Immunodominant T-cell responses to dengue virusNS3 are associated with DHFThaneeya Duangchindaa,b, Wanwisa Dejnirattisaia, Sirijit Vasanawathanac, Wannee Limpitikuld,Nattaya Tangthawornchaikulb, Prida Malasitb,e, Juthathip Mongkolsapayaa,e,1, and Gavin Screatona,1

aDivision of Immunology and Inflammation, Department of Medicine, Faculty of Medicine, Hammersmith Hospital, Imperial College London, London W120NN, United Kingdom; bMedical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and TechnologyDevelopment Agency, Pathumthani 12120, Thailand; cPediatric Department, Khon Kaen Hospital, Ministry of Public Health, Khonkaen 40000, Thailand;dPediatric Department, Songkhla Hospital, Ministry of Public Health, Songkhla 90100, Thailand; and eMedical Molecular Biology Unit, Faculty of Medicine,Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

Edited* by Brigitte A. Askonas, Imperial College London, London, United Kingdom, and approved August 19, 2010 (received for review July 26, 2010)

Dengue infections are increasingat analarming rate inmany tropicaland subtropical countries, where epidemics can put health care sys-tems under extreme pressure. The more severe infections lead todengue hemorrhagic fever (DHF), which can be life threatening. Avarietyof viral and host factors havebeen associatedwith the sever-ityofdengue infections. Because secondarydengue infection ismorecommonlyassociatedwithDHF thanprimary infections, theacquiredimmune response to dengue, both B cells and T cells have been im-plicated. In this study,we set out to study T-cell responses across theentiredenguevirusproteomeand to seewhether thesewere relatedtodisease severity in a cohort ofdengue-infected children fromThai-land. Robust responses were observed in most infected individualsagainst most viral proteins. Responses to NS3 were the most fre-quent, and there was a very strong association between the magni-tude of the response and disease severity. Furthermore, in DHF,cytokine-high CD107a-negative cells predominated.

flavivirus | immunopathogenesis | cytokine

Dengue virus is transmitted toman via the bite from an infectedmosquito, and infections are a growing threat to public health

in a number of tropical and subtropical countries. There are esti-mated to be up to 50 million dengue infections annually, and epi-demic activities have the potential to almost overwhelm healthcaresystems (1). Dengue viruses display substantial sequence diversity,allowing them to be classified into four distinct serotypes that differin primary amino acid sequence by 30–35%. This sequence di-vergence is nearly as great as the difference between dengue andothermembers of the familyflaviviridae. Importantly, the sequencedivergence means that infection with one serotype of dengue doesnot provide immunity to infection with other serotypes (2). Inmanyparts of the world, all four serotypes of dengue cocirculate,meaning that sequential or secondary dengue infections are com-mon (3–5).After the bite from the infected mosquito, there is a period of

fever that occurs during a time of high viraemia. In most cases,referred to as dengue fever (DF), the virus is controlled, the feverremits, and the patient makes an unremarkable recovery. In 1–5%of cases, the disease can become more severe with extensiveplasma leak and sometimes hemorrhage (6). This syndrome isknown as dengue hemorrhagic fever (DHF) and can be lifethreatening with a 20% fatality rate that can be reduced to wellbelow 1% with expert management that mainly relies primarily oncareful fluid management (1).Forty years ago, it was recognized that the majority of cases of

DHF occur in patients suffering secondary dengue infections (6–8), although there is also a peak of severe dengue in children ex-periencing primary infections during the first year of life (9, 10).The association with secondary infections is perhaps best exem-plified by careful epidemiological studies of dengue infection inisland populations such as Cuba (7).The pathogenesis of DHF is the subject of some debate in the

field. The etiology is likely to be multifactorial with contributions

from both the virus and host. However, there are three key obser-vations that must be borne in mind. First, as mentioned above,DHF is more common after a secondary infection (6–8). Second,a high peak virus load is associated with a higher risk of DHF (11,12). Finally, the severe symptoms of DHF tend to occur at the timeof virus control when virus loads are falling steeply (11, 12).In 1977, Halstead put forward the hypothesis of antibody-de-

pendent enhancement (ADE) to explain the link between severityand secondary infection (13). However, although patients withhigh dengue viraemia are unwell with high fever and constitutionalsymptoms, they do not appear to develop DHF until around thetime they control the virus (11, 12). The association of DHF withvirus control and a peak in inflammatory cytokine production hasled some to suggest that the disease is a form of immunopathology(14, 15), which shares some features with experimental models ofT cell-mediated immunopathology in mice (16–19).In this study, we have performed a global scan for T-cell

responses by using overlapping peptides covering the entire den-gue proteome. Robust T-cell responses can be seen after infection,and although the whole of the virus can be recognized, responsesare particularly focused on nonstructural protein 3 (NS3). A clin-ical study of children suffering dengue infection showed that theamplitude of responsewas highly associatedwith the severity of thedisease with high cytokine and low CD107a expressing T cellspredominating the response in DHF.

ResultsDetection of Dengue-Specific T Cells in PBMC from Dengue-InfectedPatients. Dengue is a positive-strand RNA virus belonging to thefamily flaviviridae. The nucleic acid is ≈11 kb in length and istranscribed as a polyprotein that is cleaved by both viral and host-encoded proteases into 10 polypeptides (20). The virus has threestructural proteins—capsid, premembrane (prM), and envelope—together with seven nonstructural (NS) proteins—NS1, NS2a,NS2b, NS3, NS4a, NS4b, andNS5. A number of T-cell epitopes fordengue have been reported (21–23), but to our knowledge, noprevious studies have looked for responses to the whole denguevirus proteome.Overlapping peptides from dengue serotype 2 strain 16681

(Den2) were aliquoted into seven pools corresponding to in-dividual dengue serotype 2 proteins (C-prM), E, NS1, (NS2a-NS2b), NS3, (NS4a-NS4b), and NS5. Staphylococcal enterotoxin

Author contributions: T.D., J.M., and G.S. designed research; T.D. and W.D. performedresearch; S.V., W.L., N.T., and P.M. contributed new reagents/analytic tools; T.D., W.D.,J.M., and G.S. analyzed data; and T.D., J.M., and G.S. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. E-mail: g.screaton@imperial.ac.uk orjmongkol@imperial.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1010867107/-/DCSupplemental.

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B (SEB; 1 μg/mL) and peptide diluent (0.8%DMSO in PBS) wereused as positive and negative controls, respectively.PBMC were collected, after informed consent, and cry-

opreserved from children seen in hospital with dengue infection.Dengue infection was determined serologically, and virus sero-type where possible was determined by serotype specific RT-PCR (24). Overlapping peptides were used to stimulate 2-wkconvalescent PBMC from 40 dengue-infected patients—18 sec-ondary DF, 22 secondary DHF; the patient details are shown inTable S1. Responses of CD4+ and CD8+ T cells were measuredby using intracellular cytokine staining (ICS) for IFN-γ and TNF-α,and the ICS strategy is outlined in Fig. 1A.Significant responses were seen in most patients with low levels

of background reactivity. A representative set of responses isshown inFig. 1B andC, where robust responses can be seen in boththe CD4+ and CD8+ T-cell subsets to a variety of viral proteins, inparticular to NS3.

NS3 Is the Immunodominant T-Cell Antigen in Dengue Infection.Next,we analyzed the data from all 40 individuals selected at random tocompare the level of responses between the different denguepeptide pools, the details of these patients are shown in Table S1.Positive responses were called when they were greater than thepercentage of cytokine-producing cells plus 3 SDs fromfive denguenonimmune volunteers.Most patients showed responses to severalepitopes, particularly patients in the DHF group where all of thepatients responded to NS3 protein (Fig. 2A). Responses to E, NS1,and NS5 proteins were also common in DHF, with >70% of DHFpatients having T-cell response to these proteins. Similar responsepatterns were found in the DF group where NS3 elicited T-cellresponses in the majority of patients (78%). Responses to E, NS1,NS2, and NS5 were found in most DF patients; 30% for E, NS1,

and NS2 and 44% for NS5. Similar patterns of reactivity were seenwhen the CD4+ and CD8+ T-cell subsets were analyzed separately(Fig. S1 A and B).Importantly, we found a highly significant association between

the magnitude of the T-cell response to dengue and disease se-verity (Fig. 2B). The sum of all of the responses across the entiredengue proteome in the DHF group was 5.22 ± 3.85% versus2.02 ± 1.59% in the DF patients, P = 0.0008 (Fig. 2C).

Comparison of T-Cell Responses to NS3 in Primary and SecondaryResponses. The analyses presented above were performed by us-ing peptides derived from the dengue serotype 2 strain 16681.The patients recruited into the study were suffering infection witha variety of dengue serotypes, meaning that in many cases, thetest antigen did not match the serotype of the current denguevirus infection. To correct for this mismatch, we synthesized ad-ditional overlapping peptides covering the immunodominantantigen NS3 from prototype strains of Den1, Den3, and Den4.In this second analysis, four groups of patients were selected, 10

primaryDFcases, 5 primaryDHFcases, 15 secondaryDFcases, and15 secondary DHF cases, and details of these patients are shown inTable S2. PBMC were incubated with an overlapping pool of NS3peptides corresponding to the serotype of the current infection asdetermined by RT-PCR. After incubation, cells were stained forCD4 and CD8 and intracellular staining performed for TNF-α andIFN-γ, and inaddition, cellswere stainedwith anti-CD107a,which isused as a surrogate measure of degranulation (25).Studies of T-cell responses in dengue have to date concentrated

on individuals suffering secondary infection in large part becauseof the relative scarcity of primary cases, and in particular, primaryDHF. T-cell responses could be readily detected in primary casesby stimulation with the NS3 overlapping peptides. Responses in

Fig. 1. T-cell responses to the dengue proteome. Shownare representative plots for CD4+ and CD8+ T-cell subsetsfrom PBMC stimulated with peptide pools covering thedengue proteome. (A) Gating strategy. Representative setsof responses for CD4 (B) and CD8 (C) T cells.

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primary DHF patients were significantly higher than in DFpatients (TNF-α, P = 0.0080, and IFN-γ, P = 0.0013) (Fig. 3A).This highly significant association between the magnitude of theT-cell response and severity was also found in secondary dengueinfections, TNF-α (P < 0.0001) and IFN-γ (P = 0.0001). Thesefindings were similar in CD4+ and CD8+ T cells (Fig. S2).

More Cytokine Production and Less Degranulation in DHF. There hasbeen much recent interest in the development of T-cell vaccinesandmeasures of their efficacy. It is becoming clear that it is not justthe magnitude of the T-cell response but also the quality of theinduced T cells that are important for function, with the sugges-tion that polyfunctional T cells are the most desirable.Analysis of the T cells induced in dengue illustrated two in-

teresting features. First, the majority of T cells are monofunc-tional with respect to IFN-γ, TNF-α secretion, and degranulation(CD107a) (Fig. S3). Second, there were clear differences in thetype of response mobilized in DF when compared with DHF.In Fig. 3B, we have broken these data down into three groups:

CD107a only, cytokine (TNF-α or IFN-γ) only, and double positivefor cytokine and CD107a. CD107a-expressing cells were relativelydistinct from cells producing TNF-α and IFN-γ, with the majorityexpressing no associated cytokine. In addition, CD107a-producingcells were rarer in cases of DHF where cytokine-producing cellswere the dominant population. In primary cases, 71% of cells wereCD107a-positive in DF compared with 13% in DHF (P= 0.0007),and similarly in secondary infections, CD107a cells accounted for45%of responding cells inDF and 18% inDHF (P= 0.0055). Thisdifference was mirrored by a commensurate rise of cytokine onlyproducing cells in both primary and secondary DHF. Both CD4+

and CD8+ T cells gave similar patterns (Fig. S4).

In a final series of experiments, we used MHC tetramerstaining to focus on an immunodominant HLA-A11–restrictedepitope from NS3 (GTS) that we have described (22). PBMCfrom 10 HLA-A*11–positive individuals (5 DF, 5 DHF, all sec-ondary infections; details shown in Table S3) were stained withHLA-A*11 tetramer refolded with the peptide corresponding tothe serotype of their secondary infection. Cells were sub-sequently stimulated with the same dengue serotype specificpeptide and stained for CD107a, IFN-γ, and TNF-α.The results of these experiments are shown in Fig. 4A, where it

can be seen that the production of IFN-γ and TNF-αwere higher inthe DHF group compared with DF; this reached statistical signif-icance for IFN-γ. Furthermore, in the DF patients, a higher pro-portion of responding cells showed CD107a staining comparedwith theDHF groupDF 60%vs. DHF 31% (P= 0.0079) (Fig. 4B).

DiscussionTo our knowledge, this is the first study that has comprehensivelyexamined T-cell responses in dengue-infected patients across theentire dengue proteome. These data from 40 cases show thatresponses to the virus are broad and can recognize most of viralproteins, particularly in cases of DHF (Fig. 2 A and B). When theresponses to individual proteins are compared, responses to NS3,a viral protease, NTPase, and helicase, are dominant. Significantresponses were also seen against envelope and NS5. These resultsconcur with the reports of a number of T-cell epitopes in dengueinfection where NS3 responses are the most frequently seen, al-though this analysis shows the response to be considerably broaderthan reported (21–23, 26). In a recent report on T-cell responses inhuman vaccines, receiving the yellow fever 17D vaccine, the NS3response was subdominant to NS5 in most individuals (27). In-

Fig. 2. Breadth and magnitude of denguespecific T-cell responses. (A) T-cell responsesagainst dengue viruswerebroad; thenumbersabove the bars represent the number ofresponding patients. (B) Magnitude of theresponses to the peptide pools in the DF andDHFpatientgroups. (C) Sumof responses to allpeptide pools for each patient. Differencesbetween the two groups were tested by usingthe nonparametric two tailed Mann–Whitneytest. Asterisks indicated P values: *P < 0.05;**P < 0.005; ***P < 0.0005.

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terestingly, in this study, there was a major dominant epitope inNS4b found in HLA-A*02-expressing individuals, with the similarresult was seen in dengue vaccines (28).The determinants of immunodominance are not fully un-

derstood but are presumed to result from a variety of events oc-curring in the generation of the T-cell response: the amount ofantigen produced, the timing of protein production, and host-specific factors such as antigen processing, MHC, and TCR rep-ertoires (29). The reason for the dominance of the NS3 responsein dengue is not clear, unlike other viruses in which programs ofantigen production lead to the emergence of different antigensearly and late during the virus lifecycle dengue is transcribed asa single polyprotein that is cleaved into its 10 constituent poly-peptides by host- and virus-specific proteases that include NS3(20). This processing presumably means that the polypeptides areproduced in equimolar concentrations. However, possible dif-ferences in intracellular targeting and stability may lead to pref-erential processing and presentation of NS3 peptides.

Extensive sequence diversity is a feature of many RNA virusesbecause of poor fidelity of the RNA polymerase enzyme. Mostsequence changes are detrimental to viral fitness, reducing theefficiency of virus replication. However, many sequence changesare tolerated, and the emergence of new viral strains is believed inpart to be driven by immune responses, leading to the emergenceof immune escape variants. This phenomenon is perhaps best ex-emplified for the antibody response by influenza and for the T-cellresponse by HIV (30–32). Sequence diversity in dengue viruses is≈30–35%, meaning that viruses differ from each other by nearly asmuch as the dengue viruses themselves differ from other membersof the flavivirus family such as Japanese encephalitis virus (50%).The sequence variation in dengue is such that the virus can bedivided into four distinct serotypeswhere by definition immunity toone serotype will not provide long-lasting immunity to the others.In dengue-endemic areas, multiple viral serotypes cocirculate,

although the relative frequencies of the four viruses vary from yearto year, perhaps driven by the heard immunity of susceptible hu-

Fig. 3. NS3-specific responses in primaryand secondary infections. (A) PBMC werestimulated with NS3 from the serotype oftheir current infection. Total respondingcells (CD107a or IFN-γ or TNF-α) or in-dividual responses to CD107a, IFN-γ, andTNF-α are shown. Differences between thetwo groups were tested by using the non-parametric two-tailed Mann–Whitney test.(B) Responding T cells from Fig. 3A weredivided into three groups: degranulation(CD107a) only, degranulation and cytokineproduction, and cytokine production only.Pie charts show the percentages of thefunctional cells to total responding T cellsfrom patients in the DF and DHF groups.The average percentages were calculatedby summing the percentages from individ-uals and dividing by number of cases.

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Fig. 4. MHC tetramer analysis of theHLA-A11-restricted NS3-GTS response.(A) PBMC from 10 secondary dengue-infected patients, 5 DF and 5 DHF (Ta-ble S3), were stained with the HLA-A*11-NS3-GTS tetramer correspondingto the serotype of their secondary in-fection. After stimulation with thesame peptide used for tetramer stain-ing and in the presence of anti-CD107aAb, cells were stained for CD8, TNF-α,and IFN-γ. (B) Pie charts showing thedetails of the tetramer-specific respon-ses by following the format of Fig. 3B.

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man hosts (3–5). Secondary dengue infections are common, andthe severe disease manifestations of DHF and shock are morecommonly seen in these secondarily infected individuals (6–8).DHF pathogenesis is likely to be multifactorial with contributionsfrom the virus and the host. High peak virus loads are associatedwith a higher risk of DHF (11, 12), and one explanation for thehigher virus loads is antibody-dependent enhancement. ADE canbe readily demonstrated in vitro and has also been demonstratedin vivo in bothmurine and primatemodels (33–36). ADEmay alsoexplain the peak in DHF seen in children during the first year oflife where passively transferred maternal antibody may enhanceinfection (10, 37).However, although there is a correlation between virus/antigen

load and severity, the severe sequaelae of dengue infection occurwhen the virus load is falling steeply and are coincidental withsharp rises in inflammatory cytokine production. Many of thesecytokines can be T cell-derived, and the coincidence of severedisease with viral clearance has led a number of investigators tosuggest the DHF may result from T cell-mediated immunopa-thology (15, 21, 38, 39). Immunopathology is now being recog-nized to be a feature of a number of viral diseases where theprotective antiviral response, although capable of controlling vi-rus, may cause considerable inflammation and tissue damage thatcan, in some instances, be fatal. In humans, immunopathology hasbeen invoked in severe influenza infections, RSV after ineffectivevaccination, severe hepatitis in hepatitis B virus infections, and theimmune reconstitution syndrome seen in individuals coinfectedwith tuberculosis and HIV upon the commencement of anti-retroviral therapy (40–43).We had described pronounced original antigenic sin in the

T-cell response to dengue virus whereby the T-cell response insecondary infection is shaped in part by the response to the pri-mary encounter with dengue. This phenomenon leads to highlycross-reactive responses that in some cases are more avid to theprimary encountered virus than to the secondary infection (22,23). In this report, we went on to examine the T-cell response toNS3 in four groups of patients: primary and secondary infectionson both DF and DHF groups; in each case, we examined the re-sponse to NS3 from the serotype of virus responsible for theircurrent infection.There were highly significant differences in both IFN-γ and

TNF-α production in the DHF groups when compared with DFgroups regardless of primary or secondary infection. This findingis in keeping with reports of correlations between the serum levelsof inflammatory cytokines such as IFN-γ, TNF-α, and interleukinsand disease severity (6). Polyfunctional analysis of T-cellresponses has recently been of great interest in the vaccine fieldwhere it is now recognized that the ability of T cells to control virusreplication does not correlate well with the overall magnitude ofthe response. Results from analysis of effective vaccines such asbacillus Calmette–Guérin, vaccinia, and yellow fever 17D virus oreffective HIV controllers versus rapid progressors suggest thatT cells with multiple functions including IFN-γ, TNF-α, CD107a,and IL-2 are the most effective at controlling virus (27, 44–47).In this study, we found that the majority of patients made

monofunctional or oligofunctional responses and cells producingcytokine together with CD107a were rare. Of great interest in thisstudy was the finding that in both primary and secondary DHF,<20% of T cells produce CD107a, whereas >80% produce cyto-kine only. This situation is reversed in primary DF where 71%produce CD107a and 29% cytokine only, and secondary DF 45%produce CD107a. CD107a expression was found on CD4+ T cellsin addition to CD8+ in DF, which fits with reports of cytotoxicityof antidengue CD4+ T-cell lines, and it is possible that this pop-ulation plays an important cytotoxic role in vivo (48, 49).These results suggest that the functional phenotype of dengue

responsive T cells, together with the magnitude of the T-cell re-sponse, may play a role in the development of severe disease. We

propose that the limited cytotoxic potential of T cells in a subset ofpatients may not allow early and effective viral control. Instead,the cytokine-only producing T cells are stimulated, which in thecontext of high virus/antigen loads will lead to excessive pro-duction of inflammatory cytokines triggering the tissue damageand leak characteristic of DHF.

Materials and MethodsSamples. Blood samples were taken from normal healthy subjects andfrom patients admitted to the Pediatric Departments of Khon Kaen andSongkhla hospitals after informed consent. The ethical committees of KhonKaen, Songkhla, and Siriraj hospitals, Thailand, as well as the Riversideethical committee in the United Kingdom approved the studies. Acutedengue infection was identified by RT-PCR-based dengue gene identifi-cation or dengue-specific IgM capture ELISA (24, 50, 51). Secondary dengueinfection (an acute infection in a patient who had previously encountereddengue on one or more occasions) was defined as a dengue-specific IgM/IgG ratio < 1.8, by IgM and IgG capture ELISA by using paired acute andconvalescent sera.

Disease severity was classified according to theWorld Health Organizationcriteria (52). PBMC were isolated from whole blood by Ficoll-Hypaque den-sity gradient centrifugation and cryopreserved for subsequent studies. Pa-tient details were made anonymous and stored on a password protecteddatabase, patients in both DF and DHF groups were selected at random. Inboth the global scan of the whole virus proteome and the targeted study ofNS3 responses, the ages and male:female ratio (in parentheses) of child-ren were as follows: for the global scan, DF = 9.4 ± 3.6 y (7:11) andDHF = 10.3 ± 2.8 y (10:12) and for NS3 responses, DF = 10.3 ± 2.2 y (6:4)and DHF = 10.8 ± 2.9 y (3:2) for primary infection, and DF = 10.4 ± 2.9 y (8:7)and DHF = 11.1 ± 0.3 y (8:7) for secondary infection. The details of thesepatients are shown in Tables S1–S3.

Dengue nonimmune controls were a group of five individuals who hadnever traveled to a dengue endemic area and who had not received eitheryellow fever or Japanese encephalitis vaccination. These were students fromthe laboratory who donated blood after informed consent and undera protocol approved by the Riverside Ethics Committee UK.

Peptides. 15-mer peptides overlapping by 5 amino acids corresponding tosequences of dengue serotype 2 strain 16681 (CprM, E, NS1, NS2, NS3, NS4,and NS5) and to the NS3 sequences of dengue serotype 1 strain Hawaii,dengue serotype 3 strain H87, and dengue serotype 4 strain H241 weresynthesized to >80% purity (Sigma Aldrich). Lyophilized peptides wereresuspended in dimethyl sulfoxide (DMSO; Sigma) at 80 mg/mL for peptidemixtures. The overlapping peptides were grouped together in pools cor-responding to individual dengue proteins (dengue 2: CprM-26, E-97, NS1-71, NS2-70, NS3-123, NS4-70, NS5-95, dengue 1 NS3-124, dengue 3 NS3-124, and dengue 4 NS3-124). The final concentration of any singlepeptide was 2 μg/mL, and the final concentration of DMSO was <1% in allexperiments.

Cell Stimulation for Multifunctional Assessment. PBMC were thawed andrested in complete RPMI media (RPMI 1640 supplemented with 10% heatinactivated FCS, L-glutamine, and antibiotics) for at least 2 h at 37 °C in a 5%CO2 incubator; viability was then examined by using Trypan blue dye ex-clusion. Cell viability was >85% for all cases. For stimulation, briefly, PBMCwere incubated in the presence of peptide mixes (final concentration ofeach peptide was 2 μg/mL) and the titrated amount of CD107a FITC (BDPharmingen) for 1 h at 37 °C in a 5% CO2 incubator, followed by an addi-tional 5 h in the presence of the protein transport inhibitors monensin (BDBiosciences) and brefeldin A (BFA) (Sigma). A negative control, PBMC in-cubated in the peptide diluents (0.8% DMSO in PBS), and a positive control,PBMC incubated with 1 μg/mL of staphylococcal enterotoxin B (SEB), wereincluded in all experiments.

Tetramer Staining and Cell Stimulation. HLA-A11 was refolded around a 10-mer epitope GTS present between positions 133 and 142 of the NS3 protein.Three separate variants of this peptide were used for refolding GTSGSPIVNRfor serotype 1, GTSGSPIVDR from serotype 2, and GTSGSPIINR, which is foundin both dengue serotypes 3 and 4. PBMCwere stained with the phycoerythrin(PE)-conjugated GTS-A*11 tetramer, which corresponded to the serotype oftheir current secondary dengue infection, at 37 °C for 20 min. Thereafter,cells were incubated with 10 μg/mL of the same GTS peptide that corre-sponded to the serotype of their current infection. Cells were stained forCD107a, CD8, and cytokine as described above and below. A negative con-

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trol, cells incubated without peptide, was included to control for sponta-neous production of cytokine and/or expression of CD107a. A positive con-trol, cells incubated with 1 μg/mL of SEB, was also included in all assays. Agate was set around the CD8+ tetramer+ cell population, and the function ofthese cells with respect to CD107a, TNF-α, and IFN-γ secretion were calcu-lated by using gates set on the negative control-unstimulated tetramerpositive cells.

Immunofluorescent Staining. Immediately after stimulation, PBMC werewashedonceandfixed/permeabilizedbyusingFACSpermeabilizationbuffer II(BD Pharmingen). For four-color analysis, the cells were washed and stainedwith the titrated amount of IFN-γAPC, TNF-α FITC, CD4 PE, and CD8 PerCP. Forfive-color analysis, the cells were stained with the titrated amount of IFN-γAPC, TNF-α PE-Cy7, CD4 PE, and CD8 APC-Cy7. The cells were thenwashed andfixed with 1% formaldehyde in PBS. All antibodies were purchased fromBD Pharmingen.

Flow Cytometric Analysis. Two hundred thousand to 500,000 events werecollected per sample. Analysis was performed by using FlowJo software(version 7; TreeStar). Background responses detected in negative controltubes were subtracted from those detected in stimulated samples for everyspecific functional combination.

Statistical Analysis. Differences between the DF and DHF groups were allevaluated by the nonparametric two-tailed Mann–Whitney test by using thePrism program (Graph Pad Software).

ACKNOWLEDGMENTS. We thank A. Chairunsri and the staff at the MedicalMolecular Biology Unit Siriraj Hospital and Khon Kaen and Songkhla hospitalsin Thailand for technical assistance and sample collection. This work wassupported by the Medical Research Council, UK; the Wellcome Trust, UK; theNational Institute for Health Research Biomedical Research Centre fundingscheme Thailand; the Thailand Tropical Disease Research Program T2; and theThailand National Centre for Genetic Engineering and Biotechnology.

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