Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein.

Abstract

The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported.
In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae.
The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.

Full text

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RESEARCHOpen Access
Identification of a conserved JEV serocomplex
B-cell epitope by screening a phage-display
peptide library with a mAb generated against
West Nile virus capsid protein
En-Cheng Sun1,2, Jing Zhao1,2, Tao Yang1, Ni-Hong Liu1, Hong-Wei Geng1, Yong-Li Qin1,2, Ling-Feng Wang1,2,
Zhi-Gao Bu1, Yin-Hui Yang3, Ross A Lunt4, Lin-Fa Wang4, Dong-Lai Wu1*
Abstract
Background: The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates
the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites
on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined
B-cell epitopes on the WNV C protein have not been reported.
Results: In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear
epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb,
designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence
KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was
conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the
minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated
that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus
(JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum.
Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of
the Family Flaviviridae.
Conclusion: The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb
generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV
serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV
serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.
Background
West Nile virus (WNV) is a positive-sense, single-
stranded RNA virus of the family Flaviviridae, genus
Flavivirus. It is a member of the Japanese encephalitis
virus (JEV) serocomplex, which is comprised of several
medically important viruses including WNV, JEV, Saint-
Louis encephalitis virus (SLEV) and Murray Valley fever
virus (MVEV) [1,2]. The close antigenic relationship of
viruses belonging to the JEV serocomplex accounts for
the serologic cross-reactivity seen in diagnostic
laboratories.
The 10.7-kilobase WNV genome is translated into a
single polyprotein, which is subsequently processed by
viral- and host-encoded proteases into structural and
nonstructural proteins. Three structural proteins (C,
prM/M and E) make up the viral particle and seven
nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A,
NS4B and NS5) are required for genome replication and
polyprotein processing [3].
The capsid (C) protein is the building block of the
nucleocapsid. The C protein is a small 12 kD protein
* Correspondence: dlwu@hvri.ac.cn
1The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State
Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China
Full list of author information is available at the end of the article
Sun et al. Virology Journal 2011, 8:100
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© 2011 Sun et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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composed of 105 amino acids, and is highly positively
charged due to a large number of lysine and arginine
residues. The charged residues are clustered at the N- and
C-terminal ends, and are separated by an extremely con-
served internal hydrophobic region which mediates mem-
brane association [4]. The nascent capsid protein also
contains a C-terminal hydrophobic anchor that serves as a
signal peptide for the endoplasmic reticulum translocation
of the membrane precursor [5]. The secondary structure
of recombinant C protein from Dengue virus (DENV) 2
and Yellow Fever virus(YFV), as determined by NMR
techniques, shows that flavivirus C proteins are predomi-
nately dimeric in solution and are composed of four alpha
helices (a1-a4), in which the N terminus (residues 1-20) is
conformationally labile or unstructured [6]. The first eluci-
dated 3 D structure of DENV C protein dimer (residues
21-100) suggested possible mechanisms for its interactions
with RNA and the viral membrane [7].
Flavivirus C proteins are targeted by host immune
responses. The specificities of a serotype-specific human
CD4+ cytotoxic T-lymphocyte clone (CTL) and a panel
of serotype cross-reactive human CD4+ CTL have been
mapped to epitopes contained within the DENV4 C pro-
tein, indicating that anti-viral T cell responses are direc-
ted against C protein-derived peptides [8]. Further, the
production and characterization of anti-DENV C antibo-
dies suggests that the N terminus region covering the
first 20 amino acids of DENV C protein is the predomi-
nant target of humoral immune responses in mice [9].
The aim of our study was to identify WNV-specific and/
or JEV serocomplex-specific B-cell epitopes on C protein
using phage display technology. Phage display has proven
to be a powerful and economic technique for epitope iden-
tification and has been used widely in epitope mapping in
flaviviruses [10-13]. The results described in this report
will facilitate the development of diagnostic tests for the
specific serological evaluation of WNV/JEV serocomplex
infection and further understanding of the antigenic struc-
ture of C protein which will benefit the rationale design of
JEV serocomplex vaccines.
Results
Production of recombinant C protein
The recombinant WNV C protein used as antigen for
monoclonal antibody generation was considered firstly.
A baculovirus expression system was used to produce
recombinant WNV C protein in Sf9 insect cells. The
recombinant C protein generated in insect cells was
recognized by antibodies contained in WNV-positive
equine serum by Western blot (WB) (Figure 1).
Production and characterization of C protein-specific mAb
Purified C protein was used to immunize BALB/c mice.
After cell fusion and screening, several hybridoma cell
lines were generated which produced C-reactive mAbs.
Among them, the antibody produced by the line desig-
nated as 6D3 was selected for strong reactivity against
recombinant C protein in WB (Figure 2, panel a) and in
an indirect ELISA (data not shown). The 6D3 mAb also
showed strong reactivity against WNV antigen slides by
an indirect immunofluorescence assay (IFA; Figure 2,
panel b). The 6D3 mAb recognized the JEV serocom-
plex viruses WNV and JEV by IFA, while no reactivity
against the non-JEV serocomplex flaviviruses DENV1-4,
YFV and Tick-borne encephalitis virus (TBEV) was seen
(data not shown).
The 6D3 mAb was composed of an IgG1 heavy chain
paired with a l-type light chain, as determined using
the Mouse MonoAb-ID Kit (HRP). The titer of antibody
in hybridoma cell culture supernatants and in ascities
was measured by indirect ELISA and was determined to
be 1:512 and 1:1,024,000, respectively.
Phage enrichment by biopanning
The purified 6D3 mAb was used to pan a phage dis-
played peptide library to determine the fine specificity of
the C protein-specific mAb. After three rounds of bio-
panning, a marked enrichment of phages was achieved
from the phage displayed 12-mer library. The output to
Figure 1 Recombinant C protein is recognized by antibodies in
WNV-positive equine serum. Purificated recombinant C protein
(lane 1) and cell lysates from insect cells infected with wild-type
baculovirus (lane 2) were evaluated for reactivity with serum from
WNV-positive horses. M: PageRuler™ Prestained Protein Ladder
(Fermentas, Canada).
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input ratio following each of the three rounds of biopan-
ning was 0.00018%, 0.024% and 0.89%.
Epitope prediction
Ten phage clones were selected for reactivity with 6D3
following enrichment of the phage display peptide library.
These selected clones were further evaluated by ELISA
for reactivity with the 6D3 mAb and a negative control
mAb (anti-porcine IFN-g). As shown in Figure 3, the 6D3
mAb reacted with each clone (F1-F10), giving optical
density readings at 492 nm (OD492 nm) greater than 1.0.
In contrast, the negative control antibody gave low
OD492 nm readings (OD492 nm < 0.2). These data indi-
cate that the 6D3 mAb specifically reacts with the ten
phage clones that were selected following three rounds of
enrichment of the peptide library with 6D3. We next
sequenced the peptide insert of the ten selected phage
clones that reacted with the 6D3 mAb. An alignment of
the peptide insert sequences indicated that six 6D3-reac-
tive clones (F1-F6) displayed a consensus peptide
sequence of KKPGGPG. The consensus sequence motif
defined by the peptide library screen are identical to the
sequence3KKPGGPG9found in WNV C protein (Figure
4), indicating that peptide library screen successfully
identified the C protein epitope recognized by 6D3.
Fine mapping of epitope
For further epitope determination, we generated a series
of truncated peptides derived from the KKPGGPG
peptide that was identified by screening the peptide
library with the 6D3 mAb. The full-length and truncated
peptides (Table 1) were generated as MPB fusion pro-
teins and were used in WB analysis with the 6D3 mAb.
We found that only the full-length KKPGGPG polypep-
tide (MBP-Hp-1) was recognized by mAb 6D3 (Figure
5). Removal of one or more amino acids at either the
amino or carboxy terminus of the polypeptide abolished
antibody binding, indicating that the polypeptide
KKPGGPG is the minimal linear epitope recognized by
6D3 (Figure 5).
WNV- and JEV-positive serum reactivity with the
identified epitope
To assess whether the minimal linear epitope was
immunogenic in the context of JEV serocomplex infec-
tion, we tested WNV- and JEV-positive equine serum
for antibodies specific for the KKPGGPG polypeptide
expressed as an MBP fusion protein (MBP-Hp-1).
Serum from WNV-positive horses (Figure 6, panel a)
and JEV-positive horses (Figure 6, panel b) reacted with
the MBP-Hp-1 fusion protein containing the KKPGGPG
epitope, but not with MBP protein alone. Serum from
DENV1-4 positive mice did not react with the MPB-
Hp-1 fusion protein (Figure 6, panels c-f). These data
were further confirmed by ELISA (data not shown).
These results demonstrate that the minimal linear B-cell
epitope is targeted by humoral immune responses in the
context of bona fide JEV serocomplex virus infection.
Figure 2 The mAb 6D3 recognizes recombinant C protein and WNV-infected C6/36 cells. (a) Recombinant C protein (lane 1) and cell
lysates from insect cells infected with wild-type baculovirus (lane 2) were evaluated for reactivity with mAb 6D3 by WB. M: PageRuler™
Prestained Protein Ladder (Fermentas, Canada). (b) Analysis of immunofluourescence produced by (i) mAb 6D3, (ii) a negative control mAb (anti-
porcine IFN-g mAb), (iii) a positive control serum (WNV-positive mouse serum), and (iv) a negative control serum (WNV-negative mouse serum)
on acetone-fixed, WNV infected C6/36 cells (WNV antigen slides).
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Sequence similarity and prediction of cross-reactivity
We then evaluated the conservation of the KKPGGPG
epitope among viruses of the JEV serocomplex. Analysis
of C protein sequences from 28 different JEV serocom-
plex isolates demonstrates that the epitope recognized by
6D3 is conserved among the JEV serocomplex, with the
exception of SLEV C protein, in which a G-to-K muta-
tion is found (Figure 7). The motif is absent in non-JEV
serocomplex members of Flaviviridae family (Figure 7).
Discussion
Monoclonal antibodies with well defined epitopes have
become a powerful tool to study protein structure and
have been widely used to diagnose and treat a variety of
infectious agents [14-18]. The binding properties of anti-
bodies can be used experimentally to define antigenic
structures of pathogen-associated proteins and to under-
stand virus-antibody interactions at a molecular level. In
this study, we described the generation and epitope
mapping of a WNV C-protein-specific mAb, and
demonstrate that the epitope is conserved among many
JEV serocomplex members. Precise analysis of WNV C
protein epitopes will lead to a better understanding of
host immune responses, the development of epitope-
based marker vaccines, and diagnostic tools for WNV
and/or JEV serocomplex infection.
Phage display is an in vitro selection technique in
which a peptide or protein is genetically fused to a coat
protein of bacteriophage, resulting in display of the
fused protein on the exterior of the phage virion. A
phage display library can consist of either random pep-
tide libraries or gene-targeted libraries, thus providing a
powerful and economic technique for epitope identifica-
tion. This technology can identify amino acids on pro-
tein antigens that are critical for antibody binding and,
further, can define peptide motifs that are both struc-
tural and functional mimotopes of both protein and
non-protein antigens [19,20].
In our current study, we generated a C protein specific
mAb, named 6D3, using recombinant C protein
expressed in insect cells by a recombinant baculovirus
system. We found that the 6D3 mAb reacted with WNV
and JEV by IFA, but not with other non-JEV serocomplex
flaviviruses, such as DENV1-4, YFV and TBEV. The lin-
ear epitope recognized by the 6D3 mAb was defined as
KKPGGPG using phage display technology to perform a
screen of a peptide library. This peptide sequence directly
corresponded to a region of WNV C protein with the
Figure 3 The mAb 6D3 specifically recognizes clones selected from the phage display peptide library. Ten clones selected after three
rounds of biopanning of a phage display peptide library used the 6D3 mAb by phage-ELISA. The anti-porcine IFN-g mAb served as a negative
control.
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sequence3KKPGGPG9. Further fine mapping using trun-
cation mutants revealed the core determinant of the
mAb binding site was KKPGGPG. The peptide was also
recognized by WNV/JEV-positive equine serum, indicat-
ing that the epitope is immunogenic in horses in the con-
text of viral infection. Consistent with analysis of cross
reactivity using IFA and WB with WNV/JEV-positive
equine and DENV1-4-positive mouse serum, sequence
alignments of JEV serocomplex sequences demonstrated
that the motif is highly conserved among JEV serocom-
plex members, but is absent in other viruses of the Flavi-
virus genus.
Conclusions
We have generated the C protein-specific 6D3 mAb and
shown that it recognizes a linear epitope that is highly
conserved among the JEV serocomplex. The 6D3 mAb
has great potential to improve JEV-serocomplex diag-
nostic tests and aid the design of robust epitope-based
vaccines.
Methods
Cell lines, plasmid and serum specimens
The myeloma cell line SP2/0 was cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Invitrogen) in a
Figure 4 Alignment of 12-mer peptide sequences from ELISA-positive clones defines a linear epitope for the mAb 6D3. The peptide
insert from ten phage clones that reacted with the mAb 6D3 were aligned. Conserved amino acid residues are boxed and the consensus
sequence motif is provided below the alignment. The matching sequence3KKPGGPG9of the WNV C protein is provided at the bottom of
alignment for comparison.
Table 1 The oligonucleotides encoding full-length and truncated versions of the KKPGGPG motif
Designations of oligonucleotides The sequences of oligonucleotidesCoding motifs (designations)
Hp-1-F
Hp-1-R
Hp-2-F
Hp-2-R
Hp-3-F
Hp-3-R
Hp-4-F
Hp-4-R
Hp-5-F
Hp-5-R
5’-AATTCaagaaaccaggagggcccggcTAAG-3’
5’-TCGACTTAgccgggccctcctggtttcttG-3’
5’-AATTCaaaccaggagggcccggcTAAG-3’
5’-TCGACTTAgccgggccctcctggtttG-3’
5’-AATTCaagaaaccaggagggcccTAAG-3’
5’-TCGACTTAgggccctcctggtttcttG-3’
5’-AATTCccaggagggcccggcTAAG-3’
5’-TCGACTTAgccgggccctcctggG-3’
5’-AATTCaagaaaccaggagggTAAG-3’
5’-TCGACTTAccctcctggtttcttG-3’
KKPGGPG (Hp-1)
KPGGPG (Hp-2)
KKPGGP (Hp-3)
PGGPG (Hp-4)
KKPGG (Hp-5)
Notes: Bases introduced to form the overhanging ends of EcoR I and Sal I after annealing the two complementary oligonucleotides are shown in capital letters;
stop codons TAA are added.
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humidified 5% CO2atmosphere at 37°C. All culture
media were supplemented with 10% heat-inactivated
fetal bovine serum (GIBCO, Invitrogen), 0.1 mg/ml
of streptomycin and 100 IU/ml of penicillin. The
WNV NY99 genome (GenBank accession number
AY842 931.3) was cloned into plasmid pMAL™ -C2x
(New England Biolabs, Inc., USA), and JEV-positive/
negative equine serum and DENV1-4-positive/negative
mouse serum were maintained in our laboratory. WNV-
positive/negative mouse serum was obtained from the
Beijing Institute of Microbiology and Epidemiology, and
the WNV-positive equine serum was from the CSIRO
Australian Animal Health Laboratory (AAHL).
Expression of recombinant C protein
Recombinant WNV C protein was prepared according
to the product instructions of the Bac-to-Bac®Baculo-
virus Expression System (Invitrogen). In brief, the C
gene from WNV NY99 strain was cloned into the pFast-
Bac™ vector. The recombinant pFastBac™ vector was
then transformed into competent DH10Bac™ E. coli
cells, which were subsequently plated on triple antibiotic
LB plates (Kanamycin, Gentamicin and tetracycline)
with BluoGal. The site-specific transposition reaction
takes place between the mini-Tn7 elements (pFastBac™
vector) and the mini-attTn7 attachment sites on the
bacmid DNA in DH10Bac™. This reaction is mediated
by a transposase, an enzyme encoded by the helper plas-
mid that is also in DH10Bac™ E. coli. This transposition
step disrupts the lacZ reading frame and allows blue/
white screening. Colonies containing the recombinant
bacmid DNA appear white, while colonies containing
the non-recombinant bacmid DNA appear blue. Bacmid
DNA was recovered from white colonies and was subse-
quently verified via PCR. Insect cells were transfected
with recombinant Bacmid DNA by using Cellfectin®.
Recombinant baculovirus supernatant was harvested 2-5
days after transfection, and was titered using the Baculo-
Titer™ Assay Kit according to manufacturer’s instruc-
tions. Recombinant protein was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) and purificated by Ni-nitrilotriacetic acid affinity
chromatography (Qiagen) according to the manufac-
turer’s instructions, then identified by WB. For WB,
recombinant C protein and Sf9 cells infected with wild-
type baculovirus were subjected to electrophoresis on
Figure 5 KKPGGPG is the minimal epitope recognized by the mAb 6D3. Full length and truncated versions of the KKPGGPG epitope were
expressed as MBP fusion proteins and assessed for reactivity with the mAb 6D3 by WB. MBP-Hp-1, KKPGGPG; MBP-Hp-2, KPGGPG; MBP-Hp-3,
KKPGGP; MBP-Hp-4, PGGPG; MBP-Hp-5, KKPGG; M: PageRuler™ Prestained Protein Ladder (Fermentas, Canada).
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10% SDS-PAGE after reduction with dithiothreitol
(DTT) at 100°C for 5 min. Samples were transferred to
a nitrocellulose membrane and were blocked overnight
with 5% skim milk powder in PBST at 4°C. The mem-
brane was incubated with WNV-positive equine sera as
the primary antibody, followed by an HRP-conjugated
rabbit anti-equine secondary antibody (LICOR Bios-
ciences). The color was developed using 3,3’-diamino-
benzidine tetrahydrochloride (DAB) substrate and was
stopped by rinsing in deionized water followed by drying
the membrane.
Preparation and characterization of mAbs against C
protein
Hybridomas secreting C protein-specific antibodies were
generated according to standard procedures with a few
modifications [21]. Briefly, six-week-old female BALB/c
mice were immunized subcutaneously with purified C pro-
tein emulsified with an equal volume of Freund’s complete
adjuvant (Sigma, St. Louis, MO, USA). Two booster injec-
tions containing purified C protein in Freund’s incomplete
adjuvant were given at 2-week intervals. A final immuniza-
tion, consisted of purified C protein without adjuvant and
was injected intraperitoneally. Three days after the final
immunization, mice were euthanized, and spleen cells were
harvested and fused with SP2/0 myeloma cells at 5-10:1
ratio using polyethylene glycol (PEG 4000, Sigma).
The hybridoma cells were seeded into 96-well plates and
selected in HAT medium (DMEM containing 20% fetal
bovine serum, 100 ug/ml streptomycin, 100 IU/ml penicil-
lin, 100 mM hypoxanthine, 16 mM thymidine and 400
mM aminopterin), and after 5 days, the medium was
removed and replaced with fresh HT-DMEM medium.
After HAT/HT selection, culture supernatants of surviving
clones were screened for reactivity and specificity by indir-
ect ELISA, WB and IFA.
The ELISA assay has been described previously [22].
Briefly, microplates were sensitized at 4°C overnight
with the affinity-purified WNV-C protein at 50 ng/ml.
The sensitized plates were incubated with test culture
supernatants from hybridomas at 37°C for 1 h, with
HRP-conjugated goat anti-mouse secondary antibodies
(LICOR Biosciences) at a 1:4,000 dilution at 37°C for 1
h, followed by color development with substrate solution
containing o-phenylenediamine (OPD).
WB was performed using mAbs as primary antibodies
and a HRP-conjugated goat anti-mouse secondary
antibody.
The IFA results were supplied by Beijing institute of
Microbiology and Epidemiology. WNV, JEV, DENV1-4,
YFV and TBEV antigen slides were prepared on porous
slides using WNV, JEV, DENV1-4, YFV and TBEV
infected and uninfected C6/36 cells. Cell suspensions
were dripped onto slides, fixed using acetone, air-dried,
Figure 6 Antibodies in WNV/JEV-positive equine sera recognize the MBP fusion protein containing the KKPGGPG epitope. MBP alone
or MBP fused with the KKPGGPG peptide epitope (MBP-Hp-1) was evaluated by WB analysis for reactivity with antibodies in (a) WNV-positive
equine serum, (b) JEV-positive equine serum, (c) DENV1-positive mouse serum, (d) DENV2-positive mouse serum, (e) DENV3-positive mouse
serum, and (f) DENV4-positive mouse serum. M, PageRuler™ Prestained Protein Ladder (Fermentas, Canada).
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and were stored at -20°C until use. Next, anti-C protein
mAbs and WNV-, JEV-, DENV1-4-, YFV- and TBEV-
positive/negative mouse serum (positive/negative con-
trol) were incubated on acetone-fixed antigen slides for
2 h. A FITC-conjugated goat anti-mouse IgG was used
as a secondary antibody, and slides were viewed at a
magnification of ×40 on a fluorescence microscope
(Leica, Germany).
The positive clones were subcloned three times by
limiting dilution. Selected clones were cultured in the
peritoneal cavities of pristine-primed BALB/c mice to
obtain ascites fluid.
The mAb titer was determined by indirect ELISA as
described above and the antibody subtype was deter-
mined using the Mouse MonoAb-ID Kit (HRP) (Invitro-
gen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. This test identifies the IgG1, IgG2a, IgG2b,
IgG3, IgA and IgM subtype classes and the ? and l light
chains using monospecific rabbit polyclonal antibodies
(Pabs).
Determination of epitopes by phage-displayed random
peptide library
The Ph.D.-12™ Phage Display Peptide Library Kit was
purchased from New England BioLabs Inc. The dodeca-
peptide library consists of 2.7×109electroporated
sequences (1.5×1013pfu/ml). All of the mAbs were puri-
fied from the ascites fluid of mice inoculated with the
hybridoma cells by affinity chromatography using rPro-
tein G (Sigma, USA) according to the manufacturer’s
instructions. The concentration of purified protein was
determined by the Bradford Protein Assay Kit (http://
www.beyotime.com/ Compatibility Chart For Bradford
Kit. Pdf).
Three successive rounds of biopanning were carried
out according to the manufacturer’s instructions (New
Figure 7 Alignment of WNV and other flavivirus C protein amino acid sequences surrounding the 6D3 epitope region. Amino acids
from the region encompassing the mAb 6D3 epitope of 22 WNV strains (including 3 Kunjin virus strains) and 14 other flavivirus strains were
aligned for analysis using Lasergene analysis software. The sequence motif recognized by the mAb 6D3 is boxed.
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England BioLabs Inc). Briefly, one well of a 96-well
microtiter plate was coated with 15 μg of purified mAb
in coating buffer (0.1 M NaHCO3, pH 8.6), followed by
blocking with blocking buffer (0.1 M NaHCO3, pH8.6,
and 5 mg/ml BSA) for 2 h at 4°C. About 1.5×1011pfu
(4×1010phages, 10 μl of the original library) were added
to the well and incubated for 1 h at room temperature
by gentle shaking. The unbound phages were removed
by successive washings with TBS buffer (50 mM Tris-
HCl, pH7.5, 150 mM NaCl) containing gradually
increasing concentrations of Tween-20 (0.1%, 0.3%, and
0.5%). The bound phages were eluted with elution buffer
(0.2 M Glycine-HCl, 1 mg ml-1BSA, pH 2.2). The
eluted phages were amplified in early-log E. coli ER2738
cells.
After three rounds of biopanning, ten individual phage
clones were selected and assayed for target binding by
sandwich ELISA as described by the manufacturer’s
instructions (New England BioLabs Inc). Briefly, 96-well
microtiter plates were coated overnight with 2 μg of the
6D3 mAb or antiporcine IFN-g mAb (Sigma, USA),
which served as a negative control. After 2 h of blocking
with blocking buffer at 4°C, phage clones were added to
the wells (2×1011pfu in 100 μl per well) and incubated
with agitation for 2 h at room temperature. Bound
phages were subjected to reaction with HRP-conjugated
anti-M13 antibody (Pharmacia, USA) for 2 h at room
temperature, followed by color development with sub-
strate solution containing o-phenylenediamine (OPD).
The DNA inserts displayed by ELISA-positive phage
clones were sequenced with the 96 gIII sequencing pri-
mer: 5’-TGAGCGGATAACAATTTCAC-3’ as described
by the manufacturer’s instructions (New England Bio-
Labs Inc).
Fine mapping of the epitope by WB
A series of complementary oligonucleotides (Table 1)
encoding for the full-length and truncated versions of
the peptide motif KKPGGPG were synthesized,
annealed, and cloned into EcoR I/Sal I sites of prokaryo-
tic expression vector pMAL™-C2x (New England Bio-
labs, Inc., USA), resulting in five recombinant plasmids.
The E. coli TB1 cells transformed with the recombinant
plasmids were induced with 0.5 mM IPTG to produce
recombinant MBP-fused polypeptides. The series of
MBP-fused polypeptides was screened by WB using the
C protein-specific mAb as described above.
Reactivity of the epitope with WNV/JEV-positive equine
serum and DENV1-4-positive mouse serum
To test whether the KKPGGPG epitope could be
detected by antibodies generated by a mammalian host
in the context of WNV/JEV infection, we evaluated the
reactivity of WNV/JEV-positive equine serum against
MBP-Hp-1 (MBP fusion containing the peptide of
KKPGGPG) by WB. WB was performed as described
above, using a HRP-conjugated rabbit anti-equine sec-
ondary antibody (LICOR Biosciences). To test whether
non-JEV serocomplex virus infection can induce antibo-
dies specific for the KKPGGPG epitope, we evaluated
the reactivity of DENV1-4-positive mouse sera against
MBP-Hp-1 by WB, using an HRP-conjugated goat anti-
mouse secondary antibody.
Homology analysis
To investigate the conservation of the epitope among
flaviviruses, sequence alignment of the epitope and
amino acid sequences from the corresponding region on
C protein of 22 WNV strains (including 3 Kunjin virus
strains) was performed using the DNASTAR Lasergene
program (Windows version; DNASTAR Inc., Madison,
WI). Alignment analysis was also performed between
the identified epitope and other associated flavivirus
strains, including the members of JEV serocomplex, and
another three antigenically related flavivirus, DENV1-4,
YFV and TBEV. Factors related to the time of virus iso-
lation and geographic region of origin of all strains were
considered.
Acknowledgements
The authors thank Dr. Peter Wilker for editing the manuscript. This study was
supported by National High-Tech Research and Development Program of
China 2007AA02Z406 and Heilongjiang Natural Science Foundation of China
ZJN-0602-01.
Author details
1The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State
Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
2Graduate School of Chinese Academy of Agricultural Sciences, Beijing
100081, PR China.3Beijing Institute of Microbiology and Epidemiology,
Beijing 100071, PR China.4CSIRO Livestock Industries, Australian Animal
Health Laboratory, Geelong, Victoria 3220, Australia.
Authors’ contributions
DLW designed the experiment. ECS and JZ carried out most of the
experiments and ECS wrote the manuscript. RAL supplied the equine sera
against WNV. YHY supplied the mouse sera against WNV, JEV, DENV, YFV
and TBEV. ZGB supplied the WNV C gene. TY, HWG, YLQ, LFW and NHL
participated part of experiments. DLW and LFW revised the manuscript. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 December 2010 Accepted: 6 March 2011
Published: 6 March 2011
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Cite this article as: Sun et al.: Identification of a conserved JEV
serocomplex B-cell epitope by screening a phage-display peptide
library with a mAb generated against West Nile virus capsid protein.
Virology Journal 2011 8:100.
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