This information is current as

of July 14, 2015.

Deletion of the C-Terminal Region of Dengue

Virus Nonstructural Protein 1 (NS1)
Abolishes Anti-NS1-Mediated Platelet
Dysfunction and Bleeding Tendency
Mei-Chun Chen, Chiou-Feng Lin, Huan-Yao Lei, Shih-Chao
Lin, Hsiao-Sheng Liu, Trai-Ming Yeh, Robert Anderson and
Yee-Shin Lin

Supplementary
Material
References
Subscriptions
Permissions
Email Alerts

http://www.jimmunol.org/content/suppl/2009/07/13/jimmunol.080067
2.DC1.html
This article cites 61 articles, 29 of which you can access for free at:
http://www.jimmunol.org/content/183/3/1797.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscriptions
Submit copyright permission requests at:
http://www.aai.org/ji/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
9650 Rockville Pike, Bethesda, MD 20814-3994.
Copyright 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

J Immunol 2009; 183:1797-1803; Prepublished online 10

July 2009;
doi: 10.4049/jimmunol.0800672
http://www.jimmunol.org/content/183/3/1797

The Journal of Immunology

Deletion of the C-Terminal Region of Dengue Virus

Nonstructural Protein 1 (NS1) Abolishes Anti-NS1-Mediated
Platelet Dysfunction and Bleeding Tendency1
Mei-Chun Chen,* Chiou-Feng Lin, Huan-Yao Lei,* Shih-Chao Lin,* Hsiao-Sheng Liu,*
Trai-Ming Yeh, Robert Anderson, and Yee-Shin Lin2*

nfection with dengue viruses (DV)3 causes disease ranging

from mild dengue fever to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (1). The
clinical features of DHF/DSS include plasma leakage, bleeding
tendency, and thrombocytopenia (2). The pathogenesis of DHF/
DSS is complicated and is the subject of active investigation. In
addition to a direct virus-mediated effect, dendritic cells, monocytes/macrophages, mast cells/basophils, T cells, Abs, cytokines, and complement may all contribute to the progression of
dengue hemorrhagic disease (311). Abs derived from a first
DV infection may enhance the secondary infection of different
serotypes, by a phenomenon called Ab-dependent enhancement
(1218). In addition, Abs generated against DV nonstructural
protein 1 (NS1) recognize common epitopes on coagulationrelated proteins, platelets, and endothelial cells (19). We have
also demonstrated the presence of Abs in patient sera which

*Department of Microbiology and Immunology, Institute of Clinical Medicine, Department of Medical Laboratory Science and Biotechnology, and Center for Gene
Regulation and Signal Transduction Research, National Cheng Kung University Medical College, Tainan, Taiwan; and Department of Microbiology and Immunology,
Dalhousie University, Halifax, Nova Scotia, Canada
Received for publication February 27, 2008. Accepted for publication May 31, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1

This work was supported by Grant NSC953112-B006 002 from the National Research Program for Genomic Medicine, National Science Council, Taiwan.

cross-react with platelets and endothelial cells (20, 21). Further

investigation showed that anti-DV NS1 Abs cause endothelial
cell apoptosis and immune activation (2125).
Bleeding tendency is a marker of hematological abnormality
in DHF/DSS patients (2). Both vascular endothelial cells and
platelets play important roles in this phenomenon, although the
pathogenic mechanisms are not fully understood. Platelet autoantibodies that cause thrombocytopenia have been reported in
some virus infections, including hepatitis C virus, cytomegalovirus, and HIV (26 28). In DV infection, anti-platelet autoantibodies induce complement-mediated cell lysis, which may, at
least in part, account for the pathogenic mechanisms of thrombocytopenia. In addition, these Abs also inhibit platelet aggregation (20). During blood vessel injury, activated platelets adhere to the injury site followed by changing shape, releasing
granule contents, and eventually aggregating together through
fibrin formation (29). Therefore, in this study, we aimed to clarify which step of platelet aggregation was influenced by antiDV NS1 Abs.
Using sequence homology alignment, we found that the C-terminal
region of DV NS1 protein contains cross-reactive epitopes with selfAgs (30, 31). To explore the pathological role of cross-reactive
epitopes in the hemorrhagic syndrome, we deleted the C-terminal region of DV NS1 protein to generate C NS1. Both full-length NS1
and C NS1 proteins were used to produce Abs, the pathogenic effects of which were compared both in vitro and in vivo.

Address correspondence and reprint requests to Dr. Yee-Shin Lin, Department of

Microbiology and Immunology, National Cheng Kung University Medical College, 1
University Road, Tainan 701, Taiwan. E-mail address: yslin1@mail.ncku.edu.tw

Materials and Methods

Mice

Abbreviations used in this paper: DV, dengue virus; DHF, dengue hemorrhagic
fever; DSS, dengue shock syndrome; NS1, nonstructural protein 1; JEV, Japanese
encephalitis virus; PF-4, platelet factor-4; PDI, protein disulfide isomerase.
Copyright 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0800672

C3H/HeN mice were obtained from The Jackson Laboratory and maintained on standard laboratory food and water in the Laboratory Animal
Center of National Cheng Kung University Medical College. Their
8-wk-old progeny were used for the experiments. Housing, breeding,

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

The mechanisms underlying dengue hemorrhagic disease are incompletely understood. We previously showed that antidengue virus (DV) nonstructural protein 1 (NS1) Abs cross-react with human platelets and inhibit platelet aggregation.
Based on sequence homology alignment, the cross-reactive epitopes reside in the C-terminal region of DV NS1. In this study,
we compared the effects of Abs against full-length DV NS1 and NS1 lacking the C-terminal aa 271 to 352 (designated C
NS1). Anti-C NS1 Abs exhibited lower platelet binding activity than that of anti-full-length NS1. Anti-full-length NS1 but
not anti-C NS1 Abs inhibited platelet aggregation, which was shown to involve integrin IIb3 inactivation. We found that
the bleeding time in full-length NS1-hyperimmunized mice was longer than that in the normal control mice. By contrast, C
NS1-hyperimmunized mice showed a bleeding time similar to that of normal control mice. Passively administered anti-DV
NS1, but not anti-C NS1, Ab level decreased markedly in serum and this decrease was correlated with Ab binding to
platelets. A transient platelet loss in the circulation was observed after anti-DV NS1, but not anti-C NS1, Ab administration.
In summary, platelet dysfunction and bleeding tendency are induced by anti-full-length DV NS1 but not by anti-C NS1 Abs.
These findings may be important not only for understanding dengue hemorrhagic disease pathogenesis but also for dengue
vaccine development. The Journal of Immunology, 2009, 183: 17971803.

1798

DENGUE NS1 C-TERMINUS IN PLATELET DYSFUNCTION

and experimental use of the animals were performed in strict accordance with
the Experimental Animal Committee in National Cheng Kung University.

Platelet preparation
Human whole blood containing the anticoagulant (29.9 mM sodium citrate,
113.8 mM glucose, 72.6 mM NaCl, and 2.9 mM citric acid (pH 6.4)) was
centrifuged at 100 g for 20 min at room temperature to obtain plateletrich plasma. The platelet-rich plasma was centrifuged at 1000 g for 10
min at room temperature and washed in EDTA/PBS buffer twice. The
washed platelets were suspended in Tyrodes solution (137 mM NaCl, 20
mM HEPES, 3.3 mM NaH2PO4, 2.7 mM KCl, 1 mg/ml BSA, and 5.6 mM
glucose (pH 7.4)) at a concentration of 108 platelets/ml.

Recombinant protein and Ab preparation

Ab binding to platelet assay

Washed platelets were fixed with 1% formaldehyde in PBS at room
temperature for 10 min and then washed with PBS. Various doses of
anti-full-length DV NS1, anti-C NS1, or anti-JEV NS1 were incubated
with platelets for 30 min. After washing, platelets were incubated with
FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 30 min. Ab binding to platelets was analyzed using flow cytometry (BD Biosciences).

Platelet aggregation assay

Platelet-rich plasma depleted whole blood was centrifuged at 1000 g for
10 min at room temperature and the supernatant was collected as plateletpoor plasma. The platelet number in platelet-rich plasma was determined
and diluted to 107 in 450 l of platelet-poor plasma. Platelets were preincubated with 25 g of anti-full-length DV NS1, anti-C NS1, or anti-JEV
NS1 Abs at 37C for 30 min, followed by addition of 20 M ADP (SigmaAldrich). Platelet aggregation was detected using an automated aggregometer PACKS-4 (Helena Laboratories).

Granule secretion and integrin activation assay

Aliquots of 107 platelets were pretreated with 25 g of anti-DV NS1,
anti-C NS1, or anti-JEV NS1 Abs for 30 min, followed by addition of
20 M ADP for 10 min. For platelet factor-4 (PF-4) detection, samples
were placed on ice for 5 min to stop the reaction and centrifuged at
12,000 g for 1 min. The supernatant was assayed for PF-4 level using
an ELISA kit (American Diagnostica). For P-selectin and active-form
integrin IIb3 expression, PE-conjugated anti-CD62 or FITC-conjugated PAC-1 Ab (BD Biosciences) was used. Stained platelets were
fixed with 1% formaldehyde for 10 min, washed twice, and detected
using flow cytometry.

Bleeding time
Bleeding time was performed by a 3-mm tail-tip transection (32, 33).
Blood droplets were collected on filter paper every 30 s for the first 3 min,
and every 10 s thereafter. Bleeding time was recorded when the blood spot
was smaller than 0.1 mm in diameter.

Ab titer determination
DV NS1, C NS1, or JEV NS1 protein was coated on 96-well plates at 0.2
g/well in coating buffer (NaCO3 1.59 g, NaHCO3 2.93 g (pH 9.6), in 1
liter H2O) at 4C overnight, blocked with 1% BSA in PBS at 4C overnight, and then washed three times with PBS. Mouse sera were pooled and
diluted serially from 1/1000 to 1/2048000. The diluted mouse sera (100 l)
were added into the protein-coated wells, and incubated at 4C overnight.
After washing three times with PBS, peroxidase-conjugated anti-mouse
IgG was added into each well (Calbiochem) and incubated for 2 h at room
temperature. After the wells were washed three times with PBS, 200 l
ABTS (Sigma-Aldrich) was added into each well and the absorbance was
measured at 405 nm.

FIGURE 1. Purification of recombinant DV2 full-length NS1, C NS1,

and JEV NS1 proteins. A, The C-terminal region of DV2 NS1 protein from
aa 271 to 352 was deleted to generate C terminus-truncated (C) DV NS1.
B, Target plasmids were digested with BamH1 and were identified using
agarose gel electrophoresis. C, His6 Tag-containing fusion proteins, overexpressed in E. coli, were purified on Ni2 columns and analyzed by 10%
SDS-PAGE.

Statistics
We used the paired t test for statistical analysis. Statistical significance was
set at p 0.05.

Results
Abs against DV NS1 lacking C terminus show lower binding
activity to human platelets than that of anti-full-length NS1
We previously found that anti-DV NS1 Abs cross-reacted with
platelets and endothelial cells (21, 22, 34), and the C-terminal region of DV NS1 protein contained cross-reactive epitopes (30, 31).
We therefore deleted the C terminus of DV NS1 protein from aa
271 to 352 (C NS1) (Fig. 1), and immunized C3H/HeN mice to
generate anti-C NS1 Abs. Using native form NS1 derived from
DV-infected BHK cells, we confirmed that anti-C NS1 Abs
showed similar binding ability to native NS1 protein as that of
anti-full-length NS1 (data not shown). The binding ability of antiC NS1 to platelets was lower than that of anti-full-length NS1
Abs (Fig. 2). Anti-JEV NS1 was used as a negative control. The
anti-NS1 Abs used in this experiment were obtained from mice
immunized for a total of five times. We also tested Abs derived
from mice after immunization with different doses of NS1. The
results showed a gradual increase of platelet binding by anti-NS1
Abs derived from mice after 2, 3, or 5 immunizations (Fig. S1A).4
This gradual increase correlates with their binding activity to the
C-terminal region of NS1 (Fig. S1B).
Anti-full-length NS1 but not anti-C NS1 Abs inhibit
ADP-induced platelet aggregation
We next examined the effect of anti-C NS1 Abs on ADP-induced
platelet aggregation. Results showed that only anti-full-length NS1
Abs inhibited platelet aggregation, while anti-C NS1 Abs did not
(Fig. 3). Anti-JEV NS1 Abs showed no effect on ADP-induced
platelet aggregation as a negative control.
Anti-DV NS1 Abs inhibit ADP-induced platelet aggregation
through integrin IIb3 inactivation
The mechanism of anti-NS1 Ab-mediated platelet aggregation inhibition was investigated. We first checked whether anti-DV NS1
4

The online version of this article contains supplemental material.

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

Japanese encephalitis virus (JEV) NS1, DV2 NS1 (New Guinea C strain)
(22), and C terminus (aa 271352)-deleted DV2 NS1 (C NS1) cDNA
were cloned into the pET28a vector with His6 Tag. Plasmids were introduced into Escherichia coli BL21. The recombinant proteins were induced
by 1 M isopropyl B-D-1-thiogalactopyranoside (Calbiochem) and purified with Ni2 columns. After purification, proteins were examined using
10% SDS-PAGE. Proteins from SDS-PAGE were excised and homogenized in adjuvant to immunize mice. Purified protein (25 g) was emulsified in CFA for the first immunization, and 2 wk later in IFA for additional 1, 2, or 4 immunizations every week. Mouse sera were collected 3
days after the last immunization, and IgG was purified using protein G
columns (Pharmacia Fine Chemicals).

The Journal of Immunology

Abs might interfere with the steps of ADP-induced platelet

granule secretion. Platelet -granules contain many proteins,
such as PF-4, platelet-derived growth factor, -thromboglobulin, fibrinogen, von Willebrand factor, and fibronectin (3538).
Moreover, following granule secretion, P-selectin undergoes redistribution from the platelet -granule membrane to the plasma
membrane (39, 40). We used P-selectin and PF-4 as granule
secretion markers (41, 42). Results showed that neither antifull-length NS1 nor anti-C NS1 Abs inhibited P-selectin expression (Fig. 4A) and PF-4 secretion (Fig. 4B).
The activation of platelet membrane glycoprotein IIb3,
plasma fibrinogen receptor, is essential for platelet aggregation
(43 45). We found that anti-full-length NS1 but not anti-C
NS1 Abs inhibited ADP-induced activation of integrin IIb3
(Fig. 4C). These results indicated that anti-NS1 Abs inhibited
ADP-induced platelet aggregation via blocking integrin IIb3
activation.
Active immunization with full-length NS1 but not C NS1
causes prolonged bleeding time in mice
Because anti-C NS1 Abs showed reduced platelet binding
ability and did not inhibit platelet aggregation compared with
anti-full-length DV NS1 Abs, we investigated their effects in
vivo following active immunization of mice with full-length
and C NS1 proteins. The bleeding tendency was determined
by measuring mouse tail bleeding time (32, 33). Results showed
that the bleeding time in full-length NS1-immunized mice was
longer than that in normal control mice. The bleeding time in
C NS1-immunized mice was similar to that of JEV NS1-immunized mice and normal control mice (Fig. 5A). However, the

FIGURE 3. Anti-DV NS1 but not anti-C NS1 Abs inhibit ADP-induced platelet aggregation. Human platelet-rich plasma was preincubated
with 25 g of anti-full-length DV NS1, anti-C NS1, anti-JEV NS1, or
control IgG at 37C for 30 min followed by stimulation with 20 M ADP,
and platelet aggregation was recorded for 6 min. A, Percentage light transmission was monitored using an aggregometer. B, Platelet aggregation was
quantified and shown as the mean SD of triplicate cultures. The normal
control, which was not pretreated with Abs, was normalized to 100% of
platelet aggregation. , p 0.01.

platelet counts in DV NS1-, C NS1-, and JEV NS1-immunized

mice were all increased (Fig. 5B), ruling out the possible mechanism that the prolonged bleeding time in DV NS1-immunized
mice might be due to reduced platelet counts. In this study, mice
were actively immunized for a total of five times. We also immunized mice for two or three doses of DV NS1, and the results
showed prolonged bleeding time in these mice, yet statistically
nonsignificant, as compared with PBS-immunized controls (Fig.
S2A). The platelet numbers were increased after mice were immunized with PBS in adjuvant for three times or with DV NS1
in adjuvant for two or three times (Fig. S2B).
We next investigated the effects of anti-full-length NS1 or
anti-C NS1 Abs after passive immunization in mice. Results
showed a marked decrease of anti-full-length NS1 Ab titers in
mouse sera by 24 h (Fig. 6A) while the titers of anti-C NS1
(Fig. 6B) and anti-JEV NS1 (Fig. 6C) showed only a slight
decrease or no change up to 48 h after Ab administration. The
decrease of serum Ab titers was correlated with their platelet
binding activity as evidenced by the presence of Abs on the
platelets isolated from mice passively immunized with anti-fulllength NS1 Abs (Fig. 6D). In addition to using Abs obtained
from mice immunized five times with DV NS1 as shown in Fig.
6, we also tested Abs from mice immunized two or three times
with DV NS1. Results showed that anti-DV NS1 Abs derived

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

FIGURE 2. Anti-C NS1 Abs show lower binding to human platelets

than that of anti-full-length DV NS1 Abs. Polyclonal Abs against fulllength DV NS1, C NS1, or JEV NS1 were generated in mice and purified
on protein G columns. A, Platelets were incubated with various Abs for 30
min, followed by FITC-conjugated anti-mouse IgG staining and analyzed
by flow cytometry. One representative histogram with 5 g Ab treatment
is shown. B, The percentages of platelet binding were quantified. Data are
presented as the mean SD of triplicate cultures. , p 0.05; , p
0.01; , p 0.001.

1799

1800

DENGUE NS1 C-TERMINUS IN PLATELET DYSFUNCTION

Discussion

FIGURE 4. Anti-NS1 Abs inhibit ADP-induced integrin IIb3 activation but not -granule secretion in platelets. Human platelet-rich
plasma was preincubated with 25 g of anti-full-length NS1, anti-C
NS1, anti-JEV NS1, or control IgG at 37C for 30 min followed by
stimulation with 10 or 20 M of ADP for 10 min. A, The platelets were
stained with PE-conjugated anti-CD62 Ab and analyzed by flow cytometry. The percentages of P-selectin-expressing cells were quantified
and shown as the mean SD of triplicate cultures. Bacitracin (BCT; 5
mM) was used as the control showing an inhibition of ADP-induced
platelet activation. B, ADP-stimulated platelet supernatants were collected to determine the concentrations of PF-4 by ELISA. C, ADPstimulated platelets were stained with FITC-conjugated PAC-1 Ab and
analyzed by flow cytometry. The percentages of active-form integrin
IIb3-expressing cells were quantified and shown as the mean SD of
triplicate cultures. , p 0.05; , p 0.01.

from sera of mice immunized two or three times with NS1 also
showed the decrease of serum Abs and the increase of platelet
binding activity (Fig. S3). Furthermore, anti-full-length NS1
Abs, but not anti-C NS1 or anti-JEV NS1, caused a transient
platelet loss in the circulation at 6 h after administration with
anti-full-length NS1 Abs (Fig. 6E).

We previously showed that anti-DV NS1 Abs cross-reacted with

human platelets and caused platelet dysfunction (20, 34). In the
present study, we further showed that anti-NS1 inhibited ADPinduced platelet aggregation via blocking integrin IIb3 activation. In addition, the major cross-reactive epitopes are located in
the C-terminal region of NS1 protein. Compared with Abs against
full-length NS1, Abs against NS1 lacking the C terminus showed
lower platelet binding ability and did not inhibit ADP-induced
platelet aggregation. These results correlated with the bleeding tendency in mice. We found prolonged bleeding times after mice were
immunized with NS1 as compared with that of mice immunized
with C NS1 or JEV NS1.
Anti-dengue Abs play various crucial roles in the development of DV-caused disease, ranging from amplifying the number of DV-infected target cells at the beginning of infection to
the later stages of immune-mediated cell or tissue damage. The
generation and titer of the anti-dengue Abs as well as the status
of primary infection or secondary infection are very important
to explain their roles in the dengue pathogenesis. In this study,
we address the role of anti-NS1 in platelet dysfunction and
bleeding tendency. Because NS1 is a nonstructural protein, antiNS1 Abs cannot enhance DV infection. However, anti-NS1 Abs
can influence dengue immunopathogenesis due to the ability of
anti-NS1 Abs to bind platelets via cross-reactive epitopes (34).
This is also a problem with using NS1 as a candidate vaccine.
In this study, we demonstrate that carboxy-truncated NS1 is
largely depleted of platelet cross-reactivity, thereby providing a
novel vaccine candidate with improved safety characteristics.
Thrombocytopenia is a common feature in patients after DV
infection. One of the possible mechanisms of DV-induced

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

FIGURE 5. Prolonged bleeding time in NS1-hyperimmunized mice. A,

C3H/HeN mice were i.p. immunized with recombinant full-length NS1
(n 10), C NS1 (n 9), or JEV NS1 (n 9) proteins or nonimmunized
as the normal control (n 10), and the bleeding time was determined 3
days after the last injection as described in Materials and Methods. B, After
the bleeding time experiment, mouse blood samples were collected and the
platelet numbers were counted using an automatic blood-cell counter. ,
p 0.01; , p 0.001.

The Journal of Immunology

thrombocytopenia is that DV impairs hematopoietic progenitor

cell growth resulting in a decrease in thrombopoiesis (46). Also,
anti-envelope protein Abs enhanced binding of DV to platelets,
supporting a role for platelet clearance in the pathogenesis of
thrombocytopenia (47). We previously demonstrated that crossreactive Abs in dengue patient sera and mouse anti-NS1 Abs
caused platelet lysis (20, 34), illustrating a further potential
mechanism for platelet loss in dengue disease. A recent study
demonstrated that anti-platelet autoantibodies elicited by DV
NS1 caused thrombocytopenia and mortality in mice (48). We
also showed that passive immunization with anti-full-length
NS1 Abs caused transient platelet loss, a common feature of
dengue disease. Most interestingly, anti-C NS1 Abs did not
cause platelet loss. At present, our data do not elucidate the
mechanism of platelet loss. Whether it is due to sequestration
from the circulation or platelet destruction or by other mechanisms remains for further investigation. We determined the NS1
Ab titer, complement C5a, and LDH level in mouse sera after
passive immunization with anti-NS1 Abs. At 6 h, anti-DV NS1
titer in mouse sera showed a decrease as compared with that at
1 h. In addition, both LDH and C5a levels were increased in the
anti-NS1-treated group as compared with the control group at
6 h (our unpublished data). Furthermore, anti-NS1 Abs can also
bind to endothelial cells and stimulate the expression of adhe-

sion molecules (24). These adhesion molecules trap platelets on

endothelial cell surface (49), which may provide another reason
for the low platelet numbers in circulation at 6 h. By 24 h,
platelet replenishment allows for platelets to reach normal
levels.
Nevertheless, we found that DV NS1-, C DV NS1-, and
JEV NS1-immunized mice all had elevated platelet counts, suggesting that the differences in bleeding times were not due to
different platelet counts. The mechanism of blood coagulation is
complicated in that, in addition to platelet number, platelet
function, blood vessel function, and coagulation factors all contribute to stop bleeding. The increased bleeding time in NS1immunized mice is very likely, at least in part, due to the dysfunction of platelet aggregation, which was demonstrated in our
in vitro studies. The increased platelet numbers observed in all
the active-immunization groups may be explained by the phenomenon of adjuvant-induced inflammation, which involves induction of proinflammatory cytokines, leading to inflammatory
thrombopoiesis (50, 51). The effect of adjuvant-induced thrombopoiesis was confirmed by immunizing mice with adjuvant
plus PBS only to give a platelet number of 1264 276
103/l (n 14) as compared with normal control of 825
83 103/l (n 10).
Mice passively administered anti-NS1 Abs showed decreased
Ab titers in the circulation as compared with those mice given
anti-C NS1 or anti-JEV NS1 Abs. Our finding that anti-NS1 (but
not anti-C NS1 or anti-JEV NS1) shows good binding to platelets
in vivo may provide a possible explanation for the observed decreases in serum Ab titers. Other than binding to platelets, the
decrease of anti-DV NS1 Abs in the circulation might also be
caused by their binding to endothelial cells as we have previously
demonstrated (21).
In the present study, NS1 and C NS1 derived from DV2 were
used. We previously reported that the levels of anti-endothelial cell
Abs were similar in patients infected by different DV serotypes
(52). These findings suggested that there is no serotype-specificity
for anti-NS1 autoantibody production. We also showed that the
cross-reactivity of DV3-infected patient sera to endothelial cells,
which led to induction of endothelial cell apoptosis, could be inhibited by DV2 NS1 preabsorption (21). We recently showed that
anti-DV2 NS1 Abs cross-reacted with liver vessel endothelium
(53). Furthermore, IgG purified from DV3-infected patient sera
caused liver injury in mice, and liver injury induced by these IgG
was inhibited by preabsorption with DV2 NS1. Therefore, antiNS1-mediated cross-reactivity shows no dengue serotype-specificity. The sequence alignment by ClustalW2 showed that the identity
of the aa 271352 sequence between DV2 and DV1 is 81.71%,
between DV2 and DV3 is 81.71%, and between DV2 and DV4
is 76.83%.
Using two-dimensional gel electrophoresis and Western blot
analysis, we have previously identified anti-DV NS1 cross-reactive proteins from endothelial cell membrane extract (31).
Among them, protein disulfide isomerase (PDI) can be expressed on the platelet surface and participate in platelet aggregation (54 56). Our unpublished results show that anti-DV NS1
can also cross-react with PDI on the platelet surface. PDI may
regulate the activation of integrin IIb3, the fibrinogen receptor, which is required for the late stage of platelet aggregation
(55, 57). Therefore, the inhibitory effect of anti-DV NS1 on the
activation of integrin IIb3 may be through the recognition of
platelet surface PDI to block its active sites and interfere with
PDI function. This hypothesis is currently under investigation.
Numerous strategies of dengue vaccine design are based on
the protective efficacy of Abs against viral E or NS1 protein

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

FIGURE 6. Passively administered anti-NS1 but not anti-C NS1

Abs are diminished in mouse sera and this decrease is correlated to the
binding of Abs to platelets. Mice were i.v. administered with 500 g of
anti-full-length NS1, anti-C NS1, or anti-JEV NS1 IgG. After 1, 24,
and 48 h, blood samples were collected. n 5/group. AC, The serum
Ab titers were analyzed by ELISA as described in Materials and Methods. D, Freshly isolated mouse platelets after Ab administration for 24 h
were washed and fixed with 1% formaldehyde in PBS, and then stained
with FITC-conjugated anti-mouse IgG. Ab binding to platelets was detected and quantified by flow cytometry. , p 0.01; , p 0.001.
E, Platelet numbers at different time points were monitored by an automatic blood-cell counter.

1801

DENGUE NS1 C-TERMINUS IN PLATELET DYSFUNCTION

(58 61). Although the E protein is responsible for eliciting

major neutralizing Abs during DV infection, it is also associated with the induction of infection-enhancing Abs. A limitation to the vaccine regimen containing NS1, however, is that
anti-NS1 Abs may cause cross-reaction with platelets. The resultant consequences of platelet lysis and impaired platelet aggregation may lead to thrombocytopenia and bleeding tendency.
The findings in this study suggest that C-terminal deletion of
NS1 protein may provide a possible strategy for dengue vaccine
development.

21. Lin, C. F., H. Y. Lei, A. L. Shiau, C. C. Liu, H. S. Liu, T. M. Yeh, S. H. Chen,

and Y. S. Lin. 2003. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J. Med. Virol. 69: 8290.
22. Lin, C. F., H. Y. Lei, A. L. Shiau, H. S. Liu, T. M. Yeh, S. H. Chen, C. C. Liu,
S. C. Chiu, and Y. S. Lin. 2002. Endothelial cell apoptosis induced by antibodies
against dengue virus nonstructural protein 1 via production of nitric oxide. J. Immunol. 169: 657 664.
23. Lin, Y. S., C. F. Lin, H. Y. Lei, H. S. Liu, T. M. Yeh, S. H. Chen, and C. C. Liu.
2004. Antibody-mediated endothelial cell damage via nitric oxide. Curr. Pharm.
Design 10: 213221.
24. Lin, C. F., S. C. Chiu, Y. L. Hsiao, S. W. Wan, H. Y. Lei, A. L. Shiau, H. S. Liu,
T. M. Yeh, S. H. Chen, C. C. Liu, and Y. S. Lin. 2005. Expression of cytokine,
chemokine, and adhesion molecules during endothelial cell activation induced
by antibodies against dengue virus nonstructural protein 1. J. Immunol. 174:
395 403.
25. Lin, C. F., S. W. Wan, H. J. Cheng, H. Y. Lei, and Y. S. Lin. 2006. Autoimmune
pathogenesis in dengue virus infection. Viral Immunol. 19: 127132.
26. Panzer, S., E. Seel, M. Brunner, G. F. Kormoczi, M. Schmid, P. Ferenci, and
M. Peck-Radosavljevic. 2006. Platelet autoantibodies are common in hepatitis C
infection, irrespective of the presence of thrombocytopenia. Eur. J. Haematol. 77:
513517.
27. Ip, H., and B. D. Corner. 1973. Thrombocytopenic purpura in cytomegalovirus
mononucleosis. Lancet 2: 621.
28. Bettaieb, A., P. Fromont, F. Louache, E. Oksenhendler, W. Vainchenker,
N. Duedari, and P. Bierling. 1992. Presence of cross-reactive antibody between
human immunodeficiency virus (HIV) and platelet glycoproteins in HIV-related
immune thrombocytopenic purpura. Blood 80: 162169.
29. Kamath, S., A. D. Blann, and G. Y. H. Lip. 2001. Platelet activation: assessment
and quantification. Eur. Heart J. 22: 15611571.
30. Wan, S. W., C. F. Lin, M. C. Chen, H. Y. Lei, H. S. Liu, T. M. Yeh, C. C. Liu,
and Y. S. Lin. 2008. C-terminal region of dengue virus nonstructural protein 1 is
involved in endothelial cell cross-reactivity via molecular mimicry. Am. J. Infect.
Dis. 4: 8591.
31. Cheng, H. J., C. F. Lin, H. Y. Lei, H. S. Liu, T. M. Yeh, Y. H. Luo, and Y. S. Lin.
2009. Proteomic analysis of endothelial cell autoantigens recognized by antidengue virus nonstructural protein 1 antibodies. Exp. Biol. Med. 234: 6373.
32. Severin, S., M. P. Gratacap, N. Lenain, L. Alvarez, E. Hollande, J. M. Penninger,
C. Gachet, M. Plantavid, and B. Payrastre. 2007. Deficiency of Src homology 2
domain-containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth. J. Clin. Invest. 117: 944 952.
33. Pergolizzi, R. G., G. Jin, D. Chan, L. Pierre, J. Bussel, B. Ferris, P. L. Leopold,
and R. G. Crystal. 2006. Correction of a murine model of von Willebrand disease
by gene transfer. Blood 108: 862 869.
34. Lin, C. F., H. Y. Lei, C. C. Liu, H. S. Liu, T. M. Yeh, R. Anderson, and Y. S. Lin.
2008. Patient and mouse antibodies against dengue virus nonstructural protein 1
cross-react with platelets and cause their dysfunction or depletion. Am. J. Infect.
Dis. 4: 69 75.
35. Kaplan, K. L., M. J. Broekman, A. Chernoff, G. R. Lesznik, and M. Drillings.
1979. Platelet -granule proteins: studies on release and subcellular localization.
Blood 53: 604 618.
36. Reed, G. L., M. L. Fitzgerald, and J. Polgar. 2000. Molecular mechanisms of
platelet exocytosis: insights into the secrete life of thrombocytes. Blood 96:
3334 3342.
37. Rendu, F., and B. Brohard-Bohn. 2001. The platelet release reaction: granules
constituents, secretion and functions. Platelets 12: 261273.
38. King, S. M., and G. L. Reed. 2002. Development of platelet secretory granules.
Semin. Cell Dev. Biol. 13: 293302.
39. Jensen, M. K., P. de Nully Brown, B. V. Lund, O. J. Nielsen, and
H. C. Hasselbalch. 2000. Increased platelet activation and abnormal membrane
glycoprotein content and redistribution in myeloproliferative disorders.
Br. J. Haematol. 110: 116 124.
40. Heijnen, H. F. G., N. Debili, W. Vainchencker, J. Breton-Gorius, H. J. Geuze, and
J. J. Sixma. 1998. Multivesicular bodies are an intermediate stage in the formation of platelet -granules. Blood 91: 23132325.
41. Morrell, C. N., K. Matsushita, K. Chiles, R. B. Scharpf, M. Yamakuchi,
R. J. A. Mason, W. Bergmeier, J. L. Mankowski, W. M. Baldwin III, N. Faraday,
and C. J. Lowenstein. 2005. Regulation of platelet granule exocytosis by Snitrosylation. Proc. Natl. Acad. Sci. USA 102: 37823787.
42. Kaplan, K. L., and J. Owen. 1981. Plasma levels of -thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 57: 199 202.
43. Du, X., and M. H. Ginsberg. 1997. Integrin IIb3 and platelet function. Thromb.
Haemost. 78: 96 100.
44. Shattil, S. J., H. Kashiwagi, and N. Pampori. 1998. Integrin signaling: the platelet
paradigm. Blood 91: 26452657.
45. Sims, P. J., M. H. Ginsberg, E. F. Plow, and S. J. Shattil. 1991. Effect of platelet
activation on the conformation of the plasma membrane glycoprotein IIb-IIIa
complex. J. Biol. Chem. 266: 73457352.
46. Murgue, B., O. Cassar, M. Guigon, and E. Chungue. 1997. Dengue virus inhibits
human hematopoietic progenitor growth in vitro. J. Infect. Dis. 175: 14971501.
47. Wang, S., R. He, J. Patarapotikul, B. L. Innis, and R. Anderson. 1995. Antibodyenhanced binding of dengue-2 virus to human platelets. Virology 213: 254 257.
48. Sun, D. S., C. C. King, H. S. Huang, Y. L. Shih, C. C. Lee, W. J. Tsai, C. C. Yu,
and H. H. Chang. 2007. Antiplatelet autoantibodies elicited by dengue virus
non-structural protein 1 cause thrombocytopenia and mortality in mice.
J. Thromb. Haemost. 5: 22912299.
49. Tailor, A., D. Cooper, and D. N. Granger. 2005. Plateletvessel wall interactions
in the microcirculation. Microcirculation 12: 275285.

Acknowledgments
We thank the Proteomic Research Core facility, Academia Sinica, Taiwan,
for preparing C terminus (aa 271352)-deleted DV2 NS1 (C NS1) plasmid. We thank Dr. Shu-Ying Sherry Wang for helping with sequence
alignment of NS1 proteins from different dengue serotypes. We also thank
Dr. S. L. Hsieh (National Yang-Ming University, Taipei, Taiwan) and Dr.
Y. L. Lin (Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan) for providing JEV NS1 plasmid.

Disclosures
The authors have no financial conflict of interest.

References
1. Henchal, E. A., and J. R. Putnak. 1990. The dengue viruses. Clin. Microbiol. Rev.
3: 376 396.
2. Gubler, D. J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev.
11: 480 496.
3. Green, S., and A. Rothman. 2006. Immunopathological mechanisms in dengue
and dengue hemorrhagic fever. Curr. Opin. Infect. Dis. 19: 429 436.
4. Clyde, K., J. L. Kyle, and E. Harris. 2006. Recent advances in deciphering viral
and host determinants of dengue virus replication and pathogenesis. J. Virol. 80:
11418 11431.
5. Halstead, S. B. 2007. Dengue. Lancet 370: 1644 1652.
6. Lei, H. Y., T. M. Yeh, H. S. Liu, Y. S. Lin, S. H. Chen, and C. C. Liu. 2001.
Immunopathogenesis of dengue virus infection. J. Biomed. Sci. 8: 377388.
7. Fink, J., F. Gu, and S. G. Vasudevan. 2006. Role of T cells, cytokines and
antibody in dengue fever and dengue haemorrhagic fever. Rev. Med. Virol. 16:
263275.
8. Pang, T., M. J. Cardosa, and M. G. Guzman. 2007. Of cascades and perfect
storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock
syndrome (DHF/DSS). Immunol. Cell Biol. 85: 43 45.
9. Kurane, I., B. L. Innis, A. Nisalak, C. Hoke, S. Nimmannitya, A. Meager, and
F. A. Ennis. 1989. Human T cell responses to dengue virus antigens: proliferative
responses and interferon production. J. Clin. Invest. 83: 506 513.
10. King, C. A., J. S. Marshall, H. Alshurafa, and R. Anderson. 2000. Release of
vasoactive cytokines by antibody-enhanced dengue virus infection of a human
mast cell/basophil line. J. Virol. 74: 7146 7150.
11. Avirutnan, P., P. Malasit, B. Seliger, S. Bhakdi, and M. Husmann. 1998. Dengue
virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J. Immunol. 161: 6338 6346.
12. Halstead, S. B., C. N. Venkateshan, M. K. Gentry, and L. K. Larsen. 1984.
Heterogeneity of infection enhancement of dengue 2 strains by monoclonal antibodies. J. Immunol. 132: 1529 1532.
13. Littaua, R., I. Kurane, and F. A. Ennis. 1990. Human IgG Fc receptor II mediates
antibody-dependent enhancement of dengue virus infection. J. Immunol. 144:
31833186.
14. Mady, B. J., D. V. Erbe, I. Kurane, M. W. Fanger, and F. A. Ennis. 1991.
Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc receptors. J. Immunol. 147: 3139 3144.
15. Morens, D. M. 1994. Antibody-dependent enhancement of infection and the
pathogenesis of viral disease. Clin. Infect. Dis. 19: 500 512.
16. Anderson, R., S. Wang, C. Osiowy, and A. C. Issekutz. 1997. Activation of
endothelial cells via antibody-enhanced dengue virus infection of peripheral
blood monocytes. J. Virol. 71: 4226 4232.
17. Huang, K. J., Y. C. Yang, Y. S. Lin, J. H. Huang, H. S. Liu, T. M. Yeh,
S. H. Chen, C. C. Liu, and H. Y. Lei. 2006. The dual-specific binding of dengue
virus and target cells for the antibody-dependent enhancement of dengue virus
infection. J. Immunol. 176: 28252832.
18. Brown, M. G., C. A. King, C. Sherren, J. S. Marshall, and R. Anderson. 2006. A
dominant role for FcRII in antibody-enhanced dengue virus infection of human
mast cells and associated CCL5 release. J. Leukocyte Biol. 80: 12421250.
19. Falconar, A. K. 1997. The dengue virus nonstructural-1 protein (NS1) generates
antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic
fever pathogenesis. Arch. Virol. 142: 897916.
20. Lin, C. F., H. Y. Lei, C. C. Liu, H. S. Liu, T. M. Yeh, S. T. Wang, T. I. Yang,
F. C. Sheu, C. F. Kuo, and Y. S. Lin. 2001. Generation of IgM anti-platelet
autoantibody in dengue patients. J. Med. Virol. 63: 143149.

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015

1802

The Journal of Immunology

50. Hill, R. J., M. K. Warren, and J. Levin. 1990. Stimulation of thrombopoiesis in
mice by human recombinant interleukin 6. J. Clin. Invest. 85: 12421247.
51. Kaser, A., G. Brandacher, W. Steurer, S. Kaser, F. A. Offner, H. Zoller, I. Theuri,
W. Widder, C. Molnar, O. Ludwiczek, et al. 2001. Interleukin-6 stimulates
thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis.
Blood 98: 2720 2725.
52. Lin, C. F., H. Y. Lei, H. S. Liu, T. M. Yeh, S. H. Chen, C. C. Liu, and Y. S. Lin.
2004. Autoimmunity in dengue virus infection. Dengue Bull. 28: 5157.
53. Lin, C. F., S. W. Wan, M. C. Chen, S. C. Lin, C. C. Cheng, S. C. Chiu,
Y. L. Hsiao, H. Y. Lei, H. S. Liu, T. M. Yeh, and Y. S. Lin. 2008. Liver injury
caused by antibodies against dengue virus nonstructural protein 1 in a murine
model. Lab. Invest. 88: 1079 1089.
54. Essex, D. W., K. Chen, and M. Swiatkowska. 1995. Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane. Blood
86: 2168 2173.
55. Essex, D. W., and M. Li. 1999. Protein disulphide isomerase mediates platelet
aggregation and secretion. Br. J. Haematol. 104: 448 454.

1803
56. Wilkinson, B., and H. F. Gilbert. 2004. Protein disulfide isomerase. Biochim.
Biophys. Acta 1699: 35 44.
57. Lahav, J., N. Gofer-Dadosh, J. Luboshitz, O. Hess, and M. Shaklai. 2000. Protein
disulfide isomerase mediates integrin-dependent adhesion. FEBS Lett. 475:
89 92.
58. Kelly, E. P., J. J. Greene, A. D. King, and B. L. Innis. 2000. Purified dengue 2
virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. Vaccine 18: 2549 2559.
59. Apt, D., K. Raviprakash, A. Brinkman, A. Semyonov, S. Yang, C. Skinner,
L. Diehl, R. Lyons, K. Porter, and J. Punnonen. 2006. Tetravalent neutralizing
antibody response against four dengue serotypes by a single chimeric dengue
envelope antigen. Vaccine 24: 335344.
60. Qu, X., W. Chen, T. Maguire, and F. Austin. 1993. Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system. J. Gen. Virol. 74: 89 97.
61. Costa, S. M., M. S. Freire, and A. M. B. Alves. 2006. DNA vaccine against the
non-structural 1 protein (NS1) of dengue 2 virus. Vaccine 24: 4562 4564.

Downloaded from http://www.jimmunol.org/ by guest on July 14, 2015