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 (firstname.lastname@example.org).
The Journal of InfectiousDiseases 2013;207:700–8
Published by Oxford University Press on behalf of the Infectious Diseases Society
of America 2012.
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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
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by guest on October 26, 2015
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
J. R. P., K. H. E., W. S., and B. L. I. are
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