of July 14, 2015.This information is current asDysfunction and
Bleeding TendencyAbolishes Anti-NS1-Mediated PlateletVirus
Nonstructural Protein 1 (NS1) Deletion of the C-Terminal Region of
DengueYee-Shin LinLin, Hsiao-Sheng Liu, Trai-Ming Yeh, Robert
Anderson and Mei-Chun Chen, Chiou-Feng Lin, Huan-Yao Lei,
Shih-Chaohttp://www.jimmunol.org/content/183/3/1797doi:
10.4049/jimmunol.0800672July 2009;2009; 183:1797-1803; Prepublished
online 10 J
ImmunolMaterialSupplementary2.DC1.htmlhttp://www.jimmunol.org/content/suppl/2009/07/13/jimmunol.080067Referenceshttp://www.jimmunol.org/content/183/3/1797.full#ref-list-1,
29 of which you can access for free at:cites 61 articles This
article Subscriptionshttp://jimmunol.org/subscriptions is online
at:The Journal of Immunology Information about subscribing to
Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright
permission requests at: Email
Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts
when new articles cite this article. Sign up at: Print ISSN:
0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights
reserved.Copyright 2009 by The American Association of9650
Rockville Pike, Bethesda, MD 20814-3994.The American Association of
Immunologists, Inc., is published twice each month by The Journal
of Immunology by guest on July 14,
2015http://www.jimmunol.org/Downloaded from by guest on July 14,
2015http://www.jimmunol.org/Downloaded from Deletion of the
C-Terminal Region of Dengue VirusNonstructural Protein 1 (NS1)
Abolishes Anti-NS1-MediatedPlatelet Dysfunction and Bleeding
Tendency1Mei-Chun Chen,* Chiou-Feng Lin,Huan-Yao Lei,* Shih-Chao
Lin,* Hsiao-Sheng Liu,*Trai-Ming Yeh,Robert Anderson,and Yee-Shin
Lin2*Themechanismsunderlyingdenguehemorrhagicdiseaseareincompletelyunderstood.
Wepreviouslyshowedthat anti-denguevirus(DV) nonstructural
protein1(NS1) Abscross-react withhumanplateletsandinhibit platelet
aggregation.Based on sequence homology alignment, the
cross-reactive epitopes reside in the C-terminal region of DV NS1.
In this
study,wecomparedtheeffectsofAbsagainstfull-lengthDVNS1andNS1lackingtheC-terminalaa271to352(designatedCNS1).
Anti-C NS1 Abs exhibited lower platelet binding activity than that
of anti-full-length NS1. Anti-full-length NS1 butnot anti-C NS1 Abs
inhibited platelet aggregation, which was shown to involve integrin
IIb3 inactivation. We found thatthe bleeding time in full-length
NS1-hyperimmunized mice was longer than that in the normal control
mice. By contrast, CNS1-hyperimmunized mice showed a bleeding time
similar to that of normal control mice. Passively administered
anti-DVNS1, but not anti-CNS1, Ablevel
decreasedmarkedlyinserumandthisdecreasewascorrelatedwithAbbindingtoplatelets.
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 ndings may
be important not only for understanding dengue hemorrhagic disease
pathogenesis but also for denguevaccinedevelopment.
TheJournalofImmunology,2009,183:17971803.Infectionwithdengueviruses(DV)3causesdiseaserangingfrom
mild dengue fever to severe dengue hemorrhagic fe-ver (DHF)
anddengueshocksyndrome(DSS) (1). Theclinical features of DHF/DSS
include plasma leakage, bleedingtendency, and thrombocytopenia (2).
The pathogenesis of DHF/DSS is complicated and is the subject of
active investigation. Inaddition to a direct virus-mediated effect,
dendritic cells, mono-cytes/macrophages, mast cells/basophils,
Tcells, Abs, cyto-kines, and complement may all contribute to the
progression ofdenguehemorrhagicdisease(311). AbsderivedfromarstDV
infection may enhance the secondary infection of
differentserotypes, by a phenomenon called Ab-dependent
enhancement(1218). Inaddition, Absgeneratedagainst
DVnonstructuralprotein1(NS1) recognizecommonepitopes
oncoagulation-relatedproteins, platelets, andendothelialcells(19).
Wehavealsodemonstratedthepresenceof Abs inpatient
serawhichcross-react with platelets and endothelial cells (20, 21).
Furtherinvestigationshowedthat
anti-DVNS1Abscauseendothelialcellapoptosisandimmuneactivation(2125).Bleeding
tendency is a marker of hematological
abnormalityinDHF/DSSpatients(2). Bothvascular endothelial
cellsandplatelets play important roles in this phenomenon, although
thepathogenic mechanisms are not fully understood. Platelet
auto-antibodiesthat causethrombocytopeniahavebeenreportedinsome
virus infections, including hepatitis C virus,
cytomegalo-virus,andHIV(2628).InDVinfection,anti-plateletautoan-tibodies
induce complement-mediated cell lysis, which may, atleast in part,
account for the pathogenic mechanisms of
throm-bocytopenia.Inaddition,theseAbsalsoinhibitplateletaggre-gation(20).Duringbloodvesselinjury,activatedplateletsad-heretotheinjurysitefollowedbychangingshape,
releasinggranulecontents,
andeventuallyaggregatingtogetherthroughbrin formation (29).
Therefore, in this study, we aimed to clar-ifywhichstepofplatelet
aggregationwasinuencedbyanti-DVNS1Abs.Using sequence homology
alignment, we found that the C-terminalregion of DV NS1 protein
contains cross-reactive epitopes with self-Ags (30, 31). Toexplore
the pathological role of cross-reactiveepitopes in the hemorrhagic
syndrome, we deleted the C-terminal re-gion of DV NS1 protein to
generate C NS1. Both full-length NS1and C NS1 proteins were used to
produce Abs, the pathogenic ef-fects of which were compared both in
vitro and in vivo.Materials and MethodsMiceC3H/HeN mice were
obtained from The Jackson Laboratory and main-tained on standard
laboratory food and water in the Laboratory AnimalCenter of
National Cheng Kung University Medical College.
Their8-wk-oldprogenywereusedfor theexperiments. Housing,
breeding,*Department of Microbiology and Immunology, Institute of
Clinical Medicine,
De-partmentofMedicalLaboratoryScienceandBiotechnology,andCenterforGeneRegulation
and Signal Transduction Research, National Cheng Kung University
Med-icalCollege, Tainan,
Taiwan;andDepartmentofMicrobiologyandImmunology,Dalhousie
University, Halifax, Nova Scotia, CanadaReceived 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
pagecharges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith
18 U.S.C. Section 1734 solely to indicate this fact.1This work was
supported by Grant NSC953112-B006002 from the National Re-search
Program for Genomic Medicine, National Science Council,
Taiwan.2AddresscorrespondenceandreprintrequeststoDr.Yee-ShinLin,DepartmentofMicrobiology
and Immunology, National Cheng Kung University Medical College,
1University Road, Tainan 701, Taiwan. E-mail address:
[email protected]: DV,
denguevirus; DHF, denguehemorrhagicfever; DSS, dengueshocksyndrome;
NS1, nonstructural protein1; JEV, Japaneseencephalitis virus; PF-4,
platelet factor-4; PDI, protein disulde isomerase.Copyright 2009 by
The American Association of Immunologists, Inc.
0022-1767/09/$2.00The Journal of
Immunologywww.jimmunol.org/cgi/doi/10.4049/jimmunol.0800672 by
guest on July 14, 2015http://www.jimmunol.org/Downloaded from and
experimental use of the animals were performed in strict accordance
withthe Experimental Animal Committee in National Cheng Kung
University.Platelet preparationHuman 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)) wascentrifuged at 100 g for
20 min at room temperature to obtain platelet-rich plasma. The
platelet-rich plasma was centrifuged at 1000 g for 10minat
roomtemperatureandwashedinEDTA/PBSbuffer twice. Thewashed platelets
were suspended in Tyrodes solution (137 mM NaCl, 20mM HEPES, 3.3 mM
NaH2PO4, 2.7 mM KCl, 1 mg/ml BSA, and 5.6 mMglucose (pH 7.4)) at a
concentration of 108platelets/ml.Recombinant protein and Ab
preparationJapanese encephalitis virus (JEV) NS1, DV2 NS1 (New
Guinea C strain)(22),
andCterminus(aa271352)-deletedDV2NS1(CNS1)cDNAwere cloned into the
pET28a vector with His6 Tag. Plasmids were intro-duced into
Escherichia coli BL21. The recombinant proteins were
inducedby1MisopropylB-D-1-thiogalactopyranoside(Calbiochem)andpuri-ed
with Ni2columns. After purication, proteins were examined
using10%SDS-PAGE.
ProteinsfromSDS-PAGEwereexcisedandhomoge-nizedinadjuvanttoimmunizemice.Puriedprotein(25g)wasemul-sied
in CFA for the rst immunization, and 2 wk later in IFA for
addi-tional 1, 2, or 4 immunizations every week. Mouse sera were
collected 3daysafterthelast immunization,
andIgGwaspuriedusingproteinGcolumns (Pharmacia Fine Chemicals).Ab
binding to platelet assayWashedplatelets
werexedwith1%formaldehydeinPBSat
roomtemperaturefor10minandthenwashedwithPBS.
Variousdosesofanti-full-length DV NS1, anti-C NS1, or anti-JEV NS1
were incubatedwith platelets for 30 min. After washing, platelets
were incubated withFITC-conjugated anti-mouse IgG (Jackson
ImmunoResearch Laborato-ries)for30min.
Abbindingtoplateletswasanalyzedusingowcy-tometry(BDBiosciences).Platelet
aggregation assayPlatelet-rich plasma depleted whole blood was
centrifuged at 1000 g for10 min at room temperature and the
supernatant was collected as platelet-poor plasma. The platelet
number in platelet-rich plasma was determinedand diluted to 107in
450 l of platelet-poor plasma. Platelets were prein-cubated with 25
g of anti-full-length DV NS1, anti-C NS1, or anti-JEVNS1 Abs at 37C
for 30 min, followed by addition of 20 M ADP (Sigma-Aldrich).
Platelet aggregationwas detectedusinganautomatedaggre-gometer
PACKS-4 (Helena Laboratories).Granule secretion and integrin
activation
assayAliquotsof107plateletswerepretreatedwith25gofanti-DVNS1,anti-C
NS1, or anti-JEV NS1 Abs for 30 min, followed by addition of20 M
ADP for 10 min. For platelet factor-4 (PF-4) detection,
sampleswereplacedonicefor
5mintostopthereactionandcentrifugedat12,000 g for 1 min. The
supernatant was assayed for PF-4 level usinganELISAkit
(AmericanDiagnostica).
ForP-selectinandactive-formintegrinIIb3expression,
PE-conjugatedanti-CD62or FITC-conju-gatedPAC-1Ab(BDBiosciences) was
used. Stainedplatelets werexedwith1%formaldehydefor 10min,
washedtwice, anddetectedusingowcytometry.Bleeding
timeBleedingtimewas performedbya3-mmtail-tiptransection(32,
33).Blood droplets were collected on lter paper every 30 s for the
rst 3 min,and every 10 s thereafter. Bleeding time was recorded
when the blood spotwas smaller than 0.1 mm in diameter.Ab titer
determinationDV NS1, C NS1, or JEV NS1 protein was coated on
96-well plates at 0.2g/well in coating buffer (NaCO3 1.59 g, NaHCO3
2.93 g (pH 9.6), in
1literH2O)at4Covernight,blockedwith1%BSAinPBSat4Cover-night, and
then washed three times with PBS. Mouse sera were pooled anddiluted
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 washingthreetimeswithPBS,
peroxidase-conjugatedanti-mouseIgG was added into each well
(Calbiochem) and incubated for 2 h at roomtemperature.
AfterthewellswerewashedthreetimeswithPBS, 200lABTS (Sigma-Aldrich)
was added into each well and the absorbance wasmeasured at 405
nm.StatisticsWe used the paired t test for statistical analysis.
Statistical signicance wasset at p 0.05.ResultsAbs against DV NS1
lacking C terminus show lower bindingactivity to human platelets
than that of anti-full-length NS1Wepreviouslyfoundthat
anti-DVNS1Abscross-reactedwithplatelets and endothelial cells (21,
22, 34), and the C-terminal re-gion of DV NS1 protein contained
cross-reactive epitopes (30, 31).We therefore deleted the C
terminus of DV NS1 protein from aa271 to 352 (C NS1) (Fig. 1), and
immunized C3H/HeN mice togenerate anti-C NS1 Abs. Using native form
NS1 derived fromDV-infected BHKcells, we conrmed that anti-CNS1
Absshowedsimilar bindingabilitytonativeNS1proteinasthat
ofanti-full-length NS1 (data not shown). The binding ability of
anti-CNS1toplateletswaslowerthanthatofanti-full-lengthNS1Abs (Fig.
2). Anti-JEV NS1 was used as a negative control.
Theanti-NS1Absusedinthisexperiment
wereobtainedfrommiceimmunizedforatotalofvetimes.
WealsotestedAbsderivedfrommiceafterimmunizationwithdifferent
dosesofNS1. Theresults showed a gradual increase of platelet
binding by anti-NS1Abs derived from mice after 2, 3, or 5
immunizations (Fig. S1A).4This gradual increase correlates with
their binding activity to theC-terminal region of NS1 (Fig.
S1B).Anti-full-length NS1 but not anti-C NS1 Abs inhibitADP-induced
platelet aggregationWe next examined the effect of anti-C NS1 Abs
on ADP-inducedplatelet aggregation. Results showed that only
anti-full-length NS1Abs inhibited platelet aggregation, while
anti-C NS1 Abs did not(Fig. 3). Anti-JEVNS1Absshowednoeffect
onADP-inducedplatelet aggregation as a negative control.Anti-DV NS1
Abs inhibit ADP-induced platelet aggregationthrough integrinIIb3
inactivationThe mechanism of anti-NS1 Ab-mediated platelet
aggregation in-hibition was investigated. We rst checked whether
anti-DV NS14The online version of this article contains
supplemental material.FIGURE1. Purication of recombinant DV2
full-length NS1, C NS1,and JEV NS1 proteins. A, The C-terminal
region of DV2 NS1 protein fromaa 271 to 352 was deleted to generate
C terminus-truncated (C) DV NS1.B, Target plasmids were digested
with BamH1 and were identied usingagarose gel electrophoresis. C,
His6 Tag-containing fusion proteins, over-expressed in E. coli,
were puried on Ni2columns and analyzed by 10%SDS-PAGE.1798 DENGUE
NS1 C-TERMINUS IN PLATELET DYSFUNCTION by guest on July 14,
2015http://www.jimmunol.org/Downloaded from Abs might interfere
with the steps of ADP-induced plateletgranulesecretion. Platelet
-granules containmanyproteins,suchasPF-4,
platelet-derivedgrowthfactor, -thromboglobu-lin, brinogen, von
Willebrand factor, and bronectin (3538).Moreover, following granule
secretion, P-selectin undergoes re-distribution from the platelet
-granule membrane to the plasmamembrane(39, 40).
WeusedP-selectinandPF-4as granulesecretionmarkers (41, 42). Results
showedthat neither
anti-full-lengthNS1noranti-CNS1AbsinhibitedP-selectinex-pression(Fig.
4A)andPF-4secretion(Fig. 4B).The activation of platelet membrane
glycoprotein IIb3,plasma brinogen receptor, is essential for
platelet aggregation(4345). Wefoundthat anti-full-lengthNS1but not
anti-CNS1AbsinhibitedADP-inducedactivationof integrinIIb3(Fig. 4C).
Theseresultsindicatedthat
anti-NS1AbsinhibitedADP-inducedplateletaggregationviablockingintegrinIIb3activation.Active
immunization with full-length NS1 but not C NS1causes prolonged
bleeding time in miceBecause anti-CNS1 Abs showed reduced platelet
bindingabilityanddidnot inhibit platelet
aggregationcomparedwithanti-full-lengthDVNS1Abs,
weinvestigatedtheir effectsinvivofollowingactive immunizationof
mice withfull-lengthandCNS1proteins.
Thebleedingtendencywasdeterminedby measuring mouse tail bleeding
time (32, 33). Results showedthat the bleeding time in full-length
NS1-immunized mice waslongerthanthat innormal control mice.
ThebleedingtimeinCNS1-immunizedmicewassimilartothatofJEVNS1-im-munized
mice and normal control mice (Fig. 5A). However, theplatelet counts
in DV NS1-, C NS1-, and JEV NS1-immunizedmice were all increased
(Fig. 5B), ruling out the possible mech-anism that the prolonged
bleeding time in DV NS1-immunizedmice might be due to reduced
platelet counts. In this study, micewere actively immunized for a
total of ve times. We also im-munized mice for two or three doses
of DV NS1, and the resultsshowed prolonged bleeding time in these
mice, yet statisticallynonsignicant, as compared with PBS-immunized
controls (Fig.S2A). The platelet numbers were increased after mice
were im-munized with PBS in adjuvant for three times or with DV
NS1inadjuvantfortwoorthreetimes(Fig. S2B).Wenext
investigatedtheeffectsof
anti-full-lengthNS1oranti-CNS1Absafterpassiveimmunizationinmice.
Resultsshowedamarkeddecreaseofanti-full-lengthNS1Abtitersinmouseseraby24h(Fig.
6A)whilethetitersofanti-CNS1(Fig. 6B) andanti-JEVNS1(Fig. 6C)
showedonlya
slightdecreaseornochangeupto48hafterAbadministration.Thedecreaseof
serumAbtiterswascorrelatedwiththeir plateletbindingactivityas
evidencedbythepresenceof Abs ontheplatelets isolated from mice
passively immunized with anti-full-lengthNS1Abs(Fig. 6D).
InadditiontousingAbsobtainedfrom mice immunized ve times with DV
NS1 as shown in Fig.6, we also tested Abs from mice immunized two
or three timeswithDVNS1. Resultsshowedthat
anti-DVNS1AbsderivedFIGURE2. Anti-C NS1 Abs show lower binding to
human plateletsthanthat of anti-full-lengthDVNS1Abs. Polyclonal
Absagainst full-length DV NS1, C NS1, or JEV NS1 were generated in
mice and puriedon protein G columns. A, Platelets were incubated
with various Abs for 30min, followed by FITC-conjugated anti-mouse
IgG staining and analyzedby ow cytometry. One representative
histogram with 5g Ab treatmentis shown. B, The percentages of
platelet binding were quantied. Data
arepresentedasthemeanSDoftriplicatecultures. ,p0.05; ,p0.01;, p
0.001.FIGURE3. Anti-DVNS1but not anti-CNS1Absinhibit ADP-in-duced
platelet aggregation. Human platelet-rich plasma was
preincubatedwith25gofanti-full-lengthDVNS1, anti-CNS1, anti-JEVNS1,
orcontrol IgG at 37C for 30 min followed by stimulation with 20 M
ADP,and platelet aggregation was recorded for 6 min. A, Percentage
light trans-mission was monitored using an aggregometer. B,
Platelet aggregation wasquantied and shown as the mean SD of
triplicate cultures. The
normalcontrol,whichwasnotpretreatedwithAbs,wasnormalizedto100%ofplatelet
aggregation. , p 0.01.1799 The Journal of Immunology by guest on
July 14, 2015http://www.jimmunol.org/Downloaded from from sera of
mice immunized two or three times with NS1
alsoshowedthedecreaseofserumAbsandtheincreaseofplateletbindingactivity(Fig.
S3). Furthermore,
anti-full-lengthNS1Abs,butnotanti-CNS1oranti-JEVNS1,causedatransientplatelet
lossinthecirculationat
6hafteradministrationwithanti-full-lengthNS1Abs(Fig.
6E).DiscussionWe previously showed that anti-DV NS1 Abs
cross-reacted withhumanplateletsandcausedplatelet dysfunction(20,
34). Inthepresent study, wefurther showedthat
anti-NS1inhibitedADP-inducedplatelet
aggregationviablockingintegrinIIb3activa-tion.Inaddition,themajorcross-reactiveepitopesarelocatedinthe
C-terminal region of NS1 protein. Compared with Abs
againstfull-length NS1, Abs against NS1 lacking the C terminus
showedlower platelet bindingabilityanddidnot inhibit
ADP-inducedplatelet aggregation. These results correlated with the
bleeding ten-dency in mice. We found prolonged bleeding times after
mice wereimmunizedwithNS1ascomparedwiththatofmiceimmunizedwith C
NS1 or JEV NS1.Anti-dengueAbsplayvariouscrucial
rolesinthedevelop-ment of DV-caused disease, ranging from
amplifying the
num-berofDV-infectedtargetcellsatthebeginningofinfectiontothe later
stages of immune-mediated cell or tissue damage. Thegeneration and
titer of the anti-dengue Abs as well as the
statusofprimaryinfectionorsecondaryinfectionareveryimportantto
explain their roles in the dengue pathogenesis. In this study,we
address the role of anti-NS1inplatelet dysfunctionandbleeding
tendency. Because NS1 is a nonstructural protein, anti-NS1 Abs
cannot enhance DV infection. However, anti-NS1 Abscan inuence
dengue immunopathogenesis due to the ability ofanti-NS1 Abs to bind
platelets via cross-reactive epitopes
(34).ThisisalsoaproblemwithusingNS1asacandidatevaccine.Inthis
study, wedemonstratethat carboxy-truncatedNS1islargely depleted of
platelet cross-reactivity, thereby providing
anovelvaccinecandidatewithimprovedsafetycharacteristics.Thrombocytopenia
is a common feature in patients after DVinfection. One of the
possible mechanisms of DV-inducedFIGURE4.
Anti-NS1AbsinhibitADP-inducedintegrinIIb3 acti-vation but not
-granule secretion in platelets. Human
platelet-richplasmawaspreincubatedwith25gofanti-full-lengthNS1,
anti-CNS1, anti-JEVNS1, or control IgGat 37Cfor
30minfollowedbystimulation with 10 or 20 M of ADP for 10 min. A,
The platelets
werestainedwithPE-conjugatedanti-CD62Abandanalyzedbyowcy-tometry.
The percentages of P-selectin-expressing cells were quantiedand
shown as the mean SD of triplicate cultures. Bacitracin (BCT; 5mM)
wasusedasthecontrol showinganinhibitionof ADP-inducedplatelet
activation. B, ADP-stimulatedplatelet
supernatantswerecol-lectedtodeterminetheconcentrations of
PF-4byELISA. C, ADP-stimulated platelets were stained with
FITC-conjugated PAC-1 Ab andanalyzedbyowcytometry. Thepercentagesof
active-formintegrinIIb3-expressing cells were quantied and shown as
the mean SD oftriplicatecultures. , p0.05; , p0.01.FIGURE5.
Prolonged bleeding time in NS1-hyperimmunized mice.
A,C3H/HeNmicewerei.p. immunizedwithrecombinant full-lengthNS1(n
10), C NS1 (n 9), or JEV NS1 (n 9) proteins or
nonimmunizedasthenormalcontrol(n10),andthebleedingtimewasdetermined3days
after the last injection as described in Materials and Methods. B,
Afterthe bleeding time experiment, mouse blood samples were
collected and theplatelet numbers were counted using an automatic
blood-cell counter.,p 0.01;, p 0.001.1800 DENGUE NS1 C-TERMINUS IN
PLATELET DYSFUNCTION by guest on July 14,
2015http://www.jimmunol.org/Downloaded from thrombocytopenia is
that DV impairs hematopoietic progenitorcell growth resulting in a
decrease in thrombopoiesis (46). Also,anti-envelope protein Abs
enhanced binding of DV to platelets,supportingaroleforplatelet
clearanceinthepathogenesisofthrombocytopenia (47). We previously
demonstrated that cross-reactiveAbsindenguepatient
seraandmouseanti-NS1Abscausedplatelet lysis (20, 34), illustratinga
further potentialmechanismforplatelet lossindenguedisease. Arecent
studydemonstratedthat anti-platelet autoantibodies
elicitedbyDVNS1causedthrombocytopeniaandmortalityinmice(48). Wealso
showed that passive immunization with
anti-full-lengthNS1Abscausedtransient platelet loss,
acommonfeatureofdenguedisease. Most interestingly,
anti-CNS1Absdidnotcauseplatelet loss. At present, our datadonot
elucidatethemechanismofplatelet loss. Whetherit
isduetosequestrationfromthecirculationorplateletdestructionorbyothermecha-nisms
remains for further investigation. We determined the NS1Abtiter,
complementC5a, andLDHlevelinmouseseraafterpassive immunization with
anti-NS1 Abs. At 6 h, anti-DV NS1titer in mouse sera showed a
decrease as compared with that at1 h. In addition, both LDH and C5a
levels were increased in
theanti-NS1-treatedgroupascomparedwiththecontrol groupat6 h (our
unpublished data). Furthermore, anti-NS1 Abs can
alsobindtoendothelialcellsandstimulatetheexpressionofadhe-sion
molecules (24). These adhesion molecules trap platelets
onendothelial cell surface (49), which may provide another
reasonfor thelowplatelet numbers incirculationat 6h. By24h,platelet
replenishment allows for platelets to reach
normallevels.Nevertheless, wefoundthat DVNS1-, CDVNS1-, andJEV
NS1-immunized mice all had elevated platelet counts,
sug-gestingthat thedifferencesinbleedingtimeswerenot duetodifferent
platelet counts. The mechanism of blood coagulation iscomplicated
in that, in addition to platelet number, plateletfunction, blood
vessel function, and coagulation factors all
con-tributetostopbleeding. TheincreasedbleedingtimeinNS1-immunized
mice is very likely, at least in part, due to the dys-function of
platelet aggregation, which was demonstrated in ourin vitro
studies. The increased platelet numbers observed in
alltheactive-immunizationgroupsmaybeexplainedbythephe-nomenon of
adjuvant-induced inammation, which involves in-duction of
proinammatory cytokines, leading to inammatorythrombopoiesis (50,
51). The effect of adjuvant-induced throm-bopoiesis was
conrmedbyimmunizingmice withadjuvantplus PBSonlytogive a platelet
number of 1264276103/l (n14) as comparedwithnormal control of
82583103/l(n10).Micepassivelyadministeredanti-NS1AbsshoweddecreasedAbtitersinthecirculationascomparedwiththosemicegivenanti-C
NS1 or anti-JEV NS1 Abs. Our nding that anti-NS1 (butnot anti-C NS1
or anti-JEV NS1) shows good binding to
plateletsinvivomayprovideapossibleexplanationfortheobservedde-creasesinserumAbtiters.
Other thanbindingtoplatelets, thedecreaseof anti-DVNS1Abs
inthecirculationmight alsobecaused by their binding to endothelial
cells as we have previouslydemonstrated (21).In the present study,
NS1 and C NS1 derived from DV2 wereused. We previously reported
that the levels of anti-endothelial
cellAbsweresimilarinpatientsinfectedbydifferent DVserotypes(52).
These ndings suggested that there is no
serotype-specicityforanti-NS1autoantibodyproduction.
Wealsoshowedthat
thecross-reactivityofDV3-infectedpatientseratoendothelialcells,which
led to induction of endothelial cell apoptosis, could be in-hibited
by DV2 NS1 preabsorption (21). We recently showed
thatanti-DV2NS1Abs cross-reactedwithliver vessel endothelium(53).
Furthermore, IgGpuriedfromDV3-infectedpatient seracaused liver
injury in mice, and liver injury induced by these
IgGwasinhibitedbypreabsorptionwithDV2NS1. Therefore,
anti-NS1-mediated cross-reactivity shows no dengue
serotype-specic-ity. The sequence alignment by ClustalW2 showed
that the
identityoftheaa271352sequencebetweenDV2andDV1is81.71%,betweenDV2andDV3is81.71%,
andbetweenDV2andDV4is 76.83%.Using two-dimensional gel
electrophoresis and Western blotanalysis,
wehavepreviouslyidentiedanti-DVNS1cross-re-active proteins
fromendothelial cell membrane extract (31).Among them, protein
disulde isomerase (PDI) can be ex-pressed on the platelet surface
and participate in platelet aggre-gation (5456). Our unpublished
results show that anti-DV
NS1canalsocross-reactwithPDIontheplateletsurface.PDImayregulatetheactivationofintegrinIIb3,thebrinogenrecep-tor,whichisrequiredforthelatestageofplateletaggregation(55,
57). Therefore, the inhibitory effect of anti-DV NS1 on
theactivation of integrinIIb3 may be through the recognition
ofplateletsurfacePDItoblockitsactivesitesandinterferewithPDIfunction.Thishypothesisiscurrentlyunderinvestigation.Numerousstrategiesofdenguevaccinedesignarebasedontheprotectiveefcacyof
Absagainst viral Eor NS1proteinFIGURE6.
Passivelyadministeredanti-NS1but not anti-CNS1Abs are diminished in
mouse sera and this decrease is correlated to thebinding of Abs to
platelets. Mice were i.v. administered with 500 g
ofanti-full-lengthNS1, anti-CNS1, oranti-JEVNS1IgG. After1, 24,and
48 h, blood samples were collected. n 5/group. AC, The serumAb
titers were analyzed by ELISA as described in Materials and
Meth-ods. D, Freshly isolated mouse platelets after Ab
administration for 24 hwere washed and xed with 1% formaldehyde in
PBS, and then stainedwith FITC-conjugated anti-mouse IgG. Ab
binding to platelets was de-tected and quantied by ow cytometry., p
0.01;, p 0.001.E,
Plateletnumbersatdifferenttimepointsweremonitoredbyanau-tomaticblood-cellcounter.1801
The Journal of Immunology by guest on July 14,
2015http://www.jimmunol.org/Downloaded from (5861). Althoughthe
Eproteinis responsible for
elicitingmajorneutralizingAbsduringDVinfection, it
isalsoassoci-atedwiththeinductionofinfection-enhancingAbs.
Alimita-tiontothevaccineregimencontainingNS1, however,
isthatanti-NS1Absmaycausecross-reactionwithplatelets. There-sultant
consequences of platelet lysis and impaired platelet ag-gregation
may lead to thrombocytopenia and bleeding
tendency.Thendingsinthisstudysuggest that C-terminal deletionofNS1
protein may provide a possible strategy for dengue
vaccinedevelopment.AcknowledgmentsWe thank the Proteomic Research
Core facility, Academia Sinica, Taiwan,for preparing C terminus (aa
271352)-deleted DV2 NS1 (C NS1) plas-mid. WethankDr.
Shu-YingSherryWangfor helpingwithsequencealignment of NS1 proteins
from different dengue serotypes. We also thankDr. S. L. Hsieh
(National Yang-Ming University, Taipei, Taiwan) and Dr.Y. L. Lin
(Institute of Biomedical Science, Academia Sinica, Taipei, Tai-wan)
for providing JEV NS1 plasmid.DisclosuresThe authors have no
nancial conict of interest.References1. Henchal, E. A., and J. R.
Putnak. 1990. The dengue viruses. Clin. Microbiol. Rev.3: 376396.2.
Gubler, D. J. 1998. Dengue and dengue hemorrhagic fever. Clin.
Microbiol. Rev.11: 480496.3.
Green,S.,andA.Rothman.2006.Immunopathologicalmechanismsindengueand
dengue hemorrhagic fever. Curr. Opin. Infect. Dis. 19: 429436.4.
Clyde, K., J. L. Kyle, and E. Harris. 2006. Recent advances in
deciphering viraland host determinants of dengue virus replication
and pathogenesis. J. Virol. 80:1141811431.5. Halstead, S. B. 2007.
Dengue. Lancet 370: 16441652.6.
Lei,H.Y.,T.M.Yeh,H.S.Liu,Y.S.Lin,S.H.Chen,andC.C.Liu.2001.Immunopathogenesis
of dengue virus infection. J. Biomed. Sci. 8: 377388.7. Fink, J.,
F. Gu, andS. G. Vasudevan. 2006. Roleof Tcells, cytokines
andantibody in dengue fever and dengue haemorrhagic
fever.Rev.Med.Virol. 16:263275.8. Pang, T., M. J. Cardosa, andM. G.
Guzman. 2007. Of cascadesandperfectstorms: theimmunopathogenesisof
denguehaemorrhagicfever-dengueshocksyndrome (DHF/DSS). Immunol.
Cell Biol. 85: 4345.9.
Kurane,I.,B.L.Innis,A.Nisalak,C.Hoke,S.Nimmannitya,A.Meager,andF.
A. Ennis. 1989. Human T cell responses to dengue virus antigens:
proliferativeresponses and interferon production. J. Clin. Invest.
83: 506513.10. King, C. A., J. S. Marshall, H. Alshurafa, andR.
Anderson. 2000.
Releaseofvasoactivecytokinesbyantibody-enhanceddenguevirusinfectionofahumanmast
cell/basophil line. J. Virol. 74: 71467150.11. Avirutnan, P., P.
Malasit, B. Seliger, S. Bhakdi, and M. Husmann. 1998. Denguevirus
infection of human endothelial cells leads to chemokine production,
com-plement activation, and apoptosis. J. Immunol. 161:
63386346.12. Halstead, S. B., C. N. Venkateshan, M. K. Gentry,
andL. K. Larsen. 1984.Heterogeneity of infection enhancement of
dengue 2 strains by monoclonal an-tibodies. J. Immunol. 132:
15291532.13. Littaua, R., I. Kurane, and F. A. Ennis. 1990. Human
IgG Fc receptor II mediatesantibody-dependent enhancement of
denguevirusinfection. J. Immunol. 144:31833186.14. Mady, B. J., D.
V. Erbe, I. Kurane, M. W. Fanger, andF. A. Ennis.
1991.Antibody-dependent enhancement of dengue virus infection
mediated by
bispe-cicantibodiesagainstcellsurfacemoleculesotherthanFcreceptors.
J. Im-munol. 147: 31393144.15. Morens, D. M. 1994.
Antibody-dependent enhancement of infectionandthepathogenesis of
viral disease. Clin. Infect. Dis. 19: 500512.16. Anderson, R., S.
Wang, C. Osiowy, andA. C. Issekutz. 1997. Activationofendothelial
cells via antibody-enhanceddengue virus infectionof peripheralblood
monocytes. J. Virol. 71: 42264232.17. 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-specic binding of
denguevirusandtargetcellsfortheantibody-dependentenhancementofdenguevirusinfection.
J. Immunol. 176: 28252832.18. Brown, M. G., C. A. King, C. Sherren,
J. S. Marshall, and R. Anderson. 2006. Adominant role for FcRII in
antibody-enhanced dengue virus infection of humanmast cells and
associated CCL5 release. J. Leukocyte Biol. 80: 12421250.19.
Falconar, A. K. 1997. The dengue virus nonstructural-1 protein
(NS1) generatesantibodiestocommonepitopesonhumanbloodclotting,
integrin/adhesinpro-teins and binds to human endothelial cells:
potential implications in haemorrhagicfever 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, andY. S.
Lin. 2001. Generationof IgManti-plateletautoantibody in dengue
patients. J. Med. Virol. 63: 143149.21. Lin, C. F., H. Y. Lei, A.
L. Shiau, C. C. Liu, H. S. Liu, T. M. Yeh, S. H. Chen,andY. S. Lin.
2003. Antibodiesfromdenguepatientseracross-reactwithen-dothelial
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
antibodiesagainst dengue virus nonstructural protein 1 via
production of nitric oxide. J. Im-munol. 169: 657664.23. 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, andadhesionmoleculesduringendothelial cell
activationinducedbyantibodies against denguevirus nonstructural
protein1. J. Immunol. 174:395403.25. Lin, C. F., S. W. Wan, H. J.
Cheng, H. Y. Lei, and Y. S. Lin. 2006. Autoimmunepathogenesis in
dengue virus infection. Viral Immunol. 19: 127132.26. Panzer, S.,
E. Seel, M. Brunner, G. F. Kormoczi, M. Schmid, P. Ferenci, andM.
Peck-Radosavljevic. 2006. Platelet autoantibodies are common in
hepatitis Cinfection, irrespective of the presence of
thrombocytopenia. Eur. J. Haematol. 77:513517.27. Ip, H., and B. D.
Corner. 1973. Thrombocytopenic purpura in
cytomegalovirusmononucleosis. 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
betweenhuman immunodeciency virus (HIV) and platelet glycoproteins
in HIV-relatedimmune thrombocytopenic purpura. Blood 80: 162169.29.
Kamath, S., A. D. Blann, and G. Y. H. Lip. 2001. Platelet
activation: assessmentand quantication. 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 isinvolved 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. Proteomicanalysisof endothelial cell
autoantigensrecognizedbyanti-dengue 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. Deciency of Src
homology 2domain-containing inositol 5-phosphatase 1 affects
platelet responses and throm-bus growth. J. Clin. Invest. 117:
944952.33. 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 diseaseby gene
transfer. Blood 108: 862869.34. 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
1cross-react with platelets and cause their dysfunction or
depletion. Am. J. Infect.Dis. 4: 6975.35.
Kaplan,K.L.,M.J.Broekman,A.Chernoff,G.R.Lesznik,andM.Drillings.1979.
Platelet -granule proteins: studies on release and subcellular
localization.Blood 53: 604618.36. Reed, G. L., M. L. Fitzgerald,
andJ. Polgar. 2000. Molecularmechanismsofplatelet exocytosis:
insightsintothesecretelifeofthrombocytes. Blood96:33343342.37.
Rendu, F., and B. Brohard-Bohn. 2001. The platelet release
reaction: granulesconstituents, 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,
andH.C.Hasselbalch.2000.Increasedplateletactivationandabnormalmembraneglycoprotein
content and redistribution in myeloproliferative disorders.Br. J.
Haematol. 110: 116124.40. Heijnen, H. F. G., N. Debili, W.
Vainchencker, J. Breton-Gorius, H. J. Geuze, andJ. J. Sixma. 1998.
Multivesicular bodies are an intermediate stage in the forma-tion
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,andC.
J. Lowenstein. 2005. Regulationof platelet granuleexocytosis
byS-nitrosylation. Proc. Natl. Acad. Sci. USA 102: 37823787.42.
Kaplan, K. L., and J. Owen. 1981. Plasma levels of -thromboglobulin
and plate-let factor 4 as indices of platelet activation in vivo.
Blood 57: 199202.43. Du, X., and M. H. Ginsberg. 1997. Integrin
IIb3 and platelet function. Thromb.Haemost. 78: 96100.44. Shattil,
S. J., H. Kashiwagi, and N. Pampori. 1998. Integrin signaling: the
plateletparadigm. Blood 91: 26452657.45. Sims, P. J., M. H.
Ginsberg, E. F. Plow, and S. J. Shattil. 1991. Effect of
plateletactivationontheconformationof
theplasmamembraneglycoproteinIIb-IIIacomplex. J. Biol. Chem. 266:
73457352.46. Murgue, B., O. Cassar, M. Guigon, and E. Chungue.
1997. Dengue virus inhibitshuman hematopoietic progenitor growth in
vitro. J. Infect. Dis. 175: 14971501.47. Wang, S., R. He, J.
Patarapotikul, B. L. Innis, and R. Anderson. 1995.
Antibody-enhanced binding of dengue-2 virus to human platelets.
Virology 213: 254257.48. Sun, D. S., C. C. King, H. S. Huang, Y. L.
Shih, C. C. Lee, W. J. Tsai, C. C. Yu,andH. H. Chang. 2007.
Antiplatelet autoantibodies elicitedbydenguevirusnon-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 interactionsin the microcirculation.
Microcirculation 12: 275285.1802 DENGUE NS1 C-TERMINUS IN PLATELET
DYSFUNCTION by guest on July 14,
2015http://www.jimmunol.org/Downloaded from 50. Hill, R. J., M. K.
Warren, and J. Levin. 1990. Stimulation of thrombopoiesis inmice 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-6stimulatesthrombopoiesisthroughthrombopoietin:
roleininammatorythrombocytosis.Blood 98: 27202725.52. 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 injurycausedbyantibodiesagainst
denguevirusnonstructural protein1inamurinemodel. Lab. Invest. 88:
10791089.54. Essex, D. W., K. Chen, and M. Swiatkowska. 1995.
Localization of protein di-sulde isomerase to the external surface
of the platelet plasma membrane. Blood86: 21682173.55.
Essex,D.W.,andM.Li.1999.Proteindisulphideisomerasemediatesplateletaggregation
and secretion. Br. J. Haematol. 104: 448454.56. Wilkinson, B.,
andH. F. Gilbert. 2004. Proteindisuldeisomerase. Biochim.Biophys.
Acta 1699: 3544.57. Lahav, J., N. Gofer-Dadosh, J. Luboshitz, O.
Hess, and M. Shaklai. 2000. Proteindisulde isomerase mediates
integrin-dependent adhesion. FEBS Lett. 475:8992.58. Kelly, E. P.,
J. J. Greene, A. D. King, and B. L. Innis. 2000. Puried dengue
2virusenvelopeglycoproteinaggregatesproducedbybaculovirusareimmuno-genic
in mice. Vaccine 18: 25492559.59. Apt, D., K. Raviprakash, A.
Brinkman, A. Semyonov, S. Yang, C.
Skinner,L.Diehl,R.Lyons,K.Porter,andJ.Punnonen.2006.Tetravalentneutralizingantibodyresponseagainst
fourdengueserotypesbyasinglechimericdengueenvelope antigen. Vaccine
24: 335344.60. Qu, X., W. Chen, T. Maguire, and F. Austin. 1993.
Immunoreactivity and pro-tective effects in mice of a recombinant
dengue 2 Tonga virus NS1 protein pro-duced in a baculovirus
expression system. J. Gen. Virol. 74: 8997.61. Costa, S. M., M. S.
Freire, and A. M. B. Alves. 2006. DNA vaccine against
thenon-structural 1 protein (NS1) of dengue 2 virus. Vaccine 24:
45624564.1803 The Journal of Immunology by guest on July 14,
2015http://www.jimmunol.org/Downloaded from