Download High-level expression of recombinant dengue

Journal of Medical Virology 65:553±560 (2001)
High-Level Expression of Recombinant Dengue
Viral NS-1 Protein and Its Potential Use as a
Diagnostic Antigen
Jau-Ling Huang,1* Jyh-Hsiung Huang,2 Ron-Hwa Shyu,1 Chao-Wei Teng,1 Yi-Ling Lin,3
Ming-Der Kuo,1 Chen-Wen Yao,1 and Men-Fang Shaio1
1
Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China
National Institute of Preventive Medicine, Division of Health, Taipei, Taiwan, Republic of China
3
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China
2
The prevalence of NS1 Ab response in patients
with dengue viral infection and the potential of
using recombinant NS1 protein as a diagnostic
antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal
half of NS1 proteins (rNS1, rNS1-C) were highly
expressed (10±30 mg/l) and further puri®ed and
refolded. The good antigenicity of the full-length
rNS1 protein was con®rmed by interaction with
19 dengue NS1-speci®c monoclonal antibodies
(MAbs) in ELISA; however, the antigenicity of
rNS1-C was relatively lower. The full-length rNS1
antigen also differentiated reliably between sera
from dengue virus-infected patients and sera
from normal controls. When rNS1 was used as an
antigen to detect human anti-NS1 IgM and IgG
Ab, the anti-NS1 Ab response was found in 15 of
17 patients (88%) with primary dengue infection
and all 16 patients (100%) with secondary dengue
infection. These results indicated that using the
full-length rNS1 whose antigenicity is restored as
ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either
primary or secondary dengue infection. This
®nding suggested that the anti-NS1 antibody
appeared not only in secondary and severe
dengue virus infection and might not correlate
the pathogenesis of dengue hemorrhagic fever.
The study also veri®ed that our puri®ed rNS1
protein showed similar immunological properties as native dengue viral proteins. Genetic
engineering production of recombinant NS1 antigen could provide a safe and valuable resource
for dengue virus serodiagnosis. J. Med. Virol.
65:553±560, 2001. ß 2001 Wiley-Liss, Inc.
KEY WORDS: ¯avivirus; nonstructural 1 protein; recombinant fragment;
ELISA; IgM
ß 2001 WILEY-LISS, INC.
INTRODUCTION
The ¯avivirus family, Flaviviridae, contains approximately 70 viruses. There are three major viral
complexes within this family: tick-borne encephalitis
virus (TBEV), Japanese encephalitis virus (JEV), and
dengue virus (DEN) [Calisher et al., 1989]. Infection
with some of these viruses has been controlled through
the availability of safe and effective vaccines [Stephenson, 1988]. However, despite years of research effort,
the development of dengue vaccine remains at the
experimental stage [Chambers et al., 1997]. Most cases
of dengue viral infection present as milder febrile
diseases. Nevertheless, some patients with dengue
viral infection may develop complications including
dengue hemorrhagic fever (DHF) or dengue shock
syndrome (DSS), especially when secondary infection
of different serotypes occurs. Today, dengue fever (DF)
and dengue hemorrhagic fever (DHF) are the most
important medical arbovirus epidemics throughout the
tropical and subtropical regions of the world [Halstead,
1988]. A rapid, sensitive, speci®c and economical
laboratory diagnostic test is needed to con®rm dengue
viral infection as well as to prevent and control DF and
DHF outbreaks.
A variety of serological tests have been used routinely
for the diagnosis of dengue viral infection [Gubler,
1998]. The hemagglutination inhibition (HI) test is
currently used as the standard serologic method by the
World Health Organization (WHO) for serologic classi®cation of dengue viral infection. However, it requires
acute and convalescent-phase serum samples and
Grant sponsor: Institute of Preventive Medicine, National
Defense Medical Center, Taipei, Taiwan, Republic of China.
*Correspondence to: Jau-Ling Huang, Division of Virology,
Institute of Prevention Medicine, National Defense Medical
Center, P.O. Box. 90048-700, SanHsia, Taipei, Taiwan.
E-mail: [email protected]
Accepted 17 April 2001
554
causes cross-reaction with other ¯aviviruses. In addition, HI and other methods, such as complement
®xation (CF) and the plaque reduction neutralization
test (PRNT), are slow and dif®cult to manipulate and
are therefore unsuitable for large-scale routine use.
Enzyme-linked immunosorbent assay (ELISA) is a
simple and rapid test, but IgG-ELISA is very nonspeci®c and exhibits broad cross-reactivity among
¯aviviruses. The IgM antibody capture ELISA (MACELISA) has become the most widely used method of
serodiagnosis and seroepidemiological survey of lengue
virus during the past few years [Gubler, 1998].
However, the diagnostic antigens used in MAC-ELISA
are prepared from dengue viruses-infected mouse brain
or cultured cells and are extracted by acetone-sucrose
gradient. Not only are these procedures laborious and
unsafe, they may also result in differences in batch
quality.
The ability to produce recombinant antigens raises
the question of whether such antigens could be used in
place of existing antigens, eliminating the problems
associated with preparation of cells and viruses. Many
recombinant proteins (fragments) that harbor immunodominant epitopes have been used successfully as
antigens in diagnostic tests for viral infection. These
proteins include the core NS3, NS4, NS5 proteins of the
hepatitis C virus [Cathy, 1997]; the gag protein of
human immunode®ciency virus-1 [Sohn et al., 1994];
the nucleocapsid protein of Hantan virus [Zoller et al.,
1993] and the prM/E protein of the Japanese encephalitis virus [Konishi et al., 1996].
Dengue virus consists of a single positive-stranded
RNA genome, which codes for three structural (C, PrM/
M, E) and seven nonstructural proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5) [Rice et al., 1985].
NS1 was found to exist as a secreted protein and as a
membrane-associated species, both of which were
highly immunogenic [Falconar and Young, 1990]. The
precise function of dengue NS1 protein remains
unclear. Two paradoxical observations of the effect of
anti-NS1 Ab have been reported. Some vaccine studies
have found that the NS1 protein could elicit a protective
immune response and was therefore a vaccine candidate; that is, anti-NS1 Ab could protect against dengue
viral infection [Schlesinger et al., 1987]. However, antiNS1 antibody has also been reported to be associated
with the immunopathogenesis of DHF [Falker et al.,
1973].
In this study, we used dengue NS1 protein with
approaching natural antigenicity as antigen to evaluate the anti-NS1 Ab response in patients with dengue
virus infection. Using 35S-label viral NS1 protein to
perform radioimmunoprecipitation (RIP), we could
detect the anti-NS1 Ab in the sera of patients with
primary dengue virus infection. In addition, an Escherichia coli-produced recombinant NS1 protein (rNS1)
with restored antigenicity was used as antigen to
perform recombinant NS1-based IgM-speci®c ELISA
and was found to be capable of differentiating the sera
of dengue virus infection from those of normal healthy
Huang et al.
adults and JEV-vaccinated children. The combined
results of rNS1-based IgM and IgG ELISA showed
a high prevalence of anti-NS1 antibody response in
patients with either primary (88%) or secondary (100%)
infection. These results showed that anti-NS1 Ab
could be detected not only in sera of severe secondary
infection, but also in sera of milder febrile patients
with primary dengue infection. High correlation of
anti-NS1 response with dengue virus infection suggests the potential use of rNS1 as a serodiagnostic
antigen.
MATERIALS AND METHODS
Human Serum Samples
The serum samples were supplied by the National
Institute of Preventive Medicine, Department of
Health, Taiwan, Republic of China. The characteristics
of dengue viral infection in patients sera used in this
study were con®rmed by several methods. Reverse
transcriptase-polymerase chain reaction (RT-PCR) and
PRNT were used to determine the subtype of dengue
virus infection. Cases were classi®ed as either primary
or secondary infections according to the WHO criteria
[1986] using the HI test. The Duo IgM/IgG capture
ELISA (Pan-Bio, East Brisbane, Australia) were also
used to differentiate between primary and secondary
dengue virus infection.
Viruses, Cell Line, and Plasmids
A local Taiwanese DEN2 strain, PL046, isolated from
infected Aedes egypti mosquitoes in 1980 was employed
in this study. The propagation of the virus was carried
out in C6/36 cells with RPMI 1640 medium containing
2% fetal calf serum. The NS1 expression plasmids were
a kind gift from Dr. Yi-Ling Lin. Brie¯y, the NS1
regions of these plasmids were ampli®ed from PL046
viral RNA by RT-PCR using primers covering different
NS1 regions. The NS1 PCR fragments with Not I
cohesive ends were inserted into Not I site of pET21b
(Novagen, Madison, WI). The NS1 sequences were inframed with the N terminal T7-Tag sequences.
Expression and Puri®cation of Recombinant
NS1 Protein
The NS1 expression plasmids were transformed to
BL21(DE3). Expression of the NS1 clone was induced
by the addition of 1 mM isopropyl b-D-thiogalactopyranpside (IPTG) to a growing culture for 2±3 hr. The
bacteria were harvested and resuspended in TE buffer
(50 mM Tris-HCl pH 8.0, 2 mM EDTA). After treatment
with lysozyme and Triton X-100, the bacterial lysate
was sonicated and centrifuged to pellet down the
inclusion bodies, which were then further puri®ed by
repeated centrifugation and washing. The insoluble
inclusion bodies were denatured with 8 M urea. The
solubilized proteins were then refolded by slowly
dialyzing them out of the denaturant with refolding
buffers (1 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl,
555
Anti-NS1 Ab in Dengue Patients
0.1 mM PMSF, 2.0 mM reduced glutathione, 0.2 mM
oxidized glutathione) containing sequentially decreased concentrations of urea (4 M, 2 M, 1 M, 0 M). Finally,
the inclusion bodies were completely solubilized in
1 PBS. The recombinant NS1 fragments were further
puri®ed with T7-Tag af®nity puri®cation chromatography (Novagen, Madison, WI). The puri®ed and refolded
proteins were analyzed with protein assay reagent (BioRad, Hercules, CA) and SDS-PAGE to determine the
protein concentration and purity. The purity was
quanti®ed by CCD software (AlphaEase Version 2.02,
Alpha Innotech, San Leandro, CA).
Radioimmunoprecipitation
C6/36 monolayers were infected with PL046 at a
multiplicity of infection (MOI) of 5. At 48 hr postinfection, the cells were starved with methionine-free and
cysteine-free RPMI medium 1640 for 1 hr and labeled
with 50 mCi of 35S-labeled Pro-Mix (Amersham Pharmacia, Buckinghamshire, UK) per 35 mm dish for 2 hr
at 378C. The cells were lysed with lysis buffer consisting
of 1% Nonidet P-40 (NP-40); 150 mM NaCl, 50 mM TrisHCl, pH 7.5; 1 mM EDTA, containing a cocktail of
protease inhibitors, including 20 mg of phenylmethylsulfonyl ¯uoride, 2 mg of leupeptin, and 2 mg of aprotinin
per ml. Aliquots of cell lysate were mixed with the
sera of infected patients or monoclonal antibodies
captured on staphylococcal protein A-coated Sepharose
(Amersham Pharmacia) for 1 hr at room temperature
(RT). The immune complexes were washed with RIPA
buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM
EDTA; 0.1% SDS; 1% Triton X-100; 1% sodium
deoxycholate), suspended in Laemmli sample buffer,
boiled and analyzed by SDS-10% PAGE, and ¯uorographed at ÿ708C.
Western Blot Analysis
IPTG-induced bacterial pellets or inclusion bodies
were lysed with SDS-sample buffer (with 2-mercaptoethanol), boiled, separated by SDS-PAGE, and transferred to nitrocellulose membranes (Amersham
Pharmacia). The blots were blocked with 5% skimmed
milk in phosphate-buffered saline (PBS) and reacted
with mouse anti-DEN2 NS1 monoclonal antibodies (Lin
et al., 1998). The secondary antibody was goat antimouse immunoglobulin conjugated with horseradish
peroxidase (HRP) (Jackson, West Grove, PA) and
developed with the enhanced chemiluminescence
(ECL) system (Amersham).
Recombinant Protein-Based IgM- and
IgG-Speci®c ELISA
Each well of Nunc immunomicrotiter plates (Nalge
Nunc, Rochester, NY) was coated with 100 ml of
recombinant NS1 proteins (200 ng/well for IgM and
100 ng/well for IgG detection) diluted in PBS. The
plates were blocked by incubation in PBS, and 1%
bovine serum albumin (BSA) at 48C overnight. After
the plates were washed (6 times with 1 PBS, 0.05%
Tween-20 and 1 time with 1 PBS), 100 ml of human
sera diluted 1:100 in dilution buffer (1 PBS, 0.05%
Tween-20, 1% BSA, 1 mg/ml normal goat serum) was
added and incubated RT for 1 hr. The plates were
washed again and antibodies were detected by adding
100 ml/well of 1:2,000 diluted HRP-conjugated goat
anti-human IgG or anti-human IgM immunoglobulin
(Jackson) in dilution buffer. After incubation for 1 hr at
RT, the plates were washed again, and then incubated
with 100 ml/well 3,30 ,5,50 -tetramethylbenzidine (TMB)
(Sigma Chemical Co., St. Louis, MO ) substrate solution
at RT for 10 min. The reaction was stopped with 2N
H2SO4. Absorbency (optical density, OD) was read at
450 nm using a Dynatech MR700 ELISA reader. The
cutoff extinction of the ELISA was determined as the
mean extinction of 20 negative serum samples plus 3
standard deviations (3 SD). Three to ®ve positive and
negative control serum samples were included on each
plate. The cutoff extinction of each plate was adjusted
according to the variation of the mean extinction of the
negative control sera. Assay results were quanti®ed in
terms of the OD (sample)/OD (cutoff) ratio. Values of
>1.0 were considered positive.
RESULTS
Expression of Recombinant Dengue2
NS1 Fragments
To determine whether the DEN nonstructural protein NS1 could be used as a diagnostic antigen, we ®rst
examined the antibody pro®les of DEN patients. The
serum of each DEN patient was reacted with 35S-Metlabeled, DEN2 virus-infected C6/36 lysate and analyzed by RIP. The mouse MAb speci®c for DEN2 NS1
was used as a positive control (Fig. 1A). The RIP results
showed that the sera of DEN patients had higher levels
of anti-NS1 antibodies compared with those of normal
sera (Fig. 1B). This suggested that differentiation of
DEN patients from the normal population could be
done using the NS1 antigen. Therefore, the high-level
E. coli expression system using T7 RNA polymerase
driving T7 promoter plasmid (pET21) was used to
obtain recombinant DEN2 NS1 proteins. We focused on
the expression of rNS1, rNS1-N and rNS1-C, three
recombinant NS1 fragments. These fragments cover
the regions of full-length, N-terminal and C-terminal of
NS1 and have predicted molecular mass of 48, 24, and
28 kDa, respectively (Fig. 2A). The amino acid sequence
for the DEN2 NS1 protein was analyzed using the
DNASTAR computer program. The potential B-cell
epitopes were displayed in parallel with the organizations of these recombinant NS1 fragments (Fig. 2B).
Immunocharacterization and Puri®cation of
Recombinant NS1 Fragments
Western blot analyses were performed to verify the
structure of the NS1 recombinant fragments. The
primary Abs used were NS1 monoclonal antibodies
556
Huang et al.
antigenicity of this rNS1 is highly similar to that of
authentic virus NS1 protein, and suggested that it
harbors more immunodominant epitopes than rNS1-C.
The high antigenicity of rNS1 protein makes it a useful
antigen for detecting human anti-NS1 immune
response to dengue viral infection.
Evaluation of Recombinant NS1-Based
Solid-Phase ELISA
Fig. 1. Detection of anti-NS1 Ab response in serum samples from
dengue virus-infected patient by radioimmunoprecipatation (RIP).
A: RIP with different mouse anti-DEN2 MAbs served as the position
control. Lysates from 35S-Met-labeled DEN2 PL046 cells were
immunoprecipitated with envelope (E), NS1, and NS3 MAbs as
indicated. The numbers on the left side denote the positions of
molecular mass standards (M). The positions of E, NS1 (monomer),
and NS3 proteins are indicated by arrows. B: RIP for detecting Ab
response of dengue virus infection. 35S-Met-labeled DEN2 cell lysates
were immunoprecipitated with serum samples from patients with
dengue infection (P5, P7) and normal adults (N). Anti-NS1, E, and
NS3 Ab were detected in the sera of these two patients.
named 13-1 and 8-2 that recognized N-terminal (aa
109±123) and C-terminal half, respectively. As shown
in Figure 3B and C, rNS1 was recognized by these two
anti-NS1 MAbs. As expected, rNS1-N was only recognized by MAb 13-1 and rNS1-C was only recognized by
MAb 8-2. These data clearly identi®ed the expressed
proteins as recombinant dengue NS1 fragments.
Among them, rNS1 and rNS1-C were highly expressed
(about 50% of total protein) and formed insoluble
inclusion bodies. However, rNS1-N was weakly expressed and more dif®cult to be puri®ed and prepared as
an ELISA diagnostic antigen (Fig. 3A). To investigate
the suitability of rNS1 and rNS1-C as diagnostic
antigens, these proteins were further puri®ed and
refolded. The purity of rNS1 and rNS1-C was enhanced
by washing the inclusion bodies thoroughly, making
the inclusion body fractions almost equal to the NS1
recombinant proteins (Fig. 4A). To restore the native
conformation of NS1 recombinant proteins, the inclusion bodies were solubilized, refolded and then further
puri®ed by T7 column chromatography. Their purities
were above 90% after these treatments (Fig. 4B).
High Immunogenicity of rNS1 Protein
The immunoreactivity of these refolded NS1 recombinant fragments was also con®rmed by ELISA, using
mouse anti-NS1 MAbs. Among the 20 mouse anti-NS1
MAbs used, 19 MAbs could react speci®cally with rNS1
and only about half of them could interact well with
rNS1-C (Table I). This ®nding indicated that the
To evaluate whether rNS1 or rNS1-C is a suitable
ELISA diagnostic antigen for dengue viral infection,
these recombinant fragments were directly coated on
ELISA plates and used to capture human anti-NS1
antibodies. Goat anti-human IgM- or IgG-conjugated
HRP were used as the secondary antibodies. To
determine the amount of antigen needed, a series
titration of recombinant NS1 fragments were coated
and used to differentiate the positive and negative
control sera for dengue virus infection. The ideal
amount of antigen to get signi®cant P/N ratio for IgG
and IgM detection were 100 ng/well and 200 ng/well,
respectively. We analyzed the anti-NS1 antibody
responses of human sera from 33 DEN patients
(17 primary and 16 secondary infection) and 20
noninfected adults. Tables II and III show the results
of rNS1-based IgM-speci®c and IgG-speci®c ELISA
(columns I and II). Both the IgM- and the IgG- speci®c
assays could differentiate signi®cantly between DEN
patients and normal adults (P < 0.0005 and P < 0.005,
respectively). This ®nding demonstrated that anti-NS1
Ab responses could be detected in the sera of dengue
virus-infected patients. When calculating the seropositive rate, the cutoff values were de®ned as the mean
extinction of sera of 20 noninfected adults plus three
standard deviations. It was found that 82% (27/33) of
the samples were anti-NS1 seropositive (OD of sample/
OD of cutoff > 1) when rNS1-based ELISA was used to
detect IgM-speci®c Ab (column I of Tables II and III). In
contrast, the IgG-speci®c ELISA had a relatively lower
seropositive rate (58%, 19/33) (column II of Tables II
and III). This may be due to the cross-reactivity of IgG
Ab in normal sera and the relatively high cutoff value of
the IgG assay. Detection of NS1-speci®c IgM Ab
reaction enhanced the positive rate, especially in
dengue patients who were in the early stage of infection. The earliest time after infection when anti-NS1
IgM antibodies became detectable was day 3. This
indicated that the combination of high antigenic NS1
Ag and IgM-speci®c Ab detection may be suitable for
early diagnosis of dengue infection.
The results of NS1-C-based IgM- and IgG-speci®c
ELISA are shown in columns III and IV of Tables II and
III. Only IgM- speci®c ELISA can differentiate signi®cantly between sera from DEN patients and those
from normal adults (P < 0.0005). The seropositive rates
of rNS1-C-based ELISA were lower than those of rNS1based ELISA (columns III and IV vs. columns I and II).
This indicated that the full-length NS1 harbors more
important immunodominant epitopes than rNS1-C
Anti-NS1 Ab in Dengue Patients
557
Fig. 2. A: Structural representation of the recombinant NS1
fragments in the T7 Tag (T7), NS1 signal peptide (S), and different
NS1 regions. rNS1 rNS1-N, and rNS1-C indicate the full-length,
N- and C-terminal half of NS1 recombinant protein, respectively.
Their molecular masses were shown. B: Computer-predicted antigenic
index of DEN2 PL046 NS1 protein. The 352-amino acid residue NS1
protein was analyzed using DNASTAR Lasergene software (Windows
3.08, DNA Star, Madison, WI). Several predicted epitopes with high
antigenic index were observed and paralleled with the number of
amino acids (top line).
Fig. 3. Immunocharacterization of rNS1 fragments with NS1 MAbs.
A: Coomassie blue staining of SDS-PAGE resolved bacterial lysates
from IPTG-induced BL21 (DE3) with different plasmids. Vector:
pET21 plasmid; NS1: pET21 plasmid with full-length sequence of
NS1; NS1-C: pET21 plasmid with sequence of C-terminal half of NS1;
NS1-N: pET21 plasmid with sequence of N-terminal half of NS1.
B: Western blotting assay of recombinant NS1 fragments with MAb
13-1 speci®c for the N-terminal of NS1 protein. The lysates described
above were blotted and reacted with MAb 13-1. This MAb speci®cally
detected rNS1 (44 kDa) and rNS1-N (24 kDa). C: Western blotting
assay of recombinant NS1 fragments with MAb 8-2 speci®c for the
C-terminal of NS1 protein. It detected both rNS1 and rNS1-C (28 kDa).
The extra band (*), which could be recognized by these two Mabs, was
a major rNS1 degradation product.
Fig. 4. Puri®cation of NS1 recombinant fragments. A: Cellular
location of the NS1 recombinant fragments. Soluble fractions (Sol.)
and inclusion bodies (Inc.) of bacterial lysates expressing rNS1 or
rNS1-C were separated and resolved by SDS-PAGE. B: Refolding and
puri®cation of NS1 recombinant fragments. The inclusion bodies were
solubilized using 8 M urea. The solubilized proteins were refolded
gradually by decreasing the concentration of urea and ran through the
T7 af®nity column. The ®nal products were completely resolved in
PBS buffer. Inc.: before treatment; T7: after treatment.
558
Huang et al.
TABLE I. Antigenicity Con®rmation of rNS1 and rNS1-C by
ELISA With Mouse Monoclonal Antibodies
Recombinant antigen
Mouse MAb
rNS1
rNS1-C
D2NS1-13-1
D2NS1-8-2
D2NS1-48-6
D2NS1-43-1
D2NS1-45-3
D2NS1-42-2
D2NS1-48-2
D2NS1-49-1
D2NS1-52-2
D2NS1-42-1
D2NS1-39-1
D2NS1-44-1
D2NS1-37-4
D2NS1-32-7
D2NS1-16-1
D2NS1-12-1
D2NS1-11-4
D2NS1-9-1
D2NS1-8-1
D2NS1-7-1
D2m-48-4a
BTBa
1.340
1.354
1.388
1.616
1.201
1.391
1.340
1.236
1.544
1.434
1.407
0.511
1.663
1.808
1.321
0.532
0.071
0.599
1.613
0.885
0.116
0.057
0.244
1.242
0.855
1.426
1.027
1.306
0.095
0.046
1.509
1.296
0.308
0.570
0.784
1.194
1.351
0.397
0.152
0.271
0.430
0.076
0.192
0.056
suggested that the NS1 proteins of dengue virus are
highly immunogenic and can induce humoral immune
response after primary dengue virus infection. Using
highly antigenic rNS1 as the diagnostic antigen, we can
detect anti-NS1 Ab response from most of the serum
samples of primary and secondary dengue virus
infection. The serum samples used in this study were
from dengue fever patients with relatively mild
syndromes, and the results suggested that the antiNS1 Ab response is not correlated with severe dengue
hemorrhagic fever. In addition, the results for 94%
(31/33) of the rNS1-ELISA correlated with the respective results of HI and commercial ELISA tests. The
high sensitivity of the assay demonstrated the feasibility of using recombinant NS1 as a diagnostic antigen.
DISCUSSION
ELISA, enzyme-linked immunosorbent assay; MAb, monoclonal antibody.
a
Non-NS1 MAbs representing the negative control.
does and serves as a more signi®cant diagnostic Ag for
dengue viral infection.
The seropositive rates by using the full-length rNS1
for IgM- or IgG-speci®c ELISA were 88% (15/17) of
patients with primary infection and 100% (16/16) of
patients with secondary infection (Table IV). This
DF and DHF are important public health problems in
urban populations throughout the tropical and subtropical regions of the world. In spite of the development of various assays for serodiagnosis of dengue viral
infection, the diagnostic antigens used in these assays
must still be prepared from tissue culture-grown viral
proteins. This task is laborious, unsafe, expensive, and
subject to batch quality variation, making it unsuitable
for routine large-scale production. Moreover, the titer
of dengue virus that can be obtained from cell culture is
relatively low, especially for dengue 3 and dengue 4.
NS1 was originally described as a soluble complement -®xing antigen. It may also be a circulating
antigen and play an important role in mediating
immunity. It elicits high-titer antibody in animals
infected or immunized with virus [Mason et al., 1990].
TABLE II. Detection of Anti-NS1 Ab Response by rNS1-Based ELISA From Patients of Primary Dengue Virus Infection
DEN patients
9393
9539
9775
9529
9528
10995
11113
9507
9442
9443
SL4
SL5
SL7
LK1
LK2
PB1
PB2
Normal meansc
Cutoff d
Serotype
Status of
infectiona
HI titer
?
?
2
3
3
1
1
3
1
?
1
1
1
1
1
?
?
Primary/day 3
Primary/day 8
Primary/day 9
Primary/day 12
Primary/day 13
Primary/day 5
Primary/day 17
Primary/day 14
Primary/day 17
Primary/day 18
Primary/day > 30
Primary/day >30
Primary/day > 30
Primary/day > 30
Primary/day > 30
Primary/day > 30
Primary/day > 30
< 10
10
10
10
80
10
320
320
1280
1280
ND
ND
ND
ND
ND
ND
ND
I
rNS1-IgM
II
rNS1-IgG
III
rNS1-C-IgM
IV
rNS1-C-IgG
0.283b
0.183
0.132
0.320b
0.282b
0.424b
0.627b
0.333b
0.460b
0.460b
0.250b
0.279b
0.543b
0.467b
0.705b
0.204
0.315b
0.150
0.216
0.268
0.312
0.216
0.379
0.192
0.333
0.413
0.583b
1.252b
1.106b
0.396
0.574b
0.522b
0.497b
0.703b
0.513b
0.808b
0.239
0.480
0.342b
0.129
0.100
0.176
0.211
0.394b
0.680b
0.339b
0.442b
0.441b
0.324b
0.293b
0.704b
0.620b
0.879b
0.314b
0.337b
0.118
0.270
0.289
0.355
0.217
0.442
0.163
0.333
0.427
0.621b
1.208b
1.098b
0.284
0.500b
0.583b
0.536b
0.704b
0.429
0.606b
0.284
0.458
ELISA, enzyme-linked immunosorbent assay; HI, hemagglutination inhibition; ND, no data.
a
IgM 1.0, IgG < 3.0 by Dengue Duo IgM capture and IgG capture ELISA (PanBio).
b
Labels showed positive results (>0.458).
c
Means of 20 non-dengue-infected normal adults' sera.
d
Means ‡3 SD.
559
Anti-NS1 Ab in Dengue Patients
TABLE III. Detection of Anti-NS1 Ab Response by rNS1-Based ELISA From Patients of Secondary Dengue Virus Infection
DEN patients
Serotype
Status of infectiona
HI titer
9802
9509
11146
11196
11167
11194
9435
9594
9331
9568
9385
9742
9386
9462
9670
PB3
Normal means
Cutoff
?
1
3
3
2
2
1,3,4
?
1
3
1
?
1,4
1,3
?
?
Secondary/day 8
Secondary/day 8
Secondary/day 9
Secondary/day 13
Secondary/day 13
Secondary/day 6
Secondary/day 9
Secondary/day 14
Secondary/day 17
Secondary/day 18
Secondary/day 26
Secondary/day 293
Secondary/day 22
Secondary/day 17
Secondary/day 23
Secondary/day > 30
10240
5120
ND
ND
ND
ND
10240
2560
20480
10240
2560
5120
2560
10240
2560
ND
I
rNS1-IgM
II
rNS1-IgG
III
rNS1-C-IgM
IV
rNS1-C-IgG
0.494b
0.278b
0.326b
0.374b
0.291b
0.305b
0.194
0.209
0.376b
0.625b
0.401b
0.693b
0.215
0.285b
0.220b
0.246b
0.150
0.216
0.302
0.405
0.324
0.266
0.454
0.690b
0.733b
0.587b
0.529b
1.251b
0.552b
1.127b
0.511b
0.677b
0.789b
0.359
0.239
0.480
0.499b
0.296b
0.496b
0.440b
0.307b
0.272b
0.170
0.155
0.351b
0.548b
0.374b
0.658b
0.214
0.194
0.412b
0.263
0.118
0.270
0.325
0.434
0.373
0.422
0.545b
0.834b
0.560b
0.697b
0.562b
1.240b
0.625b
1.200b
0.665b
0.568b
0.874b
0.376
0.284
0.458
ELISA, enzyme-linked immunosorbent assay; HI, hemagglutination inhibition.
a
IgM 1.0 and IgG 3.0 by Dengue Duo IgM capture and IgG capture ELISA (PanBio).
b
Labels showed positive results.
Previous results suggested that NS1 is an excellent
candidate that can be used in a recombinant immunodiagnostic assay. In this study, we obtained a high yield
of rNS1 and veri®ed its high antigenicity. The recoveries of these recombinant proteins were very high,
ranging within 1±3 mg/dl of culture, an amount
suf®cient to perform 5,000±30,000 tests.
In earlier studies, anti-NS1 antibody was rarely
found in patients with primary dengue infection but
was detected in most patients with secondary infection.
These observations led to the association of anti-NS1
antibody and the development of DHF [Falker et al.,
1973]. Using our highly antigenic rNS1, we could detect
an anti-rNS1 Ab response in 88% of patients with
primary dengue infection and in all patients with
secondary infection. This universal prevalence of antiNS1 Ab in patients with secondary infection has also
been previously reported [Falkler et al., 1973; Kuno
et al., 1990]. However, in this study, the prevalence of
anti-NS1 Ab in patients with primary infection was
relatively high compared with the results of Falkler
and Kuno and their colleagues, who detected anti-NS1
IgG Ab in only 2/111 and 1/9 of primary infection
samples. They speculated that the low positive rates
were due to the low sensitivity of their assay systems
(Western blot analysis or complement ®xation assay)
TABLE IV. Reactivity of Serum Samples of Dengue
Virus-Infected Patients on rNS1-ELISA
No. of positive
serum samples
No. of patients
Primary infection (17)
Secondary infection (16)
IgM
IgG
No. of
double
negative
results
14
13
9
10
2
0
and low-antibody titers of specimens commonly found
in primary infection. In the early stage of primary
infection, the IgM Ab may be induced before the
appearance of IgG. Thus, the use of high antigenic
NS1 as a diagnostic antigen in ELISA to detect IgM
speci®c Ab may enhance the detection rate in patients
with primary infection. Our results concerning the
prevalence of anti-NS1 Ab in the sera of patients with
primary dengue viral infection correspond to the
®ndings of Huang et al. These investigators used an
NS1 peptide (NS1-P1) or NS1 MAb-captured viral NS1
protein to detect an anti-NS1 Ab response in patients
with primary and secondary infections [Huang et al.,
1999; Shu et al., 2000]. Taking these together, we
conclude that anti-NS1 Ab appears in primary and
milder dengue virus infection, and may not be correlated with the pathogenesis of DHF.
Among structural and nonstructural proteins of
¯aviviral genome, NS1 is a relatively conserved
¯aviviral protein. In the present study, DEN2 rNS1
reacted with the serum samples from dengue 2 virusinfected patients. Furthermore, DEN2 rNS1 also crossreacted with serum samples of patients with other
dengue serotypes infection. This suggested that our
rNS1 protein also has epitopes which are common to all
four dengue virus serotypes [Falconar and Young,
1991]. We also evaluated the cross-reactivity of rNS1
with serum samples of other ¯avivirus infection and
found that the reaction rate was 2/7 in Japanese
encephalitis and 0/2 in yellow fever patients (data not
shown). However, using the DEN-2 rNS1 protein as an
antigen, we discovered that the anti-NS1 Ab titers from
serum samples of JEV-vaccinated children were below
cutoff values. This ®nding is important for the differentiation of dengue sera from JEV vaccine-immunized
sera in countries, such as Taiwan, where JEV vaccine
560
Huang et al.
immunization is in the routine schedule. The high
prevalence of NS1 Ab in dengue patients and relatively
low cross-reactivity with JEV-infected and JEV-vaccinated sera make DEN-2 rNS1 a suitable diagnostic
antigen.
In summary, the recombinant NS1 protein used in
this study was found to be a useful diagnostic antigen
that is easy to prepare and handle. The E. coliexpressed NS1 protein is suitable for inexpensive
massive production with low batch variation. To
explore further the immunodominant domain of the
NS1 protein, other recombinant dengue proteins will be
produced. Further research using IgM-capture recombinant NS1 fragment-speci®c ELISA and combining
other immumunodominant dengue recombinant fragments should improve the sensitivity and speci®city of
dengue recombinant cocktail antigens.
ACKNOWLEDGMENTS
The authors thank Dr. Duane J Gubler and Dr. Natth
Bhamarapravati for their helpful comments, and Mrs.
Jiunn-Jye Wey for technical assistance.
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