M A J O R A R T I C L E
Experimental Dengue Virus Challenge of
Human Subjects Previously Vaccinated With
Live Attenuated Tetravalent Dengue Vaccines
Wellington Sun,1,aKenneth H. Eckels,2,aJ. Robert Putnak,1,aArthur G. Lyons,1,aStephen J. Thomas,1,a
David W. Vaughn,1,aRobert V. Gibbons,1,aStefan Fernandez,1,aVicky J. Gunther,1,aMammen P. Mammen Jr,5,a
John D. Statler,3,aand Bruce L. Innis4,a
1Department of Virus Diseases,2Pilot Bioproduction Facility, Walter Reed Army Institute of Research, Silver Spring, Maryland;3Department of
Radiology, Walter Reed Army Medical Center, Washington, D. C.;4Vaccine Discovery and Development, GlaxoSmithKline, King of Prussia,
Pennsylvania; and5Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
(See the editorial commentary by Durbin and Whitehead on pages 697–9.)
Background.Protection against dengue requires immunity against all 4 serotypes of dengue virus (DENV).
Experimental challenge may be useful in evaluating vaccine-induced immunity.
Methods.Ten subjects previously vaccinated with a live attenuated tetravalent dengue vaccine (TDV) and 4
DENV-naive control subjects were challenged by subcutaneous inoculation of either 103plaque-forming units (PFU)
of DENV-1 or 105PFU of DENV-3. Two additional subjects who did not develop DENV-3 neutralizing antibody
(NAb) from TDV were revaccinated with 104PFU of live attenuated DENV-3 vaccine to evaluate memory response.
Results.All 5 TDV recipients were protected against DENV-1 challenge. Of the 5 TDV recipients challenged
with DENV-3, 2 were protected. All DENV-3–challenge subjects who developed viremia also developed elevated
liver enzyme levels, and 2 had values that were >10 times greater than normal. Of the 2 subjects revaccinated with
DENV-3 vaccine, 1 showed a secondary response to DENV-2, while neither showed such response to DENV-3. All
4 control subjects developed dengue fever from challenge. Protection was associated with presence of NAb, although
1 subject was protected despite a lack of measurable NAb at the time of DENV-1 challenge.
Conclusions.Vaccination with TDV induced variable protection against subcutaneous challenge. DENV-3
experimental challenge was associated with transient but marked elevations of transaminases.
Keywords.dengue; tetravalent vaccine; live virus challenge.
Dengue is an arboviral disease caused by 4 serotypes
of dengue virus (DENV). It is the leading vector-
borne disease in humans, with >50 million infections
annually worldwide, and has the potential to be rees-
tablished in parts of the United States . Existing
dengue prevention and control strategies have failed to
prevent the expansion of this disease .Dengue hem-
orrhagic fever (DHF) and dengue shock syndrome
(DSS) are the severe forms of the disease, occurring
usually from secondary infection by a heterologous
serotype, and account for the majority of fatalities .
The pathogenesis of DHF and DSS has been postulat-
ed to be mediated by antibody-dependent enhance-
ment [4, 5]. An effective dengue vaccine is urgently
needed [6, 7]. A tetravalent vaccine should simul-
taneously immunize against all 4 DENV serotypes.
Received 20 April 2012; accepted 8 August 2012; electronically published 5
aPresent affiliations: Division of Vaccines and Related Product Applications,
Office of Vaccine Research and Review, CBER, Rockville (W. S.), Pilot Bioproduc-
tion Facility (K. H. E.) and Department of Virus Diseases, Division of Communica-
ble Diseases and Immunology (J. R. P., S. J. T., R. V. G., and V. J. G.), Walter
Reed Army Institute of Research, Silver Spring, National Center for Medical Intel-
ligence/Defense Intelligence Agency, Fort Detrick (M. P. M. Jr.), and Department
of Radiology and Radiologic Sciences, Uniformed Services University of the
Health Sciences, Bethesda, Maryland (J. D. S.); Global Clinical Research and
Development (D. M. V.) and Vaccine Discovery and Development (B. L. I.),
GlaxoSmithKline Vaccines, King of Prussia, Pennsylvania (D. W. V.); Radio-
logic Associates of Fredericksburg, Fredericksburg, Virginia (J. D. S.); and Depart-
ment of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok,
Thailand (S. F.).
Correspondence: Wellington Sun, MD, Division of Vaccines and Related
Product Applications, Office of Vaccine Research and Review, CBER, 1451 Rock-
ville Pike, Rm 2302, Rockville, MD 20852 (email@example.com).
The Journal of InfectiousDiseases 2013;207:700–8
Published by Oxford University Press on behalf of the Infectious Diseases Society
of America 2012.
700 • JID 2013:207 (1 March) • Sun et al
by guest on October 26, 2015
Neutralizing antibodies (NAb) developed after infection
confer both long-lasting protection against reinfection by the
homologous serotype and transient cross-protection against
heterologous serotypes . Although the protective levels of
serotype-specific NAb have not been established, the NAb
titer measured by the 50% plaque reduction neutralization test
(PRNT50) has been generally accepted as the immune re-
sponse end point for dengue vaccine trials . Because of the
cross-reactivity of NAb among the serotypes and the phenom-
enon of original antigenic sin, it is difficult to infer the
immune status of any particular individual in whom infec-
tions with multiple DENV serotypes have occurred . Simi-
larly, in the setting of live attenuated tetravalent dengue
vaccine (TDV), it is difficult to infer vaccine-induced protec-
tion on the basis of the presence of NAb against multiple sero-
types because of the presence of cross-reactive, nonprotective
NAb. The cross-reactive, nonprotective NAb may contribute
to antibody-dependent disease enhancement [4, 5]. This has
been a major theoretic concern for dengue vaccine developers.
A suboptimally immunogenic vaccine not only might fail to
protect but also might pose an increased risk of DHF follow-
ing subsequent natural infection . Currently, the tetrava-
lent live attenuated vaccine approach is in late-phase
development [6, 7]. We have evaluated several TDV formula-
tions in clinical trials [12–20]. Here, we evaluate TDV-
induced immunity in selected previously vaccinated subjects
by experimentally challenging them with underattenuated
monovalent DENV-1 or DENV-3 .
MATERIALS AND METHODS
This was a descriptive, phase II, controlled, open-label experi-
mental challenge study. The primary study objectives were (1)
to evaluate the safety of giving either underattenuated DENV-1,
DENV-3, or attenuated DENV-3 to subjects previously vacci-
nated with TDV, (2) to determine whether DENV-1 and
DENV-3 NAb elicited by TDV protect against clinical disease
from homologous-serotype challenge virus, and (3) to deter-
mine the clinical safety and immunogenicity of DENV-3 re-
vaccination in TDV recipients who did not develop DENV-3
NAb from previous TDV vaccination. Secondary objectives
were to study cell-mediated immunity in study subjects, the
results of which are described elsewhere . Three study
arms—challenge, revaccination, and control—were used. Sub-
jects in the challenge and revaccination arms were vaccinated
with 2 doses of TDV 12–42 months before this study. Subjects
in the control arm were flavivirus naive.
All subjects were healthy adults 18–45 years of age. Subjects in
the challenge and revaccination arms were recruited from
subjects who previously participated in TDV clinical trials [15,
16, 18]. The challenge subjects had to have had a DENV-1 or
DENV-3 NAb response following vaccination to be considered
for homologous DENV-1 or DENV-3 challenge, to reduce the
potential risk of DHF following challenge. For inclusion in the
study, control subjects had to be flavivirus seronegative. All
subjects provided written informed consent and had to score
>75% on a written knowledge examination about the study, to
demonstrate their comprehension of the risks of the study.
The study was approved by institutional review boards. The
study was conducted in accordance with current good clinical
practices under Title 21 of the US Code of Federal Regula-
tions, Parts 50, 56, and 312. The study was completed in 2002,
prior to the implementation of the National Clinical Trials
All baseline evaluations were done ≤28 days before challenge.
Subjects were inoculated on day 0 and monitored for 30
minutes. A self-reported symptom and temperature diary was
kept for 3 weeks. Symptoms were graded on a scale of 0–3,
with 0 defined as none, 1 defined as mild (the symptom did
not affect normal activity, and no medication was required), 2
defined as moderate (the symptom required medication or a
change in activity level), and 3 defined as severe (the
symptom required bed rest or was unrelieved by medication).
A reactogenicity index (RI), a quantitative measurement of the
severity of clinical symptoms, was calculated for each subject on
the basis of 9 solicited dengue symptom grades and durations
. From days 4 to 17, when viremia and clinical illness were
most likely to occur, all subjects were housed in quarters and
monitored by a study physician at least twice daily, with vital
signs recorded every 8 hours. Blood samples were obtained on
days 0, 4–17 (daily), 24, 30, and 60 for laboratory tests. Subjects
were hospitalized as a precaution if they developed an oral tem-
perature of >38.0°C, thrombocytopenia (ie, a platelet count of
<100000 platelets/µL), hemoconcentration (ie, hematocrit in-
crease of >15% from baseline), or any significant clinical illness.
Follow-up after day 17 included telephone contact on day 21
and clinic visits on days 24, 30, and 60. A tourniquet test was
done on all subjects at baseline and daily on subjects who de-
veloped fever, until 2 days after defervescence.
Inclusion criteria included normal baseline results of the fol-
lowing laboratory tests: serologic analyses for human immu-
nodeficiency virus, hepatitis B virus, and hepatitis C virus;
complete blood count (CBC) with differential; and determina-
tion of aspartate transaminase (AST) level, the alanine trans-
aminase (ALT) level, international normalized ratio (INR),
prothrombin time/partial thromboplastin time (PT/PTT),
blood urea nitrogen level, blood glucose level, and creatinine
DENV Challenge After Tetravalent Vaccination • JID 2013:207 (1 March) • 701
by guest on October 26, 2015
level. Chest radiography and abdominal ultrasonography were
performed at baseline and during fever. Abdominal ultraso-
nography was repeated if the subject became febrile, to evalu-
ate for gallbladder wall thickening and ascites. Before
challenge, all control subjects were seronegative to DENV-1,
-2, -3, and -4, West Nile virus, yellow fever virus, St. Louis
encephalitis virus, and Japanese encephalitis virus by hemag-
glutination inhibition assays and were seronegative to DENV-
1, -2, -3, and -4 and yellow fever virus by PRNT50. Pregnancy
was excluded by a negative serum β human chorionic gonado-
tropin (β-HCG) test result within 48 hours of inoculation and
a negative urine β-HCG test result on day 0. On days 4–17,
CBC with differential was measured daily, and PT/PTT, AST
level, ALT level were measured every 2 days. A platelet count
of <20 000 platelets/µL and an ALT level of >1000 IU/L were
considered to be serious adverse events.
Challenge and Vaccine Viruses
The DENV-1 and DENV-3 strains were chosen because these
2 challenge strains consistently caused fever in previous clini-
cal trials. The challenge DENV-1 and DENV-3 were derived
from clinical DENV isolates, using a seed lot system under
current Good Manufacturing Practices, which involves passag-
es in cell cultures, as described by Eckels et al [12, 19, 23].
CH53489 cl 24/28 PDK-0 (DENV-3-Ch) had failed evaluation
as vaccine candidates because of their reactogenicity [19, 20].
When given at 104plaque-forming units (PFU) subcutaneous-
ly, both strains previously consistently elicited dengue fever in
4 and 5 adult subjects, respectively [20, 23, 24]. Both challenge
viruses were parental in lineage to the respective vaccine
strains in the TDV. The DENV-3 component of the TDV,
DENV-3 CH53489 cl 24/28 PDK-20 (DENV-3 PDK20), was
used in the revaccination arm. All test articles were given as
0.5 mL subcutaneous injections in the deltoid region. Residu-
als in the multidose vial were verified for potency by a plaque
assay on Vero cells. The doses of DENV-1-Ch, DENV-3-Ch,
and DENV-3 PDK20 were 103PF, 105PFU, and 104PFU,
Blood specimens collected on days 0 and 4–17 were tested for
viremia by delayed plaque assay  and by serotype-specific
quantitative real-time reverse transcription polymerase chain
reaction (RT-PCR), using ABI Prism 7900HT as previously
described . Primers and probes were based on the 5′ un-
translated region of DENV. The limit of quantitation for the
assay was determined to be 5.1× 101and 1.0 ×101genome
copies/mL for DENV-1 and DENV-3, respectively .
NAb titers were measured on days 0, 30, and 60, using
PRNT50involving a 6-well format with Vero cells . All
samples were tested in triplicate. A positive test cutoff was
defined as a 50% reduction in plaques at a dilution of 1:10.
This was a descriptive study with a small sample size and did
not formally test any hypothesis. Data are tabulated individu-
ally for each subject.
There were 135 eligible subjects from 3 previously completed
TDV phase I and II clinical trials. Thirty-nine of 135 eligible
subjects were available. Of these, 12 subjects completed
screening, and 5 (subjects TD1_1–5) were selected for DENV-
1 challenge, 5 (subjects TD3_1–5) were selected for DENV-3
challenge, and 2 (subjects TV3_1 and TV3_2) were selected
for DENV-3 monovalent revaccination. The assignment of the
serotype challenge was based on the highest postvaccination
DENV-1 and DENV-3 PRNT50 titers. Two subjects were
and Controls Who Did Not Receive Live Attenuated Tetravalent
Dengue Vaccine Before Dengue Virus (DENV) Challenge or
Demographic Characteristics of Subjects Who Did
Test Article, Study Arm,
Abbreviations: Af, African American; As, Asian; W, white.
702 • JID 2013:207 (1 March) • Sun et al
by guest on October 26, 2015
selected for DENV-3 revaccination because of their lack of
postvaccination DENV-3 antibody. The subjects were blinded
to the serotype of challenge or revaccination. The 4 control
group subjects (subjects CD1_1, CD1_2, CD3_1, and CD3_2)
were selected from 19 screened individuals. The demographic
characteristics of the subjects are shown in Table 1. All 16 sub-
jects completed the study per protocol.
Local reactions from the injections were minimal. The only
subject with an observable local sign was a DENV-3-Ch
control subject, in whom a 3.5 ×1.0-cm area of erythema
emerged at the injection site by day 4 and resolved by day
5. No subjects developed DHF or DSS, as defined by World
Health Organization criteria . Specifically, no subjects de-
veloped hemoconcentration, positive tourniquet sign, ascites,
gallbladder wall thickening, bleeding, hypotension, or labora-
tory-determined serious adverse events.
Of the 5 TDV recipients given DENV-1-Ch, none developed
dengue illness (Table 2). Only 1 vaccinee, subject TD1_5, devel-
oped fever and headache (duration, 1 day), which were due to a
tooth abscess. Both DENV-1-Ch controls, subjects CD1_1 and
CD1_2, developed fever of 39.3°C (grade 3) and 38.3°C (grade
1) orally, respectively, and had dengue symptoms that lasted 2–6
days. Subject CD1_2 had the most severe symptoms, with an RI
of 30. No vaccinees developed generalized rash. The incubation
periods in the control subjects were long, at 21 days for subject
CD1_1 and 17 days for subject CD1_2. Subject CD1_1 devel-
oped the typical maculopapular generalized rash. No subjects
who received DENV-1-Ch developed neutropenia or thrombo-
cytopenia. Subject CD1_1 was the only DENV-1-Ch subject
who developed elevated AST and ALT levels, in whom peaks of
41 IU/mL and 56 IU/mL, respectively, were observed.
Three of 5 vaccinees, subjects TD3_1, TD3_2, and TD3_4, de-
veloped dengue-like illness (Table 2). The other 2, subjects
TD3_3 and TD3_5, remained well. Subjects TD3_1 and
TD3_4 developed grade 3 fever and symptoms. The clinical
illness in TDV recipients was no worse than that in controls.
The RIs for the symptomatic vaccinees were 17 (for subject
TD3_1), 17 (for subject TD3_2), and 20 (for subject TD3_4),
and the RIs for the control subjects were 21 (for subject
CD3_1) and 63 (for subject CD3_2). Subjects TD3_2 and
TD3_4 developed the typical maculopapular generalized rash
of dengue, which lasted 4–5 days. Both control subjects devel-
oped dengue-like illness associated with grade 2 fever and gen-
eralized rash. Onset of symptoms with DENV-3-Ch occurred
on 2–5 days after inoculation. TDV recipient TD3_1–3 and
characteristic of dengue. Subject CD3_1 developed thrombocy-
topenia. Except for subject TD3_5, all subjects who were chal-
lenged with DENV-3-Ch developed elevated ALT levels
(Table 3). Subjects TD3_1, TD3_2, and CD3_1 had markedly
elevated AST and ALT levels. The ALT level peaked at 977 IU/L
(on day 9) and 428 IU/L (on day 13) in 2 of these subjects.
The elevations lasted from days 5 to 30 and days 5 to 24 in
subjects TD3_1 and TD3_2, respectively. Subject CD3_1, a
control, had elevated AST and ALT levels on days 9–24, which
peaked at 367 IU/L (for AST) and 349 IU/L (for ALT) on
days 13 and 15, respectively. The AST and ALT levels returned
to normal by day 30 in all 3 subjects. The INRs were normal
for all DENV-3-Ch subjects throughout the study. No subjects
had any evidence for DHF, such as abnormal hemoconcentra-
tion, pleural effusion, ascites, hepatomegaly, or gallbladder
The 2 TDV recipients who were revaccinated with the attenu-
ated DENV-3 component from the original TDV they re-
ceived did not develop any clinical symptoms or any clinically
significant abnormal laboratory findings.
Viremia and NAb
Of the 5 TDV recipients, all but subject TD1_2 were viremic
with DENV-1 from their previous vaccination with TDV
(data not shown). Four of the 5 TDV recipients had NAb to
DENV-1 at the time of challenge, with reciprocal PRNT50
titers of 415, 235, 451, and 198. Subject TD1_3 did not have
measurable DENV-1 NAb at the time of and after challenge,
yet he remained well after challenge. Only 1 TDV recipient,
subject TD1_4, developed viremia, which was detected only
on day 7, by delayed plaque assay but not RT-PCR, and she
did not develop dengue (Table 4). None of the TDV recipients
challenged with DENV-1-Ch developed a rise in PRNT50titer
after challenge, suggesting a lack of significant viral replication.
As expected, both controls, subjects CD1_1 and CD1_2, sero-
converted to DENV-1, but only subject CD1_2 was viremic,
with a peak viral titer of 107genome copies/mL (Table 4).
Of the 5 TDV recipients challenged with DENV-3, only
subject TD3_3 was viremic with DENV-3 after the previous
vaccination with TDV (data not shown). She was also viremic
with DENV-1 and -2. The 3 TDV recipients who developed
dengue-like illness on challenge, subjects TD3_1, TD3_2, and
TD3_4, had reciprocal DENV-3 NAb titers of <10, 19, and 16,
respectively, at the time of challenge. All 3 developed viremia
detected by both delayed plaque assay and RT-PCR (Table 4).
The magnitude of viremia was 5–7 log10genome copies/mL.
Viremia was also detected in the 2 control subjects, at levels of
DENV Challenge After Tetravalent Vaccination • JID 2013:207 (1 March) • 703
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6–8 log10 genome copies/mL. The duration of viremia was
longer in the control subjects. Subjects TD3_3 and TD3_5 re-
mained well after challenge; their prechallenge reciprocal titers
were 57 and 116, respectively. Notably, subject TD3_3 did not
have viremia detected by delayed plaque assay, but viremia
was detected by RT-PCR, at a level of 4 log10genome copies/
mL. The 3 symptomatic vaccinees and the asymptomatic
subject TD3_3 who were viremic all developed a marked rise
in DENV-3 PRNT50titers after challenge, suggesting a sec-
ondary antibody response to viral replication. Subject TD3_5,
who had the highest prechallenge titer, did not show any in-
crease in NAb titer after challenge.
Subjects TV3_1 and TV3_2 did not seroconvert to DENV-3 1
month after TDV vaccination, but subject TV3_2 did so by
the time of challenge. Neither developed viremia from DENV-
3 revaccination. Subject TV3_1 had no DENV-3 NAb at the
time of revaccination. He showed a pattern suggesting original
antigenic sin, with seroconversions to DENV-2, -3, and -4,
but had a much greater rise in DENV-2 antibody level.
Subject TV3_2 had DENV-3 antibody detected at the time of
challenge, and she did not show any rise in her antibodies
after revaccination (Table 4).
In monkeys given TDV, 1 or more DENV serotype(s) may
dominate the antibody response, resulting in incomplete pro-
tection against the less dominant serotypes when the animals
were subsequently challenged [13, 29, 30]. Similar NAb im-
balance has been seen with all TDVs tested in humans to
Tetravalent Dengue Vaccine Before Dengue Virus (DENV) Challenge or Revaccination
Solicited Systemic Adverse Events (AEs) Among Subjects Who Did and Controls Who Did Not Receive Live Attenuated
Study Day(s) With AE (No. of Days With Grade 2 or 3 Symptoms)
2, 7 (0)
11, 12 (0)
21, 22 (39.3)
21, 22 (1)
8, 10, 17–19, 22 (6)
21, 22 (1)
4, 5 (38.5)
5, 6 (39.4)
4, 5 (2)
4, 5, 6, 7 (2)
4, 5 (1)
0, 5, 6, 7–10 (1)
4, 7, 8 (0)
4, 5 (1)
1, 6–9, 18 (1)
1, 2, 4, 6–8, 9, 10,
11, 13, 14,
19, 20 (3)
6–8, 11–15 (1)
8, 14 (0)
All adverse events were self-reported by subjects in a symptom and temperature diary. Days on which symptom severity was grade 3 are in bold.
Abbreviation: RI, reactogenicity index.
aFever data in parentheses are peak temperature in°C. Grade 1 severity was defined as a temperature of 38.0°C–38.4°C; grade 2, as a temperature of 38.4°C–
38.9°C; and grade 3, as a temperature of 38.9°C–40.0°C.
704 • JID 2013:207 (1 March) • Sun et al
by guest on October 26, 2015
date [15, 16, 31–34]. Strategies to mitigate such imbalance in
the NAb response have included increasing the attenuation or
lowering the dose(s) of the dominant serotype(s) and adminis-
tering multiple doses of TDV, with various degrees of success.
While it is generally accepted that the simultaneous induction
of NAb to all 4 serotypes in each individual will likely predict
protection against all 4 serotypes, this tetravalent seroconver-
sion has been difficult to achieve. If it is assumed that serocon-
version to each serotype is independent of seroconversion to
the other serotypes, even a 90% rate for each individual sero-
type will lead to only a 66% tetravalent NAb response rate. In
fact, the observed tetravalent seroconversion rates and titers
may be due in part to cross-reactive NAb responses and there-
fore may overestimate long-term vaccine efficacy. This study is
the first experimental DENV challenge of TDV recipients and
was designed to explore the relationship between NAb from
TDV vaccination and protection against disease. The subjects,
who were living in an area where DENV is not endemic, were
not exposed to wild-type DENV after their last TDV vaccina-
tion 8–42 months ago, and therefore their antibodies at the
time of challenge were solely the result of their previous TDV
vaccinations. Previous vaccination with TDV was associated
with protection against DENV-1 challenge. Such protection
may last 42 months after vaccination, as with subjects TD1_1
and TD1_3–5. The protection against DENV-1 appears to be
mediated by sterile immunity, with no observed increase in
NAb level from the challenge. Subject TD1_4 had a single
episode of viremia, on day 7, detected by delayed plaque assay
but not by RT-PCR. This may be due to the better sensitivity
for DENV-1 exhibited by delayed plaque assay, compared
with RT-PCR (10 vs 500 PFU/mL) . In contrast, the pro-
tection against DENV-3 was variable. Three of the 5 TDV re-
cipients developed clinical disease despite developing DENV-3
NAb from 2 doses of TDV. Of the 5 TDV recipients chal-
lenged with DENV-1, 4 were viremic with DENV-1 from their
original vaccination with TDV. Of the 5 TDV recipients
Who Did Not Receive Live Attenuated Tetravalent Dengue Vaccine Before Dengue Virus (DENV) Challenge or Revaccination
Results of Clinical Laboratory Studies Within 30 Days of Challenge or Revaccination Among Subjects Who Did and Controls
Hct Increase of >15%
Nadir Platelet Count,
Peak ALT L
evel, IU/L INR
0.5 (7, 8)
Data in parentheses indicate the day after inoculation on which the peak or nadir of the laboratory value was recorded.
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; Hct, hematocrit; INR, international normalized ratio.
aA neutrophil count of <1000 neutrophils/µL was considered to be abnormal.
bA platelet count of <100 000 platelets/µL was considered to be abnormal.
DENV Challenge After Tetravalent Vaccination • JID 2013:207 (1 March) • 705
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Attenuated Tetravalent Dengue Vaccine (TDV) Before DENV Challenge or Revaccination
Reciprocal 50% Plaque Reduction Neutralization Test (PRNT50) Dengue Virus (DENV) Titers and Viremia Among Subjects Who Did and Controls Who Did Not Receive Live
Time of Challenge,
Mos. After TDV
Reciprocal DENV-1, -2, -3, -4 PRNT50Titer, Time Pointa
Study Day(s) With Viremia
Peak Viremia, GC/mL,
by RT-PCRAfter TDVBefore Challenge After Challenge By DPBy RT-PCRb
421593, <10, <10, <10
274, 42, <10, <10
65, 10, <10, <10
903, 168, 10, <10
250, <10, <10, <10
415, <10, <10, <10
235, 174, 20, <10
<10, <10, <10, <10
451, 94, 30, <10
198, 19, 42, <10
480, <10, <10, <10
59, 19, <10, <10
<10, <10, <10, <10
114, 10, <10, <10
41, <10, 10, <10
<10, <10, <10, <10
<10, <10, <10, <10
343, 59, 18, <10
3162, <10, <10, <10
0, 1, 4
963, 52, 25, <10
1529, 58, 72, <10
6400, 633, 640, 678
26, 112, 20, 86
210, 663, 67, 650
914, 233, <10, <10
144, 51, 19, <10
649, 57, 57, 39
<10, 52, 16, 18
171, 355, 116, 60
3135, 824, 1234, 66
259, 366, 2896, 58
7448, 1269, 2733, 1915
826, 1056, 1290, 977
<10, 396, 41, 72
4, 5, 6
4, 5, 6, 7, 8,c9, 10
4,c5, 6, 7, 8, 9,c10
4,c5, 6, 7, 8
<10, <10, <10, <10
<10, <10, 20, <10
<10, 43, 857, <10
<10, 68, 235, <10
4, 6, 7–11
4–6, 7, 8–10
<10, 46, <10, <10
255, <10, <10, <10
<10, <10, <10, <10
336, <10, 17, <10
<10, 1736, 61, 48
154, <10, 13, <10
Abbreviation: DP, delayed plaque assay; GC, genome copies; NA, not applicable; RT-PCR, quantitative real-time reverse transcription polymerase chain reaction.
aData for revaccinees were obtained after TDV, before revaccination, and after revaccination. Data after TDV, after challenge, and after revaccination were obtained 28–30 days after inoculation.
bDay of peak viremia is in bold.
cBelow the limit of quantitation.
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challenged with DENV-3, only subject TD3_3 was viremic
with the DENV-3 vaccine strain from her original TDV vacci-
nation (data not shown). Interestingly, subject TD3_3 again
developed DENV-3 viremia from challenge but had no associ-
ated dengue symptoms. Her serum had no culturable DENV-
3, suggesting antibody neutralization of virus. The clinical
courses of the DENV-3 breakthrough infections were consis-
tent with dengue fever and not DHF. All 3 breakthrough in-
fections, which occurred in subjects TD3_1, TD3_2, and
TD3_4, had detectable viremia by both culture and RT-PCR,
suggesting circulating infectious virus. The peak viremia titers
in these 3 subjects were not enhanced and were comparable to
titers in DENV-naive controls. Notably, the 2 TDV recipients
protected against DENV-3, subjects TD3_3 and TD3_5, did
not have viremia detected by culture, but subject TD3_3 had a
peak viremia of 4 log10genome copies/mL detected by RT-
PCR. Interestingly, these 2 protected subjects had the highest
prechallenge DENV-3 NAb titers, at 1:57 (for subject TD3_3)
and 1:116 (for subject TD3_5). The prechallenge NAb titer of
1:57 for subject TD3_3 was apparently not enough to prevent
detection of viremia by RT-PCR but was enough to prevent de-
tection of culturable virus from serum. Thus, high, long-lasting
(≥ 9 months) postvaccination titers of NAb from TDV may be
more predictive of protection. In TDV trials, the Nab titer mea-
sured 9 months or later after vaccination may be a better end-
point. This may be difficult in DENV-endemic areas where
there is circulating virus and continuous exposure.
Our study monitoredfor DHF,byvital signs, laboratorypara-
graphy. We previously showed that abdominal ultrasonography
detected subclinical pericardial, perihepatic, and perisplenic
effusions associated with DENV challenge . Findings of
abdominal ultrasonography in this study were unremarkable.
The challenge with DENV-3 was associated with marked ele-
vations of AST and ALT levels in 2 TDV recipients, subjects
TD3_1 and TD3_2, and in 1 control, subject CD3_1. Wild-type
DENV infection is known to be associated with acute hepatitis,
occasionally with markedly elevated results of liver functions
tests, both in dengue fever and DHF [36, 37]. Four of 5 TDV
recipients developed elevated AST and ALT levels. In subjects
TD3_1 and TD3_2, the peak ALT levels were 20-fold and 10-
fold, respectively, above the normal upper limit, and the onset
of the elevation corresponded to the onset of dengue symptoms.
The elevated AST and ALT levels were short-lived and resolved
by the end of the study. Importantly, in our subjects with elevat-
ed AST and ALT levels, there were neither prolonged PTs nor
bleeding, the complications associated with wild-type DENV in-
fection with hepatitis [38, 39]. Elevated AST and ALT levels
were also seen in control subject CD1_1 but not to the degree
seen in subjects TD3_1 and TD3_2. Even without evidence of
plasma leakage in subjects TD3_1 and TD3_2, the degree of ele-
vation in results of liver function tests was in the range
indicative of severe disease, according to the 2009 World Health
Organization criteria . During efficacy trials of TDV in
DENV-endemic areas, there should be close monitoring to eval-
uate any increased incidence of acute liver disease in the vacci-
nated population, compared with that in controls.
There are obvious limitations to this study, given its small
size. The subjects were selected from a limited pool of TDV
recipients who received different formulations and schedules
of TDV. The small size of the study precluded any meaning-
ful assessment of the risk of DHF, as well as any demonstra-
tion of the level of NAb as a correlate of protection. The
dose used for DENV-1 challenge was nearly 2 logs lower
than the dose for DENV-3 challenge, making direct compar-
ison of serotypes difficult. The lower DENV-1 challenge dose
PFU, compared with 105PFU for DENV-3) may
explain the delayed onset of clinical symptoms, on days 19
and 21, in the 2 DENV-1 control subjects and perhaps also
the absence of DENV-1 viremia in subject CD1_1. Another
possible limitation is the subcutaneous delivery of virus by
needle injection, which may not completely replicate infec-
tion by mosquitoes.
Our results suggest that TDV can induce long-term protection
and that long-lasting, higher, serotype-specific NAb titers from
TDV vaccination are more likely to predict vaccine-induced pro-
tective immunity. Durbin et al showed that when subjects previ-
ously vaccinated with DENV-1, -2, or -4 monovalent vaccines
were challenged with fully attenuated heterotypic DENV-1 or -2
vaccines, no significant clinical or laboratory adverse events oc-
curred . This was not the case with our near–wild-type
DENV-3 challenge, in which we observed that TDV failed to
protect and that the challenge was associated with unexpected el-
evation of transaminase levels, suggesting liver injury. Given that
a significant percentage of TDV recipients will not have protec-
tive tetravalent NAb responses, our study supports the well-
follow-up period to monitor rates of complications from wild-
type DENV infections in both the vaccinated and control groups.
The usefulness of experimental DENV challenge studies
should be further evaluated. These studies will likely be
limited in sample size but may be useful in selecting vaccine
candidates for field trials. Alternatively, experimental challenge
studies may serve as adjuncts to field efficacy trials to evaluate
protection against noncirculating dengue serotypes.
tion; the staff of the Clinical Trials Department at Walter Reed Army In-
stitute of Research and the nursing staff at Walter Reed Army Medical
Center, for their assistance in performing the study; and Dr Charles
N. Oster, for serving as medical monitor.
The views expressed herein are those of the authors and
do not necessarily represent those of the Department of the Army or
Department of Defense.
We thank the study subjects, for their participa-
DENV Challenge After Tetravalent Vaccination • JID 2013:207 (1 March) • 707
by guest on October 26, 2015
Financial support. Download full-text
Medical Research and Materiel Command.
Potential conflicts of interest.
coinventors of the multivalent dengue vaccine (US patent 6638514). This
study was conducted prior to awarding of the patent. All other authors
report no potential conflicts.
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.
This work was supported by the US Army
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