1. [Human Vaccines 2:2, e1-e8, EPUB Ahead of Print: http://www.landesbioscience.com/journals/vaccines/abstract.php?id=2555; March/April 2006]; ©2006 Landes Bioscience
Live Attenuated Chimeric Yellow Fever Dengue Type 2 (ChimeriVax™-
Research Paper
DEN2) Vaccine: Phase I Clinical Trial for Safety and Immunogenicity
Effect of Yellow Fever Pre-immunity in Induction of Cross Neutralizing
Antibody Responses to All 4 Dengue Serotypes
Farshad Guirakhoo1* ABSTRACT
Scott Kitchener2,†
E .
A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability,
Dennis Morrison3
UT
and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-
Remi Forrat4
DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adults
randomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaque
RIB
Karen McCarthy2
forming units [PFU] or low dose, 3 log PFU) or YF-VAX® by the subcutaneous route (SC).
Richard Nichols1
To determine the effect of YF preimmunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjects
IST
previously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as an
Sutee Yoksan5
open-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderate
Xiaochu Duan1
intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve
D
subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively,
Thomas H. Ermak1
seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX®
OT
Niranjan Kanesa-Thasan1
seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-
DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN
Philip Bedford1
ON
serotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with either
ChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculated
Jean Lang4
with ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of
.D
Marie-Jose Quentin-Millet4
neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after
1 year. These data demonstrated that (1) the safety and immunogenicity profile of the
Thomas P Monath1
.
CE
ChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and (2) preimmunity to
YF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a long
1Acambis, Inc..; Cambridge, Massachusetts USA
IEN
lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter
observation can have practical implications toward development of a dengue vaccine.
2Acambis Research Limited; Peterhouse Technology Park; Cambridge, UK
3Bio-Kinetic Clinical Applications; Springfield, Missouri USA
SC
INTRODUCTION
4Sanofi Pasteur; Campus Mérieux; Marcy-L'étoile, France
BIO
5Center for Vaccine Development; Institute of Sciences and Technology for Research
& Development; Mahidol University; Nakhonpathom, Thailand Dengue, a disease caused by DEN virus with four distinct species (serotypes 1–4) is the
†Present address: Centre for Military and Veterans Health; Mayne Medical School; most important vector-borne viral disease of human kind. Approximately 100 million
ES
Herston, 4006 Australia persons are affected by DEN viruses annually in tropical and subtropical regions of the
*Correspondence to: Farshad Guirakhoo; Acambis, Inc.; 38 Sidney Street; Cambridge,
world.1,2 Severe and potentially lethal forms of the disease, dengue hemorrhagic fever
ND
Massachusetts 02139 USA; Tel.: PLEASE PROVIDE; Fax: PLEASE PROVIDE; Email: (DHF) and dengue shock syndrome (DSS), are increasing in geographic distribution and
Farshad.Guirakhoo@acambis.com incidence, and are currently affecting up to 500,000 individuals. These factors have
Received 11/14/05; Accepted 12/15/05
This manuscript has been published online, prior to printing for Human Vaccines,
LA
spurred intensive efforts to construct a safe and effective DEN vaccine, but despite many
efforts spanning 50 years, no commercially available vaccine against DEN virus has been
Volume 2, Issue 2. Definitive page numbers have not been assigned. The current cita-
developed. The development of a vaccine against DEN is considered a high priority by the
05
tion is: Human Vaccines 2006; 2(2): World Health Organization.3 The pathogenesis of DHF drives the design of DEN
http://www.landesbioscience.com/journals/vaccines/abstract.php?id=2555 vaccines. DHF is an immunopathological disease, which occurs primarily in individuals
Once the issue is complete and page numbers have been assigned, the citation will
20
change accordingly.
who have sustained a prior infection with one of the DEN serotypes and are exposed to
another, heterologous serotype.4 Infection with any of the four serotypes of DEN provides
©
KEY WORDS
durable immunity to that specific serotype, based on neutralizing antibodies. However,
following infection with one DEN serotype, immunity to other heterologous serotypes, if
Live attenuated, Chimeric yellow fever-dengue 2, occurring at all, is of very short duration.5 A secondary infection with a heterologous
Phase 1 trial, Safety, Immunogenicity serotype may be enhanced by infection of target cells (monocyte macrophages) bearing Fc
ACKNOWLEDGEMENTS
receptors with immune complexes of virus with binding antibody. The infected cells are
then cleared by DEN-specific T cell responses. The results of immune clearance in the face
See page e8. of a high viral load are complement activation and the release of chemokines and cytokines
that alter vascular permeability and elicit other pathogenic events associated with DHF.6
e1 Human Vaccines 2006; Vol. 2 Issue 2

2. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
a successful vaccine against DEN must have two characteristics: Table 1 Treatment schedule
Given the immunopathogenesis of DHF, it is obvious that
simultaneously, and (2) it must induce long-lasting neutralizing Group No. Subjects YF-immune Vaccine Dose log 10 PFU
(1) it must induce immunity against all 4 DEN virus serotypes
in 0.5 mL
antibody responses (titers should not fall to a level that leaves
the subject sensitized to immunopathological events but not Double-blind, randomized
protected against future infections). These requirements, partic- 1 14 No ChimeriVax™-DEN2 5.0
ularly the latter, can only be met with a live, attenuated vaccine 2 14 No ChimeriVax™-DEN2 3.0
3 14 No YF-VAX® =≥5.04
that induces durable immunity. Empirical efforts to develop
live, attenuated vaccine candidates have demonstrated that Open
4 14 Yes ChimeriVax™-DEN2 5.0
achieving a balance between sufficient attenuation (safety) and
immunogenicity is a very complicated task. Attempts to
combine monovalent live vaccines into one vaccine uncovered
significant interactions between the four virus strains, resulting in reconstituted with sterile diluent provided (sodium chloride injection USP,
interference effects.7 containing no preservative, Batch# UB054AA and UB054AC). The formu-
ChimeriVax™-DEN2 is a live, attenuated, genetically engineered lated vaccine contained not less than 5.04 log10 PFU/0.5 ml, according to
virus prepared by replacing the genes encoding the two structural the manufacturer’s brochure.
proteins, premembrane (prM) and envelope (E), of the YF 17D Clinical study. A randomized, double-blind, single-center outpatient
study was performed. The clinical trial protocol and informed consent
vaccine virus with the corresponding genes of the DEN2 virus
forms were approved by an Institutional Review Board (IRB) according to
(strain PUO-218, isolated from a case of classical DF, Bangkok, FDA regulations, as outlined in the US Code of Federal Regulations. The
Thailand). 8 It is a monovalent component of a tetravalent vaccine
study was conducted under Good Clinical Practices and an Investigational
formulation9 currently being investigated in a Phase I study. New Drug application approved by the FDA. Written informed consent was
Preclinical studies have demonstrated that the ChimeriVax™- obtained from each subject in accordance with the ethical principles in the
DEN2 virus was is not neurovirulent when administered to adult Declaration of Helsinki at a screening visit, prior to entry into the study.
mice via the intracerebral (IC) route, is genetically stable in cell culture, The objectives of this study were to determine: (1) safety, tolerability, and
induces low levels of viremia, and protects 100% of monkeys against viremia levels following administration of ChimeriVax™-DEN2 at doses ® of
wt DEN2 heterologous challenge upon a single SC immunization. 10 3.0 and 5.0 log10 PFU, respectively, and a control vaccine (YF-VAX )
administered by the SC route to healthy adult male and female subjects,
We present results of a proof of principle study for safety, tolera-
® (2) the neutralizing antibody response against wt DEN2 virus in subjects
bility and immunogenicity of ChimeriVax™-DEN2 and YF-VAX vaccinated with ChimeriVax™-DEN2 at doses of 3.0 and 5.0 log10 PFU,
(as a control) in a randomized, double-blind, single-center outpatient respectively, (3) the effect of prior YF immunity on the immune response to
study. In order to determine if YF immunity precludes vaccination ChimeriVax™-DEN2, and (4) the duration of the neutralizing antibody
with ChimeriVax™-DEN2, a group of YF-immune subjects has been response in all subjects up to 1 year post vaccination.
included in the study to receive ChimeriVax-DEN2 as an open-label After screening, 42 healthy adults 18–49 years of age, without prior
vaccine. immunity to YF, Japanese encephalitis (JE) or tick-borne encephalitis (TBE),
This is the first study of a chimeric YF-DEN vaccine in humans were randomized equally to 3 groups receiving a single vaccination on study
that shows that the vaccine is safe and immunogenic, and that Day 1 with either low or high dose ChimeriVax™-DEN2 (3.0 or 5.0 log10
PFU, respectively) or YF-VAX®. In addition, 14 subjects previously vacci-
preimmunity to YF does not interfere with ChimeriVaxTM-DEN2
nated against YF (27–29 months prior) received high dose ChimeriVax™-
vaccination, but induces high levels of cross-reactive and long-lasting DEN2 as an open-label vaccine (Table 1). Subjects returned to the clinic on
neutralizing antibodies against other DEN serotypes. Days 2-11, 21 and 31 for safety assessments, viremia, and antibody responses,
MATERIALS AND METHODS
and 6 and 12 months post-vaccination to measure durability of antibody
response. T cell responses were also measured on Days 1 and 31.
Safety was assessed based on adverse events (AEs; as assessed by sponta-
Vaccines. ChimeriVax™-DEN2. The vaccine virus was manufactured neous reporting, structured interview, and subject diary), vital signs, physical
by BioReliance Corp. (Rockville, MD) in accordance with current Good examination, and routine laboratory investigations (haematology, blood
Manufacturing Practices (cGMP). The virus was grown in Vero (African chemistry, and urinanalysis) at screening and on Days 11, 21 and 31 after
green monkey kidney) cells from cell banks that have been tested for adven- vaccination. Treatment-emergent AEs were defined as those starting or
titious agents, according to US Food and Drug Administration (FDA) increasing in severity after vaccination. Three physicians (Data Monitoring
guidelines for mammalian cell culture derived products. Supernatant fluid Committee) independent of the trial reviewed serious AEs and other trial
from Vero cell cultures containing vaccine virus was harvested, clarified from safety issues.
cellular debris by filtration, and treated with nuclease enzyme (Benzonase®) Measurement of viremia. On Days 2–11, sera were collected and frozen
to digest nucleic acids derived from host cells. The nuclease-treated bulk below -60˚C for viremia testing. ChimeriVax™-DEN2 and YF-VAX®
virus was then concentrated by ultrafiltration and purified by diafiltration. viremia titers were measured by direct plaquing in Vero cell monolayer
The vaccine (Lot# 01H01, titer 8.1 log10 PFU/ml) was formulated with grown in 12-well plastic tissue culture plates. Plaques were enumerated by
2.5% Human Serum Albumin (HSA) and 7.5% lactose in minimal essential staining Vero cell monolayers with crystal violet. The mean duration, mean
medium Eagle’s salt (MEME) without phenol red or L-glutamine (vaccine peak, and AUC of viremia induced by ChimeriVax™-DEN2 and YF-VAX®
diluent). Prior to inoculation, the vaccine was diluted with vaccine diluent were evaluated.
(Lot# 01H02) to contain 5.0 (high dose) and 3.0 (low dose) log10 PFU/0.5 ml, Measurement of immune response. Responses against DEN2 and YF
respectively, and administered to subjects by SC injection into the deltoid 17D viruses. Antibody responses were measured on Day 1 (prevaccination),
region. Day 31, and 6 and 12 months post vaccination. Two different neutralization
YF-VAX®. Commercial YF 17D vaccine (YF-VAX®, Batch# UB 102AA methods were performed at Acambis Inc. (Cambridge, MA): (1) PRNT50
and UB 132AA) was purchased from Sanofi-Pasteur (Swiftwater, PA). The (50% plaque reduction neutralization titer, constant virus with varying
lyophilized vaccine contained sorbitol and gelatin as stabilizers, and was serum dilution) in Vero cells against ChimeriVax™- DEN2 and wt strains
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3. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
Incidence of treatmenttreatment-related AEs, in terms of no. (%)
(approximately 50 PFU/well), and used to inoculate
Table 2
of subjects in each treatment group
triplicate wells of confluent LLC-MK2 cells. After
12 hours adsorption, cells were overlaid with
ChimeriVax TM - ChimeriVax TM - YF-VAX ® ChimeriVax TM - 5% CO2 atmosphere, at which time they were fixed
methylcellulose, and incubated for 7 days at 37˚C,
DEN2 5. DEN2 3.0 DEN2 5.0 with formaldehyde and plaques were visualized by
0log 10 PFU log 10 PFU log 10 PFU staining with crystal violet. Plaques were then
YF-Immune counted, and the PRNT50 is was determined by
using log probit analysis. The percent reduction of
N
plaques at each dilution level was plotted to determine
14 14 14 14
Any
the 50% reduction titer: plaque reduction points
9 (64.3) 10 (71.4) 11 (78.6) 12 (85.7) between 15% and 85% were used. Results were
AE in ≥ 2 subjects in any group expressed as reciprocal of dilution. A serum was
Fatigue 4 (28.6) 2 (14.3) 1 (7.1) 4 (28.6) considered to be positive for the presence of neutral-
Malaise 2 (14.3) 3 (21.4) 0 (-) 2 (14.3) izing antibodies when the neutralizing antibodies
Pyrexia 1 (7.1) 0 (-) 4 (28.6) 1 (7.1) titer thus determined is ≥1:10.
Rigors 1 (7.1) 1 (7.1) 1 (7.1) 4 (28.6) T-cell studies. Collection, processing and culture
Arthralgia 0 (-) 0 (-) 1 (7.1) 3 (21.4) of peripheral blood mononuclear cells (PBMC).
Myalgia 2 (14.3) 1 (7.1) 3 (21.4) 5 (35.7) The T cell response was evaluated on Days 1 and 31
Headache 9 (64.3) 5 (35.7) 8 (57.1) 6 (42.9) by measuring the production of IFNγ by PBMC
Photophobia 0 (-) 0 (-) 2 (14.3) 0 (-) stimulated in culture with inactivated ChimeriVax™-
Diarrhea 1 (7.1) 1 (7.1) 0 (-) 2 (14.3) DEN2 virus antigen produced by glutaraldehyde
Pharyngo- laryngeal pain 1 (7.1) 2 (14.3) 0 (-) 1 (7.1) fixation as previously described.15 Whole blood was
Rash 0 (-) 1 (7.1) 0 (-) 3 (21.4) collected in Vacutainer cell preparation tubes (CPT,
Injection site mass 0 (-) 0 (-) 2 (14.3) 0 (-) BDBiosciences) and sent to Acambis, Inc., for
Injection site pain 0 (-) 0 (-) 2 (14.3) 0 (-)
isolation and cryopreservation of PBMC. Cells
Symptom Index (mean ± SD) 2.9 ± 4.0 2.4 ± 2.8 2.6 ± 2.7 4.4 ± 4.8
were washed in RPMI 1640, cryopreserved in heat-
inactivated human AB serum (SeraCare, Oceanside
CA) containing 10% DMSO, stored in liquid
of DEN2 (PR159, isolated in Puerto Rico in 1964, representing the nitrogen, and thawed immediately before testing. For measuring IFNγ
American genotype I; and JaH, isolated in Jamaica in 1982, representing the production, PBMC were cultured in RPMI 1640 containing 10% heat-
American genotype II), and (2) LNI (log neutralization index, constant serum inactivated human AB serum in 96-well flat bottom plates at 1.5 x 105 cells
with varying virus dilution; routinely used for measurement of responses to per well for 7 days at 37˚C with glutaraldehyde-inactivated Chimeri-Vax™-
many live viral vaccines including YF 17D) in Vero cells against YF 17D DEN2 virus-infected Vero cell antigen. Controls consisted of inactivated
virus. The titer is defined as LNI, which is the log10 virus titer difference mock-infected Vero cells. Inactivated viral antigen or control Vero cell antigen
between serum-virus mixture for the test sample and a negative control was added at a concentration of 1:100.15 PBMC were also stimulated with
serum.11,12 1 µg/ml ConA as an assay positive control. IFNγ production was determined
For PRNT, sera were heat-inactivated (at 56˚C for 30 minutes), serially by ELISA using culture supernatants collected on Day 7.
diluted (2-fold), mixed with an equal volume of DEN virus (approximately IFNγ ELISA. Culture supernatant samples from Day 7 were analyzed for
50 PFU/well), and used to inoculate duplicate wells of confluent Vero cells. IFNγ content at three dilutions (1:2, 1:10, 1:50) by an indirect ELISA assay
After 1 hour adsorption, cells were overlaid with methylcellulose and (OptEIATM human IFNγ Kit, BDBiosciences, San Diego, CA) according to
incubated for 4 days (37˚C, 5% CO2 atmosphere), at which time they were the manufacturer’s instructions (assay range 5–300 pg/ml). Plates were read
fixed with formaldehyde. Plaques were stained by incubation with 3H5 at 450 nm (reference wavelength 570 nm) on a VersaMax Spectrophotometer
(anti-DEN2) monoclonal antibody solution, and sequentially incubated (Molecular Devices Corp., Sunnyvale, CA). Standard curves were generated,
with goat anti-mouse IgG-alkaline phosphatase conjugate, followed by and IFNγ concentrations were calculated using Softmax Pro software.
5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (BCIP/NBT) Statistical methods. Analysis population. All subjects who received at
substrate solution. The titer (PRNT50) was calculated from the highest dilution least one vaccination were included in the safety population. The Per
of test serum reducing the mean plaque count (of duplicate wells) by ≥50%, Protocol (PP) population was defined as subjects who received at least one
compared to the mean value of a standard normal control serum.13 A serum vaccination with double-blind study medication, who provided serum
is considered to be positive for the presence of neutralizing antibodies when samples at least up to Day 31, and who had no significant protocol deviations
the neutralizing antibody titer thus determined is at least superior or equal identified prior to unblinding.
to 1:10. For LNI, 10-fold dilutions of YF17 D virus (commercial YF-VAX® Safety evaluation. Adverse events. Treatment-emergent AEs were coded
vaccine passaged once in Vero cells) were mixed with heat inactivated test according to the MedDRA (Medical Dictionary for Regulatory Activities)
sera. The mixture was used to inoculate Vero cells. Plaques were visualized version 4.0, and tabulated by system organ class, preferred term, and severity
by staining the monolayer with crystal violet.11,12 for each treatment group. The incidence of all treatment-emergent AEs and
Responses against prototype strains of DEN 1- 4. This PRNT was treatment-emergent AEs considered related to the study drug (i.e., rated
performed in LLC-MK2 cells at the Center for Vaccine Development, possibly, probably or definitely related) were described between treatment
Institute of Sciences and Technology, Mahidol University (Thailand). The groups. A symptom index was calculated for each subject as the sum of the
DEN 1-4 strains selected are those currently used to standardize the DEN maximal duration x severity scores for fever/chills, myalgia/arthralgia,
neutralization test under a research program sponsored by the World Health rash/pruritus, eye pain/conjunctivitis, headaches and malaise.
Organization. Wild type (wt) strains included: DEN1 (strain 16007, isolated Viremia. For analysis purposes, the absence of detectable virus in serum
in Thailand in 1964), DEN2 (strain 16681, isolated in Thailand in 1964), was designated as value “0”, even though the lowest detectable value based
DEN3 (strain 16562, isolated in the Philippines in 1964), and DEN4 on the test method used was 10 PFU/mL. Mean duration, mean peak, and
(strain 1036, isolated in Indonesia in 1967). The test was performed according AUC of viremia induced by ChimeriVax™-DEN2 and YF-VAX® were
to Russell et al.14 Sera were heat-inactivated (at 56˚C for 30 minutes), calculated per group. The difference between treatment groups for each
serially diluted (4-fold), mixed with an equal volume of DEN viruses measure of viremia (peak, duration, and AUC) was compared using an
e3 Human Vaccines 2006; Vol. 2 Issue 2

4. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
Summary of Viremia
Analysis of Variance (ANOVA) model.
Table 3
A residual analysis was performed in
Treatment YF-VAX ® 5.0 log 10 ChimeriVax TM - ChimeriVax TM - ChimeriVax TM -
order to check that the underlying
Group PFU a YF-naïve DEN2 3.0 log 10 DEN2 5.0log 10 DEN2 5.0 log 10
assumptions of normality and equality
PFU YF-naïve PFU YF-naïve PFU YF-immune
of variance were met within the model.
If the assumptions were violated, suitable
transformation of the data or alternative No. subjects 14 14 14 14
nonparametric modeling methods were
No. (%) subjects viremic 2 (14.3) 9 (64.3) 8 (57.1) 11 (78.6)
conducted. The Fisher’s Exact Test was
used to compare the proportion of Peak (PFUa/mL) [SD] 20.0 [51.44] 11.4 [12.31] 12. 1 [16.72] 29.3 [38.72]
subjects viremic on one or more study Duration (Days) [SD] 0.4 [1.16] 1.2 [1.42] 1.4 [1.65] 1.9 [1.23]
days between treatment groups. AUCb (PFU/mL) [SD] 44.3 [116.86] 20.0 [33.74] 20.7 [32.04] 50.4 [67.61]
Immunogenicity evaluation. Sero-
conversion rates on Day 31 to the a PFU, plaque-forming units, measured in Vero cell cultures. b Area under the curve.
respective strain were compared
between treatment groups for each of the
strains using Fisher’s Exact Test. For Table 4 Seroconversion rate (%) by treatment group, Day 31
GMT calculation, antibody level <10
was converted into the whole number Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM
minus one (i.e., 9). GMTs on Days 1, Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 -DEN2 5.0 log 10
Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune
31, 6 and 12 months were summarized
N = 13 N = 13 N = 14
by treatment group. The difference in
GMTs on Days 1 and 31 were compared
between treatment groups using an DEN2: strain 16681 0% 92.3% 100% 100%
ANOVA model. The seroconversion rate DEN2: ChimeriVax-D2 0 100 100 100
was defined as the proportion of subjects
DEN2: strain PR-159 0 84.6 92.3 100
without neutralizing antibody at
baseline (Day 1) who developed DEN2: strain JaH 0 92.3 92.3 100
PRNT50 of ≥1:10 at each timepoints (for DEN1: strain 16007 0 23.1 23.1 100
response against DEN antigen) or LNI DEN3: strain 16562 0 23.1 15.4 100
of ≥0.7 day31 minus day1 (for response
DEN4: strain 1036 0 0 0 100
against YF antigen).
YF 17D strain 92.3% 0 0 ND
RESULTS ND: Not determined
Subject population. Fifty-six subjects,
14 per treatment group, were randomized, vaccinated, and completed the reported laboratory abnormality was increased Creatine Kinase (CK) in five
31-day follow-up. All 56 subjects were included in the safety population; 53 subjects, which was noted to be coincidental with muscle strain or injury
subjects (14 in the 5.0 log10 PFU YF-immune group and 13 in each of the associated with recreational activities in all incidents. All elevations of liver
other treatment groups) were included in the PP population. Three subjects enzymes were minimal and unrelated to significant clinical symptoms.
were excluded from the PP population (one from each YF naïve group) due Viremia. Magnitude, duration, and AUC of viremia in subjects during
to prior YF immunity (i.e., LNI ≥0.7 on Day 1). the 11 days after vaccination are shown in Table 3. More YF-naïve subjects
Overall and within each group, the majority of subjects were female vaccinated with ChimeriVax™-DEN2 than with YF-VAX® developed
(41/56, 73.2%) and Caucasian (50/56, 89.3%). The treatment groups were viremia on one or more study days: 8 (57.1%) in the ChimeriVax™-DEN2
similar in respect to mean age and body mass index (BMI). 5.0 log10 PFU group and 9 (64.3%) in the ChimeriVax™-DEN2 3.0 log10
Safety. No serious AEs (SAEs) were reported during the study. Overall, PFU group, compared with 2 (14.3%) in the YF-VAX® group. Peak viremia
the most frequent individual treatment-emergent AEs (in any group) were (p = 0.038) and AUC (p = 0.039) were significantly higher, and duration of
headache (62.5%), myalgia (39.3%), and fatigue (28.6%). Table 2 presents viremia (p = 0.022) was significantly shorter in subjects vaccinated with
the incidence of treatment-related AEs and symptom index in the different YF-VAX® than with ChimeriVax™-DEN2 3.0 log10 PFU. There was no
groups. The profile of AEs following vaccination with ChimeriVax™- statistically significant difference between the two dose groups of
DEN2 was similar to that with YF-VAX®. The incidence of related AEs was ChimeriVax™-DEN2 with respect to peak (p = 0.084), duration (p = 1.000),
slightly higher in YF-immune than YF-naïve subjects vaccinated with or AUC (p = 0.98) of viremia. Slightly higher numbers of YF-immune
ChimeriVax™-DEN2 high dose (85.7 vs. 64.3%, respectively), with a subjects, compared with YF-naïve subjects, developed viremia following
higher incidence of myalgia, arthralgia, rash, and rigors, and a lower incidence vaccination with ChimeriVax™-DEN2 5.0 log10 PFU (11/14, p = 0.472).
of headache. Six subjects (1 YF-naïve, 1 YF-immune subject in the Most subjects developed viremia within 8 days after vaccination.
ChimeriVax™-DEN2 high dose group, and all 4 YF-naïve subjects vaccinated The number of subjects with viremia (p = 0.472), viremia mean peak
with YF-VAX®) presented pyrexia considered related to the study treatment. (p = 0.148), duration of viremia (p = 0.236), and AUC (p = 0.091) tended
Body temperature remained lower below 38˚C. Most AEs were of mild to to be higher in YF-immune subjects following ChimeriVax™-DEN2
moderate intensity. Two subjects, one in the ChimeriVax™-DEN2 low 5.0 log10 PFU than in YF-naïve subjects. However, none of these values
dose and one in the YF-VAX® group, experienced severe headache possibly were significant in this small sample size. The higher level of viremia in
related to the vaccine for one day. Most AEs resolved within 1–4 days. The YF-immune subjects raised concerns about an increased risk of AEs; it did
symptom index was low among different groups. There were no obvious not, however, exceed the viremia level from YF vaccination.
patterns for shifts in haematology, blood chemistry, or urinanalysis variables. Immunogenicity. Response 30 days after vaccination. All (100%) and
Ten subjects (4 YF-immune subjects (29%) in the ChimeriVax™-DEN2 12/13 (92.3%) of YF-naïve subjects inoculated with 5.0 and 3.0 log10PFU
high dose group and 2 YF-naïve (14%) each in the three treatment groups) of ChimeriVax™-DEN2, respectively, seroconverted to wt DEN2 strain
had abnormal laboratory values reported as AEs. The most commonly 16681 (Table 4) with similar GMT (Table 5). Seroconversion rates and
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5. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
GMTs for DEN PRNT tests and Mean titers for YF LNI by treatment group,
serotypes on Day 31 were significantly
Table 5
Day 31
higher in YF-immune subjects vaccinated
with ChimeriVax™-DEN2 than in YF-
Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM -
naïve subjects. For DEN1, GMTs in
Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 DEN2 5.0 log 10
YF-immune subjects, and YF-naïve
Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune
subjects vaccinated with either 5.0 or
N = 13 N = 13 N = 14
3.0 log10 PFU ChimeriVax™-DEN2
were 79.2 vs. 10.1 and 12, respectively
(p < 0.0001). Similarly, for DEN3, titers
DEN2: strain 16681 <10 365.0 358.6 383.3
were 73.2 vs. 13.2 and 11.8 (p < 0.0001)
DEN2: ChimeriVax-D2 <10 570.0 921.3 975.4 (Table 5). None of the YF-naïve subjects
DEN2: strain PR-159 <10 313.8 218.3 724.5 seroconverted to DEN4. GMT to DEN4
DEN2: strain JaH <10 227.8 240.3 463.9 in YF-immune subjects was 57.3.
Response 6 and 12 months after
DEN1: strain 16007 <10 12.0 10.1 79.2
vaccination. One hundred percent of
DEN3: strain 16562 <10 11.8 13.2 73.2 subjects vaccinated with Chimerivax™-
DEN4: strain 1036 <10 <10 <10 57.3 DEN2 were seropositive against wt
YF17D strain <11.968 <10.025 -0.064 0.676 DEN2 strain 16681 at 6 and 12
months after vaccination (Tables 6
and 8). At these timepoints, GMT
Table 6. Proportion Seropositive (%) by treatment group, 6 months
remained high, with levels between
183.3 and 744.1 at 12 months (Tables
7 and 9). Seropositivity rates and GMT
Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM - against DEN2 strains PR-159 and JAH
Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 DEN2 5.0 log 10 decreased steadily until 1 year after
Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune vaccination, and were the highest in the
N = 13 N = 13 N = 14 YF-immune group. These two strains
are from the Americas, and belong to
DEN2: strain 16681 0% 100% 100% 100 two distinct variant groups (America I
DEN2: ChimeriVax-D2 0 100 100 100 and II, respectively).
DEN2: strain PR-159 0 84.6 76.9 92.9 At 6 and 12 months, 100% of
YF-immune subjects inoculated with
DEN2: strain JaH 0 76.9 69.2 92.9
ChimeriVax™-DEN2 remained sero-
DEN1: strain 16007 0 30.8 23.1 100 positive to DEN1 and DEN3, while
DEN3: strain 16562 0 23.1 15.4 100 64.3% and 28.6% were seropositive to
DEN4: strain 1036 0 7.7 7.7 64.3 DEN4 at 6 and 12 months, respectively.
In these subjects, GMTs against DEN1
and 3 decreased steadily but remained
relatively high at 12 months (89.2
Table 7. GMTs by treatment group, 6 months and 71.8 against DEN1 and DEN3,
respectively).
Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM - T cell response. T cell responses were
Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 DEN2 5.0 log 10 evaluated in cell culture by IFNγ
Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune production in response to inactivated
N = 13 N = 13 N = 14 viral cell lysate, which has been shown
to generate primarily CD4+ T cell
DEN2: strain 16681 <10 568.6 285.1 870.2 responses to the vaccine.16
DEN2: ChimeriVax-D2 <10 606.8 303 672 IFNγ cytokine production. IFNγ
DEN2: strain PR-159 <10 55.1 49.5 160
cytokine production was compared on
Days 1 and 31 of the study (before
DEN2: strain JaH <10 29.0 24.7 72.5 vaccination and on Day 30 after vacci-
DEN1: strain 16007 <10 14.4 <10 285.1 nation) by testing the response to
DEN3: strain 16562 <10 <10 <10 268.1 inactivated ChimeriVaxTM-DEN2 virus
DEN4: strain 1036 <10 <10 <10 23.8 antigen. Comparisons between vaccina-
tion groups were made using the differ-
ence between values on Days 31 and 1
GMTs against other DEN2 strains were the highest in the YF-immune (Fig. 1). All groups responded to the inactivated antigens (IFNγ responses
group. Ninety-two (92.3%) of subjects inoculated with YF-VAX® serocon- ranged from undetected to over 7,500 pg/ml). Subjects who received 3.0 or
verted to YF 17D virus. None of YF naïve subjects inoculated with Chimeri- 5.0 log10 PFU of ChimeriVaxTM-DEN2 vaccine had equivalent IFNγ levels
Vax™-DEN2 seroconverted to YF 17D virus. In the YF-immune group (geometric mean IFNγ response of 161 and 168 pg/ml), and Chimeri-
who received ChimeriVax™-DEN2, 2 subjects had a boost in YF antibodies. VaxTM-DEN2 vaccinated subjects had slightly greater responses than YF
Low seroconversion rates (not higher than 23.1%) to heterologous DEN vaccinated subjects (geometric mean IFNγresponse of 96 pg/ml [not signif-
serotypes 1, 3, and 4 were observed in YF-naïve subjects inoculated with icant, p = 0.565 vs. 3.0 log10 PFU or p = 0.505 vs. 5.0 log10 PFU]).
ChimeriVax™-DEN2 at high or low dose. In contrast, 100% of YF- YF-immune subjects who received the 5.0 log10 PFU dose vaccine had an
immune subjects inoculated with ChimeriVax™-DEN2 seroconverted to increased number of IFNγ responders and increased geometric mean IFNγ
all heterologous DEN serotypes. GMTs against heterologous DEN levels (geometric mean IFNγ response of 578 pg/ml) relative to naïve
e5 Human Vaccines 2006; Vol. 2 Issue 2

6. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
Proportion seropositive (%) by treatment group, 12 months
subjects receiving either 3.0 log10 PFU
(not significant, p = 0.3231) or 5.0 Table 8.
Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM -
log10 PFU of ChimeriVaxTM-DEN2 (not
Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 DEN2 5.0 log 10
significant, p = 0.1827).
Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune
About 65% (9/14) of the Chimeri-
N = 13 N = 13 N = 14
VaxTM-DEN2TM and YF vaccinated
subjects had a positive IFNγ response to
the administered vaccine as test antigen,
DEN2: strain 16681 0% 100% 100% 100
whereas about 90% (13/14) of YF-
immune subjects vaccinated with DEN2: ChimeriVax-D2 0 100 100 100
ChimeriVaxTM-DEN2 had a positive DEN2: strain PR-159 0 69.2 69.2 92.9
response. Positive response was defined DEN2: strain JaH 0 61.5 53.8 85.7
as 5-fold background or ≥ 50 pg/ml on DEN1: strain 16007 0 30.8 23.1 100
Day 30, if Day 1 was less than 10 pg/ml
(below sensitivity of the ELISA assay). DEN3: strain 16562 0 23.1 7.7 100
DISCUSSION
DEN4: strain 1036 0 0 7.7 28.6
the evaluation of safety and tolera- Table 9. GMTs by treatment group, 12 months
This was the first clinical trial for
single dose vaccine of 5.0 or 3.0 log10 Virus Used in YF-VAX ® ChimeriVax TM - ChimeriVax TM - ChimeriVax TM -
bility of ChimeriVax™-DEN2 as a
Neutralization YF-naïve DEN2 3.0 log 10 DEN2 5.0 log 10 DEN2 5.0 log 10
immune, healthy subjects. There is Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune
PFU in both YF-naïve and YF-
a theoretical risk of sensitization of N = 13 N = 13 N = 14
subjects to DHF on exposure to a DEN2: strain 16681 <10 368.9 183.3 744.1
heterologous DEN serotype after DEN2: ChimeriVax-D2 <10 272.7 272.7 320.0
immunization with a monovalent DEN2: strain PR-159 <10 42.2 30.6 72.5
vaccine. This risk is mitigated by
DEN2: strain JaH <10 18.0 14.5 32.8
conducting the trial in the Midwest
of the United States, which is not DEN1: strain 16007 <10 13.1 10.1 89.2
endemic for dengue. Trials of live DEN3: strain 16562 <10 <10 <10 71.8
attenuated monovalent dengue DEN4: strain 1036 <10 <10 <10 <10
vaccines have been previously
performed in the USA, with the
same theoretical concern regarding sensitization of subjects to DHF.
They were also conducted in adults, who have a lower risk for
DHF/DSS than children living in endemic areas. To reduce risk
further, the trial excluded subjects who frequently travel to dengue
endemic areas. Subjects were advised of the potential risk of
enhanced disease if exposed to DEN viruses in the future.
The high dose of ChimeriVax™-DEN2 used in the current trial
(5.0 log10 PFU) corresponds to the routinely administered standard
dose of YF-VAX®, and was based on the expectation that
ChimeriVax™-DEN2 would have a similar replication efficiency in
humans than the YF 17D virus used as live vector for DEN prM-E
genes. The lower dose (3.0 log10 PFU) was selected based on
preclinical data in nonhuman primates: a tetravalent formulation
containing all 4 chimeric viruses required a 3-log dose of
ChimeriVax™-DEN2 and a higher dose of the other 3 viruses to Figure 1. IFNγ responses to vaccine (study Day 31 minus Day 1). YF:
YF-VAX®. CVx3: ChimeriVax-DEN2 low dose group (3.0 log10 PFU). CVx5:
avoid possible interference effects.9 ChimeriVax-DEN2 high dose group (5.0 log10 PFU). YF-CVx5: YF-immune,
Adverse events were anticipated to resemble those associated with ChimeriVax-DEN2 high dose group. Bars represent the geometric mean.
ChimeriVax™-JE. Data from Phase 1 and 2 trials of this vaccine Values ≤ 10 pg/ml (the sensitivity of the IFNγ ELISA assay) were plotted as
indicated that it was well tolerated and highly immunogenic at all 5 pg/ml.
dose levels tested (5.8 to 1.8 log10 PFU).17 We found no unexpected
safety concerns with ChimeriVax™-DEN2 vaccinations. The most commonly reported laboratory abnormality was increased CK
profile of AEs observed following ChimeriVax™-DEN2-vaccination related to muscle injury. Other liver enzyme elevations (AST and
was similar to that observed after vaccination with YF-VAX®. There ALT) were minor and unrelated to clinical syndromes.
was some evidence suggesting a dose-response relationship for The transient low level of viremia following administration of
ChimeriVax™-DEN2 tolerability, with a higher incidence of ChimeriVax™-DEN2 vaccine was not closely associated with symp-
headache and fatigue. However, most were mild to moderate in toms or signs of ill health. No differences were observed between
intensity and transient, generally resolving within 1–4 days. The viremia induced with either dose of ChimeriVax™-DEN2 viruses.
www.landesbioscience.com Human Vaccines e6

7. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
More subjects vaccinated with ChimeriVax™-DEN2 than with Similar to our observation, boosted DEN heterologous responses
YF-VAX® developed viremia. However, the magnitude and AUC were observed in YF-immune subjects vaccinated with live attenuated
(but not duration) of viremia in these subjects were lower than with DEN vaccines.22 The short-term (to Day 30) antibody responses were
YF-VAX® (not significant). The viremia profiles of the two groups demonstrated with antibody assays including neutralization, but the
(YF-naive and YF-immune) receiving 5.0 log10 PFU of Chimeri- authors concluded that evidence for protection against subsequent
Vax™-DEN2 clearly indicated that preexisting YF immunity did DEN virus infection was inconclusive. Unlike the present study, the
not hinder the replication of vaccine virus, and even the number of authors could not demonstrate conclusively the prior timing or
subjects with viremia as well as viremia levels tended to be higher in receipt of YF vaccination, long-term broad neutralization antibody
YF-immune subjects. However, this trend was not associated with an responses, or provide evidence for cross-reactive T cell responses to
increased risk of AEs. Similarly, the preimmunity to YF 17D did not DEN.23 Simultaneous administration of attenuated YF and DEN
interfere with ChimeriVax™-JE vaccination, In sum, Chimeri- vaccines to humans seemed to have modified fever and general
Vax™-DEN2 vaccine induced a low level of viremia with a transient symptoms, and had increased antibody responses to both viruses.24
duration in all subjects. Moreover, initial vaccination with YF followed by natural DEN
Immunogenicity was assessed based on the neutralizing antibody infection or administration of an attenuated DEN vaccine boosted
response, represented as the proportion of subjects seroconverted to antibodies to both viruses.25 Scott et al26 showed that subjects who
wt strain 16681 (ChimeriVax™-DEN2 groups) or YF 17D virus were previously immunized with YF and subsequently inoculated
(YF-VAX® group) by PRNT50, 30 days after vaccination. Both with a live, attenuated DEN2 vaccine had enhanced immune
doses of ChimeriVax™-DEN2 demonstrated high levels of neutral- responses to DEN2, which were also more durable (lasting 3 years)
izing antibodies against DEN2 (i.e., homologous strain), similar to than in subjects without previous YF immunity. However, because
the level of neutralizing antibodies induced by ChimeriVax™-JE the immune responses had not been measured by neutralization test,27
against JE virus17 and the level of neutralizing antibodies induced by which is the only test that predicts protective immunity, it is not clear
ChimeriVax™-WN against WN virus (data not shown). The whether the enhanced response might have been due to enhancing
magnitude was such that dose-response was not evident even against (binding, nonneutralizing) antibodies elicited to DEN2 virus by the
other wt strains of DEN2 virus (e.g., PR-159 and JaH) or from preceding YF vaccination. In addition, Scott et al did not show that
assessment of GMTs. The fact that this vaccine was highly effective YF followed by DEN2 vaccines elicited a long-lasting immune
against several important genotypes of DEN2 is very encouraging. response to the other three DEN serotypes (1, 3, or 4). In our study,
Cross-reactivities with other DEN serotypes were limited in YF naïve YF-immune subjects had been inoculated with YF vaccine 24-29
subjects. However, when subjects were primed with YF-VAX®, the months prior to inoculation with Chimerivax™-DEN2. A shorter
level of cross-reactive neutralizing antibodies against the other 3 DEN interval between these 2 vaccinations could have the same positive
serotypes was increased and lasted up to one year (the last sampling effect: in a recent study, YF vaccination only 5 months prior to
time). The highest and lowest cross-reactive neutralizing antibodies inoculation with a ChimeriVax™-DEN tetravalent vaccine also
were directed against DEN1 and DEN 4 viruses, respectively. The increased the neutralizing antibody response to DEN viruses,
GMT against DEN4 virus in YF-immune subjects was still in the compared to nonimmune subjects (unpublished data). While YF
positive range (23.8) when measured at 6 months post immunization, immunity boosted the response to DEN viruses after ChimeriVax™
but it dropped to <10 after 1 year. The anamnestic response among vaccination, the reciprocal was not true (ChimeriVax™-DEN2 did
YF-immune subjects could have great implications for the development not boost antibodies against YF virus). Of 14 YF immune subjects
of an immunization strategy for a tetravalent vaccine by avoiding the who received ChimeriVax™-DEN 2, only 2 (14%) had a boost in
potential interference problem that presenting a single YF-DEN YF antibody, and one of these had a very marginal titer at baseline
chimeric virus poses to the host. In this instance, the induction of
(LNI 0.65; data not shown).
interferon occurs in concert with virus replication and is modulated
Flavivirus cross-reactive epitopes have been located within
effectively by the YF nonstructural proteins18,19 to allow sufficient
domain II (formerly A), whereas type- or subtype-specific epitopes
replication required for immunization. Since the initial immunizing
have been found in domains I and III (formerly C and B) of TBE
agent (YF) is incapable of sensitizing the subject to DHF, there will
virus.28,29,30 Monoclonal antibodies recognizing epitopes within
be no danger that the first (priming) inoculation would leave the
subject vulnerable to this disease if the second injection was delayed domain II exhibited strong HI and neutralizing activities, whereas
or not administered. Across YF vaccines, the sharing of genes those binding to epitopes within domains I and III exhibited weak
controlling T cell immunity and the chimeric DEN vaccine containing HI and neutralizing activities.31 It has been shown that binding of
an YF vector backbone might be the postulate for the unique immu- TBE monoclonal antibodies to epitopes within domains I and III
nizing capacity of the sequential immunization regimen. enhanced (in unidirectional way) the binding of antibodies to
There are precedents for sequential immunization. In YF- domain II.28,32 It is possible that prior YF infection had created a
immune individuals who received inactivated TBE vaccine, population of B cells recognizing a broad array of cross-reactive
anti-TBE IgG antibodies appeared earlier and in higher titers than DEN epitopes. Enhancing antibodies were found in sera of YF-
in nonYF-immune subjects. In addition, a broad spectrum flavivirus immune subjects taken before vaccination with a live attenuated
hemagglutination inhibition (HI) response in all subjects and low DEN2 vaccine. The levels of seroconversion to DEN2 were signifi-
titer of neutralizing response against DEN2 in some subjects were cantly higher in YF-immune subjects with enhancing antibodies
developed.20 Price et al.21 previously described a method for sequen- compared to nonimmune subjects.27 However, the opposite was not
tial flavivirus immunization, comprising a series of three immunizations true, since prevaccination with DEN2 vaccine did not increase the
with DEN2 and two heterologous viruses (YF and JE). However, seroconversion rate against YF vaccine.8,33,34
unlike the present study, the sequence of YF followed by The T-cell responses in this clinical trial were consistent with the
DEN2, without the addition of JE immunization, failed to confer neutralizing antibody responses in that both doses of vaccine stimu-
cross-protective immunity. lated similar T cell immune responses, and prior immunity to YF
e7 Human Vaccines 2006; Vol. 2 Issue 2

8. Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine
virus did not inhibit the T cell response to ChimeriVax™-DEN2. 12. Monath TP, Nichols R, Archambault WT, Moore L, Marchesani R, Tian J, Shope RE,
Thomas N, Schrader R, Furby D, Bedford P. Comparative safety and immunogenicity of
IFNγ responses were virtually the same for the 2 doses of Chimeri- two yellow fever 17D vaccines (ARILVAX™ and YF-VAX®) in a Phase III multicenter,
Vax™-DEN2 (103 and 105 pfu). In addition, the IFNγ response to double-blind clinical trial. Am J Trop Med Hyg 2002b; 66:533-41.
ChimeriVax™-DEN2 was not diminished by prior vaccination 13. Guirakhoo F, Pugachev K, Zhang Z, Myers G, Levenbook I, Draper K, Lang J, Ocran S,
Mitchell F, Parsons M, Brown N, Brandler S, Fournier C, Barrere B, Rizvi F, Travassos A,
against YF virus and even higher numbers of responders were seen, Nichols R, Trent D, Monath T. Safety and efficacy of chimeric yellow Fever-dengue virus
suggesting a trend for enhanced T cell immunity in YF preimmune tetravalent vaccine formulations in nonhuman primates. J Virol 2004; 78(9):4761-75.
subjects. The T cell response generated to inactivated virus antigen 14. Russell PK, Nisalak A, Sukhavachana P, Vivona S. A plaque reduction test for dengue virus
neutralizing antibodies. J Immunology 1967; 99:291-6.
probably represents CD4+ responses to both DEN structural and YF
15. Kurane I, Innis BL, Nisalak A, Hoke C, Nimmannitya S, Meager A, Ennis FA. Human T
nonstructural proteins. Inactivated DEN viral lysate produces cell responses to dengue virus antigens: Proliferative responses and interferon γ production.
primarily CD4+ responses in naturally infected subjects16 and in J Clin Invest 1989; 83:506-13.
subjects who have received live attenuated DEN vaccines.35 16. Mangada MM, Ennis FA, Rothman AL. Quantitation of dengue virus specific CD4+ T
cells by intracellular cytokine staining. J Immunol Methods 2004; 284:89-97.
However, this study did not determine the specific proteins against 17. Monath TP, Guirakhoo F, Nichols R, Yoksan S, Schrader R, Murphy M, Blum P,
which the immune response was generated. Recent studies in Woodward S, McCarthy K, Mathis D, Johnson C, Bedford P. Chimeric live, attenuated
patients with secondary DEN virus infection using overlapping vaccine against Japanese encephalitis (ChimeriVax™-JE): Phase 2 clinical trials for safety
and immunogenicity, effect of vaccine dose and schedule, and memory response to chal-
DEN peptide sequences have shown that T cell responses were lenge with inactivated Japanese Encephalitis antigen. J Infect Dis 2003; 188:1213-30.
generated to both DEN structural and non-structural proteins.36 18. Liu WJ, Wang XJ, Mokhonov VV, Shi PY, Randall R, Khromykh AA. Inhibition of inter-
In conclusion, this single dose monovalent ChimeriVax™- feron signaling by the New York 99 strain and Kunjin subtype of West Nile Virus involves
blockage of STAT1 and STAT2 activation by nonstructural proteins. J Virol 2005;
DEN2 vaccine has been well tolerated, with a safety profile consistent 79:1934-42.
with that of YF-VAX®, and comparable immunogenicity to the 19. Guo JT, Hayashi J, Seeger C. West Nile Virus inhibits the signal transduction pathway of
respective target wt virus strains. Cross-reactivity to other DEN alpha interferon. J Virol 2005; 79:1343-50.
serotypes was low in YF naïve subjects immunized with ChimeriVax™- 20. Kayser M, Klein H, Paasch I, Pilaski J, Blenk H, Heeg K. Human antibody response to
immunization with 17D yellow fever and inactivated TBE vaccine. J Med Virol 1985;
DEN2. However, preimmunity to YF-VAX® enhanced the level of 17(1):35-45.
durable DEN cross-reactive neutralizing antibodies, which may be 21. Price WH. Sequential immunization as a vaccination procedure against dengue viruses. Am
useful in designing a strategy for a dengue vaccine. Nevertheless, the J Epidemiology 1968; 88:392-397.
current ChimeriVax™-DEN2 vaccine is destined to contribute to 22. Kanesa-thasan N, Sun W, Ludwig GV, Rossi C, Putnak JR, Mangiafico JA, Innis BL,
Edelman R. Atypical antibody responses in dengue vaccine recipients. Am J Trop Med Hyg
the development of a tetravalent vaccine. For this purpose, 2003; 69(Suppl 6):32-38.
ChimeriVax™-DEN2 is believed to be safe and immunogenic, 23. Kurane I, Okamoto Y, Dai LC, Zeng LL, Brinton MA, Ennis FA. Flavivirus-cross-reactive,
warranting further development towards a market-ready DEN vaccine. HLA-DR15-restricted epitope on NS3 recognized by human CD4+ CD8- cytotoxic T
lymphocyte clones. J Gen Virol 1995; 76:2243-9.
Acknowledgements 24. Dorrance WR, Frankel JW, Gordon I, Patterson PR, Schlesinger RW, Winter JW. Clinical
Authors would like to thank all clinical and laboratory staff at and serologic response of man to immunization with attenuated dengue and yellow fever
viruses. J Immunol 1956; 77(5):352-64.
Acambis, Inc., Bio-Kinetic Clinical Application, Inc., and Mahidol 25. Carey DE, Myers RM, Rodrigues FM. Two episodes of dengue fever, caused by types 4 and
University, Thailand, especially Alison Deary for clinical trial 1 viruses, in an individual previously immunized against yellow fever. Am J Trop Med Hyg
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26. Scott RM, Eckels KH, Bancroft WH, Summers PL, McCown JM, Anderson JH, Russell
Dr. Bruno Guy, Sanofi-Pasteur, for helpful discussion on T-cell studies. PK. Dengue 2 vaccine: Dose response in volunteers in relation to yellow fever immune sta-
This work was supported by Sanofi-Pasteur, Marcy-L'Etoile, France. tus. J Infect Dis 1983; 148(6):1055-60.
27. Eckels KH, Kliks SC, Dubois DR, Wahl LM, Bancroft WH. The association of enhancing
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