JGV Papers in Press. Published June 19, 2014 as doi:10.1099/vir.0.062562-0
1 2
Production of a neutralizing antibody against envelope protein of dengue virus type 2 using linear array epitope (LAE) technique
3
Peng-Yeh Laia, Chia-Tse Hsub, Shao-Hung Wangc, Jin-Ching Leed, Min-Jen Tsenga, Jaulang
4
Hwanga, e, Wen-Tsai Jia and Hau-Ren Chena,*
5
a
6
Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102,
7
Taiwan.
8
b
9
Biochemical Engineering, Kao Yuan University, Luzhu District, Kaohsiung City 82151,
Department of Life Science, Institute of Molecular Biology and Institute of Biomedical
Department of Chemical and Biochemical Engineering and Institute of Chemical and
10
Taiwan
11
c
12
University, Chiayi City 60004, Taiwan.
13
d
14
80708,Taiwan
15
e
16
11031, Taiwan.
17 18
*Correspondence to Dr. Hau-Ren Chen, Department of Life Science, National Chung Cheng University.
19
168, Sec. 1, University Road, Min-Hsiung, Chia-Yi 62102, Taiwan.
20
Tel, 886-5-2720411 ext 66503; Fax, 886-5-2722871; e-mail,
[email protected] 21
Running title: Novel antibody generated by LAE technique
22
Abstract: 245
23
Text including figure legends: 4832
24
Total 5 figures and 0 table
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Category: Animal virus-Positive-strand RNA
26
Department of Microbiology, Immunology and Biopharmaceuticals, National Chiayi
Department
of
Biotechnology,
Kaohsiung
Medical
University,
Kaohsiung
city
Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei City
27
Abstract
28
Dengue virus belongs to Flavivirus and contains a positive-stranded RNA genome
29
Binding of dengue virus to host cells was mediated through domain III of the viral
30
envelope protein. Many therapeutic monoclonal antibodies (mAbs) against domain III
31
have been generated and characterized because of its high antigenicity. We have
32
previously established a novel PCR method named linear array epitope (LAE)
33
technique for producing monoclone-like polyclonal antibodies. To prove this method
34
could be utilized to produce antibody against epitopes with low antigenicity, a region
35
of 10 amino acids (V365NIEAEPPFG374) from domain III of the envelope protein in
36
dengue virus serotype 2 (DENV2) was selected to design the primers for LAE
37
technique. A DNA fragment encoding 10 directed repeats of these 10 amino acids for
38
producing the tandem repeated peptides was obtained and fused it with
39
GST-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from
40
E. coli and used as antigen for immunizing rabbits to obtain polyclonal antibody.
41
Furthermore, this EIII antibody could recognize envelope proteins either ectopically
42
overexpressed or synthesized by DENV2 infection using immunoblot and
43
immunofluorescence assays. Most importantly, this antibody was also capable of
44
detecting DENV2 virions by ELISA assay and could block viral entry into BHK-21
45
cells as shown by immunofluorescence and qRT-PCR assays. Taken together, LAE
46
technique could be applied for production of antibody against antigen with low
47
antigenicity successfully and carries a high potential to produce antibodies with good
48
quality for academic research, diagnosis and even therapeutic application in the
49
future.
50
Abbreviations: LAE: linear array epitope, TR-PCR: template-repeated polymerase
51
chain reaction, PEIa: exotoxin receptor-binding domain A of Pseudomonas
52
531. Introduction 54
Dengue virus is a considerable threat to public health all worldwide. The outbreaks
55
and epidemics caused by virus infection become a serious public health issue in many
56
tropical and subtropical countries. Dengue viruses are classified into four serotypes
57
(DENV1 to DENV4) and are transmitted to human by mosquito vectors such as Aedes
58
aegypti and Aedes albopictus. Virus infection may cause different kinds of diseases,
59
including dengue fever (DF) with febrile illness, dengue haemorrhagic fever (DHF)
60
with plasma leakage syndrome and dengue shock syndrome (DSS). Approximately
61
100 million cases of DF and 500,000 cases of DHF and DSS occur every year, with a
62
mortality rate of 5% (Gubler, 2002; Wilder-Smith & Gubler, 2008). Therefore, it is
63
important and urgent to develop proper vaccines for prevention of the disease. In the
64
past decades, there are several approaches attempting to develop dengue virus
65
vaccines, including live attenuated viruses, chimeric viruses, recombinant subunit
66
antigens, expression vector-based vaccines and DNA vaccines (Chokephaibulkit &
67
Perng, 2013; Heinz & Stiasny, 2012; Lee et al., 2012; Leng et al., 2009; Schmitz et al.,
68
2011). However, there is currently no commercially available vaccine against dengue
69
virus.
70
Dengue virus belongs to the family of Flaviviridae, genus Flavivirus, with a
71
positive-sense single-stranded RNA genome. The RNA genome of 10.7 kb in length
72
has a 5’ cap structure but lacks 3’ poly A tail, and encodes a polyprotein which is
73
cleaved into three structural proteins (core, premembrane and envelope) and seven
74
non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by host
75
and/or viral proteases. Among the three structural proteins, the premembrane and
76
envelope proteins are the primary antigenic targets of humoral immune response in
77
human (Wan et al., 2013). The neutralizing antibodies are usually induced by the
78
envelope protein (Murphy & Whitehead, 2011). The envelope protein is consisted of
79
495 amino acids with a molecular weight of about 60 kDa. It has been suggested that
80
the major role of the envelope protein is host cell attachment and entry (Bhardwaj et
81
al., 2001). The ectodomain of envelope protein is composed of three domains,
82
including domain I, II and III (Modis et al., 2003; 2004; Rey et al., 1995). Domain I
83
contains mainly type-specific non-neutralizing epitopes and is thought to be the
84
molecular hinge region involved in low-pH-triggered conformational changes
85
(Roehrig et al., 1998). Domain II is the dimerization domain, and is involved in
86
virus-mediated membrane fusion. It contains many cross-reactive epitopes eliciting
87
neutralizing and non-neutralizing monoclonal antibodies (Rey et al., 1995; Roehrig et
88
al., 1998). Domain III is an immunoglobulin-like structure containing the most distal
89
projecting loops from the virion surface. According to X-ray crystal structure, domain
90
III contains 10 β-strands (A to G and a small extra sheet, AxCxDx). In addition,
91
domain III carries multiple type- and subcomplex-specific epitopes eliciting
92
neutralizing monoclonal antibodies that have been hypothesized to block virus
93
binding to the host cell receptor (Bhardwaj et al., 2001; Chiu & Yang, 2003; Modis et
94
al., 2003; 2004; Rey et al., 1995; Sukupolvi-Petty et al., 2007; Volk et al., 2004; Yu et
95
al., 2004). Since envelope domain III plays important roles in viral entry, it is a good
96
target for generating neutralizing antibody.
97
Linear array epitope technique (LAE) is a method for preparation of an immunogen
98
containing multiple copies of a defined peptide in a linear alignment. This method
99
was designed to overcome poor immune response induced by traditional
100
immunogenic peptide/protein antigens. The tandem repeated epitopes are able to
101
efficiently induce a strong immune response in vivo (Yi et al., 2006) and prime CD4+
102
T cells in vitro (Barratt-Boyes et al., 1999). Immunization of female rabbits with the
103
LAE-produced immunogen that contained the exotoxin receptor-binding domain A of
104
Pseudomonas (PEIa) and 12 copies of gonadotropin-releasing hormone (GnRH)
105
resulted in the generation of high-titer antibodies specific for GnRH. The anti-GnRH
106
antibodies effectively neutralized GnRH activity in vivo, as demonstrated by the
107
degeneration of the ovaries in the injected rabbits (Hsu et al., 2000). The
108
LAE-produced gastrin-releasing peptide (GRP) immunogen also induced antibody
109
potentially inhibiting breast cancer cell proliferation in vitro and in vivo (Guojun et al.,
110
2008). A rabbit anti-mouse EKLF (erythroid Krűppel-like factor) antibody induced by
111
LAE-produced immunogen could be used to detect EKLF in mouse embryonic
112
samples by Western blot and immunostaining, and was applicable for chromatin
113
immunoprecipitation (ChIP) assay (Shyu et al., 2007).
114
In this study, we used LAE technique to produce a tandem-repeated domain III region
115
(V365NIEAEPPFG374)10 of DENV2 envelope protein as the immunogen (referred as
116
EIII epitope hereafter). We demonstrated that anti-EIII antibody induced by this novel
117
immunogen could be used to detect the envelope protein either ectopically
118
overexpressed or synthesized by DENV2 infection in cells. Importantly, the antibody
119
could be used to detect dengue virions and to block dengue viral entry into BHK-21
120
cells successfully.
1212. Results 122
Epitope selection and the principle of linear array epitope (LAE) technique
123
To test whether LAE technique could be used to induce antibodies against DENV2
124
envelope domain III, we chose an epitope (V365NIEAEPPFG374) consisting of
125
β-strand and random coil secondary structures, and was not co-localized with previous
126
neutralizing epitopes (Crill & Chang, 2004; Gromowski & Barrett, 2007; Lin et al.,
127
1994; Thullier et al., 2001; Wan et al., 2013). According to the three-dimensional
128
structures
129
(www.rcsb.org/pdb), this epitope seemed to be localized on the surface of the protein
130
(Fig. S1). To synthesize repeated EIII epitope fused with GST, two oligonucleotides
131
(oligo Den E-A and Den E-B) were designed for template-repeated PCR (TR-PCR).
132
Oligo Den E-A encodes VNIEAEPPFG peptide, and its 5’ and 3’ halves are
133
complementary to those of oligo Den E-B, respectively (Figure 1). The TR-PCR
134
products containing repeated EIII epitope were used as the templates for the second
135
PCR (adapter-PCR) to introduce the restriction enzyme sites and stop codon. The
136
products of both TR-PCR and adapter-PCR showed the ladder pattern in 8% PAGE
137
(Figure 1d). The lowest band (the major one in lane 3) among the ladders was the
138
monomer, and the higher bands were multimers. The DNA products were eluted and
139
cloned into T vectors, followed by screening for positive clones. Finally, a clone with
140
10 tandem repeated epitopes was obtained.
141
Preparation of tandem repeated antigen for generating rabbit antiserum against
142
domain III of the envelope protein
143
The DNA fragment with 10 repeats of EIII epitope was subcloned into two expression
144
plasmids for producing tandem repeated antigens, generating GST-Den EIII10-His6
145
fusion protein and PEIa-Den EIII10-His6 fusion protein (data not shown) in E. coli
146
BL21(DE3). The induced proteins were purified by glutathione Sepharose and nickel
147
column, respectively. The Coomassie blue staining for purification of GST-Den
148
EIII10-His6 protein is shown in Figure 2a. Elution fractions 3 to 7 were collected as the
149
antigen to immunize rabbits. The harvested antiserum against GST-Den EIII10-His6
150
protein was named EIII antibody, and the antibody titer against recombinant
151
full-length domain III fused with His6 tag (referred as EIII-His6 hereafter) was
152
monitored by ELISA every two weeks. Even as low as 10 ng coated EIII-His6 could
of
the
envelope
protein
obtained
from
protein
data
bank
153
be significantly detected by this antibody; the OD410 values of 102- and 103-fold
154
diluted sera were much higher than those of control (Fig. 2b). The titer of the
155
antiserum achieved plateau after 6 weeks (data not shown).
156
Evaluation of the specificity of EIII antibody by various assays
157
To further confirm the specificity of the harvested EIII antibody, ELISA and
158
immunoblot assay was performed. Recombinant EIII-His6 proteins from four
159
serotypes of DENV were used to test whether EIII Ab could recognize EIII protein of
160
all serotypes. In ELISA assay, Fig. 2c showed that EIII Ab mainly detected EIII-His6
161
of DENV2 with 104-fold dilution, but cross-reacted with EIII-His6 of all four
162
serotypes with 102-fold dilution. In immunoblot assay, EIII antibody could recognize
163
purified EIII-His6, PEIa-Den EIII10-His6 and GST-Den EIII10-His6, but not the
164
unrelated proteins such as PEIa-His6 (the protein product of empty pPEIa-His6 vector),
165
TAF7-His6 (a transcriptional factor) and NS3-C-His6 (the N-terminal truncated NS3
166
protein of dengue virus), suggesting the high specificity of LAE-induced antibody
167
(Fig. 3a). We next examined if EIII antibody could detect full-length DENV2
168
envelope protein in cells. Figure 3b and 3c showed that EIII antibody could
169
specifically detect the DENV2 envelope proteins ectopically overexpressed in
170
BHK-21 or HEK293T cells by Western blot analysis and immunofluorescence assay,
171
respectively. Furthermore, we tested if the antibody could be used in
172
immunoprecipitation assay. The cell lysates from HEK293T cells transfected with
173
vector only or with pCMV-3FLAG-DENV2 envelope were incubated with EIII
174
antibody, resulting in immunoprecipitation of ectopically expressed FLAG-tagged
175
DENV2 envelope proteins (Fig. 3d).
176
Application of EIII antibody for detecting virions and blocking viral entry
177
Since EIII antibody could recognize recombinant envelope proteins in BHK-21 and
178
HEK293T cells, we examined if it could recognize the envelope protein of dengue
179
virus virions. C6/36 cells were infected with DENV2 for 10 to 12 days and the cell
180
lysates or the media were collected for Western blot analysis. EIII antibody could
181
clearly detect dengue virus premembrane/envelope heterodimer protein in
182
virus-infected cell lysates as indicated in the filled arrow (Sukupolvi-Petty et al., 2010;
183
Zhang et al., 2003). EIII antibody also detected envelope protein in the medium
184
fraction of virus-infected culture weakly as indicated in the open arrow (Fig. 4a). To
185
further confirm this result, EIII antibody was used to detect virus-infected BHK-21
186
cells at different time points after infection. As shown in Figure 4b, EIII antibody
187
could detect virus envelope proteins in the infected cells and the signal increased
188
commensurate to the infection time, which correlated with the replication cycle of
189
dengue virus. Since the envelope protein is located on the surface of the virus, the
190
antibody was tested for virions recognition. Different amounts of dengue viruses were
191
coated on the ELISA plate and serially diluted EIII antibody was used to detect the
192
virions. The results showed that EIII antibody with 10- to 103-fold dilutions could
193
detect virions in a dosage-dependent manner (Fig. 4c).
194
Finally we tested if EIII antibody could be applied for blocking dengue virus infection.
195
Dengue viruses were pre-incubated with serially diluted EIII antibody and the
196
virus-antibody mixture was used to infect BHK-21 cells. Viruses were detected for
197
NS3 by immunofluorescence and qRT-PCR assays. The immunofluorescence signals
198
were quantified by counting green fluorescence-positive cells in different fields, and
199
the results showed that EIII antibody with 10-fold dilution significantly reduced the
200
fluorescence signals (Fig. 5a and 5b). The levels of NS3 RNA were also decreased by
201
EIII antibody (Fig 5c), suggesting that viral entry might be blocked effectively. In
202
order to further verify the process, we treated EIII Ab either before or after dengue
203
virus infection and found that EIII Ab affected viral infection significantly only in
204
pretreated condition, indicating that EIII Ab blocked virus infection through blocking
205
of viral attachment (Fig. 5d). Since EIII Ab could block DENV2 infection, we further
206
test if this Ab would block viral entry of other serotypes. All fours serotypes of
207
DENV were used to evaluate the blocking ability as done in Fig. 5c. The result
208
suggested EIII Ab specifically blocked DENV2 infection although DENV4 could not
209
be determined (Fig. S3)
2103. Discussion 211
Infection by dengue virus is a global concern. However, there is still no approved
212
antiviral treatment available so far. Several candidate vaccines remain in preclinical or
213
clinical evaluation (Durbin & Whitehead, 2010). The main strategy of these candidate
214
vaccines is to induce neutralizing antibodies to block virus infection. The best target
215
for blocking virus infection is the structural proteins on the virion’s surface.
216
According to previous studies, many neutralizing antibodies were induced by the
217
envelope proteins. Based on epitope mapping data, many epitopes of the type-specific
218
neutralizing antibodies against individual flaviviruses are localized to domain III,
219
whereas neutralizing monoclonal antibodies that cross-react with other flaviviruses
220
are localized primarily to domain II (Beasley & Barrett, 2002; Cecilia & Gould, 1991;
221
Chavez et al., 2010; Crill & Chang, 2004; Crill & Roehrig, 2001; Fonseca et al., 1994;
222
Goncalvez et al., 2004a; Goncalvez et al., 2004b; Lin et al., 1994; Oliphant et al.,
223
2005; Oliphant et al., 2006; Stiasny et al., 2006). Some studies further defined contact
224
residues for serotype-specific, subcomplex-specific and cross-reactive monoclonal
225
antibodies that recognize domain III of the envelope protein in dengue virus; the
226
epitopes of serotype-specific neutralizing monoclonal antibodies are localized to the
227
BC, DE and FG loop on the lateral ridge of domain III, whereas subcomplex-specific
228
monoclonal antibodies recognize A strand of domain III (Gromowski & Barrett, 2007;
229
Gromowski et al., 2008; Lok et al., 2008; Rajamanonmani et al., 2009;
230
Sukupolvi-Petty et al., 2007; Thullier et al., 2001). To prove that LAE technique
231
could improve epitopes of poor antigenicity, we chose the epitope (V365 N I E A E P P
232
F G374) localized between E-strand and F-strand of domain III, which has not been
233
reported to induce any neutralizing antibody. During the antigen purification process,
234
there were some ladder-like minor bands located under the major bands (Fig. 2a). We
235
speculate that the tandem repeated sequences might affect antigen translation or
236
degradation. This tandem repeated antigen indeed induces EIII antibody production,
237
which could be utilized for basic biochemical assays, such as Western blot analysis,
238
immunofluorescence and co-immunoprecipitation. In addition, this antibody could
239
specifically detect EIII-His6 (Fig. 3a), ruling out the possibility that it recognized the
240
junction sequences between two repeated epitopes. More importantly, this antibody
241
could be also applied for detecting dengue virus virions and blocking viral infection
242
into BHK-21 cells. According to literature search, this is the first report of application
243
of LAE technique in virus-related research.
244
From epidemiological studies, it is generally believed that heterologous antibodies
245
that fail to neutralize the infecting serotype contribute primarily to the development of
246
DHF/DSS in both infants and adults (Halstead, 2003). Those sub-neutralizing
247
antibodies enhance viral uptake by Fcγ receptor (FcγR)-dependent or Fcγ-independent
248
mechanisms called antibody-dependent enhancement (ADE) (Halstead & O'Rourke,
249
1977; Huang et al., 2006). To reduce the risk of ADE, the neutralizing antibodies
250
must be protective against each of the four dengue virus serotypes. This might be
251
achieved by choosing a conserved epitope in different serotypes to produce tetravalent
252
neutralizing antibodies by LAE technique. Therefore, this is the second reason why
253
we chose a region of 10 amino acids (V365NIEAEPPFG374) from domain III of the
254
envelope protein in DENV2 as the target of epitope. This sequence is 100% conserved
255
in DENV1, DENV2 and DENV3 but is only with two amino acids difference in
256
DENV4 (Fig. S2a). Interestingly, even this minor divergence might reflect in the
257
generated EIII Ab: 2000-fold diluted EIII Ab recognizes recombinant EIII-His6 of
258
DENV1 to DENV3, but not DENV4 (Fig. S2b). However, 100-fold diluted EIII Ab
259
could detect all the recombinant EIII-His6 of four serotypes (data not shown).
260
Similarly, EIII Ab majorly recognizes recombinant EIII-His6 of DENV2 under low
261
concentration (104-fold dilution), but cross-reacts with EIII-His6 of all four serotypes
262
under high concentration (102-fold dilution) in ELISA (Fig. 2c). Surprisingly, Fig. 5
263
and Fig. S3 also show that EIII Ab specifically blocks DENV2 infection to BHK-21
264
cells although the epitope are identical in DENV1, DENV2 and DENV3. We
265
speculate this specific blockage might derive from higher avidity of EIII Ab with
266
envelope protein of DENV2, suggesting that the the antibody generated by LAE not
267
only recognizes the sequence but also discriminate structural arrange of corresponding
268
antigen.
269
To further delineate the process of blockage, we further examined whether that EIII
270
Ab blocked virus infection through the blockage of viral attachment to host receptor
271
(Fig. 5d). Our result is consistent with previous studies, which suggests that domain
272
III also play important roles in binding to the host cell receptors (Bhardwaj et al.,
273
2001; Chiu & Yang, 2003; Crill & Roehrig, 2001; Hung et al., 2004).
274
Taken together, LAE technique can be used to develop antigens with higher
275
antigenicity or antibodies with higher specificity through simple PCR-based method
276
even without any DNA template. Moreover, the LAE-produced polyclonal antibody
277
recognized the same epitope, displaying the property of monoclonal antibody and may
278
be considered as monoclone-like polyclonal antibody. Such antigens and antibodies
279
have high potential for uses in academic research, diagnosis and therapeutic
280
application in the future.
2814. Methods 282
Template-repeated PCR (TR-PCR) and adapter-PCR
283
KOD Hot Start DNA Polymerase (Novagen) was used in TR-PCR. Two
284
oligonucleotides were used in this reaction: Den E-A: 5’-GTT AAC ATC GAA GCC
285
GAA CCT CCT TTT GGT-3’ and Den E-B: 5’-GGC TTC GAT GTT AAC ACC
286
AAA AGG AGG TTC-3’. In TR-PCR, the two primers were also functional as
287
templates. The PCR programs were 30 cycles of denaturation at 94°C for 1 minute,
288
annealing at 44°C for 1 minute, and polymerization at 72°C for 1 minute, followed by
289
a final polymerization step at 72°C for 10 minutes. After TR-PCR, the PCR product
290
was then used as template for adapter-PCR reaction catalyzed by Taq polymerase
291
(Fermentas). Two oligonucleotides were used in this reaction: Den E-5b: 5’-GGA
292
TCC GTT AAC ATC GAA GCC-3’ and Den E-3he: 5’-GAA TTC ATT AAA GCT
293
TAC CAA AAG GAG GTT C-3’. The PCR condition were 5 cycles of denaturation at
294
94°C for 1 minute, annealing at 44°C for 1 minute, and polymerization at 72°C for 1
295
minute, followed by 10 cycles of denaturation at 94°C for 1 minute, annealing at 48°C
296
for 1 minute, and polymerization at 72°C for 1 minute. Finally the reaction was
297
terminated with polymerization step at 72°C for 10 minutes. The product of adaptor
298
PCR was cloned into T vector for sequencing. The correct construct was then
299
subcloned into expression vectors pGEX-His6 or pPEIa-His6.
300
Antigen and antibody preparation
301
Recombinant GST-Den EIII10-His6 protein was expressed in BL21 (DE3) with 1 mM
℃ for 3hrs followed by breaking cells with sonication. After centrifugation,
302
IPTG at 37
303
the soluble protein was purified with Glutathione-Sepharose 4B (Amersham
304
Pharmacia). One hundred or five hundred micrograms of purified GST-Den
305
EIII10-His6 proteins were mixed with Freund’s complete adjuvant (Chemicon) and
306
then subcutaneously injected into female New Zealand white rabbits four times with
307
two weeks intervals. Sera were collected from rabbits’ ears biweekly and titer was
308
tested by ELISA. The sera were purified by Millipore montage® antibody purification
309
kit.
310
Enzyme-linked immunosorbent assay (ELISA)
311
The recombinant EIII-His6 proteins are the courtesy of Dr. Chih-Hsiang Leng in
312
National Health Research Institute, Taiwan (Leng et al., 2009). The consensus
313
sequence for EIII from DENV1 was obtained by alignment of the amino acid
314
sequences from five different isolates of DENV1, including Brazil/97-11/1997,
315
Jamaica/CV1636/1977,
316
Thailand/AHF 82-80/1980. The consensus sequences for EIII from DENV2, DENV3
317
and DENV4 were obtained by alignment of the amino acid sequences from different
318
isolates, respectively. For DENV2, 16681-PDK53, China/D2-04, Jamaica/1409/1983,
319
Malaysia M2, Malaysia M3, Peru/IQT2913/1996, Thailand/0168/1979, Puerto
320
Rico/PR159-S1/1969,
321
Thailand/16681/84,
322
referenced. Strains for DENV3 included Philippines/H87/1956, China/80-2/1980,
323
Singapore/8120/1995, Martinique/1243/1999, and Sri Lanka/1266/2000. Strains for
324
DENV4
325
Thailand/0348/1991 Singapore/8976/1995, and Thailand/0476/1997. 10 ng, 100 ng
Nauru/West
Pac/1974,
Tonga/EKB194/1974 Thailand/NGS-C/1944,
included
Singapore/S275/1990
P27914, and
Philippines/H241/1956,
and
Thailand/PUO-218/1980,
Thailand/TH-36/1958
were
Dominica/814669/1981,
326
or 1000 ng of EIII-His6 proteins or dengue virus virion were coated on 96-well plates
327
with coating buffer (10 mM Na2CO3, 35 mM NaHCO3, pH 9.6) at 37
328
4
℃ for 2 hrs or
330
℃ overnight. After coating, samples were blocked by blocking solution (1% BSA and 0.0375% saponin in PBS) at 37℃ for 2 hrs or at 4℃ for overnight. The samples were incubated with EIII antibody at 37℃ for 2 hrs and washed with 120 μl PBS
331
three times. Then the samples were incubated with goat anti-rabbit IgG-HRP second
332
antibody at 37
333
substrate solution [2,2-azino-diethylbenzthiazoline sulphonic acid (ABTS) 0.54mg/ml,
334
0.1M citric acid pH 4.2, 3μl 30% H2O2/ml] was incubated with samples at room
335
temperature for 15 minutes. The signals were detected by multiscan ELISA reader
336
with A410 nm (Dynatech).
337
Cell cultures, transfection, virus strain and infection:
338
Baby hamster kidney (BHK-21) cells and human embryonic kidney (HEK293T) cells
339
were cultured at 37
340
medium (Gibco) supplemented with 10% fetal bovine serum (Biowest). C6/36, a
341
mosquito cell line established from Aedes albopictus was grown at 28
342
in RPMI 1640 medium (Gibco) supplemented with 10% FBS. Cells were transfected
343
with pCMV-3FLAG or pCMV-3FLAG-envelope using TurbofectTM reagent
344
(Fermentas). DENV1 (Myanmar 3886201 strain), DENV2 (PL046, NGC strain),
345
DENV3 (98TW503 strain) and DENV4 (H241 strain) stock was prepared in C6/36
346
cells and titrated by plaque assay. In infection experiments, appropriated cells were
347
seeded on culture plates and incubated at 37
329
℃ for 1 hour and washed three times with 120 ul PBS. Finally,
℃ with 5% CO in Dulbecco's modified Eagle's medium (DMEM) 2
℃ with 5% CO
2
℃ overnight. The next day, cells were
℃ for 2 hrs by adding dengue virus-containing culture supernatant of a
348
infected at 37
349
MOI of 0.02 or 12. After infection, cells were washed with PBS and cultured in fresh
350
medium.
351
Western blot and Co-immunoprecipitation (Co-IP):
352
Samples and pre-stained marker were subjected to a 10% or 12.5% polyacrylamide
353
gel for electrophoresis. The proteins were then electrophoretically transferred from gel
354
onto PVDF membrane by a semi-dry transfer system (Biometra). After transferring
355
onto the PVDF membranes and subsequent blocking, the blots were incubated with
356
the respective primary antibodies, followed by the secondary antibody conjugated
357
with horseradish peroxidase (HRP). Finally the signal was detected by adding
358
chemiluminescence reagent (PerkinElmer) and visualized using Chemi Genius2
359
CG2/D2A
360
immunoprecipitation, 150 micrograms cell lysates from HEK293T cells transfected
361
with empty vector or pCMV-3FLAG-envelope plasmid were immunoprecipitated with
362
EIII antibody using Dynabeads protein A system (Invitrogen), followed by Western
363
blot with FLAG antibody.
364
Immunofluorescence assay (IFA):
365
In dengue virus blocking assay, BHK-21 cells were seeded on cover glasses in
366
six-well plates (105 or 3X 105 cells per well) and incubated at 37°C for overnight. EIII
367
antibody at different dilutions was incubated with dengue virus with a MOI of 0.02 at
368
4°C for 1 hour. The virus-antibody mixture was then added to BHK-21 and incubated
369
for an additional hour at 4°C. Negative controls received PBS instead of antibody.
370
After infection, BHK-21 cells were washed three times with 1 ml cold PBS and
machine
(Syngene)
or
using
traditional
X-ray
film.
For
371
cultured in DMEM medium supplemented with 10% FBS. At different time points
372
after infection, the cells on cover glasses were then permeabilized with 0.25% Triton
373
X-100 followed by 100% methanol fixation at -20°C for 5 minutes. After fixation,
374
cells were incubated with rabbit-anti-Den2 NS3 antibody followed by incubation with
375
anti-rabbit-IgG-Alexa488 (Molecular Probes) secondary antibody. Finally, cover
376
glasses were mounted on slide with Vectashield mounting medium (Vector Labs) with
377
4, 6-diamidino-2-phenylindole (DAPI). The slides were observed by fluorescence
378
microscope or confocal microscope. In transfection experiment, pCMV-3FLAG or
379
pCMV-3FLAG-envelope plasmid was transfected to HEK293T cells by using
380
TurbofectTM transfection reagent (Fermentas). The primary antibody was EIII or
381
FLAG and the secondary antibody was anti-rabbit-IgG-Alexa488 or anti-mouse
382
IgG-Alexa555 (Molecular Probes) in this experiment.
383
Quantitative reverse transcription polymerase chain reaction (qRT-PCR):
384
Total RNA was isolated from mock infected or dengue virus infected cells by REzol
385
C&T reagent (PROTECH). 100 ng or 1μg RNA was used as template for qRT-PCR
386
using KAPA SYBR® FAST One-Step qRT-PCR kit (KAPA Biosystem) or ABI
387
qRT-PCR kit and ABI StepOne detection system and analysis software (Applied
388
Biosystems). Levels of NS3 were normalized to β-actin mRNA levels using a
389
comparative threshold cycle (2–[delta] Ct) method which converts differences of cycle
390
numbers to test gene/β-actin ratios.
391
Statistical Analysis
392
The data were analyzed using either t-test or Mann–Whitney U test and the results
393
with p value less than 0.05 were considered significant.
394 3955. Acknowledgements 396
We thank Dr. Wen Chang (Institute of Molecular Biology, Academia Sinica, Taiwan)
397
for providing plasmid pET-21C-EIII for expression of domain III protein and Dr.
398
Huey-Nan Wu (Institute of Molecular Biology, Academia Sinica, Taiwan) for
399
providing plasmid pMR-SpeI-prME-HA1-RzD for construction of full-length
400
envelope protein and NS3 antibody for Western blot. We highly appreciate
401
Chih-Hsiang Leng and Hsin-Wei Chen (National Institute of Infectious Diseases and
402
Vaccinolog, National Health Research Institute) for providing purified recombinant
403
EIII-His6 proteins of four serotypes DENV. We also thank Dr. Ying-Ray Lee, (Chia-Yi
404
Christian Hospital) for providing Dengue virus type 2 (PL046 strain) for infection
405
experiments. This study is based in part on sequence data from the Taiwan Pathogenic
406
Microorganism Genome Database provided by Centers for Disease Control, Ministry
407
of Health and Welfare, Taiwan (supported by National Research Program for Genome
408
Medicine 99-0324-01-F-12). The interpretation and conclusions contained herein do
409
not represent those of Centers for Disease Control, Ministry of Health and Welfare,
410
Taiwan. This work was supported by National Science Council grants
411
NSC93-2311-B-194-002 for Hau-Ren Chen.
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564
Figure legends
565 566
Fig. 1. The linear array epitope (LAE) technique includes TR-PCR and adapter-PCR steps
567
(a) Template-repeated PCR (TR-PCR): Oligo Den E-A and oligo Den E-B are used as
568
primers and templates for TR-PCR. (b) Primers designed for TR-PCR. The peptide
569
sequences of epitope chosen are VNIEAEPPFG. Oligo Den E-A encodes this peptide
570
and oligo Den E-B is partially complementary to oligo Den E-A as indicated. The 5’
571
half of oligo Den E-A (shown as a) is complementary to the 5’ half of oligo Den E-B
572
(shown as a’) and the 3’ half of oligo Den E-A (shown as b) is complementary to the
573
3’ half of oligo Den E-B (shown as b’). (c) Adapter-PCR: The products of TR-PCR
574
(3μl) are subjected to adapter-PCR as templates. Adapter primer Den E-5b and Den
575
E-3he introduced proper restriction enzyme sites, leading to have a Bam HI site at 5’
576
end, a Hind III/Eco RI site at 3’ end, and two stop codons at the end of coding region.
577
The PCR products are then subcloned into a T vector. (d) The products of TR-PCR
578
(lane 2) and adapter-PCR (lane 3) are analyzed on 8% PAGE with ethidium bromide
579
stain. Lane 1 and 4 indicate 100 bp DNA markers.
580 581
Fig. 2. Novel tandem-repeated antigen was used to generate anti-EIII antiserum from rabbit
582
(a) Recombinant GST-Den EIII10-His6 protein was expressed in E. coli BL21 (DE3)
583
and was purified through Glutathione-Sepharose affinity column. Elution fractions
584
were analyzed on 10% SDS-PAGE and stained with Coomassie blue. Lane N, total
585
lysates before induction. Lane I, total lysates after 3 hrs IPTG induction. Lane Wash,
586
wash fraction during column purification. Elution fractions 1 to 8 indicated fractions
587
eluted by 10 mM glutathione from affinity column. (b) The titer of anti-EIII antiserum
588
from rabbit was measured by ELISA. 10 ng, 100 ng and 1000 ng of EIII-His 6 proteins
589
from DENV2 PL046 were coated onto ELISA plate and serial dilutions of EIII Ab
590
were added into the plate to measure OD410. No antigen (Ag) coated, no primary (1’)
591
antibody and no secondary (2’) antibody are all negative control sets. (c) The titer of
592
anti-EIII antiserum from rabbit was measured by ELISA. 10 ng, 100 ng and 1000 ng
593
of recombinant EIII-His6 proteins from four serotypes were coated onto ELISA plate
594
and serial dilutions of EIII Ab were added into the plate to measure OD410. No antigen
595
(Ag) coated, no primary (1’) antibody and no secondary (2’) antibody are all negative
596
control sets.
597 598
Fig. 3. The specificity of EIII antibody was evaluated in various assays
599
(a) The specificity of EIII antibody was first examined by Western blot. Lane 1:
600
purified EIII-His6, lane 2: PEIa-His6, lane 3: PEIa-Den EIII10-His6, lane 4: TAF7-His6,
601
lane 5: NS3-C-His6, lane 6: GST-Den EIII10-His6. Arrow indicated PEIa-Den
602
EIII10-His6 and arrow head indicated GST-Den EIII10-His6, respectively. (b) BHK-21
603
cells were transfected with empty vector or FLAG-envelope plasmid. Then, cell
604
lysates were analyzed by Western blot using either anti-EIII or anti-FLAG antibodies.
605
(c) Overexpression of FLAG-envelope in HEK293T cells were analyzed by
606
immunofluorescence assay using either anti-EIII or anti-FLAG antibodies. (d)
607
HEK293T cells were transfected with empty vector or pCMV-3FLAG-envelope
608
plasmid. Then, 150 μg total protein of cell lysates were immunoprecipitated with 10,
609
20 or 50 μl EIII antibody, followed by Western blot using anti-FLAG antibody. Input
610
was loaded with 15 μg of total protein.
611
Fig. 4. EIII antibody could recognize viral envelope proteins and virions
612
(a) C6/36 cells were infected by dengue virus and the medium was collected post 10
613
to 12 days infection. The cells were lysed by 2X sample buffer and the proteins either
614
in medium fraction (med) or in the cell fraction were analyzed by Western blot. The
615
first two lanes indicate mock infection. The other six lanes indicate three independent
616
experiments (Exp.1, 2 and 3) for dengue virus infection. Filled Arrow, open arrow and
617
arrow head indicated the band of premembrane/envelope heterodimer, envelope and
618
NS3 proteins, respectively. (b) BHK-21 cells were infected by dengue virus (MOI: 12)
619
and cells were harvested after 12 or 24 hrs infection, followed by Western blot
620
analysis with EIII or NS3 antibody. (c) Virions with different dilution folds were
621
coated in ELISA plate and were detected by anti-EIII antibody.
622
Fig. 5. EIII antibody could block dengue viral entry
623
(a) Purified EIII antibodies with serial dilution were pre-mixed with DENV2 PL046
624
strain, and then the antibody-virus mixtures were used to infect BHK-21 cells. After
625
infection for 48 hrs, cells were fixed with methanol and then dengue viruses were
626
detected by anti-NS3 (1:200) antibody. The signals were quantified in (b) by counting
627
the green fluorescence positive cells/DAPI positive cells in different fields. The
628
percentage of green fluorescence positive cells were measured based on over 180
629
DAPI-positive cells examined). The data was analyzed by Mann–Whitney U test (*
630
p