CD8� T Cells Can Mediate Short-Term Protection against HeterotypicDengue Virus Reinfection in Mice
Raphaël M. Zellweger, William W. Tang, William E. Eddy, Kevin King, Marisa C. Sanchez, Sujan Shresta
La Jolla Institute for Allergy & Immunology, La Jolla, California, USA
ABSTRACT
Dengue virus (DENV) is a major public health threat worldwide. Infection with one of the four serotypes of DENV results in atransient period of protection against reinfection with all serotypes (cross-protection), followed by lifelong immunity to the in-fecting serotype. While a protective role for neutralizing antibody responses is well established, the contribution of T cells toreinfection is less clear, especially during heterotypic reinfection. This study investigates the role of T cells during homotypic andheterotypic DENV reinfection. Mice were sequentially infected with homotypic or heterotypic DENV serotypes, and T cell sub-sets were depleted before the second infection to assess the role of DENV-primed T cells during reinfection. Mice primed nonle-thally with DENV were protected against reinfection with either a homotypic or heterotypic serotype 2 weeks later. Homotypicpriming induced a robust neutralizing antibody response, whereas heterotypic priming elicited binding, but nonneutralizingantibodies. CD8� T cells were required for protection against heterotypic, but not homotypic, reinfection. These results suggestthat T cells can contribute crucially to protection against heterotypic reinfection in situations where humoral responses alonemay not be protective. Our findings have important implications for vaccine design, as they suggest that inducing both humoraland cellular responses during vaccination may maximize protective efficacy across all DENV serotypes.
IMPORTANCE
Dengue virus is present in more than 120 countries in tropical and subtropical regions. Infection with dengue virus can beasymptomatic, but it can also progress into the potentially lethal severe dengue disease. There are four closely related denguevirus serotypes. Infection with one serotype results in a transient period of resistance against all serotypes (cross-protection),followed by lifelong resistance to the infecting serotype, but not the other ones. The duration and mechanisms of the transientcross-protection period remain elusive. This study investigates the contribution of cellular immunity to cross-protection usingmouse models of DENV infection. Our results demonstrate that cellular immunity is crucial to mediate cross-protection againstreinfection with a different serotype, but not for protection against reinfection with the same serotype. A better understandingof the mediators responsible for the cross-protection period is important for vaccine design, as an ideal vaccine against denguevirus should efficiently protect against all serotypes.
The four serotypes of dengue virus (DENV) are the etiologicagent of dengue, a rapidly spreading arboviral disease that is
present in more than 120 countries (1–5). Recent estimates sug-gest that more than 3.5 billion people living in tropical and sub-tropical regions are at risk of infection, with 390 million infectionsper year, of which 96 million are symptomatic (1–3).
Infection with DENV is often asymptomatic (6, 7), but if dis-ease is apparent, it ranges from dengue fever to severe dengue(formerly known as dengue hemorrhagic fever [DHF] or dengueshock syndrome [DSS]) (5, 8). Dengue fever is a self-limited ill-ness characterized by headache, retro-orbital pain, nausea, muscleand joint pain, and leukopenia. Warning signs of severe denguedisease include abdominal pain, persistent vomiting, fluid accu-mulation, mucosal bleeding, lethargy, liver enlargement, in-creased hematocrit, and low platelet count (5, 8, 9). Signs of severedengue are severe plasma leakage, severe bleeding, respiratory dis-tress, and severe organ involvement (liver, kidney, heart, centralnervous system) (5, 8, 9).
Infection with one serotype of DENV results in lifelong immu-nity to that serotype due to induction of a robust serotype-specificneutralizing antibody response (10–12). Additionally, after infec-tion, there is a period of protection against heterotypic infectionwith other serotypes (cross-protection) (13–17). The duration ofthe cross-protection period remains a matter of debate (13–15,
17). A recent reanalysis of historical data suggests a duration of 8weeks (17). Based on serology and on epidemic modeling, otherestimates vary from 1 to 2 weeks to 1 year or more (13–15). Inaddition to the duration, the mechanism of cross-protection re-mains elusive too (17). The transient cross-protection is oftenassumed to rely on high titers of cross-reactive antibodies reactivefor all DENV serotypes (18–20), but experimental evidence isscarce. Therefore, the precise features of the transient cross-pro-tection remain unclear.
While the importance of a robust neutralizing antibody re-sponse for protection against DENV is undisputed (10–12, 21),less is known about the importance of T cells during reinfection, in
Received 9 January 2015 Accepted 6 April 2015
Accepted manuscript posted online 8 April 2015
Citation Zellweger RM, Tang WW, Eddy WE, King K, Sanchez MC, Shresta S. 2015.CD8� T cells can mediate short-term protection against heterotypic dengue virusreinfection in mice. J Virol 89:6494 –6505. doi:10.1128/JVI.00036-15.
particular T cells previously activated by another DENV serotype(heterotypic T cells). It has been hypothesized that altered re-sponses from heterotypic T cells can result in a “cytokine storm”and exacerbation of disease (22). However, increasing evidencesuggests a protective role for T cells during DENV infection (23–30), including for heterotypic T cells (23, 25).
A better understanding of the contribution of T cells (bothhomotypic and heterotypic) to protection against reinfection iscrucial to support DENV vaccine development. Efforts to developa vaccine that efficiently protects against all DENV serotypes arestill ongoing (31–33). Inducing a balanced neutralizing antibodyresponse against all four DENV serotypes has been the overarch-ing goal of most vaccine developers, but it is increasingly recog-nized that the presence of neutralizing antibodies (as measured by50% plaque reduction neutralization test [PRNT50]) may not bean optimal correlate of protection (34–36). A vaccine that effi-ciently induces both humoral and cellular responses may be pref-erable.
In this study, we investigated in mice the contribution of cel-lular immunity to protection during homotypic or heterotypicreinfection 2 weeks after a primary infection. 129/Sv mice lackingtype I and II interferon (IFN) receptors (AG129) or type I IFNreceptor only (IFNAR�/�) were nonlethally primed with DENVserotype 2 (DENV2) or DENV serotype 4 (DENV4) prior to chal-lenge with DENV2 2 weeks later. In order to maximize the chanceof capturing the potential contribution of T cells, we chose tochallenge with virus shortly after the peak of the T cell response(expected around days 7 to 10), but before T cell responses returnto the prepriming level (28). This approach also allowed us to bewithin the 2-week period that has been postulated as being theshortest period of cross-protection according to the most conser-vative estimates (14, 17).
Both homotypic (DENV2) and heterotypic (DENV4) primingreduced viral load, morbidity, and/or mortality upon challengewith DENV2. Depletion of CD8� T cells prior to challenge abro-gated protection in the case of heterotypic, but not homotypic,priming. CD8� T cells were likely not required for protection afterhomotypic priming due to the induction of a robust serotype-specific neutralizing antibody response. However, transfer ofDENV2 (homotypic)- or DENV4 (heterotypic)-primed CD8� Tcells both reduced viral load upon reinfection with DENV2.Therefore, while both homotypic and heterotypic CD8� T cellsefficiently reduced viral load upon reinfection, CD8� T cells werenecessary for viral load reduction only after heterotypic priming.
Taken together, our results demonstrate that 2 weeks afterpriming, CD8� T cells were necessary for protection against het-erotypic reinfection but not homotypic reinfection. This studysuggests a protective role for CD8� T cells during heterotypicreinfection, which implies that inducing both humoral and cellu-lar responses during vaccination may maximize protective effi-cacy across all DENV serotypes.
MATERIALS AND METHODSEthics statement. This study was carried out in strict accordance with therecommendations in the Guide for the Care and Use of Laboratory Animals(37), the U.S. Public Health Service Policy on Humane Care and Use ofLaboratory Animals (38), and the Association for Assessment and Accred-itation of Laboratory Animal Care International (AAALAC). All experi-mental procedures were approved and performed according to the guide-
lines set by the La Jolla Institute for Allergy and Immunology Animal Careand Use Committee (protocol number AP-28SS1-0809).
Mice. 129/Sv mice deficient in type I and II interferon receptors(AG129) and 129/Sv mice deficient in type I interferon receptor(IFNAR�/�) were housed under specific-pathogen-free (SPF) conditionsat the La Jolla Institute for Allergy and Immunology (LJI). Sex-matched 5-to 6-week-old mice were used. For survival studies, mice were sacrificedwhen moribund or at the first signs of paralysis.
Virus production. DENV serotype 2 (DENV2) strain PL046 (39),DENV2 strain S221 (28), DENV serotype 3 (DENV3) strain UNC3001(40) (obtained from Aravinda deSilva, University of North Carolina[UNC] Chapel Hill), and DENV serotype 4 (DENV4) strain H241 (pur-chased from ATCC) were amplified on C6/36 cells as described in refer-ence 41. DENV2 PL046, DENV2 S221, DENV3 UNC3001, and DENV4H241 were quantified by standard plaque assay on baby hamster kidney(BHK) cells as previously described (42). DENV2 S221 organ titers werequantified by real-time quantitative reverse transcription-PCR (qRT-PCR) as previously described (41). There are approximately 5 � 104
genomic equivalents (GE) per PFU for DENV2 strain S221.Infections. All infections were performed intravenously; virus was
diluted to a total volume of 200 �l phosphate-buffered saline (PBS) with10% fetal calf serum (FCS). When needed, the viral dose used for primingwas adjusted between experiments to account for differences in virulencebetween different strains and batches of virus.
T cell depletions. T cell-depleting antibodies 2.43 (IgG2b anti-mouseCD8), GK1.5 (IgG2b anti-mouse CD4), and the isotype control LTF2(IgG2b) were purchased from BioXCell. CD8� or CD4� T cells weredepleted by administering 250 �g of 2.43 or GK1.5 antibody intraperito-neally in a total volume of 200 �l in PBS 3 days and 1 day before challengewith virus as previously described (30). CD8� depletion efficiency wasmore than 98% as verified by flow cytometry.
Viral RNA quantification in organs. Viral RNA was quantified inorgans by real-time qRT-PCR as described previously (41).
DENV-specific IgG detection by ELISA. DENV-reactive IgG was de-tected by enzyme-linked immunosorbent assay (ELISA) on DENV S221-coated plates as previously described (30). To compare the relativeamount of DENV-reactive IgG in different sera, the optical density (OD)values of three samples were averaged and plotted against the log of thedilution factor. Subsequently, a linear regression was done with the pointsin the linear range of the curve. Based on the equation of the linear regres-sion, a dilution factor can be calculated for any desired OD in the linearrange and compared to the dilution factor of another group at the sameOD. When sera were compared, this was done for ODs of 0.3 to 0.7 with0.1 increments, and the factors obtained from those five OD measureswere averaged.
Neutralizing activity of serum. Anti-DENV neutralizing antibodies(Abs) were quantified by 50% plaque reduction neutralization test(PRNT50) as previously described (30). Serum was serially diluted 1:2 withthe starting dilution being 1:10. The highest dilution reducing more than50% of the plaques is reported as “50% neutralizing titer.”
Generation of DENV-immune serum. IFNAR�/� mice were infectedintravenously (i.v.) with 2 � 106 PFU DENV2 strain S221, and serum wascollected on day 15 and 32; fractions collected on day 15 and 32 werepooled.
Direct in vivo intracellular cytokine staining (ICS). As previouslydescribed (43–45), this method allows for detection and enumeration of Tcells that are actively producing cytokines in vivo after contact with DENVantigen. One hour after challenge with DENV2, 200 �g brefeldin A(Sigma) diluted in PBS containing 20% ethanol (EtOH) (vol/vol) wasinjected i.v. into mice in a total of 200 �l to retain cytokines in the cells.Mice were sacrificed 6 h later, and spleens were passed through 70-�m cellstrainers to obtain single-cell suspensions. Without any other stimulation,cells were stained for CD3, CD8, CD44, and CD62L (extracellularly) aswell as gamma interferon (IFN-�) and CD107a (intracellularly). Sampleswere acquired on an LSR II flow cytometer (BD Bioscience) and analyzed
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with FlowJo software (Tree Star). The following antibodies were used:anti-CD3-efluor 450 (anti-CD3 antibody conjugated to efluor 450) (clone17A2; eBioscience), anti-CD8a-PerCP-Cy5.5 (anti-CD8a antibody conju-gated to peridinin chlorophyll protein [PerCP] and Cy5.5) (clone 53-6.7;eBioscience), anti-CD62L-Alexa Fluor 700 (clone MEL-14; eBioscience),anti-CD44-PE-Cy7 (anti-CD44 conjugated to phycoerythrin [PE] and Cy7)(BD Pharmingen, clone IM7), anti-IFN-�-FITC (anti-IFN-� conjugated tofluorescein isothiocyanate [FITC]) (clone XMG1.2; Tonbo Biosciences), andanti-CD107a-PE (clone 1D4B; eBioscience).
In vivo cytotoxicity assay. IFNAR�/� recipient mice were infectedwith 1 � 105 PFU DENV2 (PL046) or DENV4 (H241). Two weeks later,donor splenocytes were obtained from naive IFNAR�/� mice. Red bloodcells (RBC) were lysed (RBC lysis buffer; eBioscience), and cells wereresuspended at 1 � 107 cells/ml in RPMI 1640 containing 2% FCS, peni-cillin, and streptomycin (Gibco). The cells were pulsed with 0.5 �g/ml ofDENV peptide NS4B99-107 (NS4B with amino acids 99 to 107) (28) or nopeptide (1 h, 37°C). The cells were washed three times and labeled withcarboxyfluoroscein succinimidyl ester (CFSE) (CellTrace; Invitrogen/Molecular Probe) in PBS with 0.1% bovine serum albumin (BSA) for 10min at 37°C. DENV peptide-pulsed cells (target cells) were labeled with 1�M CFSE (CFSEhi), and the non-peptide-pulsed cells (marker cells) werelabeled with 100 nM CFSE (CFSElo). After the cells were washed, the twocell populations were mixed at a 1:1 ratio and 3.5 � 106 cells from eachpopulation were injected i.v. into naive or infected recipient mice. After 15h, the ratio of target (CFSEhi) to marker (CFSElo) cells was determined inthe spleens of recipient mice by flow cytometry. Cytolytic activity in therecipient is expected to reduce the peptide-labeled target population, butnot the unlabeled marker population.
CD8� T cell isolation and transfer. CD8� T cells were isolated bymagnetically activated cell sorting (MACS) positive selection with a kitfrom Miltenyi Biotech (CD8a Ly-2), and total T cells were isolated byMACS negative selection with the Pan T cell isolation kit II from MiltenyiBiotech according to the manufacturer’s instructions. After the cells wereisolated, they were injected intravenously in a total volume of 200 �l PBS.
Statistical analysis. For viral RNA titers, P values were calculated withPrism (GraphPad Software) using the unpaired t test with Welch’s cor-rection. For statistical analysis, the value of the limit of detection wasattributed to the samples that were under the detection limit.
For survival curves, P values were calculated with Prism (GraphPadSoftware) using the log rank (Mantel-Cox) test.
RESULTSNonlethal DENV priming protects from heterotypic lethalDENV2 challenge in a CD8-dependent manner. To evaluate theeffect of nonlethal DENV priming on a subsequent lethal chal-lenge with a different DENV serotype (heterotypic challenge)shortly after priming, we first used a model of DENV infection in129/Sv mice lacking type I and II IFN receptors (AG129 mice). Wechose this model because some DENV strains replicate in AG129mice and cause a disease that recapitulates many features of DENVinfection in humans and can be lethal (39, 41, 46, 47).
AG129 mice were nonlethally primed with 1 � 104 PFUDENV4 (strain H241) 2 weeks prior to challenge with 1 � 105 PFUDENV2 (strain S221), and survival was monitored. A controlgroup was challenged with 1 � 105 PFU DENV2 without priming.All the nonprimed animals died by day 20, whereas 62.5% of theDENV4-primed animals survived without signs of disease untilday 25, when the experiment was terminated (Fig. 1A). Theseresults show that nonlethal DENV4 priming reduced mortalityupon DENV2 challenge.
The 2-week period between priming and challenge was chosento give enough time for the T cell response to develop (28). Inorder to maximize our chance of observing any potential effect of
T cells, we chose to challenge with virus soon after the peak, beforethe T cell levels were back to prepriming levels. Also, challenging 2weeks after priming ensured that we were challenging within theshortest duration that has been postulated for the cross-protec-tion period observed after DENV infection (14, 17).
Previous studies from our laboratory have demonstrated thatboth CD4� and CD8� T cells can contribute to protection againstDENV infection (24, 27–30). Therefore, we assessed the role of Tcells in the heterotypic protection observed 2 weeks after nonle-thal DENV4 priming. AG129 mice were primed (DENV4) andchallenged (DENV2) as described above, and CD4� or CD8� Tcells were depleted just before challenge with DENV2. Depletionof CD4� T cells before challenge had no significant effect on sur-vival after DENV2 challenge (Fig. 1B), whereas depletion of CD8�
T cells abolished the protection mediated by DENV4 priming(Fig. 1B).
To confirm the survival phenotype, we next investigated howDENV4 priming would influence the viral load after DENV2 chal-lenge. AG129 mice were primed with 1 � 105 PFU DENV4 andchallenged 2 weeks later with 1 � 104 PFU DENV2. DENV2 RNAtiters were measured in the liver 3 days after DENV2 challenge.The liver was chosen as we have previously demonstrated thathigh liver viral RNA titers on day 3 are predictive of severe diseaseand short survival time (30, 48). Compared to the nonprimedcontrol group, DENV4-primed mice had about 1,000-fold-lowerviral RNA titers in the liver 3 days after DENV2 challenge (Fig.1C). As in the previous experiment, CD4� or CD8� T cells weredepleted in some animals prior to DENV2 challenge. Consistentwith the survival data shown in Fig. 1B, depletion of CD8� T cells,but not CD4� T cells, abrogated the viral load reduction mediatedby DENV4 priming (Fig. 1C).
To confirm that a similar phenomenon would be observedafter priming with another DENV serotype, AG129 mice wereprimed with 5 � 104 PFU DENV3 (strain UNC3001) 2 weeksprior to challenge with 1 � 104 PFU DENV2 (strain S221). As inthe previous experiment, CD4� or CD8� T cells were depletedbefore challenge in some of the animals. One control group with-out priming (challenge only) was included. Three days after chal-lenge, viral RNA was present in the livers of the nonprimed micebut not detected in the livers of DENV3-primed mice (Fig. 1D). Asfound in the DENV4 priming experiments, CD8 depletion, butnot CD4 depletion, abolished the viral load reduction mediated byDENV3 priming (Fig. 1D). Taken together, these results show that2 weeks after DENV priming, CD8� T cells contribute to protec-tion against heterotypic reinfection.
CD8� T cells are required to reduce viral load upon hetero-typic, but not homotypic, reinfection. After demonstrating theimportance of CD8� T cells in protection after heterotypic prim-ing, we evaluated whether CD8� T cells were also required forprotection after homotypic DENV priming. AG129 mice werenonlethally primed with 1 � 105 PFU DENV2 (strain PL046) orDENV4 (strain H241) 2 weeks prior to challenge with 1 � 104 PFUDENV2 (strain S221). The DENV2 strain PL046 was chosen fornonlethal priming, as it is less virulent than the strain used forchallenge, DENV2 strain S221 (39). A nonprimed group (chal-lenge only) was included. Prior to challenge with DENV2, half ofthe animals were depleted of their CD8� T cell population. Liverviral RNA titers were measured on day 3 (Fig. 2A). In thenonprimed group, CD8� T cell depletion had no effect on the liverRNA titer on day 3, likely because 3 days are insufficient for CD8�
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T cells to become activated and functional after primary infection(24). In DENV2-primed mice (homotypic priming), the liverRNA titer was reduced by 100-fold or more compared to the titerin the nonprimed mice, and CD8 depletion had no effect on theviral load reduction. In comparison, in DENV4-primed mice(heterotypic priming), viral load reduction was observed in theCD8-competent, but not in the CD8-depleted animals. Similarresults were observed when viral RNA titers were measured in thespleen (Fig. 2B).
Because IFN-� production is a major function of CD8� T cells,we wanted to confirm our results in mice with intact IFN-� recep-tor signaling. Therefore, we next performed similar experimentswith 129/Sv mice deficient in type I IFN receptor only (IFNAR�/�
mice). As these mice are less immunocompromised than AG129mice, they are more resistant to DENV infection and manifestfewer signs of disease than AG129 mice. However, some DENVstrains replicate in IFNAR�/� mice to detectable levels (47), whichallows for assessment of the effect of priming on subsequentDENV challenge. Those experiments would be impossible in wild-
type (WT) mice in which DENV does not replicate to measurablelevels (47). Consistent with results obtained from experimentsusing AG129 mice, homotypic priming with 1 � 106 PFU DENV2(strain PL046) 2 weeks prior to DENV2 challenge (2 � 105 PFU,strain S221) reduced viral RNA titers in the livers of IFNAR�/�
mice upon challenge, even when CD8� T cells were depleted be-fore challenge (Fig. 2C). After heterotypic priming (DENV4 strainH241, 1 � 106 PFU), a reduction in viral RNA titer was observedupon DENV2 challenge in CD8-sufficient, but not CD8-depleted,mice (Fig. 2C). Similar results were obtained in the spleen (Fig.2D). Collectively, these results demonstrate that while both ho-motypic and heterotypic DENV priming reduce viral RNA titerupon reinfection, CD8� T cells are necessary to mediate viral loadreduction after heterotypic, but not homotypic, priming. If notstated otherwise, all subsequent experiments are performed inIFNAR�/� mice.
CD8� T cells are necessary to reduce morbidity upon hetero-typic, but not homotypic reinfection. Next, we examinedwhether the decrease in viral load observed after DENV2 challenge
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FIG 1 DENV priming protects from heterotypic lethal DENV2 challenge in a CD8-dependent manner. (A) AG129 mice were either left untreated or primed with1 � 104 PFU DENV4 (strain H241) 2 weeks prior to challenge with 1 � 105 PFU DENV2 (strain S221) (number of mice [n] � 8 to 13). The survival of nonprimedmice was significantly different (P � 0.05) from the survival of primed mice by the log rank (Mantel-Cox) test. This is indicated by an asterisk. (B) Mice wereprimed and challenged as described above for panel A, and their CD4� or CD8� T cell populations were depleted before challenge. The DENV4-primednondepleted group shown in panel A is also depicted in panel B for clarity. Survival was monitored (n � 8 to 10). The survival of mice depleted of their CD8�
T cells was significantly different (P � 0.05) from the survival of nondepleted mice by the log rank (Mantel-Cox) test. This is indicated by an asterisk. (C) AG129mice were primed with 1 � 105 PFU DENV4 (strain H241) 2 weeks prior to challenge with 1 � 104 PFU DENV2 (strain S221). Before challenge, mice weredepleted of their CD4� or CD8� T cell populations or not depleted. Viral RNA titers were measured in the livers 3 days after challenge. (D) AG129 mice wereprimed with 5 � 104 PFU DENV3 (strain UNC3001) 2 weeks prior to challenge with 1 � 104 PFU DENV2 (strain S221). Before challenge, mice were depletedof their CD4� or CD8� T cell populations or not depleted. Viral RNA titers were measured in the liver 3 days after challenge. Gray symbols represent samples thatwere under the detection limit (broken line) and therefore have no numerical value. In panels C and D, each symbol represents the value for an individual mouse,and the bar represents the mean value for the group of mice. The mean is not shown when some of the samples were under the detection limit. Values that weresignificantly different in panels C and D by unpaired t test with Welch’s correction are indicated by bars and asterisks as follows: *, P � 0.05; **, P � 0.01; ***,P � 0.001.
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in DENV2- or DENV4-primed animals correlated with a reduc-tion in morbidity. IFNAR�/� mice were primed with 1 � 106 PFUDENV2 (strain PL046) or DENV4 (strain H241) 2 weeks prior tochallenge with 2 � 106 PFU DENV2 (strain S221) in the presenceof 10 �l of DENV2-immune serum (see Materials and Methods).This elevated viral challenge dose was chosen in order to increasethe chance of observing signs of disease. DENV-immune serumwas given to the mice based on our unpublished observation thatthis serum amount causes antibody-dependent enhancement ofinfection and increases the severity of disease when administeredwith this viral challenge strain and dose. To confirm the impor-tance of CD8� T cells in heterotypic protection, CD8� T cells weredepleted in half of the mice between priming and challenge, andthe health of the mice was monitored. In the nonprimed controlgroup, all animals but one were sick (hunchback posture, ruffledfur, or reduced mobility) or dead by day 4, and more than 50%were dead by day 6 regardless of the presence of CD8� T cells (Fig.3A, top). In the DENV2-primed animals (homotypic priming),no sign of disease was observed on day 4 or 6, regardless of thepresence of CD8� T cells (Fig. 3A, middle). In DENV4-primedanimals (heterotypic priming), all animals were sick on day 4 inthe absence of CD8� T cells, whereas the CD8-competent animalswere all healthy on day 4 (Fig. 3A, bottom).
In addition to monitoring health, weight loss was recorded as asign of morbidity. IFNAR�/� mice were primed with DENV2 orDENV4 or not primed prior to challenge with DENV2 as de-scribed above. Half of the animals were depleted of their CD8� Tcell population before challenge. As shown in Fig. 3B, weight losswas observed in all nonprimed mice, regardless of the presence ofCD8� T cells. All nonprimed CD8-depleted mice and 60% of thenonprimed, CD8-sufficient mice had lost weight (20% weightloss) by day 5 and were sacrificed. Weight loss was not observed inDENV2-primed (CD8-depleted or not) animals, whereas inDENV4-primed animals, weight loss was observed in CD8-de-pleted, but not in CD8-sufficient, animals. These results show thatCD8� T cells are necessary to reduce morbidity and weight lossupon DENV2 challenge in IFNAR�/� mice primed with DENV4(heterotypic priming). However, after homotypic priming(DENV2), no sign of disease or weight loss occurred, irrespectiveof the presence of CD8� T cells.
Priming with DENV4 induces DENV2-reactive antibodies,but not DENV2 neutralizing antibodies. We analyzed the hu-moral immune response after DENV2 or DENV4 priming.IFNAR�/� mice were primed with 1 � 106 PFU DENV2 (strainPL046) or DENV4 (strain H241) 2 weeks prior to challenge with2 � 105 PFU DENV2 (strain S221). One group of mice was not
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FIG 2 CD8� T cells are necessary to reduce viral load upon heterotypic, but not homotypic, reinfection. (A and B) AG129 mice were primed with 1 � 105 PFUDENV2 (strain PL046) or DENV4 (strain H241) 2 weeks prior to challenge with 1 � 104 PFU DENV2 (strain S221). One control group was not primed. CD8�
T cells were depleted in half of the mice before challenge. Viral RNA titers (DENV2) were monitored in the liver (A) and the spleen (B) 3 days after challenge. (Cand D) IFNAR�/� mice were primed with 1 � 106 PFU DENV2 (strain PL046) or DENV4 (strain H241) 2 weeks prior to challenge with 2 � 105 PFU DENV2(strain S221). One control group was not primed. Before challenge, CD8� T cells were depleted in half of the mice. Viral RNA titers (DENV2) were monitoredin the liver (C) and the spleen (D) 3 days after challenge. Gray symbols represent samples that were under the detection limit (broken line) and therefore have nonumerical value. Each symbol represents the value for an individual mouse, and the bar represents the mean value for the group of mice. The mean is not shownwhen some of the samples were under the detection limit. Values that were significantly different by unpaired t test with Welch’s correction are indicated by barsand asterisks as follows: *, P � 0.05; **, P � 0.01; ***, P � 0.001.
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primed prior to DENV2 challenge. Three days after challenge, thelevels of DENV2-reactive IgG were measured in serum by ELISA(Fig. 4A). DENV2-reactive IgG antibodies were present in bothDENV2- and DENV4-primed animals, but the levels were 5-foldhigher in DENV2-primed mice than in DENV4-primed mice. NoDENV2-reactive IgG antibodies were detected in nonprimed(DENV2-challenged) mice on day 3, indicating that the viral chal-lenge without priming did not result in IgG production by day 3.The neutralizing activity of the serum was analyzed by 50% plaquereduction neutralization test (PRNT50) on BHK21 cells. As shown
in Fig. 4B, DENV2 neutralizing antibodies were detected inDENV2-primed, but not in DENV4-primed, animals. There-fore, both DENV2 and DENV4 priming induced production ofDENV2-reactive IgG, but only DENV2 priming induced aDENV2 neutralizing antibody response. This result implies a min-imal role for the humoral response in mediating the short-termheterotypic protection in DENV4-primed mice upon DENV2challenge.
T cells are responding to challenge with DENV2 in bothDENV2- and DENV4-primed mice. To further support our hy-
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FIG 3 CD8� T cells are necessary to reduce morbidity upon heterotypic, but not homotypic, reinfection. IFNAR�/� mice were primed with 1 � 106 PFU DENV2(strain PL046) or DENV4 (strain H241) 2 weeks prior to challenge with 2 � 106 PFU DENV2 (strain S221) in the presence of 10 �l of immune serum, an amountthat increases the severity of disease. One control group was not primed. Before challenge, CD8� T cells were depleted in half of the mice. The health (A) andweight (B) of the animals were assessed daily. The data in panel A are pooled from two experiments. The number of mice (n) was 7 to 9 for panel A and 3 to 5 forpanel B.
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pothesis that CD8� T cells can mediate heterotypic protectionduring DENV2 challenge in DENV4-primed mice, we analyzedthe T cell response during DENV2 challenge in nonprimed orDENV2- or DENV4-primed mice. IFNAR�/� mice were primedwith DENV2 (strain PL046) or DENV4 (strain H241) 2 weeksprior to challenge with DENV2 (strain S221). One control groupwas not primed. The percentage of T cells producing cytokineswas monitored by “direct” in vivo intracellular cytokine staining(ICS) (43–45) 7 h after challenge with DENV2. In vivo adminis-tration of brefeldin A (which causes retention of cytokines in theGolgi complex) 1 h after DENV2 challenge allows for detectionand enumeration of T cells that are actively producing cytokines invivo after exposure to DENV. As expected, the percentage of acti-vated CD8� T cells (characterized by CD44hi, IFN-�-positive[IFN-��], or CD107a�) was significantly higher in both DENV2-or DENV4-primed mice than in nonprimed animals (Fig. 5A).However, the percentage of IFN-�� CD8� T cells was lower inDENV4-primed mice than in DENV2-primed mice. These resultsdemonstrate that both DENV2 and DENV4 priming leads toCD8� T cell activation, but DENV4-primed CD8� T cells are lessefficient than DENV2-primed CD8� cells in producing IFN-�after DENV2 challenge.
To confirm the functionality of the CD8� T cells activated byDENV2 or DENV4 priming, the cytolytic activity of the CD8� Tcells was assessed by in vivo cytotoxicity assay in IFNAR�/� miceprimed with DENV2 (strain PL046) or DENV4 (strain H241).Equal numbers of naive splenocytes labeled with the DENV pep-tide NS4B99-107 (CFSEhi target cells) and splenocytes without pep-tide (CFSElo marker cells) were transferred into naive mice orDENV2- or DENV4-infected recipient mice 2 weeks after prim-ing. The target/marker ratio was determined by flow cytometry ofspleen cells of recipient mice 15 h after transfer. Similar cytolyticactivity was detected in both DENV2- and DENV4-infected mice,and as expected, no cytotoxic activity was detected in naive mice(Fig. 5B). Taken together, these results suggest that priming witheither DENV2 or DENV4 induced a CD8� T cell response that wasboth measurable and functional upon DENV2 challenge.
CD8� T cells from DENV2- or DENV4-primed mice are suf-ficient to reduce viral load when transferred into naive recipi-ents prior to challenge with DENV2. Finally, we investigatedwhether CD8� T cells activated by DENV2 or DENV4 primingwould be sufficient to reduce viral load upon DENV2 challenge.IFNAR�/� mice were primed with DENV2 (strain PL046) or
DENV4 (strain H241), and 2 weeks later, their CD8� T cells wereisolated (positive selection of CD8� T cells). CD8� T cells (2 �107) from either DENV2- or DENV4-primed mice were trans-ferred into naive IFNAR�/� mice 1 day prior to challenge withDENV2 (strain S221). Viral RNA titers were monitored in the liverand spleen 3 days after challenge with DENV2 (Fig. 6A and B).Recipients that received DENV2- or DENV4-primed CD8� Tcells had a lower viral RNA titer in the liver (Fig. 6A) and spleen(Fig. 6B) 3 days after DENV2 challenge compared to the mice thatdid not receive CD8� T cells. The viral RNA titer was lower inmice that received homotypic (DENV2-primed) CD8� T cellsthan in mice that received heterotypic (DENV4-primed) CD8� Tcells, which correlated with the higher frequency of IFN-�� CD8�
T cells observed in DENV2-primed mice compared to DENV4-primed mice upon challenge with DENV2 (Fig. 5). To assess theduration of protection mediated by the DENV2- or DENV4-primed T cells against DENV2 reinfection, IFNAR�/� mice wereprimed with DENV2 or DENV4, and 6 weeks later, total T cellswere isolated (MACS negative selection of total T cells), and var-ious numbers of T cells were transferred into naive AG129 recip-ients. One day later, mice were challenged with DENV2, and viraltiters were measured in the liver by qRT-PCR 3 days after chal-lenge (Fig. 6C). Both DENV2- and DENV4-primed T cells effi-ciently reduced viral load upon challenge with DENV2, demon-strating that both homotypic and heterotypic T cells can beprotective for at least 6 weeks after priming.
Collectively, these results demonstrated that both homotypic(DENV2-primed) and heterotypic (DENV4-primed) CD8� Tcells were functional and sufficient to reduce viral load uponDENV2 challenge. However, when equal numbers of CD8� T cellswere transferred, CD8� T cells activated by DENV2 (homotypicpriming) caused a larger viral RNA reduction than CD8� T cellsactivated by DENV4 (heterotypic priming).
DISCUSSION
In this study, we used a murine model to investigate the contribu-tion of T cells to protection shortly after DENV reinfection. Ourresults showed that CD8� T cells were necessary for protectionagainst heterotypic reinfection in mice that were nonlethallyprimed with one DENV serotype 2 weeks before challenge withanother DENV serotype. However, CD8� T cells were not re-quired for protection against a homotypic challenge (priming andchallenge with the same serotype).
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ENV2
FIG 4 Priming with DENV4 induces DENV2-reactive antibodies, but not DENV2 neutralizing antibodies. IFNAR�/� mice were primed with 1 � 106 PFUDENV2 (strain PL046) or DENV4 (strain H241) 2 weeks prior to challenge with 2 � 105 PFU DENV2 (strain S221). One control group was challenged but notprimed. On day 3 after challenge, serum was harvested, and DENV2-reactive IgG antibodies were measured by ELISA (n � 3) (A), and DENV2-neutralizingantibodies were quantified by PRNT50 (B). Naive serum and 5 �g of the DENV-specific neutralizing antibody 4G2 were included as negative and positivecontrols, respectively.
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Homotypic (DENV2) priming induced a robust neutralizingantibody response against the serotype used for challenge(DENV2), whereas heterotypic (DENV4) priming induced anti-bodies that bound to, but did not neutralize, DENV2. PerhapsCD8� T cells were not necessary for protection after homotypicpriming due to the presence of serotype-specific neutralizing an-tibodies but were required after heterotypic priming because no
cross-neutralizing antibodies were present. We demonstrated byadoptive transfer experiments that both homotypic and hetero-typic CD8� T cells were able to reduce viral load upon reinfection.Therefore, we hypothesize that cross-protective CD8� T cell re-sponses can efficiently compensate for the absence of cross-pro-tective antibodies upon heterotypic reinfection; in contrast, afterhomotypic priming, humoral responses are sufficient to protectfrom reinfection. Figure 7 shows a graphical summary of our find-ings.
In our experiments, we did not detect DENV2 neutralizing an-tibodies after DENV4 priming. This could be due to a lower levelof replication of the DENV4 strain used for priming compared tothe DENV2 strain. In the absence of cross-neutralizing antibodies,cross-protection relied on cellular immunity. However, it is pos-sible that under other experimental conditions (other viral strainsor higher viral doses), cross-neutralizing antibodies can be in-duced and can contribute to protection.
While the protective efficacy of a robust neutralizing antibodyresponse is undisputed (10–12, 21), we suggest that cellular im-munity can contribute to protection, especially when the antibodyresponse is not fully protective by itself, as may be the case duringheterotypic challenge early after DENV priming. The absence ofcross-neutralizing antibodies early after infection with DENV inhumans has been reported (49, 50). One study analyzed the hu-moral immune response 1 to 2 weeks after the onset of fever andreported that cross-reactive antibodies were readily detectedagainst all serotypes, whereas neutralizing antibodies were foundonly for the infecting serotype (50). In another study, antibodyfound in convalescent-phase serum was weakly neutralizing forother serotypes (49).
In all our experiments, CD8� T cell depletion always abol-ished the viral load reduction observed on day 3 after hetero-typic challenge. However, upon DENV2 challenge of IFNAR�/�
mice, signs of disease and mortality on day 6 were lower in CD8�
T cell-depleted DENV4-primed mice than in CD8� T cell-de-pleted nonprimed mice. This suggests that other protective mech-anisms (as for example innate immunity or nonneutralizing anti-bodies) also play a role besides CD8� T cells upon heterotypicpriming.
Overall, we do not exclude a protective role for cross-neutral-izing antibodies, nor do we imply that heterotypic CD8� T cellsare the exclusive mediators of cross-protection. Simply, we sug-gest (i) that heterotypic CD8� T cells can play a beneficial role ifhumoral responses fail to be fully protective and (ii) that cross-protection may not rely exclusively on humoral responses.
Cellular immunity efficiently protected from heterologous re-infection in our study. However, when equal numbers of CD8� Tcells from DENV2- or DENV4-primed mice were transferred intonaive recipients before challenge with DENV2, CD8� T cells fromDENV2-primed mice (homotypic) reduced the viral load to agreater degree than CD8� T cells from DENV4-primed mice. Thiscould be explained by at least two nonmutually exclusive hypoth-eses. First, CD8� T cells in DENV4-primed mice may have a lowerstate of activation than CD8� T cells in DENV2-primed mice dueto lower replication of the DENV4 strain used compared to theDENV2 strain. Alternatively, due to antigenic differences betweenthe DENV2 and DENV4 strains used for priming, only some of theCD8� T cells primed by DENV4 are reactive to peptides also pres-ent in DENV2, resulting in lower CD8� T cell responses uponchallenge with DENV2 in mice primed by DENV4 than in animals
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FIG 5 T cells are activated and functional during challenge with DENV2 inmice primed with homotypic or heterotypic DENV. (A) IFNAR�/� mice wereprimed with 1 � 105 PFU DENV2 (strain PL046) or DENV4 (strain H241) 2weeks prior to challenge with 2 � 105 PFU DENV2 (strain S221). One controlgroup was not primed. T cell activity was monitored by in vivo ICS 7 h afterchallenge with DENV2. (Top) Percent CD44hi cells of CD8� T cells; (middle)percent IFN-�-producing CD8� T cells; (bottom) percent CD8� T cells ex-pressing degranulation marker CD107a�. (B) The cytolytic activity of Tcells was assessed by in vivo cytotoxicity assay in IFNAR�/� mice 2 weeksafter priming with 1 � 105 PFU DENV2 (strain PL046) or DENV4 (strainH241). Equal numbers of naive splenocytes labeled with the DENV peptideNS4B99-107 (CFSEhi target cells) and unlabeled splenocytes (CFSElo markercells) were transferred into naive or DENV2- or DENV4-infected recipientmice. The target/marker ratio was determined by flow cytometry in thespleens of recipient mice 15 h after transfer.
CD8� T Cells Mediate Cross-Protection against DENV
June 2015 Volume 89 Number 12 jvi.asm.org 6501Journal of Virology
primed by DENV2. Our finding that upon challenge with DENV2,fewer cells produced IFN-� or expressed CD107a in DENV4-primed mice than in DENV2-primed mice is consistent with bothhypotheses.
Use of mice deficient in both type I and II IFN receptor(AG129) or in type I IFN receptor only (IFNAR�/�) is not ideal, asboth strains lack an important component of the immune system.Therefore, caution must be used when extrapolating our results todengue disease in humans. However, the advantage of using thesemice is that some DENV strains replicate to measurable levels andcause a dengue-like disease (39, 46), thereby allowing for investi-gation of the effect of priming on subsequent DENV infection.The experiments described here would be impossible in WT mice,in which DENV does not replicate. The lack of replication in WTmice may be explained by the fact that DENV interferes with IFNsignaling to establish infection in humans but is unable to do so inmice (51–54). Therefore, mice deficient in IFN signaling havebeen increasingly used as a model to study DENV-induced pathol-ogy and immunity (26, 30, 47, 48, 55–61).
Both the mechanism and duration of cross-protection afterDENV infection remain controversial (17). Estimates for thelength of the cross-protection period vary between 1 and 2 weeksto 1 year or longer, potentially depending on the person or viralserotype (13–17). Both viral and host factors may thus influence
the nature of cross-protection. Our study was designed to inves-tigate experimentally the role of CD8� T cells during homotypicand heterotypic DENV reinfection. The 2-week period betweenpriming and challenge was chosen because we wished to maximizeour chance of observing any potential effect of the cellular responses.Therefore, we chose to wait until after the peak cellular responses(expected around days 7 to 10 in mice [27, 28]) but before the con-traction of the T cell compartment. Our study, focusing mainly onshort-term protection, can be viewed as a proof-of-principle demon-stration of the importance of T cells for protection against heterotypicreinfection. Although a comprehensive study of the long-term pro-tective capacity of CD8� T cells is required in the future, our datasuggest that both homotypic and heterotypic cellular responses canbe protective against DENV challenge for at least 6 weeks.
A better understanding of the mechanisms that mediate short-and long-term protection against both homotypic and hetero-typic reinfection with DENV is crucial, as the ultimate goal of aDENV vaccine is to provide long-term protection against all sero-types and genotypes within a serotype. Despite years of effort, thisgoal has not yet been attained (31–34, 36). The role of cellularimmunity in mediating protection against DENV reinfection orafter DENV vaccination has not received much attention, as mostprotection studies focused mainly on neutralizing antibodies. Ourstudy does not exclude a role for cross-reactive and/or cross-neu-
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FIG 6 CD8� T cells from DENV2- and DENV4-primed mice reduce viral load when transferred into naive recipients prior to challenge with DENV2. (A and B)IFNAR�/� mice were primed with 1 � 105 PFU DENV2 (strain PL046) or DENV4 (strain H241). Two weeks later, CD8� T cells were isolated, and 2 � 107 CD8�
cells were transferred into naive IFNAR�/� mice prior to challenge with 2 � 105 PFU DENV2 (strain S221) 1 day later. Viral RNA titers were monitored in theliver (A) and spleen (B) 3 days after challenge with DENV2. (C) IFNAR�/� mice were primed with 2 � 105 PFU DENV2 (strain S221) or 1 � 106 PFU DENV4(strain H241). Six weeks later, total T cells were isolated, and various numbers of T cells were transferred into naive AG129 mice prior to challenge with 1 � 104
PFU DENV2 (strain S221) 1 day later. Viral RNA titers were monitored in the liver 3 days after challenge with DENV2.
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tralizing antibodies at other time points after heterotypic chal-lenge or under different priming conditions. However, our resultsdo suggest that early after infection, CD8� T cells are crucial tomediate protection against heterotypic reinfection if cross-neu-tralizing antibodies are absent.
Our study adds to the increasing experimental evidence sup-porting an important role for cellular immunity during DENVinfection in humans and mouse models (23, 25–30). Our data alsosupport the hypothesis that a DENV vaccine that efficiently in-duces both humoral and cellular immunity may offer better pro-tection than a vaccine that elicits humoral immune responsesalone (25–28, 30). The phase IIb and III results of the most ad-vanced DENV vaccine candidate, a live-attenuated tetravalent for-mulation developed by Sanofi Pasteur, is consistent with this hy-pothesis (31–33). The overall efficacy of the Sanofi vaccine,composed of the structural proteins of DENV and nonstructuralproteins of yellow fever 17D, ranged from 30 to 65%. In addition,the efficacy was much lower in DENV-naive individuals (35%efficacy) than in individuals who were DENV seropositive at base-line (74% efficacy) (31, 62). As the DENV structural proteins arethe major targets of anti-DENV antibody but not T cell responses,the limited efficacy of the Sanofi vaccine against certain DENVserotypes or in naive people may be due to the absence of robust Tcell responses.
In summary, our results suggest that protection against hetero-typic reinfection may not rely exclusively on antibody and showthat CD8� T cells can contribute crucially to protection duringheterotypic reinfection and/or if humoral responses alone are notfully protective. Our findings have important implications forvaccine design, as they suggest that inducing both humoral andcellular responses during vaccination may maximize protectiveefficacy across all DENV serotypes.
ACKNOWLEDGMENTS
This project was supported by NIH grants U54AI057517 from the South-eastern Regional Center of Excellence for Emerging Infectious and Biode-fense (S.S.), R56 A1085063 (S.S.), U01 AI082185 (S.S.), and NIH contractHHSN272200900042C to Alessandro Sette and an LJI Center for Infec-tious Disease Research Fellowship to R.M.Z.
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DENV2challenge
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ERM
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DENV2 CD8+ T cellsDENV4 CD8+ T cells viral load reduction
CD8-depletion
CD8-depletion
FIG 7 Graphical summary.
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CD8� T Cells Mediate Cross-Protection against DENV
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