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: yslin1@mail.ncku.edu.tw3Abbreviationsusedinthispaper: 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