Download Highly Conserved Region 141–168 of the NS1 Protein Is a New

Jpn. J. Infect. Dis., 64, 109-115, 2011
Original Article
Highly Conserved Region 141–168 of the NS1 Protein
Is a New Common Epitope Region of Dengue Virus
Promsin Masrinoul1, Magot Omokoko Diata1, Sabar Pambudi1,
Kriengsak Limkittikul3, Kazuyoshi Ikuta1,2, and Takeshi Kurosu1,2*
1
Department of Virology, Research Institute for Microbial Diseases,
Osaka University, Osaka 565-0871, Japan; and
2
Mahidol-Osaka Center for Infectious Diseases and
3
Department of Tropical Pediatrics, Faculty of Tropical Medicine,
Mahidol University, Bangkok 10400, Thailand
(Received December 15, 2010. Accepted January 12, 2011)
SUMMARY: Dengue virus (DENV) nonstructural protein 1 (NS1) is a major target of humoral immunity in
patients and is believed to be involved in DENV pathogenesis. In addition, NS1 is a diagnostic target as it is
secreted, and circulates, in patients’ plasma at an early stage of viral infection. In this study, we aimed to
identify common epitope regions for all serotypes by preparation of mouse monoclonal antibodies (MAbs)
against NS1. A total of 10 out of the 20 hybridoma clones which were specific to DENV produced MAbs that
recognized NS1. These MAbs mapped to three regions of DENV-2 NS1, namely amino acids 1–40 (epitope
region 1), 141–168 (epitope region 2), and 267–312 (epitope region 3). Epitope region 2 was recognized by
both complex-specific (2H11 and 3C4) and subcomplex-specific MAbs (4E5 and 5G12), whereas epitope
regions 1 and 3 were recognized by subcomplex-specific MAbs (5E2, 1A5, and 3F10) only. These epitope
regions were found to be highly conserved among all four serotypes of DENV by sequence analysis and
database comparison. The MAbs against these epitope regions, especially 2H11 and 3C4, could therefore be
valuable diagnostic tools.
reported that immunization with NS1 provides protection
to mice against lethal DENV, Japanese encephalitis virus
(JEV), and West Nile virus (WNV) infections, thus suggesting that NS1 could be a target for therapy or a subunit
vaccine against flaviviruses (11–14). The identification of
antigenic regions of NS1 proteins conserved among all
four DENV serotypes will be useful for the development of
subunit vaccines (15), and monoclonal antibodies (MAbs)
against these common epitope regions will be valuable
diagnostic tools.
In this study, we generated DENV NS1-specific MAbs
and identified three cross-reactive epitope regions. Epitope
region 1 (amino acids [aa] 1–40) and epitope region 3 (267–
312) were identified by subcomplex-specific MAbs (5E2,
1A5, and 3F10), which reacted with more than one serotype
of DENV but not all serotypes, and epitope region 2 was
newly identified by complex-specific MAbs (2H11 and 3C4),
which reacted with all serotypes of DENV and certain
subcomplex-specific MAbs (4E5 and 5G12). These epitope
regions, especially epitope region 2, were found to be highly
conserved among all DENV serotypes, thereby suggesting
that MAbs 2H11 and 3C4 could prove to be useful diagnostic tools.
INTRODUCTION
Dengue fever/dengue hemorrhagic fever (DHF) is the
most important human arthropod-borne viral disease, and
the incidence of severe and life-threatening forms of this
disease, namely DHF or dengue shock syndrome, is increasing (1,2). Dengue virus (DENV) has four serotypes (DENV
1–4), all of which are responsible for severe forms of the
disease. The World Health Organization currently estimates
that there are 50 to 100 million dengue infections globally
each year, more than 500,000 of which are DHF (3). However, although DENV is a major public health problem, an
effective vaccine or treatment for DENV infection has yet
to be developed.
The DENV genome contains three structural proteins
(capsid, pre-membrane [prM], and envelope) and seven
nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a,
NS4b, NS5). NS1 is a 46–50-kDa glycoprotein expressed in
infected mammalian cells in both membrane-associated
(mNS1) and secreted (sNS1) forms (4,5). Furthermore, circulating sNS1 proteins have been detected in patients’ plasma
with DENV infections. sNS1 could therefore be a target for
early diagnosis. NS1 is also known to be a major target of
humoral immunity in DENV infection (6–8). Likewise, there
is accumulating indirect evidence for an involvement of NS1
in DENV pathogenesis because high levels of NS1 have been
detected in patients’ plasma and correlated with a severe
form of the disease (9,10). In addition, several groups have
MATERIALS AND METHODS
Cells and viruses: DENV serotypes 1 (Mochizuki strain),
2 (16681 strain), 3 (H87 strain), 4 (H241 strain), and JEV
(JaGAr strain) were propagated in C6/36 cells for 7–9 days.
The culture medium was stored at –80°C before use. Myeloma PAI cells were cultured in Roswell Park Memorial
Institute 1640 medium (RPMI 1640; NACALAI TESQUE,
Kyoto, Japan) supplemented with 10% fetal calf serum
*Corresponding author: Mailing address: Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. Tel: +81 6 6879 8308, Fax:
+81 6 6879 8310, E-mail: [email protected]
109
(FCS). Human renal epithelial cell line 293T cells were
cultured in Dulbecco’s modified Eagle medium (DMEM;
Gibco, Gland Island, N.Y., USA) supplemented with 10%
FCS. Baby hamster kidney (BHK) cells were grown in
Eagle’s minimum essential medium (MEM; NACALAI
TESQUE) supplemented with 10% FCS. All cell lines were
cultured at 37°C with 5% CO2. The C6/36 mosquito cells
were grown in Leibovitz 15 medium (Gibco) supplemented
with 0.3% BactoTM Tryptose Phosphate Broth (Becton
Dickinson, Sparks Glencoe, Md., USA) and 10% FCS at
28°C. DENV, BHK cells, and C6/36 cells were kindly provided by Dr. Yoshinobu Okuno, Kanonji Institute, Research
Foundation for Microbial Diseases of Osaka University,
Kanonji, Kagawa, Japan.
Human DENV patient plasma: Plasma from patients
infected with DENV 1–4 was kindly provided by the Faculty of Tropical Medicine, Mahidol University, Bangkok,
Thailand. This study was approved by the local Ethics
Committee, and all patients provided informed consent to
participate.
Generation of dengue-specific MAbs: For the preparation of DENV 1–4 particles, the supernatants from BHK cells
individually infected with DENV 1–4 were ultracentrifuged
at 100,000 × g for 2 h. The pelleted DENV particles were
resuspended in PBS and kept at –80°C prior to injection.
The recombinant NS1 protein (rNS1) derived from DENV-2
New Guinea C strain (NGC) was expressed in Escherichia
coli BL21 strain and purified under denaturing conditions,
as described previously (16). Four-week-old female BALB/
c mice (JAPAN SLC, Shizuoka, Japan) were immunized intraperitoneally with a mixture of DENV 1–4 particles with/
without inactivation by 4% paraformaldehyde (PFA) in PBS,
or recombinant NS1 from DENV-2 NGC with complete
Freund’s adjuvant (Wako, Osaka, Japan). The immunized
mice were boosted 3–4 times with BHK cells infected with
a mixture of DENV 1–4. Three days after the final booster,
splenocytes were prepared and fused to myeloma PAI cells
using polyethylene glycol #1500 (Roche Diagnostics,
Mannheim, Germany). The fused cells were cultured in
DMEM supplemented with 15% FCS in the presence of
hypoxanthine-aminopterin-thymidine (Gibco). MAbs produced from the hybridomas were screened in an immunofluorescent assay using BHK cells infected with DENV 1–
4. Hybridomas were cloned twice by limiting dilution.
Indirect immunofluorescence assay (IFA): For the
preparation of virus antigen, BHK cells infected with DENV
1–4 or mock-infected were collected 2 days postinfection.
Cells infected separately with DENV 1–4 were mixed and
seeded onto 96-well plates for hybridoma screening. The
cells were fixed with 4% PFA in PBS, permeabilized with
1% Triton X-100 in PBS for 5 min, and then incubated with
hybridoma culture fluid or anti-DENV-specific MAbs (2H2)
(HB114, ATCC) as control for 1 h. They were then washed
three times with PBS and further treated with Alexa 488
goat anti-mouse IgG antibody (Invitrogen, Paisley, UK) at
a dilution of 1:300 for 30 min. Finally, they were washed
three times with PBS prior to observation by fluorescence
microscopy.
Determination of serotype-specificity and isotyping of
MAbs: The serotype specificities of MAbs were determined
by IFA and immunoblotting using DENV-infected BHK
cells. The isotypes of antibodies were examined by immunochromatrography using an IsoQuickTM kit (Sigma, St. Louis,
Mo., USA)
SDS-PAGE and Western blotting (WB) analysis: The
DENV-infected BHK cells were dissolved in sodium dodecyl
Table 1. Primers used for the construction of cDNA clones
Recombinant
gene
Primer
NS1-NS2A
(Full)
Fw (EcoRV)
Rv (BamH1)
5´ GCCGATATCAGATAGTGGTTGCGTTGTGAGC 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(41~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGAAACTAGCTTCAGCTATCCAGAAA 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(81~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGGAAAATGAGGTGAAGTTAACTATT3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(121~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGGCAAAAATGCTCTCTACAGAGTC 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(141~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGGCAGAATGCCCCAACACAAATAG 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(169~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGCTAAAATTGAAAGAAAAACAGGAT 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(197~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGGATATGGGTTATTGGATAGAAAGTG 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(221~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGAAAAACTGCCACTGGCCAAAATC 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(267~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGCCATGGCATCTAGGTAAGCTTGAG 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
NS1-NS2A
(313~)
Fw (EcoRV)
Rv (BamH1)
5´ GGCGATATCGTGCTGCCGATCTTGCACATTACCA 3´
5´ GCGGGATCCTCATCGGGTCCTAAGCATTTCCTCC 3´
The underlines indicate the restriction enzyme recognition sites.
110
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer without β-mercaptomethanol and heated at
100°C for 5 min. The resulting samples were separated on a
15% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, Mass.,
USA). These membranes were incubated overnight with
antibodies produced by hybridoma clones and then with
horseradish peroxidase-conjugated anti-mouse IgG (Jackson
ImmunoResearch Laboratories, West Grove, Pa., USA) for
1 h. The reactive viral proteins were visualized using ECL
WB detection reagent (GE Healthcare, Buckinghamshire,
UK).
Plasmid construction: For the construction of plasmids
expressing NS1 protein, viral RNA was extracted using a
QIAmp Viral RNA Mini kit (Qiagen, Valencia, Calif., USA),
and reverse transcription was performed using Superscript
III (Invitrogen) with random primers. The resultant cDNAs
were amplified by PCR with the set of primers (Table 1).
The PCR products were inserted into the EcoRV/BamHI sites
of pFLAG-CMVTM-3 expression vector (Sigma), then the
plasmids were transformed into E. coli XL1-blue cells and
subjected to sequence analysis. The expression of these
mutant NS1 proteins was confirmed by WB analysis using
an anti-Flag M2 antibody.
Transfection: For epitope mapping, a series of truncated
mutant NS1 plasmids was transfected into 293 T cells using
Lipofectamine 2000 (Invitrogen). At 2 days after transfection, cells were detached by treatment with 0.5 mM EDTA
in PBS and washed once with PBS. The cell precipitates
were resuspended with PBS, coated onto glass slides, and
then subjected to IFA with MAbs, human plasma infected
with DENV (HPD), or anti-Flag M2 antibody (Sigma). The
residual proteins were detected by immunoblotting.
Sequence analysis: The amino acid sequences of NS1
proteins were aligned using ClustalW, and percent identities were calculated using Bioedit version 7.0.5.
Analysis of amino acid sequence variation within
epitope regions: A total of 1,228 DENV-1 sequences, 815
DENV-2 sequences, 572 DENV-3 sequences, and 91 DENV4 sequences were collected from the NCBI protein database. The amino acid sequences within epitope regions were
analyzed by using Bioedit version 7.0.5. Variations for each
amino acid residue were displayed in an Entropy H(x) plot.
Identification of antigenic DENV proteins: Immunoblotting after SDS-PAGE was carried out with MAbs using
DENV-infected BHK cell lysate under non-reducing conditions to identify antigenic target DENV proteins. A total of
10 out of the 20 MAbs (50%) reacted with a DENV protein
at 46 kDa (NS1) (Fig. 1), whereas two MAbs (10%) reacted
with a DENV protein at 18–20 kDa (prM) (data not shown).
The target viral proteins for eight MAbs could not be identified, thus suggesting that these MAbs might recognize
conformational epitopes.
Of the 10 anti-NS1 MAbs described above, 2H11 and 3C4
were found to be complex-specific, 4G1 and 4C2 were found
to be serotype-specific, and the others (1A5, 3F10, 4E5, 5E2,
4G11, and 5G12) were found to be subcomplex-specific
(Table 2). The MAb isotypes were determined using an
immunochromatography kit, and all were found to be of the
IgG isotype. MAbs 4C2, 1A5, 3F10, 4E5, and 5E2 were
identified as IgG1,MAbs 4G1, 5G12, 4G11, and 2H11 as
IgG2, and MAb 3C4 as IgG3.
Mapping of cross-reactive MAbs to recombinant truncated forms of NS1 revealed three predominant epitope
regions: The majority of subcomplex anti-NS1 MAbs (1A5,
3F10, 4E5, 5E2, 5G12, 2H11, and 3C4) reacted with DENV2. To identify the epitope regions of these MAbs, a series of
recombinant DENV-2 NS1 truncated mutants was expressed
in 293T cells. The plasma from DENV-infected patients
reacted with all mutant NS1s except one (313–352), thus
indicating that B-cell epitopes of NS1 are spread across the
whole region of NS1. Epitope mapping using these MAbs
revealed three predominant epitope regions, with the 5E2
MAb mapping to an N-terminal region corresponding to aa
1–40 (epitope region 1). Four MAbs (2H11, 3C4, 4E5, and
5G12) mapped to the region located at aa 141–168 (epitope
region 2), and MAbs 1A5 and 3F10 mapped to a C-terminal
region located at aa 267–312 (epitope region 3) (Fig. 2). As
it has not been identified previously, region 2 can be considered to be a new B-cell epitope region.
The identified epitope regions exhibit high conservation among the four DENV serotypes: Since most of the
antibodies obtained were serotype cross-reactive antibodies (complex- and subcomplex-specific), the epitope
regions for each of the four serotypes were aligned (Fig. 3)
and the percent identities of their amino acid sequences
examined (Table 3). Epitope region 2 was found to be highly
conserved among all DENV serotypes, ranging in percent
identity from 75.0 to 82.1%. Epitope regions 1 and 3 were
also highly conserved among DENV-1, -2, -3 and DENV-2,
-3, -4, respectively, ranging in percent identities from 72.5
to 92.5% and 71.1 to 78.2%, respectively. Furthermore, these
epitope regions were found to be DENV-specific as none
of them exhibited high percent identities with JEV (values
ranging from 42.5 to 65.2%). The MAbs against these
regions, especially 2H11 and 3C4, could therefore prove to
be valuable diagnostic tools as they cross-react with all
DENV serotypes.
Highly conserved epitope regions among prevalent
DENV: To confirm the conservation of each epitope region,
their amino acid sequences were analyzed using 2,706
reference sequences for DENV 1–4 from the NCBI database (Fig. 4). For further analysis at the amino acid level, the
consensus sequences corresponding to the epitope regions
of each serotype were obtained and their peptide sequences
compared. Highly conserved regions were found at aa 12–
20 and 25–35 of epitope region 1, aa 154–161 of epitope
RESULTS
Generation and specificity of MAb against DENV: In
order to generate mouse MAb, BALB/c mice were immunized intraperitoneally with a mixture of DENV 1–4
particles with/without inactivation or recombinant DENV2 NS1 protein, and boosted with DENV-infected BHK cell
lysate. A total of 20 hybridomas were screened using BHK
cells infected with the DENV 1–4 mixture. To determine
the cross-reactivity to each serotype, the MAb culture
supernatants were examined by IFA using DENV-infected
BHK cells. Eight MAbs (3A1, 2B8, 3D8, 3A4, 3H12, 4C2,
4G1, and 6A5) reacted specifically with only one serotype
(serotype-specific), whereas six MAbs (1A5, 3F10, 4E5,
5E2, 4G11, and 5G12) reacted with more than one DENV
serotype, although not all serotypes (subcomplex-specific),
and four MAbs (2H11, 3C4, 3D2, and 6D6) reacted with all
DENV serotypes (complex-specific). Furthermore, two MAbs
(2F12 and 2H2) reacted with all DENV serotypes as well as
JEV (group-specific) (Table 2).
111
Table 2. Characterization of serotype-specific and cross-reactive specific MAbs
MAb
Reactivity by IFA
Ig isotype
(κ, λ)
DENV-1
DENV-2
DENV-3
DENV-4
JEV
IgM κ
IgG2b κ
IgG3 κ
IgM κ
IgG2a κ
IgG1 κ
IgG2a κ
IgM κ
IgG1 λ
IgG2a κ
IgG1 κ
IgG2b κ
IgG1 κ
IgG1 κ
IgG2a κ
IgG3 κ
IgG2b κ
IgG3 κ
IgM κ
IgM κ
–
–
–
–
–
–
–
–
+
+
+
+
–
–
+
+
+
+
+
+
+
–
–
–
–
–
–
–
+
–
+
+
+
+
+
+
+
+
+
+
–
–
–
–
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
+
–
+
+
+
+
+
+
+
–
–
–
–
+
+
+
+
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
+
3A1
2B8
3A4
3D8
3H12
4C2
4G1
6A5
4E5
4G11
5E2
5G12
1A5
3F10
2H11
3C4
3D2
6D6
2F12
2H2
Protein
Immunization2)
U1)
U
U
U
U
NS1
NS1
U
NS1
NS1
NS1
NS1
NS1
NS1
NS1
NS1
prM
prM
U
U
A
B
C
C
C
C
C
C
A
C
B
C
A
A
B
B
C
C
B
C
1)
: U indicates unidentified.
: Different immunizations were primarily used. A, B, and C indicate immunizations with an inactivated mixture of DENV,
a live mixture of DENV, and recombinant NS1, respectively. All of mice were boosted with DENV-infected BHK cell
lysates.
DENV-4
DENV-3
DENV-2
DENV-1
Mock
2)
region 2, and aa 267–274 and 294–302 of epitope region 3
(Fig. 4).
HPD
DISCUSSION
4G1
Three B-cell epitope regions of DENV NS1 have been
identified by using cross-reactive MAbs. These regions were
defined as epitope regions 1 (1–40), 2 (141–168), and 3 (267–
312) and were found to stretch along the whole region of
NS1. Plasma from dengue patients reacted with almost the
whole region (aa 1–312) of NS1, except for the C-terminal
region (aa 313–352). Epitope regions 1 and 3 are partly consistent with those identified in previous reports (17,18),
whereas epitope region 2 is identified for the first time in
this study. It should be noted that our immunization and
boosting schemes differ from those used in previous papers.
Thus, in this study, three different antigens, namely a mixture of DENV 1–4 particles with/without inactivation and
rNS1 protein, were used for immunization, and cell lysates
derived from DENV 1–4-infected BHK cells were used
for boosting. In contrast, in other studies, NS1 purified by
affinity column (19), rNS1 expressed in E. coli (17,20,21),
and DENV-infected mouse brain homogenate (22) were used
for immunization and for boosting. We believe that these
differences in the types of immunogen utilized may explain
why the epitope regions identified by each study do not
match completely.
The newly identified epitope region 2 is likely to be a
common region for all DENV serotypes as two MAbs (2H11
and 3C4) reacted with all such serotypes and a further two
(4E5 and 5G12) reacted with DENV-1, -2, and DENV-1, -2,
and -3, respectively. Sequence analysis using references
obtained from the NCBI database confirmed that this
region was highly conserved among DENVs, with the eight
4C2
4G11
1A5
3F10
4E5
5E2
5G12
2H11
3C4
Fig. 1. Characterization of serotype-specific and cross-reactive MAbs.
The cell lysates of C6/36 cells infected with DENV 1–4 individually
were subjected to SDS-PAGE and transferred to polyvinylidene
fluoride (PVDF) membranes. The membranes were stained with each
MAb separately. The NS1 proteins exhibited the mobility at 46
kDa.
112
A
(41-352)
(81-352)
(121-352) (141-352) (169-352) (197-352) (221-352) (267-352) (313-352)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(-)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
Mock
αFlag
(1-352)
(-)
M2
HPD
1A5
3F10
2H11
3C4
4E5
5G12
5E2
B
1
NS1
Epitope region 1
(1-40)
5E2
352
Epitope region 2
(141-168)
Epitope region 3
(267-312)
2H11
1A5
3C4
3F10
4E5
5G12
Fig. 2. Epitope mapping of cross-reactive MAbs. (A) The NS1 truncated mutants were expressed in 293T cells. The cells
were stained with anti-Flag M2 antibody, HPD, and MAbs, and followed by Alexa 488 goat anti-mouse IgG secondary
antibody. The MAbs mapped to three main regions, 1–40, 141–168, and 267–312. (B) Schematic figure of three main
epitope regions of each MAb on NS1 protein.
amino acids positioned at aa 154–161 of epitope region 2
being particularly highly conserved among the reference
sequences, even in different DENV serotypes (Fig. 3). This
region may therefore be part of a recognition site for the
complex-specific MAbs 2H11 and 3C4.
We expected to obtain cross-reactive (complex- and
subcomplex-specific) anti-NS1 MAbs more easily upon
immunization of mice with rNS1. However, this was not
the case, and MAbs generation actually improved when
mice were immunized with a live/inactivated mixture of
DENVs. Curiously, more than half of the MAbs produced
by immunization with DENV-2 rNS1 did not react with
DENV-2 NS1, although they did react with other DENV
serotypes. This suggests that the conformation of rNS1 may
not fully reflect the native form of NS1. Since DENV NS1
easily forms inclusion bodies in E. coli (23), rNS1 was
purified under denaturing conditions (16), which may have
changed the actual conformation of NS1. It is presumably
113
Flavivirus strain
Peptide sequence
Epitope
region 1
(1-40)
DENV-1-Mochizuki
DENV-2-16681
DENV-3-H87
DENV-4-H241
JEV-JaGAr
Epitope
Region 2
(141-168)
DENV-1-Mochizuki
DENV-2-16681
DENV-3-H87
DENV-4-H241
JEV-JaGAr
141 PECPDDQRAWNIWEVEDYGFGIFTTNIW 168
A...NTN....SL........V......
....SAS....V.........V......
S...NER....FL........M......
K....EH....SMQI..F....TS.RV.
DENV-1-Mochizuki
DENV-2-16681
DENV-3-H87
DENV-4-H241
JEV-JaGAr
267 PWHLGKLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKVIHEWC 312
31
........M...F.D......T.D............AS..L.T...
...........NY.......IS.N..T..........S..L.....
........I..GE.P....TIQ.D.DH.........AS..LVTQ..
..DENGIV....Y.P..K.TIT.D..K....V....DS..L.TD..
Epitope
region 3
(267-312)
1 DSGCVVNWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPK 40
......S..NK.........I.DN...........PE..S
.M...I....K.............................
.T..A.S.S.K..........IDN...........PE..A
.T..AIDITRK.MR.......H.D.EA.VDR..YLPET.R
Fig. 3. Amino acid sequence alignment of epitope regions among flaviviruses.
Table 3. Amino acid homology of epitope regions among DENV1–4 and JEV
Percent identity (%) of amino acid sequence
Epitope
Flavivirus
Epitope
region 1
(1–40)
DENV-1
DENV-2
DENV-3
DENV-4
JEV
Epitope
region 2
(141–168)
Epitope
region 3
(267–312)
DENV-1
(Mochizuki)
DENV-2
(16681)
DENV-3
(H87)
DENV-4
(H241)
JEV
(JaGAr)
1D1)
77.5
ID
92.5
75.0
ID
72.5
82.5
72.5
ID
42.5
45.0
47.5
52.5
ID
DENV-1
DENV-2
DENV-3
DENV-4
JEV
ID
75.0
ID
82.1
78.9
ID
75.0
82.1
75.0
ID
57.1
53.5
50.0
53.5
ID
DENV-1
DENV-2
DENV-3
DENV-4
JEV
ID
80.4
ID
82.6
78.2
ID
65.2
76.0
71.1
ID
56.5
65.2
63.0
63.0
ID
1)
: ID indicates identical virus strain.
not necessary to prepare purified NS1 to obtain anti-NS1
antibodies; instead, it is likely to be more efficient and convenient to immunize with virus particles and boost with cell
lysates derived from DENV-infected cells.
In summary, MAbs 2H11 and 3C4, which recognize a
newly identified epitope region (aa 141–168) of the NS1
protein, could be useful tools for early diagnosis as they
react with all DENV serotypes.
Conflict of interest None to declare.
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Acknowledgments This work was supported in part by SYSMEX
Corporation (Osaka, Japan). P.M. was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The manuscript was
proofread by Medical English Service (Kyoto, Japan).
We thank Dr. Arias J. Fernando for the critical reading of the manuscript.
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