ALYDVIQKL) se-
quences of genotype 3 was tested in all three patients. Positive
responses were elicited only by the NS4 1992–2000 peptide (data
not shown) and results were confirmed by tetramer staining, show-
ing elevated frequencies of CD8 cells specific for this epitope (Fig.
1C); staining was negative for the other tetramers containing the
four additional peptides. Levels of CD8 frequencies were in the
range of the values previously reported in the acute phase of in-
fection (11–13, 16, 21, 27, 28), suggesting that these responses
should be immunodominant for the patients studied.
Longitudinal analysis of the CD8 response
All patients developed a persistent infection and the positive T cell
responses were followed sequentially over time. The frequency of
tetramer positive cells either ex vivo or upon in vitro peptide stim-
ulation was evaluated at different time points from the acute phase
of infection throughout a follow-up ranging from 12 to 34 wk (Fig.
2). In all patients, the frequency of tetramer positive cells ex vivo
declined rapidly and became either negative (⬍0.01% of the total
CD8
⫹
cells) or barely detectable (Fig. 2B). Viremia was partially
suppressed at the time of clinical presentation or shortly after in
patients 1, 2, 3, and 6. In patient 4 the decline of viremia was less
profound but still detectable, while in patient 5 viremia levels re-
mained constantly elevated throughout the follow-up (Fig. 2A). As
previously reported, HCV-specific cells can be functionally im-
paired during the acute phase of infection and can be unable to
expand efficiently upon peptide stimulation (11, 13–15). In keep-
ing with these observations, no expansion following 10 days of in
vitro peptide stimulation was observed by tetramer staining (tet-
ramer
⫹
cells ⬍0.01% of the total CD8 cells) in most patients at
the first time point tested corresponding to the peak of ALT (Fig.
2C). The capacity to proliferate recovered few weeks later but then
declined before or concurrently with a new elevation of viremia
levels (Fig. 2, Aand C). The stimulation with control peptides
corresponding to CMV, EBV, or influenza epitopes induced a
good expansion of T cells at all time points tested with no corre-
lation with the evolution of the disease (Fig. 2C), indicating that
the impairment of the T cell responses was HCV specific.
Sequencing of the relevant HCV epitopes
Viral escape by mutations at critical positions of immunodominant
epitopes is a potential mechanism of immune evasion. Therefore,
we next investigated whether viral sequence evolution and im-
mune evasion by the selection of escape mutations could be re-
sponsible for the decline of the CD8 response. The two regions of
the HCV genome encoding the CD8 epitopes targeted by the six
FIGURE 2. Longitudinal analysis of viremia and T cell responses from the acute phase of infection. Viremia levels were analyzed at the indicated time
points (A). The frequency of tetramer positive cells was evaluated either ex vivo (B) or upon in vitro 10 days peptide stimulation (C) at different time points
from the onset of the disease throughout a follow-up ranging from 12 to 34 wk in six HLA-A2
⫹
patients with acute HCV infection. The percentage of
tetramer
⫹
CD8 cells specific for HCV NS3 1073 (F), HCV NS4 1992 (f), and HCV-unrelated viruses, such as EBV, CMV, and influenza (white symbols),
is represented.
7522 CD8 FUNCTION AND EPITOPE MUTATIONS IN ACUTE HCV INFECTION
patients studied (NS3 1073–1081 and NS4 1992–2000) were am-
plified by PCR and multiple molecular clones were sequenced
(Fig. 3). Direct sequencing and clonal analysis were performed at
three sequential time points starting from the clinical onset.
Sequencing of the immunogenic peptides indicates that in the
acute stage of infection the epitopic regions carried by the infect-
ing virus generally matched the corresponding sequences more
frequently reported among the published HCV strains of identical
genotype. In four of the seven sequences tested, the autologous
virus corresponded exactly to the sequence of the peptides used
and no mutational event occurred over time. In particular, in pa-
tient 1 the dominant infecting sequence of the NS3 1073 epitope
was identical at all time points tested from the acute phase to 21
wk later. Similar results were obtained in patients 2, 4, and 5 by
sequencing the epitope NS4 1992–2000. Although a small per-
centage of clones in patients 4 and 5 showed a mutation in the
COOH-terminal flanking region (S to C), this event was only tran-
sient and no amino acid changes within the sequence of the im-
munodominant epitope (VLSDFKTWL) were detectable at any of
the time points tested.
In contrast, in patients 2 and 3, the NS3 1073–1081 sequence
CVNGVCWTV, which was homogeneously (patient 3) or predom-
inantly (patient 2) present at the time of the ALT peak, was com-
pletely replaced over time by the sequence CINGVCWTV. In pa-
tient 2, 4 wk after clinical onset an additional mutation leading to
an amino acid change (V to A) in position 5 of the epitope emerged
transiently (Fig. 3) and was no more detectable 18 wk later.
In patient 6, sequence analysis could not be performed at the
ALT peak, due to the lack of serum samples. For this reason, the
NS4 1992–2000 epitope was first sequenced 6 wk after clinical
onset. As shown in Fig. 3, at the first time point tested six different
viral species were simultaneously present which differed at resi-
dues 3, 6, and 7 of the epitope. This region continued to evolve
over time with a mix of three different populations present at week
10 and a single viral variant corresponding to the sequence VLS-
DFKRWL detectable six weeks later.
Effect of viral mutations on CD8 responses
Two different types of experimental approaches were used to as-
sess whether epitope recognition could be affected by the emer-
gence of mutations and to compare the relative immunogenicity of
the different viral sequences detected in individual patients. First,
freshly isolated T cells of the earliest time points and T cell lines
generated upon 10 days in vitro peptide stimulation were used to
compare how efficiently peptides undergoing selection and becom-
ing prevalent over time (variant peptides) were recognized by CD8
FIGURE 3. Longitudinal evolution of the HCV sequences corresponding to the identified CD8 epitopes. PCR products and molecular clones of the HCV
epitopes NS3 1073 and NS4 1992 were sequenced at three indicated time points from clinical onset. Fifteen to 25 molecular clones were sequenced for
each time point. Top rows, The sequences most frequently reported in gene bank among the isolates of the genotype infecting each patient. Bottom rows,
The sequences of the autologous virus. The sequences of the HCV regions that did not induce significant CD8 responses are illustrated within shaded areas.
7523The Journal of Immunology
cells compared with peptides present early in infection but unde-
tectable at later time points (prototype peptides). Second, the ca-
pacity of the different sequences detected in individual patients to
induce a CD8 response by 10 days peptide stimulation in vitro was
compared at different time points after clinical onset.
In patients 2 and 3, all detected sequences of the NS3 1073–
1081 region were first tested at different peptide concentrations by
ex vivo ELISPOT analysis with PBMC of the acute phase infec-
tion when the infecting HCV quasispecies was either exclusively
or predominantly CVNGVCWTV (in patients 2 and 3, respec-
tively). Although the sequence eventually selected (CINGVC
WTV) was recognized by CD8 cells only at very high peptide
concentrations (1–10
M), significant T cell responses were al-
ready induced by the CVNGVCWTV sequence at 1–10 nM con-
centration of the peptide (Fig. 4A). Identical results were obtained
when T cell lines produced by stimulation of PBMC derived from
the acute stage of infection with the CVNGVCWTV peptide were
tested with different concentrations of CVNGVCWTV or
CINGVCWTV peptides (Fig. 4A). In addition, the new variant
(CINGACWTV) emerging transiently in patient 2 and the
CINGVCWTV peptide that was finally selected in both patients
were also less efficient than the CVNGVCWTV sequence in CD8
expansion in vitro because at peptide concentrations ⬍1
M only
the CVNGVCWTV peptide was able to induce efficient CD8 ex-
pansion and IFN-
␥
production (Fig. 5). Efficiency in induction of
CD8 responses did not change throughout the follow-up (Fig. 5).
In patient 6, the amino acid sequence of the NS4 1992–2000
epitope was already heterogeneous at the time of the first sequenc-
ing analysis (Fig. 3). Three weeks from the onset, the strongest T
cell responses in ex vivo ELISPOT analysis using serial dilutions
of the different peptides were induced by the sequences VLSD
FKTWL, followed by VLSDFKSWL and VLTDFKTWL (Fig.
4A), which disappeared at the two subsequent time points tested
during the follow-up. In contrast to the high avidity recognition of
these sequences (in the nM range), the viral variant VLSDFRTWL
that was selected among the viral quasispecies and finally re-
mained the only detectable sequence, was able to induce poor lev-
els of T cell response ex vivo (Fig. 4A). Moreover, this sequence
was recognized by CD8 cells expanded in vitro upon stimulation
with the VLSDFKTWL sequence much less efficiently than the
stimulatory peptide and only at very high concentrations (Fig. 4A).
The hierarchy of T cell stimulatory capacity by the different se-
quences detected ex vivo was confirmed by in vitro experiments of
10 days peptide stimulation and remained the same throughout the
follow-up (Fig. 5).
FIGURE 4. Titration of CD8 responses with peptides corresponding to autologous HCV sequences. CD8-mediated responses to each of the identified
epitopes were tested ex vivo by ELISPOT analysis in all six patients with acute HCV infection. Efficiency of recognition of the different sequences identified
over time in patients 2, 3, and 6 was tested also in vitro by intracellular cytokine staining on short-term T cell lines obtained by in vitro 10 days peptide
stimulation (graphs in the middle). Both ex vivo and in vitro analyses were performed on PBMC of the earliest available time point corresponding to clinical
presentation. ELISPOT results are expressed as
␦
SFC per 10
6
PBMC detected upon overnight incubation with the indicated concentrations of peptides (A).
T cell lines were induced with different concentrations of CVNGVCWTV peptide in patients 2 and 3 and tested by intracellular cytokine staining with
CVNGVCWTV, CINGVCWTV, and CINGACWTV peptides. In patient 6, T cell lines were generated by stimulation with VLSDFKTWL peptide and
tested by intracellular cytokine staining with VLSDFKTWL, VLSDFKSWL, and VLSDFRTWL peptides. Results are expressed as percentage of IFN-
␥
-
positive cells among the total CD8 population. Responses to epitopes where mutations were detected are illustrated within the shaded area; the white area
identifies the responses to epitopes that did not change over time (B).
7524 CD8 FUNCTION AND EPITOPE MUTATIONS IN ACUTE HCV INFECTION
Functional characterization of HCV-specific T cell responses ex
vivo
Although elevated frequencies of HCV-specific CD8 cells were
detectable in all individuals during the acute phase of infection (as
shown by tetramer staining in Fig. 1), mutations emerged only in
three of the seven epitopes identified. To better understand the
reason for this limited occurrence of mutations despite the appar-
ent immunodominance of the epitopes analyzed, the functional
features of HCV-specific T cell responses associated with selection
of escape mutations were compared with those associated with no
evolution of the viral sequences.
Because high avidity CD8 cells have been shown to clear the
virus more efficiently than low avidity cells and should thus exert
a stronger immune pressure (29 –32), we first compared the degree
of avidity recognition of the epitopes acquiring mutations or re-
maining unchanged over time. Ex vivo analysis of the CD8 re-
sponse to the epitopes that did not change over time in patients 1,
2, 4, and 5 (Fig. 4B) showed that the amount of peptide ligand
required to exert effector function at clinical presentation was
much higher than that required to exert the same function by CD8
cells able to express selective pressure on the epitope in patients 2,
3, and 6 (Fig. 4A).
In keeping with the results obtained in patients 2 and 3, the
avidity of the NS3 1073 CINGVCWTV peptide recognition in
patient 1 was low because concentrations ⬎100 nM were needed
to induce IFN-
␥
production ex vivo. Remarkably, identical reac-
tivity was detected in patient 1 with the CVNGVCWTV peptide
that in contrast was able to induce T cell activation at much lower
concentrations (nM range) in patients 2 and 3. The epitope NS4
1992 VLSDFKTWL displayed a low functional avidity in patients
2, 4, and 5, although the same epitope was recognized with high
avidity by CD8 cells of patient 6 and showed a great degree of
variation in this patient. Moreover, low avidity T cell responses
were detected in patient 2 also with the peptide VLTDFKTWL,
which is generally carried by genotype 1b HCV.
Low avidity recognition of the peptide ligand by high frequency
tetramer positive CD8 cells suggests that lack of emergence of
escape mutations within immunodominant CD8 epitopes is the re-
sult of an impaired CD8 function. To further address this issue, we
then tested the degranulation efficiency of CD8 cells ex vivo in
response to peptide stimulation, as shown by CD107a up-regula-
tion. Despite the high frequency of tetramer positive CD8 cells
specific for HCV epitopes that did not undergo variations over
time, the level of degranulation ex vivo in patients 1, 4, and 5 was
very poor or totally undetectable upon CD8 stimulation with the
corresponding peptide (Fig. 6). In contrast, CD107a up-regulation
was clearly more efficient in patients 2, 3, and 6 in whom muta-
tional events were observed.
A functional impairment of CD8 cells specific for the epitopes
with no evidence of mutations was further confirmed at the time of
clinical onset by the discrepancy between frequency of HCV-spe-
cific CD8 cells detected by tetramer staining and frequency of CD8
cells of the same epitope specificity able to produce ex vivo IFN-
␥
detected by ELISPOT (Fig. 7). Thus, most CD8 cells specific for
epitopes without evidence of mutations were unable to produce
IFN-
␥
, whereas the majority of CD8 cells specific for epitopes that
underwent variations produced efficiently IFN-
␥
.
Finally, we asked whether CTL escape could be facilitated by an
impaired CD4 function in view of the evidence that the lack of
CD4 help in the setting of a monospecific CD8 response in chim-
panzees can favor escape (33). The helper T cell response was
studied by ex vivo ICS for IFN-
␥
after stimulation with recombi-
nant HCV Ags only in genotype 1 infected patients 1, 2, and 3
because genotype 1 HCV proteins were available. Two genotype 1
FIGURE 5. Efficiency of the different HCV sequences identified in each patient to induce a CD8 response in vitro at different time points following
clinical onset. Short-term T cell lines were induced by 10 days stimulation with different concentrations of each peptide identified in each patient and tested
by intracellular cytokine staining after4hofstimulation with 1
M of the original stimulatory peptide; results are expressed as percentage of IFN-
␥
-positive
cells among the total CD8 population.
7525The Journal of Immunology
infected patients with a self-limited evolution of infection were
also studied in the acute phase of illness for comparison. The re-
sults indicate that IFN-
␥
production by CD4 cells was less efficient
in patients with a chronic progression of disease compared with
those with self-limited evolution of infection (Fig. 8), showing that
escape in our patients occurred in the presence of an impaired
CD4 help.
Discussion
Recognition and elimination of intracellular virus by HCV-specific
CD8 cells is believed to be essential for successful recovery from
HCV infection (11–12, 21, 28, 34). Therefore, the emergence of
mutations within immunodominant CD8 epitopes able to abrogate
protective CD8 responses represents a possible mechanism of
FIGURE 6. Ex vivo expression of CD107a in HCV-specific CD8 cells at the time of clinical onset. PBMC from patients 1 to 6 were incubated for 2 h
with or without HCV NS3 1073 or NS4 1992 specific peptides (1
M). The dot plots represent CD107a expression in gated HCV tetramer
⫹
CD8 cells
cultured in medium alone or with the relevant peptide.
7526 CD8 FUNCTION AND EPITOPE MUTATIONS IN ACUTE HCV INFECTION
virus evasion from immune surveillance that can contribute to
HCV persistence.
To investigate the role of this mechanism in HCV pathobiology,
particular attention was paid in our study to analyze the global T
cell reactivity to identify the most dominant CD8 epitopes and to
follow their evolution over time, because immunodominant CD8
responses are more likely to exert a strong selective pressure on the
virus and to drive escape (32, 35–38). Moreover, the quality of the
different epitope-specific CD8 responses was carefully character-
ized because it is increasingly clear that not all Ag-specific CD8
⫹
cells are equally effective in viral clearance and that the avidity of
these cells is one of the variables that can greatly impact on their
antiviral efficacy (29 –32). When reagents of the appropriate ge-
notype were not available to perform a comprehensive analysis of
the overall CD8 repertoire, selected peptides and tetramers were
used to identify high frequency HCV-specific CD8 responses. The
epitopes selected by these two experimental approaches in six pa-
tients with acute hepatitis C and chronic evolution of infection
were all located within HCV regions that can tolerate mutations, as
shown by their variability among HCV genotypes reported in the
published HCV sequences. Moreover, T cell responses to NS3
1073 and NS4 1992 in patients 1 and 2 were not only strong but
also narrowly focused; these features should confer optimal ability
to CD8 cells to exert selective pressure, thereby enhancing the
likelihood that selection of escape mutations can occur within the
corresponding epitopes.
When the NS3 1073–1081 and NS4 1992–2000 regions were
sequenced longitudinally from the time of clinical onset, emer-
gence of mutations was observed in three of the six patients stud-
ied. Evidence for the selection of escape mutants was provided by
the emergence of HCV sequences less efficiently recognized by
CD8 cells not only ex vivo but also in vitro by CD8 lines generated
by 10 days stimulation of PBMC derived from the earliest avail-
able time points with the epitopic sequences detectable at those
times but totally lost at subsequent determinations. Moreover, the
newly generated sequences were poorly immunogenic in terms of
capacity to induce a CD8 response in vitro. To this inefficient
priming of a new response may contribute the original antigenic
sin as suggested by the stronger responses induced by the
CVNGVCWTV variant even when the corresponding sequence
was no more carried by the infecting virus.
Patient 3 was infected by genotype 1b which more frequently
carries a CVNGVCWTV sequence in the NS3 region 1073–1081
(GenBank data). Because the infecting virus was 100% CVNGVC
WTV at the first sequencing analysis, it is very likely that this
patient was infected by a HCV strain carrying this NS31073–1081
sequence. In view of the evidence that CD8 cells of patient 3
recognize much less efficiently the CINGVCWTV than the
CVNGVCWTV variant, the CINGVCWTV sequence is likely to
be an escape variant selected by the CD8 pressure. In patient 6, the
viral quasispecies evolved from the coexistence of six different
variants of the NS4 region 1992–2000 at the first available se-
quencing to the final selection of a single viral sequence (VLSD
FRTWL). This viral evolution is consistent with escape because
the variant eventually selected was the one less efficiently recog-
nized by CD8 cells.
In patient 2, the sequence of the transmitted virus was unde-
fined. Genotyping as well as sequencing of another viral region
(NS4 1992–2000) indicate that the patient was infected by geno-
type 1a which generally carries a CINGVCWTV NS3 1073 se-
quence (GenBank data). The first available sequencing was not
homogeneously represented by a single NS31073–1081 species,
because two different NS3 1073–1081 sequences (CINGVCWTV
and CVNGVCWTV) were present at the earliest sequenced time
point. A possible interpretation is that the infecting virus carried a
CVNGVCWTV sequence because this variant was recognized
more efficiently by CD8 cells (Fig. 4) and was more potent in CD8
stimulation in vitro (Fig. 5). It is likely that the selective pressure
exerted by CD8 cells contributed to the transition from a predom-
inant CVNGVCWTV to a homogeneous CINGVCWTV variant.
The possibility that also reversion to a more fit consensus sequence
favored by a fitness advantage of the CINGVCWTV variant was
operating in driving viral evolution to CINGVCWTV cannot be
excluded, assuming a survival advantage for the CINGVCWTV
variant in the context of a genotype 1a virus.
FIGURE 7. Ratio between ex vivo frequency of CD8 cells specific for
HCV NS3 1073 or NS4 1992 detected by tetramer staining and IFN-
␥
ELISPOT. Calculations were done in each patient using IFN-
␥
ELISPOT
data obtained with the peptides of optimal length (9 mers) for CD8 rec-
ognition. The ratio was calculated normalizing the number of IFN-
␥
⫹
and
Tet
⫹
cells to total PBMC. The best concordance between tetramer staining
and IFN-
␥
ELISPOT is expressed by ratios closer to 1.
FIGURE 8. Ex vivo IFN-
␥
production by CD4 cells upon PBMC stim-
ulation with recombinant HCV proteins during the acute phase of infection
in patients with different outcome of disease. PBMC from patients with
chronic (top) or self-limited (bottom) evolution of acute HCV infection
were stimulated overnight with six recombinant HCV proteins representing
the indicated structural and nonstructural HCV Ags and tested for IFN-
␥
production by ICS. Results are expressed as percentage of IFN-
␥
⫹
CD4
cells among the total CD4
⫹
population.
7527The Journal of Immunology
In four of the seven epitopes sequenced longitudinally in six
different patients no mutations were detected during the time of the
follow-up. Because only a limited number of preselected epitopes
were tested in patients 4 and 5, we cannot exclude that in these
patients the selected epitopes were subdominant and thus unable to
drive escape, although the elevated frequencies of HCV-specific
CD8 cells detected by tetramer staining were in line with the dom-
inant responses previously reported in acute hepatitis C (11–13,
27). In patients 1 and 2, the HCV-specific response was compre-
hensively screened with a panel of peptides covering the overall
HCV sequence. The response was narrowly focused on the NS3
1073 and NS4 1992 epitopes and sustained by high frequency CD8
cell populations (3–12% of the total circulating CD8 cells). De-
spite clear immunodominance, no mutations emerged during the
time of the follow-up within NS3 1073 in patient 1 and within
1992–2000 in patient 2. A possible interpretation is that these CD8
responses were functionally impaired. The possibility was sup-
ported by three lines of evidence. First, recognition of these
epitopic regions was always sustained by low avidity CD8 cells
requiring high peptide concentrations for their activation. Because
low avidity recognition of the peptide ligand is associated with
lower effector efficiency (29 –32), low avidity CD8 cells should be
poorly effective in exerting selective pressure on the virus. Second,
a much lower proportion of tetramer positive CD8 cells specific for
the epitopes that did not show sequence changes was able to pro-
duce IFN-
␥
compared with CD8 cells specific for HCV regions
undergoing variation of their sequences. Third, degranulation ca-
pacity, a function related to the cytolytic function of CD8 cells (39,
40), was defective in tetramer positive CD8 cells specific for
epitopes that did not acquire amino acid variations.
Although the study of a limited number of epitopes requires
caution in drawing conclusions, our data are consistent with the
concept that the emergence of escape mutations in the acute stage
of HCV infection reflects the residual functional efficiency of CD8
cells of different epitope specificity. Exhaustion as well as a direct
inhibitory effect of HCV on T cell function may lead to early
suppression of some dominant CD8 responses, which may be par-
ticularly profound if CD4 T cell help is defective (33, 41, 42).
Thus, depending on the efficiency of these early strategies of T cell
silencing, emergence of mutations may act as an additional mech-
anism of virus evasion from those dominant responses which are
functionally preserved. Our results are consistent with the view
that virus persistence is the result of a combined action of different
complementary mechanisms which may operate simultaneously or
sequentially in individual patients at the level of different dominant
epitopes to silence the protective CD8 response.
Acknowledgments
We thank A. Sette and J. Sidney for helpful discussions.
Disclosures
The authors have no financial conflict of interest.
References
1. Weiner, A., A. L. Erickson, J. Kansopon, K. Crawford, E. Muchmore,
A. L. Hughes, M. Houghton, and C. M. Walker. 1995. Persistent hepatitis C virus
infection in a chimpanzee is associated with emergence of a cytotoxic T lym-
phocyte escape variant. Proc. Natl. Acad. Sci. USA 92: 2755–2759.
2. Erickson, A. L., Y. Kimura, S. Igarashi, J. Eichelberger, M. Houghton, J. Sidney,
D. McKinney, A. Sette, A. L. Hughes, and C. M. Walker. 2001. The outcome of
hepatitis C virus infection is predicted by escape mutations in epitopes targeted
by cytotoxic T lymphocytes. Immunity 15: 883– 895.
3. Tsai, S. L., Y. M. Chen, M. H. Chen, C. Y. Huang, I. S. Sheen, C. T. Yeh,
J. H. Huang, G. C. Kuo, and Y. F. Liaw. 1998. Hepatitis C virus variants cir-
cumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gas-
troenterology 115: 954 –965.
4. Chang, K. M., B. Rehermann, J. G. McHutchison, C. Pasquinelli, S. Southwood,
A. Sette, and F. V. Chisari. 1997. Immunological significance of cytotoxic T
lymphocyte epitope variants in patients chronically infected by the hepatitis C
virus. J. Clin. Invest. 100: 2376 –2385.
5. Timm, J., G. M. Lauer, D. G. Kavanagh, I. Sheridan, A. Y. Kim, M. Lucas,
T. Pillay, K. Ouchi, L. L. Reyor, J. S. Zur Wiesch, et al. 2004. CD8 epitope
escape and reversion in acute HCV infection. J. Exp. Med. 200: 1593–1604.
6. Kantzanou, M., M. Lucas, E. Barnes, H. Komatsu, G. Dusheiko, S. Ward,
G. Harcourt, and P. Klenerman. 2003. Viral escape and T cell exhaustion in
hepatitis C virus infection analysed using class I peptide tetramers. Immunol. Lett.
85: 165–171.
7. Bowen, D. G., and C. M. Walker. 2005. The origin of quasispecies: cause or
consequence of chronic hepatitis C viral infection? J. Hepatol. 42: 408 – 417.
8. Rehermann, B., and M. Nascimbeni. 2005. Immunology of hepatitis B virus and
hepatitis C virus infection. Nat. Rev. Immunol. 5: 215–229.
9. Franco, A., C. Ferrari, A. Sette, and F. V Chisari. 1995. Viral mutations, TCR
antagonism and escape from the immune response. Curr. Opin. Immunol. 7:
524 –531
10. Penin, F., J. Dubuisson, F. A. Rey, D. Moradpour, and J. M. Pawlotsky. 2004.
Structural biology of hepatitis C virus. Hepatology 39: 5–19.
11. Urbani, S., C. Boni, G. Missale, G. F. Elia, C. Cavallo, M. Massari, G. Raimondo,
and C. Ferrari. 2002. Virus-specific CD8
⫹
lymphocytes share the same effector-
memory phenotype but exhibit functional differences in acute hepatitis B and C.
J. Virol. 76: 12423–12434.
12. Thimme, R., D. Oldach, K. M. Chang, C. Steiger, S. C. Ray, and F. V. Chisari.
2001. Determinants of viral clearance and persistence during acute hepatitis C
virus infection. J. Exp. Med. 194: 1395–1406.
13. Lechner, F., D. K. Wong, P. R. Dunbar, R. Chapman, R. T. Chung,
P. Dohrenwend, G. Robbins, R. Phillips, P. Klenerman, and B. D. Walker. 2000.
Analysis of successful immune responses in persons infected with hepatitis C
virus. J. Exp. Med. 191: 1499 –1512.
14. Gruener, N. H., F. Lechner, M. C. Jung, H. Diepolder, T. Gerlach, G. Lauer,
B. Walker, J. Sullivan, R. Phillips, G. R. Pape, and P. Klenerman. 2001. Sus-
tained dysfunction of antiviral CD8
⫹
T lymphocytes after infection with hepatitis
C virus. J. Virol. 75: 5550 –5558.
15. Accapezzato, D., V. Francavilla, P. Rawson, A. Cerino, A. Cividini,
M. U. Mondelli, and V. Barnaba. 2004. Subversion of effector CD8
⫹
T cell
differentiation in acute hepatitis C virus infection: the role of the virus. Eur.
J. Immunol. 34: 438 – 446.
16. Francavilla, V., D. Accapezzato, M. De Salvo, P. Rawson, O. Cosimi, M. Lipp,
A. Cerino, A. Cividini, M. U. Mondelli, and V. Barnaba. 2004. Subversion of
effector CD8
⫹
T cell differentiation in acute hepatitis C virus infection: exploring
the immunological mechanisms. Eur. J. Immunol. 34: 427– 437.
17. Gandhi, R. T., and B. D. Walker. 2002. Immunologic control of HIV-1. Annu.
Rev. Med. 53: 149 –172.
18. Krmpotic, A., M. Messerle, I. Crnkovic-Mertens, B. Polic, S. Jonjic and
U. H. Koszinowski. 1999. The immunoevasive function encoded by the mouse
cytomegalovirus gene m152 protects the virus against T cell control in vivo. J. Exp.
Med. 190: 1285–2196.
19. Seifert, U., H. Liermann, V. Racanelli, A. Halenius, M. Wiese, H. Wedemeyer,
T. Ruppert, K. Rispeter, P. Henklein, A. Sijts, et al. 2004. Hepatitis C virus
mutation affects proteasomal epitope processing. J. Clin. Invest. 114: 250 –259.
20. Meyer-Olson, D., N. H. Shoukry, K. W. Brady, H. Kim, D. P. Olson, K. Hartman,
A. K. Shintani, C. M. Walker, and S. A. Kalams. 2004. Limited T cell receptor
diversity of HCV-specific T cell responses is associated with CTL escape. J. Exp.
Med. 200: 307–320.
21. Lauer, G. M., E. Barnes, M. Lucas, J. Timm, K. Ouchi, A. Y. Kim, C. L. Day,
G. K. Robbins, D. R. Casson, M. Reiser, et al. 2004. High resolution analysis of
cellular immune responses in resolved and persistent hepatitis C virus infection.
Gastroenterology 127: 924 –936.
22. Tester, I., S. Smyk-Pearson, P. Wang, A. Wertheimer, E. Yao, D. M. Lewinsohn,
J. E. Tavis, and H. R. Rosen. 2005. Immune evasion versus recovery after acute
hepatitis C virus infection from a shared source. J. Exp. Med. 201: 1725–1731.
23. Ray, S. C., L. Fanning, X. H. Wang, D. M. Netski, E. Kenny-Walsh, and
D. L. Thomas. 2005. Divergent and convergent evolution after a common-source
outbreak of hepatitis C virus. J. Exp. Med. 201: 1753–1759.
24. Cox, A. L., T. Mosbruger, Q. Mao, Z. Liu, X. H. Wang, H. C. Yang, J. Sidney,
A. Sette, D. Pardoll, D. L. Thomas, and S. C. Ray. 2005. Cellular immune se-
lection with hepatitis C virus persistence in humans. J. Exp. Med. 201:
1741–1752.
25. Timm, J., G. M. Lauer, D. G. Kavanagh, I. Sheridan, A. Y. Kim, M. Lucas,
T. Pillay, K. Ouchi, L. L. Reyor, J. S. Zur Wiesch, et al. 2004. CD8 epitope
escape and reversion in acute HCV infection. J. Exp. Med. 200: 1593–1604.
26. Bowen, D. G., and C. M. Walker. 2005. Mutational escape from CD8
⫹
T cell
immunity: HCV evolution, from chimpanzees to man. J. Exp. Med. 201:
1709 –14.
27. Lechner, F., N. H. Gruener, S. Urbani, J. Uggeri, T. Santantonio, A. R. Kammer,
A. Cerny, R. Phillips, C. Ferrari, G. R. Pape, and P. Klenerman. 2000. CD8
⫹
T
lymphocyte responses are induced during acute hepatitis C virus infection but are
not sustained. Eur. J. Immunol. 30: 2479 –2487.
28. Rahman, F., T. Heller, Y. Sobao, E. Mizukoshi, M. Nascimbeni, H. Alter,
S. Herrine, J. Hoofnagle, T. J. Liang, and B. Rehermann. 2004. Effects of antiviral
therapy on the cellular immune response in acute hepatitis C. Hepatology 40:
87–9.
29. Alexander-Miller, M. A. 2005. High-avidity CD8
⫹
T cells: optimal soldiers in
the war against viruses and tumors. Immunol. Res. 31: 13–24.
7528 CD8 FUNCTION AND EPITOPE MUTATIONS IN ACUTE HCV INFECTION
30. Draenert, R., C. L. Verrill, Y. Tang, T. M. Allen, A. G. Wurcel, M. Boczanowski,
A. Lechner, A. Y. Kim, T. Suscovich, N. V. Brown, et al. 2004. Persistent rec-
ognition of autologous virus by high-avidity CD8 T cells in chronic, progressive
human immunodeficiency virus type 1 infection. J. Virol. 78: 630 – 641.
31. Derby, M., M. Alexander-Miller, R. Tse, and J. Berzofsky. 2002. High-avidity
CTL exploit two complementary mechanisms to provide better protection against
viral infection than low-avidity CTL. J. Immunol. 166: 1690 –1697.
32. O’Connor, D. H., T. M. Allen, T. U. Vogel, P. Jing, I. P. DeSouza, E. Dodds,
E. J. Dunphy, C. Melsaether, B. Mothe, H. Yamamoto, et al. 2002. Acute phase
cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus
infection. Nat. Med. 8: 493– 499.
33. Grakoui, A., N. H. Shoukry, D. J. Woollard, J. H. Han, H. L. Hanson, J. Ghrayeb,
K. K. Murthy, C. M. Rice, and C. M. Walker. 2003. HCV persistence and im-
mune evasion in the absence of memory T cell help. Science 302: 659 – 662.
34. Bertoletti, A., and C. Ferrari. 2003. Kinetics of the immune response during HBV
and HCV infection. Hepatology 38: 4 –13.
35. Koibuchi, T., T. M. Allen, M. Lichterfeld, S. K. Mui, K. M. O’Sullivan,
A. Trocha, S. A. Kalams, R. P. Johnson, and B. D. Walker. 2005. Limited se-
quence evolution within persistently targeted CD8 epitopes in chronic human
immunodeficiency virus type 1 infection. J. Virol. 79: 8171– 8181.
36. Jones, N. A., X. Wei, D. R. Flower, M. Wong, F. Michor, M. S. Saag, B. H. Hahn,
M. A. Nowak, G. M. Shaw, and P. Borrow. 2004. Determinants of human im-
munodeficiency virus type 1 escape from the primary CD8
⫹
cytotoxic T lym-
phocyte response. J. Exp. Med. 200: 1243–1256.
37. Borrow, P., H. Lewicki, X. Wei, M. S. Horwitz, N. Peffer, H. Meyers,
J. A. Nelson, J. E. Gairin, B. H. Hahn, M. B. Oldstone, and G. M. Shaw. 1997.
Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs)
during primary infection demonstrated by rapid selection of CTL escape virus.
Nat. Med. 2: 205–211.
38. Yang, O. O., P. T. Sarkis, A. Ali, J. D. Harlow, C. Brander, S. A. Kalams, and
B. D. Walker. Determinant of HIV-1 mutational escape from cytotoxic T lym-
phocytes. J. Exp. Med. 197: 1365–1375.
39. Betts, M. R., J. M. Brenchley, D. A. Price, S. C. De Rosa, D. C. Douek,
M. Roederer, and R. A. Koup. 2003. Sensitive and viable identification of anti-
gen-specific CD8
⫹
T cells by a flow cytometric assay for degranulation. J. Im-
munol. Methods 281: 65–78.
40. Wolint, P., M. R. Betts, R. A. Koup, and A. Oxenius. 2004. Immediate cytotox-
icity but not degranulation distinguishes effector and memory subsets of CD8
⫹
T
cells. J. Exp. Med. 199: 925–936.
41. Gerlach, J. T., H. M. Diepolder, M. C. Jung, N. H. Gruener, W. W. Schraut,
R. Zachoval, R. Hoffmann, C. A. Schirren, T. Santantonio, and G. R. Pape. 1999.
Recurrence of hepatitis C virus after loss of virus-specific CD4
⫹
T cell response
in acute hepatitis C. Gastroenterology 117: 933–941.
42. Ulsenheimer, A., J. T. Gerlach, N. H. Gruener, M. C. Jung, C. A. Schirren,
W. Schraut, R. Zachoval, G. R. Pape, and H. M. Diepolder. 2003. Detection of
functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic
hepatitis C. Hepatology 37: 1189 –1198.
7529The Journal of Immunology
