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. Author manuscript; available in PMC: 2015 Oct 15.
Published in final edited form as: J Immunol. 2014 Sep 12;193(8):4117–4124. doi: 10.4049/jimmunol.1401597

CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice

Raphaël M Zellweger 1, William E Eddy 1, William W Tang 1, Robyn Miller 1, Sujan Shresta 1,*
PMCID: PMC4185219  NIHMSID: NIHMS621737  PMID: 25217165

Abstract

Dengue virus (DENV) causes pathologies ranging from the febrile illness dengue fever to the potentially lethal severe dengue disease. A major risk factor for developing severe dengue disease is the presence of sub-protective DENV-reactive antibodies from a previous infection (or from an immune mother), which can induce antibody-dependent enhancement of infection (ADE). However, infection in the presence of sub-protective anti-DENV antibodies does not always result in severe disease, suggesting that other factors influence disease severity. This study investigates how CD8+ T cell responses influence the outcome of antibody-mediated severe dengue disease. Mice were primed with alum-adjuvanted UV-inactivated DENV prior to challenge with DENV. Priming failed to induce robust CD8+ T cell responses, and induced non-neutralizing antibody responses that increased disease severity upon infection. Transfer of exogenous DENV-activated CD8+ T cells into primed mice prior to infection prevented antibody-dependent enhancement and dramatically reduced viral load. Our results suggest that in the presence of sub-protective anti-DENV antibodies, efficient CD8+ T cell responses reduce the risk of antibody-mediated severe dengue disease.

Introduction

Dengue virus (DENV) is the most prevalent mosquito-borne virus that affects humans (1). The four dengue serotypes (DENV1–4) can cause a spectrum of disease ranging from the self-limiting flu-like illness dengue fever (DF) to the potentially lethal severe dengue disease, during which severe bleeding, organ dysfunction, increased vascular permeability and shock can occur (2, 3). DENV is endemic in more than 100 countries in tropical and sub-tropical regions where 2.5 to 3.6 billion people are at risk of infection (4, 5). An estimated 390 million cases of DENV infection occur annually, 96 million of which are apparent, 500,000 severe, and 20,000 fatal (5, 6).

A major risk factor for developing severe dengue disease is the presence of DENV-reactive antibodies (Abs) from a previous infection with a different serotype (heterotypic infection) or, in the case of infants, acquired from an immune mother (1, 7). This epidemiological observation led to the concept of Ab-dependent enhancement of infection (ADE), which proposes that sub-protective levels of DENV-specific Abs can amplify viral infection and exacerbate disease (8, 9). However, not all secondary heterotypic infections result in severe disease (1). This suggests that while sub-protective Abs can increase disease severity, other factors may also influence disease outcome.

In vitro, numerous studies have shown increased infection due to the presence of sub-neutralizing levels of anti-DENV Abs (10–14). In non human primates, increased viremia has been demonstrated after transfer of Abs (15, 16) and, in mice, passive transfer of sub-protective amounts of Abs turned a mild illness into severe dengue-like disease (17, 18). However, to this date, there is no report of severe dengue disease being experimentally induced in vivo by priming with an inactivated virus that, in turn, generates disease-enhancing Ab responses directly in the primed host.

In the present study, we investigated the effects of priming with alum adjuvanted UV-inactivated DENV2 (al-UV-DENV2) on subsequent infection with DENV. Priming of mice with al-UV-DENV2 increased the severity of dengue disease via ADE upon challenge with DENV serotype 2 (DENV2). Priming with al-UV-DENV2 induced non-neutralizing Abs and failed to induce CD8+ T cell responses. Transfer of exogenous DENV-primed CD8+ T cells into al-UV-DENV2-primed mice prior to challenge with DENV2 prevented disease-enhancement and reduced viral load, revealing that CD8+ T cells can modulate the severity of ADE-mediated dengue disease. This suggests that in the presence of sub-protective levels of anti-DENV Abs, inefficient CD8+ T cell responses may further increase the risk of developing severe dengue disease, while efficient cellular responses reduce disease severity. Our study suggests that the quality of the CD8+ T cell response could be one of the factors that influence disease outcome when infection with DENV occurs in the presence of sub-neutralizing levels of antibody.

Material and Methods

Mice

129/Sv mice deficient in type I and II interferon receptors (AG129) and wild-type 129/Sv mice were housed under SPF conditions at the La Jolla Institute for Allergy and Immunology (LJI). Sex-matched 5 to 6 week-old mice were used. For survival studies, mice were sacrificed when moribund or at the first signs of paralysis. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All experimental procedures were approved and performed according to the guidelines set by the La Jolla Institute for Allergy and Immunology Animal Care and Use Committee (protocol number AP-28SS1-0809).

Virus production

DENV serotype 2 (DENV2) strain S221 (19) was amplified in C6/36 cells and quantified by real-time qRT-PCR as previously described (20). Viral stocks were resuspended in PBS containing 10% FCS. Based on baby hamster kidney (BHK) cell plaque assay (21), there are approximately 5×104 genomic equivalents (GE) per plaque forming unit (PFU) for S221.

UV-inactivation

In a well of a 12-well plate, 1×1012 GE DENV2 S221 were UV-irradiated in 500 μl of PBS with 500,000 μjoules in a UV Stratalinker 1800 (Stratagene). Subsequently, 500 μl of PBS containing 10% FCS (Gemini Bioproducts) were added to reach a volume of 1 ml, to which 1 ml of alum (Imject Alum, Thermo Scientific) was added dropwise while vortexing. The UV-inactivated virus/alum mix was vortexed for another 30 minutes at room temperature.

Priming and infection of mice

Mice were injected intraperitoneally twice (day −14 and −5) with 1×1011 GE UV-inactivated DENV strain S221 in alum (“al-UV-DENV2” in the text), in a volume of 200 μl per injection. For challenge with replicating virus, mice were infected intravenously with 5×108 GE of DENV strain S221 (“DENV2”) diluted in 200 μl of PBS with 10% FCS.

Antibodies

2H2 is an IgG2a reactive for the prM/M protein of DENV serotypes 1–4. 4G2 is a DENV-neutralizing IgG2a reactive for the E protein of DENV serotypes 1–4. 2H2 and 4G2 were produced as previously described (22). C1.18, an IgG2a isotype control of irrelevant specificity, was purchased from BioXCell.

Passive antibody and serum transfer

Antibodies (2H2, anti-DENV and C1.18, isotype control) were administered intraperitoneally (i.p.) in 200 μl of PBS 30 minutes prior to infection. For serum transfer experiments, 200 μl of serum were injected i.v. one day prior to challenge with DENV2.

Purification of IgG from serum

IgG was isolated from serum by protein G affinity chromatography using the Nab Protein G Spin Kit from Thermo Scientific according to the manufacturer’s instructions. After purification, the antibody was dialized against PBS, sterile filtered and the concentration was adjusted to an IgG concentration similar to the one found in the serum. DENV-reactivity of the purified IgG was confirmed by DENV ELISA and the purity was assessed by SDS-PAGE. IgG was administered i.p. in a total volume of 200 μl PBS 30 minutes prior to infection.

DENV-specific IgG detection by ELISA

DENV-reactive IgG were detected by ELISA on DENV S221-coated plates as previously described (22). Briefly, 96-well plates were coated with sucrose-purified DENV2 strain S221 (109 GE per well in 50 μl 0.1M NaHCO3); the plated virus was UV-inactivated and the plates were incubated overnight at 4°C. Subsequently, plates were blocked with 100 μl Blocker Casein in PBS (Thermo Scientific, 1 hour, room temperature). Blocking solution was flicked off and serum was titrated 1:3 over 7 titration steps, starting with an initial dilution of 1 in 30 in PBS. Serum dilutions were incubated 1.5 hr at room temperature, followed by washing of the plates three times with 0.05% (v/v) Tween 20 (Sigma) in PBS (Gibco). Bound antibody was detected using a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG (Jackson Immunoresearch) and TMB (eBioscience). Results are reported as a plot of absorption (OD 450nm) versus dilution.

Neutralizing activity of serum

Anti-DENV neutralizing Abs were quantified by 50% plaque reduction neutralization test (PRNT50) as previously described (22). Serum was serially diluted 1:2 with the starting dilution being 1:10. The highest dilution reducing more than 50% of the plaques is reported as “50% neutralizing titer”.

Viral RNA quantification in organs

Viral RNA was quantified in organs by real-time qRT-PCR as described previously (20).

Flow Cytometry and intracellular cytokine staining (ICS)

As previously described (19) with the following modifications: 2×105 splenocytes were plated for ICS, and incubation time with peptide was 6 hours. Peptide NS4B99–107 (19) was used for restimulation. The following monoclonal Abs were used: anti-CD8a-PerCP-Cy5.5 (eBioscience, clone 53-6.7), anti-CD107a-PE (eBioscience, clone 1D4B), anti-CD62L-AlexaFluor 700 (eBioscience, clone MEL-14) and anti-CD44-PE-Cy7 (BD Pharmingen, clone IM7). Samples were acquired on an LSR II flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

CD8+ T cell isolation and adoptive transfer

CD8+ T cells cells were isolated by magnetic cell sorting (MACS) using positive selection CD8a (Ly-2) beads from Miltenyi Biotech according to the manufacturer’s instructions. After isolation, the purity of the CD8+ T cell population was assessed by flow cytometry before injection into recipient mice; our preparation contained 94.9% CD8+ T cells (data not shown). A total of 5×107 CD8+ T cells were injected i.v. into each recipient mice in a total volume of 200 μl of PBS.

In vivo cytotoxicity assay

AG129 recipient mice were infected with 5×108 GE S221. Eight days later, donor splenocytes were obtained from naïve AG129 mice. RBC were lysed (RBC lysis buffer, eBioscience) and cells were resuspended at 1×107 cells/ml in RPMI containing 2% FCS, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Gibco). Cells were pulsed with 0.5 μg/ml of DENV peptide NS4B99–107 (19) or no peptide (1 hour, 37°C). Cells were washed 3 times and labeled with CFSE (Cell Trace, Invitrogen/Molecular Probe) in PBS/0.1% BSA for 10 min at 37°C. DENV peptide-pulsed cells (“target cells”) were labeled with 1 μM CFSE (CFSEhi) and the non-labeled cells (“marker cells”) with 100 nM CFSE (CFSElo). After washing excess CFSE, the two cell populations were mixed at a 1:1 ratio and 6.5×106 cells from each population were injected i.v. into naive or infected recipient mice. After 15 hours, the ratio of “target” (CFSEhi) to “marker” (CFSElo) cells was determined in the spleen of recipient mice by flow cytometry. Cytolytic activity in the recipient is expected to reduce the peptide-labeled target population, but not the unlabeled marker population.

Statistical analysis

P-values were calculated with Prism (GraphPad Software) using the unpaired t-test with Welch’s correction, except for survival curves, for which the Log-rank test was used; * for P≤0.05, ** for P≤0.01, *** for P≤0.001.

Results

Priming with al-UV-DENV2 induces non-neutralizing DENV-specific IgG

In this study, 129/Sv mice lacking type I and II IFN-receptors (AG129) were used, as DENV replicates in these mice and causes a disease that recapitulates many features of DENV infection in humans (19, 23, 24). AG129 mice were primed twice (on day −14 and −5) with 1×1011 genomic equivalents (GE) of alum-adjuvanted UV-inactivated DENV2 (al-UV-DENV2) i.p. It is important to note that this priming regimen was not chosen to emulate vaccination schedules used in clinical settings, where the goal is to maximize the neutralizing antibody response. Our d-14/d-5 rapid priming schedule was chosen because, based on observations in our laboratory, it would result in the production of DENV-specific binding antibodies, but not neutralizing antibodies. We reasoned that those conditions would be likely to induce antibody-mediated disease enhancement (14, 18), which was necessary for our study. DENV2 strain S221 (19) was UV-inactivated in order to obtain a non-replicating antigen, and alum was chosen as adjuvant because it promotes efficient Ab responses (25, 26). Analysis of the humoral immune response induced by al-UV-DENV2 shows that on day −1, anti-DENV2 IgG were present in the serum, as measured by ELISA (figure 1A), but neutralizing Ab responses were undetectable by plaque reduction neutralization test (PRNT50, figure. 1B).

Figure 1. Priming with al-UV-DENV2 induces non-neutralizing DENV-specific IgG.

Figure 1

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AG129 mice were primed with al-UV-DENV2 on days −14 and −5. On day −1, DENV-specific IgG levels were measured in the serum by ELISA (A) and the presence of neutralizing Abs was detected by PRNT50 (B).

Results were pooled from 2 independent experiments, n=9–10 total per group. The dotted line represents the background signal in A, and the detection limit in B. For B: each symbol represents one mouse, and the DENV-neutralizing Ab 4G2 was used as a positive control. Samples under the detection limit are represented in grey.

Priming with al-UV-DENV2 increases viral load and reduces survival time upon challenge with DENV2

Next, we challenged al-UV-DENV2-primed AG129 mice with 5×108 GE DENV2 strain S221 on day 0. Two non-primed control groups were included: i) one group challenged with 5×108 GE DENV2 in the presence of 15μg of anti-DENV monoclonal Ab 2H2, an amount which causes Ab-mediated severe dengue disease, shorter survival time and elevated liver viral RNA titer (ADE group) (22), and ii) one group challenged with 5×108 GE DENV2 in the presence of 15 μg of C1.18, an isotype control Ab of irrelevant specificity (isotype group). Antibodies were administered i.p. 30 minutes before infection. Compared to the non-primed isotype group, al-UV-DENV2-primed mice had a reduced survival time, similar to the survival time observed in the non-primed ADE group (figure. 2A). On day 3, compared to the non-primed isotype mice, al-UV-DENV2-primed mice had elevated liver viral RNA titers that were similar to the ones observed in the non-primed ADE mice (figure. 2B). Liver was chosen because liver viral RNA titer on day 3 correlates with disease severity (18, 22). Next, we wanted to exclude that alum or the FCS/PBS (10%) in which virus was diluted (see material and methods) caused the increase in viral RNA observed in the liver 3 days after challenge. Therefore, AG129 mice were primed on day −14 and −5 with either al-UV-DENV2 (as previously) or with FCS/PBS (10%) in alum without virus (vehicle only). On day 0, mice were challenged with DENV2, and viral RNA levels were determined in the liver on day 3. As a control, one group was left untreated until challenge on day 0. Compared to non-primed controls, viral RNA levels were elevated in the al-UV-DENV2-primed mice, but not in the mice primed with alum and FCS/PBS without virus (figure. 2C). This result demonstrates that the presence of UV-inactivated virus was required in the alum preparation to induce the viral titer increase observed in the liver on day 3 after challenge with DENV2. Therefore, we excluded a non-specific effect of the vehicle itself. In summary, priming with al-UV-DENV2 induced non-neutralizing anti-DENV Abs and resulted in reduced survival time and increased viral load upon DENV2 challenge.

Figure 2. Priming with al-UV-DENV2 exacerbates disease severity upon infection.

Figure 2

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A, B) AG129 mice were primed with al-UV-DENV2 on days −14 and −5. On day 0, mice were challenged with DENV2. Two non-immunized control groups were included: ADE group (virus with enhancing amount of anti-DENV Ab) and isotype group (virus and isotype control). Survival was monitored (A) and viral RNA levels were measured in the liver by qRT-PCR on day 3 after challenge (B).

C) AG129 mice were primed on day −14 and −5 with al-UV-DENV2 or with alum and 10% FCS/PBS but without virus (vehicle only). Mice were challenged with DENV2 on day 0, and viral RNA levels were measured by qRT-PCR in the liver on day 3.

For A: results were pooled from 2 independent experiments, n=9–10 total per group. Stars show statistical comparison between the isotype group and the ADE group or the al-UV-DENV2-primed group. For B: each symbol represents one mouse; the experiment was performed twice with similar results. For C: each symbol represents one mouse.

Passive transfer of al-UV-DENV2 immune serum increases viral titer in the liver upon challenge with DENV2

To demonstrate that Ab induced by al-UV-DENV2-priming was responsible for the disease enhancement observed above, 200 μl of serum from al-UV-DENV2-primed AG129 mice were transferred i.v. into naïve AG129 mice one day before challenge with DENV2. Two control groups that did not receive immune serum were included: the ADE and isotype groups as described above. Compared to the isotype group with no serum transfer, mice that had received al-UV-DENV2 immune serum prior to challenge had elevated liver viral RNA levels three days after challenge (figure. 3A). This suggests that the Abs present in the al-UV-DENV2 immune serum were responsible for the increase in viral RNA levels observed upon challenge in al-UV-DENV2-primed AG129 mice.

Figure 3. Passive transfer of UV-DENV2 immune serum increases viral RNA levels in the liver upon challenge with DENV2.

Figure 3

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AG129 or congenic WT mice were primed with al-UV-DENV2 on days −14 and −5. On day 0, serum was collected and 200 μl of serum from AG129 mice (A) or WT mice (B) were transferred i.v. into naïve AG129 recipient mice one day prior to challenge with DENV2. Viral RNA levels were measured in the liver on day 3 after infection. The isotype and ADE control groups (described in fig. 1) received no serum and were challenged on day 0. In C, serum from al-UV-DENV2-primed WT mice was left untreated (untr.) or heat-inactivated (inact.) before transfer into naïve AG129 mice one day prior to challenge. In one group of recipients, IgG purified from al-UV-DENV2-primed WT serum were transferred prior to challenge. Viral RNA titers were measured by qRT-PCR in the liver on day 3 post-infection.

Each symbol represents one mouse, the experiment in A was repeated twice with similar results; see also supplementary figure S1 and S2.

To confirm that the enhancement caused by passive transfer of al-UV-DENV2 immune serum from AG129 mice (lacking type I and II IFN receptors) would also occur after transfer of wildtype (WT) immune serum, WT mice were primed with al-UV-DENV2 and serum was collected on day 0. As observed with AG129 mice, non-neutralizing DENV2-specific IgG were detected in the serum of al-UV-DENV2-primed WT mice (suppl. figure S1). Transfer of al-UV-DENV2-immune WT serum into naïve AG129 mice prior to challenge with DENV2 increased liver viral RNA levels by ~10-fold three days post-infection (figure. 3B).

To exclude a role for complement proteins in the enhancement observed after serum transfer, another experiment was performed where al-UV-DENV2 immune serum from WT mice was heat-inactivated (56°C, 30 minutes) prior to transfer into AG129 recipients. Additionally, to verify that the IgG present in the transferred serum was responsible for the increase in DENV-infection, IgG was purified out of al-UV-DENV2 immune serum by protein G affinity chromatography and was transferred into naïve recipients prior to challenge with DENV2. The amount of purified IgG transferred was equivalent to the amount of IgG present in the 200 μl of serum transferred in previous experiments. The DENV-reactivity of the isolated IgG was confirmed by ELISA, and its purity by SDS-PAGE (supplementary figure S2). As shown in figure 3C, transfer of either untreated or heat-inactivated al-UV-DENV2 immune serum resulted in higher DENV2 titers in the recipients upon challenge, as did transfer of purified IgG from UV-DENV2 serum. These results confirm that priming with al-UV-DENV2 induced an Ab response that, in turn, caused Ab-mediated disease enhancement upon challenge with DENV2.

Priming with al-UV-DENV2 does not activate CD8+ T cells

Next, we hypothesized that priming with al-UV-DENV2 resulted in severe disease because of the simultaneous presence of non-neutralizing anti-DENV Abs and inefficient CD8+ T cell responses. We expected CD8+ T cell responses to be low and/or inefficient after priming with al-UV-DENV2 because i) alum skews immune responses towards humoral rather than cellular immunity (25), ii) efficient CD8+ T cell responses are not readily induced by non-replicating antigens (27) and iii) priming with al-UV-DENV2, which is non-replicating, does not result in production of non-structural proteins, which are the main targets of anti-DENV CD8+ T cell responses (19, 28, 29). Thus, the number and phenotype of CD8+ T cells were assessed in the spleen of AG129 mice that were either primed with al-UV-DENV2 on days −14 and −5 or infected with 5×108 GE DENV2 on day −8 (figure 4). Compared to naïve animals, the total number and percentage of CD8+ T cells increased in the spleen of animals infected with replicating DENV2, but not in the spleen of animals primed with non-replicating al-UV-DENV2 (figure. 4A, B). Similarly, more than 50% of the CD8+ T cells were activated (CD44hi CD62Llo) after infection with DENV2, but the percentage of activated CD8+ T cells in al-UV-DENV2-primed mice was comparable to the percentage in naïve mice (figure. 4C). These results demonstrate that priming with al-UV-DENV2 failed to induce measurable CD8+ T cell responses.

Figure 4. Priming with al-UV-DENV2 does not induce measurable CD8+ T cell responses.

Figure 4

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AG129 mice were primed with al-UV-DENV2 on days −14 and −5 or infected with DENV2 on day −8. On day 0, the total number (A) and the percentage (B) of CD8+ T cells were determined in the spleen, and the percentage of activated cells (CD44hi CD62Llo) within the CD8+ T cell population was determined (C) by flow cytometry.

Two independent experiments were pooled, n=8 total.

Transfer of DENV-primed CD8+ T cells in al-UV-DENV2-primed mice prevents Ab-mediated disease enhancement and reduces viral load upon challenge with DENV2

We hypothesized that transferring CD8+ T cells from an animal that had been infected with (replicating) DENV2 into al-UV-DENV2-primed recipients prior to challenge would prevent the Ab-induced increase in viral load. First, we verified that infection of AG129 mice with DENV2 would induce functional CD8+ T cells by assessing their ability to degranulate and lyse target cells after infection. AG129 mice were infected with DENV2 and 8 days later splenocytes were restimulated with NS4B99–107, an immunodominant DENV-peptide from the NS4B protein (19). After in vitro restimulation, CD107a+ (degranulation marker) CD8+ T cells were present in the DENV2-infected animals, but not in naïve mice (figure. 5A). In another experiment, the cytolytic activity of the T cells was assessed by in vivo cytotoxicity assay. Equal numbers of naïve splenocytes labeled with the DENV peptide NS4B99–107 (CFSEhi target cells) and splenocytes without peptide (CFSElo marker cells) were transferred into naïve or DENV2-infected recipient mice 8 days post-infection. The target:marker ratio was determined by FACS in the spleen of recipient mice 15 hours after transfer. Cytotoxic CD8+ T cells are expected to lyse peptide-labeled target cells, but not marker cells, thereby reducing the target:marker ratio. Figure. 5B shows that cytolytic activity was detected in DENV2-infected mice but not in naïve mice. Therefore, we concluded that DENV-specific CD8+ T cells from AG129 mice were functional and capable of lysing target cells after infection with DENV2.

Figure 5. Transfer of DENV-primed CD8+ T cells prevents ADE and reduces viral load after al-UV-DENV2-priming.

Figure 5

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A) AG129 mice were infected with 5×108 GE DENV2 and 8 days later the percentage of CD8+ T cells staining positive for CD107a (marker of degranulation) was determined by FACS after in vitro restimulation with the DENV peptide NS4B99–107.

B) The functionality of DENV2-primed CD8+ T cells was assessed by in vivo cytotoxicity assay: equal numbers of naïve splenocytes labeled with the DENV peptide NS4B99–107 (CFSEhi target cells) and unlabeled splenocytes (CFSElo marker cells) were transferred in naïve or infected recipient mice (infection as in A). The target:marker ratio was determined by FACS in the spleen of recipient mice 15 hours after transfer.

C) AG129 mice were primed with al-UV-DENV2 on days −14 and −5 (or not primed) and challenged on day 0 with 5×108 GE DENV2 i.v. On day −1, some mice received 5×107 CD8+ T cells from mice infected with 5×108 GE DENV2 seven days earlier. Viral RNA titers were measured on day 3 post-challenge in the liver.

For A, B and C, each symbol represents one mouse n=4–6; experiment A has been repeated twice with similar results. Samples with DENV RNA content under the detection limit are depicted in grey and have no numerical value. For statistical analysis, the value of the detection limit was attributed to samples under the detection limit in order to calculate the p-value.

Next, we tested whether transfer of CD8+ T cells from DENV2-infected donors into al-UV-DENV2-primed recipients would prevent Ab-mediated increase in viral load. To this end, 5×107 CD8+ T cells (positive selection of CD8+ T cells) from day 7 DENV2-infected AG129 mice were adoptively transferred into al-UV-DENV2-primed AG129 mice one day prior to DENV2 challenge. As a control, one group of al-UV-DENV2-primed mice did not receive CD8+ T cells. Two groups of non-primed mice (with and without transferred CD8+ T cells) were included. When no CD8+ T cells were transferred, viral RNA levels in the liver 3 days after challenge were higher in al-UV-DENV2-primed animals relative to non-primed animals (figure. 5C). Viral RNA was undetectable in non-primed animals in which CD8+ T cells had been transferred. Similarly, no viral RNA was detected in al-UV-DENV2-primed animals that had received CD8+ T cells prior to challenge.

These results demonstrate that transfer of DENV2-primed CD8+ T cells prior to challenge with DENV2 dramatically reduced viral load in both non-primed and al-UV-DENV2-primed mice. Similar results were obtained in an experiment where total T cells (negative selection of untouched total T cells) from DENV2-infected animals were transferred between al-UV-DENV2 priming and DENV2 challenge (data not shown).

Discussion

In this study, we investigated the effect of priming with alum adjuvanted UV-inactivated DENV2 (al-UV-DENV2) on subsequent infection with DENV2 in the absence or presence of exogenous DENV2-primed CD8+ T cells. Our findings are summarized in figure 6. Priming of type I and II IFN-receptor deficient AG129 mice with al-UV-DENV2 reduced survival time and increased liver viral load upon subsequent infection with DENV2. Priming with non-replicating al-UV-DENV2 induced non-neutralizing DENV2-specific IgG, which in turn mediated Ab-dependent enhancement of dengue disease (ADE) upon challenge with (replicating) DENV2 (figure 6). Our study demonstrates for the first time that priming with an inactivated virus can elicit an Ab response that can induce Ab-mediated dengue disease enhancement upon challenge. While ADE following passive transfer of immune serum or monoclonal antibodies has been widely demonstrated in animal models (15, 17, 18, 22, 30, 31) our study describes a direct phenomenon in which priming with an inactivated virus results in ADE upon challenge of the same animal, not via serum transfer.

Figure 6.

Figure 6

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Model summarizing the effect of al-UV-DENV2 priming on subsequent DENV2 infection with or without transfer of exogenous DENV2-primed CD8+ T cells.

In contrast to infection with replicating DENV2, priming with al-UV-DENV2 did not induce measurable CD8+ T cell responses. Transfer of DENV2-primed CD8+ T cells into al-UV-DENV2-primed recipients prevented antibody-dependent enhancement of disease and reduced viral load upon DENV2 challenge (figure 6). Our results suggest that in the presence of enhancing amounts of antibodies, efficient CD8+ T cell responses limit disease severity and antibody-dependent enhancement.

A caveat of our study is the use of mice deficient in type I and II IFN-receptor (AG129). As IFN is a key player of the immune system, we would suggest great caution when extrapolating our results to dengue disease in humans. However, we chose to use AG129 mice because DENV replicates to measurable levels and causes a dengue-like disease in these mice (32, 33). This allows for assessment of the effect of al-UV-DENV2 priming on a subsequent DENV infection. The experiments described here would not be feasible in WT mice, in which DENV does not replicate. The lack of replication in WT mice may be explained by the fact that DENV inhibits IFN signaling pathways to establish infection in humans, but is unable to do so in mice (34–37). While using IFN-receptor deficient mice is not ideal, AG129 mice have been increasingly used as a model to study DENV-induced pathology and immunity (17, 18, 22, 24, 30, 31, 38–40). Use of these mice permits us to measure dengue virus infection outcomes that may be relevant to humans.

The rapid priming schedule chosen in our study was not meant to emulate vaccination schedules used in clinical settings, which aim at obtaining the best possible protective immunity via optimal affinity maturation of the antibody response. In contrast, in our study, the priming regimen was chosen specifically to induce a non-neutralizing DENV-specific antibody response, which has the potential to induce antibody-dependent enhancement of disease. As this priming schedule did not induce robust endogenous CD8+ T cell responses, we were able to transfer exogenous DENV-primed CD8+ T cells and investigate their impact on the outcome of antibody-induced severe dengue disease. In humans, ADE can be observed when patients are sequentially infected with two different DENV serotypes (heterotypic secondary infections) (1, 7). However, in our study, priming with al-UV-DENV resulted in ADE upon challenge with the same serotype (homotypic challenge). The antibody response induced by priming did not reach the threshold required for neutralization. Consequently, the sub-neutralizing character of the response induced by priming was sufficient to induce ADE upon challenge, regardless of the sero-specificity. This is consistent with experimental evidence demonstrating that even neutralizing antibodies can induce ADE at lower concentrations (41, 42), both in vitro (13) and in vivo (18).

The low CD8+ T cell responses observed after priming with al-UV-DENV2 could be explained because the antigen was given in alum, which is known to skew the immune response towards humoral rather than cellular immunity (25, 26). Additionally, the priming virus was UV-inactivated and, consequently, unable to replicate. Therefore, priming did not result in production of non-structural viral proteins, which are the major CD8+ T cell targets during natural DENV infection (19, 28, 29). Furthermore, a non-replicating antigen is less likely to induce robust T cell responses compared to a replicating virus (27). Transfer of T cells from mice infected with replicating DENV2 into al-UV-DENV2-primed mice prior to viral challenge dramatically reduced viral RNA levels upon infection.

Keeping in mind the limitations of our model and the pitfalls associated with extrapolating data from animal models to human disease, we would like to speculate on some parallels between our results and what has been observed in humans. In our study, ADE seemed to be the result of the combination of sub-protective, non-neutralizing anti-DENV Abs and nonexistent anti-DENV CD8+ T cell responses. In humans, while the presence of anti-DENV Abs from a previous heterotypic infection (or from an immune mother) is a major risk factor for developing severe dengue disease, not all patients with pre-existing anti-DENV Abs develop severe disease upon heterotypic re-infection (1). In fact, only a small percentage of secondary DENV infections result in shock (1). We would like to suggest that, perhaps, the risk of developing severe dengue disease is higher in patients that have both sub-protective levels of Abs and inefficient (or nonexistent) T cell responses.

Our study shows that the absence of a robust CD8+ T cell response negatively influences disease outcome in the presence of sub-neutralizing antibodies, but that does not exclude the contribution of other factors to disease outcome. Many other host and viral factors influence disease outcome, including the quality of the antibody response, the interval between infections, differences in strain virulence, and overall immune status of the host. Therefore, while many factors can influence disease outcome, our data suggest that in the presence of antigen-induced sub-neutralizing levels of antibodies, an inefficient (or absent) CD8+ T cell response may be detrimental to the host.

In patients, the protective efficacy of T cell responses may be influenced by several factors including the HLA-phenotype. During secondary DENV infections, some alleles were associated with disease susceptibility, and others with protection (43, 44). Different HLA alleles were also associated with differential magnitude of cellular anti-DENV responses in humans (29). Furthermore, HLA alleles associated with increased risk of severe dengue disease (45, 46) were associated with weaker CD8+ T cell responses, whereas alleles associated with decreased risk of severe disease were associated with higher and polyfunctional cellular responses (29). These findings support the hypothesis that during secondary infections, disease outcome may be influenced by both the serological status of the host and the magnitude and/or quality of cellular immune responses.

In the case of infants born to DENV-immune mothers, DENV infection is more likely to evolve into severe dengue disease between 5 and 12 months of age, a time window during which passively transferred maternal DENV-specific Abs have dropped below protective levels, but have not yet disappeared completely (1, 9, 47–51). As those infants have never been exposed to DENV, they do not have DENV-specific memory T cells. Therefore, the increased susceptibility to severe dengue disease is observed in a situation where exogenous sub-protective levels of DENV-specific Abs (from the mother) are already present at the time of infection, whereas endogenous anti-DENV CD8+ T cell responses only start to be primed at the time of infection. Perhaps Abs initiate enhancement of infection before endogenous CD8+ T cell responses begin to limit viral spread. In addition, endogenous anti-DENV cellular responses may be weak in infants as their immune system is not yet fully functional (52).

Based on these observations and on our results, we hypothesize that in the presence of sub-protective humoral immunity, inefficient (or absent) CD8+ T cell responses may be an additional risk factor for developing severe dengue disease, whereas robust cellular responses may prevent Ab-mediated severe dengue disease. Our data may contribute to explain why infection in the presence of sub-protective levels of anti-DENV antibodies does not always result in severe dengue disease.

Supplementary Material

1

Acknowledgments

Funding

This project was supported by NIH grants U54AI057517 from the Southeastern Regional Center of Excellence for Emerging Infectious and Biodefense (SS), R56 A1085063 (SS), U01 AI082185 (SS), and NIH Contract HHSN272200900042C to Alessandro Sette and a LJI Center for Infectious Disease Research Fellowship to RZ.

Abbreviations

DENV

dengue virus

ADE

antibody-dependent enhancement

Abs

antibodies

al-UV-DENV2

alum adjuvanted UV-inactivated dengue virus serotype 2

References

  • 1.Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Archives of virology. 2013;158:1445–1459. doi: 10.1007/s00705-013-1645-3. [DOI] [PubMed] [Google Scholar]
  • 2.Carod-Artal FJ, Wichmann O, Farrar J, Gascon J. Neurological complications of dengue virus infection. Lancet neurology. 2013;12:906–919. doi: 10.1016/S1474-4422(13)70150-9. [DOI] [PubMed] [Google Scholar]
  • 3.Halstead SB. Dengue. Lancet. 2007;370:1644–1652. doi: 10.1016/S0140-6736(07)61687-0. [DOI] [PubMed] [Google Scholar]
  • 4.World Health Organization. Dengue guidelines for diagnosis, treatment, prevention and control. WHO Press; Geneva: 2009. New edition. [PubMed] [Google Scholar]
  • 5.Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI. The global distribution and burden of dengue. Nature. 2013 doi: 10.1038/nature12060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Beatty ME, Stone A, Fitzsimons DW, Hanna JN, Lam SK, Vong S, Guzman MG, Mendez-Galvan JF, Halstead SB, Letson GW, Kuritsky J, Mahoney R, Margolis HS, Asia P Americas Dengue Prevention Boards Surveillance Working G. Best practices in dengue surveillance: a report from the Asia-Pacific and Americas Dengue Prevention Boards. PLoS neglected tropical diseases. 2010;4:e890. doi: 10.1371/journal.pntd.0000890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Halstead SB. Immune enhancement of viral infection. Progress in allergy. 1982;31:301–364. [PubMed] [Google Scholar]
  • 8.Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses. Advances in virus research. 2003;60:421–467. doi: 10.1016/s0065-3527(03)60011-4. [DOI] [PubMed] [Google Scholar]
  • 9.Halstead SB, Nimmannitya S, Cohen SN. Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. The Yale journal of biology and medicine. 1970;42:311–328. [PMC free article] [PubMed] [Google Scholar]
  • 10.Kliks SC, Nisalak A, Brandt WE, Wahl L, Burke DS. Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. The American journal of tropical medicine and hygiene. 1989;40:444–451. doi: 10.4269/ajtmh.1989.40.444. [DOI] [PubMed] [Google Scholar]
  • 11.Boonnak K, Slike BM, Burgess TH, Mason RM, Wu SJ, Sun P, Porter K, Rudiman IF, Yuwono D, Puthavathana P, Marovich MA. Role of dendritic cells in antibody-dependent enhancement of dengue virus infection. Journal of virology. 2008;82:3939–3951. doi: 10.1128/JVI.02484-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kou Z, Quinn M, Chen H, Rodrigo WW, Rose RC, Schlesinger JJ, Jin X. Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. Journal of medical virology. 2008;80:134–146. doi: 10.1002/jmv.21051. [DOI] [PubMed] [Google Scholar]
  • 13.Mehlhop E, Ansarah-Sobrinho C, Johnson S, Engle M, Fremont DH, Pierson TC, Diamond MS. Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner. Cell host & microbe. 2007;2:417–426. doi: 10.1016/j.chom.2007.09.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Pierson TC, Xu Q, Nelson S, Oliphant T, Nybakken GE, Fremont DH, Diamond MS. The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell host & microbe. 2007;1:135–145. doi: 10.1016/j.chom.2007.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Goncalvez AP, Engle RE, St Claire M, Purcell RH, Lai CJ. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:9422–9427. doi: 10.1073/pnas.0703498104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Halstead SB. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. The Journal of infectious diseases. 1979;140:527–533. doi: 10.1093/infdis/140.4.527. [DOI] [PubMed] [Google Scholar]
  • 17.Balsitis SJ, Williams KL, Lachica R, Flores D, Kyle JL, Mehlhop E, Johnson S, Diamond MS, Beatty PR, Harris E. Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. PLoS pathogens. 2010;6:e1000790. doi: 10.1371/journal.ppat.1000790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell host & microbe. 2010;7:128–139. doi: 10.1016/j.chom.2010.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yauch LE, Zellweger RM, Kotturi MF, Qutubuddin A, Sidney J, Peters B, Prestwood TR, Sette A, Shresta S. A protective role for dengue virus-specific CD8+ T cells. Journal of immunology. 2009;182:4865–4873. doi: 10.4049/jimmunol.0801974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Prestwood TR, Prigozhin DM, Sharar KL, Zellweger RM, Shresta S. A mouse-passaged dengue virus strain with reduced affinity for heparan sulfate causes severe disease in mice by establishing increased systemic viral loads. Journal of virology. 2008;82:8411–8421. doi: 10.1128/JVI.00611-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Perry ST, Buck MD, Plummer EM, Penmasta RA, Batra H, Stavale EJ, Warfield KL, Dwek RA, Butters TD, Alonzi DS, Lada SM, King K, Klose B, Ramstedt U, Shresta S. An iminosugar with potent inhibition of dengue virus infection in vivo. Antiviral research. 2013;98:35–43. doi: 10.1016/j.antiviral.2013.01.004. [DOI] [PubMed] [Google Scholar]
  • 22.Zellweger RM, Miller R, Eddy WE, White LJ, Johnston RE, Shresta S. Role of humoral versus cellular responses induced by a protective dengue vaccine candidate. PLoS pathogens. 2013;9:e1003723. doi: 10.1371/journal.ppat.1003723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yauch LE, Shresta S. Mouse models of dengue virus infection and disease. Antiviral research. 2008;80:87–93. doi: 10.1016/j.antiviral.2008.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zellweger RM, Shresta S. Mouse models to study dengue virus immunology and pathogenesis. Frontiers in immunology. 2014;5:151. doi: 10.3389/fimmu.2014.00151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunology and cell biology. 2004;82:488–496. doi: 10.1111/j.0818-9641.2004.01272.x. [DOI] [PubMed] [Google Scholar]
  • 26.Slifka MK, Amanna I. How advances in immunology provide insight into improving vaccine efficacy. Vaccine. 2014 doi: 10.1016/j.vaccine.2014.03.078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Salek-Ardakani S, Flynn R, Arens R, Yagita H, Smith GL, Borst J, Schoenberger SP, Croft M. The TNFR family members OX40 and CD27 link viral virulence to protective T cell vaccines in mice. The Journal of clinical investigation. 2011;121:296–307. doi: 10.1172/JCI42056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Mathew A, Rothman AL. Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunological reviews. 2008;225:300–313. doi: 10.1111/j.1600-065X.2008.00678.x. [DOI] [PubMed] [Google Scholar]
  • 29.Weiskopf D, Angelo MA, de Azeredo EL, Sidney J, Greenbaum JA, Fernando AN, Broadwater A, Kolla RV, De Silva AD, de Silva AM, Mattia KA, Doranz BJ, Grey HM, Shresta S, Peters B, Sette A. Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:E2046–2053. doi: 10.1073/pnas.1305227110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ng JK, Zhang SL, Tan HC, Yan B, Maria Martinez Gomez J, Tan WY, Lam JH, Tan GK, Ooi EE, Alonso S. First Experimental In Vivo Model of Enhanced Dengue Disease Severity through Maternally Acquired Heterotypic Dengue Antibodies. PLoS pathogens. 2014;10:e1004031. doi: 10.1371/journal.ppat.1004031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Williams KL, Sukupolvi-Petty S, Beltramello M, Johnson S, Sallusto F, Lanzavecchia A, Diamond MS, Harris E. Therapeutic Efficacy of Antibodies Lacking FcgammaR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies. PLoS pathogens. 2013;9:e1003157. doi: 10.1371/journal.ppat.1003157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Johnson AJ, Roehrig JT. New mouse model for dengue virus vaccine testing. Journal of virology. 1999;73:783–786. doi: 10.1128/jvi.73.1.783-786.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Shresta S, Sharar KL, Prigozhin DM, Beatty PR, Harris E. Murine model for dengue virus-induced lethal disease with increased vascular permeability. Journal of virology. 2006;80:10208–10217. doi: 10.1128/JVI.00062-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Aguirre S, Maestre AM, Pagni S, Patel JR, Savage T, Gutman D, Maringer K, Bernal-Rubio D, Shabman RS, Simon V, Rodriguez-Madoz JR, Mulder LC, Barber GN, Fernandez-Sesma A. DENV inhibits type I IFN production in infected cells by cleaving human STING. PLoS pathogens. 2012;8:e1002934. doi: 10.1371/journal.ppat.1002934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ashour J, Morrison J, Laurent-Rolle M, Belicha-Villanueva A, Plumlee CR, Bernal-Rubio D, Williams KL, Harris E, Fernandez-Sesma A, Schindler C, Garcia-Sastre A. Mouse STAT2 restricts early dengue virus replication. Cell host & microbe. 2010;8:410–421. doi: 10.1016/j.chom.2010.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Yu CY, Chang TH, Liang JJ, Chiang RL, Lee YL, Liao CL, Lin YL. Dengue virus targets the adaptor protein MITA to subvert host innate immunity. PLoS pathogens. 2012;8:e1002780. doi: 10.1371/journal.ppat.1002780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Morrison J, Laurent-Rolle M, Maestre AM, Rajsbaum R, Pisanelli G, Simon V, Mulder LC, Fernandez-Sesma A, Garcia-Sastre A. Dengue virus co-opts UBR4 to degrade STAT2 and antagonize type I interferon signaling. PLoS pathogens. 2013;9:e1003265. doi: 10.1371/journal.ppat.1003265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Weiskopf D, Yauch LE, Angelo MA, John DV, Greenbaum JA, Sidney J, Kolla RV, De Silva AD, de Silva AM, Grey H, Peters B, Shresta S, Sette A. Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design. Journal of immunology. 2011;187:4268–4279. doi: 10.4049/jimmunol.1101970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Tan GK, Ng JK, Trasti SL, Schul W, Yip G, Alonso S. A non mouse-adapted dengue virus strain as a new model of severe dengue infection in AG129 mice. PLoS neglected tropical diseases. 2010;4:e672. doi: 10.1371/journal.pntd.0000672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Schul W, Liu W, Xu HY, Flamand M, Vasudevan SG. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. The Journal of infectious diseases. 2007;195:665–674. doi: 10.1086/511310. [DOI] [PubMed] [Google Scholar]
  • 41.Dowd KA, Pierson TC. Antibody-mediated neutralization of flaviviruses: a reductionist view. Virology. 2011;411:306–315. doi: 10.1016/j.virol.2010.12.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Pierson TC, Fremont DH, Kuhn RJ, Diamond MS. Structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development. Cell host & microbe. 2008;4:229–238. doi: 10.1016/j.chom.2008.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Appanna R, Ponnampalavanar S, Lum Chai See L, Sekaran SD. Susceptible and protective HLA class 1 alleles against dengue fever and dengue hemorrhagic fever patients in a Malaysian population. PloS one. 2010:5. doi: 10.1371/journal.pone.0013029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Stephens HA, Klaythong R, Sirikong M, Vaughn DW, Green S, Kalayanarooj S, Endy TP, Libraty DH, Nisalak A, Innis BL, Rothman AL, Ennis FA, Chandanayingyong D. HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais. Tissue antigens. 2002;60:309–318. doi: 10.1034/j.1399-0039.2002.600405.x. [DOI] [PubMed] [Google Scholar]
  • 45.Loke H, Bethell DB, Phuong CX, Dung M, Schneider J, White NJ, Day NP, Farrar J, Hill AV. Strong HLA class I--restricted T cell responses in dengue hemorrhagic fever: a double-edged sword? The Journal of infectious diseases. 2001;184:1369–1373. doi: 10.1086/324320. [DOI] [PubMed] [Google Scholar]
  • 46.Nguyen TP, Kikuchi M, Vu TQ, Do QH, Tran TT, Vo DT, Ha MT, Vo VT, Cao TP, Tran VD, Oyama T, Morita K, Yasunami M, Hirayama K. Protective and enhancing HLA alleles, HLA-DRB1*0901 and HLA-A*24, for severe forms of dengue virus infection, dengue hemorrhagic fever and dengue shock syndrome. PLoS neglected tropical diseases. 2008;2:e304. doi: 10.1371/journal.pntd.0000304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Chau TN, Hieu NT, Anders KL, Wolbers M, Lien le B, Hieu LT, Hien TT, Hung NT, Farrar J, Whitehead S, Simmons CP. Dengue virus infections and maternal antibody decay in a prospective birth cohort study of Vietnamese infants. The Journal of infectious diseases. 2009;200:1893–1900. doi: 10.1086/648407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Chau TN, Quyen NT, Thuy TT, Tuan NM, Hoang DM, Dung NT, Lien le B, Quy NT, Hieu NT, Hieu LT, Hien TT, Hung NT, Farrar J, Simmons CP. Dengue in Vietnamese infants--results of infection- enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity. The Journal of infectious diseases. 2008;198:516–524. doi: 10.1086/590117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Halstead SB, Scanlon JE, Umpaivit P, Udomsakdi S. Dengue and chikungunya virus infection in man in Thailand, 1962–1964. IV. Epidemiologic studies in the Bangkok metropolitan area. The American journal of tropical medicine and hygiene. 1969;18:997–1021. doi: 10.4269/ajtmh.1969.18.997. [DOI] [PubMed] [Google Scholar]
  • 50.Kliks SC, Nimmanitya S, Nisalak A, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. The American journal of tropical medicine and hygiene. 1988;38:411–419. doi: 10.4269/ajtmh.1988.38.411. [DOI] [PubMed] [Google Scholar]
  • 51.Simmons CP, Chau TN, Thuy TT, Tuan NM, Hoang DM, Thien NT, Lien le B, Quy NT, Hieu NT, Hien TT, McElnea C, Young P, Whitehead S, Hung NT, Farrar J. Maternal antibody and viral factors in the pathogenesis of dengue virus in infants. The Journal of infectious diseases. 2007;196:416–424. doi: 10.1086/519170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.PrabhuDas M, Adkins B, Gans H, King C, Levy O, Ramilo O, Siegrist CA. Challenges in infant immunity: implications for responses to infection and vaccines. Nature immunology. 2011;12:189–194. doi: 10.1038/ni0311-189. [DOI] [PubMed] [Google Scholar]

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