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Clinical and Diagnostic Laboratory Immunology, January 2000, p. 58-63, Vol. 7, No. 1
1071-412X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a Monoclonal Antibody and Its
Single-Chain Antibody Fragment Recognizing the Nucleoside
Triphosphatase/Helicase Domain of the Hepatitis C Virus
Nonstructural 3 Protein
Zhu-Xu
Zhang,1
Una
Lazdina,1
Margaret
Chen,1
Darrell L.
Peterson,2 and
Matti
Sällberg1,*
Divisions of Clinical Virology and Basic Oral
Sciences, Huddinge University Hospital, S-141 86 Huddinge,
Sweden,1 and Department of Biochemistry
and Molecular Biophysics, Virginia Commonwealth University,
Richmond, Virginia2
Received 6 July 1999/Returned for modification 19 August
1999/Accepted 6 October 1999
 |
ABSTRACT |
We have produced a murine monoclonal antibody (MAb), ZX10,
recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb
recognized a discontinuous epitope of the NTPase/helicase domain, of
which the linear sequence GEIPFYGKAIPL at residues
1371 to 1382 constitutes one part. cDNAs from variable
regions coding for the heavy and light chains were
cloned, sequenced, and assembled into the NS3-ScFv, which was
inserted into procaryotic and eucaryotic expression vectors.
Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity.
However, the ability to bind the peptide 1371-1382 had been
lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated
by antibodies to HCV NS4A, confirming the in vitro activity of
the NS3 ScFv. Thus, we have designed a functional NS3
NTPase/helicase domain-specific ScFv which should be evaluated
further with respect to disturbing enzymatic functions of the NS3 protein.
| |
INTRODUCTION |
The hepatitis C virus (HCV)
nonstructural 3 protein (NS3) performs vital enzymatic functions in the
HCV life cycle. The N-terminal one-third serine-like protease domain of
the NS3 protein, together with the cofactor NS4A, is required for
cleavage of the junction between NS2 and NS3 and the downstream
NS3/NS4A, NS4A/NS4B, NS4B/NSA5, and NS5A/NS5B junctions (1,
4). The C-terminal two-thirds of the NS3 protein has NTPase and
helicase functions (6, 16). Importantly, the NS3 protein is
also one of most conserved proteins of HCV. Collectively, these
properties suggest that the NS3 protein may be a suitable target for
antiviral therapy.
The variable regions of the heavy and light chains (VH and VL,
respectively) of an antibody are essential for the recognition of
antigens. Single-chain variable-region fragments (ScFvs) of antibodies, containing VH and VL DNA assembled by a flexible linker sequence, allow for intracellular expression of antibody fragments. Several reports have demonstrated that intracellularly expressed ScFvs
may reduce the susceptibility of transfected cells to viral infections
or disturb viral enzymes within the life cycles of human
immunodeficiency virus type (9, 20), tick-borne encephalitis virus (5), and murine coronavirus (8). The
production of ScFvs to the HCV core and envelope proteins was recently
described (2). This approach therefore offers an alternative
antiviral therapy by blocking vital steps in the viral life cycle
inside infected cells. We have developed a murine anti-NS3 monoclonal antibody (MAb) which recognizes the NS3 ATPase/helicase domain. From
the NS3-specific MAb we designed and produced a functional NS3-specific
ScFv by use of procaryotic and eucaryotic expression systems.
| |
MATERIALS AND METHODS |
Cells.
The SP2/0 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, (FCS), 2 mM
L-glutamine, 100 U of penicilin per ml, and 100 µg of
streptomycin per ml (GIBCO-BRL, Gaithersburg, Md.). The BHK cell
line was maintained in Dulbecco modified Eagle medium (GIBCO-BRL)
with 10% fetal calf serum and antibiotics. All cells were incubated at
37°C with 7% CO2.
rNS3 and synthetic peptides.
The expression, purification,
and enzymatic characterization of a recombinant HCV NS3 (rNS3),
genotype 1a, covering residues 1007 to 1612 has previously been
described (6). Synthetic peptides covering the
ATPase/helicase domain of HCV NS3 have been described previously
(15, 18). Additional peptides covering the identified epitope region were synthesized by using an automated synthesizer (Syro; Syntex, Tubingen, Germany) as described previously
(19).
MAb production and characterization.
BALB/c mice were
immunized and boosted with rNS3 (10 µg) three times every 2 weeks.
Three days after the last injection, spleen cells were harvested and
fused with the SP2/0 myeloma cells by standard procedures. Following
three cycles of cloning and screening by enzyme immunoassay (EIA), a
stable hybridoma, ZX10, was selected for antibody analysis and
extraction of mRNA. Purification of the ZX10 antibody was performed by
using immobilized protein A/G (Pierce, Rockford, Il.). Analysis of the
binding characteristics and the fine specificity of the ZX10 antibody
was done by using EIAs as previously described (14, 15).
Construction and expression of ScFv- and HCV NS3/NS4A-encoding
plasmids.
Total cellular mRNA was extracted from the ZX10
hybridoma by using magnetic beads coated with oligo(dT)25
(Dynal A.S., Oslo, Norway). The VH and VL of NS3 MAb ZX10 were
amplified from cDNA by PCR with a recombinant phase antibody system
(Pharmacia Biotech, Uppsala, Sweden). The assembled NS3-ScFv cDNA
fragment was directly ligated to the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.). The DNA sequence was determined with
an automated sequencer (ALF express; Pharmacia). The NS3-ScFv DNA was
reamplified to introduce the cloning sites and to omit the leader sequence.
For Escherichia coli expression, the forward primer 5'-CCG
CTC GAG CAG GTG AAG CTG CAG-3' and reverse primer
5'-CGG ATC CTA CCG TTT GAT TTC CAG-3' were used to amplify
the NS3-ScFv to introduce 5' BamHI and 3' XhoI
restriction sites (underlined). The digested fragment was inserted into
the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to
give NS3-ScFv-pET.
For eucaryotic expression, the primer pair 5'-CCG
GGT ACC
ACA GGT GAA GCT GCA G-3' and 5'-GCC
TCT AGA CTA CCG TTT GAT
TTC
CAG-3' was used to introduce
KpnI and
XbaI
restriction sites (underlined).
After PCR amplification, the
reamplified NS3-ScFv was digested
with
KpnI and
XbaI and then inserted in the pcDNA3.1/His A vector
(Invitrogen). This produces an NS3-ScFv (NS3-ScFv-pcDNA) which
should
be retained
intracellularly.
A 2.1-kb DNA fragment of HCV NS3/NS4A, encoding a region covering the
amino acid sequence from HCV position 1007 to 1711,
was amplified with
high-fidelity polymerase (Expand High Fidelity
PCR; Boehringer,
Mannheim, Germany) from a patient infected with
HCV genotype 1a and was
inserted into a
BamHI- and
XbaI-digested
pcDNA3
vector (Invitrogen). All expression constructs were sequenced
once
again to ensure that the reading frame was correct. The cloning
and
characterization of this gene will be described in detail
elsewhere
(unpublished
data).
Characterization of the NS3-ScFv.
The expression of
NS3-ScFv-pET was induced with IPTG
(isopropyl-
-D-thiogalactopyranoside) in E. coli BL21 transformants. The expressed protein was purified by
using an Ni-nitrilotriacetic acid (Ni-NTA) chelating resin under
denaturating conditions according to the recommendation of the
manufacturer (Qiagen Gmbh, Hilden, Germany). The cells were pelleted
and lysed in 8 M urea in Tris-HCl buffer, pH 8. The clarified
supernatant was loaded on the Ni-NTA resin column. The bound protein
was eluted with Tris buffer (pH 5.9, 5.0, and 4.0) containing 8 M urea
or containing 0.1 M EDTA. Protein-containing fractions were analyzed by
enzyme-linked immunosorbent assay and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Expression of NS3-ScFv-pET was confirmed by Western blot analysis. The
samples containing NS3-ScFv-pET were applied on an
SDS-PAGE minigel
(PhastSystem; Pharmacia) and transferred electrophoretically
to a
nitrocellular membrane (Pharmacia). The gel was developed
by silver
staining. The membrane was blocked with a 2% bovine
serum
albumin-phosphate-buffered saline solution for 1 h at room
temperature or overnight at 4°C prior to probing with Ni-NTA
conjugate
(Qiagen) or a goat anti-mouse Fab conjugate (A-2179; Sigma,
St.
Louis, Mo.). The bands were visualized with an insoluble substrate,
BCIP-NBT (B-5655;
Sigma).
The specificity of the purified NS3-ScFv-pET was determined by both
direct and competitive EIAs. The NS3-ScFv-pET, alone or
in mixture with
MAb ZX10, was added to plates coated with 1 µg
of rNS3 per ml. In the
direct EIA, anti-Xpress antibody (Invitrogen)
directed against an
epitope carried by the NS3-ScFv-pET fusion
protein was added prior to
addition of a goat anti-mouse antibody
conjugate (A-1047; Sigma). In
the competitive EIA, the amount
of remaining ZX10 MAb was indicated by
the goat anti-mouse antibody
conjugate (A-1047; Sigma). The enzyme
reaction was developed by
addition of 100 µl of
p-nitrophenyl phosphate (Sigma), and optical
densities were
read at 405
nm.
Coexpression of NS3-ScFv and HCV NS3/NS4A.
To further
characterize the specificity of the NS3-ScFv and the ability to
recognize coexpressed HCV NS3 in a eucaryotic system, the T7 coupled
reticulocyte lysate system (Promega, Madison, Wis.) was used. In vitro
translation of the NS3-ScFv-pcDNA and control pcDNA vectors was carried
out at 30°C with [35S]methionine (Amersham
International plc, Buckinghamshire, United Kingdom). The labeled
proteins were separated on an SDS-12% polyacrylamide gel and
visualized by exposure to X-ray film (Hyper film-MP, Amersham) for 6 to
18 h. NS3-ScFv and NS3 complex formation was analyzed by
immunoprecipitation. Protein A/G-Sepharose beads (Pierce) were incubated with the anti-Xpress antibody (Invitrogen) or anti-NS4A mouse
serum (19) at 4°C for 1 h. The gel was then washed
with 0.05 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 0.15 M NaCl, 0.25% gelatin, and 0.02% NaN3 (NET buffer). The antibody-loaded
protein A/G-Sepharose was then incubated for 2 h at 4°C under
agitation together with a mixture of separately translated NS3/4A and
NS3-ScFv or with these two proteins cotranslated. The
immunoprecipitates were washed five times with NET buffer supplemented
with 0.5 M NaCl and 0.3% SDS. The pellet was resuspended in 20 µl of
2× SDS sample buffer and heated at 100°C for 3 min. The proteins
were then analyzed by SDS-PAGE.
| |
RESULTS |
Production of a MAb (ZX10) against HCV NS3.
The specificity of
the NS3 MAb ZX10 was characterized by using rNS3 and 16-amino-acid
synthetic peptides covering the ATPase/helicase domain of NS3
(Fig. 1). The epitope of the ZX10 MAb
could be finely mapped to the linear sequence
GEIPFYGKAIPL at residues 1371 to 1382 (Fig.
2). A much lower reactivity to the linear
synthetic peptide corresponding to NS3 residues 1367 to 1382 than to
rNS3 was observed (Fig. 3). This region
is similar to the one identified by a human MAb (12). Thus,
the ZX10 MAb most likely recognizes a conformationally dependent
epitope of which the linear sequence GEIPFYGKAIPL at
residues 1371 to 1382 may be a part in composing the complete epitope.
Overall, the reactivity of the ZX10 MAb is similar to those previously
observed in immunized mice and HCV-infected humans (3, 7, 15,
18).
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FIG. 1.
Characterization of the specificity of binding of MAb
ZX10 to rNS3 and 16-amino-acid synthetic peptides with a 6-amino-acid
overlap by solid-phase EIA. OD, optical density.
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FIG. 2.
Fine mapping of the peptide-specific reactivity by using
deletion analogues of the NS3 peptide covering residues 1367 to 1382 by
solid-phase EIA. OD, optical density.
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FIG. 3.
Characterization of the preferred binding of MAb ZX10 by
testing serial dilutions of MAb ZX10 with rNS3 ( ) and the peptide
p1367 to 1382 ( ) by solid-phase EIA. OD, optical density.
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Construction of ZX10-ScFv expression vectors.
Sequence
analysis using the V BASE database with the DNAPLOT
software (http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO .html) showed that the rearranged VH and VL domains of the ZX10 MAb used sequences from the VH1 and V
III gene families (Fig.
4). The NS3-ScFv-pET and NS3-ScFv-pcDNA
vectors were constructed as outlined in Fig. 4. Sequence analysis did
not show any sequence alterations during the cloning steps
(data not shown). The DNA corresponding to complete NS3-ScFv together with the (Gly4Ser)3 linker
comprises about 780 bp and encodes a protein of 32 kDa
(Fig. 2).
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FIG. 4.
Deduced amino acid sequence of NS3-ScFv and outline for
amplification and cloning of NS3-ScFv in the pET and pcDNA3.1/His
vectors. The thin solid line indicates the VH sequence, the thick solid
line indicates the linker sequence, and the dotted line indicates the
VL sequence of the NS3-ScFv. Primers containing the indicated
restriction site are indicated by horizontal half arrows.
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E. coli-expressed NS3-ScFv contains histidine residues as an
amino-terminal leader sequence, which facilitates purification
and
detection. Both soluble and insoluble fractions were isolated,
and the
NS3-ScFv protein was located in the insoluble fraction.
The NS3-ScFv
was solubilized in Tris buffer (pH 8.0) containing
8 M urea and was
purified by use of Ni-NTA chelating resin. The
eluted fractions were
analyzed by Western blotting. A detectable
32-kDa NS3-ScFv band,
corresponding to the expected molecular
mass, was identified by using
Ni-NTA conjugate (Fig.
5).
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FIG. 5.
Analysis of NS3-ScFv-pET expressed in E. coli
by use of a silver-stained SDS minigel (right) and by Western blotting
(left). Total E. coli lysate of the pET-15b vector without
insert (lanes 1 and 7) and lysate of the E. coli pellet
containing NS3-ScFv-pET (lanes 2 and 8) or containing the NS3-ScFv-pET
eluted from the Ni-NTA column by Tris buffer with 8 M urea at pH 5.0 (lanes 3 and 9) or pH 4.0 (lanes 5 and 11) or with 0.1 M EDTA (lanes 6 and 12) are shown. Lanes 4 and 10, molecular size markers. The Western
blot was developed with an Ni-NTA conjugate and the insoluble substrate
BCIP-NBT.
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Binding activity of ZX10-ScFv.
E. coli-expressed
NS3-ScFv-pET was characterized by EIA. First, a 
chain-specific
antibody (A-1047; Sigma) did not recognize the NS3-ScFv (Fig.
6a), whereas a Fab-specific antibody
(A-2197; Sigma) bound the immobilized NS3-ScFv (Fig. 6a). Second,
serial dilutions of NS3-ScFv in renaturating buffer (20% glycerol, 0.5 M NaCl, 2 mM -mercaptoethanol, 0.05 M Tris-HCl, pH 7.4) were mixed
with a constant concentration of MAb ZX10. The NS3-ScFv inhibited the
binding of the ZX10 MAb to rNS3, confirming the specificity of the
NS3-ScFv. As a negative control, pET-15b without any insert but
expressed, purified, and diluted exactly like the NS3-ScFv was used and
had no effect on the binding of ZX10 to NS3 (Fig. 6). Moreover, despite
repeated experiments, we could not observe that the NS3-ScFv bound to
the epitope peptide of the ZX10 MAb (data not shown). Overall, these
experiments show that the recognition of rNS3 was retained but that the
fine specificity, or the avidity, of the NS3-ScFv was reduced.
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FIG. 6.
(a) E. coli-expressed ScFv in renaturating
buffer applied directly to microplates was indicated by chain- and
Fab-specific antibodies. (b) Analysis of the inhibition of MAb ZX10
binding to rNS3 by NS3-ScFv-pET or pET-15b vector without insert, both
expressed and purified from E. coli and diluted in
renaturating buffer. Residual binding of ZX10 was indicated by goat
anti-mouse immunoglobulin G. OD, optical density.
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|
The interaction between the NS3-ScFv and HCV NS3 was further evaluated
by using eucaryotic expression systems. An immunoprecipitation
assay
was used for analysis of the interaction between NS3-ScFv-pcDNA
and
NS3/4A expressed in a rabbit reticulocyte lysate in vitro
translation
system. As positive controls, anti-NS4A immunoprecipitated
in
vitro-translated NS3/4A (Fig.
7, lane 1),
whereas anti-Xpress
immunoprecipitated in vitro-translated NS3-ScFv
(Fig.
7, lane
4). Coexpressed NS3-ScFv and NS3/4A could be
immunoprecipitated
by either anti-NS4A (Fig.
7, lane 3) or anti-Xpress
(Fig.
7, lanes
2 and 7). Moreover, the mixture of separately translated
NS3/4A
and ZX10-ScFv also formed a complex that was immunoprecipitated
by anti-NS4A (Fig.
7, lane 5) or by anti-Xpress (Fig.
7, lane
6). Thus,
the NS3-ScFv-pcDNA plasmid expresses a functional single-chain
antibody
fragment that recognizes and binds coexpressed HCV NS3.
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FIG. 7.
Analysis of protein-protein interaction of the
NS3-ScFv-pcDNA and NS3/4A proteins translated in vitro by a rabbit
reticulocyte translation assay followed by immunoprecipitation. The
proteins were labeled with [35S]methionine during
translation and were then immunoprecipitated as described in Materials
and Methods. Proteins were separated by SDS-8% PAGE. The
NS3-ScFv-pcDNA and NS3/4A proteins and the sizes of the molecular mass
markers are indicated. Positive controls are NS3/4A immunoprecipitated
by anti-NS4A (lane 1) and NS3-ScFv-pcDNA immunoprecipitated by
anti-Xpress (lane 4). Coexpressed NS3/4A and NS3-ScFv-pcDNA were
immunoprecipitated by anti-NS4A serum (lane 3) and antibody against
Xpress (lanes 2 and 7). Mixtures of separately translated NS3/4A and
NS3-ScFv-pcDNA were also immunoprecipitated by anti-NS4A (lane 5) and
anti-Xpress (lane 6) antibodies.
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| |
DISCUSSION |
The HCV NS3 protein, which is most likely not present within HCV
virions, can be detected inside the infected host cell. During HCV
infection, the NS3 protein participates in the processing of the HCV
polyprotein precursor and unwinding of viral RNA helices. Therefore, it
may be possible to interfere with the enzymatic functions of NS3, which
may disturb the viral life cycle. Intracellular therapies against HCV
infection have been tested by using antisense oligonucleotides and
ribozymes (10, 17). In addition, recombinant antibody
fragments, or ScFv, expressed intracellularly have been demonstrated to
inhibit numerous viruses (5, 8, 9, 11, 20).
We first produced MAb ZX10 against the NTPase/helicase domain of HCV
NS3. The epitope of the ZX10 MAb could be finely mapped to the linear
sequence GEIPFYGKAIPL at residues 1371 to 1382. However, the ZX10 MAb
recognized rNS3 around 36 times more efficiently than the linear
synthetic peptide epitope, which suggests that the complete epitope for
the ZX10 MAb is conformationally dependent. This is not a unique
phenomenon. We have previously characterized a MAb to the hepatitis B
core antigen (HBcAg) that preferentially binds native HBcAg (13,
14). Within HBcAg one linear sequence could be precisely mapped,
which most likely constitutes a part of the complete discontinuous
epitope (13, 14). However, the peptide binding was of very
low affinity, since in solution the peptide was unable to block the
binding of the MAb to recombinant HBcAg (13). Due to the
significant similarities, we propose the same type of binding
characteristics for the HCV NS3-specific MAb ZX10. Importantly, the
ZX10 MAb seems to recognize HCV NS3 in a similar way as do humans
(3). We therefore designed an intracellular ScFV based on
the ZX10 MAb.
The obtained sequences from the VH and VL domains of the ZX10 hybridoma
showed that the rearranged sequences used the gene families VH1 and
VIII. The VH and VL cDNA fragments from the ZX10 hybridoma were
assembled by a linker sequence to the NS3-ScFv, which was cloned into
both procaryotic and eucaryotic expression vectors. E. coli-expressed NS3-ScFv retained most of the binding characteristics of the original MAb despite the recombination of the
VH, VL, and linker DNA fragments. However, we were unable to repeat the
peptide recognition exerted by the ZX10 MAb with the NS3-ScFv. This
indicates that the NS3-ScFv may have a slightly altered fine
specificity or that the affinity had been reduced. This is not
surprising, since the peptide recognition of the ZX10 MAb was rather
weak and presumably of low affinity.
Studies were performed to mimic the intracellular eucaryotic expression
of HCV NS3 and the NS3-ScFv. By in vitro translation assays we found
that the NS3-ScFv recognized and bound cotranslated NS3/NS4A, since the
complex could be immunoprecipitated by antibodies to NS4A. This
verifies that most of the binding activity of the ZX10 MAb was also
retained by the recombined VH and VL domains expressed by the
NS3-ScFv-pcDNA. We also noted that the NS3-ScFv could be expressed
transiently and stably by several cell lines, indicating that the
NS3-ScFv expression was nontoxic (data not shown). Collectively, this
suggests that we have designed a eucaryotic vector coding for a
single-chain antibody specific for the NTPase/helicase domain of HCV
NS3 which can be stably expressed by mammalian cells. Hypothetically,
intracellularly expressed NS3-ScFv may recognize endogenously expressed
NS3 protein and may thereby possibly interfere with its intracellular functions.
| |
ACKNOWLEDGMENTS |
Financial support was obtained from the Swedish Medical Research
Council (grant K98-06X-12617-01A) and funds provided by the School of
Dentistry at the Karolinska Institutet.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division
of Clinical Virology, F 68, Huddinge University Hospital,
S-141 86 Huddinge, Sweden. Phone: 46-8-5858 7939. Fax: 46-8-5858 7933. E-mail: misg{at}labd01.hs.sll.se.
| |
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Clinical and Diagnostic Laboratory Immunology, January 2000, p. 58-63, Vol. 7, No. 1
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
