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Journal of Virology, January 1999, p. 11-18, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of a Domain Containing B-Cell
Epitopes in Hepatitis C Virus E2 Glycoprotein by Using Mouse
Monoclonal Antibodies
Jae Woo
Lee,1
Kwang-mi
Kim,1
Seung-Hye
Jung,1
Ki Jeong
Lee,2
Eung-Chil
Choi,1
Young-Chul
Sung,2 and
Chang-Yuil
Kang1,*
Laboratory of Immunology, College of
Pharmacy, Seoul National University, Seoul
151-742,1 and
Laboratory of
Virol-immunology, Department of Life Science, Pohang University of
Science and Technology, Pohang 790-784,2 Korea
Received 8 July 1998/Accepted 30 September 1998
 |
ABSTRACT |
Evidence from clinical and experimental studies of human and
chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To
identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by
using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were
able to inhibit the binding of CET MAbs to the former fusion protein.
Inhibitory activity was observed in most sera tested, which indicated
that CET-1 to -6 were similar to anti-E2 antibodies in human sera with
respect to the epitope specificity. The spacial relationship of
epitopes on E2 recognized by CET MAbs was determined by surface plasmon
resonance analysis and competitive enzyme-linked immunosorbent assay.
The data indicated that three overlapping epitopes were recognized by
CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we
analyzed the reactivities of CET MAbs to six truncated forms and two
chimeric forms of recombinant E2 proteins. The data suggest that the
epitopes recognized by CET-1 to -6 are located in a small domain of E2
spanning amino acid residues 528 to 546.
| |
INTRODUCTION |
Most individuals who contact
hepatitis C virus (HCV), responsible for most cases of posttransfusion
and non-A, non-B hepatitis (4), develop a chronic infection
which is a major cause of liver cirrhosis and hepatocellular carcinoma
and more rarely leads to liver cancer (1, 33). Despite the
recognition of HCV as an important cause of morbidity throughout the
world and the advances in epidemiology and molecular virology, the
pathogenesis of this disease and the molecular mechanism of viral
persistence with high rates are not fully understood (7).
HCV, a positive-stranded RNA virus with a genomic size of about 9.5 kb,
has one large open reading frame that encodes a polypeptide of 3,011 amino acids (aa). The single polypeptide precursor processed by
cellular and viral proteases results in a core protein (C), two
glycosylated envelope proteins (E1 and E2/NS1), and nonstructural proteins (NS2 to NS5) (5, 16, 39).
Comparative genome alignments suggest that the HCV E2 protein
corresponds to the flavivirus NS1 glycoprotein and the major pestivirus
envelope protein gp53/gp55 (gp53 in bovine viral diarrhea virus and
gp55 in hog cholera virus) (26). Both flaviviral NS1 and
pestiviral gp53/55 are known to elicit protective antibodies in hosts
vaccinated with these proteins (32, 44).
In a chimpanzee model study of HCV, in vivo protection was achieved by
vaccination with recombinant HCV E1/E2 proteins, and the anti-E2
antibody titers were shown to correlate with the protection (3). In another model study of chimpanzee, antibodies
present in patient sera could prevent infection when incubated in vitro with virus prior to infection (8). In addition, HCV E2
protein expressed in Chinese hamster ovary (CHO) cells bound to human cells with high affinity, and sera from protected chimpanzees contained
antibodies which neutralized the binding of E2 protein to target cells
(31). Thus, several pieces of evidence suggest that the
envelope glycoprotein E2 is a key antigen for vaccine development
against HCV infection (21, 24, 30, 38).
Several observations suggest that hypervariable region 1 (HVR-1), which
is located at the N terminus of E2 (12, 18, 42) and contains
cytotoxic T-lymphocyte epitopes and several B-cell linear epitopes
(35, 43, 46), may be involved in the neutralization of HCV,
and antibodies directed at this region are shown to prevent binding of
viruses (9, 19, 20, 37). However, the higher genetic
variability of this region may allow virus to escape immune surveillance, and the variability of the HCV genome has posed serious
problems in development of a broadly reactive vaccine against HCV
infection (11, 17, 29, 41). In addition, some studies have
reported the existence of B-cell epitopes within the HCV E2 protein
downstream of HVR-1. However, detailed mapping of those regions has not
been done (27, 28, 40).
In this study, to identify epitopes of HCV E2 glycoprotein, we
generated six monoclonal antibodies (MAbs), CET-1 to -6, against HCV E2
antigen by using recombinant fusion proteins. To characterize the MAbs,
we evaluated the competitive reactivity to E2 protein with HCV-immune
sera and performed surface plasmon resonance (SPR) analyses. Finally,
from the relative reactivities of MAbs to chimeric and truncated forms
of E2 protein, we could identify a domain containing the epitopes of
CET MAbs in the E2 region.
| |
MATERIALS AND METHODS |
Mice and MAbs.
Five- to six-week-old BALB/c female mice
(Charles River Laboratory, Osaka, Japan) were immunized with 10 µg of
a truncated form of HCV E2 protein (amino acid residues 386 to 693)
lacking the C-terminal hydrophobic region (E2t) fused to human growth hormone (hGH) or herpes simplex virus type 1 (HSV-1) glycoprotein D
(gD) (hGHE2t or gDE2t) three times by intraperitoneal injection. The
first immunization was done with complete Freund's adjuvant (Sigma,
St. Louis, Mo.); the second and the third immunizations were done with
incomplete Freund's adjuvant (Sigma). Three days after the third
injection, splenocytes obtained from the immunized mice were fused with
SP2/0 cells as described by Galfre et al. (10). Hybridoma
cell lines were tested by enzyme-linked immunosorbent assay (ELISA).
Hybridomas producing MAbs with high affinity to E2t or gD were selected
and subcloned twice. MAbs against E2t protein were designated CET MAbs
and a MAb against gD was named 3H9. MAbs were purified from ascites
fluid by protein A-Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden)
affinity column chromatography. Biotinylation of CET MAbs was performed
with EZ-Link Sulfo-NHS (N-hydroxysuccinimide)-LC-Biotin
(Pierce, Rockford, Ill.) according to the manufacturer's instructions.
Fusion proteins.
To establish the recombinant CHO cell lines
expressing gDE2t, we constructed the expression vector pMT3-gDE2t as
follows. The cDNA encoding a C-terminally truncated gD (aa 1 to 326)
was amplified by PCR from HSV-1 (KOS strain) and then inserted into pMT3 to produce pMT3-gD. Then the cDNA of HCV-N, genotype 1b, encoding
the E2t region spanning amino acid residues 386 to 693 was obtained by
PCR amplification and fused in frame to the cDNA encoding amino acid
residue 326 of gD in pMT3-gD (23). The gDE2t protein in
culture media of the recombinant CHO cells was purified by affinity
column chromatography using CNBr-activated Sepharose-4B linked with
anti-gD MAb. hGHE2t was also expressed in CHO cells and purified as
described previously (23).
Human HCV sera.
Human sera were obtained from patients with
chronic hepatitis C who visited Korea Cancer Center Hospital and
College of Medicine, Seoul National University, Seoul, Korea. A total
of eight samples were heat inactivated at 56.5°C for 30 min prior to experiments.
ELISA.
To detect murine MAbs specifically binding to E2t or
gD, wells of a Maxisorp Immunoplate (Nunc InterMed, Roskilde, Denmark) were coated with hGHE2t, gDE2t, hGH, or gD at a concentration of 1.5 µg/ml at 4°C overnight. After blocking with 1% bovine serum albumin solution for 30 min, hybridoma culture media were added to the
wells. After incubation for 2 h at room temperature, alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulin (Ig) at a
concentration of 100 ng/ml was added to each well, and the immunoplate
was incubated for 2 h. p-Nitrophenyl phosphate was added to the wells, and optical density at 405 nm was measured.
The binding affinity of antibodies in human HCV sera to recombinant
hGHE2t was assessed by using an immunoplate coated with hGHE2t protein
at a concentration of 1.5 µg/ml. Human HCV sera at a dilution of
1/100 were added to the wells. Subsequent steps were the same as for
the method used to detect murine MAbs specifically binding to E2t
protein, but using AP-conjugated goat anti-human Ig instead of
AP-conjugated goat anti-mouse Ig.
The competitive reactivity of CET MAbs to E2t protein with antibodies
in HCV in human sera was assayed. Wells of an immunoplate were coated
with hGHE2t, and human HCV sera at a dilution of 1/20 were added. Ten
minutes later, biotinylated CET MAbs were added to each well to a final
concentration of 400 ng/ml, and then the plate was incubated for 2 h. After an additional 2-h incubation with AP-conjugated extravidin at
a concentration of 100 ng/ml, development of the reaction was performed
as described above.
To investigate epitope specificities of CET MAbs by competitive ELISA,
wells of an immunoplate were coated with hGHE2t at
a concentration of 2 µg/ml. The unlabeled CET MAbs were added
to the wells at serial
fivefold dilutions beginning at 100 µg/ml.
After incubation for 10 min, biotinylated CET MAbs were added
to each well to a final
concentration of 1 µg/ml, and the plate
was incubated for an
additional 2 h. Addition of secondary antibody
and development of
the reaction were performed as described
above.
For mapping of the epitopes, the culture supernatant samples of
transfected CHO cells or COS-7 cells expressing a chimeric,
truncated,
or wild-type form of E2t were assayed for binding to
six CET MAbs.
Primarily, the culture supernatant samples were
tested for the presence
of various types of E2t. The culture supernatant
samples of
transfectant CHO cells or COS-7 cells were added to
the wells of an
immunoplate coated with anti-gD MAb 3H9 at a concentration
of 2 µg/ml. After incubation for 4 h, human HCV sera at a dilution
of
1/500 were added to the wells. Subsequent steps were the same
as for
the methods used to assay the binding affinity of antibodies
in human
HCV sera to hGHE2t. Then the culture supernatant samples
were assayed
for binding to each MAb. The samples were added to
an immunoplate
coated with 3H9. After incubation for 24 h, each
of biotinylated
CET MAbs was added for an additional incubation
for 4 h. Addition
of secondary antibody and development of the
reaction were performed as
described
above.
SPR analysis.
Experiments were run on a BIAcore X instrument
(Biosensor, Uppsala, Sweden) at 25°C, using HEPES-buffered saline (10 mM HEPES [pH 7.4], 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20)
as flow buffer. We activated a research-grade CM-5 sensor chip
(Biosensor) by a standard amine coupling method with a 7-min pulse of
0.05 M NHS-0.2 M EDC
[N-ethyl-N'-(dimethylaminopropyl)
carbodiimide]. Immobilization of gDE2t protein on the activated sensor
chip was performed by incubation of 1 µg of gDE2t in 10 mM acetate
buffer (pH 4.8) with the sensor chip. Excessive NHS groups were
inactivated with ethanolamine. Immobilization of 4,000 resonance units
(RU) was achieved. For epitope mapping, series of successive addition of MAbs (1 µM in flow buffer) were performed. For kinetic assay of
each MAb, gDE2t immobilization of 800 RU was achieved on another sensor
chip by using standard methods described above with injection of each
MAb at a concentration of 100 nM in flow buffer at a flow rate of 20 µl/min for 4 min. Apparent association and dissociation rates were
determined by curve fitting using BIAevaluation 2.1 (Biosensor).
Construction of expression plasmids.
HCV cDNA covering the
E2 region was obtained by reverse transcription-PCR from the serum
sample of a Korean patient. The complete nucleotide sequence of the
clone was determined; the clone was classified as genotype 1b and named
HCV-N (23).
To construct an expression vector encoding a truncated form of E2
protein spanning amino acid residues 384 to 693, the TPLgDE2t
region of
pSK-TPLgDE2t was amplified by PCR. The PCR product digested
with
ClaI and
XbaI was inserted into the
Rc/CMVdhfr expression
vector to generate Rc/CMVdhfr-TPLgDE2t
(Fig.
2A) (
23).
For the construction of expression vectors encoding truncated forms of
E2t protein, five partial E2t-derived sequences (E2A,
E2B, E2C, E2D,
and E2E) were amplified by PCR from the Rc/CMVdhfr-TPLgDE2t
expression
vector by using an upstream primer (5'-GCC GCA CGA
CCA ACC GGT TCG
TGA-3') and the following downstream primers:
5'-GCT CTA GAA CAG TGC
GGC AAT AAA-3' for E2A, 5'-GTC TAG ACC
TGC GAT GCG GGT ACG-3' for E2B,
5'-GCT CTA GAC TAC GTA GGG GCA
CCG GAA-3' for E2C, 5'-GCT CTA GAA CCC
AGT GCT ATT CAT-3' for
E2D, and 5'-GCT CTA GAG CCT GTA TGG GTA GTC-3'
for E2E. We positioned
the stop codon of each truncated E2t open
reading frame not to
be located at antigenic domains deduced from the
hydrophilicity
plot of HCV E2. The PCR products were digested with
AgeI and
XbaI.
The DNA fragment encoding amino
acid residues 397 to 693 of the
E2t region in plasmid
Rc/CMVdhfr-TPLgDE2t was replaced by five
PCR products, using
AgeI and
XbaI sites in the E2t region, resulting
in expression vectors Rc/CMVdhfr-TPLgDE2A to -E.
For the construction of an expression vector encoding HVR-1-replaced
E2t protein, a DNA fragment encoding HVR-1 of E2t (amino
acid residues
384 to 411) in Rc/CMVdhfr-TPLgDE2t was replaced
by a DNA fragment
encoding amino acid residues of E2 HVR-1 from
another strain, HCV-1
(subtype 1a). A 96-bp DNA fragment for replacement
HVR-1 was
constructed as follows. Two oligonucleotides, 5'-GGA
ATT CCA TAT GGA
AAC CCA CGT CAC CGG GGG AAG TGC CGG CCA CAC TGT
GTC TGG ATT TGT T-3'
and 5'-TTT ACA AGC TGG ACG TTC TGC TTG GCG
CCT GGT GCG AGG AGG CTA ACA
AAT CCA GAC ACA GTG T-3', were partially
annealed by a 3' stretch of
complementary nucleotides, and 5'
single strands were filled by the
Klenow fragment of DNA polymerase
I (Promega, Madison, Wis.). The
108-mer DNA constructed was digested
with
NdeI and
AluI and replaced the
NdeI-
AluI DNA
fragment of
the original E2t HVR-1, resulting in the expression vector
Rc/CMVdhfr-TPLgDE2t-1.
For the construction of an expression vector encoding a chimeric
derivative of E2D, E2D sequences in Rc/CMVdhfr-TPLgDE2D were
amplified
by two rounds of PCR with three primers. The first-round
PCR used the
downstream primer for E2D (described above) and an
upstream primer,
5'-AGC TGG GGT GAA AAT GAT ACG GAC GTC TTC GTC
CTT AAC AAT ACC AGG CCA
CCG CTG GGC-3', containing the sequences
encoding amino acid residues
528 to 546 of HCV-1. The first-round
PCR product (105 bp) was used as
the downstream primer for the
second-round PCR in conjunction with the
upstream primer used
for the construction of E2t derivatives described
above. The final
PCR product was designated E2D-1, encoding E2D in
which the domain
spanning amino acid residues 528 to 546 was replaced
by that of
HCV-1. Subsequent steps were the same as for the methods
used
to construct expression vectors encoding variant forms of E2t,
resulting in the expression vector Rc/CMVdhfr-TPLgDE2D-1.
All nucleotide sequences of the PCR products were determined by the
dideoxynucleotide chain termination method (
34) using
a
Li-COR (Lincoln, Neb.) sequencer. All oligonucleotide primers
used in
this study were purchased from Bioneer Inc., Cheongwon,
Korea.
Transfection and cell lines.
CHO cells deficient in the
dihydrofolate reductase gene (CHO DHFR
cells) were
maintained in RPMI 1640 medium (GIBCO, Gaithersburg, Md.) supplemented
with 10% fetal bovine serum (GIBCO), 10 mM L-glutamine, hypoxanthine, and thymidine plus antibiotics. All DNA constructs expressing different forms of gDE2t protein were linearized by AhdI and introduced into CHO DHFR cells by
electroporation as follows. CHO DHFR cells were
resuspended in phosphate-buffered saline at a concentration of
107 cells/ml. Linearized plasmid was added to the cell
suspension at a concentration of 20 µg/ml. An electric pulse was
applied at 250 V and 500 µF with a Gene Pulser (Bio-Rad, Hercules,
Calif.). Two days after transfection, G418 (GIBCO) was added to media
at a concentration of 500 µg/ml to select for neomycin resistance. After 14 days of selection, G418 was replaced by 10 nM methorexate (Sigma). The concentration of MTX in the media was increased to a
maximum of 1 µM. When monolayers reached confluence, the expression of different forms of gDE2t protein was assayed by ELISA.
COS-7 cells were maintained in RPMI 1640 medium supplemented with 10%
fetal bovine serum and 10 mM
L-glutamine plus antibiotics.
The DNA construct Rc/CMVdhfr-TPLgDE2D-1 was introduced into COS-7
cells
by liposome-mediated transfection using Lipofectin reagent
(GIBCO). We
used 2 µg of DNA in 10 µl of Lipofectin per 2 × 10
5 cells, and performed the transfection according to the
manufacturer's
instructions. Three days after transfection, the
expression of
gDE2D-1 protein was assayed by
ELISA.
| |
RESULTS |
Generation of anti-E2 MAbs.
To produce MAbs specific for HCV
E2 protein, recombinant fusion proteins hGHE2t and gDE2t were used as
immunogens. They were expressed in CHO cells in secreted form
(23). Splenocytes from immunized BALB/c mice were used to
generate hybridomas secreting anti-E2 MAbs. The MAbs produced were
expected to bind to the E2t portion or the hGH or gD portion of the
recombinant E2 protein. Therefore, we performed ELISA to select MAbs
binding to E2t only, not to hGH or gD protein. MAbs which bound to
hGHE2t and gDE2t with considerable affinity but not to hGH or gD were
selected and named CET-1 to -6 (Table 1).
The binding activities of CET-1 to -5 to gDE2t were lower than those to
hGHE2t, and reactivity of CET-6 to gDE2t was higher than its reactivity
to hGHE2t. This observation suggested that differences in the binding
of CET MAbs might be attributed to slightly different epitope
structures of the two E2 fusion proteins. At the same time, 3H9 (Table
1), a MAb with specific affinity to gD, was developed from a fusion by
using a mouse immunized with gDE2t. All six CET MAbs and 3H9 were
identified as IgG1 isotype and 
light chain (data not shown). SPR
analyses were performed to obtain association and dissociation rate
constants for all MAbs. Calculated Kd values for
CET MAbs were evenly distributed and ranged from 6.3 × 107 to 4.8 × 108 M (data not shown).
Comparison of epitope specificities of CET MAbs with those of Abs
in HCV-infected individuals.
To compare the epitope specificities
of CET MAbs with those of Abs in HCV-immune sera, we investigated
whether the serum samples from eight individuals were able to inhibit
the binding of CET MAbs to hGHE2t by ELISA. Prior to the inhibition
assay, HCV serotyping was performed by ELISA. We tested the binding
activities of serum samples against HVR-1 peptides derived from the E2
sequence of subtype 1a or 1b. Eight samples were all reactive
preferentially to the HVR-1 peptide of subtype 1b sequence, homologous
to that of E2t fusion proteins used for generating CET MAbs (data not shown). The sera were also tested for binding to hGHE2t; seven of the
eight sera showed binding to hGHE2t (Table
2).
Inhibitory activity was observed in six of the seven sera. In case of
CET-1, most sera showed inhibitory activities. In CET-3
and CET-5, four
of seven sera showed modest or weak inhibitory
activities. In CET-2,
-4, and -6, four of seven sera showed weak
inhibitory activities. These
data suggested that antibodies similar
in epitope specificity to CET-1
prevalently existed in HCV-infected
individuals. Antibodies similar to
CET-3 and CET-5 less frequently
existed, and those similar to CET-2,
-4, and -6 were barely present
in HCV-infected
individuals.
Epitope specificities of CET MAbs determined by SPR analysis and
ELISA.
To analyze the epitopes on HCV E2 protein recognized by six
CET MAbs, we tested simultaneous binding of CET MAbs by a series of SPR
analyses (13, 14). We designed an experiment of sequential addition of five MAbs to gDE2t of which the surface binding sites were
previously saturated with one of the CET MAbs. The saturation of
binding sites on gDE2t coated on a sensor chip was accomplished by
double injections of a CET MAb and followed by the sequential addition
of the other five CET MAbs. We performed this experiment repeatedly
with all of the six CET MAbs, changing the order of addition. The
increase in RU level upon the addition of each MAb in the sensorgram
allowed us to map the epitope specificity of the six CET MAbs. The
competitive ELISA of CET MAbs confirmed the results of SPR analyses and
permitted a more comprehensive interpretation of the sensorgram data.
Figure
1A shows an example of a
sensorgram obtained from SPR analyses described above. The early stage
of the sensorgram shows
the saturation step by two consecutive
injections of CET-4. For
the sensor chip-immobilized gDE2t to be
completely bound to CET-4,
we used an excessive amount of CET-4 for the
first injection.
To avoid a false-positive response, unoccupied CET-4
sites on
gDE2t must be blocked before subsequent injections of samples.
This was accomplished by an additional injection of CET-4. The
initial
increase in RU from the baseline level and the next small
increase
confirm that the available sites for CET-4 are almost
occupied by
initial two injections of CET-4. In the next five
injections, CET-5,
-3, -2, -6, and -1 were sequentially added
to the gDE2t protein
saturated with CET-4. A significant increase
in RU level is shown only
after the injection of CET-2, indicating
that the binding of CET-2 is
not hindered by the previous binding
of CET-4. CET-4 and CET-2 MAbs
showing simultaneous binding in
the sensorgram are therefore judged to
bind to independent epitopes.
The competitive ELISA performed in a
pairwise manner showed results
equivalent to those for the SPR
analysis. CET-2 could not inhibit
the binding of CET-4, whereas CET-1,
-3, and -5 could completely
inhibit the binding of CET-4 (Fig.
1B).
Inhibitory activity was
also shown in CET-6, but to a lesser extent,
indicating that CET-4
and CET-6 bound to independent epitopes which
were adjacently
located.
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FIG. 1.
SPR analysis for determining the epitope specificities
of CET MAbs. (A) Sensorgram showing sequential addition of CET MAbs.
Experimental conditions are described in Materials and Methods. SPR
response was measured in RU. Buffer flow was maintained at 5µl/min
throughout the analysis. The saturation step was performed with two
injections of CET-4. CET-4 was bound to gDE2t by the first injection,
and the unoccupied binding sites on gDE2t were blocked by the second.
CET-5, -3, -2, -6, and -1 were added in turn. Each injection start
point and the name of corresponding MAb are indicated. The injection
volumes were 25 µl for the first injection of CET-4 and 15 µl in
all other cases. (B) Competitive ELISA for binding to hGHE2t. Various
amounts of unlabeled CET MAbs were used as competitors. hGHE2t-coated
wells were incubated with competitors for 10 min. After incubation,
biotin-labeled CET-4 was added to the wells. Percent inhibition =
100 × (A405 of CET-4 bound to hGHE2t 
A405 of CET-4 bound to hGHE2t in presence of
competitor)/A405 of CET-4 bound to hGHE2t. (C)
Reactive pattern matrix showing the binding ability of pairs of CET
MAbs to E2t protein.  , pairs of CET MAbs that bind concurrently.
 , pairs of CET MAbs that interfere with binding. (D) Two-dimensional
surface-like map of the epitopes on E2t recognized to six CET MAbs.
Numbers 1 to 6 correspond to the CET-1 to CET-6; overlapping circles
represent MAb groups within which pairs of MAb cannot bind
simultaneously.
|
|
From the interpretation of a series of sensorgram and ELISA data, we
could display the reactivity patterns for six CET MAbs
as a 6 × 6 matrix (Fig.
1C). We identified the existence of six
different epitopes
for six CET MAbs. Accordingly, we could construct
a two-dimensional
surface-like map of epitopes on E2t protein
(Fig.
1D). The diagram
demonstrated a cluster of epitopes composed
of three major epitopes:
the first recognized by CET-2, the second
recognized by CET-1, -3, -5, and -6, and the third recognized
by CET-4. The second epitope consisted
of four minor overlapping
epitopes. The first epitope recognized by
CET-2 and the third
recognized by CET-4 did not overlap at
all.
Identification of an antigenic domain on E2 protein.
For
mapping antigenic determinants on HCV E2 protein recognized by CET
MAbs, we screened E2 region by examining the reactivities of CET MAbs
to a series of different recombinant E2 proteins stably expressed in
CHO cell lines. These recombinant CHO cell lines were established by
the construction of seven recombinant expression vectors. The primary
construct was the expression plasmid Rc/CMVdhfr-gDE2t (23),
encoding E2 protein truncated to remove a C-terminal hydrophobic domain
that appeared to anchor the protein in the endoplasmic reticulum
(25, 36). For efficient expression and purification in cell
culture system, the signal sequence of E2 was replaced by the coding
region of HSV-1 gD because HCV E2 protein was shown to be secreted,
depending on the signal peptide or the secretory protein fused to the
E2 protein (23).
Six derivatives of this plasmid were constructed to express five
truncated forms of E2t with different lengths and a chimeric
form
of E2t (Fig.
2A). It was determined whether the six derivatives
of E2t
as well as the original form of E2t were present in the
culture media
by ELISA with HCV type 1b-immune sera. For all seven
constructs, the
culture supernatant of CHO cells showed positive
reactivity to
HCV-immune sera (Table
3).
To identify the epitopes on E2, we tested six CET MAbs for reactivities
with a panel of culture supernatant samples containing
different forms
of E2t. The plasmid expressing gDE2t-1 was designed
to determine
whether the epitopes recognized by CET MAbs were
present within HVR-1
of E2 protein, considered as the major neutralization
epitope
(
6). To minimize the conformational change of the whole
E2
molecule, HVR-1 was replaced with the corresponding region
of another
strain HCV-1, subtype 1a. The culture supernatant samples
of gDE2t and
gDE2t-1 bound to all CET MAbs; affinities were not
significantly
different among the MAbs. The result showed that
HVR-1 was not involved
as an epitope recognized by CET MAbs (Table
3). Whereas the culture
supernatant samples of gDE2A, gDE2B,
and gDE2C did not show binding
activity with any of CET MAbs,
the culture supernatant samples of gDE2D
and gDE2E showed binding
activities with all CET MAbs (Table
3). This
finding indicated
that the epitopes recognized by CET-1 to -6 may exist
in a region
spanning amino acid residues 527 to
560.
For precise mapping of the domain recognized by CET MAbs, we designed
an additional plasmid construct, Rc/CMVdhfr-TPLgDE2D-1,
expressing
a chimeric form of gDE2D with 19 amino acid residues
(aa 528 to 546)
replaced by the corresponding region of HCV-1
of another subtype 1a
(Fig.
2B). The culture supernatant of
recombinant
COS-7 cells expressing gDE2D-1 showed positive reactivity
to HCV-immune
serum (Table
3). However, the binding reactivity was not
shown
with any of CET MAbs. This analysis suggested that the
antigenic
determinants of CET-1 to -6 were located in a small domain
within
E2 spanning amino acid residues 528 to 546.
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FIG. 2.
Schematic diagrams of expression plasmids. (A)
Constructs expressing C-terminally truncated forms of HCV E2 differing
in length and form. All constructs were based on Rc/CMVdhfr-TPLgDE2t.
The E2t cDNA was modified by replacing the
AgeI-XbaI fragment near the full-length E2t gene
with PCR products of partially E2t-derived sequences except E2t-1, in
which the NdeI-AluI segment encoding HVR-1 of E2t
cDNA was replaced by a newly synthesized
NdeI-AluI DNA fragment (described in Materials
and Methods). Names of recombinant expression plasmids are shown at the
left. The striped boxes in E2t-1 and E2D-1 represent the regions
replaced with the corresponding sequences of HCV-1. Positions of amino
acids at both ends of derivatives of E2t cDNA are shown.
PCMV, human cytomegalovirus promoter; BGH polyA, bovine
growth hormone polyadenylation signal; TPL, adenovirus tripartite
leader. (B) Alignment of amino acid sequences of the region replaced in
E2D-1 obtained from HCV-1 (group 1a) and HCV-N (group 1b). Amino acids
are indicated in single-letter code. Positions of amino acids at both
ends of the region are shown. Horizontal bars designate common
sequences. Amino acid numbering is according to Choo et al.
(5).
|
|
| |
DISCUSSION |
It is clear that the development of a broadly reactive vaccine is
the most effective method for preventing hepatitis C. The viral
envelope E2 glycoprotein has been suggested to be responsible for
binding of the virus to target cells, and antibodies to this region
have been proposed to neutralize the virus and to drive immune
selection. Despite the strong variability of the HCV E2 sequences,
certain domains of biological importance (e.g., ligands for viral
attachment to target cells) must be preserved. Thus, determining which
region of E2 is critical in binding to host cell receptors and
identifying the genotype-conserved determinants are very important for
the development of an HCV vaccine (2, 22, 27, 28, 40, 45).
Recently, it was also reported that the neutralizing epitope(s) in HCV
E2 protein may be downstream of HVR-1 (31).
The purpose of our study was to identify the B-cell epitopes in HCV E2
protein by using MAbs specific for the E2 protein. We generated six
murine MAbs specific for HCV E2 protein by using recombinant E2
proteins expressed in CHO cells. It was previously reported that the
full-length E2 protein remained membrane anchored due to the C-terminal
hydrophobic region and that the signal peptide of E2 was not
appropriate for the efficient expression in CHO cells. Therefore, we
designed two fusion constructs, hGHE2t and gDE2t, for the efficient
expression of E2 in CHO cells, and they were detected in the culture
media of the recombinant CHO cells (23).
Since CET MAbs were generated by using nonnatural forms of the HCV E2
protein, we compare the epitope specificities of CET MAbs with those of
anti-E2 antibodies in HCV-immune sera by competitive ELISA. The serum
samples all showed the same HCV serotype, 1b. Therefore, the inhibitory
activity of sera may not be influenced by the serotype of HCV
infection. The data showed that the HCV-immune sera had the anti-E2
antibodies which could bind to the same epitopes that were recognized
by CET MAbs. We could classify the epitopes into three groups: epitopes
recognized by CET-1 (most prevalent), those recognized by CET-3 and -5 (less frequent), and those recognized by CET-2, -4, and -6 (barely detected).
The epitope specificities of CET MAbs were determined by SPR analysis.
We tested the abilities of the MAb to bind simultaneously to the gDE2t
covalently linked to a sensor chip, using the sequential multideterminant binding method. In addition, we performed the competitive ELISA in a pairwise manner. The ELISA data strongly supported the conclusions from the SPR analysis. Since the
conformational changes in the antigen due to the previous binding of
the MAb(s) and the electrostatic interactions between the MAbs may
distort the binding patterns of CET MAbs, the diagram does not
correspond to the actual physical map of the binding sites on E2. These
cases are seen as asymmetry in the reactivity pattern matrix; e.g., CET-1 could not bind to the antigen previously bound with CET-4. However, the binding of CET-1 did not influence that of CET-4 (Fig.
1C). We depict this as the overlap of two circles of different sizes in
Fig. 1D. We interpreted the proposed surface-like map of epitopes shown
in the diagram in accordance with the results of a previous epitope
specificity assay as follows. The major epitope is recognized by CET-1.
The minor epitope recognized by CET-6 neighbors the major one. These
two epitopes do not overlap and are surrounded by the other two minor
epitopes recognized by CET-2 and -4. The other minor epitopes
recognized by CET-3 and CET-5 are depicted as conformational ones and
extremely overlapped. HVR-1 of HCV E2 protein, which is the most
variable region of the HCV genome, has been known to contain linear
epitopes recognized by patient antibodies, although it is disputed
whether the antibodies against HVR-1 can actually neutralize HCV
infection (17, 20, 43). In our experiment, HVR-1 was not an
epitope recognized by CET MAbs.
We used a series of E2 proteins with C-terminal truncations to
determine the locations of the B-cell epitopes recognized by CET MAbs.
The HCV-immune sera were reactive to each truncated type and to
wild-type E2 equivalently, indicating that the overall structure
integrity of each protein is not significantly compromised as a
consequence of the truncations. The binding reactivities of CET MAbs to
the truncated E2 proteins showed an all-or-none binding pattern: all
CET MAbs showed binding activities only with E2t, E2E, and E2D. This
result indicates that the epitopes recognized by CET MAbs all exist in
a region spanning amino acid residues 527 to 560. However, it was also
possible that the epitopes were present in a region upstream of amino
acid residue 527 and that the C-terminal deletion for the construction
of E2C distorted the conformational epitopes. To determine which
explanation was appropriate, we constructed E2D-1, in which the domain
spanning amino acid residues 528 to 546 was replaced with the
corresponding domain of HCV-1, subtype 1a, and found that E2D-1 was
reactive to HCV-immune sera. If the latter explanation were the case,
CET MAbs could bind to E2D-1. However, E2D-1 did not show binding activity with any of CET MAbs, indicating that the epitopes are present
in the region spanning amino acid residues 528 to 546. We compared the
amino acid sequences of this region with those of other HCV strains and
identified an increased divergence in the alignment (Fig.
3). This observation suggests that the
B-cell epitopes in this region may be involved in the genetic drift by immunoselection. Therefore, it will be important to determine whether
the epitopes in this domain are involved in HCV infection.
View larger version (13K):
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|
FIG. 3.
Alignment of 19 amino acids (in single-letter code)
corresponding to the putative B-cell antigenic domain spanning map
positions 528 to 546. The sequence of HCV-N is compared with that from
11 different isolates. Names of strains and subtypes are indicated at
the left. Horizontal bars designate common sequences. Amino acid
numbering is according to Choo et al.(5). GenBank database
accession numbers of the sequences: HCV-1, M62321; HCV-H, M67463;
HC-J1, D10749; HCV-J, D90208; HCV-BK, M58335; HC-JK1, X61596; HC-J4,
D00832; HC-J5, D10076; HC-J6, D00944; HC-J7, D10076; HC-J8, D01221.
|
|
To date, evidence suggestive of the neutralizing role of antibodies
directed by HCV E2 protein has accumulated. However, few data are
available regarding mapping of the B-cell epitopes in E2 protein other
than HVR-1 (27, 28, 40). We report here the identification
of B-cell epitopes in a newly described domain on E2 protein by
measuring the reactivities of truncated or chimeric E2 proteins to
anti-E2 MAbs. Additional studies will be needed for the fine mapping of
the individual epitopes in the putative B-cell immunogenic domain that
we identified. Also, defining the neutralization potential of this
domain will be important for future vaccine development.
| |
ACKNOWLEDGMENTS |
We thank R. Ward, U.S. Environmental Protection Agency, for a
review of the manuscript.
This work was supported by the Korea Science and Engineering Foundation
through the Research Center for New Drug Development at Seoul National University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Immunology, College of Pharmacy, Seoul National University,
Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea. Phone: 82-2-880-7860. Fax: 82-2-885-1373. E-mail: cykang{at}plaza.snu.ac.kr.
| |
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Abstract
Introduction
