CD8+ T-cell Responses in Flavivirus-Naive Individuals Following Immunization with a Live-Attenuated Tetravalent Dengue Vaccine Candidate
+ CD8 T-cell Responses in Flavivirus-Naive Individuals Following Immunization with a Live-Attenuated Tetravalent Dengue Vaccine Candidate
Haiyan Chu 3 4
Sarah L. George 0 2 3
Dan T. Stinchcomb 3 4
Jorge E. Osorio 3 4
Charalambos D. Partidos 3 4
0 St. Louis Veterans Administration Medical Center , Missouri
1 182; Pan American Dengue Network Meeting , Belem, Brazil, 19-22 October 2014. Pharmaceuticals, 504 S. Rosa Rd, Ste 200, Madison, WI 53719
2 Department of Internal Medicine, Division of Infectious Diseases , Allergy and Immunology , Saint Louis University School of Medicine
3 Received 1 December 2014; accepted 21 April 2015; electronically published 5 May 2015. Presented in part: 63rd American Society of Tropical Medicine and Hygiene Annual Meeting , New Orleans, Louisiana, 2-6 November 2014. Abstract
4 Takeda Vaccines, Inc , Deerfield, Illinois
We are developing a live-attenuated tetravalent dengue vaccine (TDV) candidate based on an attenuated dengue 2 virus (TDV-2) and 3 chimeric viruses containing the premembrane and envelope genes of dengue viruses (DENVs) -1, -3, and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). In this study, we analyzed and characterized the CD8+ T-cell response in flavivirusnaive human volunteers vaccinated with 2 doses of TDV 90 days apart via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that TDV elicits CD8+ T cells targeting the nonstructural NS1, NS3, and NS5 proteins of TDV-2. The cells were characterized by the production of interferon-γ, tumor necrosis factor-α, and to a lesser extent interleukin-2. Responses were highest on day 90 after the first dose and were still detectable on 180 days after the second dose. In addition, CD8+ T cells were multifunctional, producing ≥2 cytokines simultaneously, and cross-reactive to NS proteins of the other 3 DENV serotypes. Overall, these findings describe the capacity of our candidate dengue vaccine to elicit cellular immune responses and support the further evaluation of T-cell responses in samples from future TDV clinical trials.
Dengue viruses (DENVs) transmitted primarily by the
Aedes aegypti mosquito cause infections that impact
public health mainly in tropical and subtropical regions of the
world [1, 2]. Recently, it was estimated that these viruses
cause approximately 390 million DENV infections
annually . DENVs circulate in nature as 4 distinct serotypes
(DENV-1 to DENV-4) that share a high degree of
homology with each other . Each serotype causes a spectrum
of diseases, including subclinical infection, dengue fever
(DF), and life-threatening dengue hemorrhagic fever or
dengue shock syndrome . Moreover, there appears to
be a distinct clinical and epidemiological pattern for each
serotype, suggesting that they exhibit variability in
virulence and pathogenesis .
Currently, there is no dengue vaccine or antiviral
therapy for DENV. Generally, infection with 1 dengue
serotype will confer homologous, long-term protection
. However, subsequent reinfection with a
heterologous serotype has the potential to cause severe disease,
which could be mediated by antibodies
(antibodydependent enhancement) and/or T cells [7–9].
Therefore, vaccine development against DENV has focused
on tetravalent formulations that simultaneously provide
immunity to all 4 serotypes . We have developed a
live-attenuated tetravalent dengue vaccine (TDV)
candidate that consists of an attenuated DENV-2 strain
(TDV-2), and 3 chimeric viruses containing the
premembrane ( prM) and envelope (E) genes of DENV-1,
-3, and -4 expressed in the context of the TDV-2
genome (TDV-1, TDV-3, TDV-4, respectively) [11–15]. TDV
(under the previous name DENVax) has been extensively tested
in preclinical studies [16–18], 2 completed Phase I clinical trials
[19, 20], and is currently being tested in Phase II clinical trials. In
the Phase I studies with healthy adult volunteers, the candidate
vaccine was shown to be generally well tolerated, and induced
neutralizing antibody responses to all 4 dengue serotypes
The humoral immune response to DENV primarily targets
the prM and E structural proteins and is predominately
composed of serotype-cross-reactive antibodies [21–23]. In contrast,
the cellular immune response to DENV mainly targets the
nonstructural (NS) proteins . DENV vaccine candidates have
been shown to elicit T-cell responses [17, 18, 25, 26], and in a
mouse model the protective role of CD8+ T cells is well
established . More recently, a comprehensive analysis of
DENVspecific T-cell responses provided evidence suggesting that a
vigorous multifunctional CD8+ T-cell response is associated
with protection from DENV disease .
In this study, we performed an analysis of the kinetics of
CD8+ T-cell responses to the backbone of the TDV candidate
vaccine in flavivirus-naive individuals that received 2 doses of
the vaccine by intradermal (ID) or subcutaneous (SC)
administration. Moreover, these responses were characterized in terms
of cytokine profile produced, their multifunctional nature, and
the targeted NS proteins they recognized.
MATERIALS AND METHODS
The construction and characterization of each TDV vaccine
strain has been previously reported . The clinical material
used for vaccination in this Phase I trial consisted of 2 × 104
plaque-forming units ( pfu) of TDV-1, 5 × 104 pfu of TDV-2,
1 × 105 pfu of TDV-3, and 3 × 105 pfu of TDV-4.
Ethical approval of the study protocol was granted by the Saint
Louis University Institutional Review Board prior to initiation
of a National Institutes of Health (NIH)–sponsored Phase I
clinical trial at the university. Informed written consent was
obtained from all study participants, and the study was registered
with clinicaltrials.gov, identifier: NCT01110551.
The development of DENV-specific CD8+ T-cell responses was
measured in peripheral blood mononuclear cells (PBMCs) of 6
individuals randomly selected from 2 cohorts of
flavivirusnaive, healthy adults. Five individuals per cohort were
vaccinated with TDV via either the SC or ID route; 1 individual from
each cohort received placebo and was included in this study
as a control. In both cohorts, TDV or placebo was administered
on days 0 and 90. The kinetics of T-cell responses were
measured in PBMCs collected on days 0, 14, 90, 104, and 270
postprimary vaccination via the SC or ID routes. Standardized
assays to measure vaccine virus RNA in serum samples
postvaccination were performed as described by Osorio et al , and
data from individuals who participated in this study as reported
 are presented in Supplementary Table 1.
Blood from each subject was collected in sodium heparin cell
preparation tubes at various time points pre- and postvaccination
(see above), and PBMCs were separated by centrifugation at room
temperature (18°C–25°C) in a horizontal rotor at 1800g (relative
centrifugal force) for 25 minutes. Cells were washed twice with
phosphate buffer saline (PBS), and cell viability was monitored
by trypan blue exclusion test. The cell pellet was resuspended in
fetal bovine serum (FBS) at a concentration of 1 × 107 cells/mL
and then was mixed with an equal volume of freezing media
(20% dimethyl sulfoxide [DMSO] (volume/volume) in
heat-inactivated FBS) to yield a final concentration of 5 × 106 cells/mL.
After gently mixing, 1 mL aliquots of cell suspension in prelabeled
Nunc Cryo Tubes were placed into a room temperature
isopropanol-filled cell freezer. The apparatus was stored at −70°C
overnight, and the next day vials were transferred into liquid
nitrogen for long-term storage.
Peptide arrays representing the entire sequence of the NS1 (aa
1–352; 47 peptides), the NS3 (aa 1–618; 83 peptides), and the
NS5 ( pool 1; 80 peptides spanning residues 1–469, and pool
2; 76 peptides spanning residues 458–900) proteins from the
DENV-2 New Guinea C (NGC) strain were used to measure
T-cell responses to the DENV-2 backbone of TDV.
Cross-reactive T-cell responses were measured using peptide arrays
representing the entire sequence of NS1, NS3, and NS5 proteins from
DENV-1 (strain; Singapore/S275/1990), DENV-3 (strain;
Philippines/H87/1956), and DENV-4 (strain; Singapore/8976/
1995). All peptide arrays used in this study were obtained
from the National Institute of Allergy and Infectious Diseases
Biodefense and Emerging Infections Research Resources
Repository (BEI Resources). The peptide length ranged from 12–20
mers, and the amino acid overlap ranged from 10–13 amino
acids and varied between peptide arrays. Detailed information
about each array is available at http://www.beiresources.org/.
Intracellular Cytokine Staining Assay
Frozen PBMCs were thawed and washed twice with warm complete
Roswell Park Memorial Institute (cRPMI)–10 (RPMI 1640, 10%
FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL
streptomycin). Cells were resuspended at 1–4 × 106/mL in cRPMI-10 and
rested for 6–7 hours. Cells were then washed once and aliquoted for
stimulation with peptide pools at 10 µM of final total peptide
concentration. Staphylococcal enterotoxin B (SEB) (3 µg/mL) was
used as positive control. Anti-CD28 (L293)/CD49d (L25) (1 µg/
mL each) and protein transport inhibitors GolgiPlug (1 µg/mL)
and GolgiStop (2 µM) were added at the beginning of peptide
stimulation. After 16 hours’ stimulation, cells were surface-stained with
Live/dead Fixable Blue dye (Life Technologies), followed with
antihuman antibodies CD3 PerCP (SK7), CD4 PE-CF-594 (RPA-T4),
and CD8 V500 (RPA-T8). Cells were then fixed and permeabilized
before staining for intracellular cytokines interferon (IFN)–γ Alexa
Fluor 700 (B27), interleukin (IL)–2 allophycocyanin (5344.111),
and tumor necrosis factor (TNF)–α fluorescein isothiocyanate
(6401.111). Stained cells were resuspended in 1%
paraformaldehyde/PBS and acquired on a 5-laser BD LSRII flow cytometer
within 24 hours. Compensation was performed using antimouse
IgG BD CompBeads according to the manufacturer’s instruction.
For the majority of tested samples, at least 400 000 total events
were collected and data were analyzed mainly with FlowJo V9.7.2.
The viability of lymphocytes was >85% for all samples. Boolean
gating was used to determine simultaneous cytokine production in
cells. SEB-stimulated samples produced all 3 cytokines. Data are
presented as the percentage of cytokine-producing CD8+ T cells
after subtracting the background cytokine level in DMSO/medium
control cultures from stimulated cells at all indicated time points.
Multifunctional T-cell data were presented using SPICE v5.3033
(National Institute of Allergy and Infectious Diseases, NIH).
Kinetics and Characterization of Memory CD8+ T-cell
Responses to the TDV Backbone
Because the backbone of our vaccine candidate is based on an
attenuated DENV-2 strain, we predominantly focused our analyses
on the NS proteins that constitute the main target of the CD8+
T-cell response . To measure the magnitude of CD8+ T-cell
responses over time, we employed peptide arrays spanning the NS1,
NS3, and NS5 proteins of DENV-2, and an intracellular cytokine
staining (ICS) assay to analyze PBMCs collected from
flavivirusnaive individuals immunized with TDV. A representative gating
for CD8+ T cells from NS3 peptide array or control
DMSO-stimulated cells (subject 5, d270) is shown in Figure 1. Following
vaccination via the SC or ID routes, an increased number of CD8+ T
cells relative to day 0 were detected on days 90, 104, and 270
postprimary immunization targeting the NS1, NS3, and NS5 proteins
of DENV-2 (Figures 2 and 3). In the majority of individuals tested,
the NS3 protein was the main target of the CD8+ T-cell response
followed by the NS5 protein represented by pool 1. CD8+ T cells
mainly produced IFN-γ (Figure 2), and to a lesser extent TNF-α
(Figure 3). Overall, percentages of cells producing IL-2 were low
(Figure 4). CD8+ T-cell responses peaked on day 90 but were still
detectable on day 270. A booster injection on day 90 had no
detectable impact on the cellular responses measured on day 104, and
none of the vaccinated individuals had detectable viremia after
boosting (data not shown). The overall CD8+ T-cell response of
subjects from both SC and ID cohorts is summarized in Table 1.
Using the same peptide arrays, CD4+ T-cell responses to TDV
backbone were inconsistent with very low magnitude of response
(ranging from 0.051%–0.14%) for some individuals (data not
shown). Just as all vaccinated individuals showed a positive
response to 1 or more peptides, all individuals vaccinated with
TDV had detectable neutralizing antibody response on day 90
(20; Supplementary Table 2). Seven of 10 subjects had detectable
viral RNA from TDV-2 (days 7–14). Among these 7 individuals,
2 also had detectable viral RNA from TDV-3 on day 9
(Supplementary Table 1). In general, there were no strict associations between
viral RNA, the magnitude of the antibody response, and the CD8+
TDV Elicits Multifunctional CD8+ T Cells
To determine the breadth of CD8+ T-cell responses, we looked
for production of specific combinations of cytokines 3 months
postprimary immunization or 14 and 180 days postboost (days
104 and 270, respectively). Figures 5A and 5B show percentages
of CD8+ T cells producing a particular combination of
cytokines for the indicated test days by both routes of
immunization. The most common CD8+ T-cell phenotypes were
IFN-γ+ or IFN-γ+/TNF-α+ at all time points postvaccination.
Pie charts illustrate the distribution of T-cell populations that
produce any given combination of cytokines at on days 90,
104, and 270 postprimary immunization.
TDV Elicits Cross-reactive CD8+ T Cells Targeting the NS3
Protein of Heterologous DENVs
To determine whether the TDV-2 backbone elicits
cross-reactive T-cell responses to the NS proteins of the other 3 DENV
serotypes, we analyzed PBMCs from an individual from the
SC cohort (subject 5). This subject gave higher IFN-γ and
TNF-α response to NS3 and NS5 protein represented by pool
1 (NS5-1) at d90, d104, and d270 as compared to the other
subjects (Figure 2 and 3). Cells from days 90 and 104 were
stimulated with peptide arrays encompassing the sequences of NS1,
NS3, and NS5 ( pools 1 and 2) from DENV-1, DENV-3, or
DENV-4. Our data indicate that TDV-2 elicits cross-reactive
CD8+ IFN-γ- or TNF-α-producing T cells recognizing
predominantly the NS3 protein of all 3 DENV serotypes (Figure 6A)
and were predominantly multifunctional (IFN-γ+/TNF-α+)
Total Response NS1 NS3 NS5-1
In this study, we sought to measure and characterize the cellular
responses to TDV in a subset of healthy flavivirus-naive
individuals that participated in a Phase I clinical trial. The first
key finding of our study was the demonstration that TDV elicits
CD8+ T-cell responses characterized mainly by the production
of IFN-γ and TNF-α cytokines. The CD8+ T-cell response was
evident 3 months postprimary vaccination, was not affected by
a booster vaccination, and was detectable 6 months after the last
immunization. Generally, following DENV infection, T-cell
response begins to develop when viremia levels start declining by
day 5 or 6 postinfection. Sustained dengue-specific IFN-γ
response correlates with protection against viremia in a human
challenge study . In the Phase I study, 79% of subjects
that received the higher dosage form of TDV had detectable
TDV viral RNA  and, of the subjects selected for this
study, 7 of 10 had detectable viral RNA to TDV-2 that started
on day 7 after prime and lasted up to day 14 postvaccination.
There were no PBMC collections between days 14 and 90 (
postprimary immunization) to precisely identify the timing of the
onset of CD8+ T-cell response to TDV. Due to the attenuated
nature of the vaccine viruses that constitute TDV, the CD8+
T-cell response may occur much later than in the case of a
wild-type DENV infection. This is supported by our previous
observations in cynomolgus macaques where T-cell responses
to TDV were detectable much later following peak viremia
. A second key observation from our study was that the
booster vaccination given on day 90 did not impact the
magnitude of CD8+ T-cell responses. In our preclinical [17, 18] and
Phase I clinical studies [19, 20], we have consistently observed
that there is limited vaccine virus viremia after the boost
when given 2 to 3 months after a primary immunization.
This suggests that due to the short window between primary
and secondary immunizations, circulating neutralizing
antibodies are efficiently controlling vaccine virus replication, and
hence boosting at the T-cell level is not apparent. When we
analyzed PBMCs collected 180 days postbooster immunization, a
strong and predominantly multifunctional IFN-γ/TNF-α
memory CD8+ T-cell response was still detectable in most of the
individuals tested from the SC or ID cohorts. This response profile
may be beneficial since a rapid recall response may occur upon
DENV infection as compared to naive individuals.
CD8+ T cells are thought to play a key role in controlling viral
infections. They recognize viral epitopes in the context of
human leukocyte antigen class I molecules on the surface of
infected cells, and following activation they kill cells directly or via
secretion of cytokines such as IFN-γ and TNF-α. In our studies,
we employed peptide arrays encompassing the whole sequences
of NS1, NS3, and NS5 of DENV-2 to identify the target proteins
of the CD8+ T-cell response elicited by TDV. A key finding
from our analysis was the demonstration that the NS3 protein
was the main target of the CD8+ T-cell response followed by the
NS5-1. Although the number of individuals tested from each
cohort was small, our data suggest that the route of vaccination
does not impact the specificity of the CD8+ T-cell response in
terms of protein recognition. However, we cannot exclude the
possibility that injection route might influence epitope selection
due to differences in resident antigen-presenting cells. With
reference to the target proteins that TDV-specific CD8+ T cells
recognize, our findings are consistent with previous
observations highlighting that NS3 and NS5 proteins of DENVs are
among the main target proteins of the T-cell response
[24, 28–30]. Differences in processing of these 2 NS proteins
may account for preferential presentation of generated epitopes
and their immunodominance over T-cell epitopes from other
structural or NS proteins. However, our analysis was limited
due to the availability of cells to screen peptide arrays from
other NS proteins.
In our studies, we also provided evidence indicating that the
vaccine backbone elicited CD8+ T cells that predominantly
recognize the NS3 protein of DENV-1, -3, and -4 in 1 individual.
This might be correlated with a highly conserved region in the
NS3 protein of all 4 DENV serotypes containing motifs and
charged residues that are essential for helicase activity and
virus replication [31, 32]. More recently, an immunodominant
CD8+ T-cell epitope encompassing residues 538–547 in the
NS3 protein was shown to be conserved (100% identical)
among the 4 DENV serotypes . Therefore, one would predict
that responses to conserved epitopes would be seen in humans
. Further studies are needed to characterize the breadth of
cross-reactive T-cell responses to the backbone of the other
DENV serotypes in more TDV-vaccinated individuals. The use
of ICS assays permits us to evaluate simultaneously the presence
of different cytokines that can delineate different subsets of T
cells having multifunctional profiles. Multifunctional T-cells
responses have been associated with protection in studies with
various pathogens, including dengue [28, 30, 35, 36]. In this study,
we found that all individuals from both cohorts produced
TDV-specific multifunctional CD8+ T cells producing at least 2
cytokines. Although quantification of the magnitude and breadth
of vaccine-induced CD8+ T-cell recall responses provides
information regarding the immunogenicity of a given vaccine
candidate, it is difficult to know what their impact would be on
controlling viral replication. Several factors (such as host genetic
determinants, immune status of the individual, kinetics and
timing of antigen-specific immune responses, DENV serotype, etc.)
could have a major impact on the protective efficacy of CD8+ T
cells. Of particular interest would be to compare and contrast the
T-cell responses to TDV with those elicited by natural infection.
Overall, these findings highlight the immunogenic profile of the
candidate dengue vaccine, TDV, and support the further
evaluation of clinical samples from ongoing clinical trials.
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Financial support. This work was supported by the National Institute of
Allergy and Infectious Diseases (NIAID), contract no. HHHSN27201300021I3C.
Potential conflicts of interest. H. C., D. T. S., C. D. P., and J. E. O. are
affiliated with Takeda Vaccine, Inc. All other authors report no potential
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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