Synthetic antigens representing the antigenic variation of human hepatitis C virus.

Abstract

Immune responses against hepatitis C virus (HCV) have been studied by numerous groups. However, details concerning the production of antibodies to antigenically variable epitopes remain to be elucidated. Since the sequences of the variable regions of several HCV proteins are different among the virus strains infecting patients, we decided to design peptide combinations that represent the theoretical maximum antigenic variation of each epitope to be used as capture antigens. We prepared six peptide mixtures (hypervariable epitope constructs; HECs) representing six different epitopes from structural and non-structural proteins of HCV from genotypes 1-6. Plasma from 300 HCV patients was tested to determine if their antibodies recognize the synthetic constructs. All the patients were chronically infected with diverse HCV genotypes and did not receive antiviral treatment. Antibodies to one or more of the HECs were detected in all of the HCV-infected individuals. Immunogenicity of the HCV HECs was also evaluated in outbred and inbred mice. Strong HEC-specific antibodies were produced, and cellular responses were also induced that were Th-1 rather than Th-2. Our results show that HCV HECs are both antigens that can be used to detect the broad cross-reactivity of antibodies from HCV-infected patients, and strong immunogens that can induce antigen-specific humoral and cellular immune responses in mice.

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Synthetic Antigens Representing the Antigenic
Variation of Human Hepatitis C Virus
Kyung Hee Kang,1Yasuhiro Yamamura,4Maria P. Carlos,1Nicolas Karvelas,1In-Sup Kim,1
Deepa Sunkara,1Rebecca Rivera,1Murray B. Gardner,5David E. Anderson,2
Francisco Diaz-Mitoma,3Jose ´ V. Torres,1and Juan P. Marquez1,6
Abstract
Immune responses against hepatitis C virus (HCV) have been studied by numerous groups. However, details
concerning the production of antibodies to antigenically variable epitopes remain to be elucidated. Since the
sequences of the variable regions of several HCV proteins are different among the virus strains infecting pa-
tients, we decided to design peptide combinations that represent the theoretical maximum antigenic variation of
each epitope to be used as capture antigens. We prepared six peptide mixtures (hypervariable epitope con-
structs; HECs) representing six different epitopes from structural and non-structural proteins of HCV from
genotypes 1–6. Plasma from 300 HCV patients was tested to determine if their antibodies recognize the syn-
thetic constructs. All the patients were chronically infected with diverse HCV genotypes and did not receive
antiviral treatment. Antibodies to one or more of the HECs were detected in all of the HCV-infected individuals.
Immunogenicity of the HCV HECs was also evaluated in outbred and inbred mice. Strong HEC-specific
antibodies were produced, and cellular responses were also induced that were Th-1 rather than Th-2. Our results
show that HCV HECs are both antigens that can be used to detect the broad cross-reactivity of antibodies from
HCV-infected patients, and strong immunogens that can induce antigen-specific humoral and cellular immune
responses in mice.
Introduction
H
themajority ofthecases,theimmuneresponsegeneratedisnot
able to eliminate the infection, and a portion of these patients
eventually develop cirrhosis and hepatocellular carcinoma.
HCV was classified as non-A, non-B hepatitis (NANBH)
until it was identified in 1989 by isolating its RNA genomic
sequence from experimental chimpanzee plasma using ran-
dom primers (11). Since identified as the causative agent,
HCV is now recognized as one of the most serious public
health problems, infecting an estimated 3% of the world’s
population (about 170 million people worldwide). HCV is a
major cause of chronic liver infection that can lead to cir-
rhosis and hepatocellular carcinoma (HCC) (32). HCV is an
enveloped, single-stranded positive-sense RNA virus that
uman hepatitis C virus causes chronic infection in
approximately 70% of patients exposed to the virus. In
belongs to the Flaviviridae family. HCV encodes a single
open reading frame (ORF) of about 9600bp nucleotides in
length, flanked by a 50and a 30untranslated region (UTR).
The ORF encodes a polyprotein precursor that is processed
post-translationally by cellular and viral proteases to pro-
duce structural and nonstructural proteins, respectively. The
structural proteins consist of core, two envelope proteins
called E1 and E2, and the non-structural proteins NS2, NS3,
NS4A, NS4B, NS5A, and NS5B (4).
Based on sequence variation, HCV has been classified into
six major genotypes that differ by approximately 30% from
one another (24,26,32). Within each genotype of HCV, there
are several subtypes with nucleotide differences of approxi-
mately 20–25% (24,32). Multiple viral variants present in the
blood of a given individual (quasispecies) can differ by as
much as 10% (24). Epitope variability has been observed in
the structural E1 and E2 envelope glycoproteins, as well as in
1Department of Medical Microbiology and Immunology, and5Department of Medical Pathology and Laboratory Medicine and Center of
Comparative Medicine, School of Medicine, University of California–Davis, Davis, California.
2Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts.
3Division of Virology, Children’s Hospital of Eastern Ontario and University of Ottawa, Ottawa, Ontario, Canada.
4AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico.
6Sonora Cancer Research Center, Sonora, Mexico.
VIRAL IMMUNOLOGY
Volume 23, Number 5, 2010
ª Mary Ann Liebert, Inc.
Pp. 497–508
DOI: 10.1089/vim.2010.0043
497

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the non-structural NS3, NS4, and NS5 proteins (4,10,36–38).
Hypervariability exists mostly in the amino-terminal portion
of the E2 protein, in a region named the first hypervariable
region (HVR1) (17,26,34). HVR1 is the major neutralizing
epitope of HCV, and consists of 27 amino acids (26). A second
hypervariable region (HVR2), in the carboxyl-terminal region
of the E2 glycoprotein, consists of nine amino acids (34).
Hypervariable epitope constructs (HEC) are synthetic
peptide mixtures that contain multiple variants of a given
epitope based on hypervariable regions of viruses that
mutate their genomic sequences frequently to evade im-
mune responses. The method for designing the HEC based
on theseregionshas been
(2,3,8,9,22,23). Our previous work demonstrated that HECs
based on hypervariable regions of simian immunodefi-
ciency virus (SIV) or human immunodeficiency virus (HIV)
induce broadly reactive humoral as well as T-helper cell
responses in rodents and non-human primates (2,3,8,22,23).
Here, we apply the same principle to develop immunogens
that could be part of a diagnostic test or vaccine candidate
against HCV.
HCV HECs are composed of six antigenic variable epi-
topes representing the six major genotypes and their sub-
types circulating in the HCV-infected population. Over 300
HCV protein sequences were obtained from the Genbank
database. Design of the HEC was based on analysis of amino
acids present among antigenically variable sequences of
HCV from subtypes 1a, 1b, 2a, 2b, 3, and 4-6, with the
number of sequences from each HCV subtype representing
approximately 30%, 25%, 15%, 15%, 10%, and 5% of the total
number of sequences, respectively. Two epitopes are from
the E2 envelope glycoprotein (HVR1 and HVR2 HEC), two
epitopes are from the NS3 protein (NS3-1 and NS3-2 HEC),
and two epitopes are from the NS4 protein (NS4-1 and NS4-2
HEC). HCV HECs were used as capture antigens to detect
antibodies in patients with HCV chronic infection, and as
immunogens for examination of induction of specific im-
mune responses in mice.
Single-sequence peptides (termed analogs) derived from
the HVR1 and HVR2 regions of the E2 envelope glycoprotein
were prepared to determine if antibodies from mice immu-
describedelsewhere
nized with HCV HEC could also recognize each region. Each
HVR1 and HVR2 analog represents a specific epitope from a
single viral isolate of each genotype. HVR1 and HVR2 ana-
logs contain sequences from genotypes 1 to 6 (1a, 1b, 2a, 2b,
2e/f, 3a, 4a, 5a, and 6a).
Numerous groups have been studying the immune re-
sponses against HCV, but the production and breadth of
reactivity of antibodies to antigenically variable epitopes
representing diverse genotypes and subtypes remain to be
elucidated. In the present study, we assessed if HECs are
recognized by antibodies from individuals infected with di-
verse genotypes and subtypes of HCV (1a, 1b, 1a/b, 2a, 2b,
2a/c, and 3a). Plasma from two different groups of patients
with chronic HCV infection was tested as follows: (1) group
1 consisted of 228 HCV patient samples that were tested for
antibody binding to HVR1 and HVR2 HECs, and (2) group 2
consisted of 49 HCV patient samples tested for antibody
binding to the six HCV HECs. We also tested the antigenicity
of HCV HEC by assessing induction of humoral and cellular
immune responses in mice. Different combinations of HCV
HECs were used to immunize mice, and HEC-specific anti-
body responses, lymphocyte proliferation, and cytokine
production were determined.
Materials and Methods
Plasma samples
Archived patient plasma samples were obtained from the
AIDS Research Program at the Ponce School of Medicine in
Puerto Rico and confirmed positive for HCV by RT-PCR.
HCV genotypes were determined by line probe assay (Inno-
LiPA HCV II; Innogenetics, Ghent, Belgium). All the plasma
samples were from patients chronically infected with dif-
ferent genotypes of HCV (1a, 1b, 1a/b, 2a, 2b, 2a/c, and 3a),
and had never received HCV antiviral treatment (i.e., IFN-a
and ribavirin). All the plasma samples were first screened by
the Abbott HCV EIA 2.0 test (Abbott Laboratories, Abbott
Park, IL), that utilizes recombinant antigens c100-3, HC-31,
and HC-34. HCV viral loads were determined by the Am-
plicor HCV Monitor procedure (Roche Diagnostics Corp.,
Indianapolis, IN).
Table 1. Sequence of the HECs Synthesized to Represent Antigenic Variable Epitopes of HCV Proteins
HCV HECPosition (aa) Sequencea
Variants
HVR1 383–406
ATTYVTGGAAARATAGLASLFSPG
---H------------F-N-L-L-
ISYANGSGPDQRPY
--H-D-G---H---
LEVIKGGRHLIFCHSKKKC
IAT-R--------------
AWYELTPAETTVRLRAYLNTPGLPVC
----M-----S------M--------
IILSGKPAVVPDREVLYQEFDEMEEC
-H---R------K----RA-------
CASHIPYIEQGMQLAEQFKQKAL
------L--E-QR----L-----
32
HVR2 472–48516
NS3-11382–1400 16
NS3-21526–15518
NS4-11686–171132
NS4-21711–173332
aA bar in the HEC sequence indicates an identical amino acid.
Positions where two amino acids were added are shown, as well as the individual sequence variants expected in each of the final
constructs.
Abbreviations: HEC, hypervariable epitope construct; HCV, hepatitis C virus; HVR, hypervariable region.
498KANG ET AL.

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HEC synthesis
Peptides representing six antigenic variable epitopes of six
HCV genotypes and their subtypes (1a, 1b, 2a, 2b, 3, and 4-6),
including the hypervariable regions of E2, NS3, and NS4,
were synthesized. The sequences of HCV HECs are shown in
Table 1. The procedure of HEC synthesis has been previ-
ously described (8–9). In brief, all HCV HECs were synthe-
sized by 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry, a
solid-phase peptide synthesis method utilizing high-capacity
(0.7mmol/g) Knorr resin (Advanced ChemTech, Louisville,
KY) (8). Synthesis was performed using an automatic peptide
synthesizer (PS-3; Rainin Protein Technologies, Woburn, MA).
The appropriate molar amounts of amino acids were added
based on frequencies calculated for a given position. The final
peptides were cleaved and deprotected by addition of tri-
fluoroacetic acid with scavengers. The peptide mixture was
suspended, lyophilized, and dialyzed in distilled water at
least three times to remove acid residues from cleaved pep-
tide. Amino acid analysis was performed on all HECs to en-
sure that they had the appropriate amino acid content.
HVR1 and HVR2 HEC analog peptide synthesis
Eleven single-sequence peptides derived from both the
HVR1 and HVR2 regions of the E2 protein from a single
genotype of each viral isolate were synthesized and used as
capture antigen on ELISA plates. We designated these single-
sequence peptides as HVR1 analog and HVR2 analog to
distinguish these from the HECs, and the sequences are
shown in Table 2. Each peptide represents epitopes from
different genotypes and subtypes of HCV (1a, 1b, 2a, 2b, 2e/
f, 3a, 4a, 5a, and 6a). Two peptides from the same genotype
(i.e., 1a and 1b) came from different viral isolates with dif-
ferent amino acid sequences. The synthesis procedure was
the same as that described above.
HCV viral purification from patient plasma
HCV viral particles were purified using an HCV purifi-
cation kit (BioVintage, San Diego, CA), according to the
manufacturer’s instructions. Briefly, HCV viral particles
were released from HCV-infected patient plasma by three
freeze/thaw cycles. Supernatants were applied to mini-spin
filter columns and centrifuged at 2000g for 5min. The col-
umns were washed with wash buffer, followed by collec-
tion of HCV viral particles from the columns with elution
buffer. Viral protein concentration was measured by Brad-
ford assay.
Enzyme-linked immunosorbent assay (ELISA)
Binding of antibodies from HCV-infected patients to HCV
HEC.
ELISA was performed to quantitate antibodies pres-
ent in patient plasma that bound to HCV HEC, as previously
described (9). Briefly, all the HCV patient plasma samples
were heat-inactivated at 568C for 30min before being used in
assays. Flat-bottomed microtiter plates (Corning Costar
9018) were coated with HEC individually and held at 378C
overnight at a concentration of 5mg/well in triplicate. Non-
fat milk (10%) was added and the plates were kept for 2h at
378C to block non-specific binding, then the plates were
washed with buffer before HCV patient plasma samples
were added and incubated for 2h at 378C. Plasma from HCV-
infected patients and three control human plasma samples
negative for HBsAg, HCV, HIV-1, and HTLV-1 were tested
Table 2. HCV Epitope Analogs Used to Evaluate the Breadth of Humoral Immunity to the E2
Hypervariable Regions (HVR) in Vaccinated Animals
IsolateCountryGenbank accession no.AA positionPeptide sequence
HCV HVR1 analogs aa 383–406
1aJapan
1aUSA
1bGermany
1bUnknown
2aUSA
2bJapan
2e/2fJapan
3aAustralia
4aUK
5a USA
6aUK
HCV HVR2 analogs aa 472–485
1aJapan
1aUSA
1b Germany
1bUnknown
2a USA
2b Japan
2e/2fJapan
3a Australia
4aUK
5a USA
6a UK
BAA00705
AAB67038
CAB53095
AAD44718
AAF01178
AAF59945
BAA09891
AAC03058
CAA72338
AAC61696
CAA72801
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
aa 383–406
AETIVSGGQAARAMSGLVSLFTPG
AETHVTGGNAGRTTAGLVGLLTPG
GGTYVTGGTMAKNTLGITSLFSPG
GHTLTTGGHAARLTSGFAGLFTPG
ARTHTVGGSAAQTTGRLTSLFDMG
ATTYSTGAQVGQTVSGFAGMFRSG
ASTYVASSVSQATSGLVSLFSAGA
AVTYTTGGSAAHATRGLTSLFSVG
EAQTMIAHGVSQTTSGFASLLTPG
ANTRTVGGSAAQGARGLASLFTPG
EAQTMIAHGVSQTTSGFASLLTPG
BAA00705
AAB67038
CAB53095
AAD44718
AAF01178
AAF59945
BAA09891
AAC03058
CAA72338
AAC61696
CAA72801
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
aa 472–485
ISHANGSGPDQRPY
ISYANGSGLDERPY
ITYNESHSSDQRPY
ITYAEPTKDPDQRP
LQYEDNVTNPEDMR
LEYETNVTNDEDMR
ITYANISGPAVEKP
LTDANISGPSDDKP
LGVANISGSSDDRP
ISYAAVSGPSDDKP
ITHKNVSGPSDDRP
Abbreviations: HEC, hypervariable epitope construct; HCV, hepatitis C virus.
ANTIGENS REPRESENTING THE ANTIGENIC VARIATION OF HCV 499

Page 4

at 1:100, 1:500, 1:1000, 1:5000, and 1:10,000 dilutions. Two of
the control plasma samples were obtained from the Sacra-
mento Blood Bank, and the third control was commercially-
available normal human plasma (Biocell Laboratories, Inc.,
Rancho Dominguez, CA). Following incubation with pri-
mary antibody, the plates were washed and alkaline-
phosphatase-labeled secondary antibody (goat anti-human
IgG AP; Southern Biotechnology Associates, Birmingham,
AL) was added and incubated for 1h at 378C. After the
plates were washed, p-nitrophenyl phosphate substrate
(KPL) was added and incubated for 2h at room temperature.
Optical density (OD) was measured spectrophotometrically
at 405nm with an automatic plate reader (VERSAmax; Mo-
lecular Devices, Sunnyvale, CA) according to the manufac-
turer’s instructions (SOFTmax PRO; Molecular Devices).
End-point antibody titer was defined as the greatest dilution
of sample that maintained an OD at least twice that of
the average of control plasma tested at the same dilution.
The mean standard deviation of the OD readings was
calculated.
Immunogenicity of HCV HEC in mice.
sera was performed similarly to that described above. HCV
HEC was coated individually on the plate at a concentration
of 5mg/well in triplicate. Mice sera were added as primary
antibody, followed by alkaline-phosphatase-labeled second-
ary antibody (goat anti-mouse IgG AP, Southern Bio-
technology Associates). The rest of the procedure was
performed as described above.
ELISA for mouse
Antibody response of mice immunized with HCV HEC
to HCV viral particles purified from HCV-infected patient
plasma.
ELISA plates were coated with 10mg/well of HCV
viral particles purified from plasma of HCV-infected patients.
The rest of the procedure was performed as described above.
Mouse immunization
BALB/c (4 mice per group) and CFW (6 mice per group)
mice were immunized with a single HCV HEC and in
combination. For each immunization, 100mg of the peptides
in 50mL of sterile PBS was mixed at a 1:1 ratio with the
adjuvant Montanide ISA-51 in a final volume of 100mL.
Immunizations were administered a total of four times at
monthly intervals. All immunizations were delivered sub-
cutaneously at the base of the tail.
Collection of spleens and lymph nodes from mice
Mice were euthanized 7 days after the final immuniza-
tion. Spleen and lymph nodes (inguinal, axillary, and mes-
entery lymph nodes) were collected and pushed through a
70-mm cell strainer (BD Falcon; BD Biosciences Pharmingen,
Franklin Lakes, NJ) using sterilized phosphate Dulbecco’s
saline solution with 2% bovine calf serum (HyClone;
Thermo Fischer Scientific, Rochester, NY), and lymphocytes
were isolated and separated from red blood cells on Ficoll-
Hypaque (ICN Biomedicals Inc., Costa Mesa, CA). Cells
were counted by the trypan blue exclusion method and re-
suspended in RPMI 1640 (HyClone) supplemented with 10%
bovine calf serum and 1% antibiotic-antimycotic (Invitrogen
Corp., Carlsbad, CA).
Lymphoproliferation assay
Determination of antigen-specific lymphocyte prolifera-
tion was performed by the thymidine incorporation method.
Lymphocytes from spleens and lymph nodes (2?105cells/
well) were seeded on round-bottom 96-well tissue culture
plates in triplicate in the presence of HCV HEC individually
at a concentration of 5mg/mL. As negative control, an HEC
representing an epitope of the envelope glycoprotein of SIV
and antigenically unrelated to HCV was used at the same
concentration. The cells were incubated with the HCV HEC
at 378C in 5% CO2 for 3d. Then 1mCi of [3H]thymidine
(Amersham Biosciences, Piscataway, NJ) was then added to
each well in 50mL of RPMI 1640 supplemented with 10%
bovine calf serum and 1% antibiotic-antimycotic. Following a
12- to 14-h incubation period, the plates were harvested us-
ing a PHD cell harvester, and the amount of incorporated
tritiated thymidine was measured with a Beckman LS 6000IC
scintillation counter. The results are expressed as counts per
minute (cpm).
Cytokine ELISA
Secretion of IFN-g, IL-2, and IL-4 by HEC-specific lym-
phocytes was determined by ELISA. The lymphocytes were
seeded the same way as for the lymphoproliferation as-
say method described above, except for the number of
cells (5?105cells/well). The cells were incubated at 378C in
5% CO2for 2d. Supernatants were harvested after 48h of
stimulation with a 5-mg/mL concentration of individual
HEC. Antibody pairs and standards were purchased from
eBioscience (San Diego, CA). Cytokine capturing antibody-
coated ELISA plates were blocked with 5% bovine calf serum
(BCS)/PBS before supernatants and standard dilutions were
added at 100mL per well. Following washing six times with
PBS in 0.05% Tween-20, biotinylated detecting antibodies
were added at 100mL per well in 1% BCS/PBS. After the
plates were washed, streptadivin-conjugated horseradish
peroxidase (Zymed Laboratories, Carlsbad, CA) was added
at 100mL per well and incubated for 30min. Following a final
wash, TMB Microwell peroxidase substrate (1-component;
KPL Inc., Gaithersburg, MD) was added. Stop solution (KPL)
was added after 30min of incubation, and the plates were
read at 450nm with an automatic plate reader (VERSAmax;
Molecular Devices), according to the manufacturer’s in-
structions.
Statistical analysis
The difference between the groups was calculated by us-
ing ANOVA for normally distributed variables (GraphPad
InStat version 3.0; GraphPad Software, San Diego, CA).
Results
Binding of patient antibodies to HVR1 and HVR2 HECs
Two well-known hypervariable regions are present in the
E2 envelope protein (6,17). The HVR1 and HVR2 HECs
represent 32 (HVR1) and 16 (HVR2) different variants based
on those two regions. To determine if HCV HVR1 and HVR2
HECs were recognized by antibodies from HCV-infected
patients, 228 plasma samples (group 1) from patients in-
fected with different genotypes and subtypes of HCV (1a, 1b,
500KANG ET AL.

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2a, 2b, and 3a) were screened by ELISA. The HCV patient
plasma collection included 104 samples from patients in-
fected with genotype 1a, 76 samples with genotype 1b, 20
samples with genotype 2a, 12 samples with genotype 2b, and
16 samples with genotype 3a. The results demonstrate that
more than 80% of plasma from 228 patients contained anti-
bodies that bound to HCV HVR1 (84%) and HVR2 (80%)
HECs (Table 3). The results also showed that antibody reac-
tivity to HCV HEC was not restricted to one specific geno-
type, but was broad, including all the HCV genotypes tested
(Fig. 1). Interestingly, the end-point antibody titers to HVR1
HEC from patients with different genotypes and subtypes
of HCV tested was higher than that to HVR2 HEC, which
suggests that more antibodies specific to the HVR1 region
were produced than to the HVR2 region when patients were
chronically infected with HCV. It also suggests that a stronger
cross-reactive antibody response was induced against the se-
quence variation of the HVR1 region than to the HVR2 region
in patients with persistent chronic HCV infection.
Binding of patient antibodies to six HCV HECs
Based on the results obtained with patient group 1, we
decided to test a second group of patients (group 2) for an-
tibody reactivity to NS3-1, NS3-2, and NS4-1, NS4-2 HCV
HECs, in addition to HVR1 and HVR2 HECs. Antibody re-
activity to each HEC and the percentages of patients that
responded to each HEC are shown in Fig. 2 and Table 4,
respectively. We determined the end-point titer of each
plasma sample by ELISA up to a 1:10,000 antibody dilution.
Group 2 consisted of plasma from 50 randomly-selected
HCV-positive patients infected with diverse genotypes and
subtypes of HCV (1a, 1b, 1a/b, 2b, 2a/c, 3a, and 4c/d). The
HCV genotype and subtype distribution were 20 (1a), 7 (1b),
2 (1a/b), 9 (unknown subtype of genotype 1), 3 (2b), 2 (2a/c),
6 (3a), and 1 (4c/d). Viremia in circulating blood was
Table 3. Recognition of HCV HVR1 and HVR2 HECs
by Patients Infected with Diverse Genotypes of HCV
Genotype Number of patients HVR1 HECHVR2 HEC
1a
1b
2a
2b
3a
Total
104
76
20
12
16
228
82 (79%)
61 (80%)
20 (100%)
12 (100%)
16 (100%)
191 (84%)
75 (72%)
67 (88%)
17 (85%)
11 (92%)
13 (81%)
183 (80%)
Abbreviations: HEC, hypervariable epitope construct; HCV, hepa-
titis C virus; HVR, hypervariable region.
FIG. 1.
showing relative OD values obtained when individual plasma samples from HCV patients were tested for antibodies to
HVR1 and HVR2 HECs. All sera were diluted 1:100. The samples were considered positive if their optical density at 405nm
was more than twice that of the negative sera. The horizontal line indicates the OD value that is twice the average of four
HCV-negative serum samples. The sera did not react with a negative control HEC based on HIV-1 (data not shown).
Relative antibody titers to the HVR1 and HVR2 HECs in plasma of HCV-positive patients (group 1). Dot plot
ANTIGENS REPRESENTING THE ANTIGENIC VARIATION OF HCV501

Page 6

determined by RT-PCR. The viremia of all patients selected
was at least 315,000IU/mL. Viremia of 32 out of the 50 pa-
tients was more than 850,000IU/mL, and more than
500,000IU/mL in 11 patients.
There were no significant differences in the recognition of
epitopes from envelope proteins (HVR1 and HVR2) and non-
structural proteins (NS3-1, NS4-1, and NS4-2), except NS3-2.
More than 72% of HCV-positive patients had antibody re-
sponses to one of the HCV HECs, and 36% to NS3-2, as
shown in Table 4. Patients infected with genotype 1 showed
lower antibody titers to NS3-2 HEC than to the other HECs.
This outcome could be due to the weak antibody response to
the natural epitope represented by NS3-2 HEC. These results
suggest that the NS3-2 HEC epitope may induce a cellular
rather than a humoral response against HCV. Compared to
the antibody binding to the NS3-2 HEC, the NS3-1 HEC was
the most recognizable epitope (86%) among the HCV patient
plasma samples tested. However, 80% of patients (34/43)
had low antibody titers (1:500). This implies that the NS3-1
HEC epitope might induce production of constant but low
levels of antibodies, in addition to cellular responses against
HCV.
The NS4, NS4-1, and NS4-2 HECs represent 32 sequence
variants of the epitope. Some patients had very strong anti-
body responses (1:10,000 titer) directed at NS4-1 (14%) or
NS4-2 (8%). Interestingly, the genotype of these HCV-infected
FIG. 2.
of HVR1, HVR2, NS3-1, NS3-2, NS4-1 and NS4-2 HCV HECs by patients infected with different genotypes of HCV (1a, 1b,
1a/b, 2b, 2a/c, 3a, and 4c/d). Each dot represents an individual plasma sample from an HCV patient. Antibody end-point
titers for binding to HCV HEC were determined based on the OD value of normal human sera used as negative control.
Relative antibody titers to six HCV HECs in 50 plasma samples from HCV-positive patients (group 2). Recognition
502KANG ET AL.

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patients was 1 (1a, 1b, or unknown subtype of 1), and the
viremia was more than 500,000IU/mL. In addition, NS4-1
HEC was preferentially recognized over NS4-2 HEC by pa-
tients with chronic HCV infection (Fig. 2).
The antigen-specific humoral response to HCV HECs
Immunogenicity of HEC constructs was also studied in
mice. Individual HEC-specific antibody responses from mice
immunized with a mixture of six HECs (HVR1, HVR2, NS3-
1, NS3-2, NS4-1, and NS4-2) were analyzed. Sera obtained
before immunization were used as negative controls.
The antibody response to HVR2 HEC was very strong
(1:100,000 titer), up to 100 times more than the antibody titer
to HVR1 HEC. The antibody responses to HECs derived
from non-structural proteins NS3 and NS4 were also stron-
ger (1:4,000 antibody titer) than to HVR1, but not as high as
that of HVR2 HEC. These results suggest that all of the HCV
HECs induce strong antibody responses in mice, and that
HVR2 HEC represents an immunodominant epitope in terms
of antibody response (Fig. 3A).
Next, we assessed if antibodies from mice immunized
with HCV HEC were able to recognize HCV circulating in
patients with HCV chronic infection. The use of recombinant
E2 protein and synthetic HCV virus-like particles was re-
placed with virus particles purified from plasma of patients
infected with HCV. In order to test mouse antibody binding
activity to viral particles derived from HCV patients with
different genotypes (1a, 1b, 2a, and 3a), 10mg of particles per
well were plated as coating antigen on ELISA plates. Mouse
serum samples obtained before immunization were used as
negative controls to determine the titer. Interestingly, viral
particles from all genotypes tested were recognized by sera
from mice immunized with a mixture of the six HCV HECs,
up to 1:1000 dilution in the case of genotype 3a viral particles
(Fig. 3B).
Cross-reactivity of antibodies from mice immunized with
a mixture of six HCV HECs was also assessed (Fig. 3C and
D). Pre-bleed serum samples were used as negative controls
to determine antibody titer. Mouse serum samples were
tested and diluted until the antibody titer was up to 1:20,000
in cases requiring further dilution. We concluded that sera
from mice immunized with a mixture of HCV HECs had
cross-reactive antibodies that could recognize all the HVR1
and HVR2 analogs (antibody titers to 1:1000). In addition,
cross-reactivity of antibodies against HVR2 analogs was
superior to that against HVR1 analogs, except for three of the
analogs tested (2a, 3a, and 5a).
Lymphocyte proliferation
HCV HEC-specific lymphocyte proliferation was deter-
mined by thymidine incorporation. Six outbred mice (CFW)
per group were immunized with two HECs combined, or a
mixture of all six HECs. The two-HEC combinations were (1)
HVR1 and HVR2, (2) NS3-1 and NS3-2, and (3) NS4-1 and
NS4-2. An unrelated peptide (SIV) was used as a negative
control. In the group immunized with the HVR1 and HVR2
HECs (Fig. 4A), the proliferation of HVR1-specific lympho-
cytes was approximately three times stronger than that of
HVR2 HEC and SIV HEC. Antibody-binding studies in mice
showed that the epitope represented by HVR2 HEC induced
a stronger humoral response than that represented by HVR1
HEC (Fig. 3A). These two sets of results correlate, suggesting
that the weak humoral response is not due to the absence of
antigen-specific antibodies, but is because the immune re-
sponse is skewed toward a cellular response. It could also
suggest that the HCV HECs are strong immunogens capable
of inducing cellular as well as humoral responses.
Although both NS3-1 and NS3-2 HECs induced antigen-
specific lymphocyte proliferative responses, the NS3-2 HEC
was a more dominant epitope than the NS3-1 HEC (Fig. 4B).
Both NS4-1 and NS4-2 HECs induced strong cellular re-
sponses (Fig. 4C). The level of proliferation (in cpm) was at
least four times higher than that of the SIV negative control
(Fig. 4C).
HEC-specific lymphocyte proliferation was also tested in
the group immunized with a mixture of all six HECs (Fig.
4D). A response to each individual HEC was detected (at
least twice than the negative control). The same pattern
of proliferation responses was observed as in the group im-
munized with HEC pairs. The response to HVR1 HEC was
stronger than that to HVR2 HEC, that to NS3-2 HEC was
stronger than that to NS3-1 HEC, and that to NS4-1 HEC
was stronger than that to NS4-2 HEC. However, prolifera-
tion against non-structural protein 4 in the group immunized
with a mixture of HECs was not as strong as in the group
immunized only with the NS4-1 and NS4-2 HECs.
Detection of cytokine production
We assessed IFN-g, IL-2, and IL-4 cytokine secretion from
HEC-specific T lymphocytes by cytokine ELISA. We ob-
served similar results to those obtained from HEC-specific
proliferation responses. IFN-g and IL-2 were detected in re-
sponse to HVR1, NS3-2, NS4-1, and NS4-2 HECs in the
groups immunized with the two-HEC combinations. The
amount of IFN-g and IL-2 detected in the HVR2, the NS3-1,
and the SIV negative control was either undetectable or less
than 35pg/mL. IL-4 was not detected in any group except
for HVR1 HEC (14.9pg/mL) in the group immunized with
the two-HEC combinations (Fig. 5A, B, and C). In the mice
immunized with a mixture of all HECs, IFN-g and IL-2 were
Table 4. HCV-Positive Patient (Group 2) Antibody
Binding to the Six HCV HEC Constructs
Genotype
Number
of
patients HVR1 HVR2 NS3-1 NS3-2 NS4-1 NS4-2
1a
2b
3c
4c/d
Total
Percentage (%)
38
5
6
1
50
26
4
5
1
36
72%
30
5
6
1
42
84%
33
3
6
1
43
86%
13
2
3
0
18
36%
30
4
5
1
40
80%
27
4
5
1
37
74%
aIncludes subtypes 1a, 1b, 1a/b, and genotype 1 of unknown
subtype.
bIncludes subtypes 2b and 2a/c.
cIncludes subtype 3a only.
Abbreviations: HEC, hypervariable epitope construct; HCV, hepa-
titis C virus; HVR, hypervariable region.
ANTIGENS REPRESENTING THE ANTIGENIC VARIATION OF HCV 503

Page 8

detected in individual HECs, except for the HVR2 HEC, in
which the amount of IFN-g was the same as the negative
control (about 25pg/mL) (Fig. 5D). These results suggest
that HEC-specific immune responses were Th-1-mediated
(cellular), and not Th-2-mediated (humoral) responses, be-
cause IL-4 secretion was either minimal or undetectable.
Discussion
Numerous studies have contributed over several decades
to our understanding of cellular immune responses during
both chronic persistent and self-resolved HCV infections
(5,16,30,40). Although a protective CD8þCTL response plays
an important role in the clearance of HCV infection, it is
strongly suggested that a robust CD4þT-helper response is
also essential in control of viral replication and for the pro-
motion of effective antiviral CD8þCTL responses (1,19,33).
In addition, HCV-specific cellular responses during antiviral
treatment in patients with HCV chronic infection have been
extensively studied (7,13). In contrast to the detailed under-
standing of cellular immune responses upon HCV infection,
humoral immune responses for the control of HCV infection
have not been well analyzed. Humoral immune responses
produce antibodies that are broadly cross-reactive to anti-
genic variants of epitopes. Chronic HCV infection is usually
established regardless of the antibody production that tar-
gets various epitopes of the HCV structural and non-struc-
tural proteins. Neutralizing antibodies play an important
role in controlling hepatitis C viral infection and dissemi-
nation by directly blocking attachment and entry of HCV
to host target cells (25,35,41). A recent study showed that
human monoclonal antibodies were broadly reactive to
neutralize heterologous HCV isolates. This study also dem-
onstrated protection against HCV quasispecies infection by
neutralizing antibodies in a human liver chimeric mouse
model (20).
To what extent antibodies from an individual HCV patient
are broadly reactive at the epitope level among different
FIG. 3.
NS3-2, NS4-1, and NS4-2 HECs. The HCV HEC-specific antibody responses were determined by ELISA. BALB/c mice
immunized with a mixture of HCV HECs produced antibodies that recognized each individual HEC. Sera were obtained 7d
after the final immunization. Serum samples obtained before immunization were used as negative controls. (B) Recognition
of HCV particles (purified from plasma of patients infected by different HCV genotypes) by antibodies from mice immunized
with a mixture of 6 HCV HECs. Pre-bleed sera obtained before immunization were used as negative controls. (C and D)
Cross-reactivity of sera from mice immunized with HVR1, HVR2, NS3-1, NS3-2, NS4-1, and NS4-2 HECs. HVR1 (C) and
HVR2 (D) analogs representing genotypes from 1a to 6a were used as capturing antigen in triplicate. Sera from three mice
were evaluated with antibody dilution rates of 1:100, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16,000, and 1:20,000. Data shown
are the mean?SD of the antibody titer (in 1 per dilution rate) as assessed by ELISA with serum from four individual mice
and analyzed in triplicate. The data in Fig. 3A were regarded as statistically highly significant (***p<0.001).
Immunogenicity of HCV HECs in BALB/c mice. (A) ELISA of sera from mice immunized with HVR1, HVR2, NS3-1,
504KANG ET AL.

Page 9

genotypes and subtypes, and their specificity for epitopes
derived from different regions of the viral proteins remains
unclear. Also the clinical significance of these anti-HCV an-
tibody responses remains unknown. Our study showed that
high antibody titers to HECs representing variable antigenic
epitopes of both envelope and non-structural proteins were
observed in all HCV patients. This implies that patients with
HCV chronic infection develop broadly cross-reactive anti-
bodies against all viral proteins, but these antibodies are not
able to control the infection. These data may suggest that
significant amounts of tissue destruction and viral lysis occur
in vivo, and that the structural viral proteins that are released
could drive these antibody responses. Based on these results
we are unable to determine to what extent antibody re-
sponses are protective and driving immune pressure against
intact virions, or are simply a consequence of tissue de-
struction due to immune pressure mediated by CTLs, or
both. However, the data indicating that the majority of pa-
tients with chronic HCV infection had antibodies to the hy-
pervariable epitopes of HCV envelope protein suggest that
sequence mutation of HVR1 epitopes occurs as a result of
immune pressure–induced cross-reactive antibodies against
different variants of the HVR1 epitope. This resulted in
higher antibody titers to the corresponding sequences re-
presented within the HVR1 HEC designed for our study.
The NS3 protein has two enzymatic functions: serine
protease at the amino-terminal (180 amino acids) (31), and
NTPase/RNA helicase at the carboxy-terminal (450 amino
acids) (18). Although the HCV genome is highly variable,
core and NS3 proteins undergo less antigenic variability than
the two envelope glycoproteins (E1 and E2) and other non-
structural proteins (27). A report suggested that NS3 inter-
rupts TLR3 pathways by degradation of toll/IL-1 receptor
domain-containing adapter inducing IFN-b (TRIF) using
NS4A as a serine protease cofactor facilitating HCV evasion
of the immune response (21). Therefore, lysis of cells ex-
pressing NS3-specific epitopes could be involved in viral
clearance by the host immune response. A strong cellular
immune response induced against NS3 is considered to play
an important role in viral clearance. A previous study found
FIG. 4.
group were immunized with a combination of two HECs, or with all six HECs together. The two-HEC combinations were (1)
HVR1 and HVR2, (2) NS3-1 and NS3-2, and (3) NS4-1 and NS4-2. An unrelated peptide derived from SIV was used as a
negative control (NC). Lymphocytes from lymph nodes were stimulated in vitro with each HCV HEC at 5mg/mL for 3d.
Following 14h of incubation with 1mCi of tritiated thymidine per well, the incorporated thymidine was determined and is
presented as counts per minute (cpm). (A) Mice immunized with the HVR1 and HVR2 HECs. (B) Mice immunized with the
NS3-1 and NS3-2 HECs. (C) Mice immunized with the NS4-1 and NS4-2 HECs. (D) Mice immunized with all six HECs
combined. Data shown are the mean?SD of the value of incorporated [3H]thymidine from six individual mice and analyzed
in triplicate. Statistical analysis was conducted using one-way ANOVA (Dunnett’s multiple comparison test: *p<0.05;
**p<0.01).
HCV HEC-specific lymphocyte proliferation as measured by thymidine incorporation. Six outbred mice (CFW) per
ANTIGENS REPRESENTING THE ANTIGENIC VARIATION OF HCV505

Page 10

that the strongest and most consistently detected CD4þ
T-helper cell response was to NS3 protein in patients with
self-resolved HCV infection (14). This study also suggested
that NS3 protein contains identified dominant epitopes for
CD4þT-helper responses in both humans and chimpanzees
with resolved infection (30,39). In addition, one of the CD4þ
T-cell epitopes recognized by more than 30% of HCV pa-
tients with spontaneously resolved infection as determined
by cell proliferation assays and IFN-g detection was also
from the same region as our HCV NS3-2 HEC (15,29). It was
interesting to detect low titers of antibodies that bind NS3-1
and NS3-2 HECs in patients with chronic HCV infection.
More interestingly, our NS3-1 and NS3-2 HECs contained
similar sequences of identified CD4þT-cell epitopes from
NS3 to those used by other groups (15,30,39). We concluded
that the reason for low antibody titers to NS3-1 and NS3-2
HECs was because these HECs could be epitopes for CD4þ
T-cell responses. Another possibility could be that weak or
absent CD4þT-cell responses against NS3 protein in patients
with chronic HCV infection affect production of large
amounts of NS3-specific antibodies, and thus have low af-
finity antibodies to NS3 epitopes.
We designed NS4-1 and NS4-2 HECs to represent two
different regions of the NS4 proteins. NS4-1 and NS4-2 HECs
are composed of the C-terminal part of the NS4a protein (aa
1686–1711), and the N-terminal part of the NS4b protein (aa
1711–1733), respectively. Although we designed our own
antigenic variable epitopes from NS4 using Genbank se-
quences, we found that other laboratories designated those
two specific regions of the NS4 protein as region 5-1-1 (10).
When sera from patients in group 2 were tested for binding
to all six HCV HECs, we found that the antibodies were
more reactive with NS4-1 HEC than NS4-2 HEC. According
to a previous study, the peptide variants of NS4a region
5-1-1 were broadly recognized by patients infected with
different genotypes, whereas the peptide variants of NS4b
were only bound by antibodies from patients infected with
the same genotype (10). Interestingly, these peptide variants
contained the sequence PDREVLYQEFDE, that is also part of
our NS4-1 HEC. This may explain why antibodies to NS4-1
HEC were more frequently detected than to NS4-2 HEC in
plasma from HCV-infected patients. Another group also
noted that antibodies to NS4 were more frequently found in
patients infected with genotype 1 than other genotypes (12).
Epitopes derived from NS4 were considered among the most
immunogenic for induction of cellular responses against
HCV, when tested for induction of proliferation of human
cells from patients with chronic HCV infection (28). This
FIG. 5.
HEC-specific T lymphocytes were quantitated by cytokine ELISA. In each group, 5?105lymphocytes from the spleens were
stimulated in vitro with each HCV HEC at 5mg/mL for 2d, and the supernatants were collected to measure cytokine levels.
An HCV-unrelated peptide (SIV) was used as a negative control (NC). (A) Mice immunized with HVR1 and HVR2 HECs. (B)
Mice immunized with NS3-1 and NS3-2 HECs. (C) Mice immunized with NS4-1 and NS4-2 HECs. (D) Mice immunized with
all six HECs combined. Data shown are the mean?SD of cytokine concentration values in picograms per milliliter from three
to four individual mice and analyzed in triplicate. Statistical analysis was conducted using two-way ANOVA (Bonferroni’s
post-test: *p<0.05; **p<0.01; ***p<0.001).
Detection of cytokines secreted from HCV HEC-specific lymphocytes. IFN-g, IL-2, and IL-4 cytokines secreted from
506KANG ET AL.

Page 11

might explain why patient antibody binding to NS4 was
higher than to the other epitope variants tested in the pres-
ent study.
We showed that HCV HECs are strong immunogens that
induce both humoral and cellular immune responses in mice.
The antibody response induced by HCV HEC in mice sug-
gests that HVR2 HEC is an immunodominant epitope that
drives a humoral response. In addition, HVR2 HEC-specific
antibodies produced in mice immunized with a mixture of
six HCV HECs were broader and stronger than the response
to the HVR1 analog. Lymphocyte proliferation measured
with the thymidine incorporation assay and cytokine ELISA
to assess levels of IFN-g, IL-2, and IL-4 support our conclusion.
In addition, HEC-specific cellular responses were also de-
tected in CFW outbred mice that represent individual MHC
polymorphisms. Specific lymphocyte proliferation to HVR1
HECs was three times higher than negative controls, and
twice that to HVR2 HECs. HVR1 HEC-specific IFN-g and IL-
2, but not HVR2 HEC-specific cytokine production, was
observed. The results indicate that HVR1 is a more immu-
nogenic epitope in mice than HVR2 for induction of cellular
immune responses as represented by the E2 envelope gly-
coprotein-derived HCV HECs. In addition, the frequency of
CD69þCD4þactivated T cells in mice immunized with a
mixture of HCV HECs showed that HVR1 HECs induced
more activated T cells than HVR2 (data not shown). In
terms of non-structural protein-derived HCV HECs, NS3-2
and NS4-1 were more immunogenic epitopes than NS3-1
and NS4-2 as measured by cellular responses. In the study
designed to determine HEC-specific cytokine production,
IFN-g and IL-2 production were detected, but not IL-4,
suggesting that HCV HEC-specific T-cell responses were
biased to a Th-1 rather than to a Th-2 response.
Due to the unavailability of lymphocytes from HCV-in-
fected patients, we were not able to study cellular responses
to these epitopes after incubation with HCV HECs in vitro.
At this point, we do not know if the epitope represented by
HVR1 HECs might also induce stronger cellular responses
than those represented by HVR2 in HCV patients, as ob-
served in the mouse studies. However, there could be a
correlation between our studies with human samples and
immunogenicity of HCV HECs in mice. Our results showed
that antibodies from HCV patients showed less recognition
of NS3-2 HECs than other HCV HECs. Previous studies that
have reported T-cell responses induced by epitopes derived
from the NS3 region (15,30,39) correlate with our mouse data
showing that NS3-2 HECs induce less immunogen-specific
lymphocyte proliferation and Th-1-biased cytokine produc-
tion than other HCV HECs.
Overcoming antigenic variation with a vaccine represent-
ing only a single genotype of HCV will probably be impos-
sible. However, HECs can represent many variant forms of
viral isolates rather than having to synthesize a large number
of peptides representing each divergent form of each anti-
genic epitope. Like other peptide-based vaccines, reversion to
virulence, cold preservation requirements, and other negative
implications associated with live-attenuated virus vectors are
not a concern. In addition, HECs designed at our laboratory
are strong synthetic peptide immunogens inducing both hu-
moral and cellular immune responses directed to the most
variable HCV epitopes. We were not able to perform an HCV
challenge efficacy study with animals immunized with HCV
HECs due to the lack of susceptible small animal models for
HCV. Neutralization assays were not performed due to the
lack of appropriate detection antibodies. However, all of the
human and mouse data obtained suggest that HECs rep-
resent antigenic epitopes being recognized by more than
80% of HCV patients infected with diverse genotypes, and
could have potential as peptide components of vaccine
candidates, as they induce both humoral and cellular im-
mune responses specific to HCV. Therefore, HECs could
be used to develop potential peptide vaccine components
to overcome antigenic variation against viruses with mu-
tating genomes such as HCV.
Author Disclosure Statement
The authors have no conflicts with regard to financial in-
terests. This material has not been previously reported and is
not under consideration for publication elsewhere.
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Address correspondence to:
Dr. Jose ´ V. Torres
University of California, School of Medicine
Department of Medical Microbiology and Immunology
Tupper Hall, Room 3134
Davis, CA 95616
E-mail: jvtorres@ucdavis.edu
Received April 6, 2010; accepted June 23, 2010.
508KANG ET AL.