Review
Progress towards a dengue vaccine
Lancet Infect Dis 2009; 9: 678–87
See Newsdesk page 662
Department of Infectious
Diseases and Microbiology,
John Radcliffe Hospital, Oxford,
UK (D P Webster FRCPath);
Hospital for Tropical Diseases,
Wellcome Trust Major Overseas
Programme, Oxford University
Clinical Research Unit, Ho Chi
Minh City, Vietnam
(Prof J Farrar FRCP); and Human
Immunology Unit, The
Weatherall Institute of
Molecular Medicine, University
of Oxford, Oxford, UK
(Prof S Rowland-Jones FRCP)
Correspondence to:
Dr Daniel P Webster, Department
of Infectious Diseases and
Microbiology, John Radcliffe
Hospital, Oxford OX3 9DU, UK
danwebster@doctors.org.uk
678
Daniel P Webster, Jeremy Farrar, Sarah Rowland-Jones
The spread of dengue virus throughout the tropics represents a major, rapidly growing public health problem with
an estimated 2·5 billion people at risk of dengue fever and the life-threatening disease, severe dengue. A safe and
effective vaccine for dengue is urgently needed. The pathogenesis of severe dengue results from a complex
interaction between the virus, the host, and, at least in part, immune-mediated mechanisms. Vaccine development
has been slowed by fears that immunisation might predispose individuals to the severe form of dengue infection.
A pipeline of candidate vaccines now exists, including live attenuated, inactivated, chimeric, DNA, and viral-vector
vaccines, some of which are at the stage of clinical testing. In this Review, we present what is understood about
dengue pathogenesis and its implications for vaccine design, the progress that is being made in the development
of a vaccine, and the future challenges.
Introduction genus.13 Aedes aegypti, which exploits peridomestic water
Dengue virus infection represents a major, growing public containers as larval habitats, is by far the predominant
health problem with an estimated 2·5 billion people at risk vector of dengue, although Aedes albopictus can also
of infection in tropical and subtropical countries, mainly sustain transmission.14 A aegypti are highly adapted to
southeast and south Asia, Central and South America, and urban environments, and uncontrolled urbanisation is
the Caribbean.1 There are few data on the incidence of the rarely accompanied by investment in basic infrastructure
disease available from Africa. This growth is largely and public health measures,15 particularly in Asia and
explained by increases in human population, rapid Latin America. Containers used for water storage are
urbanisation, the continued challenge of vector control, important aedes breeding sites alongside building
and international travel.2 There are about 50 million to projects, discarded plastics, and increasing numbers of
100 million cases of dengue infection each year, with an unused tyres.13 This increase in available breeding sites,
estimated 500 000 cases of life-threatening disease in the along with daytime feeding and resting in sheltered dark
form of severe dengue—including dengue haemorrhagic spaces in houses, makes aedes an efficient vector.13
fever and dengue shock syndrome.3,4 Children have Although urgently needed, a licensed vaccine for
historically borne the brunt of the disease burden and dengue is not yet available. Control of dengue by
severe dengue has been a leading cause of admission to widespread vaccination has been a priority of WHO for
hospital and death of children in several southeast Asian three decades.16 Vaccines are available for related viruses
countries.2 The age profile of dengue is changing in Asia such as yellow fever, Japanese encephalitis virus, and
and in Latin America, however, and adults are increasingly tick-borne encephalitis virus.17 In this Review, we aim to
being affected.5 Dengue is associated with major urban present an up-to-date understanding of dengue
epidemics that have substantial economic, political, and pathogenesis, how this understanding is influencing the
social effects,6 such as those seen in Delhi (1996),7 development of vaccines, and what progress has been
Cuba (1977–79 and 1997),8 Taiwan (2002),9 and Brazil (2008).5 made in developing a safe and effective dengue vaccine.
Dengue is now also a leading cause of morbidity in
American and European travellers and military Clinical features
personnel.10 Dengue viruses cause a range of well-described clinical
There are four serotypes of dengue virus (designated illnesses.18–20 Infection ranges from an asymptomatic
DENV1–4) belonging to the Flavivirus genus of the infection to a self-limiting febrile illness, dengue fever, to
Flaviviridae family. The dengue viruses fall into a distinct severe dengue, a clinical syndrome that typically presents
group among the flaviviruses, showing a phylogenetic with capillary permeability and can lead to dengue shock
association with the Japanese encephalitis virus group syndrome and dengue haemorrhagic fever. Less common
and more distantly with yellow fever, tick-borne features of severe dengue are encephalitis, hepatitis, and
encephalitis, and hepatitis C viruses.11 The dengue virion renal dysfunction. The WHO classification scheme for
is spherical and 40–50 nm in diameter. The nucleocapsid dengue was developed in the 1970s and is at present
is covered by a lipid bilayer envelope containing the under review because of concerns that it might lead to
envelope and membrane proteins. The genome is single- under-reporting of severe dengue.18,21 What defines the
stranded positive-sense RNA of about 11 kb in length that clinical outcome of dengue infection is not completely
encodes for three structural proteins (the capsid protein, understood, although numerous hypotheses exist.
the membrane protein precursor, and the envelope Epidemiological observations from studies in Thailand
protein) and seven non-structural proteins (NS1, NS2A, in the 1960s showed that greater than 85% of cases of
NS2B, NS3, NS4A, NS4B, and NS5).12 severe dengue happened in people with pre-existing
Dengue viruses are transmitted to human beings heterotypic dengue virus antibodies, and this has led to
through the bite of infected mosquitoes of the Aedes research into dengue pathogenesis focusing on the
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immune response.22 Additional evidence from infant
studies with maternal antibodies lends weight to the
hypothesis of immune involvement in the development
of severe dengue.23 However, severe dengue can also
happen in primary dengue infection, and only a small
proportion of secondary infections progress to severe
disease, which suggests roles for other factors. There is
evidence for links between disease severity and nutritional
status,24 ethnicity,25 virulence of infecting viral strain,8,26,27
and host genetic background.28,29
Naturally acquired immunity
The envelope glycoprotein plays the most important part
in protective immunity to dengue viruses, and contains
the main epitopes recognised by neutralising antibodies.30
Robust neutralising antibody responses develop after
dengue infection and are believed to provide lifelong
protection against reinfection with the same dengue
serotype and short-lived protection, of several months,
against a heterologous dengue serotype.27,31 This naturally
acquired immunity provides optimism for the feasibility
of a dengue vaccine.
The role of dengue-specific cellular immunity in
protection against reinfection is less clearly defined.
T cells might limit the expansion of infection by killing
infected cells and secreting inflammatory cytokines, and
are likely to have a role in viral clearance.32,33 The important
protective features of the cellular response include a high
avidity, homologous response of a T-helper-1-type cell
secreting interferon γ.34,35 However, cellular immunity
cannot be isolated from the antibody response, and any
model of protection against dengue or pathogenesis of
severe disease must take into consideration both arms of
the immune response.36,37
Immunopathogenesis
Antibodies
Epidemiological studies showing an increased risk of
severe dengue in people who had been infected with
the dengue virus in the past led to the hypothesis of
antibody-dependent enhancement of infection.
Antibody-dependent enhancement is believed to
happen because pre-existing non-neutralising
concentrations of antibodies and the infecting dengue
virus form complexes that bind to Fc receptor-bearing
cells, leading to increased virus uptake and replication
thereby increasing viraemia and consequent disease
severity.38–40 Antibody-dependent enhancement is readily
shown in vitro using dengue immune sera or
monoclonal antibodies and cells of monocytic and
B-lymphocytic lineages bearing Fc receptors.38,41,42
Antibody-dependent enhancement has also been shown
in monkeys infused with human dengue immune
serum.43 Further support for the role of antibody-
dependent enhancement in complex disease is provided
by results of infection-enhancement assays in
Vietnamese infants23 implicating maternally derived
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Review
subneutralising levels of dengue antibody as a crucial
risk factor for severe dengue during infancy.
Antibody-enhanced dengue virus infection has been
linked to heightened production of vasoactive cytokines
and chemokines that have roles in the vascular
dysfunction associated with severe dengue.44–47 Antibody-
enhanced infection has also been associated with a
caspase-dependent apoptosis of target cells.48 However, it
is rare for a patient with dengue experiencing a secondary
infection to progress to more severe forms of the disease,
which indicates that other factors must play a part in
pathogenesis.49 In addition, it might be that not all cases
of severe disease result from secondary infection.50 If so,
it follows that antibody-dependent enhancement is
neither sufficient nor entirely necessary for severe
disease. Indeed, in some studies enhancing antibody
activity in pre-illness plasma has been shown not to
predict subsequent disease severity or viraemia in
secondary infection.51 Whatever the role of antibody-
dependent enhancement, it seems that a vaccine inducing
a long-lived neutralising antibody response against all
four serotypes simultaneously should not induce any risk
in this respect.52–54
Cell-mediated immunity
The possible involvement of T cells in the pathogenesis
of dengue was suggested more than two decades ago and
developments in T-cell immunology have refined our
understanding of the key events and mechanisms
involved.55–57 In this model, following the antibody-
dependent enhancement of viral replication in monocytes
and macrophages, viral antigens are presented, in
conjunction with human lymphocyte antigen molecules,
to CD4 and CD8 memory T cells, sensitised during a
previous infection. This leads to cellular proliferation
and release of proinflammatory cytokines such as tumour
necrosis factor α (TNFα) that act directly upon vascular
endothelial cells and result in plasma leakage (figure).58
The T cells generated during a primary dengue infection
respond to variant peptides during the secondary
infection. In a detailed study in Thai children,59 these
T-cell responses showed a lower affinity for the infecting
virus and a higher affinity for another virus serotype,
thus mimicking so-called original antigenic sin. This
leads to a selective expansion of lower avidity cross-
reactive memory T cells that might out-compete the naive
T cells with higher avidity for the infecting serotype.59,60
This response would be less efficient in eliminating
newly encountered dengue virus serotypes and could
lead to increased virus replication and more severe
disease.
The extent of T-cell activation is related to disease
severity23,48 and cross-reactive T cells have been shown to
produce much higher levels of both type 1 and certain
type 2 cytokines, many previously implicated in dengue
pathogenesis.61–64 There is also evidence that previous
infection with other flaviviruses that commonly circulate
679
 Review
Monocyte Interferon γ
HLA- T-cell
peptide receptor
Antigen Memory T-cell
presentation
Fcγ receptor
Non-neutralising antibody Secretion of
vasoactive
Antibody-dependent cytokines:
enhancement TNFα
Interleukin 2
Interferon γ
Platelet
Dengue virus
Endothelium
Dengue virus and antibody complexing with
platelets and activating clotting cascade Plasma leakage
Figure: Schematic representation of the immunopathogenesis of severe dengue disease
with dengue might affect the clinical course of subsequent
infections with other members of the family. CD4 T-cell
clones from patients with dengue infection have been
shown to have substantial epitope cross-reactivity among
flaviviruses.65
Regulatory T cells have been studied in acute infection
because T-cell immunopathology is thought to play a
major part in the pathogenesis of severe disease.
Although regulatory T cells are fully functional, able to
suppress the production of vasoactive cytokines, and
expand in acute dengue infection, this expansion is likely
to be insufficient or happen too slowly to control the
immune response in patients with severe disease.66
Cytokines
Studies have suggested that the capillary permeability
resulting from dengue infection is caused by the
malfunction of vascular endothelial cells induced by
cytokines or chemical mediators rather than by
destruction of the small vessels.30,35,36 Following massive
activation of memory T cells, a cytokine storm targeting
vascular endothelial cells might lead to the crucial
pathological event of capillary permeability and hence
plasma fluid and protein leakage. Patients with severe
disease have higher concentrations of cytokines including
interferon γ, TNFα, and interleukins 2, 6, 1, 8, and 10.30
These findings have been supported by recent studies of
sera from severe dengue patients in Vietnam,67 India,68
and Cuba.69
Vaccines
There is an urgent need for a safe and effective vaccine
for dengue and the features of an ideal dengue vaccine
are outlined in the panel. The association of increased
680
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disease severity in people with heterotypic immunity
necessitates a vaccine that will confer long-term
Apoptosis of protection against all four serotypes.70 All vaccines in
naive T cells development are multivalent and aimed at producing
immunity against all four serotypes. However, whether a
vaccine needs all four serotypes remains an unanswered
question, and will depend on the degree of longlasting
Expansion of
low affinity
cross protection between serotypes. Vaccine-induced
cross-reactive enhancement of disease is not without precedent and
T cells
has been seen in respiratory syncytial virus infection of
children previously immunised with a formalin-
inactivated vaccine.71 Additionally, multiple serotypes
commonly circulate together in a particular geographical
region, with each serotype being capable of causing
disease (although people very rarely have more than one
bout of severe illness, which suggests that there might be
more cross-serotypic protection than has been assumed),
and serotypes can change unpredictably from one season
to the next.72,73 A further challenge for vaccinologists has
been the lack of a suitable animal model with the same
pathological features of human infection. Replication
and immunogenicity can be studied in rhesus macaques,
acknowledged as the best available primate model,
although levels of viraemia are substantially lower than
in human beings and the clinical syndrome is different,
with little or no shock, making extrapolation of research
findings difficult.
Recent advances in molecular biology and vaccinology
have given rise to new hopes with the development of
live attenuated vaccines at present in clinical trials and a
pipeline of candidate inactivated, subunit protein, DNA,
and viral-vector vaccines are in preclinical development.
Progress is being made and phase 1 and 2 studies of
promising vaccine candidates have taken place. Extended
phase 2 and phase 3 trials will start in populations
exposed to dengue, and new guidelines for evaluating
these vaccines have been established by WHO.74 These
guidelines emphasise the need for strong regulatory
infrastructure as well as the importance of establishing
relevant study populations, defined trial clinical and
laboratory endpoints, consistent classification of dengue
syndromes, defined immune features that relate to
protection (including the relative protection afforded by
neutralising antibody vs cellular immunity), and
characterisation of inhibitory or enhancing effects of
earlier flavivirus infections.
Correlates of immune-mediated protection to most
vaccines are still unknown, and this slows the
development of new vaccines. However, recent studies
clarifying the protective mechanisms of the successful
yellow fever vaccine will be invaluable in vaccine
development. Early gene signatures that predict immune
responses in people vaccinated for yellow fever have been
identified that might allow vaccine efficacy to be
established prospectively.75 Additionally, the use of
functional genomics has identified a pattern of immune
responses to yellow fever vaccine that show that the
www.thelancet.com/infection Vol 9 November 2009

immune response to a successful vaccine is preceded by
a coordinated induction of transcription factors leading
to the development of a broad, polyfunctional, and
persistent immune response that integrates all effector
cells of the immune system.76 The candidate vaccines for
dengue that are in various stages of development are
discussed below and summarised in the table.
Chimeric vaccines
The leading candidate vaccine in clinical trials at present
is the ChimeriVax dengue vaccine. Using a new
technology, the premembrane and envelope genes of
yellow fever 17D virus are replaced with those of each
wild-type dengue virus serotype. ChimeriVax dengue
vaccine viruses are then prepared by electroporation of
Vero cells with RNA transcripts prepared from viral
cDNA.77 Three independent, phase 1 clinical trials have
been done in Australia and USA in animals unexposed
or immune to flaviviruses. As in previous studies in non-
human primates (in which they afford protection against
virulent virus challenge), the vaccines were shown to be
safe in both groups and induced interferon γ responses
for both CD4 and CD8 T cells. This vaccine is in phase 2
and soon to enter phase 3 trials.54,77,78
Live attenuated vaccines
The cost effectiveness, safety, long-term immunity, and
efficacy associated with the live attenuated vaccines
against the flaviviruses yellow fever virus and Japanese
encephalitis virus serve as a model for the feasibility of a
live attenuated dengue vaccine.
Live attenuated vaccines have been among the leading
vaccine candidates for dengue and could provide an
economically viable approach to inducing durable
humoral and cellular immunity.88,98 Neutralising
antibodies are known to mediate protection from
reinfection with a homologous serotype, and long-term
immunity to dengue viruses has been described.99
Different strategies have been adopted to develop
monovalent live attenuated vaccines against each of the
four dengue serotypes that balance attenuation and
immunogenicity. The virus must be able to replicate
sufficiently well in vivo to provoke an immune response,
but must be sufficiently restricted in systemic replication
to avoid inducing any of the dengue-associated symptoms.
The vaccines need to induce a broad neutralising antibody
response against each serotype, because if the four vaccine
viruses interfere with virus replication they could result in
preferential antibody responses to the better replicating
components, leading to disease susceptibility.100,101
For over 20 years, monovalent live attenuated vaccine
candidates, propagated and attenuated in primary and
diploid cell cultures have been evaluated in human
beings.79–81 Initially, many candidates were either under-
attenuated with unacceptable side-effect profiles or over-
attenuated and lacking desirable immunogenicity. An
acceptable balance was ultimately achieved by Halstead
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Review
Panel: Characteristics of the ideal dengue vaccine
• Good safety profile
• Rapid immunisation regime requiring a single vaccine or
two that fit in with established vaccine programmes
• Balance between reactogenicity and immunogenicity
• Suitable for use in target age groups
• Genetically stable
• Stimulates neutralising antibodies and T-helper-1
cell-mediated immunity
• Provide longlasting immunity
• Generates neutralising immunity to all four serotypes
• Does not contribute to immunopathogenesis
(vaccine-induced enhancement)
• Easily stored and transported
• Affordable and cost effective
and Marchette82 who used primary dog kidney cells. Several
monovalent vaccines passaged through primary dog
kidney cells were developed and combined into several
tetravalent formulations that have been tested in phase 1
and 2 trials in American adult volunteers or Thai adults
and children where they were shown to be safe and
immunogenic.52,53,80,81,83,102–104 However, the leading live
attenuated vaccine candidates have encountered problems
and plans for phase 3 studies have been suspended.
Reverse genetics allows the recovery of well-defined,
genetically homogeneous virus populations from cloned
cDNA in tissue-culture cells.100 Using recombinant DNA
technology, panels of mutant DENV4 viruses with engine-
ered deletions of varying lengths in the 3´ untranslated
region were generated.105 One such deletion mutant,
lacking nucleotides 10 478–10 507 from the 3´untranslated
region (now referred to as rDEN4Δ30), does not produce
detectable viraemia after inoculation of rhesus monkeys
but does induce high levels of neutralising antibodies.105,106
Importantly however, it replicates to very high titres in
Vero cells that are suitable for vaccine production.100 The
vaccine was found to be safe and highly immunogenic
even at low doses. However, asymptomatic rash, neutro-
penia, and raised serum aminotransferase concentrations
were noted in some vaccinees receiving the highest dose
tested.84 A further mutation has been introduced to
generate the vaccine rDEN4Δ30-200,201 that shows
retained immunogenicity with an improved safety
profile.88
Since the Δ30 mutation in DENV4 is in the non-
structural gene regions of the virus, this provides a
genetic DENV4 backbone for the creation of chimeric
viruses containing the structural genes encoding the
capsid, premembrane, and envelope glycoproteins of the
other dengue serotypes.85,86 These have been shown to be
safe and immunogenic although studies are needed to
evaluate tetravalent combinations.87 Low doses are needed
to induce immunity that could make these vaccines
economical to manufacture.87
681
 Review

Details Phase of Comment

clinical trial
Chimeric
ChimeriVax (Acambis/ Recombinant infectious cDNA clone of yellow fever 17D vaccine 2b Leading candidate; safe and immunogenic in
Sanofi Pasteur)54,77,78 strain as a backbone, substituting membrane precursor protein human trials
and envelope protein genes with those of dengue viruses
Live attenuated
Mahidol University Passage in primary cell culture 2* Monovalent vaccines show good immune
(Sanofi Pasteur)52,53,79–83 responses; difficulties with tetravalent
formulations
WRAIR (GSK)52,53,79–83 Passage in primary cell culture 2* Monovalent vaccines show good immune
responses; difficulties with tetravalent
formulations
Infectious clone
rDEN4Δ30 (NIAID)84–87 30 nucleotide deletion from DENV4 3’ untranslated region as 1 Monovalent vaccines show promise; tetravalent
genetic backbone for vaccines with structural genes from other formulations to be evaluated
serotypes
rDEN4Δ30-200,201 Further mutation in rDEN4Δ30 construct 1 Retained immunogenicity of rDEN4Δ30, but
(NIAID)88 with improved safety profile
Inactivated
WRAIR89 Whole purified inactivated virus Preclinical Safe and immunogenic in rhesus macaques with
evidence of efficacy
Replication-incompetent
RepliVax (Novartis)90 Capsid gene-deleted WNV with membrane precursor protein and Preclinical Immunogenicity and efficacy shown in mice
envelope protein genes substituted for dengue genes
Protein
r80E (Hawaii Amino-terminal 80% of the DENV2 envelope with adjuvants Preclinical Immunogenic with non-sterilising immunity in
Biotechnology)89 mice
cEDIII (IPK/CIGB)91 Consensus dengue virus envelope protein domain III of all four Preclinical Immunogenic in mice
serotypes
DNA
US Navy92–95 Several encoding membrane precursor protein and envelope Preclinical Immunogenic with very short-lived protection
protein genes
Virus vector
Adenovirus Tetravalent formulation combining two bivalent adenovirus Preclinical Neutralising antibody responses and short-term
(GenPhar Inc)96 constructs and long-term protection against challenge
from each serotype in rhesus macaques
Measles virus (CNRS)97 Expression of a DENV1 antigen by a vector derived from live- Preclinical Long-term production of dengue neutralising
attenuated Schwarz measles vaccine antibody in mice

*Development suspended.

Table: Candidate dengue vaccines in development

Inactivated vaccines protection against viraemia (by bioassay) in a virulent

Inactivated vaccines have attracted less attention than
live attenuated vaccines because they are likely to induce
a less robust immune response (failing to induce
T-helper-1 cellular immunity) that is less durable, are
likely to need multiple inoculations, and are more
expensive to produce. However, unlike vaccine
formulations consisting of four different attenuated
viruses, they are likely to induce a more reliably consistent
immune response (reducing fears of vaccine-induced
enhancement of disease) and would be safe in people
that are immunocompromised. The safety, immuno-
genicity, and protective efficacy of a whole purified
inactivated virus formulated with one of five different
adjuvants has been evaluated in rhesus macaques. High
concentrations of neutralising antibodies were observed
after booster immunisation with nearly complete
dengue challenge, although the vaccine did not confer
sterile immunity.89 To date, there have been no clinical
trials of inactivated dengue vaccines in people.
Replication-incompetent vaccines
The new approach of creating replication-incompetent
(encapsidation-defective) viruses has been taken to resolve
the problem of increasing the immunogenicity of
inactivated vaccines while maintaining their safety profile.90
These are capsid gene-deleted viruses, named RepliVAX,
that undergo only a single cycle of infection in vaccinated
hosts. A DENV2 vaccine has been generated by replacing
the premembrane and envelope genes of RepliVAX WN
(a West Nile virus vaccine construct). After passage the
vaccine developed further mutations enabling efficient
growth in suitable cell lines (named RepliVAX DEN2.2)

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and has been shown to have good immunogenicity and
efficacy in an established immunodeficient mouse model
of dengue.90 Similar vaccines have been produced for
West Nile virus and Japanese encephalitis virus, which are
potent and efficacious in animals, showing potential use of
this technology for the production of third-generation
flavivirus vaccines that should be potent, economical to
produce, and safe in immunocompromised people.107
Subunit vaccines
The use of recombinant DNA technology as a means of
generating subunit protein vaccines in various expression
systems represents a substantial advance. However,
many of these non-infectious, subunit vaccines are poorly
immunogenic and need potent immunostimulatory
adjuvants that activate the innate immune system and
thereby elicit antigen-specific immune responses.108–111
Aluminium-containing adjuvants are an important
component of many vaccines, but few new adjuvants
have received approval because of concerns of adverse
effects.111 Monophosphoryl lipid A is a detoxified form of
the endotoxin lipopolysaccharide, and is among the first
of a new generation of toll-like receptor agonists likely to
be used as vaccine adjuvants on a mass scale in human
populations.109
A recombinant subunit protein containing the amino-
(N)-terminal 80% of the DENV2 envelope protein (named
r80E) has been studied in formulations with one of five
different adjuvants based on aluminium, fraction QS-21
of saponin, and monophosphoryl lipid A. In mice, all
formulations were immunogenic with evidence of non-
sterilising protective efficacy.89
A new tetravalent protein vaccine candidate has been
developed that comprises a consensus dengue virus
envelope protein domain III (cED III) obtained by
alignment of aminoacid sequences from different isolates
of all four serotypes. Formulation of recombinant cED III
with aluminium phosphate induces cross-neutralising
antibodies and memory immunity after challenge with
dengue virus in Balb/c mice.91
DNA vaccines use plasmids of double-stranded DNA
in which the genetic sequence for a protein or series of
epitopes has been inserted under the control of a
mammalian promoter. They are usually given
intramuscularly and are taken up by muscle and dendritic
cells, where the genetic information for the encoded
protein is translated.108 Several dengue DNA vaccines
(encoding premembrane and envelope genes) have been
studied in mice and monkeys.92–95 In aotus monkeys, a
DENV3 DNA vaccine elicited a neutralising antibody
response and partly protected against live virus
challenge;95 a DENV1 DNA vaccine was capable of
inducing neutralising antibodies in rhesus macaques
after multiple inoculations but with very short-lived
protection.94 Although DNA vaccines showed early
promise in other disease models in animals,112 DNA
vaccination alone generally elicits only weak immune
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Review
responses in people113,114 although they are effective at
priming cellular immune responses, including those of
T-helper-1-type CD4 T cells and CD8 cytotoxic T cells, for
boosting by recombinant viruses.115,116 DNA vaccines are
expensive to produce and are likely to need complicated
immunisation regimens; therefore they are unlikely to
be of real use for prophylactic vaccination in resource-
poor settings.
Several different virus vectors have been investigated
as flaviviral vaccines. Poxvirus vectors, such as modified
vaccinia virus Ankara and fowlpox are attractive
candidates because they are able to tolerate the insertion
of large amounts of foreign genetic material and are safe
and immunogenic (particularly in heterologous prime-
boost combinations) in the study of other major
worldwide infectious diseases such as malaria,117,118
tuberculosis,119 and HIV.120 However, an attenuated
vaccinia virus (NYVAC) and a canarypox vector (ALVAC)
when used to generate Japanese encephalitis virus
vaccines were insufficiently immunogenic.121
Subsequently, research efforts have targeted replication-
deficient adenoviruses that are safe and easy to produce
and store.96 A tetravalent vaccine formulated by mixing
two bivalent adenovirus constructs (named CAdVax-
Den12 and CAdVax-Den34) elicited a neutralising
antibody response to all four serotypes and provided both
short-term and long-term protection against challenges
from each of the four serotypes in rhesus macaques.96 To
bypass pre-existing immunity to human adenoviruses
and to improve vaccine efficacy, vaccine vectors based on
simian adenoviruses have been generated and found to
be immunogenic for antibodies and T cells in mice.122
However, a major setback for adenovirus vaccines came
with the results of the STEP HIV adenovirus vaccine trial
in which the vaccine appeared to increase the rate of HIV
infection in people that had already acquired immunity
to the adenovirus vector.123 Whether the failure of this
trial is attributable to something specific about the Merck
vector or whether all adenovirus vectors will face similar
problems remains to be seen.
The measles vaccine is another potential vaccine vector.
Over the past 30 years, it has been given to hundreds of
millions of children and has proved to be both effective
and safe. This attenuated live virus induces lifelong
immunity after only one or two injections and can be
produced on a large scale and at a low cost.124 A new
vaccine has been developed based on the expression of a
minimal dengue antigen (of DENV1) by a vector derived
from paediatric live-attenuated Schwarz measles vaccine.
Immunisation of mice resulted in a long-term production
of DENV1 serotype-specific neutralising antibodies.97 The
protective efficacy of this vaccine should be further
evaluated in non-human primates. A combined measles–
dengue vaccine might provide an opportunity to
immunise children against both diseases where they
coexist, although immunological interference would
need to be evaluated in people.97
683
 Review
Search strategy and selection criteria
Data for this Review were identified by searching PubMed for
relevant articles. Search terms included “dengue”,
“epidemiology”, “vaccine”, “pathogenesis”,
“immunopathogenesis”, “antibody”, “T-cell”, and “cell-
mediated”. Articles were selected on the basis of their relevance
to dengue vaccine development. No date restrictions were
used. Although all included publications were in English, the
search was not restricted to English language articles.
Unanswered questions
Substantial progress has been made with dengue vaccine
development and yet there remain a number of questions
that need to be answered before the widespread use of
any candidate vaccine. Preclinical and clinical studies are
needed to address these issues and well-designed vaccine
studies could also contribute to our understanding.
Preclinical
Questions that need answering through preclinical
studies relate to the immune correlates of protection and
severe disease, and if virus-to-virus interference in
tetravalent formulations inhibits the antibody response
to one or more serotype in the vaccine when used in
endemic areas. With respect to the immune correlates,
appropriate and improved laboratory tests need to be
developed to find what constitutes protective immune
responses and which responses might predispose to
severe disease.
Clinical
Clinical studies are needed to answer many further
questions, such as the duration of protection afforded by
candidate vaccines. Mass immunisation with a vaccine
that only affords short-lived protection (even if this is
only to one serotype) could simply make whole
populations susceptible to the more severe forms of
disease. Further clinical questions relate to the effect of
pre-existing immunity to other flaviviruses (through
natural exposure or immunisation), the safety of
candidate vaccines to infants (so far there have been no
studies in infants in whom safety profiles and immune
response kinetics might differ from adults), the
appropriate age to immunise, and if these vaccines are
safe for people infected with HIV.
Implementation
Beyond questions for preclinical and clinical studies are
questions about the logistics of a vaccine implementation
strategy. Answers are needed on how long follow-up data
will be needed to confirm long-term safety, the age of the
targeted vaccinees, the proportion of vaccine coverage
needed for herd immunity, and, crucially, what the
vaccine will do—will it stop all disease or just severe
disease? Other questions relate to the need to regularly
684
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update the vaccine as dengue viruses evolve and if this
will drive evolution, whether the vaccine can be produced
at a cost acceptable to endemic countries, and the cost-
effectiveness of dengue prevention through vaccination.
Comparisons need to be made of different vaccination
strategies and vector control programmes as well as the
estimated cost-effectiveness of dengue prevention
compared with potential treatment with new antiviral
drugs.
Conclusion
Progress with the development of dengue vaccines has
accelerated but, alongside this, the scale of the problem
has rapidly grown and a safe effective vaccine remains
an urgent worldwide priority. Several candidate vaccines
have been developed that have shown promise in
phase 2 clinical studies. The leading candidate must
now be taken forward into larger studies in appropriate
populations in endemic areas. A coordinated approach
is needed to ensure consistency, with defined endpoints
that can be extrapolated between different studies and
that will also contribute to our understanding of dengue
immunity and pathogenesis. Ideally, a defined structure
linking organisations involved in vaccine development
and coordinated by WHO would allow the sharing of
information from vaccine studies particularly in relation
to adverse events, safety, and knowledge gained on the
potential for immune enhancement during trials.
Convincing arguments need to be in place for
introducing dengue vaccine initiatives. The
socioeconomic effect of dengue is substantially larger
than that attributable to severe dengue alone and this
effect needs to be considered in the case for vaccine
licensure and use alongside the cost-effectiveness of
other approaches.74
The opportunity now exists to bring effective and safe
dengue vaccines to populations at greatest risk, and there
is unprecedented momentum to bring about a
coordinated worldwide effort.
Contributors
The content and structure of this Review were agreed by all authors.
DPW searched the published work and prepared the first draft.
Contributions were received from JF and SR-J. All authors edited the
final draft.
Conflicts of interest
We declare that we have no conflicts of interest.
Acknowledgments
DPW is an employee of the Oxford Radcliffe Hospitals NHS Trust, UK.
JF is funded by the Wellcome Trust. SR-J is funded by the Medical
Research Council, UK.
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